CH2 Region Research Articles

The mouse monoclonal antibody 4E3 is specific for the G1m17 allotype of human IgG1

Allotype is an amino acid variation within the immunoglobulin isotypes. Four allotypes have been described for human IgG1 and two of them (G1m3 and G1m17) are located at position 214 in the CH1 region of the gamma chain. Various diseases have been associated with allotype expression, making the allotype research an interesting field in immunology. However, allotype-specific reagents are rare. In the present study the specificity of a widely used and commercially available IgG1-specific monoclonal antibody named 4E3, described as binding to the hinge region of IgG1, was evaluated. Using the ImmunoCAP™ assay technology, surprisingly no IgG1 was measured in 13 of 23 human serum and plasma samples when 4E3 was used in an antibody-enzyme conjugate as detection reagent. Further evaluation of 4E3 using eight IgG1 myeloma paraproteins revealed that 4E3 did not bind to three of them. No association was seen between the binding pattern and myeloma light chain glycosylation or the kappa or light chain use. By comparing the myeloma paraprotein binding patterns of 4E3 and TM14 (a monoclonal antibody with known G1m3 specificity), it was indicated that 4E3 only bound to myeloma paraproteins expressing the G1m17 allotype. This was confirmed using recombinant human antibodies expressing either the G1m3 or G1m17 allotype. The G1m17 bias of 4E3 seen using ImmunoCAP was also observed in microtiter plate-based enzyme-linked immunosorbent assays. The antibody 4E3 has a G1m17 bias limiting its use in assays to measure IgG1 antibodies. However, it may be a valuable allotype-specific reagent.

Bovine ultralong CDR-H3 derived knob paratopes elicit potent TNF-α neutralization and enable the generation of novel adalimumab-based antibody architectures with augmented features.

In this work we have generated cattle-derived chimeric ultralong CDR-H3 antibodies targeting tumor necrosis factor α (TNF-α) via immunization and yeast surface display. We identified one particular ultralong CDR-H3 paratope that potently neutralized TNF-α. Interestingly, grafting of the knob architecture onto a peripheral loop of the CH3 domain of the Fc part of an IgG1 resulted in the generation of a TNF-α neutralizing Fc (Fcknob) that did not show any potency loss compared with the parental chimeric IgG format. Eventually, grafting this knob onto the CH3 region of adalimumab enabled the engineering of a novel TNF-α targeting antibody architecture displaying augmented TNF-α inhibition.

Chimerism of avian IgY-scFv and truncated IgG-Fc: A novel strategy in cross-species antibody generation and enhancement.

Chicken single-chain fragment variable (IgY-scFv) is a functional fragment and an emerging development in genetically engineered antibodies with a wide range of biomedical applications. However, scFvs have considerably shorter serum half-life due to the absence of antibody Fc region compared with the full-length antibody, and usually requires continuous intravenous administration for efficacy. A promising approach to overcome this limitation is to fuse scFv with immunoglobulin G (IgG) Fc region, for better recognition and mediation by the neonatal Fc receptor (FcRn) in the host. In this study, engineered mammalian ΔFc domains (CH2, CH3, and intact Fc region) were fused with anti-canine parvovirus-like particles avian IgY-scFv to produce chimeric antibodies and expressed in the HEK293 cell expression system. The obtained scFv-CH2, scFv-CH3, and scFv-Fc can bind with antigen specifically and dose-dependently. Surface plasmon resonance investigation confirmed that scFv-CH2, scFv-CH3, and scFv-Fc had different degrees of binding to FcRn, with scFv-Fc showing the highest affinity. scFv-Fc had a significantly longer half-life in mice compared with the unfused scFv. The identified ΔFcs are promising for the development of engineered Fc-based therapeutic antibodies and proteins with longer half-lives. The avian IgY-scFv-mammalian IgG Fc region opens up new avenues for antibody engineering, and it is a novel strategy to enhance the rapid development and screening of functional antibodies in veterinary and human medicine.

Sequence and structure statistics of the nanobody database and molecular dynamics simulation analysis of the nanobody VHH

Antibodies are immunoglobulins produced in vivo by immune cells stimulated by exogenous molecules, and nano-antibodies are a class of molecules that are similar to conventional antibodies but smaller in size. Originally discovered as an antibody in camelidsand cartilaginous fishes, they are called heavy chain antibodies due to the lack of light and heavy chain constant regions in the CH1 region except for the retained heavy chain, and their binding region to the antigen consists of the heavy chain variable region only, making them the smallest antibodies available with complete antibody functional properties. In this experiment, we analyzed the commonalities and differences of the nanobodies by extracting all their amino acid sequences, performed protein modeling of the relevant nanobodies of coronaviruses among them, and then analyzed the data of RMSD and RMSF by using molecular dynamics simulation, and finally predicted and analyzed the protein conformation and the structures of VHH at all levels.

Dissecting phospho-motif-dependent Shc1 interactome using long synthetic protein fragments.

Activated receptor tyrosine kinases (RTKs) rely on the assembly of signaling proteins into high-dimensional protein complexes for signal transduction. Shc1, a prototypical scaffold protein, plays a pivotal role in directing phosphotyrosine (pY)-dependent protein complex formation for numerous RTKs typically through its two pY-binding domains. The three conserved pY sites within its CH1 region (Shc1CH1) hold particular significance due to their substantial contribution to its functions. However, how Shc1 differentially utilizes these sites to precisely coordinate protein complex assembly remains unclear. Here, we employed multiple peptide ligation techniques to synthesize an array of long protein fragments (107 amino acids) covering a significant portion of the Shc1CH1 region with varying phosphorylation states at residues Y239, 240, 313, and S335. By combining these phospho-Shc1CH1 fragments with integrated proteomics sample preparation and quantitative proteomic analysis, we were able to comprehensively resolve the site-specific interactomes of Shc1 with single amino acid resolution. By applying this approach to different cancer cell lines, we demonstrated that these phospho-Shc1CH1 fragments can be effectively used as a diagnostic tool to assess cell type-specific RTK signaling networks. Collectively, these biochemical conclusions help to better understand the sophisticated organization of pY-dependent Shc1 adaptor protein complexes and their functional roles in cancer.

Subclass Effects on Self-Association and Viscosity of Monoclonal Antibodies at High Concentrations.

The effects of a subclass of monoclonal antibodies (mAbs) on protein-protein interactions, formation of reversible oligomers (clusters), and viscosity (η) are not well understood at high concentrations. Herein, we quantify a short-range anisotropic attraction between the complementarity-determining region (CDR) and CH3 domains (KCDR-CH3) for vedolizumab IgG1, IgG2, or IgG4 subclasses by fitting small-angle X-ray scattering (SAXS) structure factor Seff(q) data with an extensive library of 12-bead coarse-grained (CG) molecular dynamics simulations. The KCDR-CH3 bead attraction strength was isolated from the strength of long-range electrostatic repulsion for the full mAb, which was determined from the theoretical net charge and a scaling parameter ψ to account for solvent accessibility and ion pairing. At low ionic strength (IS), the strongest short-range attraction (KCDR-CH3) and consequently the largest clusters and highest η were observed with IgG1, the subclass with the most positively charged CH3 domain. Furthermore, the trend in KCDR-CH3 with the subclass followed the electrostatic interaction energy between the CDR and CH3 regions calculated with the BioLuminate software using the 3D mAb structure and molecular interaction potentials. Whereas the equilibrium cluster size distributions and fractal dimensions were determined from fits of SAXS with the MD simulations, the degree of cluster rigidity under flow was estimated from the experimental η with a phenomenological model. For the systems with the largest clusters, especially IgG1, the inefficient packing of mAbs in the clusters played the largest role in increasing η, whereas for other systems, the relative contribution from stress produced by the clusters was more significant. The ability to relate η to short-range attraction from SAXS measurements at high concentrations and to theoretical characterization of electrostatic patches on the 3D surface is not only of fundamental interest but also of practical value for mAb discovery, processing, formulation, and subcutaneous delivery.

Enhanced neutralization potency of an identical HIV neutralizing antibody expressed as different isotypes is achieved through genetically distinct mechanisms

Antibodies with the same variable region can exist as multiple isotypes with varying neutralization potencies, though the mechanism for this is not fully defined. We previously isolated an HIV-directed IgA1 monoclonal antibody (mAb), CAP88-CH06, and showed that IgA1 and IgG3 isotypes of this antibody demonstrated enhanced neutralization compared to IgG1. To explore the mechanism behind this, hinge region and constant heavy chain (CH1) chimeras were constructed between the IgA1, IgG3 and IgG1 mAbs and assessed for neutralization activity, antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC). Hinge chimeras revealed that the increased neutralization potency and phagocytosis of the IgG3 isotype was attributed to its longer hinge region. In contrast, for IgA1, CH1 chimeras showed that this region was responsible both for enhanced neutralization potency and decreased ADCP, though ADCC was not affected. Overall, these data show that the enhanced neutralization potency of CAP88-CH06 IgG3 and IgA1, compared to IgG1, is achieved through distinct mechanisms. Understanding the influence of the hinge and CH1 regions on Fab domain function may provide insights into the engineering of therapeutic antibodies with increased neutralization potency.

Dual-Targeting Polymer Nanoparticles Efficiently Deliver DNA Vaccine and Induce Robust Prophylactic Immunity against Spring Viremia of Carp Virus Infection.

ABSTRACTSpring viremia of carp virus (SVCV) is highly contagious and lethal to most cyprinid fish, causing serious economic losses to the carp aquaculture industry. Although DNA vaccines can generate long-term humoral and cellular immune responses, which provide protective immunity against SVCV, the major drawback of DNA vaccines is their low immunogenicity in clinical tests. Here, we construct a dual-targeted polymer DNA vaccine delivery platform (MCS-PCHG) by using mannosylated chitosan to encapsulate the poly(d,l-lactide-co-glycolide)-loaded DNA vaccine containing the heavy-chain CH3 region (CH3) of common carp IgM and the antigenic domain (G131c). The developed nanovaccine delivery platform showed good biocompatibility in vivo and in vitro. With the modification of the mannose moiety and the modification of CH3, the constructed MCS-PCHG could efficiently activate the maturation of antigen-presenting cells. Moreover, we observe significantly high level of immune-related genes expression, serum antigen-specific IgM, SVCV-neutralizing antibody titers in fish vaccinated with MCS-PCHG. Next, the protective efficacy of MCS-PCHG was further evaluated by challenge test. The highest survival rate (ca. 84%) was observed in fish vaccinated with MCS-PCHG after challenging with SVCV. This study presents a novel design for smart, dual-targeted polymer nanoparticles, which are inherently biocompatible, promising for targeted vaccine delivery.IMPORTANCE Spring viremia of carp virus (SVCV) affects global cyprinid fish farming industry, with no available commercial vaccine. Herein, we developed a dual-targeting polymer nanovaccine (MCS-PCHG) by using mannose and common carp IgM heavy chain CH3 region (CH3) as antigen presenting cell (APCs) recognition moiety, attaining the effective delivery of antigen. This dual-targeting polymer vaccine can efficiently activate the APCs, and further induce robust and durable adaptive immune response with good protection against SVCV infection. Our study provides valuable theoretical basis for developing efficient vaccine against infectious diseases in aquaculture.

Enzymatic basis of the Fc-selective intra-chain disulfide reduction and free thiol content variability in an antibody produced in Escherichia coli

BackgroundEscherichia coli (E. coli) is a promising host for production of recombinant proteins (including antibodies and antibody fragments) that don’t require complex post-translational modifications such as glycosylation. During manufacturing-scale production of a one-armed antibody in E. coli (periplasmic production), variability in the degree of reduction of the antibody’s disulfide bonds was observed. This resulted in variability in the free thiol content, a potential critical product quality attribute. This work was initiated to understand and prevent the variability in the total free thiol content during manufacturing.ResultsIn this study, we found that the reduction in antibody’s disulfide bonds was observed to occur during homogenization and the ensuing homogenate hold step where in the antibody is exposed to redox enzymes and small molecule reductants present in homogenate. Variability in the downstream processing time between the start of homogenization and end of the homogenate hold step resulted in variability in the degree of antibody disulfide bond reduction and free thiol content. The disulfide bond reduction in the homogenate is catalyzed by the enzyme disulfide bond isomerase C (DsbC) and is highly site-specific and occurred predominantly in the intra-chain disulfide bonds present in the Fc CH2 region. Our results also imply that lack of glycans in E. coli produced antibodies may facilitate DsbC accessibility to the disulfide bond in the Fc CH2 region, resulting in its reduction.ConclusionsDuring E. coli antibody manufacturing processes, downstream processing steps such as homogenization and subsequent processing of the homogenate can impact degree of disulfide bond reduction in the antibody and consequently product quality attributes such as total free thiol content. Duration of the homogenate hold step should be minimized as much as possible to prevent disulfide bond reduction and free thiol formation. Other approaches such as reducing homogenate temperature, adding flocculants prior to homogenization, using enzyme inhibitors, or modulating redox environments in the homogenate should be considered to prevent antibody disulfide bond reduction during homogenization and homogenate processing steps in E. coli antibody manufacturing processes.

Vibrio cholerae Chromosome Partitioning without Polar Anchoring by HubP.

Partition systems are widespread among bacterial chromosomes. They are composed of two effectors, ParA and ParB, and cis acting sites, parS, located close to the replication origin of the chromosome (oriC). ParABS participate in chromosome segregation, at least in part because they serve to properly position sister copies of oriC. A fourth element, located at cell poles, is also involved in some cases, such as HubP for the ParABS1 system of Vibrio cholerae chromosome 1 (ch1). The polar anchoring of oriC of ch1 (oriC1) is lost when HubP or ParABS1 are inactivated. Here, we report that in the absence of HubP, ParABS1 actively maintains oriC1 at mid-cell, leading to the subcellular separation of the two ch1 replication arms. We further show that parS1 sites ectopically inserted in chromosome 2 (ch2) stabilize the inheritance of this replicon in the absence of its endogenous partition system, even without HubP. We also observe the positioning interference between oriC1 and oriC of ch2 regions when their positionings are both driven by ParABS1. Altogether, these data indicate that ParABS1 remains functional in the absence of HubP, which raises questions about the role of the polar anchoring of oriC1 in the cell cycle.

Application of frankincense and rice starch as eco-friendly substances for the resizing of paper as a conservation practice

Smart, environmentally friendly alternatives, i.e., frankincense and rice starch, are recommended for usage in modern paper conservation processes during the re-sizing process treatments. Different concentrations of frankincense and rice starch were applied to paper samples before and after ageing. Multiple analysis methods were performed to ensure the effectiveness of these materials. Promising results were found, but at varying degrees according to the type and concentration of the materials. Scanning electron microscopy illustrated that the frankincense particles were completely absorbed into the cell walls after ageing. Results indicated that there was no considerable change in pH before and after treatment or ageing; the best results for decreasing the acidity utilized a treatment with a mixture of frankincense and rice starch in a 2 to 1 ratio (F2S1). Fourier transform infrared spectroscopy illustrated an increased CH2 region and decreased OH stretching as a result of the bonds formed from the starch and crystals formed by frankincense, which agreed with the increased coating and strength of the paper fibers. The total color change values of all the treated samples after ageing were less than 4.5. Frankincense was found to provide strength in supporting wood fibers.

2O Penpulimab, an IgG1 anti-PD-1 antibody with Fc-engineering to eliminate effector functions and with unique epitope and binding properties

Currently marketed anti-PD-1 antibodies are typically IgG4 isotype, of which bindings to FcγRs and CH3 region instability might lead to compromise on efficacy and safety. Penpulimab is a humanized IgG1 anti-PD-1 antibody engineered at the Fc region to eliminate binding to FcγRs and C1q, and thus avoid antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) to minimize lymphocyte loss, and to avoid antibody dependent cytokine release (ADCR) such as IL-8 and IL-6, which are known to associate with irAEs and poor prognosis in checkpoint blocking immunotherapy.

Molecular characterisation of parvorder Platyrrhini IgG sub-classes

Non-human primates (NHP) are essential in modern biomedical research; New World monkeys (NWM) are mainly used as an experimental model regarding human malaria as they provide useful information about the parasite’s biology and an induced immune response. It is known that a vaccine candidate’s efficacy is mediated by a protection-inducing antibody response (IgG).Not enough information is available concerning IgG subclasses’ molecular characteristics regarding NHP from parvorder Platyrrhini. Understanding the nature of the humoral immune response and characterising the IgG subclasses’ profile will provide valuable information about the immunomodulator mechanisms of vaccines evaluated using an NHP animal model.This article has characterised IgG subclasses in NWM (i.e. genera Aotus, Cebus, Ateles and Alouatta) based on the amplification, cloning and sequencing of the immunoglobulin heavy constant gamma (IGHG) gene’s CH1 to CH3 regions. The resulting sequences enabled elucidating IGHG gene organisation; two IgG variants were found in the Aotus and Ateles monkey group and three IgG variants in the Cebus and Alouatta group. The sequences were highly conserved in Platyrrhini and had a similar structure to that reported for monkeys from parvorder Catarrhini. Such information will help in developing tools for a detailed characterisation of the humoral immune response in an NWM experimental animal model.

Site-Specific Antibody Conjugation to Engineered Double Cysteine Residues.

Site-specific antibody conjugations generate homogeneous antibody-drug conjugates with high therapeutic index. However, there are limited examples for producing the site-specific conjugates with a drug-to-antibody ratio (DAR) greater than two, especially using engineered cysteines. Based on available Fc structures, we designed and introduced free cysteine residues into various antibody CH2 and CH3 regions to explore and expand this technology. The mutants were generated using site-directed mutagenesis with good yield and properties. Conjugation efficiency and selectivity were screened using PEGylation. The top single cysteine mutants were then selected and combined as double cysteine mutants for expression and further investigation. Thirty-six out of thirty-eight double cysteine mutants display comparable expression with low aggregation similar to the wild-type antibody. PEGylation screening identified seventeen double cysteine mutants with good conjugatability and high selectivity. PEGylation was demonstrated to be a valuable and efficient approach for quickly screening mutants for high selectivity as well as conjugation efficiency. Our work demonstrated the feasibility of generating antibody conjugates with a DAR greater than 3.4 and high site-selectivity using THIOMABTM method. The top single or double cysteine mutants identified can potentially be applied to site-specific antibody conjugation of cytotoxin or other therapeutic agents as a next generation conjugation strategy.

Maria: Accurate Prediction of MHC-II Peptide Presentation with Deep-Learning and Lymphoma Patient MHC-II Ligandome

Background: The presentation of antigens by Major Histocompatibility Complex Class II (MHC-II) is an essential component of adaptive immune response. By combining whole exome sequencing and tandem mass spectrometry (LC-MS/MS), we recently demonstrated that MHC-II presented immunoglobulin neoantigens are common recognition targets in mantle cell lymphoma (MCL) [Khodadoust et al 2017 Nature]. While patient proteomic data can be difficult to obtain, computational methods can learn from these data to predict cancer neoantigen presentation informing personalized immunotherapeutic strategies across cancers. Unfortunately, current tools for predicting peptide presentation by MHC-II have major limitations due to the complexity of presentation pathways and the promiscuity of binding motifs for MHC-II alleles. We hypothesized that a method trained on naturally presented MHC-II ligandomes integrating both sequence and gene expression features could better predict presentation of tumor neoantigens.Method: We trained a recurrent neural network (RNN) model on 19 mantle cell lymphoma MHC-II ligandomes (>30,000 sequences) to build MARIA (MHC Analysis with RNN Integrated Architecture). MARIA is a deep learning algorithm that predicts peptide MHC-II presentation probabilities based on peptide sequences, neighboring context in each protein (cleavage signatures), patient MHC alleles, and gene expression levels. We evaluated the performance of MARIA with 10-fold cross-validation and also using held out data from both B-cell lymphoma and melanoma patients.Results: Gene expression levels and cleavage signatures of corresponding peptides have a profound influence on MHC-II peptide presentation but are not incorporated in standard prediction algorithms (Figure 1a). MARIA presentation scores achieved over 0.93 AUC under cross-validation on validated MHC-II ligands from our lymphoma dataset (Figure 1a). In comparison, predicted binding scores alone gave only 0.70 AUC, and conventional shallow neural network models (e.g., NetMHCIIpan3.1) gave 0.87 AUC when trained on the same dataset. When tested on held-out lymphoma and melanoma empirical ligandome data, MARIA sustained over 70% sensitivity with 90% specificity for detection of MHC-II ligands. Though MARIA was exclusively trained on non-immunoglobulin human sequences, it correctly predicted IgM presentation hot spots discovered by direct antigen presentation profiling using LC-MS/MS (Figure 1b), as well as hotspots in alpha-gliadin, a known Celiac Disease antigen, in an HLA-restricted fashion.Conclusion: MARIA enables high throughput antigen screening with higher accuracy than other methods. It can be applied to immunology applications such as vaccine design, patient profiling, and neo- and auto-antigen identification.Figure 1. Performance of MARIA predicting human MHC-II peptide presentation. a) Five different predictors of MHC-II peptide presentation were used to differentiate 3290 validation MHC-II peptides from 7500 random human decoy peptides. MARIA scores that incorporate sequence information, gene expression levels, binding scores, and cleavage signatures outperformed other methods with an aggregate AUC=0.93. b) MARIA predicted MHC-II presentation of lymphoma IgM (left) compared to experimentally recovered MHC-II peptides (right). MARIA highlighted MHC-II presentation hot spots on IgM FR3 and CH2 regions, consistent with the experimental heat-map (Spearman R=0.63, p-value<0.0001). [Display omitted] DisclosuresDavis:Vir Biotechnology: Consultancy, Equity Ownership, Honoraria; PACT Bio: Consultancy, Equity Ownership, Honoraria; Adicet Inc: Consultancy, Equity Ownership, Honoraria; Chuga Pharmabody: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Atreca: Consultancy, Equity Ownership, Honoraria; Juno: Consultancy, Equity Ownership, Honoraria. Altman:Karius: Consultancy; Personalis: Consultancy; Pfizer: Consultancy.

Development of monoclonal antibody against IgT of a perciform fish, large yellow croaker (Larimichthys crocea) and characterization of IgT+ B cells

Teleost immunoglobulin T (IgT) is considered to be a primitive immunoglobulin class specialized in mucosal immunity. In the present study, a recombinant protein containing the CH2 region of large yellow croaker (Larimichthys crocea) IgT heavy chain was expressed, purified, and used as an immunogen to produce a monoclonal antibody (mAb) against large yellow croaker IgT. Western blotting results indicated that the obtained mouse anti-IgT mAb could specifically recognize a 45 kDa protein in the skin mucus of large yellow croaker, which was identified as the IgT heavy chain by mass spectrometric analysis. Immunofluorescence assay (IFA) analysis further demonstrated that this mouse anti-IgT mAb could recognize membrane-bound IgT (mIgT) molecules on large yellow croaker IgT+ leukocytes. This mAb also could be used for sorting of large yellow croaker IgT+ B cells by flow cytometry sorting technology. Then, flow cytometric immunofluorescence analysis (FCIA) results showed that the percentages of IgT+ B cells in skin, gills, gut, spleen, head kidney and peripheral blood lymphocytes were 27.553% ± 3.312%, 12.588% ± 3.538%, 12.355% ± 3.352%, 13.075 ± 2.258%, 5.552 ± 3.275%, and 2.600 ± 0.521%, respectively, indicating that mucosal tissues (skin, gills, and gut) contained a high ratio of IgT+ B cells. Accordingly, the high protein levels of IgT were also detected in these mucosal tissues, suggesting that IgT may play a role in mucosal immunity in large yellow croaker. Taken together, our data demonstrated that the mouse anti-IgT mAb developed in this study could be used for characterizing IgT+ B cells and studying the functions of IgT in large yellow croaker.

Engineered human FcγRIIa fusion: A novel strategy to extend serum half-life of therapeutic proteins.

The immunoglobulin G (IgG) molecule has a long circulating serum half-life (~3 weeks) through pH- dependent FcRn binding-mediated recycling. To hijack the intracellular trafficking and recycling mechanism of IgG as a way to extend serum persistence of non-antibody therapeutic proteins, we have evolved the ectodomain of a low-affinity human FcγRIIa for enhanced binding to the lower hinge and upper CH2 region of IgG, which is very far from the FcRn binding site (CH2-CH3 interface). High-throughput library screening enabled isolation of an FcγRIIa variant (2A45.1) with 32-fold increased binding affinity to human IgG1 Fc (equilibrium dissociation constant: 9.04 × 10-7 M for wild type FcγRIIa and 2.82 × 10-8 M for 2A45.1) and significantly improved affinity to mouse serum IgG compared to wild type human FcγRIIa. The in vivo pharmacokinetic profile of PD-L1 fused with engineered FcγRIIa (PD-L1-2A45.1) was compared with that of PD-L1 fused with wild type FcγRIIa (PD-L1-wild type FcγRIIa) and human PD-L1 in mice. PD-L1-2A45.1 showed 11.7- and 9.7-fold prolonged circulating half-life (t1/2 ) compared to PD-L1 when administered intravenously and intraperitoneally, respectively. In addition, the AUCinf of PD-L1-2A45.1 was two-fold higher compared to that of PD-L1-wild type FcγRIIa. These results demonstrate that engineered FcγRIIa fusion offers a novel and successful strategy for prolonging serum half-life of therapeutic proteins.

Single Point Mutations Modulate Dynamical Allostery in IgG4 Monoclonal Antibodies

A novel computational method [1] is employed to investigate dynamical allostery within a set of IgG4 antibody mutants. Single point mutations in the CH3-CH3 interfacial region are known to decrease stability of the bivalent form [2,3]. We analyze long-range dynamical couplings that appear in the native protein and in mutants: F405R, T394D, Y349D and L368W. A covariance matrix is constructed from 2μs molecular dynamics trajectories from coarse-grained simulations using the Martini force field. An effective elastic network model is constructed based on inverting the covariance matrix at the residue level. The effective elastic network is subsequently perturbed at targeted residues, which increases or decreases the elastic strength between all residue pairs for residues within 10 Angstroms from the perturbation site. Upon perturbation, a response in the mean square fluctuation (MSF) is monitored at all distal residues. A stabilizing or destabilizing response is respectively identified when MSF decreases or increases by a statistically significant amount, and all IgG4 proteins exhibit long-range couplings. Destabilizing response occurs in CDR loops based on perturbations in the constant CH2 region. In the reverse direction, stabilizing perturbations in CDR loops produce destabilizing responses in the CH2 regions. Both of these regions are critical for function in antibody-dependent cell-mediated cytotoxicity, suggesting the presence of dynamical allostery. Similar response profiles group in two pairs of mutants in corroboration with the experiments [3], where the mutations F405R and T394D increase the half-antibody population, while Y349D and L368W establish monomer-dimer equilibrium. Furthermore, the allosteric pathways are modulated by the mutations, which has important ramifications for protein design and formulation. [1] Azhagiya Singam E.R., et. al. J. Am. Chem. Soc. 139(48) 17508-17517 (2017). [2] Shan, L., et. al. PLoS ONE 11:e0160345 (2016). [3] Wilkinson, I.C., et. al. MAbs 5:406-417 (2013).

Genotypic and heat stress effects on leaf cuticles of field pea using ATR-FTIR spectroscopy.

ATR-FTIR spectroscopy in combination with uni- and multivariate analysis was used to quantify the spectral-chemical composition of the leaf cuticle of pea, investigating the effects of variety and heat stress. Field pea (Pisum sativum L.) is sensitive to heat stress and our goal was to improve canopy cooling and flower retention by investigating the protective role of lipid-related compounds in leaf cuticle, and to use results in the future to identify heat resistant genotypes. The objective was to use Attenuated Total Reflection (ATR)-Fourier Transform Infrared (FTIR) spectroscopy, a non-invasive technique, to investigate and quantify changes in adaxial cuticles of fresh leaves of pea varieties that were subjected to heat stress. Eleven varieties were grown under control (24/18°Cday/night) and heat stress conditions (35/18°Cday/night, for 5days at the early flowering stage). These 11 had significant spectral differences in the integrated area of the main lipid region, CH2 region, CH3 peak, asymmetric and symmetric CH2 peaks, ester carbonyl peak, and the peak area ratio of CH2 to CH3 and ester carbonyl to CH2 asymmetric peak, indicating that cuticles had spectral-chemical diversity of waxes, cutin, and polysaccharides. Results indicated considerable diversity in spectral-chemical makeup of leaf cuticles within commercially available field pea varieties and they responded differently to high growth temperature, revealing their diverse potential to resist heat stress. The ATR-FTIR spectral technique can, therefore, be further used as a medium-throughput approach for rapid screening of superior cultivars for heat tolerance.

Influence of Length and Amino Acid Composition on Dimer Formation of Immunoglobulin based Chimera.

The dimeric immunoglobulin (Ig) chimeras used for drug targeting and delivery are preferred biologics over their monomeric forms. Designing these Ig chimeras involves critical selection of a suitable Ig base that ensures dimer formation. In the present study, we systematically analyzed several factors that influence the formation of dimeric chimera. We designed and predicted 608 cytokine-Ig chimeras where we tested the contributions of (1) different domains of Ig constant heavy chain, (2) length of partner proteins, (3) amino acid (AA) composition and (4) position of cysteine in the formation of homodimer. The sequences of various Ig and cytokines were procured from Uniprot database, fused and submitted to COTH (CO-THreader) server for the prediction of dimer formation. Contributions of different domains of Ig constant heavy chain, length of chimeric proteins, AA composition and position of cysteine to the homodimer formation of 608 cytokine-Ig chimeras were tested. Various in silico approaches were adopted for validating the in silico findings. Experimentally we also validated our approach by expressing the chimeric design of shorter cytokine with Ig domain in CHO cells and analyzing the protein by SDS-PAGE. Our results advocate that while the CH1 region and the Hinge region of Ig heavy chain are critical, the length of partner proteins also crucially influences homodimer formation of the Ig-based chimera. We also report that the CH1 domain of Ig is not required for dimer formation of Ig based chimera in the presence of larger partner proteins. For shorter partner proteins fused to CH2-CH3, careful selection of partner sequence is critical, particularly the hydrophobic AA composition, cysteine content & their positions, disulphide bond formation property, and the linker sequences. We validated our in silico observation by various bioinformatics tools and checked the ability of chimeras to bind with the receptors of native protein by docking studies. As a proof of concept, we have expressed the chimeric proteins in CHO cells and found that our design favors the synthesis of dimeric proteins. Our structural prediction study suggests that extra amino acids in the range of 15-20 added to the CH2 domain of Ig is a critical requirement to make homodimer. This information from our study will have implication in designing efficacious homodimeric chimera.