Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses.

To address the ongoing SARS-CoV-2 pandemic and prepare for future coronavirus outbreaks, understanding the protective potential of epitopes conserved across SARS-CoV-2 variants and coronavirus lineages is essential. We describe a highly conserved, conformational S2 domain epitope present only in the prefusion core of ß-coronaviruses: SARS-CoV-2 S2 apex residues 980-1006 in the flexible hinge. Antibody RAY53 binds the native hinge in MERS-CoV and SARS-CoV-2 spikes on the surface of mammalian cells and mediates antibody-dependent cellular phagocytosis and cytotoxicity against SARS-CoV-2 spike in vitro. Hinge epitope mutations that ablate antibody binding compromise pseudovirus infectivity, but changes elsewhere that affect spike opening dynamics, including those found in Omicron BA.1, occlude the epitope and may evade pre-existing serum antibodies targeting the S2 core. This work defines a third class of S2 antibody while providing insights into the potency and limitations of S2 core epitope targeting.

Can we find a possible structural explanation for antibody-dependent enhancement of dengue virus infection resulting in hemorrhagic fever?

Dengue virus infection is one of the most prevalent mosquito-borne illnesses worldwide, affecting as many as 400 million persons annually. Most people experience a self-limited viral illness, but some experience life-threatening disease. Subsequent infection with other dengue virus serotypes increases the risk of development of severe dengue disease with plasma leakage with or without hemorrhage and end organ impairment. Antibody-dependent enhancement of dengue virus infection has been implicated in the development of severe dengue disease, previously referred to as dengue hemorrhagic fever and dengue shock syndrome. We propose a structural explanation for the role of non-neutralizing antibodies in the development of antibody-dependent enhancement of dengue virus infection via complement fixation or binding to Fcγ receptors facilitating entry into target cells.

A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care.

Can we use DNA triple helices as treatment for systemic lupus erythematosus?

Systemic lupus erythematosus (SLE) is a chronic disease characterized by a variable clinical course and is associated with the presence of numerous autoantibodies. Autoantibodies against double-stranded DNA are highly specific for SLE and are directly associated with distinct clinical manifestations of the disease, specifically lupus nephritis. Examination of the sequences and the three-dimensional structures of autoantibodies specific for nucleic acids, confirms the presence of positively charged amino acids which could interact with the phosphate groups of self DNA. We hypothesize that DNA triple-helices, which can be constructed using short DNA sequences, may be useful in decreasing the clinical manifestations of SLE by inhibiting anti-dsDNA autoantibodies.

Can we explain the allergenicity of peanuts on the basis of the three-dimensional structure of its allergens and use the information to devise means of eliminating peanut allergy?

Helical bundles are found in all the known structures of peanut allergens. The peptide fragments, which survive gastrointestinal digestion of the allergens and are absorbed intact, are hypothesized to reassociate and form stable helical bundles in the circulation, which could elicit a specific IgE response resulting in peanut allergy. The hypothesis is supported by the finding of a diminished allergenicity of an isoform of the peanut allergen, Ara h 3, which has a major deletion. A very probable consequence of this deletion is the reduced tendency to form a stable helix bundle. The discovery of structurally disrupted isoforms of the other peanut allergens and the breeding of plants that contain only those isoforms could lead to the elimination of peanut allergy.

Analysis of the antibody structure based on high-resolution crystallographic studies.

High-resolution structures of liganded and unliganded antibody molecules were analyzed in terms of the interaction between the antibody with ligand, between the residues in the contact between the variable domains, and between the framework and the complementarity-determining regions of the antibody. The solvent accessibilities of the residues in the variable domains were also analyzed. The structural information is useful in the engineering of antibodies for therapeutic and other purposes.

BM-ca is a newly defined type I/II anti-CD20 monoclonal antibody with unique biological properties.

Rituximab (chimeric anti-CD20 mAb) is currently used in the treatment of B-NHL and B cell malignancies, alone or in combination with chemotherapy. However, subsets of patients do not initially respond and/or develop resistance to additional treatments. Hence, there is a need to develop more effective anti-CD20 mAbs that may improve clinical response. BM-ca is a novel humanized anti-CD20 mAb that was tested against several B-NHL cell lines and was compared to several anti-CD20 mAbs (Rituximab, ofatumumab, 2H7, B1 and B-Ly1). BM-ca was shown to strongly induce both homotypic cell aggregation and redistribution of CD20 to membrane lipid rafts. BM-ca was also able to induce programmed cell death (apoptosis) without the need for cross-linking and demonstrated potent complement-dependent cytotoxicity (CDC). BM-ca was more cytotoxic than rituximab even in malignant B cells expressing low amounts of membrane CD20. Type I anti-CD20 mAbs typically induce minimal levels of homotypic cell aggregation and apoptosis but strong localization of CD20 to lipid rafts and potent CDC. Type II anti-CD20 mAbs typically exert the reverse activities. Noteworthy, BM-ca exhibits properties that are shared by both type I and type II anti-CD20 mAbs, which may reflect the recognition of a new CD20 epitope and/or exhibit different molecular signaling. Overall, the present data show that BM-ca is a novel anti-CD20 mAb that may be classified as a type I/II. The therapeutics efficacy of BM-ca awaits its use in clinical trials.

Using simple artificial intelligence methods for predicting amyloidogenesis in antibodies.

BACKGROUND: All polypeptide backbones have the potential to form amyloid fibrils, which are associated with a number of degenerative disorders. However, the likelihood that amyloidosis would actually occur under physiological conditions depends largely on the amino acid composition of a protein. We explore using a naive Bayesian classifier and a weighted decision tree for predicting the amyloidogenicity of immunoglobulin sequences. RESULTS: The average accuracy based on leave-one-out (LOO) cross validation of a Bayesian classifier generated from 143 amyloidogenic sequences is 60.84%. This is consistent with the average accuracy of 61.15% for a holdout test set comprised of 103 AM and 28 non-amyloidogenic sequences. The LOO cross validation accuracy increases to 81.08% when the training set is augmented by the holdout test set. In comparison, the average classification accuracy for the holdout test set obtained using a decision tree is 78.64%. Non-amyloidogenic sequences are predicted with average LOO cross validation accuracies between 74.05% and 77.24% using the Bayesian classifier, depending on the training set size. The accuracy for the holdout test set was 89%. For the decision tree, the non-amyloidogenic prediction accuracy is 75.00%. CONCLUSIONS: This exploratory study indicates that both classification methods may be promising in providing straightforward predictions on the amyloidogenicity of a sequence. Nevertheless, the number of available sequences that satisfy the premises of this study are limited, and are consequently smaller than the ideal training set size. Increasing the size of the training set clearly increases the accuracy, and the expansion of the training set to include not only more derivatives, but more alignments, would make the method more sound. The accuracy of the classifiers may also be improved when additional factors, such as structural and physico-chemical data, are considered. The development of this type of classifier has significant applications in evaluating engineered antibodies, and may be adapted for evaluating engineered proteins in general.

Does the electrical activity of neurons contribute to the pathogenesis of Alzheimer's Disease?

Alzheimer's Disease is believed to be caused by the formation of amyloid fibrils formed by peptides of around 40 amino acid residues resulting from enzymatic cleavage of amyloid precursor protein (APP). The major components of those fibrils have been found to have a mainly alpha-helix conformation in apolar solutions and a looser structure in aqueous environments, while the amyloid fibrils are mostly beta-sheet. Major changes in secondary structure have been observed in other systems and are caused by various factors, including temperature, pH, and electric fields. The APP fragments, which form the amyloid fibrils, are known to migrate into the interneuronal synapses where they would be subjected to electric fields due to neuronal activity. We propose that the electrical activity of neurons causes a structural destabilization of the fragments, which could lead to fibril formation, and thereby contributes to the pathogenesis of Alzheimer's Disease.

The pandemic 2009 (H1N1) swine influenza virus is mild compared to the pandemic 1918 (H1N1) virus because of a proline-to-serine substitution in the receptor-binding site of its hemagglutinin - a hypothesis.

The relative mildness of the pandemic 2009 (H1N1) swine influenza virus compared to the 1918 pandemic (H1N1) virus may be due to a variety of possible causes, including the existence of effective immunity in the host, the lessened ability of the virus to bind to target cells or to replicate in them, a diminished secretion of molecules that could cause further complications like pneumonia, etc. A comparison of the hemagglutinin sequences from the pandemic 2009 (H1N1) viruses with that of the 1918 (H1N1) virus reveals a difference in the residues occupying position 200, which has been shown to be involved in receptor binding. In all the pandemic 2009 (H1N1) hemagglutinin sequences available in the NCBI database, position 200 is occupied by serine. In the hemagglutinin of the 1918 (H1N1) virus, position 200 is occupied by proline. A proline-to-serine substitution could introduce a significant structural change in the receptor-binding site of the hemagglutinin, which could reduce the receptor-binding ability of the 2009 (H1N1) virus. It is proposed that this substitution is the cause of the relative avirulence of the 2009 (H1N1) virus compared to the 1918 (H1N1) virus.

Novel humanized anti-CD20 monoclonal antibodies with unique germline VH and VL gene recruitment and potent effector functions.

The anti-CD20 chimeric monoclonal antibody (mAb) rituximab is the most widely used therapeutic antibody for B-cell malignancies. However, approximately 50% of non-Hodgkin's lymphoma (B-NHL) patients respond to treatment with this antibody. Novel humanized antibodies target membrane CD20 with enhanced effector properties should improve treatment for a broader patient population with relapsed and refractory disease. A novel chimerized form of the murine anti-CD20 1K1791 exerts more potent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities and induces cell death by a non-caspase dependent process. Humanized mAbs derived from 1K1791 were designed using four different humanization techniques and characterized. In contrast to rituximab or 2F2 (human anti-CD20 mAb), several of these exhibited superior ADCC, CDC, inhibition of cell growth and cell death. There was a wide range of functional differences among the humanized forms of 1K1791 despite a modest replacement of amino acid residues in the CDRs. To determine whether the superior activities exhibited by parental murine mAb 1K1791 were due to differences in VH and VL rearrangement, we analyzed its germline and compared it to other anti-CD20 mAbs. A remarkable conservation of VH and Vk (VL kappa) gene usage was observed in the murine anti-CD20 mAbs. 18/23 used the same germline gene J558.42 and 4/23 used closely related genes of the 'J558' group. Thus, 22/23 belonged to VH1 family. One exception was the mAb 1K1791, which was derived from the VH9.12 germline gene. 1K1791 was also unique in its use of a Vk19/28 family gene whereas most other mAbs (21/23) used Vk4/5 family genes. A formal relationship between the particular germline gene recruitment and antibody functionality has not been established, however, the present findings identified humanized mAbs with functional activities that were superior to rituximab and 2F2. These in vitro results support future in vivo animal testing and subsequent clinical trials.

Why is there a greater incidence of allergy to the tropomyosin of certain animals than to that of others?

Tropomyosin is a major allergen in various foods, implicated in a spectrum of mild to life threatening systemic reactions. The incidence of allergy to tropomyosin varies greatly by species, with sensitivity to crab, shrimp, cockroach, and dust mite tropomyosins, among others, being the highest, while tropomyosins in vertebrate species are considered non-allergenic. We have analyzed the possible fragments which may result from Pepsin A digestion of tropomyosins from various species and find that larger fragments of the tropomyosins from crab, shrimp, cockroach, and especially, dust mites will probably survive gastric digestion, compared to those from, for example, chicken, cattle, rabbit, or fish. These larger peptide fragments may enter the bloodstream and assume a three-dimensional structure whose stability approaches that of the intact molecule. Antibodies, including IgE, would be expected to be produced specifically against stable regions of the tertiary structure. We propose that this is a plausible explanation for the greater ability of the larger molecules derived from invertebrate tropomyosins to trigger an immediate hypersensitivity response.

A study of the structural correlates of affinity maturation: antibody affinity as a function of chemical interactions, structural plasticity and stability.

Mutations introduced in an antibody germline sequence as a result of somatic hypermutation could cause its derivatives to have an altered affinity for its target. Affinity maturation favors the selection of the antibodies which exhibit increased affinity. The mutations in 80 high affinity anti-thyroid peroxidase sequences derived from six germlines were analysed in terms of the physicochemical properties of the replacement residues, namely hydrophilicity, size and polarizability, and charge and polarity, in the context of its position and probable solvent accessibility. The effects of these substitutions were evaluated in terms of the resultant increased chemical interactivity potential of the affinity-matured antibodies relative to the germline. The results of the analysis would be useful in the rational design of antibodies and of other proteins for improved binding properties.

Water molecules in the antibody-antigen interface of the structure of the Fab HyHEL-5-lysozyme complex at 1.7 A resolution: comparison with results from isothermal titration calorimetry.

The structure of the complex between hen egg-white lysozyme and the Fab HyHEL-5 at 2.7 A resolution has previously been reported [Cohen et al. (1996), Acta Cryst. D52, 315-326]. With the availability of recombinant Fab, the X-ray structure of the complex has been re-evaluated at 1.7 A resolution. The refined structure has yielded a detailed picture of the Fab-lysozyme interface, showing the high complementarity of the protein surfaces as well as several water molecules within the interface that complete the good fit. The model of the full complex has improved significantly, yielding an R(work) of 19.5%. With this model, the structural results can be compared with the results of isothermal titration calorimetry. An attempt has been made to estimate the changes in bound waters that accompany complex formation and the difficulties inherent in using the crystal structures to provide the information necessary to make this calculation are discussed.

SDR grafting of a murine antibody using multiple human germline templates to minimize its immunogenicity.

The humanization of mAbs by complementarity-determining region (CDR)-grafting has become a standard procedure to improve the clinical utility of xenogeneic Abs by reducing human anti-murine Ab (HAMA) responses elicited in patients. However, CDR-grafted humanized Abs may still evoke anti-V region responses when administered in patients. To minimize anti-V region responses, the Ab may be humanized by grafting onto the human templates only the specificity-determining residues (SDRs), the residues that are essential for the surface complementarity of the Ab and its ligand. Typically, humanization of an Ab, whether by CDR or SDR grafting, involves the use of a single human template for the entire VL or VH domain of an Ab. We hypothesized, however, that the homology between the human template sequences and mAb to be humanized may be maximized by using templates from multiple human germline sequences corresponding to the different segments of the variable domain. This could be more advantageous in reducing the potential immunogenicity of the humanized Ab. This report describes the SDR grafting of the murine anti-carcinoembryonic antigen (CEA) mAb COL-1 using three different human germline V-kappa sequences as templates for the VL CDRs and another human template for the VL frameworks. In competition RIAs, the SDR-grafted COL-1 (HuCOL-1SDR) completely inhibited the binding of radiolabeled murine COL-1 (mCOL-1) to CEA, and showed that its binding affinity is comparable to that of the CDR-grafted Ab (HuCOL-1). The HuCOL-1SDR showed similar binding reactivity to the CEA expressed on the surface of a tumor cell line as the HuCOL-1. More importantly, compared to HuCOL-1 and the "abbreviated" CDR-grafted Ab, HuCOL-1SDR showed lower reactivity to patients' sera carrying anti-V region Abs to mCOL-1. HuCOL-1SDR, which shows a lower sera reactivity than that of the parental Abs while retaining its Ag-binding property, is a potentially useful clinical reagent. To the best of our knowledge, this is the first time a VL or VH domain of an Ab has been humanized by grafting the SDRs onto a human template comprised of several Ab sequences. We have shown that humanization of an Ab can be optimized using multiple human templates for a single variable domain of an Ab. This approach maximizes the homology between the target Ab and the human templates in both the frameworks and the CDRs by choosing as the template the human sequence that displays the highest local sequence identity to the frameworks and to each of the CDRs of the target Ab.

In vitro affinity maturation of a specificity-determining region-grafted humanized anticarcinoma antibody: isolation and characterization of minimally immunogenic high-affinity variants.

PURPOSE: HuCC49V10 (V10), a humanized anticarcinoma monoclonal antibody (Ab) CC49, was generated by grafting only the specificity-determining regions (SDRs) of CC49 onto the variable light and variable heavy frameworks of the human Abs LEN and 21/28'CL, respectively. SDRs are those residues of the complementarity-determining regions that are most critical for antigen (Ag) binding. Compared with HuCC49, which was developed by conventional complementarity-determining region grafting, V10 has lower reactivity to the sera from patients who were previously given murine CC49 in clinical trials, although its Ag-binding affinity is 2-3-fold lower than that of HuCC49. To generate variants of V10 with higher Ag-binding affinity and lower sera reactivity, in vitro affinity maturation of V10 was carried out using phage display technique. EXPERIMENTAL DESIGN: A limited library of Fabs was generated by replacing some of the SDRs with all possible residues located at the corresponding positions in human Abs. The library was enriched, by several rounds of panning, in Fabs that have high affinity for the TAG-72 Ag. The clones encoding the best binders were expressed in insect cells as whole Abs that were purified and characterized. RESULTS: Competition radioimmunoassay and surface plasmon resonance measurements showed that two of the isolates, V14 and V15, have higher binding affinity than that of V10. In addition, the surface plasmon resonance analysis showed that the variants V14 and V15, compared with the parental V10, have lower reactivity to the anti-V region Abs using sera from patients who received murine CC49. CONCLUSIONS: The two isolates, V14 and V15, which show higher Ag-binding reactivity and lower sera reactivity than the parental V10 Ab, are potentially more useful clinical reagents. These results demonstrate that phage display can be used to isolate variants of an Ab that are potentially less immunogenic in patients than the parental Ab from which they are derived.

Minimizing immunogenicity of the SDR-grafted humanized antibody CC49 by genetic manipulation of the framework residues.

The murine mAb CC49 specifically recognizes a tumor-associated glycoprotein (TAG)-72, which is expressed on the majority of human carcinomas. This Ab has potential applications in the diagnosis and treatment of human carcinomas. However, patients receiving murine CC49 generate human anti-murine Ab (HAMA) responses, preventing repeated administration of the Ab for effective treatment. To minimize the HAMA response, two versions of humanized CC49 (HuCC49) were developed: (a) HuCC49 and (b) HuCC49V10 (V10). HuCC49 was developed by grafting the CC49 CDRs, while V10 was generated by grafting only the specificity determining residues (SDRs) of the CC49 onto the frameworks of the human Abs. During the generation of both HuCC49 and V10, a few murine framework residues that were believed to be essential for the integrity of the Ag-binding site were retained. However, the indispensability of these residues for the Ag-binding activity of CC49 has not been experimentally validated. In this study, an array of V10 variants were generated by replacing, by site-specific mutagenesis, the murine framework residues that were retained in the humanized Ab with their counterparts in the human templates. The variants were tested for their (a) Ag-binding activity and (b) reactivity to sera from patients who were previously administered murine CC49 in a clinical trial. One such variant, V59, compared to the parental V10, shows a significant decrease in its reactivity to the anti-variable region Abs present in the patients' sera, while it binds to the TAG-72 Ag with a slightly higher affinity. Variant 59, which is expected to be minimally immunogenic because of its low sera reactivity, is a potentially useful clinical reagent against human carcinomas. In this study, we show for the first time that experimental validation rather than reliance on the protein data bank (PDB) should be the criterion for the indispensability of framework residues for the humanization of any murine Ab to retain its Ag-binding property and reduce its immunogenicity in patients.

Grafting of "abbreviated" complementarity-determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody.

Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinical studies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problem of immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. To minimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the "abbreviated" CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surface complementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding of radiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding ability to the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variant showed lower reactivity to patients' sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retains its Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype for the development of a potentially useful clinical reagent.