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Background: The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested.Methods: The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes.Results: The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model.Conclusion: The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial. [ABSTRACT FROM AUTHOR]

T-cell bispecific antibodies (TCBs) are a novel class of engineered immunoglobulins that unite monovalent binding to the T-cell receptor (TCR) CD3e chain and bivalent binding to tumor-associated antigens in order to recruit and activate T-cells for tumor cell killing. In vivo, T-cell activation is usually initiated via the interaction of the TCR with the peptide-HLA complex formed by the human leukocyte antigen (HLA) and peptides derived from intracellular proteins. TCR-like antibodies (TCRLs) that recognize pHLA-epitopes extend the target space of TCBs to peptides derived from intracellular proteins, such as those overexpressed during oncogenesis or created via mutations found in cancer. One challenge during lead identification of TCRL-TCBs is to identify TCRLs that specifically, and ideally exclusively, recognize the desired pHLA, but not unrelated pHLAs. In order to identify TCRLs suitable for TCRL-TCBs, large numbers of TCRLs have to be tested in the TCB format. Here, we propose a novel approach using chimeric antigen receptors (CARs) to facilitate the identification of highly selective TCRLs. In this new so-called TCRL-CAR-J approach, TCRL-candidates are transduced as CARs into Jurkat reporter-cells, and subsequently assessed for their specificity profile. This work demonstrates that the CAR-J reporter-cell assay can be applied to predict the profile of TCRL-TCBs without the need to produce each candidate in the final TCB format. It is therefore useful in streamlining the identification of TCRL-TCBs.

Monoclonal antibody-based therapeutics are an integral part of treatment of different human diseases, and the selection of suitable antibody candidates during the discovery phase is essential. Here, we describe a novel, cellular screening approach for the identification and characterization of therapeutic antibodies suitable for conversion into T cell bispecific antibodies using chimeric antigen receptor (CAR) transduced Jurkat-NFAT-luciferase reporter cells (CAR-J). For that purpose, we equipped a Jurkat-NFAT reporter cell line with a universal CAR, based on a monoclonal antibody recognizing the P329G mutation in the Fc-part of effector-silenced human IgG1-antibodies. In addition to scFv-based second generation CARs, Fab-based CARs employing the P329G-binder were generated. Using these anti-P329G-CAR-J cells together with the respective P329G-mutated IgG1-antibodies, we established a system, which facilitates the rapid testing of therapeutic antibody candidates in a flexible, high throughput setting during early stage discovery. We show that both, scFv- and Fab-based anti-P329G-CAR-J cells elicit a robust and dose-dependent luciferase signal if the respective antibody acts as an adaptor between tumor target and P329G-CAR-J cells. Importantly, we could demonstrate that functional characteristics of the antibody candidates, derived from the anti-P329G-CAR-J screening assay, are predictive for the functionality of these antibodies in the T cell bispecific antibody format., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

CD20 is a B-lymphocyte specific integral membrane protein, an activated-glycosylated phosphoprotein expressed on the surface of B-cells and a clinically validated target of monoclonal antibodies such as rituximab, ocrelizumab, ofatumumab and obinutuzumab in the treatment of all B cell lymphomas and leukemias as well as autoimmune diseases. Here, we report the extraction and purification of native CD20 from SUDHL4 and RAMOS cell lines. To improve the protein yield, we applied a calixarene-based detergent approach to solubilize, stabilize and purify native CD20 from HEK293 cells. Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation show that purified CD20 was non-aggregated and that CD20 oligomerization is concentration dependent. Negative stain electron microscopy and atomic force microscopy revealed homogenous populations of CD20. However, no defined structure could be observed. Interestingly, micellar solubilized and purified CD20 particles adopt uniformly confined nanodroplets which do not fuse and aggregate. Finally, purified CD20 could bind to rituximab and obinutuzumab as demonstrated by SEC, and Surface Plasmon Resonance (SPR). Specificity of binding was confirmed using CD20 antibody mutants to human B-cell lymphoma cells. The strategy described in this work will help investigate CD20 binding with newly developed antibodies and eventually help to optimize them. This approach may also be applicable to other challenging membrane proteins.

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8 + T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Recombinant human IgG antibodies (hIgGs) completely devoid of binding to Fcγ receptors (FcγRs) and complement protein C1q, and thus with abolished immune effector functions, are of use for various therapeutic applications in order to reduce FcγR activation and Fc-mediated toxicity. Fc engineering approaches described to date only partially achieve this goal or employ a large number of mutations, which may increase the risk of anti-drug antibody generation. We describe here two new, engineered hIgG Fc domains, hIgG1-P329G LALA and hIgG4-P329G SPLE, with completely abolished FcγR and C1q interactions, containing a limited number of mutations and with unaffected FcRn interactions and Fc stability. Both 'effector-silent' Fc variants are based on a novel Fc mutation, P329G that disrupts the formation of a proline sandwich motif with the FcγRs. As this motif is present in the interface of all IgG Fc/FcγR complexes, its disruption can be applied to all human and most of the other mammalian IgG subclasses in order to create effector silent IgG molecules., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells. Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono- and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis. Mol Cancer Ther; 15(5); 946-57. ©2016 AACR., (©2016 American Association for Cancer Research.)

Several novel anti-CD20 monoclonal antibodies are currently in development with the aim of improving the treatment of B cell malignancies. Mutagenesis and epitope mapping studies have revealed differences between the CD20 epitopes recognized by these antibodies. Recently, X-ray crystallography studies confirmed that the Type I CD20 antibody rituximab and the Type II CD20 antibody obinutuzumab (GA101) differ fundamentally in their interaction with CD20 despite recognizing a partially overlapping epitope on CD20. The Type I CD20 antibodies rituximab and ofatumumab are known to bind to different epitopes. The differences suggest that the biological properties of these antibodies are not solely determined by their core epitope sequences, but also depend on other factors, such as the elbow hinge angle, the orientation of the bound antibody and differential effects mediated by the Fc region of the antibody. Taken together, these factors may explain differences in the preclinical properties and clinical efficacy of anti-CD20 antibodies.

CD20 is a cell-surface marker of normal and malignant B cells. Rituximab, a monoclonal antibody targeting CD20, has improved the treatment of malignant lymphomas. Therapeutic CD20 antibodies are classified as either type I or II based on different mechanisms of killing malignant B cells. To reveal the molecular basis of this distinction, we fine-mapped the epitopes recognized by both types. We also determined the first X-ray structure of a type II antibody by crystallizing the obinutuzumab (GA101) Fab fragment alone and in complex with a CD20 cyclopeptide. Despite recognizing an overlapping epitope, GA101 binds CD20 in a completely different orientation than type I antibodies. Moreover, the elbow angle of GA101 is almost 30° wider than in type I antibodies, potentially resulting in different spatial arrangements of 2 CD20 molecules bound to a single GA101 or rituximab molecule. Using protein tomography, different CD20 complexes were found to be associated with the 2 antibodies, and confocal microscopy showed different membrane compartmentalization of these subpopulations of the cellular CD20 pool. Our findings offer a possible molecular explanation for the different cellular responses elicited by type I and II antibodies.

CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall survival in patients. Here, we report the development of GA101 as the first Fc-engineered, type II humanized IgG1 antibody against CD20. Relative to rituximab, GA101 has increased direct and immune effector cell-mediated cytotoxicity and exhibits superior activity in cellular assays and whole blood B-cell depletion assays. In human lymphoma xenograft models, GA101 exhibits superior antitumor activity, resulting in the induction of complete tumor remission and increased overall survival. In nonhuman primates, GA101 demonstrates superior B cell-depleting activity in lymphoid tissue, including in lymph nodes and spleen. Taken together, these results provide compelling evidence for the development of GA101 as a promising new therapy for the treatment of B-cell disorders.

Unlabelled: Recessive congenital methaemoglobinaemia (RCM) due to NADH-cytochrome b5 reductase (cytb5r) deficiency is a very rare disorder. We report on two unrelated patients (4 and 2.5 years old) with RCM type 2. Developmental delay was obvious at the age of 4 months. On follow-up, both children showed severe tetraspastic cerebral palsy, profound cognitive impairment, strabismus, impressive secondary microcephaly and failure to thrive. One novel mutation in the DIA1gene was identified. Prenatal diagnosis was successfully done in both families by mutation analysis in chorionic villi or measurement of cytb5r in fetal amniotic cells., Conclusion: Due to the severity of this disease and its 25% recurrence risk, prenatal diagnosis should be made available to all affected families.

We have adapted the protein fragment complementation assay (PCA) to the screening and selection of antibodies in the single-chain Fv (scFv) format. In this assay, two interacting proteins (target and antibody) are genetically fused to the two halves of the dissected enzyme dihydrofolate reductase. Binding of the two partners reassembles this enzyme and reconstitutes its activity, thus allowing growth on minimal medium. We have optimized this system with regard to linker length and orientation, and can reach an efficiency for antigen/antibody interactions similar to that with fused leucine zippers. Using several model antibodies specific for peptides and proteins, we show that cognate interactions give rise to about seven orders of magnitude more colonies than non-specific interactions. When transforming mixtures of plasmids encoding different antigens and/or antibodies, all colonies tested contained plasmids encoding cognate pairs. We believe that this system will be very powerful as a routine system for generating antibodies, especially in functional genomics, since it does not require purification and immobilization of the antigen. The identification of an antibody specific for a cDNA or EST-encoded protein will require only cloning, transformation and plating of bacteria., (Copyright 2001 Academic Press.)

Thioredoxin constitutes the prototype of the thiol-disulfide oxidoreductase family. These enzymes contain an active-site disulfide bridge with the consensus sequence Cys-Xaa-Xaa-Cys. The more N-terminal active-site cysteine is generally a strong nucleophile with an abnormal low pK(a) value. In contrast, the more C-terminal cysteine is buried and only little is known about its effective pK(a) during catalysis of disulfide exchange reactions. Here we have analyzed the pK(a) values of the active-site thiols in wild type thioredoxin and a 400-fold more oxidizing thioredoxin variant by NMR spectroscopy, using selectively (13)C(beta)-Cys-labeled proteins. We find that the effective pK(a) of the buried cysteine (pK(b)) of the variant is increased, while the pK(a) of the more N-terminal cysteine (pK(N)) is decreased relative to the corresponding pK(a) values in the wild type. We propose two empirical models which exclusively require the knowledge of pK(N) to predict the redox properties of thiol-disulfide oxidoreductases with reasonable accuracy.

The thiol/disulfide oxidoreductase DsbA is the strongest oxidant of the thioredoxin superfamily and is required for efficient disulfide bond formation in the periplasm of Escherichia coli. To determine the importance of the redox potential of the final oxidant in periplasmic protein folding, we have investigated the ability of the most reducing thiol/disulfide oxidoreductase, E.coli thioredoxin, of complementing DsbA deficiency when secreted to the periplasm. In addition, we secreted thioredoxin variants with increased redox potentials as well as the catalytic a-domain of human protein disulfide isomerase (PDI) to the periplasm. While secreted wild-type thioredoxin and the most reducing thioredoxin variant could not replace DsbA, all more oxidizing thioredoxin variants as well as the PDI a-domain could complement DsbA deficiency in a DsbB-dependent manner. There is an excellent agreement between the activity of the secreted thioredoxin variants in vivo and their ability to oxidize polypeptides fast and quantitatively in vitro. We conclude that the redox potential of the direct oxidant of folding proteins and in particular its reactivity towards reduced polypeptides are crucial for efficient oxidative protein folding in the bacterial periplasm.

Thiol/disulfide oxidoreductases like thioredoxin, glutaredoxin, DsbA, or protein disulfide isomerase (PDI) share the thioredoxin fold and a catalytic disulfide bond with the sequence Cys-Xaa-Xaa-Cys (Xaa corresponds to any amino acid). Despite their structural similarities, the enzymes have very different redox properties, which is reflected by a 100,000-fold difference in the equilibrium constant (K(eq)) with glutathione between the most oxidizing member, DsbA, and the most reducing member, thioredoxin. Here we present a systematic study on a series of variants of thioredoxin from Escherichia coli, in which the Xaa-Xaa dipeptide was exchanged by that of glutaredoxin, PDI, and DsbA. Like the corresponding natural enzymes, all thioredoxin variants proved to be stronger oxidants than the wild-type, with the order wild-type < PDI-type < DsbA-type < glutaredoxin-type. The most oxidizing, glutaredoxin-like variant has a 420-fold decreased value of K(eq), corresponding to an increase in redox potential by 75 mV. While oxidized wild-type thioredoxin is more stable than the reduced form (delta deltaG(ox/red) = 16.9 kJ/mol), both redox forms have almost the same stability in the variants. The pH-dependence of the reactivity with the alkylating agent iodoacetamide proved to be the best method to determine the pKa value of thioredoxin's nucleophilic active-site thiol (Cys32). A pKa of 7.1 was measured for Cys32 in the reduced wild-type. All variants showed a lowered pKa of Cys32, with the lowest value of 5.9 for the glutaredoxin-like variant. A correlation of redox potential and the Cys32 pKa value could be established on a quantitative level. However, the predicted correlation between the measured delta deltaG(ox/red) values and Cys32 pKa values was only qualitative.

A combined evaluation effort of the Boehringer Mannheim Research and Development and Evaluation Departments and the University Hospital Rotterdam is described regarding the new, fully automated Enzymun-Test PSA assay for prostate-specific antigen. The study consisted of an analytical and a clinical part. At both sites, the vast majority of intra-assay coefficients of variation ranged from 2 to 3% above prostate-specific antigen = 1 microgram/l. Below that concentration higher coefficients of variation were measured. Comparable results were obtained for the interassay imprecision. The analytical sensitivity (lower limit of detection) was found to be 0.02 microgram/l at both sites. Regarding the linearity of the assay no systematic drift to either elevated or lower values which increasing dilution was found. Deviations remained well in the range between 100 +/- 10%. The correlation with the Abbot IMx PSA assay as performed with a large set of clinical specimens revealed: y (= Enzymun) = 1.16x (= IMx) + 0.0; r = 0.985; n = 245. In this comparison study small differences between benign prostatic hyperplasia patients and prostate cancer patients were detected, perhaps partly based on the differences in recognition patterns of various molecular prostate-specific antigen forms in both assays. A follow-up after radical prostatectomy with 17 patients (50 serum samples) also showed a good comparability between the Enzymun-Test and the IMx assay. The limited check of the reference range resulted in data comparable to what can be found in the literature: out of 100 samples originating from healthy males, aged 20-60 years, 99 had prostate-specific antigen values lower than 4 micrograms/l. Based on our findings it can be concluded that the new Enzymun-Test PSA assay meets the current state-of-the-art criteria in prostate-specific antigen methodology.

Antibodies against placental alkaline phosphatase (PAP) share antigenic determinants with the intestinal isoenzyme (IAP) and vice versa. Both isoenzymes can be found as part of the total activity of alkaline phosphatase (AP) in the serum. Using antibody-coated polystyrene tubes, a simple and sensitive immunoassay was developed which allows the quantitative determination of IAP or PAP without interference of the cross-reacting isoenzyme. The presence and amount of IAP depends on the ABO blood group, secretory status and the oral fat intake. The serum IAP in healthy fasted individuals was found up to 8 U/I in secretors of blood group O and B and below 1 U/I in non-secretors and blood group A donors. In screening tests of various pathological sera. IAP was found elevated up to 100 U/I in idiopathic hyper-AP-aemia and some liver cirrhosis patients.

Using immunochemical techniques, alkaline phosphatase isoenzymes were determined in tissue samples of breast carcinomas and carcinomas of the gastrointestinal tract. In breast carcinomas only 19% of the patients expressed significant placental alkaline phosphatase activity, compared with 78% in gastrointestinal tumours. The intestinal isoenzyme was found in 50% of the breast carcinomas and in nearly all of the other examined tissues. The two isoenzymes usually represent 1% of the total alkaline phosphatase activity, but in a few cases they may constitute between 10 and 90%. In the serum of the patients under examination, elevated total alkaline phosphatase activity was found in only 7%, and elevated placental alkaline phosphatase in 6% of the cases. No cases of elevated serum intestinal alkaline phosphatase were found. We therefore consider that serum placental alkaline phosphatase is a poor tumour marker for a general screening.