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The Immunoglobulins type-M (IgMs) are one of the first antibody classes mobilized during immune responses against pathogens and tumor cells. Binding to specific target antigens enables the interaction with the C1 complex which strongly activates the classical complement pathway. This biological function is the basis for the huge therapeutic potential of IgMs but due to their high oligomeric complexity,in vitroproduction, biochemical and biophysical characterizations are challenging. In the present study, we present recombinant production of two IgM models (IgM617 and IgM012) in pentameric and hexameric states and the evaluation of their polymer distribution using different biophysical methods (AUC, SEC-MALLS, Mass Photometry and Transmission Electron Microscopy). Each IgM oligomer has individual specific expression pattern and yield with different protein quality likely due to intrinsic IgM properties and patterning. Nevertheless, the purified recombinant IgMs retain their ability to activate complement in a C1q dependent manner. And more importantly, a new method to evaluate their functional quality attribute by characterizing the kinetics of C1q binding to recombinant IgM has been developed using BioLayer Interferometry (BLI). We show that recombinant IgMs possess similar C1q binding properties as IgMs purified from human plasma.

Twelve subjects with positive SARS-CoV-2 neutralization test (NT) titers (>1:10) identified in a seroprevalence study with 1655 working adults were followed up for one year. Here we report that 7 of these 12 individuals (58%) still had NT titers ≥1:50, S1-specific IgG concentrations ≥50 BAU/ml and ≥26% ACE2 receptor binding inhibition, measured with surrogate virus NT one year after mild COVID infection. Furthermore, NT_50 titers >1:10 and S1-specific IgG levels >60 BAU/ml present at three months post-infection persisted at detectable levels for 1 year and correlated with circulating S1-specific memory B-cells. Vaccine-induced SARS-CoV2 immune responses decline at similar rates as those after infection; thus the describes threshold of 60 BAU/ml at three months post infection might also be relevant for assessment of Ab persistence after vaccination.

CD19 is among the most relevant targets in cancer immunotherapy. However, its extracellular domain (ECD) is prone to aggregation and misfolding, representing a major obstacle for the development and analysis of CD19-targeted therapeutics. Here, we engineered stabilized CD19-ECD (termed SuperFolder) variants, which also showed improved expression rates and, in contrast to the wild type protein, they could be efficiently purified in their monomeric forms. Despite being considerably more stable, these engineered mutants largely preserved the wild type sequence (>98.8%). We demonstrate that the variant SF05 enabled the determination of the monovalent affinity between CD19 and a clinically approved FMC63-based CAR, as well as monitoring and phenotypic characterization of CD19-directed CAR-T cells in the blood of lymphoma patients. We anticipate that the SuperFolder mutants generated in this study will be highly valuable tools for a range of applications in basic immunology and CD19-targeted cancer immunotherapy.

Immunoglobulin M (IgM) antibodies are reckoned as promising tools for therapy and diagnostic approaches. Nevertheless, the commercial success of IgMs is hampered due to bottlenecks in recombinant production and downstream processing. IgMs are large, complex and highly glycosylated proteins that are only stable in a limited range of conditions. To investigate these sensitive IgM antibodies we optimized the elution conditions for a commercially available IgM affinity matrix (CaptureSelect™). Applying a small-scale screening system, we optimized our single step purification strategy for high purity, high yield and retained antigen binding capacity. Here we show that IgMs are sensitive to aggregation at very acidic conditions (pH ≤ 3.0) despite often being used for affinity chromatography. We combined pH 3.5 with a high salt concentration to prevent aggregation during elution. The elution strategy presented in this paper will improve IgM processes for further applications. The herein used IgMs were produced in Chinese hamster ovary (CHO) cells. We present the first detailed glycan analysis of IgM produced in CHO cells with predominantly complex type structures at Asn171, Asn332 and Asn395 and oligomannosidic structures at Asn402 and Asn563 similar to human serum-IgM.

Immunoglobulin M (IgM) antibodies are considered as promising biopharmaceutical drugs in the future despite recombinant production is quite challenging as incomplete polymer formation or nucleic acid adherence can decrease the quality of the IgM preparation. Therefore, we defined densitometric and chromatographic methods as suitable tools to analyze the polymer distribution and the remaining nucleic acid content after initial IgM purification. Additionally, the quality of the glycosylation pattern is an important parameter for biological safety and efficacy.

Humanized monoclonal antibodies (mAbs) are among the most promising modern therapeutics, but defined engineering strategies are still not available. Antibody humanization often leads to a loss of affinity, as it is the case for our model antibody Ab2/3H6 (PDB entry 3BQU). Identifying appropriate back-to-mouse mutations is needed to restore binding affinity, but highly challenging. In order to get more insight, we have applied molecular dynamics simulations and correlated them to antibody binding and expression in wet lab experiments. In this study, we discuss six mAb variants and investigate a tyrosine conglomeration, an isopolar substitution and the improvement of antibody binding towards wildtype affinity. In the 3D structure of the mouse wildtype, residue R94h is surrounded by three tyrosines which form a so-called ‘tyrosine cage’. We demonstrate that the tyrosine cage has a supporting function for the CDRh3 loop conformation. The isopolar substitution is not able to mimic the function appropriately. Finally, we show that additional light chain mutations can restore binding to wildtype-comparable level, and also improve the expression of the mAb significantly. We conclude that the variable light chain of Ab2/3H6 is of underestimated importance for the interaction with its antigen mAb 2F5., Protein Engineering, Design & Selection, 31 (7-8), ISSN:1741-0126, ISSN:1741-0134, ISSN:0269-2139, ISSN:1460-213X

Immunoglobulin A (IgA) is the most abundant antibody class in the human body and has a unique role in mediating immunity. The ever-increasing knowledge about the potential of IgAs has renewed interest in this antibody class for therapeutic use against a variety of infectious and malignant diseases, and as a preventive agent for mucosal pathogens. Despite the considerable therapeutic potential of IgA the exploration thereof has often been hampered due to difficulties in producing and purifying desired quantities. Large amounts of pure IgA will be required for in vivo studies. This work reviews current achievements and bottlenecks in upstream and downstream processing of recombinant IgA from a biotechnological point of view. We also highlight recent accomplishments with diverse expression systems and presents different affinity techniques for the capture of recombinant IgA to compare their purification potential.

The efficient production of recombinant proteins such as antibodies typically involves the screening of an extravagant number of clones in order to finally select a stable and high-producing cell line. Thereby, the underlying principles of a powerful protein machinery, but also potential expression limitations, often remain poorly understood. To shed more light on this topic, we applied several different techniques to investigate a previously generated cell line (4B3-IgA), which expressed recombinant immunoglobulin A (IgA) with an unusually low specific productivity. Results were compared to the host cell line and to another recombinant CHO cell line (3D6-IgA) expressing another IgA that binds to an overlapping epitope. The low specific productivity of clone 4B3-IgA could not be explained by GCN or mRNA levels, but insufficiencies in protein maturation and/or secretion were determined. Despite the presence of free light chain polypeptides, they occasionally failed to associate with their heavy chain partners. Consequently, heavy chains were misassembled and accumulated to form intracellular aggregates, so-called Russell bodies. These protein deposits evoked the expression of increased amounts of ER-resident chaperones to combat the induced stress. Despite bottlenecks in protein processing, the cells' quality checkpoints remained intact, and predominantly correctly processed IgA was exported into the culture medium. The results of our study demonstrated that recombinant protein expression was impaired by heavy chain aggregation despite the presence of a disposable light chain and revealed elevated chaperone formation in combination with limited antibody assembly. Our studies suggest that the primary amino acid sequence and consequently the resulting structure of an expressed protein need to be considered as a factor influencing a cell's productivity.

Single-chain fragment variable-fragment crystallizable antibody constructs (scFv-Fc) are homodimeric proteins representing valuable alternatives to heterotetrameric full-length IgG molecules to study protein properties and product-dependent cellular behavior. In contrast to naturally occurring antibodies, these artificial molecules are assembled from functional antibody domains to reduce molecule complexity and enhance antibody expression levels. The scFv-Fc format retains critical antibody functions such as antigen binding affinity and antibody effector functions. Here, we present a protocol to convert the full-length anti-HIV-1 IgG1 antibody 2F5 into a scFv-Fc construct. Variable and constant regions are amplified by conventional PCR reactions and assembled by a single overlap-extension PCR reaction. The amplified product is then cloned into a mammalian expression vector suitable for high-titer transient gene expression. This workflow can be applied to any antibody sequence by adapting the specific primer sequences to the antibody of choice.

For many years, the potential of immunoglobulin M (IgM) antibodies was not fully understood because of characteristics different to the well-known immunoglobulin G type like low target affinity, cross reactivity and complex protein structure. In the meanwhile IgMs have been positively evaluated for their use as therapeutic agent in the mucosal environment but also in serum to eradicate upcoming tumor cells and invading antigens. Therefore IgM class of antibodies will play a significant role in clinical applications but also diagnosis in the future. To evaluate the full potential of this kind of antibody molecules large amounts of high quality product will be needed. In this review the focus is set on the biotechnological aspect of producing IgM class antibodies recombinantly in mammalian cells. Current achievements in expression and purification of this molecule are highlighted and compared.

Broadly neutralizing monoclonal antibody (MAb) 2F5 targets a linear epitope within the highly conserved membrane proximal external region (MPER) of the HIV-1 envelope protein gp41 integral subunit. Prospective vaccine developments warrant efforts currently underway to unveil the mechanistic and structural basis of its mode of action. One open question relates to the putative role that membrane phospholipids might play in the neutralization process. In this work, we establish experimental conditions that allow monitoring 2F5 insertion into lipid bilayers. Then, we compare the abilities of 2F5-based MAb, Fabs, and 2F5-specific antibodies recovered from immunized rabbits to directly penetrate into lipid bilayers and block the lytic activity of MPER-derived peptides. Antibody insertion induced membrane perturbation, which was blocked on interacting with the peptide epitope, thereby suggesting that such phenomenon was primarily mediated by the epitope-binding site. The long, hydrophobic complementarity-determining region (CDR)-H3 loop contributed little to this effect. In contrast, the CDR-H3 loop was required for blocking the lytic activity of MPER-based peptides and viral neutralization. Thus, our results suggest that core epitope binding plus association with lipid bilayers are not in conjunction sufficient to support viral neutralization by 2F5. Moreover, they support a role for the CDR-H3 loop in establishing secondary interactions with lipids and/or gp41 that would block the membrane-perturbing activity of MPER during fusion.

Due to the inherent immune evasion properties of the HIV envelope, broadly neutralizing HIV-specific antibodies capable of suppressing HIV infection are rarely produced by infected individuals. We examined the feasibility of utilizing genetic engineering to circumvent the restricted capacity of individuals to endogenously produce broadly neutralizing HIV-specific antibodies. We constructed a single lentiviral vector that encoded the heavy and light chains of 2G12, a broadly neutralizing anti-HIV human antibody, and that efficiently transduced and directed primary human B cells to secrete 2G12. To evaluate the capacity of this approach to provide protection from in vivo HIV infection, we used the humanized NOD/SCID/γ c null mouse model, which becomes populated with human B cells, T cells, and macrophages after transplantation with human hematopoietic stem cells (hu-HSC) and develops in vivo infection after inoculation with HIV. The plasma of the irradiated NOD/SCID/γ c null mice transplanted with hu-HSC transduced with the 2G12-encoding lentivirus contained 2G12 antibody, likely secreted by progeny human lymphoid and/or myeloid cells. After intraperitoneal inoculation with high-titer HIV-1 JR-CSF , mice engrafted with 2G12-transduced hu-HSC displayed marked inhibition of in vivo HIV infection as manifested by a profound 70-fold reduction in plasma HIV RNA levels and an almost 200-fold reduction in HIV-infected human cell numbers in mouse spleens, compared to control hu-HSC-transplanted NOD/SCID/γ c null mice inoculated with equivalent high-titer HIV-1 JR-CSF . These results support the potential efficacy of this new gene therapy approach of using lentiviral vectors encoding a mixture of broadly neutralizing HIV antibodies for the treatment of HIV infection, particularly infection with multiple-drug-resistant isolates.

Polyspecific human IgG preparations are indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. In addition, intraveneous IgG (IVIG) is used to treat patients with autoimmune and systemic inflammatory diseases. Lectin chromatography on Sambucus nigra agglutinin stood at the cradle of the hypothesis that the anti-inflammatory properties depend on sialylation of the N-glycans in the Fc region of IgG. A detailed analysis of fractions obtained by lectin chromatography revealed that binding of IVIG is essentially mediated by Fab glycosylation. Moreover, experiments with a monoclonal antibody from a human cell line and IVIG Fc fragments indicated that at least two sialic acids in the Fc region of an antibody are required for lectin binding. Such glycoforms contain either two monosialylated glycans or a disialylated glycan and constitute 1% or less of the total human IgG. Arguably this small proportion holds the entire anti-inflammatory potency. A new mass spectrometric quantification method of IgG subclass ratio revealed that the IVIG Fc preparation essentially consists of IgG1. This observation may be relevant when studying the effect of human Fc in murine models of inflammation because mouse IgG subclasses differ substantially in their interaction with receptors.

Anti-idiotypic antibody (Ab2) 3H6 is directed against the human monoclonal antibody 2F5, which is one of a few neutralising antibodies against HIV-1. Since the binding epitope of 2F5 is cryptic and no neutralising immune response could be elicited by several potential vaccines comprising this region, Ab2/3H6 represents a potent vaccine candidate for active immunisation. Here we describe the molecular features of Ab2/3H6 after changing the antigen binding specificity by single point mutations in the complementarity-determining region 3 of the Ab2/3H6 heavy chain. The resulting Ab2/3H6 mutants were compared in several experimental settings to the wild type Ab2/3H6 Fab fragment. Moreover, we report about an immunisation study with Ab2/3H6 Fab variants, which elicited a specific 2F5-like humoral immune response in BALB/c mice.

Antibody 2G12 is one of a small number of human immunoglobulin G (IgG) monoclonal antibodies exhibiting potent and broad human immunodeficiency virus-1 (HIV-1)-neutralizing activity in vitro, and the ability to prevent HIV-1 infection in animal models. It could be used to treat or prevent HIV-1 infection in humans, although to be effective it would need to be produced on a very large scale. We have therefore expressed this antibody in maize, which could facilitate inexpensive, large-scale production. The antibody was expressed in the endosperm, together with the fluorescent marker protein Discosoma red fluorescent protein (DsRed), which helps to identify antibody-expressing lines and trace transgenic offspring when bred into elite maize germplasm. To achieve accumulation in storage organelles derived from the endomembrane system, a KDEL signal was added to both antibody chains. Immunofluorescence and electron microscopy confirmed the accumulation of the antibody in zein bodies that bud from the endoplasmic reticulum. In agreement with this localization, N-glycans attached to the heavy chain were mostly devoid of Golgi-specific modifications, such as fucose and xylose. Surprisingly, most of the glycans were trimmed extensively, indicating that a significant endoglycanase activity was present in maize endosperm. The specific antigen-binding function of the purified antibody was verified by surface plasmon resonance analysis, and in vitro cell assays demonstrated that the HIV-neutralizing properties of the maize-produced antibody were equivalent to or better than those of its Chinese hamster ovary cell-derived counterpart.

A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic N-glycosylation. A major concern is the presence of beta 1,2-xylose and core alpha 1,3-fucose residues on complex N-glycans as these nonmammalian N-glycan residues may provoke unwanted side effects in humans. In this study we have investigated the potential antigenicity of plant-type N-glycans attached to a human monoclonal antibody (2G12). Using glyco-engineered plant lines as expression hosts, four 2G12 glycoforms differing in the presence/absence of beta 1,2-xylose and core alpha 1,3-fucose were generated. Systemic immunization of rabbits with a xylose and fucose carrying 2G12 glycoform resulted in a humoral immune response to both N-glycan epitopes. Furthermore, IgE immunoblotting with sera derived from allergic patients revealed binding to plant-produced 2G12 carrying core alpha 1,3 fucosylated N-glycan structures. Our results provide evidence for the adverse potential of nonmammalian N-glycan modifications present on monoclonal antibodies produced in plants. This emphasizes the need for the use of glyco-engineered plants lacking any potentially antigenic N-glycan structures for the production of plant-derived recombinant proteins intended for parenteral human application.

Background:The broadly neutralizing recombinant human HIV-1 antibodies 4E10, 2F5 and Igh1b12 are reported to have autoreactive potential, which is significant for HIV-1 vaccine development and passive immunotherapy using these antibodies.Objective:To investigate the clinical relevance of these findi

In recent years, plants have become an attractive alternative for the production of recombinant proteins. However, their inability to perform authentic mammalian N-glycosylation may cause limitations for the production of therapeutics. A major concern is the presence of beta1,2-xylose and core alpha1,3-fucose residues on complex N-linked glycans, as these N-glycan epitopes are immunogenic in mammals. In our attempts towards the humanization of plant N-glycans, we have generated an Arabidopsis thaliana knockout line that synthesizes complex N-glycans lacking immunogenic xylose and fucose epitopes. Here, we report the expression of a monoclonal antibody in these glycan-engineered plants that carry a homogeneous mammalian-like complex N-glycan pattern without beta1,2-xylose and core alpha1,3-fucose. Plant and Chinese hamster ovary (CHO)-derived immunoglobulins (IgGs) exhibited no differences in electrophoretic mobility and enzyme-linked immunosorbent specificity assays. Our results demonstrate the feasibility of a knockout strategy for N-glycan engineering of plants towards mammalian-like structures, thus providing a significant improvement in the use of plants as an expression platform.

We have expressed the anti-idiotypic antibody 3H6 Fab directed against the HIV-1 broadly neutralising antibody 2F5 in methylotrophic yeast Pichia pastoris. The chimeric human/mouse Fab fragment was expressed under control of the inducible AOX1 promoter and secreted via the alpha mating factor leader of Saccharomyces cerevisiae. Bioreactor experiments showed the ability of the recombinant P. pastoris clone to secrete up to 260 mg/L Fab fragment in the culture supernatant during a five days cultivation time. Codon optimisation of the Fab expression cassette gave no further improvement of specific productivity when comparing 12 clones of each construct. The subsequent purification of Fab containing supernatants was done by anion exchange and size-exclusion chromatography with a recovery resulting in 70% of the recombinant protein. For verification of the suitability of the expression system we characterised the expressed protein with respect to both, its specificity and binding affinity and could not detect any significant difference between products from yeast derived and the hybridoma derived product. Finally we tested the implicit requirement of the carbohydrate moiety in the H2 loop of the original 3H6 antibody by introducing an asparagine to alanine replacement and, in a second experiment, inhibition of N-glycosylation by tunicamycin treatment. Biochemical analysis confirmed that the N-glycosylation does not contribute to the binding properties of 3H6.

We report the production of an important human therapeutic antibody in plant cell suspension cultures and the functional analysis of that antibody, including a comparison with the same antibody produced in CHO cells. We established transgenic tobacco BY2 suspension cell cultures expressing the human monoclonal antibody 2F5, which shows broadly neutralizing activity against HIV-1. The antibody was directed to the endoplasmic reticulum of the plant cells and was isolated by cell disruption, followed by protein A chromatography. The plant- derived antibody was shown to be largely intact by SDS- PAGE and immunoblot. Antigen binding activity was investigated by electrophoretic mobility shift assay and quantitatively determined by ELISA and Biacore biosensor technology. Ligand binding properties were analyzed using the ectodomain of human Fc gamma RI for kinetic analysis. The plant- derived antibody showed similar kinetic properties and 89% of the binding capacity of its CHO-derived counterpart, but was only 33% as efficient in HIV- 1 neutralization assays. Our results show that plant suspension cultures can be used to produce human antibodies efficiently and that the analysis methods used in this study, including biosensor technology, provide useful functional data about antibody performance. This highlights important issues raised by the use of plant systems to produce human biologics. Sack, M., Paetz, A., Kunert, R., Bomble, M., Hesse, F., Stiegler, G., Fischer, R., Katinger, H., Stoeger, E., Rademacher, T. Functional analysis of the broadly neutralizing human anti-HIV-1 antibody 2F5 produced in transgenic BY-2 suspension cultures.

Since the first use of Chinese hamster ovary (CHO) cells for recombinant protein expression, production processes have steadily improved through numerous advances. In this review, we have highlighted several key milestones that have contributed to the success of CHO cells from the beginning of their use for monoclonal antibody (mAb) expression until today. The main factors influencing the yield of a production process are the time to accumulate a desired amount of biomass, the process duration, and the specific productivity. By comparing maximum cell densities and specific growth rates of various expression systems, we have emphasized the limiting parameters of different cellular systems and comprehensively described scientific approaches and techniques to improve host cell lines. Besides the quantitative evaluation of current systems, the quality-determining properties of a host cell line, namely post-translational modifications, were analyzed and compared to naturally occurring polyclonal immunoglobulin fractions from human plasma. In summary, numerous different expression systems for mAbs are available and also under scientific investigation. However, CHO cells are the most frequently investigated cell lines and remain the workhorse for mAb production until today.

One challenge in biotechnology industry is to produce recombinant proteins with prolonged serum half-life. One strategy for enhancing the serum half-life of proteins includes increasing the molecular weight of the protein of interest by fusion to the Fc part of an antibody. In this context, we have expressed a homodimer fusion protein in CHO cells which consists of two identical polypeptide chains, in which our target protein, recombinant human erythropoietin (rhEpo), is N-terminally linked with the Fc part of a human IgG(1) molecule. In the present study, culture supernatant of a stable clone was collected and purified by affinity chromatography prior characterization. We emphasized product quality aspects regarding the fusion protein itself and in addition, post-translational characterization of the subunits in comparison to human antibodies and rhEpo. However, overproduction of recombinant proteins in mammalian cells is well established, analysis of product quality of complex products for different purposes, such as product specification, purification issues, batch to batch consistency and therapeutical consequences, is required. Besides product quantification by ELISA, N-acetylneuraminic acid quantification in microtiterplates, quantitative isoform pattern and entire glycan profiling was performed. By using these techniques for the characterization of the recombinant human Epo-Fc (rhEpo-Fc) molecule itself and furthermore, for the separate characterization of both subunits, we could clearly show that no significant differences in the core glycan structures compared to rhEpo and human antibody N-glycans were found. The direct comparison with other rhEpo-Fc fusion proteins failed, because no appropriate data were found in the literature.

The screening for antigen-specific hybridoma cells with adequate production rates is still a time-, labour- and money-consuming procedure. A reduction in cell culture testing by specifically selecting those fused cells that produce antibody could therefore make hybridoma technology more attractive, even for small research groups or for newly discovered proteins at an early stage of research. Additional problems, such as the requirement to produce sufficient amounts of the unknown protein at a purity that allows specific immunisation of mice and testing of the resulting hybridoma clones, also need to be overcome. Here we present a new strategy to isolate rapidly and efficiently monoclonal antibodies against new proteins, for which only sequence information at the DNA level is known. The strategy consists of fusion of the protein to a hexa-His-tag to allow easy purification, production in yeast and insect cells to reduce background immunisation with host cell proteins and the selection of IgG-producing hybridoma cells by flow-cytometric cell sorting using the affinity matrix secretion assay technique.

Successful treatment of stage 4 neuroblastoma remains a major challenge in pediatric oncology. In order to improve the outcome, passive immunotherapy using human-mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 has been evaluated in early phase clinical trials with promising results in progressing stage 4 neuroblastoma patients. In preparation of European phase III clinical trial (HR-NBL-1/ESIOP), the cell line used for production of ch14.18 was changed. Specifically, the plasmid encoding for ch14.18 antibody was recloned into CHO cells. Here, we report the in vitro and in vivo anti-neuroblastoma activity of antibody ch14.18 produced in CHO cells (ch14.18/CHO) compared to that of ch14.18 manufactured from SP2/0 (ch14.18/SP2/0) and NS0 cells (ch14.18/NS0). First, we demonstrate identical binding of ch14.18/CHO to the nominal antigen disialoganglioside GD2 in vitro compared to ch14.18/SP2/0 and ch14.18/NS0. Binding was GD2-specific, since all precursor- and metabolite-gangliosides of GD2 tested were not recognized by ch14.18/CHO. Second, the functional properties of ch14.18/CHO were determined in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) reactions against GD2 positive neuroectodermal tumor cell lines in vitro. There was no difference in CDC mediated specific tumor cell lysis among the three different ch14.18 antibody preparations. Interestingly, ch14.18/CHO showed superior ADCC activity at low antibody concentrations. Third, the efficacy of ch14.18/CHO was evaluated in the NXS2 neuroblastoma model in vivo. Importantly, the ch14.18/CHO preparation was effective in suppression of experimental liver metastasis in this model. In vivo depletion of NK-cells completely abrogated this effect, suggesting that the mechanism involved in the ch14.18/CHO induced anti-neuroblastoma effect is mediated by NK-dependent ADCC.

SummaryBroadly neutralizing monoclonal antibodies to HIV-1 are rare but invaluable for vaccine design. 4E10 is the broadest neutralizing antibody known and recognizes a contiguous and highly conserved epitope in the membrane-proximal region of gp41. The crystal structure of Fab 4E10 was determined at 2.2 Å resolution in complex with a 13-residue peptide containing the gp41 core epitope (NWFDIT). The bound peptide adopts a helical conformation in which the key contact residues, TrpP672, PheP673, IleP675, and ThrP676, map to one face of the helix. The peptide binds in a hydrophobic pocket that may emulate its potential interaction with the host cell membrane. The long CDR H3 of the antibody extends beyond the bound peptide in an orientation that suggests that its apex could contact the viral membrane when 4E10 is bound to its membrane-proximal epitope. These structural insights should assist in the design of immunogens to elicit 4E10-like neutralizing responses.

The conserved membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 is a target of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is an important lead for vaccine design. However, immunogens that bear MPER epitopes so far have not elicited neutralizing antibodies in laboratory animals. One explanation is that the immunogens fail to recreate the proper molecular environment in which the epitopes of 2F5 and 4E10 are presented on the virus. To explore this molecular environment, we used alanine-scanning mutagenesis across residues 660 to 680 in the MPER of a pseudotyped variant of HIV-1 JR-FL , designated HIV-1 JR2 , and examined the ability of 2F5 and 4E10 to neutralize the Ala mutant viruses. The results show that the only changes to produce neutralization resistance to 2F5 occurred in residue D, K, or W of the core epitope (LEL DKW ANL). Likewise, 4E10 resistance arose by replacing one of three residues; two (W and F) were in the core epitope, and one (W) was seven residues C-terminal to these two (N WF DISNWL W ). Importantly, no single substitution resulted in resistance of virus to both 2F5 and 4E10. Surprisingly, 8 out of 21 MPER Ala mutants were more sensitive than the parental pseudovirus to 2F5 and/or 4E10. At most, only small differences in neutralization sensitivity to anti-gp120 monoclonal antibody b12 and peptide T20 were observed with the MPER Ala mutant pseudoviruses. These data suggest that MPER substitutions can act locally and enhance the neutralizing activity of antibodies to this region and imply a distinct role of the MPER of gp41 during HIV-1 envelope-mediated fusion. Neutralization experiments showing synergy between and T20 and 4E10 against HIV-1 are also presented. The data presented may aid in the design of antigens that better present the MPER of gp41 to the immune system.

The human monoclonal antibody 2F5 neutralizes primary human immunodeficiency virus type 1 (HIV-1) with rare breadth and potency. A crystal structure of a complex of 2F5 and a peptide corresponding to its core epitope on gp41, ELDKWAS, revealed that the peptide interacts with residues at the base of the unusually long (22-residue) third complementarity-determining region of the heavy chain (CDR H3) but not the apex. Here, we perform alanine-scanning mutagenesis across CDR H3 and make additional substitutions of selected residues to map the paratope of Fab 2F5. Substitution of residues from the base of the H3 loop or from CDRs H1, H2, and L3, which are proximal to the peptide, significantly diminished the affinity of Fab 2F5 for gp41 and a short peptide containing the 2F5 core motif. However, nonconservative substitutions to a phenylalanine residue at the apex of the H3 loop also markedly decreased 2F5 binding to both gp41 and the peptide, suggesting that recognition of the core epitope is crucially dependent on features at the apex of the H3 loop. Furthermore, substitution at the apex of the H3 loop had an even more pronounced effect on the neutralizing activity of 2F5 against three sensitive HIV-1. These observations present a challenge to vaccine strategies based on peptide mimics of the linear epitope.

We have previously generated human monoclonal anti-human immunodeficiency virus type 1 (anti-HIV-1) antibodies 2F5IgG and 2G12IgG with an exceptional cross-clade neutralizing potential. 2F5IgG and 2G12IgG passively administrated to macaques were able to confer complete protection from both intravenous and mucosal challenge with pathogenic HIV-simian immunodeficiency virus chimeric strains and have shown beneficial effects in a phase-1 clinical trial. We now class-switched 2F5 and 2G12 to the immunoglobulin M (IgM) or IgA isotype, to enforce features like avidity, complement activation, or the potential to neutralize mucosal transmission. For this purpose we expressed functional polymeric 2F5 and 2G12 antibodies in CHO cells and evaluated their anti-HIV-1 activity in vitro. The class switch had a strong impact on the protective potential of 2F5 and 2G12. 2G12IgM inhibited HIV-1 infection of peripheral blood mononuclear cell cultures up to 28-fold-more efficiently than the corresponding IgG and neutralized all of the primary isolates tested. The 2F5 and 2G12 antibodies of all isotypes were able to interact with active human serum to inhibit viral infection. Furthermore, we demonstrated that polymeric 2F5 and 2G12 antibodies but not the corresponding IgGs could interfere with HIV-1 entry across a mucosal epithelial layer in vitro. Although polymeric 2F5 antibodies had only limited potential in the standard neutralization assay, the results from the mucosal assay suggest that 2F5 and 2G12 antibodies may have a high potential to prevent natural HIV-1 transmission in vivo.

The human monoclonal antibody 2F5 is a potent neutralizer of most clades of HIV-1 and possesses protective effects against viral transmission. It recognizes the linear epitope ELDKWAS of the viral envelope protein gp41. As structural information about epitope recognition may help to develop an HIV-1 vaccine we initiated crystallographic analyses of mAb 2F5 and its epitope complex. We now report the preparation of the corresponding Fab fragments, complexation with the epitope peptide, and crystallization of free mAb 2F5 Fab as well as the peptide complex. Both crystal forms are well suited for high-resolution structural analysis.

The human monoclonal antibody (humAb) 2F5 is a potent candidate for immunotherapy of HIV-1. The xenohybridoma derived humAb 2F5 is of IgG3 kappa isotype. Generally, IgG1 isotype has a longer half-life (beta-clearance) in humans than IgG3. Therefore the isotype was switched to an IgG1 by ligation of the 2F5 heavy chain variable region to an IgG1 constant region and expressed as IgG1 kappa in CHO cells. CHO clones have been established, which stably express humAb 2F5 at high levels. The specificity, affinity and in vitro function of both isotypes were identical.

Some of the problems encountered with human or human-mouse heterohybridomas, such as low growth rates and high serum requirements, have led to the increased use of recombinant cell lines for production of human antibodies. To evaluate the suitability of such alternative cell lines for the production of human antibodies we have analysed several subclones with differing specific production rates of a recombinant CHO cell line. Gene copy number and site of chromosomal integration for the light and heavy chain and the dhfr gene were determined by in-situ hybridisation. Specific mRNA content was analysed by Northern blot. In addition the intracellular content in light and heavy chain was measured by flow cytometry and the specific secretion rates were determined. The stability of gene expression was followed in the highest producing subclone for over a year. As previously seen in heterohybridoma cells a high expression rate of light chain is beneficial in speeding up secretion rates of whole antibody. When grown in the presence of G418 and methotrexate the amplified gene copies in the genome of recombinant CHO cells were stable over more than 100 passages. However, the expression of light chain, and with it the secretion rate, decreased with time. The low intracellular concentration of light chain resulted in accumulation of heavy chain in the endoplasmic reticulum due to retention by chaperones. The specific secretion rate decreased by 50% after 100 passages. When no G418 or methotrexate were present 75% of the gene copies were lost after 100 passages.

The economic importance of obtaining high-producing subclones for large scale production of pharmaceutical proteins is self-evident. However, few papers have studied the changes that occur during subclone development. This information would be important for further improvement of screening and subcloning protocols. We have therefore compared subclones of a human-mouse heterohybridoma cell line producing a human antibody againt HIV-1. Three subclones with low, medium and high specific production rates were selected for this study and their light and heavy chain mRNA content, the intracellular content of light and heavy chain and the specific secretion rates compared. In addition the long time stability of antibody expression in the highest producing subclone was analysed for one year. For the three subclones a correlation between the intracellular content in light chain and the secretion rate was found, while the intracellular content in heavy chain was the same for all three subclones. These results indicate that the assembly in the endoplasmic reticulum (ER) is one of the major rate limiting factors in antibody production. During long time cultivation of the heterohybridoma cell line a continuous decrease in light and heavy chain production was seen without the appearance of a non producing sub-population.

We have stabilized a panel of 33 hybridomas producing human monoclonal antibodies (MAbs) against HIV-1 gp160 and p24. Five of these antibodies were able to neutralize different HIV-1 isolates, and two of them (2F5 and 2G12) revealed remarkable potential to neutralize primary virus isolates of different clades in several in vitro tests. To determine whether a structural basis for neutralization could be identified, we analyzed the antibodies at the molecular level. This study reports the primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chain V segments (VH, Vkappa) of the neutralizing MAbs (1B1, 1F7, 2F5, 2G12, and 3D5) and the nonneutralizing anti-gp41 MAb 3D6. Aligning the V segments with the nearest related germline genes illustrated the occurrence of somatic mutations. The neutralizing MAbs show mutational rates comparable to those of antibodies that appear in patients in whom the immune system is under constant antigenic pressure over a long period of time. In contrast, 3D6, which recognizes the immunodominant region on gp41, displays homologies as high as 97 and 98% compared with its VH and Vkappa germline genes. The diversity segments [D(H)] of 1B1, 1F7, 3D5, and 3D6 were assigned to single D(H) segments on the chromosomal D(H) locus. 2F5 presents a D(H) segment 52 nucleotides in length, which could be explained by fusion of two segments on the immunoglobulin heavy chain locus that have not yet been described as rearranged regions. 2G12 D(H) shows best homologies to a D(H) segment between D3-22 and D4-23. This D(H) segment could be the reason for the rare occurrence of antibodies competing with 2G12. Since this nearest related chromosomal region on the D(H) locus does not display recombination signals at the flanking regions, this segment is normally not taken into consideration as a site for immunoglobulin heavy chain rearrangement.

The production of human monoclonal antibodies for therapeutic use is of increasing importance for treatment of viral infections such as AIDS. As human x mouse heterohybridomas rarely reach the growth rates and cell specific production rates of mouse hybridomas the transfection of standard cell lines, such as CHO or BHK, is a promising alternative. This has the additional advantage that the IgG subtype can be changed to suit the desired application. However, the use of a cell line that has not originally developed to produce antibodies, as lymphocytes and myeloma cells have, might have unrecognised drawbacks. This will be especially significant in the case of antibodies as each molecule consists of 4 chains linked by disulphide bonds which require specific intracellular factors to be properly folded and processed (Heavy chain binding protein, Protein Disulfide Isomerase a.o.). In this study we have therefore compared two cell lines: a human x mouse heterohybridoma producing IAM-2F5, a human IgG(3) antibody specific for HIV-1 with neutralising properties and a Chinese Hamster Ovary cell transfected with dihydrofolate reductase and with the heavy and light chain genes of IAM-2F5 modified to IgG(1). From each cell line three subclones were selected with low, medium and high specific production rates. Batch cultures were performed and the following cellular parameters analysed by flow cytometry; 1) total RNA content (translational activity); 2) total protein content; 3) cell cycle phase distribution; 4) concentration of light and heavy chains; 5) concentration of helper proteins such as BiP and PDI. The production rate of heterohybridoma cells was best reflected in the intracellular concentration of kappa chain, while the gamma chain concentration was comparable for all three subclones. In the CHO cells the gamma chain expression and thus gene copy number appeared to be the limiting factor. The GRP78/BiP concentration in CHO remained unchanged in spite of a 5-fold higher concentration of gamma chain in the high producing subclone. The PDI concentration in CHO cells was much lower compared to the heterohybridoma cells, irrespective of production rates.

13 p. The membrane proximal external region (MPER) of the fusogenic HIV-1 glycoprotein-41 harbors the epitope sequence recognized by 2F5, a broadly neutralizing antibody isolated from an infected individual. Structural mimicry of the conserved MPER 2F5 epitope constitutes a pursued goal in the field of anti-HIV vaccine development. It has been proposed that 2F5 epitope folding into its native state is attained in the vicinity of the membrane interface and might involve interactions with other viral structures. Here we present results indicating that oligomeric complexes established between MPER and the conserved amino-terminal fusion peptide (FP) can partition into lipid vesicles and be specifically bound by the 2F5 antibody at their surfaces. Cryo-transmission electron microscopy of liposomes doped with MPER:FP peptide mixtures provided the structural grounds for complex recognition by antibody at lipid bilayer surfaces. Supporting the immunogenicity of the membrane-bound complex, these MPER:FP peptide-vesicle formulations could trigger cross-reactive anti-MPER antibodies in rabbits. Thus, our observations suggest that contacts with N-terminal regions of gp41 may stabilize the 2F5 epitope as a membrane-surface antigen. J.L.N. is supported by grants BIO2011-29792 and GIU-06/42 from The Ministerio de Ciencia e Innovacion (Spanish MICINN) and the Basque Government, respectively. J.M.V. is supported by The Ministerio de Ciencia e Innovacion (MICINN) grant BFU2010-15703/BMC and CAM (community of Madrid) grant S2009MAT-1507. A.A. received a pre-doctoral fellowship from the Basque Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The human monoclonal antibody (mAb) 2G12 was isolated from an asymptomatic HIV-1 infected patient in 1990. In 1994, its neutralizing activity against HIV-1 strains and binding to the glycoprotein 120 (gp120) was described for the first time (Buchacher et al. 1994; Purtscher et al. 1994; Trkola et al. 1995). At that time a recombinantly expressed 2G12 IgG1 protein was already available (see below) and characterization was exclusively done with this recombinant molecule so that a confusion between hybridoma derived and recombinantly expressed 2G12 could be precluded.

The anti-idiotypic antibody (Ab) Ab2/3H6 (Kunert et al. 2002) is directed against the human monoclonal Ab (mAb) 2F5, which broadly and potently neutralizes primary HIV-1 isolates (Wolbank et al. 2003). Ab2/3H6 is able to mimic the antigen recognition site of the mAb2F5 making it an attractive candidate antigen for HIV-1 vaccine purposes. In this study the mouse variable regions of Ab2/3H6 were subjected to various humanizing approaches using three different methods. For the CDR-grafted variants, an “aggressive” graft harboring less backmutations and a “conservative” graft with more backmutations were designed. In the Superhumanization approach we grafted the murine CDRs to a human framework (FR) which was most related concerning the canonical structure class. The Resurfacing method substitutes mouse amino acids (aa) that are surface exposed in the murine FR by residues usually found in equivalent positions in human Abs. The different Ab2/3H6 variants were characterized by competition experiments with mAb 2F5. The resurfaced and the “conservative” grafted variants showed similar binding properties to mAb 2F5 when compared to the murine Ab2/3H6 version, while the “aggressive” grafted Ab showed less affinity and the superhumanized type lost the ability to bind to mAb 2F5.

Interactions between the Fc segment of IgG and FcγRs on a variety of cells are likely to play an important role in the anti-HIV activity of Abs. Because the nature of the glycan structure on the Fc domain is a critical determinant of Fc–FcγR binding, proper Fc glycosylation may contribute to Ab-mediated protection. We have generated five different glycoforms of the broadly HIV-1–neutralizing mAb 2G12 in wild-type and glycoengineered plants and Chinese hamster ovary cells. Plant-derived 2G12 exhibited highly homogeneous glycosylation profiles with a single dominant N-glycan species. Using flow cytometry with FcγR-expressing cell lines, all 2G12 glycoforms demonstrated similar binding to FcγRI, FcγRIIa, and FcγRIIb. In contrast, two glycoforms derived from glycoengineered plants that lack plant-specific xylose and core α1,3-fucose, and instead carry human-like glycosylation with great uniformity, showed significantly enhanced binding to FcγRIIIa compared with Chinese hamster ovary or wild-type plant-derived 2G12. Using surface plasmon resonance, we show that binding of 2G12 to FcγRIIIa is markedly affected by core fucose, irrespective of its plant-specific α1,3 or mammalian-type α1,6 linkage. Consistent with this finding, 2G12 glycoforms lacking core fucose (and xylose) mediated higher antiviral activity against HIV-1 or simian immunodeficiency virus as measured by Ab-dependent cell-mediated virus inhibition. This is, to our knowledge, the first demonstration that specific alterations of Fc glycosylation can improve antiviral activity. Such alterations may result in better immunotherapeutic reagents. Moreover, biasing vaccine-induced immune responses toward optimal Fc glycosylation patterns could result in improved vaccine efficacy.

Anti-idiotypic antibodies could represent an alternative vaccination approach in human therapy. The anti-idiotypic antibody Ab2/3H6 was generated in mouse and is directed against the human monoclonal antibody 2F5, which broadly and potently neutralizes primary HIV-1 isolates. Ab2/3H6 is able to mimic the antigen recognition site of 2F5 making it a putative candidate for HIV-1 vaccine purposes. In order to reduce immunogenicity of therapeutic proteins, humanization methods have been developed. The mouse variable regions of Ab2/3H6 were subjected to three different humanization approaches, namely resurfacing, complementarity determining region (CDR)-grafting and superhumanization. Four different humanized Ab2/3H6 variants were characterized for their binding affinity to 2F5 in comparison to the chimeric Ab2/3H6. The resurfaced and the 'conservative' CDR-grafted variants showed similar binding properties to 2F5 when compared to the chimeric version, while the 'aggressive' CDR-grafted antibody showed reduced affinity and the superhumanized type lost its binding ability. In this study, we developed humanized Ab2/3H6 variants that retained the same affinity as the parental antibody, and are therefore of potential interest for future clinical trails.

Recently we were able to show that the murine anti-idiotypic antibody (Ab2) 3H6 (Kunert et al., 2002; Antiidiotypic Antibody Inducing HIV-1 Neutralizing Antibodies Pending) significantly blocked the binding of the human mAb 2F5 to its synthetic epitope ELDKWA as well as to gp160. Furthermore, 3H6 was also capable of decreasing the in vitro neutralisation potency of 2F5 in a dose-related way. Finally, the murine 3H6 Fab fragments were capable to induce Ab1-like neutralising immune responses after applying the Ab2 to mice. Thus, Ab2/3H6 successfully mimics a neutralising epitope on gp41, which is either not or only poorly immunogenic on the native HIV-1 during natural infection. In the present study the murine Ab2/3H6 was partially humanized (the murine variable regions were fused with the corresponding human constant regions) and recombinantly expressed either as whole IgG1 (using two different expression strategies) or Fab fragment in CHO cells. Crude Ab2/3H6 IgG1 and Ab2/3H6 Fab expression supernatants were effectively purified using either protein A for Ab2/3H6 IgG1 (up to 90% recovery) or a combination of anion-exchange and size-exclusion chromatography in case of Ab2/3H6 Fab (up to 75% recovery). The purified Ab2/3H6 versions were further characterized by several specificity studies and competition experiments with neutralizing monoclonal antibody nMAb 2F5.

The human monoclonal antibody 2G12 is a member of a small group of broadly neutralizing antibodies against human immunodeficiency virus type 1. 2G12 adopts a unique variable heavy domain-exchanged dimeric configuration that results in an extensive multivalent binding surface and the ability to bind with high affinity to densely clustered high mannose oligosaccharides on the "silent" face of the gp120 envelope glycoprotein. Here, we further define the amino acids responsible for this extraordinary domain-swapping event in 2G12.

The human monoclonal antibody 2F5 (Buchacher et al., 1994) is a potent candidate for immunotherapy of HIV-1 (Katinger et al., 1995). The hybridoma derived humAb is of IgG3 kappa isotype. Since the IgGl isotype has a longer half-life in human than IgG3, we switched the subclass-type to IgGl by ligation of the 2F5 heavy chain variable region to an IgGl constant region and expressed the IgGl kappa molecule in CHO-cells. Stable recombinant 2F5-IgGl expressing CHO-cells were isolated and the recombinant protein compared to the hybridoma derived immunoglobulin. Here we confirm, that specificity and affinity of different isotypes has not changed. Stability assays of the recombinant 2F5 IgGl clone were performed with and without selection pressure.

Two LC-ESI-MS methods for the analysis of antibody glycosylation are presented. In the first approach, tryptic glycopeptides are separated by RP chromatography and analyzed by ESI-MS. This “glycopeptide strategy” allows a protein- and subclass-specific quantitation of both neutral and sialylated glycan structures. Additional information about under- or deglycosylation and the protein backbone, e.g., termini, can be extracted from the same data. In the second LC-ESI-MS method, released oligosaccharides are separated on porous graphitic carbon (PGC). A complete structural assignment of neutral and sialylated oligosaccharides occurring on antibodies is thereby achieved in one chromatographic run. The two methods were applied to polyclonal human IgG, to commercial mAb expressed in CHO cells (Rituximab, Xolair, and Herceptin), in SP2/0 (Erbitux and Remicade) or NS0 cells (Zenapax) and the anti-HIV antibody 4E10 produced either in CHO cells or in a human cell line. Both methods require comparably little sample preparation and can be applied to SDS-PAGE bands. They both outperform non-MS methods in terms of reliability of peak assignment and MALDI-MS of underivatized glycans with regard to the recording of sialylated structures. Regarding fast and yet detailed structural assignment, LC-MS on graphitic carbon supersedes all other current methods.

To date only a few neutralizing antibodies against HIV-1 exist. Since these neutralizing antibodies are only rarely found in sera of HIV-1 infected individuals an active vaccine is required. We recently developed murine anti-idiotypic antibody Ab2/3H6 against monoclonal antibody (mAb) 2F5, which is one of the most prominent neutralizing antibodies. Anti-idiotypic antibody Ab2/3H6 has been partially humanized and expressed as whole immunoglobulin G in Chinese hamster ovary cells in order to minimize the human anti-mouse antibody response. Here we describe the expression, purification, and immunohistochemical characterization of the chimeric Ab2/3H6 Fab fragment, which was finally used beside the whole IgG1 as an antigen for immunization of guinea pigs. The crude sera were screened for specific antibodies against the epitope of mAb 2F5 ELDKWA as well as for reactivity against HIV-1 gp41.

The monoclonal antibody 2F5 neutralizes a broad range of human immunodeficiency virus-1 isolates via a conserved epitope on the viral glycoprotein gp41. The conformation of the principal epitope is a type I beta-turn centered on gp41 residues (664)DKW(666); in addition, binding studies indicate that residues N- and C-terminal to this core as well as structurally more distant parts of gp41 also contribute to the interaction. Ab2/3H6 is an anti-idiotypic antibody that inhibits the interaction between 2F5 and gp41 and as such, Ab2/3H6 may, in principle, possess a paratope that mimics the gp41 epitope. To establish the potential of Ab2/3H6 to serve as a guide for the design of vaccine components against human immunodeficiency virus, we investigated the crystal structure of the heterodimeric complex of Ab2/3H6 F(ab) and 2F5 F(ab)'. Ab2/3H6 F(ab) binds to 2F5 F(ab)' via a helix-like protrusion formed by residues (58(H))RYSPSLNTRL(67(H)) of the 2F5 F(ab)' variable domain and proximal to but not overlapping with the gp41 (664)DKW(666) epitope-binding pocket. This defines Ab2/3H6 as an anti-idiotypic antibody of the Ab2gamma class, i.e., an antigen-inhibitable idiotype that does not carry the internal image of the linear primary gp41 (662)ELDKWAS(668) epitope.

We recently developed a murine anti-idiotypic antibody (Ab2/3H6) versus the human monoclonal antibody 2F5, one of a few antibodies yet known to neutralize a broad range of HIV-1 primary isolates. Ab2/3H6 was not only able to bind to the paratope of mAb 2F5 but also significantly inhibited the binding of 2F5 to its synthetic epitope ELDKWA on gp41. In the present work we describe the partial humanization, expression, and characterization of Ab2/3H6 variants followed by several corresponding interaction studies with 2F5. The results of these studies support the high specificity of the recombinantly expressed Ab2s to the idiotype. Apparent affinities were designated by end point measurement and were similar compared to the murine Ab2/3H6. Moreover, the inhibition potency of chimeric Ab2/3H6 analyzed by in vitro studies could be shown to be the same as that detected for the hybridoma-derived murine Ab2/3H6.

Recombinant mammalian cells for the production of glycoproteins and antibodies are mainly established by gene amplification techniques, using selection and screening methods based on specific productivity and growth rate. In the present study we will show the selection and screening process of a recombinant CHO cell line expressing a Fc fusion protein by analysis of gene copy numbers, mRNA-levels, specific productivity and integration locus.

Antibody engineering allows proper design of improved properties in antibody products such as a high binding affinity, stable biophysical properties and low immunogenicity. Besides these obvious features improved by elegant design the primary amino acid sequence also has tremendous effects on the performance of the expression host itself influencing cell culture parameters including specific productivities and growth rates therefore having major impact on the finally obtained antibody expression titers [1]. By understanding the underlying mechanisms how certain primary sequence combinations influence culture performance in conjunction with biophysical features of the antibody protein, we might rationally improve the production process of these expensive but essential biotherapeutic products. Many different antibody variants were expressed in our group in different projects based on various isotypes. For several antibody variable regions we could observe a direct correlation of advantageous cell performance and a high degree of similarity to the closest human germline sequence of the respective mature antibody leading to higher specific productivities. However, the fundamental principles of these consistent observations and the correlation of cell behavior and biophysical product properties remain quite elusive. Therefore, in this project we aim to set the proper foundations to investigate the generalization of a concept to improve cell performance and antibody expression based on the primary sequence at a high germinality degree (Figure ​(Figure1).1). To have proper control of transgene integration locus, gene copy numbers and mRNA transcript level a suitable host for recombinase-mediated cassette exchange (RMCE) was developed to compare different antibody variants under isogenic conditions. Open in a separate window Figure 1 Observed correlation of specific productivity and sequence identity to the closest human germline sequence (germinality) (A). Antibody expression levels are influenced by several factors. Recombinase-mediated cassette exchange was applied to control for gene copy number and mRNA transcript level. Low expressing antibody variants (2F5, 4B3 and 2G12) were compared to its closest human germline sequence together with a therapeutic antibody (Ustekinumab) with low degree of germinality (B). Respective germline antibody variants were constructed by combination of germline V-, D- and J-segments.

It has been proposed that HIV-1-neutralizing 2F5 and 4E10 monoclonal antibodies recognize gp41 ectodomain pretransmembrane sequences in the context of the membrane interface. With the aim of evaluating lipid-bilayer surface effects on recognition, we use phospholipid model systems to investigate the ability of 2F5 and 4E10 to transfer into membrane interfaces and bind epitope sequences immersed therein. The experimental data support the predicted tendencies for partitioning and recognition of membrane-bound epitopes, albeit with lower affinity in the case of 2F5. Our findings support the existence of two different recognition mechanisms at membrane surfaces: the association of 2F5 with the interface possibly prevents epitope immersion into the bilayer, and 4E10 membrane association might allow recognition of the membrane-bound epitope. We discuss the relevance of these observations for the design of immunogens aimed at eliciting 2F5- and 4E10- like humoral responses.

The conserved 664DKWASLWNWFNITNWLWYIK683 (preTM) sequence preceding the transmembrane anchor of human immunodeficiency virus (HIV-1) gp41 glycoprotein subunit is accessible to the broadly neutralizing 4E10 antibody and, therefore, constitutes a potential target for vaccine design. Recently reported structural data are compatible with preTM insertion into the viral external membrane monolayer in the gp41 pre-fusion state (Zhu, P., Liu, J., Bess, J., Chertova, E., Lifson, J. D., Grise, H., Ofek, G. A., Taylor, K. A., and Roux, K. H. (2006) Nature 441, 847-852). Here we demonstrate that the broadly neutralizing 4E10 antibody is able to specifically block the membrane-restructuring activity of a peptide mimic inserted into membranes. Recognition and restructuring blocking occurred in the presence of cholesterol, whereas transmembrane versions as those promoted in 1-palmitoyl-2-oleoylphosphatidylcholine:sphingomyelin mixtures could not be effectively arrested. Spectrofluorimetric assays using rhodamine-labeled peptides revealed that recognition correlated better with pore-formation blocking than with membrane-fusion inhibition. The capacity of the antibody to recognize preTM peptides in a raft-like environment was further corroborated employing planar-supported lipid layers and fluorescence microscopy. These data support that membrane-bound epitope recognition by 4E10 results in clustering reorganization of preTM at the membrane interface. We propose that this process might interfere with the formation of fusion-competent complexes at the low spike densities existing in the HIV-1 membrane. This work comprises the first experimental report on a lipid-modulated antibody capacity to bind a membrane-bound epitope sequence and arrest its restructuring activity.