A platform for phenotypic discovery of therapeutic antibodies and targets applied on Chronic Lymphocytic Leukemia.

Development of antibody drugs against novel targets and pathways offers great opportunities to improve current cancer treatment. We here describe a phenotypic discovery platform enabling efficient identification of therapeutic antibody-target combinations. The platform utilizes primary patient cells throughout the discovery process and includes methods for differential phage display cell panning, high-throughput cell-based specificity screening, phenotypic in vitro screening, target deconvolution, and confirmatory in vivo screening. In this study the platform was applied on cancer cells from patients with Chronic Lymphocytic Leukemia resulting in discovery of antibodies with improved cytotoxicity in vitro compared to the standard of care, the CD20-specific monoclonal antibody rituximab. Isolated antibodies were found to target six different receptors on Chronic Lymphocytic Leukemia cells; CD21, CD23, CD32, CD72, CD200, and HLA-DR of which CD32, CD200, and HLA-DR appeared as the most potent targets for antibody-based cytotoxicity treatment. Enhanced antibody efficacy was confirmed in vivo using a patient-derived xenograft model.

Acoustic formation of multicellular tumor spheroids enabling on-chip functional and structural imaging.

Understanding the complex 3D tumor microenvironment is important in cancer research. This microenvironment can be modelled in vitro by culturing multicellular tumor spheroids (MCTS). Key challenges when using MCTS in applications such as high-throughput drug screening are overcoming imaging and analytical issues encountered during functional and structural investigations. To address these challenges, we use an ultrasonic standing wave (USW) based MCTS culture platform for parallel formation, staining and imaging of 100 whole MCTS. A protein repellent amphiphilic polymer coating enables flexible production of high quality and unanchored MCTS. This enables high-content multimode analysis based on flow cytometry and in situ optical microscopy. We use HepG2 hepatocellular carcinoma, A498 and ACHN renal carcinoma, and LUTC-2 thyroid carcinoma cell lines to demonstrate (i) the importance of the ultrasound-coating combination, (ii) bright field image based automatic characterization of MTCS, (iii) detailed deep tissue confocal imaging of whole MCTS mounted in a refractive index matching solution, and (iv) single cell functional analysis through flow cytometry of single cell suspensions of disintegrated MTCS. The USW MCTS culture platform is customizable and holds great potential for detailed multimode MCTS analysis in a high-content manner.

Does diet intervention in line with nutrition recommendations affect dietary carbon footprint? Results from a weight loss trial among lactating women.

BACKGROUND/OBJECTIVES: Results from studies evaluating the sustainability of diets combining environmental and nutritional aspects have been diverse; thus, greenhouse gas emissions (that is, carbon footprint (CF)) of diets in line with dietary recommendations in free-living individuals warrants further examination. Here, changes in dietary CF related to changes in food choice during a weight loss trial among lactating women who received a 12-week diet intervention based on the Nordic Nutrition Recommendations (NNR) 2004 were analyzed. The objective of this study was to examine if a diet intervention based on NNR 2004 results in reduced dietary CF. SUBJECTS/METHODS: Changes in dietary CF were analyzed among 61 lactating women participating in a weight loss trial. Food intake data from 4-day weighed diet records and results from life cycle analyses were used to examine changes in dietary CF across eight food groups during the intervention, specified in the unit carbon dioxide equivalent (CO2eq/day). Differences in changes in dietary CF between women receiving diet treatment (D-group) and women not receiving it (ND-group) were compared. RESULTS: There was no difference in change in dietary CF of the overall diet between D- and ND-group (P>0.05). As for the eight food groups, D-group increased their dietary CF from fruit and vegetables (+0.06±0.13 kg CO2eq/day) compared with a decrease in ND-group (-0.01±0.01 kg CO2eq/day) during the intervention, P=0.01. CONCLUSIONS: A diet intervention in line with NNR 2004 produced clinically relevant weight loss, but did not reduce dietary CF among lactating women with overweight and obesity. Dietary interventions especially designed to decrease dietary CF and their coherence with dietary recommendations need further exploration.

Multiple independent IgE epitopes on the highly allergenic grass pollen allergen Phl p 5.

BACKGROUND: Group 5 allergens are small proteins that consist of two domains. They belong to the most potent respiratory allergens. OBJECTIVE: To determine the binding sites and to study allergic patients' IgE recognition of the group 5 allergen (Phl p 5) from timothy grass pollen using human monoclonal IgE antibodies that have been isolated from grass pollen allergic patients. METHODS: Using recombinant isoallergens, fragments, mutants and synthetic peptides of Phl p 5, as well as peptide-specific antibodies, the interaction of recombinant human monoclonal IgE and Phl p 5 was studied using direct binding and blocking assays. Cross-reactivity of monoclonal IgE with group 5 allergens in several grasses was studied and inhibition experiments with patients' polyclonal IgE were performed. RESULTS: Monoclonal human IgE showed extensive cross-reactivity with group 5 allergens in several grasses. Despite its small size of 29 kDa, four independent epitope clusters on isoallergen Phl p 5.0101, two in each domain, were recognized by human IgE. Isoallergen Phl p 5.0201 carried two of these epitopes. Inhibition studies with allergic patients' polyclonal IgE suggest the presence of additional IgE epitopes on Phl p 5. CONCLUSIONS & CLINICAL RELEVANCE: Our results reveal the presence of a large number of independent IgE epitopes on the Phl p 5 allergen explaining the high allergenic activity of this protein and its ability to induce severe allergic symptoms. High-density IgE recognition may be a general feature of many potent allergens and form a basis for the development of improved diagnostic and therapeutic procedures in allergic disease.

Human IgE against the major allergen Bet v 1--defining an epitope with limited cross-reactivity between different PR-10 family proteins.

BACKGROUND: The interaction between IgE and allergen is a key event at the initiation of an allergic response, and its characteristics have substantial effects on the clinical manifestation. Despite this, the molecular details of the interaction between human IgE and the major birch allergen Bet v 1, one of the most potent tree allergens, still remain poorly investigated. OBJECTIVE: To isolate Bet v 1-specific human monoclonal IgE and characterize their interaction with the allergen. METHODS: Recombinant human IgE were isolated from a combinatorial antibody fragment library and their interaction with Bet v 1 assessed using various immunological assays. The structure of one such IgE in the single-chain fragment variable format was determined using X-ray crystallography. RESULTS: We present four novel Bet v 1-specific IgE, for one of which we solve the structure, all with their genetic origin in the IGHV5 germline gene, and demonstrate that they target two non-overlapping epitopes on the surface of Bet v 1, thereby fulfilling the basic criteria for FcεRI cross-linkage. We further define these epitopes and for one epitope pinpoint single amino acid residues important for the interaction with human IgE. This provides a potential explanation, at the molecular level, for the differences in recognition of isoforms of Bet v 1 and other allergens in the PR-10 protein family displayed by IgE targeting this epitope. Finally, we present the first high-resolution structure of a human allergen-specific IgE fragment in the single-chain fragment variable (scFv) format. CONCLUSIONS AND CLINICAL RELEVANCE: We here display the usefulness of allergen-specific human monoclonal IgE as a tool in studies of the crucial molecular interaction taking place at the initiation of an allergic response. Such studies may aid us in development of better diagnostic tools and guide us in the development of new therapeutic compounds.

Diversity of immunoglobulin E-encoding transcripts in sinus mucosa of subjects diagnosed with non-allergic fungal eosinophilic sinusitis.

BACKGROUND: The role of allergy in the aetiopathogenesis of chronic rhinosinusitis (CRS) remains controversial. For example, in some cases with sinus fungal infections allergy can be demonstrated by standard tests. In other cases, such signs can be absent despite elevated levels of IgE-positive cells in sinus tissue and the presence of IgE and eosinophils in the sinus mucous. OBJECTIVE: To define the nature of molecular diversity in antibodies of the IgE isotype at the site of local inflammation in subjects diagnosed with non-allergic fungal eosinophilic sinusitis (NAFES). METHODS: The local occurrence and sequence characteristics of IgE-encoding transcripts in NAFES patients were investigated and compared with sequences found in subjects diagnosed with CRS featuring systemic allergy. These sequences have also been compared with other reported IgE-encoding transcriptomes. Results IGHV genes derived from major subgroups 1, 3, 4 and 5 and a diverse set of IGHD and IGHJ genes were shown to create the IgE repertoire in patients diagnosed with NAFES and CRS. The average lengths of the third hypervariable loop in these populations were 15.8 and 14.6 residues. The sequences showed evidence of extensive somatic hypermutation (mutation frequency: NAFES, 6.4 ± 3.2%; CRS, 7.0 ± 4.4%) with substitutions targeted to complementarity-determining regions. These sequence collections thus show extensive similarities to those found in other polyclonal Ig repertoires including those encoding IgE. CONCLUSION AND CLINICAL RELEVANCE: We conclude that sinus IgE-encoding transcripts in subjects diagnosed with NAFES show evidence of conventional IgE responses and we suggest that allergens with characteristics of classical antigens should be investigated for a role in the local response occurring in NAFES. This investigation illustrates that assessment of local immunity might be an important diagnostic tool in conditions like NAFES with no systemic signs of allergy to identify or rule out an allergic component of the patient's disease.

A carbohydrate binding module as a diversity-carrying scaffold.

The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.

The immune diversity in a test tube--non-immunised antibody libraries and functional variability in defined protein scaffolds.

Technologies to develop and evolve the function of proteins and, in particular, antibodies have developed rapidly since the introduction of phage display. Importantly, it has become possible to identify molecules with binding properties that cannot be found by other means. A range of different approaches to create general libraries that are useful for the selection of such molecules specific for essentially any kind of target have emerged. We herein review some of the most prominent approaches in the field and in particular discuss specific features related to the development of antibody libraries based on single antibody framework scaffolds. This approach not only permits identification of a range of specific binders, but also facilitates further evolution of initially derived molecules into molecules with optimised functions.

A central core structure in an antibody variable domain determines antigen specificity.

Antibody binding sites provide an adaptable surface capable of interacting with essentially any molecular target. Using CDR shuffling, residues important for the assembly of mucin-1 specific paratopes were defined by random recombination of the complementarity determining regions derived from a set of mucin-1 specific clones, previously selected from an antibody fragment library. It was found that positions 33 and 50 in the heavy chain and 32, 34, 90, 91 and 96 in the light chain were conserved in many of the clones. These particular residues seem to be located centrally in the binding site as indicated by a structure model analysis. The importance of several of these conserved residues was supported by their presence in a mouse monoclonal antibody with a known structure and the same epitope specificity. Several of these corresponding residues in the mouse monoclonal antibody are known to interact with the antigen. In conclusion, critical residues important for maintaining a human antigen-specific binding site during the process of in vitro antibody evolution were defined. Furthermore, an explanation for the observed restricted germline gene usage in certain antibody responses against protein epitopes is provided.

Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries.

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.

Complementarity-determining region (CDR) implantation: a theme of recombination.

A novel technology in the area of antibody engineering has been developed which allows for the creation of new types of antibody molecules. It is called complementarity-determining region (CDR) implantation and permits the random combination of CDR sequences formed in vivo into a single master framework. Thus, totally new gene combinations can be produced and used in selection processes. The result is a genetic variability which is extremely large, even exceeding the natural variability found in the immune system. In this commentary, CDR implantation is presented and the technology is discussed.

Effect of cysteine substitutions on dimerization and interfragment linkage of human cytomegalovirus glycoprotein B (gp UL55).

Experiments were carried out to analyze the function of cysteine residues at amino acid positions 506 (cI), 550 (cII), 573 (cIII), and 610 (cIV), in dimerization and/or disulfide linkage of human cytomegalovirus (HCMV) glycoprotein B (gB). Single c-codons or pairs were substituted in the gB sequence of constructs which were used for transfection and selection of stable transfectants. Analysis of gB expression products revealed that single substitutions of cIII or cIV, but neither single nor double substitutions of cI or/and cII prevented gB dimerization. All substituted gB derivatives were, however, no longer processed by proteolytic cleavage. After deletion of the membrane anchor domain, correct proteolytic processing was again observed for anchorless gB forms. Substitutions of cI or cI/cII in secretory gB appeared to interfere with disulfide linkage between gB cleavage fragments. In the case of anchorless gB with substitutions of cII, cIII, or cIII/cIV, however, extracellular gB forms were not recovered. Using the Sindbis expression system recovery of all anchorless gB forms with cysteine substitutions was achieved. Analysis verified involvement of cI/II substitutions in intrachain disulfide linkage between cleavage fragments of HCMV gB.

Insertions and deletions in hypervariable loops of antibody heavy chains contribute to molecular diversity.

Antibody diversity, a molecular feature which allows these proteins to specifically interact with a diverse set of targets, is created at the genetic level by a variety of means. These include germline gene segment recombination, junctional diversity and single basepair (bp) substitution. We here demonstrate that a human high affinity antibody specific for an exogenous protein antigen carry three amino acid residues immediately adjacent to the first hypervariable loop of the heavy chain. These additional residues are shown not to be encoded by the germline repertoire. We also describe the characteristics of insertions and deletions, not found in any known germline sequence, within the first and second hypervariable loops of other previously described antibody-encoding genes. These findings demonstrate that insertions or deletions of entire codons provide yet another approach by which the human antibody repertoire is diversified in vivo. Since these major genetic modifications occur within or immediately adjacent to loops contributing to the antigen-binding site, they are likely to affect the binding properties of the mutated antibodies.

Exploiting sequence space: shuffling in vivo formed complementarity determining regions into a master framework.

A novel approach in molecular design is presented, where in vivo formed complementarity determining regions (CDR) from antibody genes were shuffled into a specific framework region. A synthetic gene library of soluble VH-fragments was created and the complexity of the library was determined by sequencing. The synthetic genes were diverse and contained random combinations of CDR from different germlines. All CDR were randomised in one step and by using in vivo formed CDR, the length, sequence and combination were varied simultaneously.

New approach to steroid separation based on a low affinity IgM antibody.

IgM antibodies are often of low affinity (dissociation constant (Kd) > 10(-5) M) and therefore they are usually neglected as tools in, e.g., immunoassays. Previous studies have shown that low affinity biological interactions can be studied and exploited in affinity chromatography, biosensor technology and capillary electrophoresis. In this study we have demonstrated that IgM can be a useful ligand for analytical separation of antigens in weak affinity chromatography (WAC). A low affinity human monoclonal IgM antibody, directed at digoxin, was produced in a hybridoma cell culture, purified to homogeneity and immobilized onto an HPLC support. The IgM HPLC column displayed specific weak affinity retention in the 0.01-0.1 mM range as evaluated with digoxin and ouabain. The specificity was not affected when samples of ouabain in a crude environment of diluted serum were separated on the IgM column. These findings suggest an approach in immunoadsorbent technology where biomolecules can be analyzed and separated with weak affinity chromatography using IgM as a general affinity ligand.

Three-dimensional structure of a human Fab with high affinity for tetanus toxoid.

BACKGROUND: The wide range of antibody specificity and affinity results from the differing shapes and chemical compositions of their binding sites. These shapes range from discrete grooves in antibodies elicited by linear oligomers of nucleotides and carbohydrates to shallow depressions or flat surfaces for accommodation of proteins, peptides and large organic compounds. OBJECTIVES: To determine the Fab structure of a high-affinity human antitoxin antibody. To explore structural features which enable the antibody to bind to intact tetanus toxoid, peptides derived from the sequence of the natural immunogen and antigenic mimics identified by combinatorial chemistry. To explain why this Fab shows a remarkable tendency to produce crystals consistently diffracting to d spacings of 1.7-1.8 A. To use this information to engineer a strong tendency to crystallize into the design of other Fabs. STUDY DESIGN: The protein was crystallized in hanging or sitting drops by a microseeding technique in polyethylene glycol (PEG) 8000. Crystals were subjected to X-ray analysis and the three-dimensional structure of the Fab was determined by the molecular replacement method. Interactive computer graphics were employed to fit models to electron density maps, survey the structure in multiple views and discover the crystal packing motif of the protein. RESULTS: Exceptionally large single crystals of this protein have been obtained, one measuring 5 x 3 x 2 mm (l x w x d). The latter was cut into six irregular pieces, each retaining the features of the original in diffracting to high resolution (1.8 A) with little decay in the X-ray beam. In an individual Fab, the active site is relatively flat and it seems likely that the protein antigen and derivative peptides are tightly held on the outer surface without significant penetration into the interior. There is no free space to accommodate even a dipeptide between VH and VL. One of the unique features of the B7-15A2 Fab is a large aliphatic ridge dominating the center of the active site. The CDR3 of the H chain contributes significantly to this ridge, as well as to adjoining regions projected to be important for the docking of the antigen. Both the ease of crystallization and the favorable diffraction properties are mainly attributable to the tight packing of the protein molecules in the crystal lattice. DISCUSSION: The B7-15A2 active site provides a stable and well defined platform for high affinity docking of proteins, peptides and their mimotopes. The advantages for future developments are suggested by the analysis of the crystal properties. It should be possible to incorporate the features promoting crystallization, close packing and resistance to radiation damage into engineered human antibodies without altering the desired specificities and affinities of their active sites.

Evaluation of novel control elements by construction of eukaryotic expression vectors.

A novel mammalian eukaryotic expression vector for the production of immunoglobulin heavy chain (IgH) genes has been designed. This expression vector contains the variable heavy chain (VH) promoter, the IgH intron enhancer (muE) and the IgH 3' enhancer (3'E). This construct, designated pTIF-1, was stably transfected into the myeloma cell line J558L. A fivefold increase in the expression level of a rearranged IgH gene was observed when using the pTIF-1 vector containing the 3'E compared to an expression vector lacking this enhancer. Interestingly, this positive effect on the expression level of the 3' enhancer appears to be position independent. The introduction of two recently identified Ig control elements, HS3 and HS4, to the vector cassette did not further elevate the expression level in the cell line tested. The pTIF-1 vector can be used for expression of any antibody specificity, using PCR amplification of the VDJ region of interest. Furthermore, the constant region can easily be exchanged, which further facilitates studies to dissect different effector functions of IgH constant genes.

Altered gene expression associated with apoptosis in a pre-B-leukemic cell line following cross-linking of MHC class I.

The major histocompatibility complex class I (MHC-I) has recently been shown not only to present antigens to the immune system but also to mediate transmembrane signaling, resulting in activation, inactivation, or apoptosis. Such signaling has been observed in both normal and malignant cells of the B and T cell lineage. Cross-linking of MHC-I on the pre-B-acute-lymphocytic cell line KM-3 induces an apoptotic process, which becomes evident after approximately 12 h. In order to better understand the mechanisms regulating this apoptotic process, we have investigated both gene expression and the effect of cross-linking on certain intracellular events. Differential display PCR was used to isolate two gene fragments whose level of expression was associated with the induction of apoptosis as they were downregulated in KM-3 cells following MHC-I cross-linking. These genes encode novel molecules whose function remains to be elucidated. It was further demonstrated that the apoptotic process was not accompanied by changes in [Ca2+]i, the level of activation of NF-kappaB, or changes in protein kinase C activity and that the initiation of apoptosis could be prevented by phorbol ester treatment. It is thus suggested that multiple, fine-tuned molecular events determine the outcome of cross-linking of MHC-I in this pre-B-lymphocytic cell line.

Cytomegalovirus glycoprotein B-specific antibody analysis using electrochemiluminescence detection-based techniques.

An electrochemiluminescence technique was used to develop versatile and sensitive assay strategies for determination of seroreactivities against biologically important cytomegalovirus neutralization epitopes expressed on glycoprotein B. Indirect binding assays showed wide linear assay ranges and revealed that serum samples diluted in parallel with a monoclonal antibody-based standard, simplifying quantitative analytical assessments.

Selection of binders from phage displayed antibody libraries using the BIAcore biosensor.

In this report we show that phage displayed antibodies can be selected based on dissociation rate constants, using a BIAcore biosensor. To demonstrate the principle, two Fab phage stocks displaying antibodies specific for hen egg lysozyme or phenyloxazolone were mixed in a ratio of 1:10 and injected over the biosensor chip containing immobilized lysozyme. Antigen-specific bound phages were eluted and analysed for specificity and phage titer. This procedure enriched for phages carrying specific antibodies. Selection of high affinity binders from phage libraries was then demonstrated with the BIAcore when phages were eluted and collected at different time points. Soluble antibody fragments were subsequently expressed and their kinetic parameters were determined. The time of elution was directly proportional to the affinity, due to decreased dissociation rate constants. This procedure offers a rapid and simple approach for selecting binders from phage libraries differing in antibody dissociation rate constants.