RUBY® - a tetravalent (2+2) bispecific antibody format with excellent functionality and IgG-like stability, pharmacology and developability properties.

Despite the large number of existing bispecific antibody (bsAb) formats, the generation of novel bsAbs is still associated with development and bioprocessing challenges. Here, we present RUBY, a novel bispecific antibody format that allows rapid generation of bsAbs that fulfill key development criteria. The RUBYTM format has a 2 + 2 geometry, where two Fab fragments are linked via their light chains to the C-termini of an IgG, and carries mutations for optimal chain pairing. The unique design enables generation of bsAbs with mAb-like attributes. Our data demonstrate that RUBY bsAbs are compatible with small-scale production systems for screening purposes and can be produced at high yields (>3 g/L) from stable cell lines. The bsAbs produced are shown to, in general, contain low amounts of aggregates and display favorable solubility and stress endurance profiles. Further, compatibility with various IgG isotypes is shown and tailored Fc gamma receptor binding confirmed. Also, retained interaction with FcRn is demonstrated to translate into a pharmacokinetic profile in mice and non-human primates that is comparable to mAb controls. Functionality of conditional active RUBY bsAbs is confirmed in vitro. Anti-tumor effects in vivo have previously been demonstrated, and shown to be superior to a comparable mAb, and here it is further shown that RUBY bsAbs penetrate and localize to tumor tissue in vivo. In all, the RUBY format has attractive mAb-like attributes and offers the possibility to mitigate many of the development challenges linked to other bsAb formats, facilitating both high functionality and developability.

Bispecific antibodies targeting CD40 and tumor-associated antigens promote cross-priming of T cells resulting in an antitumor response superior to monospecific antibodies.

BACKGROUND: Indications with poor T-cell infiltration or deficiencies in T-cell priming and associated unresponsiveness to established immunotherapies represent an unmet medical need in oncology. CD40-targeting therapies designed to enhance antigen presentation, generate new tumor-specific T cells, and activate tumor-infiltrating myeloid cells to remodel the tumor microenvironment, represent a promising opportunity to meet this need. In this study, we present the first in vivo data supporting a role for tumor-associated antigen (TAA)-mediated uptake and cross-presentation of tumor antigens to enhance tumor-specific T-cell priming using CD40×TAA bispecific antibodies, a concept we named Neo-X-Prime. METHODS: Bispecific antibodies targeting CD40 and either of two cell-surface expressed TAA, carcinoembryonic antigen-related cell adhesion molecule 5 (CEA) or epithelial cell adhesion molecule (EpCAM), were developed in a tetravalent format. TAA-conditional CD40 agonism, activation of tumor-infiltrating immune cells, antitumor efficacy and the role of delivery of tumor-derived material such as extracellular vesicles, tumor debris and exosomes by the CD40×TAA bispecific antibodies were demonstrated in vitro using primary human and murine cells and in vivo using human CD40 transgenic mice with different tumor models. RESULTS: The results showed that the CD40×TAA bispecific antibodies induced TAA-conditional CD40 activation both in vitro and in vivo. Further, it was demonstrated in vitro that they induced clustering of tumor debris and CD40-expressing cells in a dose-dependent manner and superior T-cell priming when added to dendritic cells (DC), ovalbumin (OVA)-specific T cells and OVA-containing tumor debris or exosomes. The antitumor activity of the Neo-X-Prime bispecific antibodies was demonstrated to be significantly superior to the monospecific CD40 antibody, and the resulting T-cell dependent antitumor immunity was directed to tumor antigens other than the TAA used for targeting (EpCAM). CONCLUSIONS: The data presented herein support the hypothesis that CD40×TAA bispecific antibodies can engage tumor-derived vesicles containing tumor neoantigens to myeloid cells such as DCs resulting in an improved DC-mediated cross-priming of tumor-specific CD8+ T cells. Thus, this principle may offer therapeutics strategies to enhance tumor-specific T-cell immunity and associated clinical benefit in indications characterized by poor T-cell infiltration or deficiencies in T-cell priming.

Allergome-wide peptide microarrays enable epitope deconvolution in allergen-specific immunotherapy.

BACKGROUND: The interaction of allergens and allergen-specific IgE initiates the allergic cascade after crosslinking of receptors on effector cells. Antibodies of other isotypes may modulate such a reaction. Receptor crosslinking requires binding of antibodies to multiple epitopes on the allergen. Limited information is available on the complexity of the epitope structure of most allergens. OBJECTIVES: We sought to allow description of the complexity of IgE, IgG4, and IgG epitope recognition at a global, allergome-wide level during allergen-specific immunotherapy (AIT). METHODS: We generated an allergome-wide microarray comprising 731 allergens in the form of more than 172,000 overlapping 16-mer peptides. Allergen recognition by IgE, IgG4, and IgG was examined in serum samples collected from subjects undergoing AIT against pollen allergy. RESULTS: Extensive induction of linear peptide-specific Phl p 1- and Bet v 1-specific humoral immunity was demonstrated in subjects undergoing a 3-year-long AIT against grass and birch pollen allergy, respectively. Epitope profiles differed between subjects but were largely established already after 1 year of AIT, suggesting that dominant allergen-specific antibody clones remained as important contributors to humoral immunity following their initial establishment during the early phase of AIT. Complex, subject-specific patterns of allergen isoform and group cross-reactivities in the repertoires were observed, patterns that may indicate different levels of protection against different allergen sources. CONCLUSIONS: The study highlights the complexity and subject-specific nature of allergen epitopes recognized following AIT. We envisage that epitope deconvolution will be an important aspect of future efforts to describe and analyze the outcomes of AIT in a personalized manner.

Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy.

BACKGROUND: Specific immunotherapy (SIT) is the only treatment with proved long-term curative potential in patients with allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B-cell repertoires are not well understood. OBJECTIVE: We sought to characterize the IgE sequences expressed by allergen-specific B cells and track the fate of these B-cell clones during SIT. METHODS: We used high-throughput antibody gene sequencing and identification of allergen-specific IgE with combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and the nasal mucosa from aeroallergen-sensitized subjects before and during the first year of subcutaneous SIT. RESULTS: Of 52 distinct allergen-specific IgE heavy chains from 8 allergic donors, 37 were also detected by using high-throughput antibody gene sequencing of blood samples, nasal mucosal samples, or both. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships. CONCLUSION: In the future, combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies might aid assessment of SIT mechanisms and efficacy.

A folded and immunogenic IgE-hyporeactive variant of the major allergen Phl p 1 produced in Escherichia coli.

BACKGROUND: Group 1 grass pollen allergens are a major cause of allergic disease. Specific immunotherapy involving controlled administration of allergens can be used as a disease-modifying treatment for such disease. Recombinant allergen variants with reduced IgE binding capacity may be used as component in such vaccines, as they may induce fewer treatment side effects than materials currently in use. A mutated variant of the immunodominant C-terminal domain of the group 1 grass pollen allergen Phl p 1 was recently established through an approach that used a set of human monoclonal IgE as a guide to identify mutations that disturbed IgE-allergen interactions. Further analysis of this domain is required to establish its potential for use in treatment. METHODS: GST-tagged wild-type and mutated C-terminal domains of Phl p 1 were produced in Escherichia coli TUNER(DE3). The products were purified by affinity chromatography on immobilized glutathione. GST was removed by enzymatic cleavage and tag-free products were purified by size exclusion chromatography. Products were assessed by SDS-PAGE, circular dichroism spectroscopy, differential scanning fluorimetry and dynamic light scattering. Rats were immunized with GST-tagged and tag-free mutated C-terminal domain of Phl p 1. Antigen-binding properties of induced antibodies were assessed by immunochemical analysis. RESULTS: The mutated domain has a structure very similar to that of the wild-type domain as determined by circular dichroism, but a reduced thermal stability. Immunization of rats demonstrates that this IgE-hyporeactive domain, despite its three sequence modifications (K8A, N11A, D55A), is able to induce antibodies that substantially block the binding of allergic subjects' IgE to the wild-type allergen. CONCLUSIONS: It is concluded that this IgE-hyporeactive molecule can be produced in folded form and that it is able to induce an antibody response that efficiently competes with IgE recognition of Phl p 1. These findings suggest that it, or a further evolved variant thereof, is a candidate for use as a component in specific immunotherapy against grass pollen allergy.

The human IgE repertoire.

IgE is a key mediator in allergic diseases. However, in strong contrast to other antibody isotypes, many details of the composition of the human IgE repertoire are poorly defined. The low levels of human IgE in the circulation and the rarity of IgE-producing B cells are important reasons for this lack of knowledge. In this review, we summarize the current knowledge on these repertoires both in terms of their complexity and activity, i.e. knowledge which despite the difficulties encountered when studying the molecular details of human IgE has been acquired in recent years. We also take a look at likely future developments, for instance through improvements in sequencing technology and methodology that allow the isolation of additional allergen-specific human antibodies mimicking IgE, as this certainly will support our understanding of human IgE in the context of human disease in the years to come.

Phl p 1-specific human monoclonal IgE and design of a hypoallergenic group 1 grass pollen allergen fragment.

Detailed understanding of how Abs of the IgE isotype interact with allergen at the onset of an allergic reaction is of great importance for deciphering mechanisms involved in the development of disease and may aid in the design of hypoallergenic variants. In this study, we have used a set of human monoclonal IgE Abs derived from the repertoires of allergic individuals, specific for the major timothy grass pollen allergen Phl p 1, to gain detailed information on the interaction between Abs and allergen. These allergen-specific IgE are to varying degrees cross-reactive toward both different allergen isoforms and various group 1 allergens originating from other grass species. The usage of human monoclonal IgE, as an alternative to polyclonal preparations or mouse Abs, allowed us to locate several important IgE-binding epitopes on the C-terminal domain of Phl p 1, all clustered to an IgE-binding "hot spot." By introducing three mutations in the IgE-binding area of the C-terminal domain we were able to significantly reduce its reactivity with serum IgE. In conclusion, our study shows the great potential of using human monoclonal IgE as a tool for studies of the molecular interactions taking place during allergic responses. Furthermore, we present a novel IgE-hyporeactive fragment with the potential to be used as a safer hypoallergenic alternative in specific immunotherapy than the pollen extracts used today.

Inconclusive evidence for or against positive antigen selection in the shaping of human immunoglobulin E repertoires: a call for new approaches.

BACKGROUND: The mechanisms driving the development of immunoglobulin E (IgE) antibody repertoires are a matter of debate. Alternatives to the classical view on antibody development, involving somatic mutation and antigen-driven selection of high-affinity variants in germinal centers, have been proposed. METHODS: We have re-analyzed the pattern of mutations in previously isolated and characterized human clonally unrelated IgE-encoding transcripts using the validated focused binomial methodology to find evidence in such genes of antigen-specific selection. RESULTS: As expected there is a selection against replacement mutations in IgE framework regions. In contrast, in all examined cases but one (assessing IgE repertoires of parasite-infected individuals) there was no evidence in favor of either positive or negative selection in complementarity determining regions. Importantly, however, the validated method also failed to detect selection for replacement mutations in two, non-IgE, hypermutated antibody populations targeting tetanus toxoid and vaccinia virus, respectively. CONCLUSIONS: Current methodology is unable to define with certainty, using commonly assessed IgE repertoire sizes, whether antigen selection is or is not a major driving force in the establishment of human IgE. New approaches are needed to address this matter.