To harness the cytotoxic capacity of immune cells for the treatment of solid tumors, we developed tetravalent, bispecific tandem diabody (TandAb) antibodies that recognize EGFRvIII, the deletion ...variant III of the epidermal growth factor receptor (EGFR), and CD3 on T-cells, thereby directing immune cells to eliminate EGFRvIII-positive tumor cells. Using phage display, we identified scFv antibodies selectively binding to EGFRvIII. These highly EGFRvIII-specific, fully human scFv were substantially improved by affinity maturation, achieving
s in the picomolar range, and were used to construct a set of bispecific EGFRvIII-targeting TandAbs with a broad range of binding and cytotoxic properties. These antibodies exhibited an exquisite specificity for a distinguished epitope in the N-terminal portion of EGFRvIII, as shown on recombinant antigen in Western Blot, SPR, and ELISA, as well as on antigen-expressing cells in FACS assays, and did not bind to the wild-type EGFR. High-affinity EGFRvIII/CD3 TandAbs were most potent in killing assays, displaying cytotoxicity toward EGFRvIII-expressing CHO, F98 glioma, or human DK-MG cells with EC
values in the range of 1-10 pM
. They also demonstrated dose-dependent growth control
in an EGFRvIII-positive subcutaneous xenograft tumor model. Together with the tumor-exclusive expression of EGFRvIII, the EGFRvIII/CD3 TandAbs' high specificity and strictly target-dependent activation with no off-target activity provide an opportunity to target tumor cells and spare normal tissues, thereby reducing the side effects associated with other anti-EGFR therapies. In summary, EGFRvIII/CD3 TandAbs are highly attractive therapeutic antibody candidates for selective immunotherapy of EGFRvIII-positive tumors.
Baker's asthma is a serious problem for a significant proportion of workers in bakeries, confectionaries, and the food industry. Although several wheat allergens related to baker's asthma have been ...described, standardized reagents for a reliable diagnosis are not yet available.
To clone novel wheat allergens related to baker's asthma and investigate the cross-reactive potential of their maize and human homologues.
A wheat cDNA phage display library was screened with sera from bakers with occupational asthma for IgE-binding structures. Homologous sequences from maize and human thioredoxins were amplified from corresponding cDNA libraries.
Within the enriched wheat cDNA repertoire we identified, among others, the sequence encoding wheat thioredoxin-
hB (
Triticum aestivum allergen 25 Tri a 25). The recombinant protein displayed enzymatic activity, and we observed a sensitization rate of 47% among bakers with occupational asthma and of 35% among patients with grass pollen allergy, but without a clinical history of cereal allergy. Furthermore, the previously characterized maize thioredoxin-
h1 (
Zea mays allergen 25 Zea m 25), sharing 74% identity with Tri a 25, exhibited distinct IgE cross-reactivity with its wheat homologue. Two bakers also showed sensitization to human thioredoxin, which shares 29% identity with Tri a 25. In a comparative study, we included recombinant α-amylase inhibitor 0.19, showing a sensitization rate of 65% in individuals with baker's asthma.
Thioredoxins represent a novel family of cross-reactive allergens that might contribute to the symptoms of baker's asthma and might in addition be related to grass pollen allergy, as indicated by the reactivity of grass pollen allergic patients to cereal thioredoxins.
The recombinant cereal thioredoxins will, together with the already reported wheat allergens, contribute to a more reliable diagnosis of baker's asthma and, perhaps, become a tool for the development of component-resolved immunotherapy.
Abstract
Bispecific antibodies that redirect the lytic activity of cytotoxic immune effector cells, such as T- and NK cells, onto tumor cells have emerged as a highly attractive and clinically ...validated treatment modality for hematological malignancies. Advancement of this therapeutic concept into solid tumor indications, however, is hampered by the scarcity of targetable antigens that are surface-expressed on tumor cells but demonstrate only limited expression on healthy tissues. To overcome this limitation, the concept of dual-targeting, i.e. the simultaneous targeting of two tumor-expressed surface antigens with limited co-expression on non-malignant cells, with multispecific antibodies has been proposed to increase tumor selectivity of antibody-induced effector cell cytotoxicity. Here, a novel CD16A (FcγRIIIa)-directed trispecific, tetravalent antibody format, termed aTriFlex, is described, that is capable of redirecting NK cell cytotoxicity to two surface-expressed antigens. Using a BCMA/CD200-based in vitro model system, the potential use of aTriFlex antibodies for dual-targeting and selective induction of NK cell-mediated target cell lysis was investigated. Bivalent bispecific target cell binding was found to result in significant avidity gains and up to 17-fold increased in vitro potency. These data suggest trispecific aTriFlex antibodies may support dual-targeting strategies to redirect NK cell cytotoxicity with increased selectivity to enable targeting of solid tumor antigens.
Steady‐state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during ...the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well‐defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re‐evaluated and re‐assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal‐induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed.
The phage surface display technique was used to identify Borrelia burgdorferi antigens. By affinity selection with immunoglobulin G from pooled sera of six Lyme borreliosis (LB) patients, the ...ribosomal protein L25 was identified. The diagnostic value of L25 was investigated by an enzyme-linked immunosorbent assay, using sera from 80 LB patients and 75 controls, and the use of the protein resulted in a specificity of 99% and a 23% sensitivity, which qualify L25 as a useful antigen when combined with others.
Development of antibody scaffolds to directly engage cytotoxic effector cells such as T-cells for therapeutic applications is limited by the scarcity of surface antigens which are expressed ...exclusively on tumor cells and show limited or no expression on non-malignant cells. We have therefore designed a novel antibody format to selectively retarget effector cell cytotoxicity to tumor cells co-expressing two surface antigens. NK-cells play an important role in the innate immune response to multiple myeloma (MM) and are known to contribute to the efficacy of novel therapeutics. We, therefore, utilized a MM-based model system to generate proof-of concept data demonstrating antibody-mediated NK-cell retargeting to cell lines co-expressing two MM-expressed surface antigens with increased selectivity ('dual-targeting').
B-cell maturation antigen (BCMA/CD269) is widely considered to be a promising target antigen for antibody-based therapies of MM due to its almost universal expression on patient myeloma cells and its restricted surface expression on cells outside of the haematological lineage. However, low levels of expression on healthy tissue, including skin, has been reported, which could account for potential side effects associated with BCMA-targeted antibody therapies due to effector cell activation in these organs.
To increase selectivity of antibody-induced, effector cell-mediated cytotoxicity towards malignant tissue, we developed a trispecific antibody format capable of selectively engaging NK-cells through bivalent binding to CD16A (FcγRIIIa) and monovalent binding to both BCMA and CD200, a second MM-expressed surface antigen found in the majority of MM patients. Using an in vitro model system, we demonstrated that binding to BCMA+/CD200+ cell lines and the resulting increase in avidity leads to preferential lysis of antigen double-positive cells compared with antigen single-positive cells. These data suggest that dual-targeting may increase the therapeutic window compared to approaches targeting only one antigen, thereby improving safety of BCMA-directed antibody therapeutics for MM. In addition to the MM-based model system used here, the novel trispecific antibody scaffolds we have developed may be adapted to alternative target combinations within MM or in other tumor indications. Moreover, they could be used to target phenotypically distinct tumor cell clones to induce deeper and more prolonged antitumor responses. Consequently, dual-targeting of effector cells to tumors using the described antibody technology could also be applied to increase safety of T-cell engaging antibodies in the absence of exclusively tumor-expressed target antigens.
Gantke:Affimed GmbH: Employment. Weichel:Affimed GmbH: Employment. Reusch:Affimed: Employment, Patents & Royalties: Patents. Ellwanger:Affimed GmbH: Employment. Fucek:Affimed GmbH: Employment. Griep:AbCheck s.r.o.: Employment. Molkenthin:AbCheck s.r.o.: Employment. Kashala:Affimed Inc.: Employment. Treder:Affimed: Employment.
Introduction: CD19 is expressed by B cells from early development through differentiation into plasma cells, and represents a validated target for the development of therapeutic antibodies to treat B ...cell malignancies such as Non Hodgkin Lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Different CD19-targeting T-cell engagers are investigated in clinical studies for the treatment of NHL or ALL, including Affimed's AFM11, a bispecific CD19/CD3 TandAb antibody, which is currently investigated in a phase 1 dose escalation study. Indeed, Affimed's bispecific tetravalent platform comprises not only T-cell engaging TandAbs with two binding sites for CD3, but also NK-cell recruiting TandAbs with two binding sites for CD16A. In the present study, Affimed's AFM11, was characterized and compared in in vitro and in vivo studies with the CD19/CD16A TandAb AFM12.
Methods: Analogous to the CD19/CD3 TandAb AFM11, a bispecific tetravalent TandAb AFM12 was constructed with two binding sites for CD19 and two sites for CD16A. Both TandAbs were characterized side by side for their biophysical properties, binding affinities to CD19+ tumor target cells and to their respective effector cells by flow cytometry. Kinetics and dose-response characteristics were evaluated in in vitro cytotoxicity assays. Potency and efficacy of both TandAbs were compared on different CD19+ tumor target cell lines using primary human effector cells. To compare the efficacy of AFM11 and AFM12 a patient-derived tumor xenograft model was developed.
Results: AFM12 mediated efficacious target cell lysis with a very fast on-set in vitro. Lysis induced by AFM11 was less efficacious (lower specific lysis than AFM12) but reproducibly more potent (lower EC50 value). In addition to the potency and efficacy of AFM11 and AFM12, different aspects of safety, such as effector cell activation in the presence and absence of target cells were investigated and will be described.
Conclusions:
Affimed's CD19/CD3 and CD19/CD16A TandAbs with identical anti-CD19 tumor-targeting domains but different effector cell-recruiting domains represent interesting molecules to study T-cell- or NK-cell-based immunotherapeutic approaches. The comparison of AFM11 and AFM12 demonstrated that AFM12-mediated lysis was fast and efficacious, whereas AFM11 showed a higher potency. In summary, the NK-cell recruiting TandAb AFM12 represents an alternative to T-cell recruiting molecules, as it may offer a different side effect profile, comparable to that of AFM13, the first NK-cell TandAb clinically investigated.
No relevant conflicts of interest to declare.
The cloning, purification, and biophysical characterization of the first eukaryotic cold shock protein homologue, Cla h 8, expressed as single functional polypeptide is reported here. It was ...discovered as a minor allergen of the mold Cladosporium herbarum by phage display using a library selectively enriched for IgE-binding proteins. Based on the sequence homology of Cla h 8 with bacterial cold shock proteins (CSPs), a homology-based computer model of the allergen was computed indicating an all-β structure of Cla h 8. This major structural feature was confirmed by CD spectroscopy. Despite the structural similarities with bacterial CSPs, the DNA-binding and unfolding behavior of Cla h 8 exhibited unique and previously undescribed characteristics. High affinities of Cla h 8 for single-stranded DNA as well as for double-stranded DNA corresponding to the human Y-box were detected. The affinity for double-stranded DNA increased significantly with decreasing temperature, which was paralleled by an increase in the β sheet content of the protein. Temperature-dependent fluorescence anisotropy and far-UV CD measurements revealed different unfolding transitions at 28 and at 35.7 °C, respectively, indicating a multistate transition, which is uncommon for CSPs. The enhanced affinity for DNA at low temperatures together with the low unfolding transition refer to the functional significance of Cla h 8 at reduced temperatures.
Fungal allergies: a yet unsolved problem Crameri, Reto; Weichel, Michael; Flückiger, Sabine ...
Chemical immunology and allergy,
2006, Letnik:
91
Journal Article
Recenzirano
Airborne fungal spores have been implicated as causative factors in respiratory allergy, particularly asthma. However, the prevalence of fungal sensitization is not known mainly due to the lack of ...standardized fungal extracts and to the overwhelming number of fungal species able to elicit IgE-mediated reactions. Recent work based on high-throughput cloning of fungal allergens revealed that fungi are able to produce extremely complex repertoires of species-specific and cross-reactive allergens. There is evidence that fungal sensitization also contributes to auto-reactivity against self-antigens due to shared epitopes with homologous fungal allergens. Detailed studies at structural and immunological level indicate molecular mimicry as a basic mechanism involved in perpetuation of severe chronic allergic diseases. The real challenge at present is not related to cloning or production of a large number of different fungal allergens but rather to the assessment of the clinical relevance of each single structure. To date, substitution of complex extracts presently used in the diagnosis of fungal allergy by single, perfectly standardized components seems feasible in contrast to specific immunotherapy which is still not developed. Recombinant fungal allergens might create new perspectives in diagnosis and therapy of fungal allergy.
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