Detection and quantification of Duffy antigen on bovine red blood cell membranes using a polyclonal antibody

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Multiscale sensing of antibody - antigen interactions by organic transistors and single-molecule force spectroscopy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Towards antibody-mediated targeting of dendritic cells for cancer immunotherapy with multivalent polymer-antigen conjugates

Der Fokus dieser Arbeit lag auf der definierten Synthese multifunktioneller Polymer-Konjugate zur Anwendung in der Krebs-Immunotherapie. Durch gezielte Variation der Kon-jugationsbedingungen wurde Zusammensetzung, Größe und Aggregationsverhalten in Zell-medium sowie in humanem Serum untersucht. Nach definierter physikalisch-chemischer Charakterisierung wurde dann die induzierte Antigen-Präsentation zur Aktivierung der T-Zellproliferation analysiert.rnDafür wurden zwei verschiedene polymere Carrier-Systeme gewählt, lineares Poly-L-lysin und eine Polylysinbürste (PLL-Bürste). Es wird vermutet, dass die PLL-Bürste aufgrund der anisotropen Form eine bessere Verteilung im Körper und eine verlängerte Zirkulationsdauer zeigen wird. Die zu konjugierenden biologisch aktiven Komponenten waren der antiDEC205-Antikörper (aDEC205) für die gezielte Adressierung CD8-positiver dendritischer Zellen (DC), ein Ovalbumin (OVA)-spezifisches Antigen mit der Kernsequenz SIINFEKL für die Spezifität der Immunantwort gegen Krebszellen, die dieses Antigen tragen, und ein immunaktivieren-der TLR9-Ligand, CpG1826. Die Effizienz dieses Konjugates dendritische Zellen zu aktivieren, welche wiederum eine Immunantwort gegen OVA-exprimierende Krebszellen induzieren, wurde durch die Konjugation aller Komponenten am identischen Trägermolekül deutlich höher erwartet.rnLineares Poly-L-lysin diente als Modellsystem um die Konjugationschemie zu etablieren und dann auf die zylindrische Polylysinbürste zu übertragen. Anhand dieser polymeren Träger wurde das Verhalten der verschiedenen Topologien des Knäuels und der Bürste im Hinblick auf den Einfluss struktureller Unterschiede sowohl auf Konjugationsreaktionen als auch auf das in situ und in vitro Verhalten untersucht.rnFluoreszenzmarkiertes Antigen und der CpG Aktivator konnten jeweils aufgrund einer Thiol-Modifizierung an die Thiol-reaktive Maleimidgruppe des heterobifunktionellen Linkers Sulfo-SMCC an PLL-AlexaFluor48 konjugiert werden. Anschließend wurde aDEC205-AlexaFluor647 an PLL gekoppelt, entweder durch Schiff Base-Reaktion des oxidierten Antikörpers mit PLL und anschließender Reduzierung oder durch Click-Reaktion des PEG-Azids modifizierten An-tikörpers mit Dicyclobenzylcyclooctin (DIBO)-funktionalisiertem PLL. Die Konjugation der biologisch aktiven Komponenten wurde mit Durchflusszytometrie (FACS) und konfokaler Laser Scanning Mikroskopie (CLSM) untersucht und die Zusammensetzung des Konjugatesrnmittels UV/Vis-Spektroskopie bestimmt. Die PLL-Bürste alleine zeigte eine hohe Zytotoxizität bei HeLa und JAWS II Zelllinien, wohingegen lineares PLL und PLL-Konjugate sowie die PLL Bürsten-Konjugate keine ausgeprägte Zytotoxizität aufwiesen. Die Polymer-Konjugate wie-sen keine Aggregation in Zellmedium oder humanem Serum auf, was mittels winkelabhängi-ger dynamischer Lichtstreuung bestimmt wurde. CLSM Aufnahmen zeigten Kolokalisation der an die einzelnen Komponenten gebundenen Fluoreszenzfarbstoffe in dendritischen Zel-len, was die erfolgreiche Konjugation und Internalisierung der Konjugate in die Zellen bele-gen konnte. FACS Messungen ergaben eine geringfügig erhöhte Aufnahme des adressierten PLL-Antigen-Antikörper-Konjugates verglichen mit dem PLL-Antigen-Konjugat. Experimente mit dem „Specific Hybridization Internalization Sensor“ (SHIP) zeigten jedoch nur Aufnahme der PLL-Konjugate in CD8+ unreife DC, nicht in reife DC, die nicht mehr unspezifisch, sondern nur noch über Rezeptoren internalisieren. Dies bewies die unspezifische Aufnahme des Kon-jugates, da Antikörper-Konjugation keine Rezeptor-vermittelte Endozytose in reife DC indu-zieren konnte. T-Zell-Proliferationsassays ergaben eine Aktivierung von CD8+ T-Zellen indu-ziert durch Antigen-tragende Konjugate, wohingegen Konjugate ohne Antigen als Negativ-kontrollen dienten und keine T-Zell-Proliferation erzielten. Es konnte jedoch kein Unter-schied zwischen adressierten und nicht adressierten Konjugaten aufgrund der unspezifischen Aufnahme durch das Polymer beobachtet werden. Lösliches SIINFEKL alleine bewirkte schon bei geringeren Konzentrationen eine T-Zell-Proliferation.rnEs war somit möglich, drei biologischen Komponenten an einen polymeren Träger zu konju-gieren und diese Konjugate im Hinblick auf Zusammensetzung, Größe, Internalisierung in dendritische Zellen und Aktivierung der T-Zell-Proliferation zu untersuchen. Außerdem wur-de die Konjugationschemie erfolgreich von dem Modellsystem des linearen PLL auf die PLL-Bürste übertragen. Die Polymer-Konjugate werde unspezifisch in DC aufgenommen und in-duzieren T-Zellproliferation, die mit Antigen-Präsentationsassays nachgewiesen wird. Es konnte jedoch durch Konjugation des Antikörpers keine Rezeptor-vermittelte Aufnahme in CD8+ DC erzielt werden.rnDiese Studien stellen einen erfolgsversprechenden ersten Schritt zur Entwicklung neuer Na-nomaterialien für die Anwendung in Krebs-Immuntherapie dar.

Development of a highly potent bispecific antibody format targeting the novel tumor-specific antigen CLDN18.2

Due to multiple immune evasion mechanisms of cancer cells, novel therapy approaches are required to overcome the limitations of existing immunotherapies. Bispecific antibodies are potent anti-cancer drugs, which redirect effector T cells for specific tumor cell lysis, thus enabling the patient’s immune system to fight cancer cells. The antibody format used in this proof of concept study–bispecific ideal monoclonal antibodies termed BiMAB–is a tailor-made recombinant protein, which consists of two fused scFv antibodies recognizing different antigens. Both are arranged in tandem on a single peptide chain and the individual variable binding domains are separated by special non-immunogenic linkers. The format is comprised of a scFv targeting CLDN18.2–a gastric cancer tumor associated antigen (TAA) –while the second specificity binds the CD3 epsilon (CD3ε) subunit of the T cell receptor (TCR) on T cells. For the first time, we compared in our IMAB362-based BiMAB setting, four different anti-CD3-scFvs, respectively derived from the mAbs TR66, CLB-T3, as well as the humanized and the murine variant of UCHT1. In addition, we investigated the impact of an N- versus a C-terminal location of the IMAB362-derived scFv and the anti-CD3-scFvs. Thus, nine CLDN18.2 specific BiMAB proteins were generated, of which all showed a remarkably high cytotoxicity towards CLDN18.2-positive tumor cells. Because of its promising effectiveness, 1BiMAB emerged as the BiMAB prototype. The selectivity of 1BiMAB for its TAA and CD3ε, with affinities in the nanomolar range, has been confirmed by in vitro assays. Its dual binding depends on the design of an N-terminally positioned IMAB362 scFv and the consecutive C-terminally positioned TR66 scFv. 1BiMAB provoked a concentration and target cell dependent T cell activation, proliferation, and upregulation of the cytolytic protein Granzyme B, as well as the consequent elimination of target cells. Our results demonstrate that 1BiMAB is able to activate T cells independent of elements that are usually involved in the T cell recognition program, like antigen presentation, MHC restriction, and co-stimulatory effector molecules. In the first in vivo studies using a subcutaneous xenogeneic tumor mouse model in immune incompetent NSG mice, we could prove a significant therapeutic effect of 1BiMAB with partial or complete tumor elimination. The initial in vitro RIBOMAB experiments correspondingly showed encouraging results. The electroporation of 1BiMAB IVT-RNA into target or effector cells was feasible, while the functionality of translated 1BiMAB was proven by induced T cell activation and target cell lysis. Accordingly, we could show that the in vitro RIBOMAB approach was applicable for all nine BiMABs, which proves the RIBOMAB concept. Thus, the CLDN18.2-BiMAB strategy offers great potential for the treatment of cancer. In the future, administered either as protein or as IVT-RNA, the BiMAB format will contribute towards finding solutions to raise and sustain tumor-specific cellular responses elicited by engaged and activated endogenous T cells. This will potentially enable us to overcome immune evasion mechanisms of tumor cells, consequently supporting current solid gastric cancer therapies.

Delivery of the p67 sporozoite antigen of Theileria parva by using recombinant Salmonella dublin: secretion of the product enhances specific antibody responses in cattle

The p67 sporozoite antigen of Theileria parva has been fused to the C-terminal secretion signal of Escherichia coli hemolysin and expressed in secreted form by attenuated Salmonella dublin aroA strain SL5631. The recombinant p67 antigen was detected in the supernatant of transformed bacterial cultures. Immunization trials in cattle revealed that SL5631 secreting the antigen provoked a 10-fold-higher antibody response to p67 than recombinant SL5631 expressing but not secreting p67. Immunized calves were challenged with a 80% lethal dose of T. parva sporozoites and monitored for the development of infection. Two of three calves immunized intramuscularly with the p67-secreting SL5631 strain were found to be protected, whereas only one of three animals immunized with the nonsecreting p67-expressing SL5631 strain was protected. This is the first demonstration that complete eukaryotic antigens fused to the C-terminal portion of E. coli hemolysin can be exported from attenuated Salmonella strains and that such exported antigens can protect cattle against subsequent parasite challenge.

Antigen binding forces of individually addressed single-chain Fv antibody molecules

Antibody single-chain Fv fragment (scFv) molecules that are specific for fluorescein have been engineered with a C-terminal cysteine for a directed immobilization on a flat gold surface. Individual scFv molecules can be identified by atomic force microscopy. For selected molecules the antigen binding forces are then determined by using a tip modified with covalently immobilized antigen. An scFv mutant of 12% lower free energy for ligand binding exhibits a statistically significant 20% lower binding force. This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.

Impaired CD19 expression and signaling, enhanced antibody response to type II T independent antigen and reduction of B-1 cells in CD81-deficient mice

The tetraspanin CD81 is ubiquitously expressed and associated with CD19 on B lymphocytes and with CD4 and CD8 on T lymphocytes. Analysis of mice with disrupted CD81 gene reveals normal T cells but a distinct abnormality in B cells consisting of decreased expression of CD19 and severe reduction in peritoneal B-1 cells. CD81-deficient B cells responded normally to surface IgM crosslinking, but had severely impaired calcium influx following CD19 engagement. CD81-deficient mice had increased serum IgM and IgA and an exaggerated antibody response to the type II T independent antigen TNP-Ficoll. These results suggest that CD81 is important for CD19 signaling and B cell function.

Crystal structure of an anti-carbohydrate antibody directed against Vibrio cholerae O1 in complex with antigen: Molecular basis for serotype specificity

The crystal structure of the murine Fab S-20-4 from a protective anti-cholera Ab specific for the lipopolysaccharide Ag of the Ogawa serotype has been determined in its unliganded form and in complex with synthetic fragments of the Ogawa O-specific polysaccharide (O-SP). The upstream terminal O-SP monosaccharide is shown to be the primary antigenic determinant. Additional perosamine residues protrude outwards from the Ab surface and contribute only marginally to the binding affinity and specificity. A complementary water-excluding hydrophobic interface and five Ab–Ag hydrogen bonds are crucial for carbohydrate recognition. The structure reported here explains the serotype specificity of anti-Ogawa Abs and provides a rational basis toward the development of a synthetic carbohydrate-based anti-cholera vaccine.

High-resolution microPET imaging of carcinoembryonic antigen-positive xenografts by using a copper-64-labeled engineered antibody fragment

Rapid imaging by antitumor antibodies has been limited by the prolonged targeting kinetics and clearance of labeled whole antibodies. Genetically engineered fragments with rapid access and high retention in tumor tissue combined with rapid blood clearance are suitable for labeling with short-lived radionuclides, including positron-emitting isotopes for positron-emission tomography (PET). An engineered fragment was developed from the high-affinity anticarcinoembryonic antigen (CEA) monoclonal antibody T84.66. This single-chain variable fragment (Fv)-CH3, or minibody, was produced as a bivalent 80 kDa dimer. The macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N,N′,N′′, N′′′-tetraacetic acid (DOTA) was conjugated to the anti-CEA minibody for labeling with copper-64, a positron-emitting radionuclide (t1/2 = 12.7 h). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA positive) and C6 rat glioma (CEA negative) xenografts. Five hours after injection with 64Cu-DOTA-minibody, microPET imaging showed high uptake in CEA-positive tumor (17.9% injected dose per gram ± 3.79) compared with control tumor (6.0% injected dose per gram ± 1.0). In addition, significant uptake was seen in liver, with low uptake in other tissues. Average target/background ratios relative to neighboring tissue were 3–4:1. Engineered antibody fragments labeled with positron-emitting isotopes such as copper-64 provide a new class of agents for PET imaging of tumors.

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant Kd = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin–biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 105–107 yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

Protective long-term antibody memory by antigen-driven and T help-dependent differentiation of long-lived memory B cells to short-lived plasma cells independent of secondary lymphoid organs

Memory is a hallmark of immunity. Memory carried by antibodies is largely responsible for protection against reinfection with most known acutely lethal infectious agents and is the basis for most clinically successful vaccines. However, the nature of long-term B cell and antibody memory is still unclear. B cell memory was studied here after infection of mice with the rabies-like cytopathic vesicular stomatitis virus, the noncytopathic lymphocytic choriomeningitis virus (Armstrong and WE), and after immunization with various inert viral antigens inducing naive B cells to differentiate either to plasma cells or memory B cells in germinal centers of secondary lymphoid organs. The results show that in contrast to very low background levels against internal viral antigens, no significant neutralizing antibody memory was observed in the absence of antigen and suggest that memory B cells (i) are long-lived in the absence of antigen, nondividing, and relatively resistant to irradiation, and (ii) must be stimulated by antigen to differentiate to short-lived antibody-secreting plasma cells, a process that is also efficient in the bone marrow and always depends on radiosensitive, specific T help. Therefore, for vaccines to induce long-term protective antibody titers, they need to repeatedly provide, or continuously maintain, antigen in minimal quantities over a prolonged time period in secondary lymphoid organs or the bone marrow for sufficient numbers of long-lived memory B cells to mature to short-lived plasma cells.

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.

Costimulatory protein B7-1 enhances the cytotoxic T cell response and antibody response to hepatitis B surface antigen.

There is a need for more effective therapy for chronic virus infections. A principle natural mechanism for elimination of virus-infected host cells is activation of viral antigen-specific cytotoxic T lymphocytes (CTL). In an effort to develop methods of inducing virus-specific CTL responses that might be utilized in therapy of virus infections, we have investigated the effect of B7, a costimulatory factor for T-cell activation. In this study we show that delivery of genes encoding human B7-1 and a viral antigen in the same recombinant viral vector to cells of mice induces a greater viral antigen-specific CTL response than does similar delivery of the viral antigen gene alone. Two recombinant adenovirus vectors were constructed with the foreign genes inserted in the early region 3. One of them (Ad1312) directed expression of the surface antigen gene of hepatitis B virus (HBS); the other (Ad1310) directed coexpression of HBS and human B7-1 (CD80) by means of an internal ribosomal entry site placed between the two coding sequences. When inoculated into BALB/c mice, both vectors induced a viral surface antigen-specific CTL response. The response induced by Ad1310 was stronger than that by Adl312 as measured by a chromium release assay for CTL activity and limiting dilution analysis for CTL precursor frequency, indicating that the B7-1 gene co-delivered with the HBS gene had an enhancing effect on the CTL response against surface antigen. Ad1310 also induced a higher titer of antibody against surface antigen than did Ad1312. This result suggests that expression of a costimulatory protein and a viral antigen in the same cells in vivo induces stronger immune responses than expression of the antigen alone. This could be a novel strategy for development of both preventive and therapeutic vaccines against infectious agents.

Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.

A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands. For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN. Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule. The two Fab fragments of the antibody were found to bind independently and with equal probability. The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.

A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells.

Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.