Classical mAbs Research Articles

UAV trajectory planning in NOMA-aided UAV-mounted RIS networks: A budgeted Multi-armed bandit approach

Abstract Reconfigurable Intelligent Surface (RIS) and Non-Orthogonal Multiple Access (NOMA) can be used as supporting technologies for driving the effective development of Unmanned Aerial Vehicles (UAV) communication systems. Herein, both technologies are combined to widen the millimeter-wave Base Stations (mmWave-BS) coverage range. In this work, we consider UAV Trajectory Planning (UTP) problem to enhance the performance of a NOMA-based UAV-mounted RIS communication network. The problem is formulated using two Budgeted Multi-Armed Bandits (BMAB) schemes. BMAB is a variant of the classical MAB algorithm that considers the random cost of selecting an arm. The UAV performs as a bandit player attempting to maximize its achievable rate (i.e., reward) via accessing different grids in its trajectory. This reward is accomplished simultaneously while minimizing the energy/cost of flying the UAV from one grid to another to preserve its battery lifetime. Hence, two variants of the Budgeted upper confidence bound (BUCB), namely the BUCB1 and BUCB2, are investigated, where the simulations show that both algorithms outperform other benchmarks.

Nanobody-peptide-conjugate (NPC) for passive immunotherapy against SARS-CoV-2 variants of concern (VoC): a prospective pan-coronavirus therapeutics.

The COVID-19 crisis, incited by the zoonotic SARS-CoV-2 virus, has quickly escalated into a catastrophic public health issue and a grave threat to humankind owing to the advent of mutant viruses. Multiple pharmaceutical therapies or biologics envision stopping the virus from spreading further; however, WHO has voiced concerns about the variants of concern (VoCs) inability to respond. Nanobodies are a new class of antibody mimics with binding affinity and specificity similar to classical mAbs, as well as the privileges of a small molecular weight, ease of entry into solid tissues, and binding cryptic epitopes of the antigen. Herein, we investigated multiple putative anti-SARS-CoV-2 nanobodies targeting the Receptor binding domain of the WHO-listed SARS-CoV-2 variants of concern using a comprehensivecomputational immunoinformatics methodology. With affinity maturation via alanine scanning mutagenesis, we remodeled an ultrapotent nanobody with substantial breadth and potency, exhibiting pico-molar binding affinities against all the VoCs. An antiviral peptide with specificity for ACE-2 receptors was affixed to make it multispecific and discourage viral entry. Collectively, we constructed a broad-spectrum therapeutic biparatopic nanobody-peptide conjugate (NPC) extending coverage to SARS-CoV-2 VoCs RBDs. We PEGylated the biparatopic construct with 20kD maleimide-terminated PEG (MAL-(PEG)n-OMe) to improve its clinical efficacy limiting rapid renal clearance, and performed in silico cloning to facilitate future experimental studies. Our findings suggest that combining biparatopic nanobody conjugate with standard treatment may be a promising bivariate tool for combating viral entry during COVID-19 illness.

Ballooning multi-armed bandits

In this paper, we introduce ballooning multi-armed bandits (BL-MAB), a novel extension of the classical stochastic MAB model. In the BL-MAB model, the set of available arms grows (or balloons) over time. In contrast to the classical MAB setting where the regret is computed with respect to the best arm overall, the regret in a BL-MAB setting is computed with respect to the best available arm at each time. We first observe that the existing stochastic MAB algorithms result in linear regret for the BL-MAB model. We prove that, if the best arm is equally likely to arrive at any time instant, a sub-linear regret cannot be achieved. Next, we show that if the best arm is more likely to arrive in the early rounds, one can achieve sub-linear regret. Our proposed algorithm determines (1) the fraction of the time horizon for which the newly arriving arms should be explored and (2) the sequence of arm pulls in the exploitation phase from among the explored arms. Making reasonable assumptions on the arrival distribution of the best arm in terms of the thinness of the distribution's tail, we prove that the proposed algorithm achieves sub-linear instance-independent regret. We further quantify explicit dependence of regret on the arrival distribution parameters. We reinforce our theoretical findings with extensive simulation results. We conclude by showing that our algorithm would achieve sub-linear regret even if (a) the distributional parameters are not exactly known, but are obtained using a reasonable learning mechanism or (b) the best arm is not more likely to arrive early, but a large fraction of arms is likely to arrive relatively early.

An asymptotically optimal strategy for constrained multi-armed bandit problems

This note considers the model of “constrained multi-armed bandit” (CMAB) that generalizes that of the classical stochastic MAB by adding a feasibility constraint for each action. The feasibility is in fact another (conflicting) objective that should be kept in order for a playing-strategy to achieve the optimality of the main objective. While the stochastic MAB model is a special case of the Markov decision process (MDP) model, the CMAB model is a special case of the constrained MDP model. For the asymptotic optimality measured by the probability of choosing an optimal feasible arm over infinite horizon, we show that the optimality is achievable by a simple strategy extended from the \(\epsilon _t\)-greedy strategy used for unconstrained MAB problems. We provide a finite-time lower bound on the probability of correct selection of an optimal near-feasible arm that holds for all time steps. Under some conditions, the bound approaches one as time t goes to infinity. A particular example sequence of \(\{\epsilon _t\}\) having the asymptotic convergence rate in the order of \((1-\frac{1}{t})^4\) that holds from a sufficiently large t is also discussed.

Conjugation Site Analysis by MS/MS Protein Sequencing.

In-depth knowledge about the site of drug-linker conjugation is important for the understanding of the conjugation efficiency and the exact locations of payloads for antibody-drug conjugates (ADCs). Here we describe a peptide mapping-based protocol, covering sample preparation procedure, LC-MS/MS setup, and data processing (auto and manual), to determine the locations of drug-linker attachment on mAbs. In comparison with classical mAb peptide mapping, some improvements will be highlighted for maintaining hydrophobic drug-loaded peptides in solution, enabling efficient chromatographic separation and mass spectrometric detection, and allowing for their unambiguous identification in LC-MS/MS map by using diagnostic fragmentation ions of the payload.

Abstract 559: Preclinical characterization of the bispecific EGFR/CD16A innate immune cell engager AFM24 for the treatment of EGFR-expressing solid tumors

Abstract Affimed has engineered AFM24 based on its Redirected Optimized Cell Killing (ROCK®) platform to redirect innate immune cells to EGFR-positive tumor cells with the potential for activation of a broad anti-tumor immune response. This fully human tetravalent bispecific EGFR and CD16A binding antibody was designed for the treatment of EGFR-expressing malignancies with the potential to overcome the resistance to other EGFR-targeting agents and offering a better safety profile. The efficacy of current standard of care (SOC), including EGFR-targeting monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs), depends on intrinsic or acquired mutations that impact EGFR signal transduction. AFM24 exerts a mutation-independent mechanism of action (MOA), redirecting innate immunity via NK cells or macrophages to EGFR-positive tumor cells. PK analyses of AFM24 revealed a half-life of ~14 days in mice and ~5 days in cynomolgus monkeys. Using radioactively labeled AFM24 in mice, tumor-specific accumulation in subcutaneous human tumors was observed. While binding to CD16A by classical mAbs has low affinity and is further impaired by competition with serum IgG, no substantial changes in the very high binding affinity of AFM24 to NK cells were observed in the presence of IgG. Anti-tumor efficacy has been shown in in vitro cytotoxicity assays and in in vivo tumor models. Due to its MOA, AFM24 was also active against tumor cell lines expressing mutated Ras in in vitro assays. Antibody-dependent cellular phagocytosis (ADCP) of tumor cell lines expressing high levels of EGFR was comparable between AFM24 and classical or Fc-enhanced mAbs, but ADCP of tumor cells with lower EGFR expression mediated by AFM24 was more pronounced. Owing to their MOA of blocking EGFR-induced signaling, the therapeutic use of current SOC is associated with severe side effects such as skin toxicity, potentially limiting its use due to dosing interruptions and/or treatment termination. AFM24 is designed to exhibit a reduced potential to inhibit EGFR signaling, aiming for an improved safety profile. In a 4-week toxicology study in cynomolgus monkeys, no skin or organ toxicity was observed at AFM24 doses between 24 and 75 mg/kg i.v. once weekly. In toxicology studies investigating EGFR-targeting mAbs, e.g. cetuximab, with a similar experimental design, severe toxicities have been observed at those dosages. In summary, AFM24 is a novel, differentiated bispecific innate immune cell engager binding to EGFR and CD16A, thereby redirecting tumor cell killing by NK cells and macrophages to EGFR-expressing tumors. Its preclinical characterization described here, encourages AFM24’s development for the treatment of EGFR-expressing tumors with the potential to overcome resistance to current SOC while conferring a favorable safety profile. IND-enabling preclinical development is ongoing. Citation Format: Michael Kluge, Uwe Reusch, Stefan Knackmuss, Torsten Haneke, Susanne Wingert, Michael Damrat, Michael Tesar, Martin Treder. Preclinical characterization of the bispecific EGFR/CD16A innate immune cell engager AFM24 for the treatment of EGFR-expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 559.

Preclinical Characterization of AFM26, a Novel B Cell Maturation Antigen (BCMA)-Directed Tetravalent Bispecific Antibody for High Affinity Retargeting of NK Cells Against Myeloma

Natural killer (NK) cells are crucial effector cells of the innate immune system capable of rapidly recognizing and eliminating infected, stressed and malignant cells. NK cells are also the prime mediators of antibody-dependent cell-mediated cytotoxicity (ADCC), a potent mechanism of anti-viral immunity that has been applied to cancer therapy by targeting tumor-expressed surface antigens using monoclonal antibodies (mAbs). Classical ADCC is mediated by low affinity Fc-mediated engagement of NK cells via FcγRIIIA (CD16A) and is modulated by differences in target antigen expression levels. While high potency of therapeutic mAbs is achieved when target antigen is available at high density, potency and efficacy decrease substantially when copy numbers are low. Classical ADCC also needs to overcome the inhibitory effect of competing serum IgG and is negatively affected by a low affinity polymorphism of CD16A (158F) that is prevalent in approximately 8 of 10 individuals. Hence, classical Fc-mediated ADCC does not fully utilize the therapeutic potential of NK cell cytotoxicity.B cell maturation antigen (BCMA) has emerged as a promising target for treatment of multiple myeloma (MM) due to its near universal expression on tumor cells and restricted expression in non-malignant tissues. Numerous therapeutic approaches are currently investigated clinically and pre-clinically and target BCMA, however, none of these are aimed at fully utilizing NK cell-mediated ADCC. Low copy numbers of BCMA (approx. 40-15,000) might limit the activity of classical mAbs against BCMA, especially in the presence of high serum concentrations of paraprotein. In addition, NK cells may be ideally suited to target minimal residual disease immediately before or after autologous stem cell transplantation. Consequently, by efficiently redirecting NK cell cytotoxicity to BCMA+ myeloma, AFM26 used alone or in combination with other approaches may provide a novel, optimized treatment strategy.Here we describe development of AFM26, a BCMA and CD16A-directed tetravalent bispecific antibody that selectively engages CD16A+ effector cells, including NK cells, and is designed to overcome the limitations of classical ADCC. AFM26 is based on the recently launched Redirected, Optimized Cell Killing (ROCK) antibody platform and combines high affinity CD16A-directed effector cell engagement with IgG-like pharmacokinetics and manufacturability. We demonstrate that AFM26 interacts with NK cells with high avidity independently of CD16A polymorphism and in presence of competing IgG. NK cell-mediated lysis of BCMA+ target cell lines induced by AFM26 is largely independent of BCMA expression levels with high potency and efficacy observed at low copy numbers (<200), as confirmed by autologous lysis of primary MM cells in vitro. Despite more potent and efficacious in vitro lysis, release of inflammatory cytokines is comparable with classical antibody formats. We further demonstrate anti-tumor activity of AFM26 in combination with adoptive transfer of primary human NK cells in vivo using human IL-15-transgenic NOG mice.AFM26 therefore is a promising agent currently in preclinical development to fully unlock NK cell cytotoxicity for BCMA-directed immunotherapy of MM. DisclosuresRoss:Affimed: Employment. Reusch:Affimed: Employment. Wingert:Affimed: Employment. Haneke:Affimed: Employment. Klausz:Affimed: Research Funding. Otte:Affimed: Research Funding. Schub:Affimed: Research Funding. Knackmuss:Affimed: Employment. Müller:Affimed: Employment. Ellwanger:Affimed: Employment. Fucek:Affimed: Employment. Schniegler-Mattox:Affimed: Employment. Koch:Affimed GmbH: Employment. Valerius:Affimed: Research Funding. Gramatzki:Affimed: Research Funding. Peipp:Affimed: Research Funding. Tesar:Affimed: Employment. Rajkovic:Affimed: Employment. Treder:Affimed GmbH: Employment.

Abstract 2786: Pharmacokinetics and in vitro/in vivo characterization of high-affinity bispecific EGFR/CD16A NK cell engagers for the treatment of EGFR-expressing tumors

Abstract The epidermal growth factor receptor (EGFR) is a validated target for the treatment of several solid tumor types. Current EGFR-targeting monoclonal antibodies (mAbs) and tyrosine kinase inhibitors function mainly through blocking of signal-transduction. Moreover, treatment with these agents is dependent on the receptor's mutational status which may cause treatment resistance in a large number of patients. In addition, EGFR-targeting therapies have been associated with side effects considered to impact prescription rates, in particular in the U.S. A natural killer (NK) cell-based EGFR-targeting approach has the potential to widen the therapeutic window and overcome intrinsic and acquired resistance. Therefore, it offers a promising and well differentiated therapeutic option. We describe the engineering of different bispecific NK cell engagers designed to redirect NK cell-mediated killing to EGFR-positive tumors. Different bispecific EGFR/CD16A antibody constructs targeting human and cynomolgus EGFR and CD16A were designed using novel human Fv antibody binding domains from a toolbox of formats. The Fv domains were evaluated in terms of their pharmacokinetic (PK) properties. Furthermore, the antibody constructs were characterized in antigen and cell binding assays, as well as in in vitro cytotoxicity assays against tumor cell lines expressing Raswt or mutated Ras. PK parameters were determined in CD1 mice and in vivo efficacy of selected antibodies was investigated in an A-431 tumor model in humanized mice. We generated antibody constructs with varying half-lives, including antibodies with a PK profile which allows for dosing comparable to IgG-based antibodies, while aiming at an improved safety profile compared to other EGFR-targeting therapies. Different high affinity tetravalent, bispecific antibodies binding to CD16A and the extracellular domain of EGFR were characterized. While binding to CD16A of classical mAbs is impaired by serum IgG, no substantial changes in binding affinity of our constructs to NK cells were observed. The antibodies showed superior potency and efficacy compared to mAbs or Fc-enhanced antibodies when tested in cytotoxicity assays. In vivo efficacy of selected antibodies was demonstrated in an A-431 tumor model in humanized mice. We have successfully designed and characterized several novel, highly potent and differentiated tetravalent bispecific antibody constructs to redirect NK cell-mediated cell killing to EGFR-positive tumor cells. These drug candidates are suitable for the treatment of EGFR-expressing malignancies with the potential to overcome resistance to other EGFR-targeting agents and offering a potentially improved safety profile. These candidates are currently in development as monotherapy and are also explored in combination with immune activating agents. Citation Format: Michael Kluge, Michael Tesar, Uwe Reusch, Stefan Knackmuss, Torsten Haneke, Kristina Ellwanger, Ivica Fucek, Thomas Mueller, Ute Schniegler-Mattox, Martin Treder. Pharmacokinetics and in vitro/in vivo characterization of high-affinity bispecific EGFR/CD16A NK cell engagers for the treatment of EGFR-expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2786.

Abstract 5671: AFM26 - A novel CD16A-directed bispecific TandAb targeting BCMA for multiple myeloma

Abstract Multiple myeloma (MM) is the second most common haematological cancer and is characterized by the accumulation of neoplastic plasma cells in the bone marrow and production of high levels of monoclonal immunoglobulin (M-protein). While historically considered incurable, recent approvals and ongoing clinical trials with monoclonal antibodies (mAbs) targeting surface antigens promise greatly improved outcomes and have heralded a new era of MM treatment in which immunotherapies are expected to take center stage. However, an unmet need remains as patients eventually relapse and/or become refractory to currently available treatments. Consequently, novel immunotherapeutic approaches are needed to provide improved treatment options to MM patients. Among the currently explored targets, B-cell maturation antigen (BCMA, CD269) is considered to be particularly attractive due to its limited expression on healthy tissues and almost universal expression on myeloma cells in the majority of patients. Natural killer (NK) cells are cytotoxic effectors of the innate immune system capable of rapidly eradicating infected and transformed cells. The cytolytic activity of NK-cells can be used therapeutically to induce tumor cell lysis by direct engagement of the activating receptor CD16A (FcγRIIIa) using mAbs (ADCC). Despite similar mechanisms of target cell lysis, activation of NK-cells is not associated with the systemic symptoms of high level cytokine release as seen with direct T-cell engagement. Hence, it is considered a potent immunotherapeutic approach with reduced toxicity and a well-manageable safety profile. NK-cells readily infiltrate bone marrow and are thought to contribute to the efficacy of current myeloma treatments. Therefore, redirecting NK-cell cytotoxicity to malignant plasma cells appears to be a suitable therapeutic approach for MM. Here we describe the characterization of AFM26, a novel tetravalent bispecific tandem diabody (TandAb) that specifically targets BCMA and CD16A with high affinity and induces potent and efficacious myeloma cell lysis. AFM26 incorporates an affinity-improved anti-CD16A domain and interacts bivalently with NK-cells, resulting in high avidity and prolonged cell surface retention that is largely unaffected by the presence of polyclonal IgG. Notably, AFM26 potently induces NK-cell-mediated in vitro lysis of target cells expressing low levels of BCMA at low effector:target ratios, even in presence of polyclonal IgG. This may suggest that AFM26, in contrast to classical mAbs, retains ADCC activity at low antibody concentrations in presence of serum IgG and despite high levels of IgG M-protein occurring in about half of MM patients. AFM26 exhibits high protein stability, full cross-reactivity with cynomolgus antigens (BCMA and CD16A) and does not bind APRIL and TACI, two functionally related receptors. These data suggest that TandAb AFM26 is a promising and highly potent drug candidate for MM treatment. Citation Format: Thorsten Gantke, Uwe Reusch, Kristina Ellwanger, Ivica Fucek, Michael Weichel, Martin Treder. AFM26 - A novel CD16A-directed bispecific TandAb targeting BCMA for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5671. doi:10.1158/1538-7445.AM2017-5671

Characterization of Factor VIII Immune Complexes By Analytical Ultracentrifugation

Inhibitory polyclonal IgG antibodies (inhibitors) to factor VIII (fVIII) represent the most significant complication in patients with congenital hemophilia A. FVIII also is the most frequently targeted coagulation factor in autoimmunity. Antibodies recognizing epitopes in the fVIII A2 and C2 domains are present in most inhibitor patients. In the current study, we characterized the hydrodynamic properties of fVIII immune complexes formed by murine anti-human anti-A2 and anti-C2 fVIII monoclonal antibodies (MAbs) 4A4 and 3D12. 4A4 is representative of the most frequently identified group of anti-A2 MAbs identified in the murine hemophilia A immune response to human fVIII. 3D12 is a classical anti-C2 MAb that inhibits the binding of fVIII to von Willebrand factor (VWF) and phospholipid membranes. Velocity sedimentation of immune complexes formed by varying ratios of 4A4 and 3D12 with a high-expression fVIII construct designated ET3 was conducted at 55,000g and 20 °C by measuring protein absorbance at 280 nm in a Beckman XL-I analytical ultracentrifuge. Sedimentation coefficient (s20,w) distributions of fVIII, MAbs and immune complexes were determined using SEDFIT. The sedimentation coefficients of fVIII in the absence of MAbs and of the MAbs in the absence of fVIII were 7.7 S and 6.4 S, respectively. Under conditions of excess MAb (equimolar 4A4 and 3D12 each in five-fold molar excess over fVIII), a 10.3 S immune complex was observed, representing singly-ligated MAbs (Figure, red trace). Under conditions of excess fVIII (fVIII in four-fold molar excess over equimolar 4A4 and 3D12), 11.9 S doubly-ligated MAb complexes were observed (Figure, green trace). A mixture containing equimolar fVIII and 4A4/3D12 MAb binding sites produced a dominant 14.0 S species and a minor 18.8 S species, indicative of cross-linked 3D12-fVIII-4A4 immune complexes (Figure, blue trace). Indefinite association or immunoprecipitation was not observed. These results demonstrate that a biclonal, bivalent anti-fVIII antibody population can form higher-order immune complexes. These complexes may be a driving factor in the immune response to fVIII by promoting B cell activation and/or antigen presentation. Additionally, these results indicate that analytical ultracentrifugation is a useful tool to characterize fVIII immune complexes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract B054: Evaluation of NY-BR-1 as a suitable antigen for CAR based immunotherapy

Abstract Despite the tremendous clinical success of CAR-T-cell therapy in leukemia, translation of this treatment option to solid tumors still remains challenging. Off-target toxicity of CAR-T-cells against low-level antigen expressing healthy tissue is considered to be the most dangerous scenario when treating cancer patients yielding eventually to at least one reported fatal SAE. To avoid such fatalities a straight assessment of target antigen expression including all known isoforms of the respective protein in tumors as well as healthy tissues must be performed before drug application. The breast differentiation antigen NY-BR-1 (ANKRD30A) is known to serve as a perfect antigen for active immunotherapy of female breast cancer due to absence of expression in any female healthy tissue except for mammary gland. Here we report on the critical evaluation of NY-BR-1 as a target antigen for CAR-T-cells using bioinformatics, staining procedures and functional CAR tests. While bioinformatic analyses and stainings using the anti-NY-BR-1 monoclonal antibody “clone2” confirmed previous findings, we observed different results when the respective “clone2 scFv” was used. This construct not only shows a higher binding capacity to NY-BR-1 but also a modest but detectable binding to the isoform NY-BR1.1 (ANKRD30B). Additionally, a CAR-construct based on “clone2 scFv” yielded into a cross-reactivity of T cells against NY-BR1.1. Due to the known expression of NY-BR1.1 in normal brain tissue in combination with the also known ability of CAR-T-cells to cross the blood brain barrier, the use of a “clone2 CAR” for the treatment of advanced breast cancer must be judged as dangerous. Our findings show that a critical evaluation of a candidate target antigen for CAR therapy shall be performed by the use of the respective scFv rather than the classical mAb approach. Citation Format: Patrick Schmidt, Claudia Luckner Minden, Claudia Ziegelmeier, Isabella Gosch, Niels Halama, Frank Momburg, Rosa Eurich, Sarah Schott, Frederik Marmé, Andreas Schneeweiß, Claus Peter Heußel, Dirk Jäger, Inka Zörnig. Evaluation of NY-BR-1 as a suitable antigen for CAR based immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B054.

Surface Plasmon Resonance Binding Kinetics of Alzheimer’s Disease Amyloid β Peptide-Capturing and Plaque-Binding Monoclonal Antibodies

Several different monoclonal antibodies (mAbs) have been actively developed in the field of Alzheimer's disease (AD) for basic science and clinical applications; however, the binding kinetics of many of the mAbs with the beta-amyloid peptides (Abeta) are poorly understood. A panel of mAbs with different Abeta recognition sites, including our plaque-binding antibody (IgG4.1), a peptide-capturing antibody (11A50), and two classical mAbs (6E10 and 4G8) used for immunohistochemistry, were chosen for characterization of their kinetics of binding to monomeric and fibrillar forms of Abeta40 using surface plasmon resonance and their amyloid plaque binding ability in AD mouse brain sections using immunohistochemistry. The plaque-binding antibody (IgG4.1) with epitope specificity of Abeta(2-10) showed a weaker affinity (512 nM) for monomeric Abeta40 but a higher affinity (1.5 nM) for Abeta40 fibrils and labeled dense core plaques better than 6E10 as determined by immunohistochemistry. The peptide-capturing antibody (11A50) showed preferential affinity (32.5 nM) for monomeric Abeta40 but did not bind to Abeta40 fibrils, whereas antibodies 6E10 and 4G8 had moderate affinity for monomeric Abeta40 (22.3 and 30.1 nM, respectively). 4G8, which labels diffuse plaques better than 6E10, had a higher association rate constant than 6E10 but showed similar association and dissociation kinetics compared to those of 11A50. Enzymatic digestion of IgG4.1 to the F(ab')(2)4.1 fragments or their polyamine-modified derivatives that enhance blood-brain barrier permeability did not affect the kinetic properties of the antigen binding site. These differences in kinetic binding to monomeric and fibrillar Abeta among various antibodies could be utilized to distinguish mAbs that might be useful for immunotherapy or amyloid plaque imaging versus those that could be utilized for bioanalytical techniques.

A Transmembrane Tight Junction Protein Selectively Expressed on Endothelial Cells and Platelets

Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells. We obtained two monoclonal antibodies, 1G8 and 4C10, that stain endothelial cell contacts and recognize a protein of 55 kDa. Purification and identification by mass spectrometry of this protein revealed that it contains two extracellular Ig domains, reminiscent of the JAM family, but a much longer 120-amino acid cytoplasmic domain. The antigen is exclusively expressed on endothelial cells of various organs as was analyzed by immunohistochemistry. Immunogold labeling of ultrathin sections of brain as well as skeletal muscle revealed that the antigen strictly colocalizes in capillaries with the tight junction markers occludin, claudin-5, and ZO-1. Upon transfection into MDCK cells, the antigen was restricted to the most apical tip of the lateral cell surface, where it colocalized with ZO-1 but not with beta-catenin. In contrast to JAM-1, however, the 1G8 antigen does not associate with the PDZ domain proteins ZO-1, AF-6, or ASIP/PAR-3, despite the presence of a PDZ-binding motif. The 1G8 antigen was not detected on peripheral blood mouse leukocytes, whereas similar to JAM-1 it was strongly expressed on platelets and megakaryocytes. The 1G8 antigen supports homophilic interactions on transfected Chinese hamster ovary cells. Based on the similarity to the JAM molecules, it is plausible that the 1G8 antigen might be involved in interendothelial cell adhesion.