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Redirection of immune cells to efficiently eliminate tumor cells holds great promise. Natural killer cells (NK), macrophages, or T cells are specifically engaged with target cells expressing markers after infection or neoplastic transformation, resulting in their activation and subsequent killing of those targets. Multiple strategies to redirect immunity have been developed in the past two decades, but they have technical hurdles or cause undesirable side-effects, as exemplified by the T cell-based chimeric antigen receptor approaches (CAR-T therapies) or bispecific T cell engager platforms. Our first-in-class bispecific antibody redirecting innate immune cells to tumors (AFM13, a CD30/CD16A-specific innate immune cell engager) has shown signs of clinical efficacy in CD30-positive lymphomas and the potential to be safely administered, indicating a wider therapeutic window compared to T cell engaging therapies. AFM13 is the most advanced candidate from our fit-for-purpose redirected optimized cell killing (ROCK®) antibody platform, which comprises a plethora of CD16A-binding innate immune cell engagers with unique properties. Here, we discuss aspects of this modular platform, including the advantages of innate immune cell engagement over classical monoclonal antibodies and other engager concepts. We also present details on its potential to engineer a fit-for-purpose innate immune cell engager format that can be equipped with unique CD16A domains, modules that influence pharmacokinetic properties and molecular architectures that influence the activation of immune effectors, as well as tumor targeting. The ROCK® platform is aimed at the activation of innate immunity for the effective lysis of tumor cells and holds the promise of overcoming limitations of other approaches that redirect immune cells by widening the therapeutic window.

Epidermal growth factor receptor (EGFR)-targeted cancer therapy such as anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors have demonstrated clinical efficacy. However, there remains a medical need addressing limitations of these therapies, which include a narrow therapeutic window mainly due to skin and organ toxicity, and primary and secondary resistance mechanisms of the EGFR-signaling cascade (e.g., RAS-mutated colorectal cancer). Using the redirected optimized cell killing (ROCK®) antibody platform, we have developed AFM24, a novel bispecific, IgG1-scFv fusion antibody targeting CD16A on innate immune cells, and EGFR on tumor cells. We herein demonstrate binding of AFM24 to CD16A on natural killer (NK) cells and macrophages with KD values in the low nanomolar range and to various EGFR-expressing tumor cells. AFM24 was highly potent and effective for antibody-dependent cell-mediated cytotoxicity via NK cells, and also mediated antibody-dependent cellular phagocytosis via macrophages in vitro. Importantly, AFM24 was effective toward a variety of EGFR-expressing tumor cells, regardless of EGFR expression level and KRAS/BRAF mutational status. In vivo, AFM24 was well tolerated up to the highest dose (75 mg/kg) when administered to cynomolgus monkeys once weekly for 28 days. Notably, skin and other toxicities were not observed. A transient elevation of interleukin-6 levels was detected at all dose levels, 2–4 hours post-dose, which returned to baseline levels after 24 hours. These results emphasize the promise of bispecific innate cell engagers as an alternative cancer therapy and demonstrate the potential for AFM24 to effectively target tumors expressing varying levels of EGFR, regardless of their mutational status. Abbreviations: ADA: antidrug antibody; ADCC: antibody-dependent cell-mediated cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; AUC: area under the curve; CAR: chimeric-antigen receptor; CD: Cluster of differentiation; CRC :colorectal cancer; ECD: extracellular domain; EGF: epidermal growth factorEGFR epidermal growth factor receptor; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; Fc: fragment, crystallizableFv variable fragment; HNSCC: head and neck squamous carcinomaIL interleukinm; Ab monoclonal antibody; MOA: mechanism of action; NK :natural killer; NSCLC: non-small cell lung cancer; PBMC: peripheral blood mononuclear cell; PBS: phosphate-buffered saline; PD: pharmacodynamic; ROCK: redirected optimized cell killing; RSV: respiratory syncytial virus; SABC: specific antibody binding capacity; SD: standard deviation; TAM: tumor-associated macrophage; TKI: tyrosine kinase inhibitor; WT: wildtype

The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. Multiple peptide resistance factor (MprF) is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistant Staphylococcus aureus (MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to antimicrobial agents and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding of bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin, and it impaired MRSA survival in human phagocytes. Thus, MprF inhibitors are recommended for new antivirulence approaches against MRSA and other bacterial pathogens.

The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. MprF is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistantStaphylococcus aureus(MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to both, human antimicrobials and antibiotics and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding into bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin. Moreover, it impaired MRSA survival in human phagocytes, which recommends MprF inhibitors for new anti-MRSA approaches. MprF-directed monoclonal antibodies provide a proof of concept for development of precisely targeted anti-virulence approaches, which block bacterial antimicrobial resistance mechanisms.

AFM13 is a bispecific tetravalent innate cell engager (ICE®) with two binding sites for CD16A and CD30 on immune cells and tumor cells, respectively. AFM13 is designed to potently activate anti-tumoral responses of natural killer (NK) cells, through antibody-dependent cellular cytotoxicity (ADCC) and macrophages via antibody-dependent cellular phagocytosis (ADCP) towards CD30 + tumor cells. It is widely known that the number of NK cells and their anti-tumor activity are often compromised in patients with cancer, including CD30 + malignancies such as Hodgkin lymphoma (HL) (Reiners, K.S. et al., Mol Ther 2013;21:895-903). Accumulating evidence suggests that the selective, high affinity binding of ICE® to CD16A enables stable pre-complexing with NK cells to enhance ADCC. Early clinical studies with AFM13 have demonstrated safety and efficacy, both as mono- and combination therapy with an anti-PD-1 checkpoint inhibitor (pembrolizumab) (Bartlett, N.L. et al., Blood 2020;136(21):2401-2409), in patients with relapsed or refractory (R/R) HL or peripheral T cell lymphoma (PTCL). Currently, AFM13 monotherapy is being evaluated in a registration-directed Phase 2 study to treat patients with PTCL (NCT04101331). Additionally, a Phase 1b/2a study in patients with PTCL (NCT03192202) previously demonstrated that NK cell numbers pre- and post-dosing of AFM13 may positively correlate with anti-tumoral response (Sawas, A. et al., Blood 2020;136(1):25-26). Therefore, the safety and efficacy of freshly prepared cord blood-derived allogeneic NK cells pre-complexed with AFM13, followed by three weekly infusions of AFM13 monotherapy, is currently being evaluated in patients with R/R CD30 + lymphomas in a Phase 1 dose escalation study (NCT04074746). The first four enrolled patients, all with HL, have shown a response rate of 100% without any safety concerns (Rezvani, K. et al., presentation AACR 2021). To further develop this CAR-like NK cell immunotherapeutic towards an off-the-shelf product, we evaluated the feasibility of cryopreservation of pre-complexed NK cells and assessed the biological activity of such cryopreserved and pre-complexed NK cells after thawing. In this study, the cell surface retention, suitability for NK cell preloading, and cytotoxic activity of AFM13 and other CD30-specific antibody formats was assessed. Furthermore, the compatibility of these formats with cryopreservation of the resulting CAR-like NK cell product was investigated. Cell surface retention assays on primary NK cells confirm substantially slower dissociation kinetics of the tetravalent bispecific ICE® compared to corresponding IgG1 or Fc-enhanced IgG1, enabling efficient and durable pre-complexing of NK cells with ICE®. Surface plasmon resonance analyses confirm long retention of ICE® independent of the CD16A polymorphism. More importantly, these assays demonstrate that the high ADCC potency and efficacy of NK cells, pre-complexed with CD16A-specific tetravalent ICE®, is maintained after one freeze-thaw cycle. These data suggest that high-affinity pre-complexing of adoptive NK cells with bispecific, CD16A-selective ICE® could be a novel cryopreserved off-the-shelf NK cell product for the effective depletion of tumor cells without the limitations and potential risks associated with the CAR-NK cell technology. Disclosures Reusch: Affimed: Current Employment, Current holder of stock options in a privately-held company. Ellwanger: Affimed: Current Employment, Current holder of stock options in a privately-held company. Fucek: Affimed: Current Employment, Current holder of stock options in a privately-held company. Müller: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Schniegler-Mattox: Affimed: Current Employment, Current holder of stock options in a privately-held company. Pahl: Affimed: Current Employment, Current holder of stock options in a privately-held company. Tesar: Affimed: Current Employment, Current holder of stock options in a privately-held company. Koch: Affimed: Current Employment, Current holder of stock options in a privately-held company.

So-called collaborative robots are a current trend in industrial robotics. However, they still face many problems in practical application such as reduced speed to ascertain their collaborativeness. The standards prescribe two regimes: (i) speed and separation monitoring and (ii) power and force limiting, where the former requires reliable estimation of distances between the robot and human body parts and the latter imposes constraints on the energy absorbed during collisions prior to robot stopping. Following the standards, we deploy the two collaborative regimes in a single application and study the performance in a mock collaborative task under the individual regimes, including transitions between them. Additionally, we compare the performance under "safety zone monitoring" with keypoint pair-wise separation distance assessment relying on an RGB-D sensor and skeleton extraction algorithm to track human body parts in the workspace. Best performance has been achieved in the following setting: robot operates at full speed until a distance threshold between any robot and human body part is crossed; then, reduced robot speed per power and force limiting is triggered. Robot is halted only when the operator's head crosses a predefined distance from selected robot parts. We demonstrate our methodology on a setup combining a KUKA LBR iiwa robot, Intel RealSense RGB-D sensor and OpenPose for human pose estimation., 8 pages, 6 figures

Immunotherapy has the potential to support and expand the body's own armamentarium of immune effector functions, which have been circumvented during malignant transformation and establishment of cancer and is presently considered to be the most promising treatment option for cancer patients. Recombinant antibody technologies have led to a multitude of novel antibody formats, which are in clinical development and hold great promise for future therapies. Among these formats, bispecific antibodies are extremely versatile due to their high efficacy to recruit and activate anti-tumoral immune effector cells, their excellent safety profile, and the opportunity for use in combination with cellular therapies. This review article summarizes the latest developments in cancer immunotherapy using immuno-engagers for recruiting T cells and NK cells to the tumor site. In addition to antibody formats, malignant cell targets, and immune cell targets, opportunities for combination therapies, including check point inhibitors, cytokines and adoptive transfer of immune cells, will be summarized and discussed.

The epidermal growth factor receptor (EGFR) is a prime target for cancer therapy across many solid tumor types, including CRC, NSCLC, breast, esophageal cancer, and SCCHN. Binding of EGFR by its ligand EGF induces cell proliferation, a process which is uncontrolled or constitutively active in many cancers. The development of anti-EGFR therapies such as monoclonal antibodies and tyrosine kinase inhibitors enabled clinicians to treat specific patient populations. Nevertheless, prognoses for patients remain unfavorable due to occurrence of intrinsic or acquired resistance via mutations of EGFR and/or downstream signaling factors such as KRAS and BRAF which are drivers of tumorigenesis. Inhibitors that target specific KRAS mutations, such as the G12C mutation (i.e. 14% of the most frequently observed in NSCLC and up to 5% in CRC), have shown early promising efficacy. While such drugs hold promise for specific mutations, there continues to be a need for new treatments with more differentiated and novel modes of action that utilize the power of the innate immune system, such as antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). Affimed has engineered the tetravalent bispecific EGFR- and CD16A-binding antibody AFM24 to engage and redirect innate immune cells such as NK cells and macrophages to EGFR+ tumor cells. In contrast to currently available EGFR-targeted therapies (e.g. cetuximab), this innate cell engager has been designed to exhibit a significantly reduced inhibition of EGFR signaling aiming for an improved safety profile, i.e. avoiding skin and gastro-intestinal toxicities. In vitro, AFM24 demonstrated high affinity binding to immune cells regardless of CD16A variants and potent killing of multiple EGFR+ tumor cell lines, carrying relevant KRAS or BRAF mutations via a different mode of action, namely ADCC and ADCP, rather than EGFR signaling inhibition. It was shown that AFM24's activity is independent of both, the EGFR cell surface expression level and the cellular origin of the respective cell lines. Cynomolgus toxicity studies revealed a very good safety profile, and no toxicity was observed after repeated administration (q7x28d, in total 5 infusions) of AFM24 up to 75 mg/kg. In contrast, EGFR-targeting mAbs (e.g. cetuximab) exhibited severe toxicities particularly in skin in toxicology studies when investigated with a similar dosing schedule. Due to its potent activation of innate immunity and its beneficial safety/tolerability profile, AFM24 has the potential to become a novel treatment option for patients suffering from EGFR-expressing cancers and to overcome limitations of available EGFR-targeted therapies such as resistance and/or associated toxicities. AFM24 has cleared the IND and clinical investigations are planned in patients with cancers known to express EGFR. Citation Format: Uwe Reusch, Michael Damrat, Susanne Wingert, Stefan H.J. Knackmuss, Thomas Mueller, Ivica Fucek, Ute Schniegler-Mattox, Kristina Ellwanger, Torsten Haneke, Andras Strassz, Wolfgang Fischer, Erich Rajkovic, Michael Tesar. AFM24, a bispecific EGFR/CD16A innate cell engager with the potential to overcome resistance to current targeted treatments for EGFR-positive malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5659.

Stefan R. Schmidt consolidates the hugely diverse field of fusion proteins and their application in the creation of biopharmaceuticals. The text is replete with case studies and clinical data that inform and intrigue the reader as to the myriad possibilities available when considering the creation of a fusion protein. This valuable text will serve the novice as a broad introduction or the seasoned professional as a thorough review of the state of the art. The first marketed therapeutic recombinant protein was human insulin (Humulin® R). Its approval in 1982 was followed by other such products, including erythropoietin (EPO), interferon (IFN), and tissue plasminogen activator (tPa). Since the 1980s, the number and general availability of recombinant products that replace natural proteins harvested from animal or human sources has increased considerably. Following the initial success, researchers started de novo designs of therapeutic proteins that do not occur in nature. The first of these new drugs to be ...

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.

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 ( AFM26 therefore is a promising agent currently in preclinical development to fully unlock NK cell cytotoxicity for BCMA-directed immunotherapy of MM. Disclosures Ross: 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.

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.

Based on the recent development of NanoLuc Luciferase a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct western blotting. The new NanoLuc Luciferase protein fusion represents a “swiss army knife solution” for today and future high throughput antibody drug screenings.

Tumor-associated antigens for effective and safe T-cell engagement are very limited, leaving a need to open up the therapeutic target space. Targeting disease-specific MHC/peptide complexes with bispecific T-cell-recruiting antibodies is a highly attractive strategy to address this need, but so far, generation of antibodies against these peptides has been reported to be challenging. Immatics’ unique target discovery engine XPRESIDENT® holds the promise of identifying novel tumor-associated MHC/peptide complexes by providing direct and quantitative evidence for their presence on a large collection of primary human tumor and normal tissue specimens. By this approach, MMP1-003, an HLA-A*02-binding peptide originating from matrix metallopeptidase 1 (MMP1), was identified as a promising therapeutic target presented by several tumor types, including colorectal and lung cancer, but absent on normal tissues. These findings are underlined by RNAseq analysis of the source antigen which also points to MMP1 being a highly attractive tumor-associated target. Consequently, a fully human antibody phage display library was screened to identify highly specific single chain antibodies, which were shown to recognize the purified HLA-A*02/MMP1-003-complex in ELISA assays as well as on peptide-pulsed HLA-A*02+ T2 cells. The best candidates were reformatted into bispecific tetravalent TandAbs® through Affimed´s proprietary platform using a human/cyno-cross-reactive CD3-binding domain for T-cell engagement. Specific target recognition was confirmed for the TandAbs in binding and cytotoxicity assays on peptide-pulsed T2 cells. HLA-A*02/peptide-complexes selected from the broad normal tissue immunopeptidome with a high degree of sequence similarity to the HLA-A*02/MMP1-003-complex served as controls to confirm the specificity and hence the low risk of off-target binding. The most promising candidates were tested on a panel of endogenously target-expressing cancer cell lines covering MMP1 +/- and HLA-A*02 +/- expression profiles, as well as the source proteins for the most closely related control peptides. The lead TandAb showed excellent target specificity across a wide range of peptide-pulsed and endogenously expressing cell lines as well as potent cytotoxicity with picomolar EC50. In summary, we have identified a tumor-associated MMP1-derived peptide in an HLA-A*02 context by exploiting the knowledge of tumor and healthy tissue immunopeptidomes using XPRESIDENT®. Overcoming the existing barrier of developing antibodies targeting specific MHC/peptide complexes, we generated and characterized highly specific and potent T-cell-recruiting TandAbs. These hold the potential to open up the therapeutic target space for T-cell engagement by providing access to intracellular proteins that are presented in a disease-specific manner as MHC/peptide complexes. Citation Format: Toni Weinschenk, Erich Rajkovic, Uwe Reusch, Michael Weichel, Kristina Ellwanger, Ivica Fucek, Michael Tesar, Dominik Hinz, Vera Molkenthin, Sebastian Bunk, Norbert Hilf, Oliver Schoor, Dominik Maurer, Kerstin Mock, Carsten Reinhardt, Martin Treder. Identification of antibodies against a novel tumor-associated MHC/peptide-target and generation of highly specific and potent HLA-A*02MMP1-003/CD3 TandAbs [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 3753. doi:10.1158/1538-7445.AM2017-3753

Five specific single-chain antibodies recognizing the human vascular endothelial growth factor receptor-2 (VEGFR-2/KDR) were selected from a V-gene phage display library constructed from mice immunized with the extracellular domain of VEGFR-2 (Ig-like domain 1-7). All five scFv antibodies (A2, A7, B11, G3, and H1) bound to the purified native antigen in enzyme-linked immunosorbent assay and Dot Blot, and showed no crossreactivity to the human VEGF-receptor 1 (VEGFR-1). The selected antibodies recognize a conformation-dependent epitope of the native receptor and do not recognize denatured antigen in Western blots, as well as linear overlapping peptides comprising the sequence of the human VEGFR-2. The five scFv antibodies bind to the surface of endothelial cells overexpressing human VEGFR-2 c-DNA (PAE/VEGFR-2 cells) as detected by surface immunofluorescence using confocal microscopy. In addition scFv A7 specifically detected VEGFR-2 expressing endothelial cells in the glomerulus of frozen human kidney tissue sections. Therefore, A7 has potential clinical application as a marker for angiogenesis in cryosections of different human tissues. Additionally, two recombinant scFvs (A2 and A7) very efficiently recognize VEGFR-2 on PAE/VEGFR-2 cells and freshly prepared human umbilical vein endothelial cells by fluorescence-activated cell sorter (FACS) analysis. The scFv fragment A7, which was the most sensitive antibody in FACS analysis, recognizes human CD34+VEGFR-2+ hematopoietic immature cells within the population of enriched CD34+ cells isolated from human cord blood. The dissociation constant of A7 was determined to be K(d) = 3.8 x 10(-9) M by BIAcore analysis. In conclusion, scFv fragment A7 seems to be an important tool for FACS analysis and cell sorting of vascular endothelial cells, progenitor cells and hematopoitic stem cells, which are positive for VEGFR-2 gene expression.

Summary A strategy determined Westprinting (for Westernblot and immunological fingerprinting) has been proposed for the immunological identification of the authentic genus Pseudomonas (rRNA-DNA homology group I; Proteobacteria gamma-3 subclass) over a wide phylogenetic range. For that purpose an antiserum has been raised which generated genus- and species-specific protein profiles of all members of the phylogenetic group after sodium dodecyl-sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) and immunoblotting. Cluster analyses based on Westprints of different Pseudomonas type strains revealed similar interspecies and intra-species relationships comparable to 16S rRNA gene sequence analysis. Differentiation of several strains belonging to the same species was shown for P. fluorescens, as well as for seven genomovars of P. stutzeri which have been well characterized by DNA-DNA similarity analysis and 16S rRNA gene-sequencing. The method has also been used for a rapid and reliable screening assay and multiple environmental isolates were identified to the genus and species level. On the basis of the Westprinting technology Pseudomonas- characteristic antigens were identified and monoclonal antibodies were raised against outer membrane protein (Opr) H2 and a 11 kDa protein band, which reacted against all examined pseudomonads belonging to rRNA-DNA homology group I.

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.

Ebner, Martin [Hrsg.]; Schön, Sandra [Hrsg.]: L3T. Lehrbuch für Lernen und Lehren mit Technologien. 2. Auflage. 2013, [9] S., Interaktive und multimediale Lernmaterialien gehören heute im Lehralltag zum guten Ton wie seinerzeit die Kreidetafel, die heute allerdings in vielen Disziplinen immer noch nicht wegzudenken ist. Eine ungeheure Vielfalt an Gestaltungs- und Einsatzmöglichkeiten von Lernmaterialien macht die Wahl und Erstellung nicht gerade einfacher. Dieses Kapitel möchte durch eine Einführung und Übersicht über diese Thematik erste Orientierungshilfen geben. Aufgrund dieses Einführungsgedankens besteht kein Anspruch auf Vollständigkeit - zu umfangreich und komplex ist die Materie und viele weitere Aspekte gehören in der Praxis berücksichtigt. Zu Beginn geben wir eine Einführung in das Thema, welche aufzeigt, was möglich wäre und welche Überlegungen zu einem möglichen Einsatz angestellt werden sollten. Die ersten Reflexionsaufgaben dienen Ihrer persönlichen Orientierung und Evaluierung Ihrer Vorhaben. In weiterer Folge betrachten wir die drei Medien Bild, Audio und Video im Detail und geben so einen Überblick über Einsatzmöglichkeiten. Wie diese Materialien erstellt werden können und vor allem mit welchen Software-Produkten erfahren Sie ebenso. Abgerundet wird das Kapitel mit einer ausführlichen Betrachtung von interaktiven Lernmaterialien und im Speziellen der Thematik Videoannotationen. (DIPF/Orig.)

Hepatitis A virus (HAV) cDNAs encoding the P3 region proteins were expressed in vivo and in vitro to characterize the HAV 3D protein and to identify the cleavage site between 30 and 3D. Protein coding sequences were placed under control of a T7 promoter and an EMCV translational initiation signal. T7 RNA polymerase was provided by simultaneous infection of transfected BS-C-1 cells with a recombinant vaccinia virus vTF7-3 (T. R. Fuerst et al., Proc. Natl. Acad. Sci. USA 83, 8122-8126, 1986). Efficient synthesis and processing of P3 proteins occurred to yield 3CD (78 kDa), 3D (54 kDa), 3ABC (33 kDa), 3BC (25 kDa), and 3C (23 kDa). Similar products were produced by translation of T7 transcripts in a rabbit reticulocyte lysate in vitro. The 3C/3D cleavage site was mapped by comparing the mobility of 3D in SDS-PAGE with 3D proteins engineered to begin at each of the two proposed cleavage sites; in addition, direct N-terminal sequencing of radiolabeled 3D protein from translation in vitro was performed. The results showed that 3D was formed by cleavage at the glutamine-arginine (Q/R) pair at position 1738 and 1739 of the HAV polyprotein. HAV 3D protein produced by autocatalytic cleavage of P3 precursor proteins in BS-C-1 cells is virtually completely insoluble and sediments after low-speed centrifugation. This is in contrast to the poliovirus 3D protein, produced from a similar construct, a significant portion of which remains soluble. Extracts containing the poliovirus 3D protein manifested high levels of RNA-dependent RNA polymerase activity, whereas those containing the HAV 3D protein showed no detectable activity by the same assay.

Hepatitis A virus (HAV) is distinguished from other picornaviruses by its tropism for the liver in infected hosts, a nonlytic infection in hepatocytes, and a slow and nonlytic growth cycle in cultured cells. Although the genome structure and organization of HAV appear to be similar to those of the other picornaviruses, the viral proteins synthesized in infected cells have not been previously characterized. We have utilized specific antisera raised in rabbits to recombinant HAV proteins expressed in Escherichia coli in an effort to identify both structural and nonstructural proteins in BS-C-1 cells throughout the course of a viral replication cycle. Replication was monitored by dot blot hybridization of viral genomes. Structural proteins VP0, VP1, VP2, and VP3 were found to accumulate during the infection cycle as did viral RNA. Nonstructural proteins 2C and 3D were not detected on immunoblots, although a minor amount of 2C could be detected by immunoprecipitation of lysates of radiolabeled, infected cells. The relative sensitivities of the various antisera were determined, and the failure to observe nonstructural proteins was shown not to be due to decreased sensitivity of the detection reagents. Thus, it appears that HAV nonstructural proteins do not accumulate in infected cells to levels comparable to those of capsid proteins.

Foot-and-mouth disease virus protease 3C is essential for the processing of the viral precursor polyprotein. It is shown here to also inhibit gene expression in baby hamster kidney cells after transient expression from transfected cDNA fragments. Protease 3C could not be detected by indirect immunofluorescence in contrast to other cDNA-encoded virus proteins, but protein synthesized de novo 16 hr after transfection could be detected by radioimmuno-precipitation. The cellular translation apparatus was, therefore, not inhibited. The enzyme, although produced as part of a fusion protein, was in size indistinguishable from that found in virus-infected cells. This suggested that the enzyme was released by autocatalysis from the recombinant fusion protein and from viral precursor protein in a similar manner. Transcription of protease 3C-encoding cDNA fragments as well as that of cotransfected fragments, which do not encode protease 3C, was inhibited as determined by hybridization assays. The shut off of transcription which was one of the cytopathic effects observed in virus-infected cells therefore correlates with the production of transactive protease 3C. The inhibitory molecular mechanism may involve truncation of the nuclear protein histone H3 at its N-terminus since this protein was found similarly truncated in virus-infected cells and after transfer of 3C-encoding cDNA fragments.

The Human Combinatorial Antibody Library (HuCAL) was screened for antibodies specific to human leukocyte antigen-DR (HLA-DR) that induce programmed death of lymphoma/leukemia cells expressing the target antigen. The active Fab fragments were affinity-matured, and engineered to IgG(4) antibodies of sub-nanomolar affinity. The antibodies exhibited potent in vitro tumoricidal activity on several lymphoma and leukemia cell lines and on chronic lymphocytic leukemia patient samples. They were also active in vivo in xenograft models of non-Hodgkin lymphoma. Cell death occurred rapidly, without the need for exogenous immunological effector mechanisms, and was selective to activated/tumor-transformed cells. Although the expression of HLA-DR on normal hematopoietic cells is a potential safety concern, the antibodies caused no long-lasting hematological toxicity in primates, in vivo. Such monoclonal antibodies offer the potential for a novel therapeutic approach to lymphoid malignancies.

The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.

Although much information on metabolic pathways within individual organisms is available, little is known about the pathways operating in natural communities in which extensive sharing of nutritional resources is the rule. In order to analyse such a consortium pathway, we have investigated the flow of 4-chlorosalicylate as carbon substrate within a simple chemostat microbial community using 13C-labelled metabolites and isotopic ratio mass spectrometric analysis of label enrichment in immunocaptured member populations of the community. A complex pathway network of carbon sharing was thereby revealed, involving two different metabolic routes, one of which is completely novel and involves the toxic metabolite protoanemonin. The high stability of the community results, at least in part, from interdependencies based on carbon sharing and the rapid removal of toxic metabolites.

The genus Caulobacter is composed of prosthecate bacteria often specialized for oligotrophic environments. The taxonomy of Caulobacter has relied primarily upon morphological criteria: a strain that visually appeared to be a member of the Caulobacter has generally been called one without challenge. A polyphasic approach, comprising 16S rDNA sequencing, profiling restriction fragments of 16S-23S rDNA interspacer regions, lipid analysis, immunological profiling and salt tolerance characterizations, was used to clarify the taxonomy of 76 strains of the genera Caulobacter. Brevundimonas, Hyphomonas and Mycoplana. The described species of the genus Caulobacter formed a paraphyletic group with Caulobacter henricii, Caulobacter fusiformis, Caulobacter vibrioides and Mycoplana segnis (Caulobacter segnis comb. nov.) belonging to Caulobacter sensu stricto. Caulobacter bacteroides (Brevundimonas bacteroides comb. nov.), C. henricii subsp. aurantiacus (Brevundimonas aurantiaca comb. nov.), Caulobacter intermedius (Brevundimonas intermedia comb. nov.), Caulobacter subvibrioides (Brevundimonas subvibrioides comb. nov.), C. subvibrioides subsp. albus (Brevundimonas alba comb. nov.), Caulobacter variabilis (Brevundimonas variabilis comb. nov.) and Mycoplana bullata belong to the genus Brevundimonas. The halophilic species Caulobacter maris and Caulobacter halobacteroides are different from these two genera and form the genus Maricaulis gen. nov. with Maricaulis maris as the type species. Caulobacter leidyia was observed to cluster with species of the genus Sphingomonas. Caulobacter crescentus is synonymous with C. vibrioides and C. halobacteroides is synonymous with Maricaulis maris as determined by these analyses and DNA-DNA hybridization. Biomarkers discerning these different genera were determined. The necessary recombinations have been proposed and a description of Maricaulis is presented.

We have previously described a monoclonal antibody (mAb), 10C3, directed against the gene-3 protein (g3p) of filamentous phage M13, which was produced to study g3p fusion protein expression in Escherichia coli and its incorporation in the phage capsid [Tesar, M., Beckmann, C., Rottgen, P., Haase, B., Faude, U., Timmis, K., 1995. Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems. Immunology 1, 53-54]. In this study we report mapping of the antigenic epitope of the mAb 10C3, by means of short overlapping peptide-sequences [Frank, R., Overwin, H., 1996. Spot synthesis. In: Morris, G.E. (Ed.), Methods in Molecular Biology, Vol. 66: Epitope Mapping Protocols. Humana Press, Totowa, NJ, pp. 149-169.] comprising the C-terminal half of the g3-protein. A minimal recognizable peptide was found which is represented in the 11 amino acid sequence from positions 292 to 302 of g3p [Wezenbeek van, P.M.G.P., Hulsebos, T.J.M., Schoenmakers, J.G.G., 1980. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene 11, 129-148]. In order to use the antibody also for detection and purification of recombinant proteins, such as single chain antibodies, the epitope was introduced as a tag sequence into the phagemid pHEN1 [Hoogenboom, H.R., Griffith, A.D., Johnson, K., Chiswell, D.J., Hudson, P., Winter, G., 1991. Multi-subunit proteins on the surface of the filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acid Res. 19, 4133-4137; Nissim, A., Hoogenboom, H.R., Tomlinson, I.M., Flynn, G., Midgley, C., Lane, D., Winter, G., 1994. Antibody fragments from a single pot phage display library as immunochemical reagents. EMBO J. 13 (3) 692-698]. Purified single chain antibodies containing this tag were detectable down to a concentration of 2 ng ml(-1) under non-denaturing conditions (ELISA) or 4 ng per lane on immunoblots. The high sensitivity of the antibody for the peptide tag was reflected in the antibody affinity constant K(D) of 6.80 x 10(-10) M, which was determined by real time biomolecular interaction analysis (BIA) based on surface plasmon resonance (SPR) [Karlsson, R., Falt, A., 1997. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J. Immunol. Methods 200, 121-133]. Finally, recombinant proteins in E. coli periplasmic extracts could be purified in a single step by affinity purification using immobilized mAb 10C3. These studies demonstrated that the new peptide-tag and its corresponding mAb represents a versatile tool for the detection of recombinant proteins selected by phage display technology.

A monoclonal antibody directed against the gene 3 product (pIII) of filamentous phage M13 was produced to study pIII-fusion protein expression in E. coli and its incorporation in the phage capsid. The protein was gel-purified from E. coli expression cultures harboring the genetic information of pill under the control of an inducible lac promoter. To study pIII-fusion protein expression, phage display systems were applied in which either the whole pIII or the C-terminal half was used (McCafferty et al. (1990) Nature (London) 348, 552–554; Szardenings and Collins (1990) Gene 94, 1–7; Barbas and Lerner (1991) In: Methods: Companion to Methods in Enzymology, Combinatorial Immunoglobulin Libraries on the Surface of Phage (Phabs): Rapid Selection of Antigen-Specific Fabs, Vol. 2, Academic Press, Orlando, pp. 119–124). In all cases, the monoclonal antibody was able to detect the native and the recombinant protein in E. coli and on the phage tip using non-denaturing (ELISA) and denaturing (SDS-PAGE, immunoblot analysis) conditions. All selected pIII-specific monoclonal antibodies were found to be directed against epitopes within amino acids 198 to 406 of pIII, which is necessary for capsid incorporation and therefore included in all pIII-mediated phage display designs.

The VP4 capsid protein of several picornaviruses has been shown to be myristoylated at an N-terminal glycine residue. Myristoylation occurs after removal of an initial methionine residue or a leader peptide, resulting in the exposure of an N-terminal eight amino acid myristoylation signal including a consensus G-x-x-x-T/S motif. Analysis of the amino acid sequence of hepatitis A virus (HAV) capsid protein reveals a potential myristoylation site beginning at position 5 of the VP4 sequence. To assess the significance of this apparent myristoylation signal, mutations were engineered (G to A; T to N) to alter the consensus sequence as well as the potential cleavage site that would be required to remove the short leader. An additional mutant was constructed in which the proposed leader was deleted. Expression of these HAV sequences in BS-C-1 cells showed that leader cleavage did not occur in the wild-type or mutant proteins, although the threonine to asparagine mutation resulted in reduced translation and processing efficiency. Transfection of BS-C-1 or FRhK-4 cells with transcripts derived from the wild-type or mutagenized cDNA clones gave rise to infectious virus, with no detectable incorporation of myristate. The results indicate that HAV does not require leader cleavage and myristoylation of VP4 for growth in cultured cells.

Foot-and-mouth disease virus (FMDV) O1 Kaufbeuren-specific cDNA fragments were subcloned into the E. coli expression vector pRIT·2T. Fusion proteins thus produced in bacteria were purified by affinity chromatography and inoculated into rabbits. Three sera thus obtained were found to be monospecific for FMDV proteins 3A, 3C, and 3D, respectively. Two others were prevalently directed against protein 2C, but in addition, either to protein 2B or to protein 3A. Five out of six mature nonstructural virus proteins can therefore be separately investigated in FMDV-infected cells, either by indirect immunofluorescence or by radioimmunoprecipitation. Immunofluorescence shows all investigated proteins to be located exclusively in the cytoplasm. One of them, protein 2C, transiently forms aggregates at the periphery of cells. Radioimmunoprecipitation confirmed current knowledge on maturation of FMDV proteins. It was further used to characterize postinfectional sera with regard to FMDV-specific antibodies. Cattle and guinea pig were found to have responded differently to FMDV nonstructural antigens. Furthermore, antigenicity of yet to be described FMDV polypeptides was observed in the guinea pig.