CD16A is a potent cytotoxicity receptor on human natural killer (NK) cells, which can be exploited by therapeutic bispecific antibodies. So far, the effects of CD16A-mediated activation on NK cell effector functions beyond classical antibody-dependent cytotoxicity have remained poorly elucidated. Here, we investigated NK cell responses after exposure to therapeutic antibodies such as the tetravalent bispecific antibody AFM13 (CD30/CD16A), designed for the treatment of Hodgkin lymphoma and other CD30 lymphomas. Our results reveal that CD16A engagement enhanced subsequent IL2- and IL15-driven NK cell proliferation and expansion. This effect involved the upregulation of CD25 (IL2Rα) and CD132 (γ) on NK cells, resulting in increased sensitivity to low-dose IL2 or to IL15. CD16A engagement initially induced NK cell cytotoxicity. The lower NK cell reactivity observed 1 day after CD16A engagement could be recovered by reculture in IL2 or IL15. After reculture in IL2 or IL15, these CD16A-experienced NK cells exerted more vigorous IFNγ production upon restimulation with tumor cells or cytokines. Importantly, after reculture, CD16A-experienced NK cells also exerted increased cytotoxicity toward different tumor targets, mainly through the activating NK cell receptor NKG2D. Our findings uncover a role for CD16A engagement in priming NK cell responses to restimulation by cytokines and tumor cells, indicative of a memory-like functionality. Our study suggests that combination of AFM13 with IL2 or IL15 may boost NK cell antitumor activity in patients by expanding tumor-reactive NK cells and enhancing NK cell reactivity, even upon repeated tumor encounters. .

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.

Glycosylation is the most frequent PTM and contributes significantly to the function of proteins depending on the type of glycosylation. Especially glycan structures like the glycosaminoglycans are considered to constitute themselves the major function of the glycoconjugate which is therefore termed proteoglycan. Here we review recent views on and novel tools for analysing the proteoglycanome, which are directly related to the type of glycanation under investigation. We define the major function of the proteoglycanome to be its interaction with various proteins in many different (patho‐)physiological conditions. This is exemplified by the differential glycosaminoglycan‐interactome of healthy versus arthritic patient sera.

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, IgG 1 -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 K D 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

Immunotherapy of B-cell malignancies with bispecific antibodies is an emerging treatment option. However, not all patients benefit from these therapies, presumably due to pretreatment regimens. Therefore, we determined the effect of different treatment lines on the activity of T cells and their responsiveness to AFM11. AFM11 is a tetravalent, bispecific CD19/CD3 immunoengager based on Affimed’s ROCK platform, currently being investigated in phase I clinical trials for non-Hodgkin lymphoma and acute lymphoblastic leukemia. T cells from B-cell lymphoma patients treated with either rituximab+bendamustine (R-Benda), rituximab+CHOP (R-CHOP), or with high-dose BEAM chemotherapy (HD-BEAM) and autologous HSCT were compared with T cells from healthy donors. Overall, in these patients, T-cell numbers were significantly reduced. To determine whether distinct chemotherapy affects AFM11 efficacy, functional T-cell assays were performed. It is interesting to note that, only in assays that combine target cell lysis, cytokine production and proliferation over 4 days at an effector to target ratio of up to 1:25 significant differences could be detected between the different treatment groups: T cells after R-CHOP showed only modest decrease in their functionality when compared with healthy controls, whereas R-Benda and HD-BEAM had a profound effect on AFM11-induced T-cell cytotoxicity. In conclusion, T cells from lymphoma patients are reduced in number and have functional defects following treatment with certain chemotherapy regimens, also reducing AFM11 efficacy. Importantly, AFM11 was still able to trigger B-cell-directed T-cell immunity in all treatment groups.

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. 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.

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