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
-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
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
. Importantly, AFM24 was effective toward a variety of EGFR-expressing tumor cells, regardless of EGFR expression level and KRAS/BRAF mutational status.
, 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.
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.
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.
To harness the cytotoxic capacity of immune cells for the treatment of solid tumors, we developed tetravalent, bispecific tandem diabody (TandAb) antibodies that recognize EGFRvIII, the deletion ...variant III of the epidermal growth factor receptor (EGFR), and CD3 on T-cells, thereby directing immune cells to eliminate EGFRvIII-positive tumor cells. Using phage display, we identified scFv antibodies selectively binding to EGFRvIII. These highly EGFRvIII-specific, fully human scFv were substantially improved by affinity maturation, achieving
s in the picomolar range, and were used to construct a set of bispecific EGFRvIII-targeting TandAbs with a broad range of binding and cytotoxic properties. These antibodies exhibited an exquisite specificity for a distinguished epitope in the N-terminal portion of EGFRvIII, as shown on recombinant antigen in Western Blot, SPR, and ELISA, as well as on antigen-expressing cells in FACS assays, and did not bind to the wild-type EGFR. High-affinity EGFRvIII/CD3 TandAbs were most potent in killing assays, displaying cytotoxicity toward EGFRvIII-expressing CHO, F98 glioma, or human DK-MG cells with EC
values in the range of 1-10 pM
. They also demonstrated dose-dependent growth control
in an EGFRvIII-positive subcutaneous xenograft tumor model. Together with the tumor-exclusive expression of EGFRvIII, the EGFRvIII/CD3 TandAbs' high specificity and strictly target-dependent activation with no off-target activity provide an opportunity to target tumor cells and spare normal tissues, thereby reducing the side effects associated with other anti-EGFR therapies. In summary, EGFRvIII/CD3 TandAbs are highly attractive therapeutic antibody candidates for selective immunotherapy of EGFRvIII-positive tumors.
BackgroundNatural killer (NK) cells are a nascent cellular immunotherapy for hematologic malignancies. Target recognition of NK cell-resistant cancers remains a substantial barrier to broad ...application of NK cell therapy. One solution are bispecific engagers that trigger NK cells via an NK activating receptor when simultaneously engaging a tumor-specific antigen.MethodsHere, we investigated single NK cell responses stimulated by the tetravalent bispecific innate cell engager (ICE®) AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on several types of NK cells.ResultsMultidimensional mass cytometry revealed heterogeneity within AFM13-directed conventional (c)NK cell responses, as well as consistent polyfunctional activation of mature terminally differentiated NK cells across donors. The source of NK cells also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from Hodgkin lymphoma patients. IL-12, IL-15, and IL-18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared to cNK cells. Cord-blood expanded NK cells that were pre-activated with IL-12, IL-15 and IL-18 also exhibited enhanced killing with AFM13 stimulation, via upregulation of signaling pathways related to NK cell effector function. These cells were stably pre-loaded with AFM13 enhancing responses to CD30+ lymphomas in vitro and in vivo in immunodeficient NSG mouse models.ConclusionsCollectively, these data identify promising combinations of AFM13 with cytokine-activated adult blood or cord blood NK cells against CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.
Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific ...engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation.
We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood-derived NK cells was investigated
and
.
We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30
lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and
expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13-NK complex cells exhibited enhanced responses to CD30
lymphomas
and
.
We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30
hematologic malignancies, warranting clinical trials with these novel combinations.
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.
.
Introduction
Affimed has developed high affinity tetravalent bispecific immune cell engagers for redirected optimized cell killing (ROCK platform). Using anti-CD16A and anti-tumor target-specific ...antibody domains, the engagers activate NK cells to efficiently kill target cells. The most advanced ROCK-based immune cell engager, AFM13, targeting CD30 on tumor cells and CD16A on immune effectors, is currently being evaluated in several clinical trials to treat CD30-positive malignancies.
Based on the fact that CD16A is not exclusively expressed on NK cells, but also on macrophages, we hypothesized that CD16A-specific immune cell engagers would also be able to activate CD16A expressing macrophages through antibody-dependent cellular phagocytosis (ADCP) contributing to anti-tumor response.
Macrophages are an essential component of the innate immune system and are a major constituent of normal tissues. They can be broadly classified into different subtypes including M1 (classically activated, generally characterized as pro-inflammatory and immuno-supportive) and M2 (alternatively activated, primarily of an anti-inflammatory profile) subtypes. Those subtypes greatly differ in their phenotype and function and appear to be highly plastic. While M1 macrophages are generally considered to be tumoricidal, M2 macrophages are mostly tumorigenic, depending on their context within the tumor microenvironment. Therapeutic agents focusing on macrophages such as the CD47/SIRPa axis, CSF-1R antibodies and elimination of tumor-associated macrophages (TAMs) have recently come into focus in immuno-oncology.
Methods
Peripheral monocytes derived from primary human hematopoietic cells of healthy donors were used to generate various macrophage subtypes (unpolarized macrophages, M1, M2a, M2c) in vitro using well-defined cytokine cocktails. These subtypes were characterized phenotypically for their CD16A expression and a wide number of additional markers. Subsequently, they were used to investigate the ability of a number of different CD16A-specific immune cell engagers derived from Affimed´s ROCK platform and control antibodies in vitro to activate and induce ADCP of target cells by flow cytometry and microscopy.
Results
We demonstrated that all of the macrophage subtypes generated in this study expressed CD16A and mediated ADCP of tumor cells. In addition, we showed that ADCP of tumor cells by several CD16A-specific engagers was both fast and robust for all investigated macrophage subtypes. Specifically, ADCP was detected as early as 2 hours after co-incubation of tumor cells with M1 or M2 macrophages and CD16A-specific immune cell engagers. Using appropriate control antibodies, it was demonstrated that ADCP mediated by CD16A-specific immune cell engagers was selective and at least as potent as ADCP mediated by classical monoclonal antibodies pan-specific for Fc-gamma receptors.
Summary and conclusion:
We have demonstrated a new mechanism whereby Affimed´s CD16A-specific immune cell engaging antibodies eliminate tumor cells by ADCP, mediated by different subsets of macrophages. Our data suggest that these antibodies may have the potential to boost tumoricidal function within the tumor microenvironment. Future directions of leveraging innate immunity as a therapeutic option in immuno-oncology will be presented.
Wingert:Affimed: Employment. Reusch:Affimed: Employment. Beez:Affimed: Employment. Pahl:Affimed: Research Funding. Cerwenka:Affimed: Research Funding; Dragonfly Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment.
Abstract
Bispecific antibodies that redirect the lytic activity of cytotoxic immune effector cells, such as T- and NK cells, onto tumor cells have emerged as a highly attractive and clinically ...validated treatment modality for hematological malignancies. Advancement of this therapeutic concept into solid tumor indications, however, is hampered by the scarcity of targetable antigens that are surface-expressed on tumor cells but demonstrate only limited expression on healthy tissues. To overcome this limitation, the concept of dual-targeting, i.e. the simultaneous targeting of two tumor-expressed surface antigens with limited co-expression on non-malignant cells, with multispecific antibodies has been proposed to increase tumor selectivity of antibody-induced effector cell cytotoxicity. Here, a novel CD16A (FcγRIIIa)-directed trispecific, tetravalent antibody format, termed aTriFlex, is described, that is capable of redirecting NK cell cytotoxicity to two surface-expressed antigens. Using a BCMA/CD200-based in vitro model system, the potential use of aTriFlex antibodies for dual-targeting and selective induction of NK cell-mediated target cell lysis was investigated. Bivalent bispecific target cell binding was found to result in significant avidity gains and up to 17-fold increased in vitro potency. These data suggest trispecific aTriFlex antibodies may support dual-targeting strategies to redirect NK cell cytotoxicity with increased selectivity to enable targeting of solid tumor antigens.
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