Engaging innate immunity for targeting the epidermal growth factor receptor: Therapeutic options leveraging innate immunity versus adaptive immunity versus inhibition of signaling.

The epidermal growth factor receptor (EGFR) is a key player in the normal tissue physiology and the pathology of cancer. Therapeutic approaches have now been developed to target oncogenic genetic aberrations of EGFR, found in a subset of tumors, and to take advantage of overexpression of EGFR in tumors. The development of small-molecule inhibitors and anti-EGFR antibodies targeting EGFR activation have resulted in effective but limited treatment options for patients with mutated or wild-type EGFR-expressing cancers, while therapeutic approaches that deploy effectors of the adaptive or innate immune system are still undergoing development. This review discusses EGFR-targeting therapies acting through distinct molecular mechanisms to destroy EGFR-expressing cancer cells. The focus is on the successes and limitations of therapies targeting the activation of EGFR versus those that exploit the cytotoxic T cells and innate immune cells to target EGFR-expressing cancer cells. Moreover, we discuss alternative approaches that may have the potential to overcome limitations of current therapies; in particular the innate cell engagers are discussed. Furthermore, this review highlights the potential to combine innate cell engagers with immunotherapies, to maximize their effectiveness, or with unspecific cell therapies, to convert them into tumor-specific agents.

A BCMA/CD16A bispecific innate cell engager for the treatment of multiple myeloma.

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089 induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.

Preclinical evaluation of AFM24, a novel CD16A-specific innate immune cell engager targeting EGFR-positive tumors.

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.

Redirected optimized cell killing (ROCK®): A highly versatile multispecific fit-for-purpose antibody platform for engaging innate immunity.

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.

Functionally Defective T Cells After Chemotherapy of B-Cell Malignancies Can Be Activated by the Tetravalent Bispecific CD19/CD3 Antibody AFM11.

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.

CD16A Activation of NK Cells Promotes NK Cell Proliferation and Memory-Like Cytotoxicity against Cancer Cells.

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 (γc) 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. Cancer Immunol Res; 6(5); 517-27. ©2018 AACR.

Bacterial expression and functional reconstitution of human heparanase.

Human heparanase is a heparan sulfate degrading enzyme located in the extracellular matrix playing a decisive role in angiogenesis and tumor metastasis. Translated as a 65 kDa inactive prae-form, the protein is processed into an 8 kDa and a 50 kDa subunit which form a non-covalently associated active heterodimer. We have expressed the two subunits separately in Escherichia coli which yielded active human heparanase upon reconstitution. The two purified subunits folded independently and secondary structure analysis by far-UV CD spectroscopy gave 33.1/11.1% α/ß content for the 50 kDa subunit and 6.9/49% α/ß content for the 8 kDa subunit. This heparanase expression system is easy and can be used for efficient screening for enzyme inhibitors.