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

Abstract

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.

Keywords: ADCC; CD16A; NK cell; ROCK®; cellular therapy; immuno-engager; immuno-oncology; innate immunity; recombinant antibodies; tetravalent bispecific antibody.

Figure 1.

SPR analysis of different anti-CD16 scFv. SPR sensorgrams of normalized relative binding signals of anti-CD16 binding scFv antibodies (Ab16^hi, Ab16^mid, 3G8, Ab16^lo) compared to wildtype IgG1, and engineered human IgG1 Fc (S239D/I332E), enhancing binding to human CD16A-158V (a) and human CD16A-158F (b). Analytes were measured at a single concentration of 312.5 nM.

Figure 2.

Tyrosine (Y) in position 140 is crucial for formation of a conformational epitope and CD16A-specific reactivity of scFv antibodies. Different recombinant CD16 variants expressed as fusion proteins of ECD sequences with monomeric Fc or membrane-anchor were analyzed. (a) Protein spots containing defined CD16 antigen variants on nitrocellulose membranes were tested for their reactivity with the indicated CD16 binding scFvs. (b) Reactivity of different anti-CD16 scFv, control scFv, or mAbs with CD16 antigen variants or EGFR antigen expressed on CHO cells anchored via fusion to the EGFR transmembrane domain or GPI, was analyzed by antibody staining and flow cytometry. (c) Binding of different anti-CD16 antibodies to CD16 antigen variants after separation by SDS-PAGE and Western blotting.

Figure 3.

ROCK® platform overview.

Figure 4.

SPR sensorgrams of normalized relative binding dissociation phases of diverse CD16A bivalently binding ROCK® engagers. Bivalent anti-CD16A domain containing scFv-IgAb_C, Db-Fc_A, scFv-IgAb_D, KiH-scDb-Fc_A, TandAb_C were compared to IgAb_E (Fc-enhanced; S239D/I332E) and monovalent anti-CD16A fragment scFv Ab16^hi on immobilized (a) human CD16A-158V and (b) cynomolgus CD16. Legend and percentage of antibodies remaining on the receptor after 3 h of dissociation are shown in (c). Analytes were measured at a single concentration of 50 nM.

Figure 5.

CD16A apparent affinity in ROCK® formats is tunable by variable domains, positioning and connector lengths. Binding of soluble CD16 antigen to ROCK® antibodies containing different CD16 binding Fvs (Ab16^mid (open blue dots), Ab16^hi (filled blue dots)) in different positions (N: anti-CD16 Fvs N-terminal of Fc, C: anti-CD16 Fvs C-terminal of Fc), antibody formats, and different domain orders was analyzed in ELISA. Representative pictograms with CD16-binding Fvs depicted in blue are shown below each group. Summary of CD16A apparent affinity by (a) Fab or scFv-based CD16 engagement, (b) diabody (Db)-based CD16 engagement, or (c) C-terminal scFv-based CD16 engagement comprising different connector lengths (10aa or 30aa) and domain orders of anti-CD16 Fv (HL: scFv domain order VH-VL, LH: scFv domain order VL-VH). All analyzed antibodies contain silenced Fc or lack Fc in the case of fusion to C-terminus of Fab. Binding specificities depicted in black or dark gray comprised antibody domains targeting BCMA, CD19, CD20, EGFR, HSA or RSV.

Figure 6.

Binding of ROCK® antibodies to primary human NK cells in the presence or absence of human IgG. Primary human NK cells were stained with increasing concentrations of the indicated (a) Fc-less ROCK®, (b) Fc fusion ROCK®, (c) IgG-like ROCK® constructs in the presence or absence of 10 mg/mL polyclonal human IgG at 37°C. Cell bound antibodies were detected by flow cytometry, and median fluorescence intensities (MFI) were used for calculation of apparent affinities (K_D) by non-linear regression.

Figure 7.

Comparative analysis of various ROCK® antibody formats regarding NK fratricide in vitro. In vitro calcein-release cytotoxicity assays of enriched primary human NK cells in the presence of different ROCK® antibody formats toward autologous NK cells after 4 h co-incubation at an E:T of 1:1. Mean EC₅₀ values of several independent experiments are summarized and plotted as individual dots. The ROCK® antibody formats are categorized based on the position of the anti-CD16 domain and the format/domain order. Only constructs containing the high affinity anti-CD16 domain and silenced Fc are included in the graph. HL: scFv domain order VH-VL, LH: scFv domain order VL-VH. N-term. = N-terminal, C-term. = C-terminal.

Figure 8.

Comparison of the pharmacokinetics of different ROCK® engager formats: concentration of antibodies over time (1 or 3 weeks observation period) after single intravenous administration of 300 µg test item.

Figure 9.

In vitro cytotoxicity of enriched primary human NK cells in the presence of several ROCK® antibodies toward cell lines expressing their corresponding tumor target. (a) Representative sigmoidal dose-response curves of the indicated BCMA-targeting ROCK® antibodies in 4 h calcein-release cytotoxicity assays with NK cells and target cell lines expressing differential levels of BCMA as indicated by the SABC values (mean of ≥3 assays) at an E:T ratio of 5:1. (b) Representative sigmoidal dose-response curves of the indicated EGFR-targeting ROCK® antibodies, comparators, or monovalently binding controls in 4 h calcein-release cytotoxicity assays with NK cells and SW-982 target cells at an E:T ratio of 5:1. (c) Correlation of ROCK® antibodies regarding NK cell-binding affinity and cytotoxic potency toward tumor target cells in vitro. Apparent affinities (K_D) of ROCK® antibody formats on primary human NK cells are shown. EC₅₀ values of the same ROCK® antibodies were determined in 3 h calcein-release cytotoxicity measurements with NK cells and BCMA-expressing RPMI-8226 target cells at an E:T ratio of 2:1 (left) or in 4 h calcein-release cytotoxicity assays with NK cells and BCMA-expressing NCI-H929 target cells at an E:T ratio of 5:1 (right). SABC, specific antibody-binding capacity.