A challenge when developing therapeutic antibodies is the identification of candidates with favorable pharmacokinetics (PK) early in development. A key determinant of immunoglobulin (IgG) serum ...half‑life in vivo is the efficiency of pH-dependent binding to the neonatal Fc receptor (FcRn). Numerous studies have proposed techniques to assess FcRn binding of IgG-based therapeutics in vitro, enabling prediction of serum half-life prior to clinical assessment. FcRn high-performance liquid chromatography (HPLC) assays FcRn binding of therapeutic IgGs across a pH gradient, allowing the correlation of IgG column retention time to the half‑life of a therapeutic IgG in vivo. However, as FcRn retention time cannot be directly compared to an in vivo parameter, modifications to FcRn-HPLC are required to enable interpretation of the data within a physiological context, to provide more accurate estimations of serum half-life. This study presents an important modification to this method, FcRn-pH-HPLC, which reproducibly measures FcRn dissociation pH, allowing correlation with previously established half-lives of therapeutic antibodies. Furthermore, the influence of incorporating various antibody modifications, binding modules, and their orientations within IgGs and bispecifics on FcRn dissociation pH was evaluated using antibodies from the redirected optimized cell killing (ROCK®) platform. Target and effector antigen-binding domain sequences, their presentation format and orientation within a bispecific antibody alter FcRn retention; tested Fc domain modifications and incorporating stabilizing disulfide bonds had minimal effect. This study may inform the generation of mono-, bi- and multi-specific antibodies with tailored half-lives based on FcRn binding properties in vitro, to differentiate antibody-based therapeutic candidates with optimal developability.
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.
During primary neurulation, the separation of a single-layered ectodermal sheet into the surface ectoderm (SE) and neural tube specifies SE and neural ectoderm (NE) cell fates. The mechanisms ...underlying fate specification in conjunction with neural tube closure are poorly understood. Here, by comparing expression profiles between SE and NE lineages, we observed that uncommitted progenitor cells, expressing stem cell markers, are present in the neural plate border/neural fold prior to neural tube closure. Our results also demonstrated that canonical Wnt and its antagonists, DKK1/KREMEN1, progressively specify these progenitors into SE or NE fates in accord with the progress of neural tube closure. Additionally, SE specification of the neural plate border via canonical Wnt signaling is directed by the grainyhead-like 3 (Grhl3) transcription factor. Thus, we propose that the fate specification of uncommitted progenitors in the neural plate border by canonical Wnt signaling and its downstream effector Grhl3 is crucial for neural tube closure. This study implicates that failure in critical genetic factors controlling fate specification of progenitor cells in the neural plate border/neural fold coordinated with neural tube closure may be potential causes of human neural tube defects.
•Neural plate border/neural fold possesses stem cell-like characters during primary neurulation.•Canonical Wnt and its antagonists progressively specify progenitors into surface or neural fates upon neural tube closure.•Fate specification into surface ectoderm in the neural fold is directed by the Grhl3 transcription factor.•Fate specification of uncommitted progenitors in the neural plate border is intimately coupled to neural tube closure.
Innate cell engager (ICE
) constructs are bispecific tetravalent antibodies targeting specific tumor antigens and simultaneously engaging natural killer (NK) cell and macrophage receptors for the ...destruction of tumor cells. Pre-complexing of ICE
constructs with adoptive NK cells is a novel approach to enhance NK cell activity. The suitability of such complexes for cryopreservation, whilst retaining the biological activity and specificity, may enable the development of off-the-shelf NK cell products. This study investigates the binding affinity of ICE
constructs targeting EpCAM and NK cell receptors CD16A, NKG2D, or NKp46 to the corresponding antigens, the ICE
antitumor activity, and feasibility of cryopreservation. Cell surface retention assays using primary NK cells confirmed a substantially slower ICE
construct dissociation kinetics compared with control molecules, suggesting the formation of durable complexes independently of the CD16A polymorphism. The high-affinity NK cell and EpCAM/CD16A ICE
complexes were superior to those engaging NKG2D or NKp46 receptors when tested for the NK-cell-mediated elimination of EpCAM-expressing tumor cells. Moreover, the potency and efficacy of these complexes were unaffected after a single freeze-thaw cycle. CD16A-selective ICE
drug candidates complexed with NK cells hold promise as novel cryopreserved off-the-shelf NK cell products with chimeric antigen receptor-like NK cell properties, capable of effective depletion of tumor cells.
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.
Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary ...complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.
Dickkopf1 (Dkk1) is a Wnt/β-catenin inhibitor that participates in many processes during embryonic development. One of its roles during embryogenesis is to induce head formation, since Dkk1-null mice ...lack head structures anterior to midbrain. The Wnt/β-catenin pathway is also known to regulate different aspects of ventral midbrain (VM) dopaminergic (DA) neuron development and, in vitro, Dkk1-mediated inhibition of the Wnt/β-catenin pathway improves the DA differentiation in mouse embryonic stem cells (mESC). However, the in vivo function of Dkk1 on the development of midbrain DA neurons remains to be elucidated. Here we examined Dkk1(+/-) embryos and found that Dkk1 is required for the differentiation of DA precursors/neuroblasts into DA neurons at E13.5. This deficit persisted until E17.5, when a defect in the number and distribution of VM DA neurons was detected. Furthermore, analysis of the few Dkk1(-/-) embryos that survived until E17.5 revealed a more severe loss of midbrain DA neurons and morphogenesis defects. Our results thus show that Dkk1 is required for midbrain DA differentiation and morphogenesis.
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