William Dall'acqua

MEDI-563, a humanized anti-IL-5 receptor alpha mAb with enhanced antibody-dependent cell-mediated cytotoxicity function

BACKGROUND Peripheral blood eosinophilia and lung mucosal eosinophil infiltration are hallmarks of bronchial asthma. IL-5 is a critical cytokine for eosinophil maturation, survival, and mobilization. Attempts to target eosinophils for the treatment of asthma by means of IL-5 neutralization have only resulted in partial removal of airway eosinophils, and this warrants the development of more effective interventions to further explore the role of eosinophils in the clinical expression of asthma. OBJECTIVE We sought to develop a novel humanized anti-IL-5 receptor alpha (IL-5Ralpha) mAb with enhanced effector function (MEDI-563) that potently depletes circulating and tissue-resident eosinophils and basophils for the treatment of asthma. METHODS We used surface plasmon resonance to determine the binding affinity of MEDI-563 to FcgammaRIIIa. Primary human eosinophils and basophils were used to demonstrate antibody-dependent cell-mediated cytotoxicity. The binding epitope of MEDI-563 on IL-5Ralpha was determined by using site-directed mutagenesis. The consequences of MEDI-563 administration on peripheral blood and bone marrow eosinophil depletion was investigated in nonhuman primates. RESULTS MEDI-563 binds to an epitope on IL-5Ralpha that is in close proximity to the IL-5 binding site, and it inhibits IL-5-mediated cell proliferation. MEDI-563 potently induces antibody-dependent cell-mediated cytotoxicity of both eosinophils (half-maximal effective concentration = 0.9 pmol/L) and basophils (half-maximal effective concentration = 0.5 pmol/L) in vitro. In nonhuman primates MEDI-563 depletes blood eosinophils and eosinophil precursors in the bone marrow. CONCLUSIONS MEDI-563 might provide a novel approach for the treatment of asthma through active antibody-dependent cell-mediated depletion of eosinophils and basophils rather than through passive removal of IL-5.

B cell depletion in vitro and in vivo with an afucosylated anti-CD19 antibody

The pan B-cell surface antigen CD19 is an attractive target for therapeutic monoclonal antibody (mAb) approaches. We have generated a new afucosylated anti-human (hu)CD19 mAb, MEDI-551, with increased affinity to human FcγRIIIA and mouse FcγRIV and enhanced antibody-dependent cellular cytotoxicity (ADCC). During in vitro ADCC assays with B-cell lines, MEDI-551 is effective at much lower mAb concentrations than the fucosylated parental mAb anti-CD19-2. Furthermore, the afucosylated CD19 mAb MEDI-551 depleted B cells from normal donor peripheral blood mononuclear cell samples in an autologous ADCC assay, as well as blood and tissue B cells in human CD19/CD20 double transgenic (Tg) mice at lower concentrations than that of the positive control mAb rituximab. In huCD19/CD20 Tg mice, both macrophage-mediated phagocytosis and complement-dependent cytotoxicity contribute to depletion with rituximab; MEDI-551 did not require complement for maximal B-cell depletion. Furthermore, extended B-cell depletion from the blood and spleen was achieved with MEDI-551, which is probably explained by bone marrow B-cell depletion in huCD19/CD20 Tg mice relative to the control mAb rituximab. In summary, MEDI-551 has potent B-cell-depleting activity in vitro and in vivo and may be a promising new approach for the treatment of B-cell malignancies and autoimmune diseases.

The TGF-β inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan

ABSTRACT Excessive transforming growth factor (TGF)-β is associated with pro-fibrotic responses in lung disease, yet it also plays essential roles in tissue homeostasis and autoimmunity. Therefore, selective inhibition of excessive and aberrant integrin-mediated TGF-β activation via targeting the α-v family of integrins is being pursued as a therapeutic strategy for chronic lung diseases, to mitigate any potential safety concerns with global TGF-β inhibition. In this work, we reveal a novel mechanism of inhibiting TGF-β activation utilized by an αvβ8 targeting antibody, 37E1B5. This antibody blocks TGF-β activation while not inhibiting cell adhesion. We show that an N-linked complex-type Fab glycan in H-CDR2 of 37E1B5 is directly involved in the inhibition of latent TGF-β activation. Removal of the Fab N-glycosylation site by single amino acid substitution, or removal of N-linked glycans by enzymatic digestion, drastically reduced the antibodys ability to inhibit latency-associated peptide (LAP) and αvβ8 association, and TGF-β activation in an αvβ8-mediated TGF-β signaling reporter assay. Our results indicate a non-competitive, allosteric inhibition of 37E1B5 on αvβ8-mediated TGF-β activation. This unique, H-CDR2 glycan-mediated mechanism may account for the potent but tolerable TGF-b activation inhibition and lack of an effect on cellular adhesion by the antibody.

PET-guided evaluation and optimization of internalized antibody-drug conjugates targeting erythropoietin producing hepatoma A2 receptor

The erythropoietin-producing hepatoma A2 receptor (EphA2) is a tyrosine kinase overexpressed by tumor stroma and cancer cells. A high expression level of EphA2 predicts poor prognosis, correlating with disease progression and metastasis. Therefore, EphA2 is a relevant therapeutic target for human cancer. Antibodies, selectively bound to EphA2, can induce rapid receptor phosphorylation that results in antibody internalization and degradation. This internalization mechanism has been exploited with the development of antibody–drug conjugates (ADCs) for cancer chemotherapy. In this study, we used PET imaging to study the pharmacokinetics and tumor delivery of a panel of anti-EphA2 monoclonal antibodies (mAbs) with and without drug conjugates. Methods: A library of human anti-EphA2 mAbs were screened and evaluated for EphA2 internalization rate, binding affinity, epitope binding, and hydrophobicity. We chose 3 of these antibodies, denoted as 1C1, 3B10, and 2H7, which recognize different epitopes, for further evaluation. ADCs were generated by S239C mutation to give a ratio of 2 drug molecules per antibody. Native mAbs and ADCs were characterized, after conjugation to a DFO chelator and 89Zr radiolabeling, in assays including cell uptake, internalization, hydrophobicity, and in vivo imaging using PET. Results: All 3 mAbs had high affinities for EphA2 but exhibited different internalization rates following the order of 1C1 > 3B10 > 2H7. Internalization rate is only 1 factor that affects in vitro cell uptake and in vivo tumor accumulation. Interestingly, the hydrophobicity of the mAbs, which followed the order of 2H7 > 1C1 > 3B10, had a strong correlation with in vivo tumor uptake measured by PET, with the least hydrophobic antibody, 3B10, showing the highest tumor uptake. ADC significantly reduced the in vivo uptake of all 3 mAbs. Conclusion: Tumor uptake of mAb is a complex process that is affected by multiple parameters, including internalization, hydrophobicity, and chemical modification. Our results suggest that the addition of drug molecules to mAb increases the clearance of the mAb presumably due to the increased hydrophobicity. Understanding the complexity of antibody-based tumor delivery may help improve ADC engineering for better tumor targeting and reduced side effects.