Contributions
Abstract: P1143
Type: Poster
Abstract Category: Therapy - disease modifying - Immunomodulation/Immunosuppression
Background: Anti-CD20 therapies have shown significant clinical efficacy in MS patients. Ofatumumab is a human IgG1 anti-CD20 antibody that depletes circulating peripheral B cells. Novartis is currently initiating two Phase 3 clinical trials in relapsing MS.
Objective: Elucidate the characteristics of ofatumumab to potentially differentiate from other anti-CD20 antibodies.
Methods: Ofatumumab binding to human CD20 protein was determined using overlapping 15-mer peptide Pepscan-based ELISA. Binding EC50 values were determined using peripheral blood mononuclear cells (PBMCs) via flow cytometry. Dissociation rates were measured via 125I-labeled antibodies in the presence of azide/2-deoxyglucose to prevent internalisation. Ofatumumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) was performed with 51Cr-labeled B cells incubated with whole blood. Complement dependent cytotoxicity (CDC)-mediated B-cell lysis was quantified via incubation with fresh-frozen pooled human serum. Immunogenicity of ofatumumab and rituximab chimeric anti-CD20 antibody was studied in silico for potentially immunogenic T-cell epitopes (Epibase profiling).
RESULTS: Ofatumumab binds to two distinct epitopes within the large and small extracellular loops of CD20 protein. This binding motif is towards the N-terminal. Ofatumumab bound to human (EC50=287ng/mL) PBMCs and stained B cell-rich regions within lymphoid tissues. Strong differences in off-rates were observed between ofatumumab and rituximab. Rituximab dissociated rapidly whereas a slow off-rate and long residence time of ofatumumab resulted in long-lasting effector function and potent B-cell depletion. In vitro whole blood assays showed superiority of ofatumumab over rituximab in lysing CD20-expressing cells, primarily due to enhanced CDC activity. ADCC activity was similar. Ofatumumab binding to target cells did not directly induce apoptosis. In silico profiling showed that ofatumumab has a low number of epitopes capable of inducing an anti-antibody response.
Conclusions: Ofatumumab binds a distinct CD20 epitope on B cells in blood and lymphoid tissues. The slow CD20-binding dissociation and enhanced CDC-mediated lysis (vs rituximab) results in potent effector functions. B-cell depletion is mediated by a combination of CDC and ADCC. These characteristics, together with low potential for anti-antibody response, makes ofatumumab suitable for testing in clinical trials as a low-dose subcutaneous treatment for relapsing MS.
Disclosure: This abstract is supported by Novartis Pharma AG, Basel, Switzerland.
All the authors are employees of Novartis Pharma AG, Switzerland.
Abstract: P1143
Type: Poster
Abstract Category: Therapy - disease modifying - Immunomodulation/Immunosuppression
Background: Anti-CD20 therapies have shown significant clinical efficacy in MS patients. Ofatumumab is a human IgG1 anti-CD20 antibody that depletes circulating peripheral B cells. Novartis is currently initiating two Phase 3 clinical trials in relapsing MS.
Objective: Elucidate the characteristics of ofatumumab to potentially differentiate from other anti-CD20 antibodies.
Methods: Ofatumumab binding to human CD20 protein was determined using overlapping 15-mer peptide Pepscan-based ELISA. Binding EC50 values were determined using peripheral blood mononuclear cells (PBMCs) via flow cytometry. Dissociation rates were measured via 125I-labeled antibodies in the presence of azide/2-deoxyglucose to prevent internalisation. Ofatumumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) was performed with 51Cr-labeled B cells incubated with whole blood. Complement dependent cytotoxicity (CDC)-mediated B-cell lysis was quantified via incubation with fresh-frozen pooled human serum. Immunogenicity of ofatumumab and rituximab chimeric anti-CD20 antibody was studied in silico for potentially immunogenic T-cell epitopes (Epibase profiling).
RESULTS: Ofatumumab binds to two distinct epitopes within the large and small extracellular loops of CD20 protein. This binding motif is towards the N-terminal. Ofatumumab bound to human (EC50=287ng/mL) PBMCs and stained B cell-rich regions within lymphoid tissues. Strong differences in off-rates were observed between ofatumumab and rituximab. Rituximab dissociated rapidly whereas a slow off-rate and long residence time of ofatumumab resulted in long-lasting effector function and potent B-cell depletion. In vitro whole blood assays showed superiority of ofatumumab over rituximab in lysing CD20-expressing cells, primarily due to enhanced CDC activity. ADCC activity was similar. Ofatumumab binding to target cells did not directly induce apoptosis. In silico profiling showed that ofatumumab has a low number of epitopes capable of inducing an anti-antibody response.
Conclusions: Ofatumumab binds a distinct CD20 epitope on B cells in blood and lymphoid tissues. The slow CD20-binding dissociation and enhanced CDC-mediated lysis (vs rituximab) results in potent effector functions. B-cell depletion is mediated by a combination of CDC and ADCC. These characteristics, together with low potential for anti-antibody response, makes ofatumumab suitable for testing in clinical trials as a low-dose subcutaneous treatment for relapsing MS.
Disclosure: This abstract is supported by Novartis Pharma AG, Basel, Switzerland.
All the authors are employees of Novartis Pharma AG, Switzerland.