Ofatumumab Exhibits Enhanced In Vitro and In Vivo Activity Compared to Rituximab in Preclinical Models of Mantle Cell Lymphoma

Abstract

Purpose: Mantle cell lymphoma (MCL) is a mature B-cell lymphoma considered to be incurable with current treatments, including first-line rituximab in combination with multiagent chemotherapy and for those eligible, high-dose chemotherapy and stem cell support or rituximab maintenance. On the other hand, achieving a complete remission by high-sensitive flow cytometry is associated with prolonged duration of remission, stressing the need to develop and/or incorporate novel agents into the management of MCL. To this end, we examined the activity of ofatumumab, an anti-CD20 monoclonal antibody with distinct binding and immunologic properties compared to rituximab, in MCL preclinical models.

Experimental design: MCL cells were labeled with (51)Cr before incubation with rituximab or ofatumumab (10 μg/mL) plus human serum or effector cells. (51)Cr-release was measured and the percentage of lysis was calculated. Surface CD20, CD55, and CD59 were measured by Imagestream analysis. SCID mice inoculated subcutaneously with Z138 cells were assigned to control versus four doses of ofatumumab or rituximab (10 mg/kg/dose).

Results: Ofatumumab exhibited enhanced in vitro complement-dependent cytotoxicity activity compared with rituximab in MCL cell lines, despite a high degree of in vitro resistance to rituximab associated with low CD20 levels and/or high expression of complement inhibitory proteins. Ofatumumab also delayed tumor progression and prolonged survival in a murine model of MCL.

Conclusions: Our results demonstrate that ofatumumab is more effective than rituximab in MCL preclinical models, including in the presence of rituximab resistance, and support the clinical investigation of ofatumumab in combination with standard systemic chemotherapy in MCL (NCT01527149).

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

©2015 American Association for Cancer Research.

Figures

Figure 1

Ofatumumab induces significantly higher levels of CDC-associated cell lysis than rituximab. MCL tumor cell lines were tested for CDC cell lysis using a 51Cr release assay with human serum from normal healthy donors as a source of complement. In all MCL cell lines tested, with the exception of Granta, ofatumumab was more effective than rituximab at inducing CDC lysis at a dose of 10 μg/mL. (*, P < 0.05).

Figure 2

Ofatumumab and rituximab induce a similar degree of ADCC-associated cell lyis. MCL tumor cell lines were tested for ADCC cell lysis using a 51Cr release assay using effector cells from normal healthy donors at a ratio of 40:1. In all MCL cell lines tested, ofatumumab and rituximab induced similar levels of ADCC lysis at a dose of 10 μg/mL.

Figure 3

In MCL cells derived from patient samples, ofatumumab induced similar or more cell lysis by CDC compared with rituximab. B cells were isolated from pretreatment tumor samples (n = 4) obtained from MCL patients and tested for monoclonal antibody response by CellTiter Glo assay using rituximab or ofatumumab at a concentration of 10 μg/mL in the presence of human serum as a source of complement. Two of the four samples exhibit a statistically significant decrease in viability following ofatumumab exposure as compared to rituximab. The remaining samples show a similar response to each antibody.

Figure 4

Ofatumumab and rituximab exhibit modest direct antitumor activity against MCL cell lines. MCL cell lines were utilized in alamarBlue reduction assays to investigate the direct antilymphoma activity of each antibody. Each antibody exhibited a time-dependent decrease in MCL cell viability following exposure to 10 μg/mL of each respective antibody in the presence of a cross-linking IgG antibody. With the exception of the Mino cell line at the 72-hour time point, all cell lines demonstrated similar degrees of direct antitumor activity with ofatumumab and rituximab at the dose tested (*, P < 0.05).

Figure 5

Expression of CD20, CD55, and CD59 in MCL cell lines compared with rituximab-sensitive Raji and rituximab-resistant Raji 4RH cell lines. The surface density of CD20, CD55, and CD59 was determined using Imagestream. MCL cell lines exhibit similar density of CD20 expression on their cell membrane when compared with the rituximab-sensitive Raji cell line. However, the complement inhibitory proteins CD55 and CD59 are present at increased surface density compared with Raji cells, similar to the rituximab-resistant Raji 4RH cell line.

Figure 6

Ofatumumab delayed tumor growth and prolonged survival in an in vivo SCID mouse model of MCL. SCID mice were inoculated subcutaneously on the posterior flank with 10 × 106 Z-138 cells. Mice with established tumors were assigned to control, ofatumumab 10 mg/kg or rituximab 10 mg/kg for 4 doses on days 0, 3, 7, and 10 following tumor establishment. Tumor volume was determined by caliper measurement. Ofatumumab slowed early tumor growth when compared with rituximab with a significant difference in tumor volume noted days 3 and 8 following the start of treatment. Mice were sacrificed when the subcutaneous tumor grew to 2 cm in its largest diameter. Using this time-point as the survival endpoint in a Kaplan–Meier analysis, ofatumumab (median survival not reached) significantly prolonged survival compared with rituximab (median = 127 days; *, P < 0.05).