Properties and structure-function relationships of veltuzumab (hA20), a humanized anti-CD20 monoclonal antibody

Abstract

Veltuzumab is a humanized anti-CD20 monoclonal antibody with complementarity-determining regions (CDRs) identical to rituximab, except for one residue at the 101st position (Kabat numbering) in CDR3 of the variable heavy chain (V(H)), having aspartic acid (Asp) instead of asparagine (Asn), with framework regions of epratuzumab, a humanized anti-CD22 antibody. When compared with rituximab, veltuzumab has significantly reduced off-rates in 3 human lymphoma cell lines tested, as well as increased complement-dependent cytotoxicity in 1 of 3 cell lines, but no other in vitro differences. Mutation studies confirmed that the differentiation of the off-rate between veltuzumab and rituximab is related to the single amino acid change in CDR3-V(H). Studies of intraperitoneal and subcutaneous doses in mouse models of human lymphoma and in normal cynomolgus monkeys disclosed that low doses of veltuzumab control tumor growth or deplete circulating or sessile B cells. Low- and high-dose veltuzumab were significantly more effective in vivo than rituximab in 3 lymphoma models. These findings are consistent with activity in patients with non-Hodgkin lymphoma given low intravenous or subcutaneous doses of veltuzumab. Thus, changing Asn(101) to Asp(101) in CDR3-V(H) of rituximab is responsible for veltuzumab's lower off-rate and apparent improved potency in preclinical models that could translate into advantages in patients.

Figure 1

Comparison of off-rates from live cells. Daudi (A), Ramos (B), and Raji (C-E) cells were stained with PE-labeled rituximab (▵), veltuzumab (blue filled square), cA20 (red triangle), D101N (green filled circle), 1F5 (purple open circle) or B1 (gray open square). The labeled mAbs were incubated at 37°C with (A-D) or without (E) excess veltuzumab Fab′-NEM, and the cells were analyzed by flow cytometry over time. The off-rate was determined by nonlinear regression (one-phase exponential decay) and P values were generated by F test using GraphPad Prism software.

Figure 2

Ex vivo depletion of B cells and lymphoma cells. (A) The effect of veltuzumab on peripheral blood lymphocytes from healthy volunteers was evaluated in vitro using flow cytometry. Decrease in the percentage of CD19⁺ cells present in the lymphocyte gate after a 2-day incubation of heparinized whole blood of healthy volunteers with veltuzumab is shown. Each line represents a different blood donor. Error bars, standard deviation. (B) The effects of veltuzumab and rituximab on peripheral blood B cells and Raji lymphoma cells are shown as the number of CD19⁺ events relative to untreated cell mixtures. B cells are derived as the CD19⁺ cells in the lymphocyte gate, while Raji cells are located in the monocytes gate. Error bars indicate standard deviation.

Figure 3

Evaluation of in vivo efficacy in mouse models. (A) Survival of mice in a disseminated Burkitt lymphoma xenograft model was compared for veltuzumab treatment via intraperitoneal versus subcutaneous administration. SCID mice were administered 1.5 × 10⁷ Daudi cells intravenously on day 0. Therapy with veltuzumab began on day 1 with mice receiving either a single intraperitoneal or single subcutaneous injection of veltuzumab at does of 60, 20, or 5 μg. Control mice received an intraperitoneal injection of either saline or 60 μg hMN-14 IgG (labetuzumab, anti-CEACAM5 isotype-matched antibody). (B) The minimal effective dose of veltuzumab was determined in a disseminated Burkitt lymphoma xenograft model. SCID mice were administered 1.5 × 10⁷ Daudi cells intravenously on day 0. Therapy with veltuzumab began on day 1 with a single intraperitoneal injection of veltuzumab. Doses administered were 0.5, 0.25, 0.1, or 0.05 μg veltuzumab. Control mice received a 200-μL intraperitoneal injection of saline. (C) Survival of mice bearing disseminated follicular cell lymphoma was examined for treatment with decreasing doses of veltuzumab. SCID mice were administered 2.5 × 10⁶ WSU-FSCCL cells intravenously on day 0. On day 5, mice received a single intraperitoneal injection of veltuzumab at a dose of 35, 3.5, 0.35, or 0.035 μg. Control mice received only saline.

Figure 4

In vivo effects of veltuzumab compared with rituximab and after NK/neutrophil depletion in Raji lymphoma model. (A) Comparison of therapeutic effects on survival of RPCI-SCID mice bearing Raji lymphoma cells treated with 10 mg/kg veltuzumab or rituximab (or untreated control) on days 5, 10, 15, and 20 after tumor inoculation intravenously (N = 15 per group), indicating significantly improved survival (P = .005) of the veltuzumab group compared with the rituximab group. (B) The effect of depleting NK cells and neutrophils on anti-lymphoma activity in SCID mice. Veltuzumab therapy consisted of 200 μg given intravenously on days 3, 5, 7, and 11; control mice received 100 μL saline. Depletion of NK cells and neutrophils abrogated the anti-lymphoma activity of veltuzumab.