Anti-CD22 and anti-CD79b antibody-drug conjugates preferentially target proliferating B cells

Abstract

Background and purpose: CD22 and CD79b are cell-surface receptors expressed on B-cell-derived malignancies such as non-Hodgkin's lymphoma (NHL). An anti-mitotic agent, monomethyl auristatin E, was conjugated to anti-CD22 and anti-CD79b antibodies to develop target-specific therapies for NHL. The mechanism of action (MOA) and pharmacological and pharmacokinetic (PK) profiles of these antibody-drug conjugates (ADCs) were investigated in cynomolgus monkeys.

Experimental approach: Animals were administered anti-CD22 or anti-CD79b ADCs, respective unconjugated antibodies or vehicle. Pharmacodynamic effects on total and proliferating B cells and serum PK were then assessed. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of the ADCs were evaluated in vitro.

Key results: Depletion of B cells was observed after administration of either ADC or the respective unconjugated antibodies. An extended duration of depletion was observed in animals administered ADCs. Similarly, preferential depletion of proliferating B cells in blood and germinal centre B cells in spleen were only observed in animals administered ADCs. Serum PK profiles of ADCs and respective unconjugated antibodies were comparable. In vitro, anti-human CD22 and anti-human CD79b antibodies showed no or only moderate ADCC activity, respectively; neither antibody had CDC activity.

Conclusions and implications: The findings support the proposed MOA: initial depletion of total B cells by antibody-mediated opsonization, followed by preferential, sustained depletion of proliferating B cells by the auristatin conjugate due to its anti-mitotic action. Delivering potent anti-mitotic agents to B cells via the specificity of monoclonal antibodies provides a means to eliminate pathogenic B cells in NHL with improved risk-benefit profiles over traditional chemotherapeutics.

Figure 1

Anti‐CD22 antibody binding to CD20⁺ B cells of cynomolgus monkeys of Chinese, Cambodian, Mauritian and Indonesian origins. Representative flow cytometry scatter plots of anti‐CD22 antibody (upper panel), isotype control (middle panel) or Hu8G10 (lower panel) on gated CD20⁺ B cells in the peripheral blood from cynomolgus monkeys of different origins are presented.

Figure 2

Depletion of CD20⁺ B cells following administration of anti‐CD22 or anti‐CD79b ADCs. Peripheral blood CD20⁺ B cells (panels A and B) or CD3⁺ T‐cells (panels C and D) in animals administered either anti‐CD22 (panels A and C) or anti‐CD79b (panels B and D) ADCs (n = 4); respective unconjugated antibodies (Ab) (n = 4) or vehicle (n = 4) over time are presented. Data presented are group mean absolute counts expressed as a percentage of baseline (% of BL) at each time point. Baseline was calculated as the average value obtained at two pre‐dose time points (shown). Error bars represent SEM. The arrow denotes administration of the test substances.

Figure 3

Baseline percentage of peripheral blood Ki‐67⁺ B cells in cynomolgus monkeys of Indonesian, Chinese and Mauritian origins. Percentage of peripheral blood CD20⁺Ki‐67⁺ B cells expressed as a percentage of total CD20⁺ B cells in cynomolgus monkeys is presented. (A) Representative cytogram of CD20⁺Ki‐67⁺ B cells gated per respective isotype control. (B) Box plot with the individual animal distribution within each animal origin. The upper, middle and lower lines of each box represent the 75th quartile, median and 25th quartile respectively. Mean percentage (±SD), median and range of peripheral blood CD20⁺Ki‐67⁺ B cells tabulated by animal origin are shown. (C) Single i.v. doses of vehicle (left panel), 3 mg·kg⁻¹ anti‐CD22 ADC (middle panel), or 3 mg·kg⁻¹ unconjugated anti‐CD22 antibody (Ab) (right panel) were administered to cynomolgus monkeys. Absolute numbers of Ki‐67⁺ and Ki‐67⁻ B cells are plotted as a percentage of the average pre‐dose value for each animal (symbols) on Day 15 (upper panel) and Day 22 (lower panel). Boxes show the individual animal distribution within each group, where the upper, middle and lower lines represent the 75th quartile, median and 25th quartile respectively. (D) Single i.v. doses of vehicle (left panel), 3 mg·kg⁻¹ anti‐CD79b ADC (middle panel), or 3 mg·kg⁻¹ unconjugated anti‐CD79b antibody (Ab) (right panel) were administered to cynomolgus monkeys. Absolute numbers of Ki‐67⁺ and Ki‐67⁻ B cells are plotted as a percentage of the average pre‐dose value for each animal (symbols) on Day 15 (upper panel) and Day 22 (lower panel). Boxes show the individual animal distribution within each group, where the upper, middle and lower lines represent the 75th quartile, median and 25th quartile respectively.

Figure 4

Depletion of germinal centers in lymphoid tissues of cynomolgus monkeys administered anti‐CD22 ADC or anti‐CD79b ADC. (A) Representative H&E‐stained section of spleen from animals administered single i.v. doses of either vehicle (upper panel) or 6 mg·kg⁻¹ anti‐CD22 ADC (lower panel) on Day 8 is presented. (B) Representative H&E‐stained section of spleen from animals administered four i.v. doses of vehicle (upper panel) or 5 mg·kg⁻¹ anti‐CD79b ADC (lower panel) on Day 71 (7 days following fourth dose) is presented.

Figure 5

Systemic exposure of anti‐CD22 antibody is slightly higher than that of anti‐CD79b antibody. Group mean (±SD) serum concentrations of antibody following a single i.v. administration of 3 mg·kg⁻¹ unconjugated anti‐CD22 antibody (Ab) or anti‐CD22 ADC (A) or 3 mg·kg⁻¹ unconjugated anti‐CD79b antibody or anti‐CD79b ADC (B) are presented. Red solid line represents serum concentration of total (both conjugated and unconjugated) antibody in animals administered ADC. Black dashed line represents serum concentration of unconjugated antibody in animals administered the respective unconjugated antibody.

Figure 6

ADCC activity of anti‐CD22 and anti‐huCD79b antibodies. Anti‐CD22 antibody, anti‐huCD79b antibody and rituximab (positive control) were evaluated for their ability to induce ADCC in human BJAB. Each figure represents the results from three independent experiments conducted with each antibody using peripheral blood mononuclear cells from different donors. The % ADCC was plotted against concentration of the antibodies and data fitted with a four‐parameter model.