Exhaustion of cytotoxic effector systems may limit monoclonal antibody-based immunotherapy in cancer patients

Abstract

The CD20 mAb ofatumumab (OFA) induces complement-mediated lysis of B cells. In an investigator-initiated phase II trial of OFA plus chemotherapy for chronic lymphocytic leukemia (CLL), OFA treatment promoted partial CLL B cell depletion that coincided with reduced complement titers. Remaining CLL B cells circulated with bound OFA and covalently bound complement breakdown product C3d, indicative of ongoing complement activation. Presumably, neither complement- nor effector cell-based mechanisms were sufficiently robust to clear these remaining B cells. Instead, almost all of the bound OFA and CD20 was removed from the cells, in accordance with previous clinical studies that demonstrated comparable loss of CD20 from B cells after treatment of CLL patients with rituximab. In vitro experiments with OFA and rituximab addressing these observations suggest that host effector mechanisms that support mAb-mediated lysis and tumor cell clearance are finite, and they can be saturated or exhausted at high B cell burdens, particularly at high mAb concentrations. Interestingly, only a fraction of available complement was required to kill cells with CD20 mAbs, and killing could be tuned by titrating the mAb concentration. Consequently, maximal B cell killing of an initial and secondary B cell challenge was achieved with intermediate mAb concentrations, whereas high concentrations promoted lower overall killing. Therefore, mAb therapies that rely substantially on effector mechanisms subject to exhaustion, including complement, may benefit from lower, more frequent dosing schemes optimized to sustain and maximize killing by cytotoxic immune effector systems.

Figure 1. Correlative studies on patients for the first 30 days of the trial

OFA was infused on days 1 (300 mg), 8 (1 g) and 29 (1 g). Blood samples were obtained immediately before, and 2, 6 and 24 hrs after starting OFA infusions. Results are normalized to pre-treatment values for absolute lymphocyte counts (ALC), CD20, and CH50. Bound OFA is normalized to the 2 hr mark (first infusion), usually the maximum amount bound; values for bound C3d are normalized to maximum amount bound, observed at 2 or 6 hrs. Absolute values for these parameters are provided in Table I, along with representative uncertainties (SD). A., ALC; B., B cell surface levels of CD20; C., cell-bound OFA; D., complement titers (CH50 determinations); E., C3d deposition on B cells. The complement titer of patient N1 was low throughout the study (1–4 day 1; 7–8 day 29) and is not plotted in panel D. Results in B, C and E are based on duplicate determinations; CH50 titers were determined in triplicate.

Figure 2. High concentrations of Al488 OFA, but not Al488 RTX, promote substantial CDC of CLL cells

A. CLL cells from 10 patients (V1 to V10) seen at the University of Virginia (UVA) Hospital were isolated and tested for CDC in the presence of 100 μg/mL mAb, 50% NHS, for 15 min at 37°C. Means and SD (n=2) are displayed. Longer incubation periods did not lead to increased CDC. B–C. Dose-response experiments for OFA binding in media reveal that OFA concentrations as low as 10 μg/mL are sufficient to saturate the cells, even for cell densities of 10⁸ cells/mL. Means and SD (n=2) are displayed for cells from patient V2 (B) and V6 (C). D–G. Dose-response experiments reveal that for high densities of CLL cells (from patients V2 (D,E) and V6 (F,G), optimal OFA-mediated CDC and C3b deposition require adequate complement (50% NHS) and OFA, but under comparable conditions RTX-mediated CDC and C3b deposition is modest. For each condition, final OFA and RTX concentrations of 10 or 100 μg/mL were examined. These results are similar to the findings for all 10 patients. Differences in Al488 OFA binding (B,C) and in CDC for cells reacted in 50% NHS vs 25% NHS were compared for significance, as noted. *, p<0.05; **, p< 0.01; ***, p< 0.001.

Figure 3. At cell densities as high as 10⁸ cells/mL, CDC mediated by ALM, RTX or OFA at 100 μg/mL can be quite effective but at the highest cell densities complement activity is substantially depleted

A. Results for Wien cells. In these experiments CDC was maximized at only ~70% in the presence of ALM, because a fraction of the Wien cells did not express CD52. B,C. Results for CLL cells of patients V11 (B) and V12 (C) (representative of results for cells from UVA patients V11–V16). Moderate to large reductions in complement titers occur when high densities of CLL cells are subjected to CDC mediated by ALM, RTX, or OFA, but only ALM and OFA promoted high levels of CDC. Cetuximab (CET) which does not bind to or induce CDC in B cells, was used as a second negative control and no complement was consumed (not shown).

Figure 4. Two-step experiment: Lower mAb concentrations in Step 1 are more effective in promoting overall CDC of the combined cell populations

A–C. Step 1: Unlabeled Daudi cells (1.8 × 10⁷ cells/mL in 50% NHS), were reacted (one hr at 37°C) with OFA (0 to 100 μg/mL). In step 2 an equal number of PKH26-labeled Daudi cells was added, + 100 μg/mL additional OFA, and after incubation, CDC and C3b deposition was determined. A,B. The %CDC of unlabeled cells after step 1 (open circles), and of PKH26 labeled cells after step 2 (filled circles), is provided. C. The summation of %CDC for unlabeled cells after step 1 plus %CDC for PKH26 cells in step 2 is a bell-shaped curve. The decrease in CDC at high OFA concentrations is not due to final high OFA concentrations (200 μg/mL). In a separate one-step control, varying amounts of OFA were added to Daudi cells (3.6 × 10⁷ cells/mL in 50% NHS); CDC was 85% for OFA concentrations of 100–400 μg/mL. D–E. C3b deposition on unlabeled cells after step 1 or on PKH26-labeled cells after step 2. F–H. Similar experiment, except OFA added in step 2 was 100 μg/mL or 20 μg/mL. Close to maximal C3b deposition and CDC is achieved for OFA concentrations of 4 μg/mL in step 1 and 20 μg/mL in step 2. I–K similar to A–C, except for RTX. Means and SD (n = 2) are displayed; usually SD are smaller than symbols. Each figure represents two or more experiments.

Figure 5. Generalization of the findings in Fig. 4 to a likely in vivo scenario

High dose mAb treatment may result in excess complement consumption (depletion of available complement). Complement depletion will lead to inadequate killing of a second target cell challenge even in the presence of additional excess mAb. Tailoring mAb doses to optimal levels (e.g. alternative dose or frequency of treatment) should be aimed at preserving complement-mediated killing activity and maintaining adequate complement levels. This paradigm may allow design of future mAb-based immunotherapy regimens in which effector function is retained over time to achieve increased overall killing and potentially increase efficacy.