Treatment of Relapsed/Refractory CLL Patients With PI3Kδ Inhibitor and Anti-CD20 Antibody Rapidly Decreases Tumor Burden but Could Induce Resistance

Therapeutic unconjugated anti-CD20 monoclonal antibodies (mAb) and small molecule inhibitors of the B cell receptor (BCR) signaling pathway and B cell lymphoma-2 (BCL2) have greatly improved therapy for patients with progressive chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL). However, these therapies are not curative, and their efficacy can be compromised by side effects and acquired drug resistance, especially with indefinite duration regimens [1]. To address these problems, we tested a limited–duration triple combination targeted therapy for relapsed/refractory CLL patients (U2-VEN, ClinicalTrials.gov NCT03379051) that involved three drugs with different mechanisms of action, which may increase effectiveness and reduce the likelihood of resistance (Figure 1A [2]). We chose: (1) ublituximab (UBL), a glycoengineered chimeric monoclonal antibody (mAb) targeting a unique epitope on CD20 antigen (Ag) on surface of CLL cells, which destroys CLL cells primarily by antibody-dependent cellular phagocytosis (ADCP); (2) umbralisib (UMB), a novel small molecule B cell receptor (BCR) signaling inhibitor that is highly selective for phosphoinositide 3-kinase delta (PI3Kδ, shown) and casein kinase 1 epsilon, which causes CLL cell death by preventing cell survival signals; and (3) venetoclax (VEN), a small molecule anti-apoptosis inhibitor that blocks cell survival promoted by BCL2, which stops mitochondria from initiating apoptosis. An initial 12 week treatment with UBL and UMB was utilized to allow the BCR signal inhibiting drug to mobilize CLL cells into the circulation where they are more susceptible to mAb cytotoxicity and thus decrease the CLL tumor burden and risk of VEN-induced tumor lysis syndrome when it is added in the 13th week of therapy (Figure 1B).

FIGURE 1

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UBL and UMB treatment in U2-VEN clinical trial results in rapid loss of CLL cells and CD20 levels. (A) Multi-drug combination targeting three different molecules to overcome single-agent resistance in relapsed/refractory CLL patients. (B) Diagram of U2-VEN clinical study design (ClinicalTrials.gov NCT03379051) [2]. Treatment consists of initially 12 cycles of 28 days. Ublituximab (purple) was administered intravenously on indicated days of first six cycles. Umbralisib (green) was orally administered daily for 12 cycles. Venetoclax was added for cycles 4–12. If minimal residual disease (MRD) was detectable in peripheral blood (PB) or bone marrow (BM), Umbralisib was continued for an additional 12 cycles. Tumor lysis syndrome (TLS) was assessed at regular intervals. (C) U2-VEN sampling timeline in first three cycles. Ublituixmab (UBL, purple) was administered intravenously (dose in mg shown) on indicated days. Umbralisib (UMB, 800 mg/d, green) was administered daily (qd). Vertical black arrows indicate blood sampling before (Pre) and after (Post) treatment on the indicated days (labeled as cycle # and day # Pre or Post, e.g., “C1D1 Pre” and “C1D1 Post”). (D) CLL decreases after day 1 treatment, rebounds on day 2, and decreases to low level after 2 months. CLL cell count in PB from all patients (n = 25) for each sampled time point was determined by flow cytometry (median +/− 95% distribution-free confidence interval [CI]). (E) CLL patient lymph node (LN) sizes decrease after 3 months treatment (24/25). LN sizes assessed by SPD of CT scans were determined at baseline and after 3 months. The percentage change in size from baseline is shown for each patient. (F) CLL CD20 levels decrease immediately after treatment and remain low throughout. CLL CD20 molecules per cell (median +/− 95% distribution-free CI) were measured by flow cytometry for 18 patients at all sampled time points. (G) CLL CD20 levels decrease relative to CD19. CLL CD20:CD19 gMFI ratio was calculated for each sampled time point (median +/− 95 distribution-free CI, n = 18). Baseline ratio if CD20 and CD19 levels remained relatively the same is shown (dotted line). Low baseline (C1D1 Pre) CLL cell CD20 levels correlate with smaller percent changes in CLL counts from baseline (H, Pearson correlation (r²) = −0.47, one-sided p-value = 0.03, n = 17) at subsequent timepoints as shown at day 8 Pre (I, r² = −0.48, one-sided p-value = 0.04, n = 17). (J) Within day CLL cell count percent change (y-axis), blood draw dates (labeled as cycle # and day #, x-axis) and log CD20 level (color gradient from red [high] to blue [low]) are shown. On C1D1, the largest CLL count decreases coincide with highest CLL CD20 levels (red/orange). As time goes on CD20 levels decrease (blue), and within day CLL count changes become more variable. High CD20 levels (red/orange) tend to coincide with larger CLL cell count decreases, although at C1D2 there are some mid CD20 level expressers (green) that have large CLL increases, and conversely at C1D8, a low CD20 expresser (dark blue) has the largest CLL cell count percentage decrease.

To study the initial effect of UBL and UMB on circulating CLL cell counts and CD20 levels, peripheral blood samples were collected immediately prior to (Pre) initiation and after (Post) infusion of UBL and UMB treatment on days 1, 2, 8, and 15 of cycle 1, and on day 1 of cycles 2 and 3 (Figure 1C, C1D1, C1D2, C1D8, C1D15, C2D1, C3D1) after informed consent from a cohort of relapsed/refractory CLL patients followed at the Wilmot Cancer Institute in the University of Rochester Medical Center (n = 25, Table S1). Most patients had advanced Rai stage with high-risk genetic features and had failed a median of two treatment regimens. Blood samples were analyzed by clinical laboratory automated cell counter and stained for flow cytometry analysis (Figure S1).

Treatment with UMB and UBL caused a rapid decease in CLL tumor burden. Baseline pre-treatment (C1D1 Pre, n = 25) CLL cell counts ranged from 0.5 to 246 × 10⁹/L (median 26.6, Figure 1D, Table S2). CLL counts decreased by a median of 60% from baseline to 6.4 × 10⁹/L after the first dose UMB (started at 800 mg by mouth every 12 h) and UBL (150 mg IV). On day 2 (C1D2), median pre-treatment CLL cell counts had rebounded to 62% of baseline (11.5 × 10⁹/L) and then decreased after the second dose of UBL (750 mg IV) to 44% of baseline (7.3 × 10⁹/L). The median CLL cell count progressively decreased from 1.6 × 10⁹/L (9% of baseline) at day 8 (C1D8) to 0.16 × 10⁹/L (1.2% of baseline) after 8 weeks of therapy (C3D1). Serial CT scans showed that 24 patients had a concomitant decrease (median 64%) in calculated lymph node volume after 12 weeks of therapy (Figure 1E).

UMB and UBL therapy was also associated with a rapid and sustained decrease in CLL surface CD20 levels. Median baseline CLL cell membrane CD20 levels (27 500 molecules/cell C1D1 Pre, n = 18) decreased to 2330 CD20 molecules/cell (8% of baseline) after administration of 150 mg of UBL (Figure 1F, Table S2). Median CD20 levels continued to decrease over time to 1783 molecules/cell (C1D2 Pre), 1374 molecules/cell (C1D2 Post), 820 molecules/cell (C1D8), and 528 molecules/cell by C3D1. In contrast, CLL cell membrane CD19 levels were not decreased by treatment as shown by the CLL CD20:CD19 ratio plot (Figure 1G). This severe decrease in CLL CD20 antigen levels could diminish the effectiveness of UBL anti-CD20 mAb treatment.

The highest levels of CLL cell CD20 were measured in CLL cells prior to treatment and the largest percentage clearance of CLL cells compared to baseline occurred after the first day of treatment. Analysis of the correlation between pre-treatment circulating cell CD20 levels and treatment response measured as the percentage decrease in circulating CLL count at day 1 (Figure 1H) and day 8 (Figure 1I) showed a significant association with higher pre-treatment CD20 levels and larger treatment response. Within day CLL, count decreases tended to be greater at higher CD20 levels (Figure 1J, orange dots, C1D1, C1D2, and C1D8 timepoints). After C1D8, CLL cell CD20 levels were generally low (Figure 1J, blue dots) and exhibited minimal change after each UBL infusion (Figure 1F, Table S2). These data suggest that CD20 levels decreased at these later timepoints to an extent where UBL is no longer effective.

One major mechanism for the loss of surface CD20 on CLL cells is by antibody-dependent trogocytosis, a “nibbling” or “shaving” process that removes the mAb-antigen complex from the surface of target CLL cells by a non-lethal mechanism. Standard doses of other CD20 targeting mAbs, such as rituximab and ofatumumab, have shown similar loss of CLL CD20 surface antigen, resulting in acquired resistance to mAb therapy. This immediate loss of CD20 antigen was largely due to mAb-dependent trogocytosis of surface CD20 antigen by myeloid or natural killer cells and not due to internalization of the mAb-Ag complex by the target CLL cell. Further understanding of antibody-dependent trogocytosis may help to reduce acquired resistance to mAb therapy.

A second mechanism for the decrease in CLL CD20 expression could be due to UMB inhibition of BCR signaling. BCR signaling is an important activator of transcription of MS4A1 coding for CD20. However, the effects of highly selective inhibition PI3Kδ and casein kinase 1 epsilon by UMB on CD20 expression remains to be formally proven. We were not able to measure this effect in this study because of the simultaneous initiation of therapy with both UMB and UBL. Moreover, BCR signaling inhibitors may also decrease mAb-mediated cytotoxicity, such as ADCP. The PI3Kδ inhibitor idelalisib decreased ADCP in vitro, whereas the highly selective PI3Kδ inhibitor UMB did not, supporting the choice of UMB in our clinical trial [3].

Therapeutic mAb clear CLL from the circulation by activation of innate immune cytotoxicity, with the most effective mechanism being ADCP by fixed macrophages in the liver and spleen. The number of opsonized targets cells cleared from the circulation by the innate immune system after each dose of mAb is determined by the finite cytotoxic capacity of the innate immune system which is subject to exhaustion [4]. We have recently shown that administration of 25 mg of IV rituximab as initial therapy for progressive CLL in therapy naïve patients results in clearance of over 80% of circulating cells within 1 h of starting therapy with minimal subsequent clearance during the other 25 mg of the first dose suggesting “exhaustion” of innate immune cytotoxicity [5]. In contrast, circulating CLL cell CD20 levels decreased to 68% of baseline at 1 h and continue to decrease to 37% of baseline at the end of the infusion. These data suggest that trogocytosis is initiated by administration of rituximab and continues after the exhaustion of innate immune cytotoxicity. A long duration of innate immune exhaustion with continued mAb-mediated trogocytosis inducing loss of surface CD20 could be mitigated by use of smaller mAb doses sufficient to fully activate innate immune cytotoxicity but not to support ongoing additional trogocytosis. We and others have tested this hypothesis by use of a high frequency (2–3 times per week) low-dose (20 mg/m² IV or 50 mg subcutaneous) rituximab regimen which is effective and does not cause long-term decreases in CLL cell surface CD20 [6]. The data from this study supports such an approach in future treatment regimens.

Limited duration multidrug targeted therapy is highly effective treatment for CLL. In this study, we confirm that initiating treatment with the combination of UMB and UBL rapidly achieved the intended aim of decreasing tumor burden and decreasing the risk of venetoclax-induced tumor lysis syndrome [2]. However, our study shows that UBL efficacy at decreasing circulating CLL cells is limited to the first two doses of the drug, after which there was minimal additional CLL cell clearance. The immediate value of the data generated by this study was diminished by the withdrawal of UMB from the market and the decision not to further develop UBL. However, our data remains important for designing future clinical trials in CLL utilizing therapeutic anti-CD20 mAb monotherapy or in combination with BCR signaling inhibitors, such as BTK or PI3Kδ inhibitors. In particular, the therapeutic benefit of anti-CD20 mAb in combination with a BCR pathway inhibitor could be optimized by treatment regimens designed to minimize loss of CLL cell surface CD20.