Baig, Nisar A.

Background Daratumumab (DARA), a human IgG1 K monoclonal antibody targeting CD38, is used to treat refractory multiple myeloma patients. CD38 is expressed on many cell types (RBCs, granulocytes, lymphocytes, etc.), and thus, DARA can interfere with serological tests. Information regarding how DARA affects anti‐granulocyte antibody (AGA) testing and optimal neutralization of DARA will help laboratories perform accurate testing. Methods Screening of AGA was performed by the granulocyte agglutination test (GAT) and the flow cytometric granulocyte immunofluorescence test (Flow‐GIFT). Samples were tested from patients on DARA ( n = 7), non‐transfused blood donors (healthy controls, n = 7) and AGA reactive samples (positive controls, n = 5). Two neutralization experiments, CD38 removal with DTT and DARA epitope blockage with mouse anti‐CD38, were evaluated. Results Positive reactivity of human IgG binding was observed in 5/7 DARA cases when tested by Flow‐GIFT; however, all 7 cases had negative GAT agglutination results. Further studies by Flow‐GIFT revealed DARA concentrations >0·63 μg/ml bound to granulocytes. DARA binding was negated by DTT though a reduced Flow‐GIFT sensitivity was observed in positive control samples due to increased background detection of human IgG. Mouse anti‐CD38 neutralized the detection of human IgG observed in DARA‐treated patient serum without effecting controls. Conclusion We established that DARA can interfere with AGA testing, leading to false positive Flow‐GIFT results without causing GAT agglutination. DTT treatment increased background binding of secondary antibodies causing a decrease in Flow‐GIFT sensitivity. In comparison, blockage of the DARA binding epitope using mouse anti‐CD38 antibody was effective in neutralizing DARA interference while maintaining Flow‐GIFT sensitivity.

Abstract Abstract 2836 Monoclonal antibodies (mAb) are an effective but non-curative treatment of CLL. Although the mechanisms of action of mAb in vivo are not fully defined, complement dependent cytotoxicity (CDC) appears to play an important role. We have previously shown that addition of ofatumumab (OFA) significantly increases alemtuzumab (ALM) CDC in vitro and identified a subpopulation of CLL cells that are intrinsically resistant to activated complement. We propose that resistance to CDC could be an important cause of mAb treatment failure in CLL patients. To test the hypothesis that CLL cells treated in vivo with OFA would be resistant to subsequent OFA retreatment in vitro, we studied CLL cells and serum from 10 previously untreated patients with progressive CLL treated with pentostatin, cyclophosphamide, and ofatumumab (PCO). Methods. Patients received 300 mg of OFA followed by 2 mg/m2 of pentostatin and 600 mg/m2 of cyclophosphamide on day 1 of therapy. Samples were taken prior to treatment (S1), immediately after the OFA infusion and prior to chemotherapy (S2), and post-chemotherapy before the second dose of OFA on day 2 (S3). Mononuclear blood cells isolated from EDTA anticoagulated blood by Ficoll-Paque centrifugation were purified to achieve a concentration of > 90% CLL. CDC was assayed at a concentration of 2 × 106/ml CLL cells with 10 mg/ml of ALM (Genzyme), OFA (G.S.K.) or rituximab (RTX)(Genentech) in AIM V medium (Invitrogen, CA) and 10% normal human serum (10%NHS)(Sigma, MO) as a source of complement for 1 hour at 37°C. Absolute viable cell counts were measured by flow cytometry using counting beads (Trucount, BD, CA) and propidium iodide staining (Sigma, MO) with a FACSCalibur (BD, CA) and CellQuest Pro software (BD, CA). Percent CDC was calculated relative to counts for CLL cells treated with only 10%NHS. Binding of mAb and C3b to CLL cells was measured by flow cytometry with mouse anti-human Fc antibody FITC-HB43 and anti-C3b antibody FITC-7C12. C5 deficient serum (C5-serum)(Sigma, MO) was used to examine mAb and C3b binding without CDC. Results were expressed as delta mean fluorescent intensity (dMFI) relative to cells treated without mAb in 10%NHS or C5-serum as appropriate. Complement (CH50) was measured based on lysis of opsonized sheep red cells using standard methods. OFA concentrations in serum samples were determined by measuring the level of binding to Daudi cells compared to standards as previously described. Results: S1 CLL cells were significantly more susceptible to in vitro OFA CDC (median 30%) than S2 and S3 cells (both 0%, p=0.002)(Fig. 1). In contrast, high levels of CDC (median 86 – 88%) were induced in vitro by ALM in all these samples and prior in vivo exposure to OFA did not affect susceptibility to ALM CDC in vitro (p>0.1). CLL cells from S2&S3 had low levels of OFA binding both prior to and post in vitro exposure to OFA (median dMFI 2 and 3 respectively) which was significantly less than OFA binding for S1 (median dMFI 38 p=0.002) likely reflecting in vivo trogocytosis of bound OFA and CD20, as previously described for RTX. In vitro ALM binding was higher than OFA (median dMFI>150) in all specimens. C3b binding was low in S2&S3 both before and after in vitro OFA exposure (median dMFI 3 and 4 respectively) with significantly higher levels in S1 (median dMFI 94, p=0.002). C3b binding was higher in all CLL cells treated with ALM (median dMFI>358). OFA treatment resulted in marked decreases in serum complement levels in S2 (median 86%, range 65–98%) and S3 (median 78% range 61–88%) compared to S1. The median serum OFA concentration was 18.5 μg/ml (range 7.3–50.4) in S2 and 19.8 μg/ml (range 0–32.3) in S3. Conclusions: Circulating CLL cells from patients treated with OFA are resistant to in vitro OFA CDC primarily because of low levels of expression of CD20. The reduction in complement titers could limit in vivo CDC but we found no evidence that low levels of OFA were important. Cells surviving OFA in vivo do retain sensitivity to ALM CDC. Our data support the previous description of trogocytosis in CLL patients treated with RTX and suggest that lower doses of these mAb, that promote far lower levels of trogocytosis, could be more effective in sustaining CDC. In addition, our study provides pre-clinical data to support a clinical trial of combination therapy with OFA and ALM for CLL. Acknowledgment: This study was supported by funding from GlaxoSmithKline and the University of Iowa/Mayo Clinic Lymphoma SPORE (CA097274). Disclosures: Taylor: Genmab: Consultancy; Glaxo Smith Kline: Research Funding. Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

Abstract Abstract 3575 Unconjugated monoclonal antibodies (MoAb) have proven value in the therapy of CLL, and alemtuzumab (ALEM) has efficacy in patients with purine analog refractory/p53 defective disease. Although the mechanisms of action of ALEM in vivo are not fully defined, complement dependant cytotoxicity (CDC) appears to play an important role. ALEM CDC resistance in a subpopulation of CLL cells in vivo could thus contribute to treatment failure. We hypothesized that the CD20 targeted complement activating human MoAb ofatumumab (OFA) would increase complement activation (as manifested by C3b deposition) as well as CDC in CLL cells treated with ALEM in vitro. Methods: We have completed experiments on 4 of 15 specimens collected from patients with progressive untreated CLL. CLL cells were isolated from blood by density gradient centrifugation and negative selection with magnetic beads. CD52 and CD20 expression were measured with fluorescent beads (Quantum MESF, Bangs Laboratories, IN). CLL cells were treated at a concentration of 2 × 106/ml with 10 mcg/ml of ALEM (Genzyme) and/or OFA (G.S.K.) in AIM V medium (Invitrogen CA) and 10% standard normal human serum (NHS) (Sigma, MO) as a source of complement for 1 hour at 37C. Absolute viable cell counts were assayed with counting beads (TruCOUNT, BD, CA) and propidium iodide (PI) staining (Sigma) using a FACSCalibur (BD) and CellQuest Pro software (BD). CDC was determined by comparing counts to CLL cells treated with NHS alone. Membrane levels of CD59, bound MoAb, and deposited C3b on the CLL cells membranes were measured by flow cytometry using using the anti-CD59 antibody FITC-CD59 (BD), mouse anti-human Fc antibody FITC-HB43, and the anti-C3b antibody FITC-7C12. To study ALEM CDC resistant cells, CLL cells were first treated with ALEM and NHS as detailed above. Surviving viable cells were isolated using density gradient centrifugation and then retreated. C5 deficient serum (Sigma) was used in experiments to determine MoAb binding and C3b deposition in the entire CLL cell population. Results: Binding of either ALEM or OFA to the CLL cells in NHS resulted in activation of complement and deposition of large amounts of membrane C3b on the cells. However, ALEM induced higher levels of C3b deposition (delta MFI 220) and caused more CDC (73% vs. 18%) compared to OFA. This correlated well with the higher levels of expression of CD52 vs. CD20 expression in CLL cells (143 ×103, range 131 ×103 – 159 ×103 vs. mean 20 ×103 molecules/cell, range 10 ×103 – 28 ×103). Studies with the anti-Fc antibody showed that only a small subpopulation of CLL cells had weak MoAb binding. Addition of OFA to ALEM increased complement-mediated C3b deposition. In three patients with high initial ALEM CDC, addition of OFA did not appreciably increase CDC (81% to 87%). However, in one patient with cells that were less sensitive to ALEM CDC, addition of OFA did appreciably increase CDC (51% to 86%). There was no relationship between levels of CD59 expression and CDC. All four CLL specimens studied contained a subpopulation of cells that were resistant to CDC (mean 13%, range 9–18%) despite robust C3b deposition. Conclusions: These data suggests that there are multiple mechanisms of resistance to MoAb-mediated CDC including: 1. Low MoAb binding because of low target antigen expression; 2. Insufficient C3b deposition after MoAb ligation and; 3. Intrinsic resistance in some cells in cells in which high levels of C3b deposition were clearly demonstrable. OFA can increase MoAb binding, C3b deposition, and CDC in CLL cells treated with ALEM. However, addition of OFA does not overcome the intrinsic resistance of a small subpopulation of CLL cells to CDC. Future studies will be needed to examine these resistance mechanisms and to develop interventions to disrupt them. In addition, membrane C3b could be a target for complement dependent cellular cytotoxity in these CDC resistant cells. Disclosures: Taylor: G.S.K.: Research Funding; Genmab: Research Funding. Zent:Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.

Abstract Abstract 3115 Background. Rituximab significantly improves the outcome of patients with B-cell non-Hodgkin lymphomas, but it does not completely eradicate residual B-cell populations in the bone marrow (BM) and lymph nodes (LN). The architecture and gene expression profile of LN stromal cells in diffuse large cell lymphoma correlates with outcome following treatment with R-CHOP (Lenz et al. 2008). Interestingly, multiple studies have demonstrated the importance of B-cells adhesion and pro-survival signaling mediated by integrin alfa-4-beta-1 (VLA-4/CD49d), which is constitutively expressed on malignant B-cells. Although several studies have demonstrated that adhesion to stromal cell cultures or ligand coated surfaces can protect malignant B-cells from apoptosis induced by chemotherapy drugs (cell adhesion-mediated drug resistance (CAM-DR); Dalton, 2002), a similar mechanism of resistance to rituximab has not, to our knowledge, been described. Hypothesis. In this study we tested the hypothesis that interactions with the microenvironment protect malignant B-cells from rituximab induced apoptosis, and that targeting these interactions with natalizumab, a humanized monoclonal anti-alfa-4 antibody approved for treatment of multiple sclerosis and Crohn's disease, can overcome this protection. Methods. Lymphoma B-cell lines were cultured on either a plastic surface, confluent HS-5 cells or a fibronectin (alfa-4-beta-1 ligand) coated surface. HS-5 is an immortalized human bone marrow stromal cell line and a well characterized model for a component of the BM microenvironment (Roecklein and Torok-Storb, 1995). Cells were treated with rituximab (10 ug/mL; Genentech), natalizumab (10ug/mL; Biogen IDEC), control IgG (10ug/mL, Sigma-Aldrich). Cell viability was measured by flow cytometry using Annexin V-FITC, propidium iodide in malignant B-cell population (anti-CD19 antibody, BD PharMingen). Results. We studied ten CD20-positive B-cell lymphoma cell lines and the CD20-positive MEC-1 cell line (derived from prolymfocytoid transformation of CLL) for rituximab induced apoptosis. The four most rituximab sensitive cell lines were used for further experiments (Karpas-422, Raji, DOHH2, SUDHL-4). Cell lines were co-cultured for 24hrs with confluent HS-5 stromal cells and subsequently treated for 24hrs with rituximab. The percentage of apoptotic cells was significantly lower (17-23% vs. 30–42%, p<0.05) for cells co-cultured with HS-5 cells compared to control cells cultivated under the same conditions on the plastic surface for all four cell lines. Similar results were obtained for several primary CLL samples (3-5% vs. 13–18%, p<0.05). We next examined if natalizumab could disrupt cell adhesion to fibronectin and overcome the stromal cell-mediated resistance to rituximab. Natalizumab reduced the number of lymphoma cells that adhered to fibronectin by 75–95% (p<0.05). Moreover, adherent cells cultivated on fibronectin coated surface completely lost the adhesion morphology after the addition of natalizumab to the cultivation media. The combination of natalizumab and rituximab versus rituximab alone increased by 26–32% (p<0.05) the number of apoptotic B-cells in co-culture with HS-5 for three of four rituximab-responding cell lines. Conclusion. We have shown that human bone marrow stromal cells (HS-5) protect lymphoma B cells from rituximab induced apoptosis suggesting existence of stromal cell adhesion-mediated antibody resistance (CAM-AR) analogous to CAM-DR. Targeting integrin alfa-4-beta-1 with natalizumab partially overcomes this cell adhesion-mediated resistance to rituximab. Research supported by P50CA97274-8 LYMPHOMA SPORE, Genentech, MSMT-MSM0021622430 and IGAMZCR NT11218-3/2010. Disclosures: Zent: Genentech: Research Funding; Genzyme: Research Funding; Novartis: Research Funding.

Summary Unconjugated monoclonal antibodies (mAbs) are an important component of effective combination therapies for chronic lymphocytic leukaemia (CLL). Antibody-dependent phagocytosis (ADP) is a major mediator of mAb cytotoxicity, but there is limited knowledge of the determinants of ADP efficacy. We used macrophages derived in vitro from autologous circulating monocytes to test the effects of mAb structure and concentration, target : effector cell ratio, duration of co-incubation and CLL cell CD20 expression on ADP. Next-generation anti-CD20 mAbs (ofatumumab, ublituximab, obinutuzumab, ocaratuzumab) were significantly more effective at inducing ADP compared to rituximab, but none were as effective as the anti-CD52 mAb alemtuzumab. Ofatumumab (10 μg/ml) used as a representative next-generation anti-CD20 mAb achieved an ADP plateau at 3 h co-incubation with a target : effector ratio of 10 : 1 (mean = 2·1 CLL cells/macrophage, range = 1·5–3·5). At 0·156 μg/ml (the lowest concentration tested) ofatumumab ADP was significantly higher than alemtuzumab. However, ofatumumab-induced ADP did not increase significantly at higher mAb concentrations. We show that anti-CD20 mAb ADP efficacy is determined by the mAb characteristics, target : effector ratio and incubation time. We suggest that preclinical evaluation of anti-CD20 mAbs to understand the determinants of ADP could be useful in designing future combination therapies for CLL.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) patients with purine analog refractory disease or TP53 dysfunction still have limited treatment options and poor survival. Alemtuzumab‐containing chemoimmunotherapy regimens can be effective but frequently cause serious infections. We report a Phase II trial testing the efficacy and tolerability of a short‐duration regimen combining pentostatin, alemtuzumab, and low‐dose high‐frequency rituximab designed to decrease the risk of treatment‐associated infections and to limit the loss of CD20 expression by CLL cells. The study enrolled 39 patients with progressive CLL that was either relapsed/refractory (n = 36) or previously untreated with 17p13 deletion (17p13−) (n = 3). Thirteen (33%) patients had both 17p13− and TP53 mutations predicted to be dysfunctional, and eight patients had purine analog refractory CLL without TP53 dysfunction. Twenty‐six (67%) patients completed therapy, with only five (13%) patients having treatment‐limiting toxicity and no treatment‐related deaths. Twenty‐two (56%) patients responded to treatment, with 11 (28%) complete responses (four with incomplete bone marrow recovery). Median progression‐free survival was 7.2 months, time to next treatment was 9.1 months, and overall survival was 34.1 months. The majority of deaths (82%) were caused by progressive disease, including transformed diffuse large B‐cell lymphoma (n = 6). Correlative studies showed that low‐dose rituximab activates complement and natural killer cells without a profound and sustained decrease in expression of CD20 by circulating CLL cells. We conclude that pentostatin, alemtuzumab, and low‐dose high‐frequency rituximab is a tolerable and effective therapy for CLL and that low‐dose rituximab therapy can activate innate immune cytotoxic mechanisms without substantially decreasing CD20 expression. Am. J. Hematol. 89:757–765, 2014. © 2014 Wiley Periodicals, Inc.

Abstract Ofatumumab (OFA), a human CD20-targeting mAb, kills B lymphocytes using the innate immune system including complement-dependent cytotoxicity (CDC). The efficacy of OFA in patients with chronic lymphocytic leukemia (CLL) is limited by drug resistance, which is not well characterized. To better understand mechanisms of resistance, we prospectively studied CLL cells isolated from blood samples collected before and after in vivo exposure to the initial dose of OFA therapy in 25 patients undergoing their first treatment for progressive CLL. As previously reported, OFA therapy rapidly decreased the absolute lymphocyte count, CD20 expression by CLL cells, and serum complement levels. We now show that after administration of the first dose of OFA, there was a modest rebound in the absolute lymphocyte count and serum complement levels, but substantial ongoing loss of CD20 expression by CLL cells. These post-OFA treatment CLL cells were highly resistant to OFA-mediated CDC but retained sensitivity to alemtuzumab-mediated CDC in vitro. Posttherapy serum OFA levels correlated inversely with both the amount of pretreatment circulating cell-bound CD20 and with the decrease in this value following treatment. In vitro OFA-mediated CDC did not predict clinical responses, and the patients with first-dose reactions to OFA did not have markers of increased complement activation in vivo. We propose that optimal efficacy of CD20- targeted therapy for CLL requires determining an mAb dose size and frequency that optimizes CLL killing without exceeding the capacity of the cytotoxic mechanisms and thus minimizes loss of CD20 expression in the surviving CLL cells.