Retreatment with Polatuzumab Vedotin in Combination with Rituximab Increases Antitumor Activity Against Polatuzumab Vedotin-Resistant DLBCL Cells

Introduction. Polatuzumab vedotin (Pola) is a monoclonal antibody against B-cell antigen CD79b conjugated to monomethyl auristatin E (MMAE), and is approved in combination with bendamustine and rituximab (Rit) in relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL) in various countries. Moreover, in the POLARIX study, Pola-R-CHP (cyclophosphamide, doxorubicin, and prednisone) achieved a significantly improved progression-free survival compared to R-CHOP (CHP plus vincristine) in previously untreated DLBCL. The treatment landscape of DLBCL have continued to change dynamically in recent years with the development of various agents. Although the detailed mechanism of resistance to Pola is not clear, understanding it is important for the development of next-line treatment to follow Pola-containing therapy, including retreatment with Pola. To address this, we established the Pola-resistant cells using Pola-sensitive human DLBCL and assessed the combination efficacy of Pola with Rit, the key component in the treatment of DLBCL to determine the efficacy of retreatment with Pola.

Methods. We established Pola-resistant DLBCL cells from STR-428 and SU-DHL-4 cells through long-term exposure to Pola. The cell cytotoxicity to Pola was assessed by WST-8 assay. The in vitro combination efficacy of Pola plus Rit was assessed by measuring the sensitivity to complement-dependent cytotoxicity (CDC), rituximab's potent mechanism of action. In CDC assays, calcein-AM stained tumor cells were incubated with or without Rit in the medium containing 15% normal human serum. After incubating for 4 hours at 37℃, the fluorescence intensity was measured. Expression levels of protein were determined by flow cytometry or immunoblotting. For in vivo experiments, Pola-resistant STR-428 cells were injected subcutaneously into C.B-17/Icr-scid/scidJcl mice. Human IgG, Pola, or Rit was administered intravenously on Day 1.

Results. Pola-resistant cells established from SU-DHL-4 cells (SU-DHL-4-Pola-R) showed a 2.3-fold higher IC50 value than parental cells. In SU-DHL-4-Pola-R cells, there was an increase in the expression of MDR1 (1.8-fold), a multidrug resistance pump mediating intracellular MMAE efflux. Interestingly, treatment with 1 μg/mL of Pola for 7 days further increased the IC50 value (16.8-fold) and the expression of MDR1 (3.6-fold）in SU-DHL-4-Pola-R cells.MDR1 inhibitor verapamil increased sensitivity to Pola, indicating that overexpression of MDR1 is one of the mechanisms of resistance to Pola in these cells. We then investigated the efficacy of combination therapy with Rit against Pola-resistant cells. Pretreatment with Pola or anti-CD79b antibody before the assays enhanced Rit-induced amount of membrane attack complex during complement activation and significantly increased CDC sensitivity in SU-DHL-4-Pola-R cells.

Other Pola-resistant cells derived from STR-428 cells (STR-428-Pola-R) displayed an IC50 value 17.6-fold higher than the parental cells. STR-428-Pola-R cells did not exhibit Pola-resistant factors such as MDR1 upregulation or loss of CD79b expression, but showed decreased expression of Bim, an apoptosis-inducing factor. Stable knockdown of Bim reduced the sensitivity to Pola in parental STR-428 cells, indicating decreased Bim expression could be the resistant mechanism to Pola in these cells. In STR-428-Pola-R cells, 3 days pretreatment with Pola or MMAE significantly increased Rit-induced CDC sensitivity. Immunoblotting analysis revealed that pretreatment with Pola or MMAE downregulated the expression of Mcl-1, which is known to attenuate CDC sensitivity. Mcl-1 inhibitor AZD5991 significantly increased CDC sensitivity, suggesting that Pola-mediated downregulation of Mcl-1 could upregulate the sensitivity to CDC in these cells. In addition, treatment with Pola (1 mg/kg) plus Rit (10 mg/kg) significantly enhanced antitumor activity in mouse xenograft models derived from STR-428-Pola-R cells.

Conclusion. Pretreatment with Pola enhanced Rit-induced CDC sensitivity in two Pola-resistant cells with distinct mechanisms of resistance: increased MDR1 or decreased Bim expression. Combination treatment of Pola with Rit enhanced antitumor activity in STR-428-Pola-R xenografted mice. These results indicate that retreatment with Pola in combination with Rit could be a therapeutic option in patients with Pola-resistant tumors.

Kawasaki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Tomita:Chugai Pharmaceutical Co., Ltd.: Current Employment. Yamashita-Kashima:Chugai Pharmaceutical Co., Ltd.: Current Employment. Yoshimura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Yoshiura:Chugai Pharmaceutical Co., Ltd.: Current Employment.