Combining milatuzumab with bortezomib, doxorubicin, or dexamethasone improves responses in multiple myeloma cell lines

Abstract

Purpose: The humanized anti-CD74 monoclonal antibody, milatuzumab, is in clinical evaluation for the therapy of multiple myeloma (MM). The ability of milatuzumab to increase the efficacy of bortezomib, doxorubicin, and dexamethasone was examined in three human CD74+ MM cell lines, CAG, KMS11, KMS12-PE, and one CD74-MM cell line, OPM-2.

Experimental design: Activity of milatuzumab as a monotherapy and combined with the drugs was evaluated by studying in vitro cytotoxicity, signaling and apoptotic pathways, and in vivo therapeutic activity in severe combined immunodeficient (SCID) mouse models of MM.

Results: Given as a monotherapy, cross-linked milatuzumab, but not milatuzumab alone, yielded significant antiproliferative effects in CD74+ cells. The combination of cross-linked milatuzumab with bortezomib, doxorubicin, or dexamethasone caused more growth inhibition than either cross-linked milatuzumab or drug alone, producing significant reductions in the IC(50) of the drugs when combined. Efficacy of combined treatments was accompanied by increased levels of apoptosis measured by increases of activated caspase-3 and hypodiploid DNA. Both milatuzumab and bortezomib affect the nuclear factor-kappaB pathway in CAG MM cells. In CAG- or KMS11-SCID xenograft models of disseminated MM, milatuzumab more than doubled median survival time, compared with up to a 33% increase in median survival with bortezomib but no significant benefit with doxorubicin. Moreover, combining milatuzumab and bortezomib increased survival significantly compared with either treatment alone.

Conclusions: The therapeutic efficacies of bortezomib, doxorubicin, and dexamethasone are enhanced in MM cell lines when given in combination with milatuzumab, suggesting testing these combinations clinically.

Figures

Figure 1. Effects of milatuzumab and GAH second antibody on proliferation of cell lines

Anti-proliferative effects of milatuzumab were determined by MTT assays. Cells were cultured with 5 μg/ml milatuzumab with or without 20 μg/ml second antibody for crosslinking to mimic the role of effector cells or crosslinking molecules present in vivo. Percent inhibition is calculated relative to untreated control cells. Error bars represent standard deviations of 4 replicates. *, not determined.

Figure 2. In vitro cytotoxicity of bortezomib, dexamethasone, and doxorubicin with and without milatuzumab in CAG

Cells were cultured with the drugs, milatuzumab (5 μg/ml), and GAH second antibody (20 μg/ml) as shown, then assayed using MTT. Percent inhibition of absorbance at 570 nm, relative to media only, was measured. ○, solid line, drug only; △, dashed line, drug + milatuzumab; □, dotted line, drug + GAH; ●, solid line, drug + milatuzumab + GAH. Error bars represent standard deviations of 4 replicates.

Figure 3. Apoptotic effect of mAbs on MM cell lines

Induction of apoptosis was evaluated by flow cytometry determination of (A) hypodiploid DNA and (B) activated caspase-3. Cells were incubated with drugs alone, milatuzumab alone, or combinations of drug + milatuzumab with and without a second antibody for cross-linking for 48 h. For determination of hypodiploid DNA, staining was done with propidium iodide. Changes in the intracellular levels of cleaved caspase-3 were measured using FITC–conjugated rabbit anti-activated caspase-3. Analyses were performed on the FACSCalibur. ○, solid line, drug only; △, dashed line, drug + milatuzumab; □, dotted line, drug + GAH; ●, solid line, drug + milatuzumab + GAH. Error bars represent standard deviations of 3 replicates, and at some points are hidden by the symbol.

Figure 3. Apoptotic effect of mAbs on MM cell lines

Induction of apoptosis was evaluated by flow cytometry determination of (A) hypodiploid DNA and (B) activated caspase-3. Cells were incubated with drugs alone, milatuzumab alone, or combinations of drug + milatuzumab with and without a second antibody for cross-linking for 48 h. For determination of hypodiploid DNA, staining was done with propidium iodide. Changes in the intracellular levels of cleaved caspase-3 were measured using FITC–conjugated rabbit anti-activated caspase-3. Analyses were performed on the FACSCalibur. ○, solid line, drug only; △, dashed line, drug + milatuzumab; □, dotted line, drug + GAH; ●, solid line, drug + milatuzumab + GAH. Error bars represent standard deviations of 3 replicates, and at some points are hidden by the symbol.

Figure 4. Western blot analyses of lysates of CAG cell treated with milatuzumab and bortezomib

CAG cells were cultured in the presence or absence of the indicated reagents for 30 min or 3 h, pelleted, then washed three times in PBS, and lysed in ice cold RIPA buffer containing 100 μg/ml PMSF and 1 μg/ml aprotinin. Milatuzumab was used at 5 μg/ml, GAH at 20 μg/ml, and bortezomib at 1 ng/ml. Equivalent amounts of protein were analyzed by Western blots and actin levels were compared as a control for protein loading. Lanes are as follows: 1, untreated control cells; 2, milatuzumab; 3, milatuzumab + GAH; 4, milatuzumab + GAH + bortezomib; and 5, bortezomib.

Figure 5. Therapeutic efficacy of bortezomib plus milatuzumab in MM-bearing SCID mice

A, SCID mice were immunosuppressed by pretreatment with fludarabine phosphate and cyclophosphamide 3 d before injection of CAG cells. Treatments were given twice weekly for 4 wk, starting 1 d after injection of the CAG cells. SCID mice bearing CAG- (B) or KMS11- (C) were treated with bortezomib, milatuzumab, and mixtures of milatuzumab + bortezomib. Treatments for experiments shown in panels B and C were given as 2 i.p. doses/wk for 3 wk, initiated on day 5 after injection of tumor cells.

Figure 5. Therapeutic efficacy of bortezomib plus milatuzumab in MM-bearing SCID mice

A, SCID mice were immunosuppressed by pretreatment with fludarabine phosphate and cyclophosphamide 3 d before injection of CAG cells. Treatments were given twice weekly for 4 wk, starting 1 d after injection of the CAG cells. SCID mice bearing CAG- (B) or KMS11- (C) were treated with bortezomib, milatuzumab, and mixtures of milatuzumab + bortezomib. Treatments for experiments shown in panels B and C were given as 2 i.p. doses/wk for 3 wk, initiated on day 5 after injection of tumor cells.

Figure 5. Therapeutic efficacy of bortezomib plus milatuzumab in MM-bearing SCID mice

A, SCID mice were immunosuppressed by pretreatment with fludarabine phosphate and cyclophosphamide 3 d before injection of CAG cells. Treatments were given twice weekly for 4 wk, starting 1 d after injection of the CAG cells. SCID mice bearing CAG- (B) or KMS11- (C) were treated with bortezomib, milatuzumab, and mixtures of milatuzumab + bortezomib. Treatments for experiments shown in panels B and C were given as 2 i.p. doses/wk for 3 wk, initiated on day 5 after injection of tumor cells.