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Keywords:

  • multiple myeloma;
  • CD48;
  • monoclonal antibody;
  • antibody therapy;
  • xenograft model

Summary

  1. Top of page
  2. Summary
  3. Methods
  4. In vivo xenograft mouse models
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author Contributions
  9. References

Monoclonal antibody (mAb) drugs are desirable for the improvement of multiple myeloma (MM) treatment. In this study, we found for the first time that CD48 was highly expressed on MM plasma cells. In 22 out of 24 MM patients, CD48 was expressed on more than 90% of MM plasma cells at significantly higher levels than it was on normal lymphocytes and monocytes. CD48 was only weakly expressed on some CD34+ haematopoietic stem/progenitor cells, and not expressed on erythrocytes or platelets. We next examined whether CD48 could serve as a target antigen for mAb therapy against MM. A newly generated in-house anti-CD48 mAb induced mild antibody-dependent cell-mediated cytotoxicity and marked complement-dependent cytotoxicity against not only MM cell lines but also primary MM plasma cells in vitro. Administration of the anti-CD48 mAb significantly inhibited tumour growth in severe combined immunodeficient mice inoculated subcutaneously with MM cells. Furthermore, anti-CD48 mAb treatment inhibited growth of MM cells transplanted directly into murine bone marrow. Finally and importantly, we demonstrated that the anti-CD48 mAb did not damage normal CD34+ haematopoietic stem/progenitor cells. These results suggest that the anti-CD48 mAb has the potential to become an effective therapeutic mAb against MM.

The past decade has seen major advances in the treatment of multiple myeloma (MM) (Kyle & Rajkumar, 2004; Raab et al, 2009). Thalidomide (Singhal et al, 1999), bortezomib (Richardson et al, 2003) and lenalidomide (Dimopoulos et al, 2007) have all emerged as very effective drugs for MM. In addition, autologous haematopoietic stem cell transplantation has improved complete response rates and has prolonged median overall survival for MM patients (Attal et al, 1996). However, MM is still not a curable disease, and that novel therapeutic drugs continue to be needed to cure MM patients (Hideshima et al, 2007).

Monoclonal antibody (mAb) therapy has had a major effect on the therapy of haematological malignancies. An0074i-CD20 mAb (rituximab) has emerged as a therapeutic agent and has resulted in improved prognosis for B cell lymphoma (Maloney et al, 1994). Given that the mechanisms of cytotoxicity by mAb therapy are quite different from those of chemotherapeutic drugs, mAb therapy can work synergistically with chemotherapy. In fact, administration of rituximab in combination with CHOP chemotherapy (cyclophosphamide, doxorubicin, vincristine, prednisolone) for B cell lymphoma patients resulted in major improvement in patient prognosis (Coiffier et al, 2002). These findings suggest the importance of developing therapeutic mAbs against MM to improve the prognosis of MM patients. However, as yet there are no approved immunotherapeutic options for MM. A number of therapeutic mAbs against MM, such as anti-CD40 (Tai et al, 2005), CD38 (Stevenson et al, 1991; Ellis et al, 1995; van der Veer et al, 2010; de Weers et al, 2011), CD138 (Tassone et al, 2004a), CD56 (Tassone et al, 2004b), β2 microglobulin (Yang et al, 2006), FGFR3 (Hadari & Schlessinger, 2009) and CS-1 mAb (Hsi et al, 2008; Tai et al, 2008), have demonstrated significant anti-tumour activity in preclinical models in vivo, and some of them are now being tested in clinical trials (Ocio et al, 2008).

Ideally, targets for therapeutic mAbs should be specifically expressed on malignant cells but not on normal cells. In reality, the paucity of tumour-specific antigens has led to the development of mAbs directed against cell surface molecules characteristic of the lineage from which the malignant cells derive, as in the case with mAbs against CD20 (rituximab) (Maloney et al, 1994) for B cell lymphoma and CD52 (alemtuzumab) for chronic lymphocytic leukaemia (CLL) (Keating et al, 2002). As these mAbs are not specific for malignant cells and deplete normal haematopoietic cells together with malignant cells, haematological toxicity is inevitable. In particular, treatment with alemtuzumab, the mAb against CD52, which is broadly expressed by mature leucocytes, has reportedly led to severe immune-deficiency (Ghobrial et al, 2003; Kluin-Nelemans et al, 2008). However, alemtuzumab can still be tolerated if accompanied by appropriate prophylaxis for virus infection (O’Brien et al, 2008) and thus can benefit CLL patients (Moreton et al, 2005; Hillmen et al, 2007), because normal haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs), which are negative for CD52, can reestablish a normal haematopoietic system after discontinuation of the mAb treatment. These findings suggest that cell surface molecules expressed on plasma cells but not on HSCs and HPCs constitute potential candidates as targets for therapeutic mAbs against MM.

CD48 is a 47-kD glycophosphatidylinositol-linked glycoprotein that is expressed on mature lymphocytes and monocytes, but not on non-haematopoietic tissues (Vaughan et al, 1983). Because it was known that CD48 is expressed in lymphoid leukaemia and lymphoma, the potentials of anti-CD48 mAb as a therapeutic tool against these diseases were previously tested. Murine IgM anti-CD48 mAb was used in the treatment of patients with B cell-chronic lymphocytic leukaemia, and transient clinical responses were observed (Greenaway et al, 1994). Murine IgG2a and chimeric mAb against CD48 showed significant anti-tumour activity against Raji B cell leukaemia cell line (Sun et al, 1998, 2000). In the present study, we shed light on CD48 as a novel target for therapeutic mAb against MM. CD48 expression on MM or normal plasma cells had not been reported previously. We demonstrated that the cell surface protein CD48 is constitutively expressed on almost all MM plasma cells at significantly higher levels than on CD34+ HSCs/HPCs and normal leucocytes. Furthermore, we generated a novel anti-CD48 mAb that could induce cytotoxicity against MM cells in vitro. The in vivo anti-MM potency of the anti-CD48 mAb was tested in mouse xenograft models. We also examined whether the anti-CD48 mAb damages normal HSCs and HPCs.

Methods

  1. Top of page
  2. Summary
  3. Methods
  4. In vivo xenograft mouse models
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author Contributions
  9. References

Patient samples

Bone marrow (BM) cells from MM patients were collected from iliac bone after informed consent had been obtained. Mononuclear cells were purified using Ficoll Paque (GE Healthcare, Piscataway, NJ, USA), and subjected to analyses. The research was approved by the institutional review boards of Osaka University School of Medicine and of all the hospitals participating in this study.

Cell lines

OPM2 cells (Katagiri et al, 1985) were kindly gifted from Yuzuru Kanakura (Osaka University, Japan). MM1S cells were kindly gifted from Hiroshi Yasui (Sapporo Medical College, Japan). RPMI8226 cells and U266 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Flow cytometry and cell sorting

Single cell suspensions from BM were stained with CD48-fluorescein isothicyanate (FITC) (MEM-102; Biolegend, San Jose, CA, USA or ebio156-4H9; eBioscience, San Jose, CA, USA) or CD52-FITC (eBioscience), and other fluorochrome-conjugated mAbs. Analysis and cell sorting were performed on FACS Aria (BD Biosciences, San Jose, CA, USA). Annexin V-allophycocyanin (APC) (Biolegend) was used to identify apoptotic cells.

Generation of anti-CD48 mAbs

BaF3 cells expressing human CD48 (hCD48) cDNA (Toyobo, Tokyo, Japan) were generated by retrovirus transduction. Six-week-old Balb/c mice (CLEA Japan, Tokyo, Japan) were immunized by footpad injection of hCD48-expressing BaF3 cells. Lymphocytes from popliteal lymph nodes were fused with SP2/0 mouse myeloma cells by using polyethylene glycol (Roche Applied Science, Basel, Switzerland). To identify anti-hCD48 mAbs secreting hybridoma clones, NIH3T3 cells expressing hCD48-ires-GFP were stained first with hybridoma supernatants, then with phycoerythrin (PE)-conjugated anti-mouse IgG antibody (eBioscience), and analysed by fluorescence-activated cell sorting (FACS).

In vitro cytotoxicity assay

Antibody-dependent cell-mediated cytotoxicity (ADCC) was measured by a 51Cr release assay. Target cells labelled with 51Cr were incubated with either anti-CD48 mAb (1B4) or isotype control (10 μg/ml) at 37°C for 15 min. Splenocytes from severe combined immunodeficient (SCID) mice (CLEA Japan, Inc., Tokyo, Japan) were then added as effector cells, and incubated at 37°C for 4 h. Finally, the 51Cr released in the supernatants was counted with a gamma counter. Complement-dependent cytotoxicity (CDC) was also measured with a 51Cr-release assay. The aliquots of the labelled cells were incubated with either anti-CD48 mAb (1B4) or isotype control (10 μg/ml) for 15 min. Cells were then incubated in RPMI 1640 medium supplemented with 25% baby rabbit complement (Cedarene, Burlington, Canada) for 1·5 h at 37°C. The percentage of specific lysis was calculated according to the following formula: percentage of specific lysis = (E − S)/(M − S) × 100, where E is the experimental release, S is the spontaneous release, and M is the maximum release by 1% Triton X-100. Purification of CD138+ plasma cells from BM cells from MM patients or CD8+ T cells from peripheral blood of healthy individuals was performed using CD138 micro beads (Miltenyi Biotec, Gladbach, Germany) or IMag human CD8 T lymphocyte enrichment set (BD Bioscience), according to the manufacturers’ instructions.

In vivo xenograft mouse models

  1. Top of page
  2. Summary
  3. Methods
  4. In vivo xenograft mouse models
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author Contributions
  9. References

Six-to-eight-week-old female SCID mice (CLEA Japan, Inc.), Rag2−/−γc−/− mice (Goldman et al, 1998) or non-obese diabetic (NOD)/SCID mice (CLEA Japan, Inc.) were subcutaneously inoculated with 5 × 106 OPM2 cells into the lower left flank. Tumour volume was calculated by means of caliper measurements using the following formula: L × W × H/2, where L (length) is the longest side of the tumour in the plane of the animal’s flank, W (width) is the longest measurement perpendicular to the length and in the same plane, and H (height) is taken at the highest point perpendicular to the flank of the animal. Intra-BM transplantation was performed as previously reported (Wang et al, 2003) with OPM2 MM cells. Eight-week-old female Rag2−/−γc−/− mice irradiated with 200cGy 4–24 h before transplantation were injected with OPM2 MM cells into the left tibia.

Colony forming assay

Methylcellulose culture assays were performed in Methocult H4334 (Stem Cell Technologies, Vancouver, BC, Canada). Colonies were counted and scored on culture day 14.

Statistical analysis

Student’s t-test was used to determine statistical significance for all the analyses in this study. Differences were defined as statistically significant when P < 0·05.

Results

  1. Top of page
  2. Summary
  3. Methods
  4. In vivo xenograft mouse models
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author Contributions
  9. References

CD48 is constitutively expressed at high levels on almost all MM plasma cells, but at low levels on HSCs and HPCs

CD48 protein expression on CD38++ CD138+ MM plasma cells and CD34+ HSCs/HPCs was analysed by means of FACS in BM of MM patients (Fig 1A). CD48 was highly expressed on all CD38++CD138+ MM plasma cells, but only weakly on CD34+CD38 HSCs and CD34+CD38+ HPCs. CD48 was highly expressed on more than 90% of the CD38++ plasma cells in 22 of the 24 MM patients whose BM was analysed (Fig 1B). In BM of MM patients, CD48++ cells overlapped with CD38++ plasma cells in most cases (Fig 1C).

image

Figure 1.  CD48 is constitutively expressed on MM plasma cells at much higher levels than on HSCs and HPCs. (A) Flow cytometric analysis of CD48 expression on CD38++ CD138+ MM plasma cells, CD34+CD38+ HPCs and CD34+CD38 HSCs in BM of an MM patient (UPN1). (B) Percentages of CD48+ cells in the CD38++ CD138+ plasma cell populations from MM patients. Each bar represents a single patient. UPN: unique patient number. (C) FACS analyses of CD38 and CD48 expression on BM cells from MM patients. Gates for CD38++CD48++ plasma cells are shown.

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CD48 expression levels on MM plasma cells were significantly higher than on normal leucocytes

In normal peripheral blood (PB), CD48 was expressed on CD19+ B cells, CD3+ T cells and SSCloCD13+ monocytes. The expression levels of CD48 were compared between MM plasma cells and normal PB leucocytes by FACS (Fig 2A). CD48 expression levels on plasma cells were higher than those on lymphocytes or monocytes. Mean fluorescent intensities of MM plasma cells [1574 ± 727 (mean ± standard error (SE), n = 3)] were significantly (P < 0·05) higher than those on normal T, B, and monocytes [137 ± 32, 197 ± 69, 291 ± 156, respectively (n = 3)] (Fig 2B). CD48 expression levels on granulocytes were lower than on lymphocytes and monocytes. Importantly, CD48 was not expressed on Glycophorin-A+ erythrocytes or on CD41a+ platelets.

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Figure 2.  CD48 expression levels on MM plasma cells are much higher than those on normal leucocytes. (A) Flow cytometric analysis of CD48 expression on CD38++CD138+ plasma cells from a MM patient (UPN21), and CD3+T cells, CD19+B cells, CD13+SSClo monocytes (Mo), CD13+SSChigh granulocytes (Gr), CD235+ Red blood cells (RBC) or CD41+platelets (Plt) of peripheral blood (PB) cells from a healthy donor. Gating strategies for the subpopulations are shown on the left. (B) Mean fluorescent intensities (MFI) of CD48-fluorescein isothiocyanate (FITC) for plasma cells from MM patients, and T cells, B cells and monocytes from peripheral blood of healthy donors (n = 4). Bars represent standard errors (SE). *P < 0·05. (C) Flow cytometric analysis of CD48 and CD52 expression in subpopulations of BM cells from an MM patient. Results of analysis of a representative MM patient (UPN5) are shown.

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The expression profiles of CD48 on BM cells from MM patients were compared with those of CD52, which is the target of alemtuzumab and known to be expressed extensively on mature leucocytes. Three MM samples were analysed and a representative finding is shown in Fig 2C. In all cases, both CD48 and CD52 were expressed on CD19+ B cells, CD3+ T cells, and CD13+ myeloid cells, while no or only slight expression of the two molecules was detected on CD34+ HSCs and HPCs. All CD38++ MM plasma cells highly expressed CD48 but not CD52.

Novel anti-CD48 mAb can induce cytotoxicity against MM cells in vitro

Anti-human CD48 (hCD48) mAbs were prepared by immunizing Balb/c mice with hCD48-expressing mouse B cell line BaF3. Of three isolated hybridoma clones secreting anti-hCD48 mAbs, one with IgG2a isotype (clone 1B4) was subjected to further analysis, because the IgG2a isotype is reportedly preferable for ADCC and inhibiting tumour growth in xenograft models (Herlyn & Koprowski, 1982).

CD48 was expressed on all MM cell lines tested (OPM2, U266, MM1S, and RPMI8226) (Fig 3A). In vitro ADCC induced by the anti-CD48 mAb 1B4 was examined by using OPM2 and U266 as the targets and splenocytes of an SCID mouse as effector cells. ADCC induced by the anti-CD48 mAb against MM cell lines was weak but certainly detectable. By incubation with the anti-CD48 mAb and effector cells (E:T ratio = 50:1), 5·6 ± 0·8% of OPM2 and 10·8 ± 2·1% of U266 MM cells were killed, while background killing with the isotype control accounted for 2·5 ± 0·9% and 1·5 ± 1·6%, respectively (Fig 3B). The difference in cytotoxicity between the isotype control and the anti-CD48 mAb was statistically significant (P < 0·05). CDC induced by the anti-CD48 mAb against MM cell lines was very strong. By incubation with the anti-CD48 mAb and complement, 49·9 ± 0·7% of OPM2, 51·0 ± 7·3% of U266, 73·7 ± 1·9% of MM1S and 83·2 ± 12·6% of RPMI8226 cells were killed, while killing with the isotype control accounted for significantly (P < 0·05) lower percentages (Fig 3C). To examine whether lack of complement inhibitory protein was the reason for the high sensitivity of MM cell lines to the anti-CD48 mAb-induced CDC, expressions of CD55 and CD59 were examined. CD55/59 was variably expressed on all MM cell lines tested (Fig 3D). All MM cell lines were sensitive to the anti-CD48 mAb-induced CDC independently of the expression levels of CD55 or CD59. Thus, loss of CD55 or CD59 is unlikely to be the reason for the high level of CDC activity of the anti-CD48 mAb against MM cell lines. We also performed a CDC assay with the anti-CD48 mAb against plasma cells purified from three MM patients, all of whom were resistant to chemotherapy and/or bortezomib. Anti-CD48 mAb-induced CDC killed 73·0 ± 10·0%, 69·3 ± 6·4% and 67·3 ± 5·9% of the MM plasma cells, while killing with the isotype control accounted for lower percentages (Fig 3E). Direct cytotoxic effects of the anti-CD48 mAb on MM cells without effector cells or complement were also examined. OPM2 MM cells were cultured in the presence of the anti-CD48mAb or isotype control for 48 h, followed by counting of apoptotic cells by means of Annexin V/propidium iodide staining. No significant difference in the number of Annexin V+ cell was observed between treatment with anti-CD48mAb and that with the isotype control (Fig 3F).

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Figure 3.  A new anti-CD48 mAb, 1B4, induces cytotoxicity against MM cells in vitro. (A) Flow cytometric analysis of CD48 expression on OPM2, U266, MM1S, and RPMI8226 MM cell lines. (B, C) Analysis of ADCC (B) and CDC (C) induced by administration of a new anti-CD48 mAb (1B4) using MM cell lines as target cells. The mean plus standard error (SE) of triplicate wells from one representative of three experiments is shown. *P < 0·05. E:T ratio in the ADCC analysis was 50:1. (D) Expression of CD55 and CD59 on MM cell lines. (E) 1B4-induced CDC against FACS-sorted MM plasma cells from patient samples. Error bars represent SEs of triplicate wells. *P < 0·05. (F) Annexin V/propidium iodide (PI) staining of OPM2 cells after incubation with the anti-CD48 mAb or isotype control, but without effector cells or complement, for 48 h.

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In vivo anti-tumour activity of the anti-CD48 mAb in MM xenograft models

In vivo anti-MM activity of the anti-CD48 mAb 1B4 was tested in a subcutaneous MM tumour model, which has been used in many studies for testing the efficacy of mAb against MM (Tassone et al, 2004a,b; Yang et al, 2006; Tai et al, 2008). OPM2 MM cells were subcutaneously inoculated into SCID mice, which lacked T and B cells but had natural killer (NK) cell activity. Two weeks after tumour inoculation, mice with tumours approximately 30mm3 in size were selected and treated three times a week with either the anti-CD48 mAb or control mouse IgG antibody (10 mg/kg body weight) for 2 weeks (Fig 4A). Tumour growth was significantly (P < 0·05) inhibited in the mice treated with the anti-CD48 mAb, while in the mice treated with the control IgG antibody the tumour volume increased exponentially (Fig 4B). After 2 weeks of treatment, the average tumour volume was 136 ± 63 mm3 in the anti-CD48 mAb-treated group (n = 5), but 897 ± 340 mm3 in the control IgG-treated group (n = 5). Rag2−/−γc−/− mice that lacked T, B and NK cells were also used as recipients of subcutaneous OPM2 xenografts. Ten days after tumour inoculation, mice with tumours approximately 100 mm3 in size were selected and treated three times a week with either the anti-CD48 mAb or the control mouse IgG antibody (10 mg/kg body weight) for 2 weeks. After 2 weeks of treatment, the average tumour volume was 380 ± 152 mm3 in the anti-CD48 mAb-treated group (n = 5), but 3567 ± 857 mm3 in the control IgG-treated group (n = 5) (Fig 4B). These results clearly show that the anti-CD48 mAb significantly (P < 0·05) inhibited growth of MM in vivo in the absence of NK cells. To examine whether CDC is a major mechanism for MM growth inhibition by 1B4 mAb in vivo, OPM2 MM cells were also first inoculated in to NOD/SCID mice, which lacked not only lymphocyte but also complement activity, and then treated with 1B4 mAb. The inhibitory effect of 1B4 mAb was more prominent in SCID mice than in NOD/SCID mice (Fig 4B), suggesting that CDC is a major inhibitory mechanism. However, it should be noted that 1B4 mAb also significantly (P < 0·05) reduced tumour growth in NOD/SCID mice, indicating that mechanisms other than CDC may be involved. The anti-MM effect of the anti-CD48mAb was found to be dependent on the dosage of mAb (Fig 4C). Anti-tumour effect of the anti-CD48 mAb was also observed when MM1S MM cells were used as targets (Fig 4D). Taken together, these findings demonstrate that the anti-CD48 mAb is highly active for controlling growth of MM in murine xenograft models.

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Figure 4. In vivo anti-tumour activity of the anti-CD48 mAb in subcutaneous MM xenograft models (A) Experimental design for anti-CD48 mAb treatment of severe combined immunodeficient (SCID) mice subcutaneously inoculated with MM cells. Mice with tumours were treated with the anti-CD48 mAb or control mouse IgG. (B) Tumour volumes of OPM2 cells in SCID, Rag2−/−γc−/−, or non-obese diabetic (NOD)/SCID mice treated with the anti-CD48mAb or control mouse IgG (n = 5 for each type of mouse). Bars represent SE and asterisks show significant differences in tumour size (P < 0·05). (C) Rag2−/−γc−/− mice inoculated with OPM2 MM cells were treated with different doses of the anti-CD48mAb (D) Tumour volumes of MM1S cells in SCID mice treated with either the anti-CD48 mAb or control IgG.

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In vivo effect of the anti-CD48 mAb against MM cells inoculated into BM microenvironment

Since MM cells usually grow in BM but not in skin, the effect of the anti-CD48 mAb against MM cells that resided in a BM microenvironment was examined. OPM2 MM cells (3 × 105 cells) were directly injected into the left tibia of Rag2−/−γc−/− mice. Ten days after the injection, the chimerisms of human CD38 (hCD38)+ OPM2 MM cells in BM of the left tibias were examined. This was followed by intravenous injection every other day of either the anti-CD48 mAb or control mouse IgG antibody at a dose of 5 mg/kg body weight. After the third mAb injection, the mice were sacrificed and examined for the chimerisms of hCD38+ OPM2 MM cells in BM of the left tibias (Fig 5A). Human CD38+ MM cells had decreased in three out of four mice treated with the anti-CD48 mAb, whereas MM cells in BM had expanded exponentially in all the mice treated with mouse IgG (n = 4) (Fig 5B, C). To exclude the possibility that MM cells had migrated to other sites from the injected bone, we investigated whether CD38+ OPM2 MM cells could be detected in BM of the left femurs, right femurs and tibias, but no hCD38+ MM cells were found. These results show that the anti-CD48 mAb treatment can inhibit growth of MM cells engrafted in a BM microenvironment.

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Figure 5. In vivo effect of the anti-CD48 mAb against MM cells inoculated in a BM microenvironment. (A) Experimental design for anti-CD48 mAb treatment of Rag2−/−γc−/− mice transplanted with OPM2 MM cells into bone marrow (BM) of the left tibia. iBMT denotes intra-bone marrow transplantation. Mice were intravenously inoculated with the anti-CD48 mAb (n = 4) or control IgG (n = 4). (B) Representative results of FACS analyses of BM cells from the left tibias on day 10 (pre-treatment) and day 16 (post-treatment). Human CD38 (hCD38)+ cells represent OPM2 MM cells in mouse BM. (C) Chimerisms of hCD38+ OPM2 MM cells in BM of the mice pre- and post-mAb treatment. Each dot corresponds to a treated mouse.

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Anti-CD48 mAb treatment did not induce CDC against CD34+ HSCs/HPCs

The cytotoxic effect of the anti-CD48 mAb 1B4 against normal lymphocytes was investigated. In vitro CDC induced by the anti-CD48 mAb against CD8+ T cells from a healthy individual was compared with that against the OPM2 MM cell line or purified MM plasma cells from a patient. Anti-CD48 mAb-induced CDC killed 74·2 ± 2·7% of the OPM2 cells, 77·8 ± 2·6% of patient plasma cells and 60·4 ± 0·1% of CD8+ T cells, while background killing with isotype control antibody accounted for lower percentages (1·3 ± 0·0%, 36·2 ± 4·5% and 0·3 ± 0·3%) (Fig 6A). CDC against normal CD19+ B cells was also tested. After incubation with the anti-CD48mAb and complement, 73·0 ± 5·8% of patient plasma cells and 67·8 ± 14·6% of CD19+ B cells were killed, while background killing rates with isotype control antibody were lower (36·7 ± 14·3% and 44·0 ± 10·6%) (Fig 6A). These results indicate that normal T and B cells are also sensitive to in vitro CDC induced by the anti-CD48 mAb.

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Figure 6.  The anti-CD48 mAb does not damage CD34+ HSCs/HPCs. (A) (Left) Analysis of CDC induced by the anti-CD48 mAb (1B4) using the OPM2 MM cell line, CD138+ MM plasma cells form a MM patient (UPN4) and CD8+T cells from a healthy donor as target cells. (Right) A CDC assay using MM plasma cells from a MM patient (UPN7) and CD19+B cells from a healthy donor. Shown is mean plus SE of triplicate wells. *P < 0·05. (B) CD34+ cells from cord blood were incubated with baby rabbit complement and either the anti-CD48 mAb or isotype control, and then subjected to a colony-forming assay. Mean numbers of colonies produced from 100 CD34+ cells are shown with error bars representing SEs of triplicate plates from one representative of two independent experiments. CFU-GEMM, mixed lineage colony-forming units (CFU); CFU-GM, granulocyte-macrophage CFU; BFU-E, erythroid burst-forming units; N.S.: no significant differences. (C) Unfractionated BM cells from an MM patient were incubated with phosphate-buffered saline (PBS), isotype control or the anti-CD48 mAb in medium supplemented with complement and then subjected to FACS analysis. Numbers represent percentages of gated cells among all cells analysed. Note that the percentages of CD38++ MM plasma cells, but not of CD34+ HSCs/HPCs, decreased significantly by anti-CD48mAb treatment. Representative results from two independent experiments are shown.

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Weak CD48 expression was detected on CD34+ HSCs/HPCs (Fig 1A). To test whether CD48 expression on CD34+ HPCs caused CDC as a result of anti-CD48 mAb administration, FACS-sorted CD34+ HPCs from cord blood were incubated with the complement and either the anti-CD48 mAb or isotype control (10 μg/ml) for 1·5 h, and then subjected to haematopoietic colony-forming assays. The numbers of mixed lineage colony-forming units (CFU-GEMM), granulocyte-macrophage CFU (CFU-GM) and erythroid burst-forming units (BFU-E) colonies formed from CD34+ cells treated with the anti-CD48 mAb were similar to those formed from CD34+ cells treated with isotype control (Fig 6B), indicating that anti-CD48 mAb did not induce CDC against CD34+ HPCs. Furthermore, CDC induced by the anti-CD48 mAb against un-fractionated BM cells from MM patients was examined (Fig 6C). After incubation with the anti-CD48 mAb (10 μg/ml) and complement for 1·5 h, CD38++CD138+ MM plasma cells, but not CD34+ HSCs/HPCs, significantly decreased. This demonstrated that anti-CD48 mAb could selectively kill MM plasma cells, but not normal HSCs/HPCs by induction of CDC.

Discussion

  1. Top of page
  2. Summary
  3. Methods
  4. In vivo xenograft mouse models
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author Contributions
  9. References

The present study has provided evidence that CD48 is highly expressed on almost all plasma cells in the majority of MM patients, and that a new anti-CD48 mAb, 1B4, can induce significant cytotoxic effects against MM cells both in vitro and in vivo. CD48 expression levels on CD34+ HSCs and HPCs were much lower than those on MM plasma cells and the anti-CD48mAb did not cause cytotoxicity. CD48 is not expressed on erythrocytes, platelets or any non-haematopoietic tissues (Vaughan et al, 1983), suggesting that anaemia, thrombocytopenia and tissue toxicity are not major concerns. CD48++ cells in BM of MM patients overlapped with cells expressing high levels of CD38, which is a promising candidate antigen for therapeutic mAb against MM (Stevenson et al, 1991; Ellis et al, 1995; Stevenson, 2006; Tai et al, 2009; van der Veer et al, 2011; de Weers et al, 2011), while CD34+ HPCs were CD38+CD48low/−. Targets of clinically effective mAbs against haematological malignancies, such as CD20 and CD52, are constitutively expressed on entire target malignant cells at high levels, but not expressed on non-haematopoietic tissues. Similarly, CD48 is expressed on almost all MM plasma cells, but not on non-haematopoietic tissues. It has been reported that a soluble form of CD48 exists, but its concentration in serum was as low as that of soluble CD20 or CD52 (Smith et al, 1997; Giles et al, 2003). Taken together, these findings indicate that CD48 is a good candidate for a therapeutic target against MM.

Sintes et al (2008), in a study that used the anti-CD48 mAb clone 99A, which has thus far not been available to us, showed that CD48 was expressed on normal human CD34+ HSCs and HPCs, while expression levels of CD48 on CD34+ cells were unclear. We were able to show clearly in the present study, that CD48 expression levels on CD34+ HSCs and HPCs were much lower than those on MM plasma cells, and confirmed these results by staining with three different clones of anti-CD48 mAb (HuLy-m3, MEM-102, ebio156-4H9). Importantly, we demonstrated that the anti-CD48 mAb did not induce CDC against CD34+ HSCs or HPCs, suggesting that faint CD48 expression on CD34+ HSCs and HPCs is not a major obstacle to the development of anti-CD48 mAb as a therapeutic mAb.

The in vivo anti-MM effects of the anti-CD48 mAb were remarkable in subcutaneous MM tumour models. Furthermore, we demonstrated that the anti-CD48 mAb treatment was effective against MM cells engrafted in a BM microenvironment. The inhibitory effect on MM was much more prominent in SCID mice than NOD/SCID mice, suggesting that CDC is likely to be a major mechanism of these more prominent anti-MM effects. In addition, the fact that 1B4 mAb was still effective against MM cells in NOD/SCID mice suggest that other mechanisms may be involved. While ADCC induced by the mouse anti-CD48 mAb in vitro was not very strong, the potential for inducing ADCC against MM plasma cells will need to be assessed after the humanized mAb is developed. In this regard, it was reported that a chimeric anti-CD48 mAb could induce significant ADCC against Raji B cell lymphoma cells (Sun et al, 2000).

A major concern regarding CD48 as a therapeutic target is its broad expression on normal lymphocytes and monocytes, which may cause severe cytopenia and immunosupression when anti-CD48 mAb is used as a therapeutic drug. In fact, normal T and B cells are also sensitive to in vitro CDC induced by the anti-CD48mAb. Normal mature lymphocytes and monocytes may be depleted together with MM cells as a result of anti-CD48 mAb treatment, whereas normal CD34+ HSCs or HPCs are not damaged and the normal haematopoietic system is re-established after discontinuation of the mAb treatment. The fact that a mAb against CD52, which is also widely expressed on normal leucocytes, has been used in clinics suggests that an antigen that is widely expressed on normal leucocytes still has the potential to serve as a target of therapeutic mAb. However, it has been reported that alemtuzumab causes pancytopenia (Keating et al, 2002; Enblad et al, 2004) as well as severe virus infections (Keating et al, 2002; Ghobrial et al, 2003; Herbert et al, 2003; Kluin-Nelemans et al, 2008), while immunosuppression induced by alemtuzumab can be tolerated if accompanied by appropriate prophylaxis for virus infection (Hillmen et al, 2007; Gribben & Hallek, 2009; Stilgenbauer et al, 2009). The potential haematological toxicity of anti-CD48mAb should therefore be very carefully tested at the pre-clinical stage.

Anti-CD48mAb may not be suitable for long-term maintenance therapy because of haematological toxicities. For induction therapy, however, we may be able to take advantage of the broad and high CD48 expression on all MM plasma cells for the total eradication of MM plasma cells. In addition, consolidation therapy with anti-CD48 mAb may also benefit MM patients. Recent progress in MM therapy has resulted in complete response or good partial response in many patients (Palumbo & Anderson, 2011). However, these patients are rarely cured because a sub-fraction of MM cells remains resistant to the therapies currently in use. Anti-CD48 mAb may have the potential to eradicate such resistant MM cells. These indicate that anti-CD48 mAb may well turn out to be an effective tool for the survival improvement of MM patients.

Acknowledgements

  1. Top of page
  2. Summary
  3. Methods
  4. In vivo xenograft mouse models
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author Contributions
  9. References

We wish to thank Irving L Weissman (Stanford University) for his kind gifts of Rag2−/−γc−/− mice and the SP2/0 myeloma cell line, Yuzuru Kanakura (Osaka University, Japan), Hiroshi Yasui (Sapporo Medical College, Japan) and Teru Hideshima (Dana Farber Cancer Institute) for their kind gifts of MM cell lines, Eui Ho Kim and Masaki Murakami for collecting MM samples, and Takafumi Kimura (Kyoto University, Japan) for technical advice. This work was supported by the Knowledge Cluster Initiative (stage-II) established by the Ministry of Education, Culture, Sports, Science and Technology/Senri Life Science Foundation, by the Sagawa Foundation for Promotion of Cancer Research and the Uehara Memorial Foundation (to N.H.).

Author Contributions

  1. Top of page
  2. Summary
  3. Methods
  4. In vivo xenograft mouse models
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Author Contributions
  9. References

NH designed the research study, performed research, analysed data and wrote the paper. HI, AM, YA, YF, SK, YM, HN, MK, TY, SF, HT, TN, SN, AT, SI, MH, YO and YO performed the research. HS wrote the paper.

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  8. Author Contributions
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