Thymoglobulin targets multiple plasma cell antigens and has in vitro ...

Leukemia (2006) 20, 1863–1869 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu

ORIGINAL ARTICLE Thymoglobulin targets multiple plasma cell antigens and has in vitro and in vivo activity in multiple myeloma MM Timm, TK Kimlinger, JL Haug, MP Kline, PR Greipp, SV Rajkumar and SK Kumar Department of Internal Medicine, Division of Hematology, Mayo Clinic, Rochester, MN, USA

Multiple myeloma is characterized by the proliferation of clonal plasma cells that have a heterogeneous expression of various cell surface markers, precluding successful use of monoclonal antibodies for therapeutic targeting of the tumor cell. Thymoglobulin (rabbit-derived polyclonal anti-thymocyte globulin), by virtue of its method of preparation, contains antibodies against several B-cell and plasma cell antigens and offers an attractive option for immunotherapy of myeloma. Here, we demonstrate potent anti-myeloma activity of the rabbit anti-thymocyte globulin preparation Thymoglobulin in vitro and in vivo in an animal model of myeloma. Thymoglobulin was able to induce dose- and time-dependent apoptosis of several myeloma cell lines, including those resistant to conventional anti-myeloma agents. Importantly, the anti-myeloma activity was preserved even when myeloma cells were grown with different cytokines demonstrating the ability to overcome microenvironmentmediated resistance. Thymoglobulin induced apoptosis of freshly isolated primary myeloma cells from patients. Using a competitive flow cytometric analysis, we were able to identify the potential antigen targets for Thymoglobulin preparation. Finally, in a plasmacytoma mouse model of myeloma, Thymoglobulin delayed the tumor growth in a dose-dependent manner providing convincing evidence for continued evaluation of this agent in the clinic in patients with myeloma, either alone or in combination with other agents. Leukemia (2006) 20, 1863–1869. doi:10.1038/sj.leu.2404359; published online 17 August 2006 Keywords: multiple myeloma; apoptosis; plasma cell antigens; rabbit anti-thymocyte globulin; polyclonal antibody

Introduction Multiple myeloma (MM) is a plasma cell malignancy that affects over 16 000 individuals in United States each year and results in over 12 000 related deaths during the same period. The median survival for the individuals affected by myeloma has been estimated to be 3–5 years from initiation of therapy. Although new therapies have improved the response rates and possibly the overall survival of these patients, it remains incurable with the current approaches. High-dose therapy and stem cell transplantation, while improving survival compared to combination chemotherapy still fails to cure the disease or provide lasting remissions. Better understanding of the disease biology over the past several years is starting to translate into more effective therapies. Novel approaches, particularly combinations of active agents, are urgently needed to attack this disease and improve the outcome of patients with myeloma. Targeting cell surface antigens using monoclonal antibodies represents a very attractive approach for treatment of malignancies Correspondence: Dr SK Kumar, Department of Internal Medicine, Division of Hematology, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA. E-mail: [email protected] Received 25 May 2006; accepted 15 June 2006; published online 17 August 2006

that has been borne out by the success of Rituximab in patients with non-Hodgkin’s lymphoma. Monoclonal antibodies, especially humanized antibodies, are usually very well tolerated with very minimal side effects as has been the experience with Rituximab. The ability to target tumor cells with minimal bystander effect is of particular advantage with this approach. Unfortunately, myeloma cells represent a heterogeneous population in terms of the expression of various cell surface antigens. Myeloma cells express various surface antigens, including CD38, CD138, CD45, adhesion molecules such as VLA4, and other lymphocyte antigens such as CD52 depending on the stage of maturation with considerable variation from patient to patient. This heterogeneity makes use of monoclonal antibodies in the treatment of myeloma, particularly challenging, as has been demonstrated by the lack of benefit for therapies targeting any one of these antigens. This calls for the use of antibody preparations that can target multiple antigens at the same time, possibly a polyclonal preparation. Rabbit-derived anti-thymocyte globulin (rATG, Thymoglobulin; Genzyme/Sangstat) is prepared by immunizing rabbits with cells derived from fragments of thymus glands. Thymoglobulin has been used extensively in the setting of solid organ transplantation as well as in the hematopoietic stem cell transplant for immunosuppression. Additionally, it has also been used for the treatment of refractory graft-versus-host disease following allogeneic stem cell transplants. Unlike the earlier monoclonal preparations directed against specific T-cell antigens, Thymoglobulin is a polyclonal preparation containing antibodies to a variety of T- and B-cell antigens. Studies have shown that the thymus, in addition to the thymocytes which usually express the T-cell antigens, also have 5–10% plasma cells. This results in a preparation that also contains antibodies against plasma cell/B-cell antigens. This was first noticed when patients undergoing solid organ transplants receiving Thymoglobulin had a significantly decreased risk of Epstein–Barr virus (EBV)-related lymphoproliferative disorder, which was determined to be related to the presence of antibodies in the preparation directed against the B-cell antigens, leading to their destruction. Also noted in the renal transplant recipients was the depletion of plasma cells and a decrease in the immunoglobulin levels. Previous studies using the polyclonal thymocyte preparations had demonstrated their ability to deplete B cells as well. Given this scenario, Thymoglobulin has been evaluated in vitro in the setting of myeloma.

Materials and methods

MM cell lines Dexamethasone sensitive (MM1.S) and resistant (MM1.R) human MM cell lines were obtained from Dr Steven Rosen (Northwestern University, Chicago, IL, USA). Doxorubicin

Thymoglobulin in multiple myeloma MM Timm et al

1864 resistant (Dox 40)-, mitoxantrone resistant (MR20)- and melphalan resistant (LR5)- Rosewell Park Memorial Institute medium (RPMI) 8226 human MM cells and sensitive RPMI 8226 cell lines were obtained from Dr William Dalton (Moffitt Cancer Center, Tampa, FL, USA). OPM1 and OPM2 cell lines were obtained from Dr Lief Bergsagel (Mayo Clinic, AZ, USA) and the U266 cell line was obtained from American Type Culture Collection (Rockville, MD, USA). All cell lines were cultured in RPMI 1640 media (Sigma Chemical, St Louis, MO, USA) that contained 10% fetal bovine serum (FBS), 2 mmol/l L-glutamine (GIBCO, Grand Island, NY, USA), 100 U/ml penicillin and 100 mg/ml streptomycin.

Thymoglobulin Thymoglobulin was supplied by Genzyme Corporation (Cambridge, MA, USA) as a sterile, non-pyrogenic, freeze-dried powder to be reconstituted with sterile water for injection, USP, for intravenous administration. Each product package contains two 7 ml vials: vial 1 containing freeze-dried Thymoglobulin (contains 25 mg anti-thymocyte rabbit immunoglobulin, glycine, mannitol and sodium chloride), and vial 2 containing sterile water for injection, USP (5 ml). The 5 ml of diluent is added slowly to the powder using sterile technique. The vial is gently rotated until all powder is completely dissolved. For animal studies, the required dose was diluted in sterile saline for injection and mixed gently before administration.

Cell proliferation and cytotoxicity assays

Myeloma cells (2–3 104 cells/well) were incubated in 96-well culture plates (Costar, Cambridge, MA, USA) in media alone or with Thymoglobulin for 48 h at 371C. To evaluate the effect of growth factors, recombinant interleukin-6 (IL-6) (10 ng/ml), insulin-like growth factor (IGF)-1 (50 ng/ml) or vascular endothelial growth factor (VEGF) (25 ng/ml) were added. The cellular cytotoxicity of Thymoglobulin against myeloma cells was assessed using colorimetric assays with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT; Chemicon International Inc., Temecula, CA, USA). Cells from 48-h cultures were pulsed with 5 mg/ml MTT (10 ml/well); 96-well plates were incubated at 371C for 4 h, followed by 100 ml isopropanol containing 0.04 N HCl. Absorbance at a wavelength of 570 nm (with correction using readings at 630 nm) were measured on a spectrophotometer (Molecular Devices Corp., Sunnyvale, CA, USA). Background absorbance (obtained from wells containing media alone) was subtracted from the observed data; the mean of triplicate readings was used.

Detection of apoptosis in myeloma cell lines and patient myeloma cells Apoptosis of myeloma cells was detected by staining of the cells with Annexin fluorescein isothiocyanate (FITC) and propidium iodide (PI). Briefly, myeloma cells (1 106) were cultured in media alone, or with varying concentrations of Thymoglobulin, and harvested every 6 h for 30 h. Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and resuspended (1 106 cells/ml) in Annexin-binding buffer (10 mmol/l HEPES (pH 7.4), 140 mmol/l NaCl and 2.5 mmol/l CaCl2). Myeloma cells (1 105) were incubated with Annexin V-FITC (5 ml) and PI (5 mg/ml) for 15 min at room temperature. The apoptotic fraction was identified as Annexin V-positive and PI-negative cells, Leukemia

analyzed using Cellquest software (BD Biosciences, San Jose, CA, USA) on a FACSCalibur flow cytometer. For evaluation of patient myeloma cells, bone marrow aspirates were subjected to ACK lysis and mononuclear cells were separated. Myeloma cells were separated by positive selection using CD138 coated magnetic beads in a Robosep system. The tumor cells were suspended in RPMI 1640 media containing 20% FBS, 2 mmol/l L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin, placed in 24-well plates and Thymoglobulin added at different concentrations. Patient cells were cultured for 48 h, harvested and washed twice with PBS, and stained with FITC-conjugated CD45 monoclonal antibody and 7-AAD for identification of apoptotic cells. The cells were then analyzed using DIVA software on an FACSCanto flow cytometer (BD Biosciences).

Determination of plasma cell targets for Thymoglobulin MM1.S, OPM2, U266 and RPMI 8226 cells were plated into sixwell plates at a concentration of 10 million cells/well, with or with out Thymoglobulin added at a concentration of 1 mg/ml. After 24 h, the cells were harvested and washed once with PBS. Three tubes were set up with each of the following markers: g1 fitc, g1 pe, cd45 fitc, cd20 fitc, cd27 fitc, cd52 fitc, cd40 fitc, cd38 pe, cd22 pe, cd19 pe, cd138 pe, cd184, cd56, cd49d, cd126, cd154, and anti-rabbit fitc. Into tube 2 was also aliquoted 20 ml of ATG (1:10 of 5000 mg/ml stock). Untreated cells were distributed into tubes 1 (no ATG control series) and 2 (ATG þ labeled reagent competitive series), whereas 24 h ATG-treated cells were added to tube 3. The cells were incubated for 15 min in the dark at room temperature. Two milliliters of PBS was added to each tube and they were centrifuged for 5 min at 300 g. The pellets were resuspended in 0.5 ml of 1% paraformaldehyde and stored in the dark at 41C until run on the BD FACSCanto.

Mouse model CB17 black severe combined immunodeficient (SCID) mice were obtained from Harlan Laboratories and maintained in a mouse barrier facility. RPMI 8226 cells were grown in RPMI 1640 media with 10% FBS as described previously. RPMI 8226 cells were harvested, washed twice with sterile PBS and re-suspended in sterile PBS at a concentration of 10 million cells per 500 ml. The mice were prepared by removing hair from a 1 cm2 area of the right flank. The RPMI 8226 cell suspension was injected subcutaneously over the right flank of the mice, each mouse receiving 10 million cells. The animals were closely followed until tumor growth was established. Once the tumors were at least 0.25 cm2 in size, they were randomized into three groups of 11 mice each. Group A received no treatment, group B mice received Thymoglobulin intraperitoneally (i.p.) at 5 mg/kg and group C received Thymoglobulin 10 mg/kg by the same route. Both groups received five daily injections of Thymoglobulin at these concentrations. The mice were followed regularly and tumors measured every 3 days. The tumor volume was calculated as length (breadth)2/2 and the mice were killed once the tumor volume reached 2.5 cm3. The mean tumor volumes across the three groups were compared and the survival of the mice in the three groups was compared using Kaplan–Meier curves and log rank statistics.


1865 Results

Thymoglobulin is cytotoxic to myeloma cell lines Thymoglobulin was cytotoxic to several different myeloma cell lines in a dose- and time-dependent manner as measured using MTT colorimetric assay for cell viability (Figure 1). Thymoglobulin was cytotoxic to more than 50% of the cells at a concentration of approximately 0.5 mg/ml for most of the cell lines. In addition, we also evaluated the effect of Thymoglobulin on the IL-6-dependent cell line KAS-6/1 and found a similar degree of cytotoxicity following treatment with Thymoglobulin. Most importantly, Thymoglobulin was able to induce cytotoxicity at similar concentrations in cell lines known to be resistant to common anti-myeloma drugs like MM1.R (dexamethasone resistant), Dox 40 (doxorubicin resistant) and LR5 (melphalan resistant) (Figure 1b). Maximum cytotoxic effect was seen at 72 h of incubation with only slightly lower activity at 48 h of incubation (Figure 1c).

Thymoglobulin induces apoptosis of myeloma cell lines and primary myeloma cells We examined the ability of Thymoglobulin to induce apoptosis of myeloma cell lines using flow cytometric evaluation of Annexin/PI staining. Thymoglobulin induced a time- (data not shown) and dose-dependent apoptosis of myeloma cell lines as shown by the increase in the Annexin-positive, PI-negative population of cells (Figure 2a). The proportion of dead necrotic (Annexin and PI positive) cells also increased with time and dose. We then treated freshly isolated primary myeloma cells with increasing concentrations of Thymoglobulin. Thymoglobulin induced apoptosis in primary myeloma cells as determined by 7-AAD staining of the cells following incubation with the drug for 36–48 h (Figure 2b). Several samples were tested and heterogeneity was seen in the sensitivity of primary myeloma cells to Thymoglobulin.

Thymoglobulin overcomes the effect of microenvironment and enhances the activity of conventional chemotherapy agents We next examined if Thymoglobulin can overcome the resistance observed when myeloma cells are cultured in conditions simulating the tumor microenvironment. When myeloma cells were grown in the presence of IL-6, VEGF or IGF, cytokines known to be important for myeloma cell growth, Thymoglobulin was able to induce comparable cytotoxicity reflecting the ability of the antibody to overcome the resistance afforded by the tumor microenvironment (Figure 3a). We examined the effect of combining Thymoglobulin with common anti-myeloma therapies including melphalan, adriamycin, dexamethasone and velcade. When myeloma cells were treated simultaneously with Thymoglobulin and each of these drugs, an additive effect was noted with melphalan (Figure 3b) and dexamethasone (not shown) across all the dose ranges.

Thymoglobulin targets multiple cell surface antigens on myeloma cells We examined the potential antigenic targets for Thymoglobulin on myeloma cells by a competitive flow cytometric method. Four different myeloma cell lines (MM1.S, RPMI 8226, U266 and OPM2) were examined to assess the maximum number of potential antigenic targets. When cells were treated with

Figure 1 Thymoglobulin is cytotoxic to myeloma cell lines. Different myeloma cell lines were cultured with or without varying concentrations of Thymoglobulin for 48 h and cell viability was measured by a colorimetric assay utilizing MTT. A dose-dependent decrease in the cell viability was observed following treatment of myeloma cell lines with Thymoglobulin (a). A similar effect was also observed with cell lines known to be resistant to conventional anti-myeloma drugs, namely Dox 40 (doxorubicin resistant), LR5 (melphalan resistant) and MM1.R (dexamethasone resistant) at comparable concentrations (b). There was a time-dependent increase in the cytotoxic effect with maximum cytotoxicity seen at 72 h of incubation (c). Thymoglobulin concentrations (mg/ml) are indicated on the x axis and viability (as a percentage of the control) is indicated on the y axis. Error bars represent one s.d.

Thymoglobulin and compared to untreated cells, a significant decrease in the detection of several B cell and plasma cell antigens was observed, as measured by a gate shift or intensity Leukemia


1866

a

b 104

4

10%

3

10

10

19% R1

3

29%

7AAD

Propidium Iodide

10

102 9%

101

101 100 100

R2

102

101

102

103

100 100

104

101

annexin 104

14%

3

10

10

3

10

2

21%

103

104

R1

35% 2

10

12%

7AAD

Propidium Iodide

104

101 100 100

102

SSC-Height

R2

101

101

102

103

100 100

104

101

annexin

102

103

104

SSC-Height

104

104

33%

31%

103

102 24% 101 100 100

7AAD

Propidium Iodide

R1 103

44% R2

102 101

101

102

annexin

103

104

100 100

101

102

103

104

SSC-Height

Figure 2 Thymoglobulin induces apoptosis of myeloma cell lines and primary myeloma cells. A dose-dependent increase in apoptosis was observed when RPMI 8226 cells were incubated with varying concentrations (none, 0.5 and 1 mg/ml) of Thymoglobulin for 24 h. The apoptotic myeloma cells are represented by the Annexin-positive, PI-negative cells (lower right quadrant) on flow cytometry (a). A similar dose-dependent increase in the apoptotic and necrotic cells were observed when freshly isolated patient myeloma cells were treated with increasing doses of Thymoglobulin (none, 0.5 or 1 mg/ml; b). The apoptotic cells are represented by the middle population (R2) and the necrotic cells by the upper population (R1) when stained with 7-AAD and examined by flow cytometry. The respective percentages of cells in each quadrant are shown on the respective panels.

of detection. The antigens whose detection shifted in the treated samples, reflecting binding of Thymoglobulin, included CD138, CD38, CD45, CD126, CD49d (VLA4), as well as CD20 in the cell lines expressing the respective antigens (Figure 4).

Thymoglobulin slows tumor growth in an animal model of myeloma Six-week-old SCID mice were injected with RPMI 8226 cells subcutaneously as described in the Methods section. Once tumors were established, mice received no injections (control group), or Thymoglobulin 5 mg/kg i.p. (group B) or Thymoglobulin 10 mg/kg i.p. (group C). When the animals were followed serially, there was a significant difference in the tumor growth between treated mice and the untreated mice, with group C receiving the maximum benefit from the Thymoglobulin (Figure 5a). Survival analysis using Kaplan–Meier product limit method demonstrated a significantly shorter survival for the untreated animals compared to the treated animals demonstratLeukemia

Figure 3 Thymoglobulin overcomes the effect of microenvironment and enhances the activity of conventional chemotherapy agents. RPMI 8226 cells were cultured in the presence of different cytokines (10 ng/ ml IL-6, 25 ng/ml VEGF or 50 ng/ml IGF-1) with or without increasing doses of Thymoglobulin and cell viability measured using MTT colorimetric assay. The median cytotoxic doses remained fairly similar to that seen in the absence of the cytokines (a). RPMI 8226 cells were then cultured with varying concentrations of Thymoglobulin with or without different concentrations of melphalan and cell viability measured at 48 h using MTT assay. An additive effect was noted at each level of Thymoglobulin concentration to increasing doses of melphalan (b).

ing a slowing of tumor growth owing to Thymoglobulin treatment.

Discussion ATG and anti-lymphocyte globulin (ALG) have been used as potent immunosuppressive agents following solid organ transplants for over two decades. In addition, it has been extensively used for the treatment of aplastic anemia and more recently is increasingly becoming a part of reduced-intensity conditioning regimens for allogeneic stem cell transplants. Traditionally, the ATGs have been thought to exert their activity through their ability to target and destroy T-cells. In recent years, attention has been brought to bear on the potent anti-B-cell activity of several of these preparations, especially that of the rabbit-derived polyclonal ATG. Several key clinical observations have led to studies demonstrating the presence of antibodies directed against B-cell antigens. In patients receiving cardiac transplants and the monoclonal antibody OKT3 for immunosuppression,


1867

Figure 4 Thymoglobulin targets multiple cell surface antigens on myeloma cells. MM1S, OPM2, U266 and RPMI 8226 cells were incubated with or without Thymoglobulin (1 mg/ml) for 4 h. The intensity of expression of various plasma cell and B-cell antigens were determined on the treated and untreated cells by flow cytometry using conjugated antibodies to the respective antigen. (a) Decreased expression of CD45, CD38, CD138, CD126, CD184, CD56, CD49d, CD126 and CD20 (RPMI 8226 only) following incubation with Thymoglobulin. The numbers represent intensity of expression expressed as a percentage of the untreated sample. (b) Representative plots demonstrating the shift in expression of various antigens following treatment with Thymoglobulin. The expression of control samples and the treated sample are as indicated.

the incidence of lymphoproliferative disorders was higher compared to a control group who received ALG.1 BonnefoyBerard et al.2 expanded on this observation and demonstrated that various ALG preparations, irrespective of the source of lymphocytes (thoracic duct lymphocytes, thymocytes or lymphocyte cell lines) used for immunization, contained antibodies directed against various B-cell antigens in addition to the antiT-cell antibodies. In addition, the authors observed that ALG had an inhibitory effect on various ligands known to induce B-cell differentiation and proliferation including pokeweed

mitogen and CD40L. They also observed an inhibitory effect of ALG on several lymphoma cell lines as well as other EBV-transformed cells. More recently, Shah et al.3 reported on the efficacy of polyclonal rabbit ATG (Thymoglobulin) in treating antibodymediated renal allograft rejection. This led to additional studies with the Thymoglobulin preparation, which clearly demonstrated its ability to induce apoptosis of naive and activated human B cells and plasma cells.4 The authors went on to demonstrate the presence of antibodies directed against various Leukemia


1868

Figure 5 Thymoglobulin slows tumor growth in an animal model of myeloma. When mice-bearing subcutaneous tumors (RPMI 8226 myeloma cell line) were injected i.p. with Thymoglobulin (10 mg/kg for 5 days), a significant decrease in the rate of growth of tumor size was noted compared to the untreated animals (a). This reduction in the growth translated into a better survival for the mice receiving Thymoglobulin as demonstrated by the Kaplan–Meier curves. A dose effect was noted with the 10 mg/kg dose being more effective compared to the 5 mg/kg dose (b).

B cell and plasma cell antigens in the Thymoglobulin preparation explaining these findings. Studies on pediatric thymi clearly confirm that approximately 6% of the cells are CD138 þ plasma cells and CD20 þ B cells. Thus, the Thymoglobulin preparation offers the potential to target multiple plasma cell antigens using a single preparation and represents an attractive strategy for therapeutic use in plasma cell malignancies. In this study, we clearly demonstrate the ability of Thymoglobulin to induce cytotoxicity in myeloma cell lines as well as patient-derived myeloma cells. A dose-dependent induction of apoptosis was confirmed by flow cytometry on myeloma cell lines as well as patient-derived myeloma cells. These results confirm previous observations that suggested induction of apoptosis when treated with 0.1–1 mg/ml of Thymoglobulin.5,6 Previous studies have implicated various mechanisms for the anti-myeloma activity of Thymoglobulin. A role for complement has been suggested by previous studies that showed enhanced cytotoxicity of Thymoglobulin when cells are incubated in the presence of complement.4,6 In addition, Zand et al.4 demonstrated decreased activity when cells were treated with the F(ab)2 fragments instead of the whole antibody, an effect that was reversed by Fc ligation using other antibodies. The cytotoxic Leukemia

effects of the Thymoglobulin on myeloma cells are also clearly observed in the absence of complement as in this study.4–6 Importance of the caspases has been highlighted in a previous study where the cytotoxicity was significantly reduced in the presence of caspase inhibitors5 and a second study that demonstrated activation of caspases 3, 8 and 9 in several myeloma cell lines.6 The current study found that Thymoglobulin was able to induce apoptosis of myeloma cells resistant to conventional anti-myeloma agents, demonstrating non-overlapping mechanisms of action. More importantly, it was able to induce apoptosis of myeloma cells when they were grown in the presence of IL-6, VEGF or IGF, which are important for tumor cell survival and proliferation in the marrow microenvironment. We then examined the specific antigens on the plasma cells that are being targeted by the Thymoglobulin preparation to further understand the anti-myeloma activity of the polyclonal Thymoglobulin preparation as well as to gauge the clinical utility of this approach. Using a competitive flow cytometry assay, we have identified some of the relevant targets on the myeloma cells. The spectrum of antibodies gives an important insight into the potential impact of using Thymoglobulin as a therapeutic agent for myeloma. Antibodies against syndecan-1 (CD138) and CD38, antigens commonly present on the plasma cell surface, may be responsible for at least some of the antimyeloma activity. Antibodies against the common leukocyte antigen (CD45), IL-6 receptor alpha chain (CD126), VLA4 (Integrin a4, CD49d), and B-cell antigen CD20, also likely play a role in the anti-myeloma activity seen with Thymoglobulin. Syndecan-1 (CD138) is a heparan sulfate bearing proteoglycan found on various epithelial cells as well as on B lineage cells depending on the stage of development. Syndecan-1 (CD138) is abundantly expressed by plasma cells, especially myeloma cells. The extracellular domain along with the heparan sulfate side chains can be cleaved off of the cell surface and can be detected in the serum as soluble syndecan-1. Syndecan-1 has been shown to be an independent prognostic factor in patients with MM and in vitro it can promote myeloma cell growth through different mechanisms. CD138 has been proposed as a therapeutic target for treatment of myeloma.7 CD38 is a type II transmembrane protein that is uniformly present on the surface of myeloma cells and has been the focus of novel therapeutic approaches for myeloma.8–10 In addition, this antigen is also present on thymic T-cells as well as activated lymphocytes. IL-6 is one of the most important cytokines for proliferation and survival of myeloma cells and myeloma cells uniformly express the receptor for IL-6 which consists of an alpha chain (CD126) and a beta chain (CD130).11,12 VLA4 is an important adhesion molecule that is present on the myeloma cell surface and is believed to play an important role in adhesion-mediated drug resistance as well as interaction with the stromal cell that is crucial for myeloma cell survival.13–18 Targeting VLA4 may be able to enhance the activity of other myeloma drugs and strengthens the rational for combining Thymoglobulin with these therapies.19 CD20 expression is variable on myeloma cells and anti-CD20 monoclonal antibody-based therapies have limited utility in myeloma. However, some recent data suggest that the early myeloma cells may have higher expression of CD20 and Thymoglobulin may provide a mechanism to target these cells.20 The expression of various antigens are highly variable in myeloma cells, and by virtue of the polyclonal nature of the product, Thymoglobulin has the ability to target a wide spectrum of myeloma cells both in terms of intra- and interpatient heterogeneity.21 In conclusion, we demonstrate the in vivo activity of Thymoglobulin in a mouse model of myeloma and provide


1869 convincing evidence for clinical evaluation of this preparation in patients with myeloma. The animal studies also point towards a dose response given the difference between the two groups, again suggesting anti-myeloma activity of the preparation. Higher daily doses or longer treatment schedule may have resulted in increased activity, but was not pursued in this study. Given the phenotypic heterogeneity of the myeloma cells and the limitations with regard to the total amount of Thymoglobulin that can be administered secondary to the immunosuppressive effects, we hypothesize that a combination of Thymoglobulin with another effective myeloma agent such as an alkylating agent is likely to have better success than Thymoglobulin alone. The results presented here will form the framework for a proposed clinical trial of Thymoglobulin and melphalan in patients with relapsed myeloma.

Acknowledgements

8

9 10

11

12

This study was supported in part by Mayo Clinic Hematological Malignancies program (SK), and a grant from Genzyme Corp., Cambridge, MA (SK).

13

References

14

1 Swinnen LJ, Costanzo-Nordin MR, Fisher SG, O’Sullivan EJ, Johnson MR, Heroux AL et al. Increased incidence of lymphoproliferative disorder after immunosuppression with the monoclonal antibody OKT3 in cardiac-transplant recipients. N Engl J Med 1990; 323: 1723–1728. 2 Bonnefoy-Berard N, Flacher M, Revillard JP. Antiproliferative effect of antilymphocyte globulins on B cells and B-cell lines. Blood 1992; 79: 2164–2170. 3 Shah A, Nadasdy T, Arend L, Brennan J, Leong N, Coppage M et al. Treatment of C4d-positive acute humoral rejection with plasmapheresis and rabbit polyclonal antithymocyte globulin. Transplantation 2004; 77: 1399–1405. 4 Zand MS, Vo T, Huggins J, Felgar R, Liesveld J, Pellegrin T et al. Polyclonal rabbit antithymocyte globulin triggers B-cell and plasma cell apoptosis by multiple pathways. Transplantation 2005; 79: 1507–1515. 5 Ayuk FA, Fang L, Fehse B, Zander AR, Kroger N. Antithymocyte globulin induces complement-dependent cell lysis and caspasedependent apoptosis in myeloma cells. Exp Hematol 2005; 33: 1531–1536. 6 Zand MS, Vo T, Pellegrin T, Felgar R, Liesveld JL, Ifthikharuddin JJ et al. Apoptosis and complement-mediated lysis of myeloma cells by polyclonal rabbit antithymocyte globulin. Blood 2006; 107: 2895–2903. 7 Tassone P, Goldmacher VS, Neri P, Gozzini A, Shammas MA, Whiteman KR et al. Cytotoxic activity of the maytansinoid

15 16

17 18

19

20 21

immunoconjugate B-B4-DM1 against CD138+ multiple myeloma cells. Blood 2004; 104: 3688–3696. Stevenson FK, Bell AJ, Cusack R, Hamblin TJ, Slade CJ, Spellerberg MB et al. Preliminary studies for an immunotherapeutic approach to the treatment of human myeloma using chimeric anti-CD38 antibody. Blood 1991; 77: 1071–1079. Goldmacher VS, Bourret LA, Levine BA, Rasmussen RA, Pourshadi M, Lambert JM et al. Anti-CD38-blocked ricin: an immunotoxin for the treatment of multiple myeloma. Blood 1994; 84: 3017–3025. Peng KW, Donovan KA, Schneider U, Cattaneo R, Lust JA, Russell SJ. Oncolytic measles viruses displaying a single-chain antibody against CD38, a myeloma cell marker. Blood 2003; 101: 2557–2562. Perez-Andres M, Almeida J, Martin-Ayuso M, Moro MJ, Martin-Nunez G, Galende J et al. Clonal plasma cells from monoclonal gammopathy of undetermined significance, multiple myeloma and plasma cell leukemia show different expression profiles of molecules involved in the interaction with the immunological bone marrow microenvironment. Leukemia 2005; 19: 449–455. Szczepek AJ, Belch AR, Pilarski LM. Expression of IL-6 and IL-6 receptors by circulating clonotypic B cells in multiple myeloma: potential for autocrine and paracrine networks. Exp Hematol 2001; 29: 1076–1081. Uchiyama H, Barut BA, Chauhan D, Cannistra SA, Anderson KC. Characterization of adhesion molecules on human myeloma cell lines. Blood 1992; 80: 2306–2314. Okada T, Hawley RG, Kodaka M, Okuno H. Significance of VLA4-VCAM-1 interaction and CD44 for transendothelial invasion in a bone marrow metastatic myeloma model. Clin Exp Metastasis 1999; 17: 623–629. Sanz-Rodriguez F, Ruiz-Velasco N, Pascual-Salcedo D, Teixido J. Characterization of VLA-4-dependent myeloma cell adhesion to fibronectin and VCAM-1. Br J Haematol 1999; 107: 825–834. Vacca A, Di Loreto M, Ribatti D, Di Stefano R, GadaletaCaldarola G, Iodice G et al. Bone marrow of patients with active multiple myeloma: angiogenesis and plasma cell adhesion molecules LFA-1, VLA-4, LAM-1, and CD44. Am J Hematol 1995; 50: 9–14. Sanz-Rodriguez F, Teixido J. VLA-4-dependent myeloma cell adhesion. Leukemia Lymphoma 2001; 41: 239–245. Sanz-Rodriguez F, Hidalgo A, Teixido J. Chemokine stromal cellderived factor-1alpha modulates VLA-4 integrin-mediated multiple myeloma cell adhesion to CS-1/fibronectin and VCAM-1. Blood 2001; 97: 346–351. Damiano JS, Cress AE, Hazlehurst LA, Shtil AA, Dalton WS. Cell adhesion mediated drug resistance (CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell lines. Blood 1999; 93: 1658–1667. Matsui W, Huff CA, Wang Q, Malehorn MT, Barber J, Tanhehco Y et al. Characterization of clonogenic multiple myeloma cells. Blood 2004; 103: 2332–2336. Lin P, Owens R, Tricot G, Wilson CS. Flow cytometric immunophenotypic analysis of 306 cases of multiple myeloma. Am J Clin Pathol 2004; 121: 482–488.

Leukemia