Immunotherapy of Multiple Myeloma With a Monoclonal Antibody Directed Against a Plasma Cell-Speciﬁc Antigen, HM1.24

interleukin (IL)-6 is a major growth factor for myeloma cells functioning by an autocrine/ were paracrine fashion, immunotherapy targeting the IL-6–signal-purchased ing system has also been reported. 13-16 Clinical trials of anti-tained IL–6 MoAb showed transient tumor cytostasis, did not result in tumor cure. 13,14 Thus, the efﬁcacy of immunotherapy for multiple myeloma has been disappointing. against human my-Department eloma xenografts in SCID mice. Our results indicate that the antigen serves a target immunotherapy of IL-2 (day 0). In the cells for 50 days either once or twice a week. As shown in absence of anti-HM1.24 MoAb, natural killing activity of Fig 3, anti-HM1.24 MoAb inhibited the tumor growth in a spleen cells was not observed. At day 14 after tumor inocula- dose-dependent manner. These results show that the effect tion, ADCC activity was not decreased, although the effect of anti-HM1.24 MoAb is dose-dependent and that higher was not enhanced by IL-2. In mice with advanced RPMI doses of the antibody can be effective in mice with advanced 8226 tumors (day 70), ADCC activity of spleen cells was tumors. There were no treatment-related deaths with the anti-signiﬁcantly reduced in the presence or absence of IL-2. body. vivo analysis observations have been reported in animal models of lymphoma in which immunotherapy had limited therapeutic efﬁcacy on solid tumors due to poor biolocalization of antibodies into the tumors. 25 Thus, anti-HM1.24 MoAb may only transient suppressive effect on normal Ig production. colony-stimulating factor (M-CSF), 36 has been shown to inT, crease the levels of ADCC by the stimulation of effector anti-interleukin-6 cells, combinations of these cytokines along with the anti-myeloma. body may further potentiate the effect of the antibody. inhibits cells can be used for immunotherapy cal using desired

variety of chemotherapeutic protocols, the overall survival isotype-matched myeloma protein UPC-10 (Cappel, Malvern, PA) has not improved over the past three decades. 1 Myeloablative were purified from the ascites fluid by ammonium sulfate precipitachemotherapy followed by allogeneic or autologous hematotion and a protein A-affinity chromatography kit (Ampure PA, Amerpoietic stem cell transplantation has increased the incidence sham Japan, Tokyo, Japan). F(ab) 2 fragment was prepared by pepsin of complete remission, 2,3 but relapses are still observed even to cell surface antigens on myeloma cells, using anti-CD38 cells, which do not express the HM1.24 antigen, were used as control antibody, 6-8 anti-CD54 antibody, 9 or soluble CD16. 10 Howtumors. The cells were cultured in RPMI 1640 medium suppleever, because these molecules are also expressed on normal mented with 10% fetal bovine serum, 100 U/mL penicillin and 100 tissues including hematopoietic stem cells, 11,12 these apmg/mL streptomycin. Before inoculation to mice, cells were washed proaches may induce side effects related to crossreactivity and resuspended in sterile phosphate-buffered saline (PBS) at a conin vivo. Based on the fact that interleukin (IL)-6 is a major centration of 1 1 10 8 cells/mL. growth factor for myeloma cells functioning by an autocrine/ Animals. Five-to 6-week-old female C. B-17 SCID mice were paracrine fashion, immunotherapy targeting the IL-6-signalpurchased from Clea Japan Co, Inc. (Osaka, Japan) and were maining system has also been reported. 13  inoculation. In further experiments, immunotherapy was started when high levels of human IgG had been detected in the sera or the † P õ .005.
subcutaneous tumors had grown to approximately 5 mm in diameter.
Mice received intraperitoneal injections of anti-HM1.24 MoAb (100 mg/dose) once or twice a week. All mice were observed daily for the appearance of solid tumors and duration of survival. or control IgG were added to wells. Effector cells were then added Human immunoglobulin assay. The levels of human IgG in the to the plates at various effector to target (E/T) ratios. After 4 hours culture supernatants and in the sera of SCID mice with ARH-77 incubation, supernatants were removed and counted in a gamma tumors were measured by enzyme-linked immunosorbent assay counter. The percentage of cell lysis was determined as above. (ELISA) as described previously. 19 Statistical analysis. The statistical significance in the data of in Cell proliferation assay. The effect of anti-HM1.24 MoAb on vitro experiments was determined by the unpaired t-test. The sigthe proliferation of human myeloma cell lines was determined using nificant differences in survival data were evaluated using a log-rank a 3 H-thymidine incorporation assay. The cells were cultured in 96test. well plates at 1 1 10 5 cells/mL in 100 mL/well and incubated with various amounts of anti-HM1.24 MoAb or control IgG for 24 hours. For personal use only. on August 21, 2017. by guest www.bloodjournal.org From mice inoculated subcutaneously. The course of serum concentrations of human IgG is shown in Fig 2. The levels of serum human IgG were increased in control mice inoculated intravenously or subcutaneously with ARH-77 cells, and the mice died within 53 { 21 or 55 { 9 days, respectively. In contrast, weekly treatment with the antibody reduced the levels of serum human IgG and prolonged the survival for up to 180 days in four of five mice bearing disseminated tumors and three of five mice bearing localized tumors. Although three of five mice bearing disseminated tumors showed an initial increase in serum human IgG, only one of single intraperitoneal injection of 20 mg anti-HM1.24 MoAb 1 day after tumor inoculation completely inhibited the development of disseminated myeloma. Serum human IgG levels were not increased in these mice until the end of the experimental period at day 180. When the treatment was delayed until day 15, serum levels of human IgG were significantly lower than those of control mice at day 28. However, serum IgG levels of anti-HM1.24 IgG-treated mice (n Å 3) were increased to the same levels as those of control mice (n Å 3) at day 42 (337 { 91 and 333 { 61, respectively) and no prolongation of survival was observed. In mice inoculated subcutaneously with ARH-77 cells, the same treatment had no antitumor effect on either serum level of human IgG or survival. We next determined whether the multiple injections of anti-HM1.24 MoAb could exhibit antitumor activity against localized RPMI 8226 tumors (Fig 1). After subcutaneous injection of RPMI 8226 cells, all mice developed localized tumors in 22 { 4 (mean { standard deviation [SD]) days. When mice were treated with 20 mg anti-HM1.24 MoAb 1 day after tumor inoculation, the mean tumor-free time was extended by 41 { 18 days and survival was significantly prolonged compared with control IgG-treated group (P õ .025). When the antibody was administered to mice for 4 consecutive days starting 1 day after tumor inoculation, the local tumor development was completely inhibited for 180 days (P õ .005). In contrast, F(ab) 2 fragment of anti-HM1.24 MoAb had no therapeutic effect in vivo. The control IgG preparation did not show any tumor inhibitory effect compared with untreated mice. On the other hand, anti-HM1. 24 MoAb did not improve the survival of SCID mice bearing HPB-ALL cells (T-cell line), which do not express HM1.24 antigen, showing that the antitumor effect of the antibody is mediated by a tumor-specific immune response.
We next evaluated the therapeutic efficacy of anti-HM1.24 MoAb in SCID mice with advanced tumors. Weekly injec-  For personal use only. on August 21, 2017. by guest www.bloodjournal.org From mice inoculated intravenously with ARH-77 cells, tumor cells proliferated mainly in the vertebral bodies and invaded the spinal cord and perivertebral tissues. In contrast, no tumor cells were found in the anti-HM1.24 MoAb-treated mice, surviving for 180 days. In SCID mice with advanced localized tumors, there was no evidence of metastases in other organs such as the liver, spleen, kidneys, lung, heart, stomach, and intestine by immunohistologic analysis using anti-HM1.24 MoAb. In the models with localized RPMI 8226 cells, tumor cells invaded the surrounding tissues including the muscle and showed typical morphologic features of neoplastic plasma cells, ie, irregular nuclear profiles with prominent nucleoli and abundant cytoplasm. Mitotic figures of the tumor cells were also identified. Neither necrotic areas nor infiltrating cells were found in the tumors of control groups (Fig 4A). In contrast, the tumors of anti-HM1. 24 MoAb-treated mice showed many shrunk cells with picnotic nuclei. Necrotic areas and infiltrating cells were also observed in the tumors (Fig 4B). No tissue damage was found in the liver, spleen, kidney, lung, or heart in mice treated with anti-HM1.24 MoAb.
In vitro effects of anti-HM1. 24 MoAb. Treatment with anti-HM1.24 MoAb alone did not inhibit either the proliferation of ARH-77 and RPMI 8226 cells or the IgG secretion from ARH-77 cells in culture even at a concentration of 100 mg/mL (data not shown). the complement-dependent cytotoxicity (59.0 { 5.8) against ARH-77 cells (complement dilution, 1:5). Anti-HM1. 24 MoAb was tested for ADCC activity against RPMI 8226 them died within 50 days, and serum human IgG started to cells using spleen cells from SCID mice as effector cells. decrease by 70 days of tumor inoculation in the remaining Although control IgG and F(ab) 2 fragment of anti-HM1.24 two mice (Fig 2A). In localized tumor models, serum human had no ADCC activity in the presence of spleen cells, anti-IgG restarted to increase by 50 days in two of five mice after HM1.24 IgG mediated ADCC activity and the extent of initial increase, and these mice died at day 114 and 115 (Fig cytotoxicity was dependent on the E/T ratio (Fig 5). ADCC 2B). Serum levels of human IgG at day 180 were undetectactivity (43.8 { 6.5) against ARH-77 cells was also observed able (õ0.1 mg/mL) in all the surviving mice, resulting in a (E/T ratio, 50). The complement-dependent cytotoxicity and complete remission rate of 80% and 60%, respectively.
ADCC were mediated by anti-HM1.24 IgG even at a low These effects on survival were statistically significant in both concentration of 0.01 mg/mL. The serum from myeloma pamodels as compared with the control groups (P õ .01).
tients caused no blocking of binding of anti-HM1.24 MoAb Finally, we examined the dose-response of the effect of the in ADCC assay (data not shown). We examined the differantibody on in vivo antitumor activity in mice with more ences of ADCC activity of spleen cells in SCID mice after advanced RPMI 8226 tumors. The antibody treatment (100 tumor inoculation (Fig 6). ADCC activity of spleen cells mg/dose) was started 22 days after injection of RPMI 8226 was augmented by the stimulation with IL-2 (day 0). In the cells for 50 days either once or twice a week. As shown in absence of anti-HM1.24 MoAb, natural killing activity of Fig 3, anti-HM1. 24 MoAb inhibited the tumor growth in a spleen cells was not observed. At day 14 after tumor inoculadose-dependent manner. These results show that the effect tion, ADCC activity was not decreased, although the effect of anti-HM1.24 MoAb is dose-dependent and that higher was not enhanced by IL-2. In mice with advanced RPMI doses of the antibody can be effective in mice with advanced 8226 tumors (day 70), ADCC activity of spleen cells was tumors. There were no treatment-related deaths with the antisignificantly reduced in the presence or absence of IL-2. body.
Histological analysis. The in vivo antitumor activity of DISCUSSION anti-HM1. 24 MoAb was further studied by histologic analysis of tumor-bearing mice that had been treated with either In this report, we provide evidence that human myeloma in SCID mice can be successfully treated with a MoAb control IgG or anti-HM1. 24   Although several investigators have developed plasma directed against a human plasma cell-specific antigen, cell-specific antibodies, 21-23 the use of antibodies for immu-HM1. 24. In vivo antitumor activity of anti-HM1.24 MoAb in notherapy of multiple myeloma has not been tested in vivo. SCID mice was observed in both disseminated and localized In a similar SCID mouse model, Huang et al 9 reported that human myeloma models. Treatment with a single injection treatment with four consecutive injections of anti-CD54 of anti-HM1. 24 MoAb was effective if the antibody was MoAb could prevent the development of disseminated ARHadministered early in the course of disease. Moreover, tu-77 tumors partially and prolong the survival of mice. Howmors were cured by multiple injections of the antibody even ever, CD54 is not a tumor-specific antigen and antitumor when therapy was started at an advanced stage of disease. mechanisms were not explored. Because anti-HM1. 24 MoAb is highly specific for human plasma cells, immunotherapy with this antibody has the potential to provide a new concern is that Ig secretion from plasma cells might be af- The precise mechanisms whereby the anti-HM1.24 MoAb exerts its antitumor activity remain elusive. Previous studies have reported that ADCC mediated by NK cells and macrophages with MoAb is the most important mechanism for antitumor effect in animal models. 24 We have shown that anti-HM1.24 IgG can mediate complement-dependent cytotoxicity and ADCC against human myeloma cells in vitro. In contrast, F(ab) 2 fragment of the antibody had no antitumor activity either in vitro or in vivo. These results indicate that Fc-mediated effector functions are responsible for the antitumor activity and support the hypothesis that ADCC and complement-dependent cytotoxicity are important for the antitumor effect of the antibody in vivo. We have ruled out the possibility that antitumor activity is due to antiproliferative effects of the antibody because anti-HM1. 24 MoAb had no effect on 3 H-thymidine incorporation in ARH-77 and RPMI 8226 cells in vitro. However, the possibility cannot be ruled out that other mechanisms may be involved to exert the powerful in vivo antitumor effects of the antibody.
Vascularization of tumors, permeability of the antibody, infiltration of effector cells, and immune status of the treated animals may play an important role in the outcome of immunotherapy. We found that the therapeutic effect of anti- lymphoma in which immunotherapy had limited therapeutic efficacy on solid tumors due to poor biolocalization of antibodies into the tumors. 25 Thus, anti-HM1. 24 MoAb may only transient suppressive effect on normal Ig production.
have limited value in the therapy of bulky solid tumors. In fact, single injection of anti-HM1. 24 MoAb inhibited the However, in view of the fact that myeloma cells in the bone increase of serum human IgG in ARH-77 myeloma models marrow are readily accessible to the antibody, immunotherwithout killing all tumor cells. It may be possible to allow apy with anti-HM1.24 MoAb can become a useful measure B cells to recover by using a small amount of the antibody. Therefore, another indication of anti-HM1. 24 MoAb could for the treatment of multiple myeloma.