Cancer Therapy: Preclinical

Therapy of Advanced B-Lymphoma Xenografts with a Combination of 90Y-anti-CD22 IgG (Epratuzumab) and Unlabeled Anti-CD20 IgG (Veltuzumab) M. Jules Mattes,1Robert M. Sharkey,1Habibe Karacay,1Myron S. Czuczman,2 and David M. Goldenberg1

Abstract Purpose: Antibodies are effective therapeutic agents in cancer, but cures are rarely if ever obtained. Combination therapies are likely to be more effective than a single agent. In this study, the combination of a new unconjugated humanized anti-CD20 IgG, veltuzumab, with a 90Y-conjugated humanized antibody to CD22 (epratuzumab) was evaluated for the treatment of B-cell lymphoma in a nude mouse model system. Experimental Design: Nude mice were grafted with the Ramos human B-lymphoma and treatment initiated when tumors were >0.1cm3. In most experiments, mice were injected first with unconjugated anti-CD20, then with 90Y-anti-CD22 1day later. Additional weekly injections of the unconjugated veltuzumab were administered for 3 weeks. Controls included a single agent only and a nonreactive control radiolabeled antibody. Results: Unconjugated anti-CD20 veltuzumab alone did not have a significant therapeutic effect, even at a total dose of 2.5 mg per mouse.The 90Y-anti-CD22 epratuzumab alone induced marked regressions of all tumors, but they regrew in a few weeks. The combination of these agents cured f80% of the mice. A nonreactive control antibody labeled with 90Y, used without veltuzumab, had no therapeutic effect. The therapeutic effect of 90Y-epratuzumab required the maximum tolerated dose of radioactivity, which was 160 ACi per mouse. Conclusions: These studies illustrate how combinations of unconjugated and radioconjugated antibodies against different B-cell markers can improve therapeutic outcome, and offer a new therapeutic paradigm for the treatment of B-cell lymphomas.

Treatment of non–Hodgkin’s lymphoma with rituximab role in non–Hodgkin’s lymphoma management as rituximab. constitutes a paradigm shift in therapy. Rituximab therapy A potential limitation of current protocols for the use of results in a f50% initial response rate (f10% complete radiolabeled anti-CD20 IgG is that a large dose of unlabeled response) in patients with previously treated follicular non– anti-CD20 Ab is injected before the radioconjugate, called Hodgkin’s lymphoma, and although it is not curative, disease predosing. This was found to be necessary to improve uptake of progression can be forestalled with retreatment or maintenance the radiolabeled Ab in the tumor (8), presumably because it therapy (1–3). As with other antibodies (Ab) in current clinical blocks the large amount of antigen present on normal B cells use, rituximab is most effective when combined with other in patients. However, despite careful selection of the dose of therapeutic modalities, such as chemotherapy (4), and may the unconjugated Ab used, it is evident that predosing may also also be complemented by other biologicals, such as the anti- inhibit uptake into the tumor of the subsequently injected CD22 Ab, epratuzumab (5), and the anti-CD80 Ab, galiximab radiolabeled Ab. For example, in a mouse model, Gopal et al. (6). CD20-based radioimmunotherapy, with 90Yor131I (9) showed that a predose of rituximab inhibited the binding conjugates, has shown better objective response rates than of and therapy with radiolabeled rituximab, but not with a naked anti-CD20 Abs (3, 7), yet has not achieved as extensive a radiolabeled anti-CD45 IgG. Clinical studies have shown encouraging antitumor responses with an 90Y-labeled anti- CD22 Ab, epratuzumab (10, 11), which has a more restricted specificity for B cells than an anti-CD45 Ab. Therefore, we Authors’Affiliations: 1Garden State Cancer Center, Center for Molecular Medicine evaluated the combination of an anti-CD20 Ab, veltuzumab, and Immunology, Belleville, NewJersey and 2Roswell Park Cancer Institute, Buffalo, with a radiolabeled anti-CD22 Ab, epratuzumab. In vitro NewYork studies suggested that tumor CD22 expression can be up- Received 2/14/08; revised 4/23/08; accepted 5/7/08. regulated by prior treatment with rituximab (5), which might Grant support: NIH grant PO1CA103985 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page enhance uptake of the radiolabeled epratuzumab. charges. This article must therefore be hereby marked advertisement in accordance Other factors make this combination promising. The high- with 18 U.S.C. Section 1734 solely to indicate this fact. energy h-particle emitted by 90Y has a long tissue path length, Requests for reprints: David M. Goldenberg, Center for Molecular Medicine and enabling cell killing even if the Ab does not fully penetrate the Immunology, 520 Belleville Avenue, Belleville, NJ 07109. Phone: 973-844-7000; Fax: 973-844-7020; E-mail: [email protected]. tumor, and even if there are antigen-negative subpopulations. F 2008 American Association for Cancer Research. However, also because of the long path length of the radiation doi:10.1158/1078-0432.CCR-08-0404 emitted, single cells or micrometastases are not good targets for

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90Y-Ab therapy (12). In contrast, single cells are suitable targets therapy seems to be advantageous, and potentially curative, in for unconjugated Abs because they are readily accessible. Thus, this animal model. the two forms of treatment can act cooperatively in reducing larger disease burdens, as well as scavenging smaller pockets of disease. Materials and Methods The mechanism of action of unconjugated anti-CD20 Abs, including veltuzumab, has been extensively investigated, but Cell lines, Abs, and radiolabeling. Ramos cells were obtained from is not fully resolved (2, 4, 13), probably partly because the the American Type Culture Collection and propagated as described different animal models used by various investigators may previously (16). While these studies were ongoing, their identity was depend on different mechanisms. In humans, an important confirmed by DNA ‘‘fingerprinting’’ done by DSMZ. They were also tested routinely for Mycoplasma contamination, with the Mycotect kit role for Ab-dependent cellular cytotoxicity has been estab- (Invitrogen), and found to be negative. The humanized IgG1 Abs lished, there is evidence that complement activation may be veltuzumab (5), epratuzumab (17), and labetuzumab (hMN-14) were in vitro important (13, 14), and data suggest that apoptosis provided by Immunomedics, Inc. Epratuzumab and labetuzumab were induction may play a role. In any case, for our present purpose, conjugated to the metal chelator 1,4,7,10-tetra-azacyclododecane and it is sufficient to note that the mechanism of action of the labeled with 90Y at a ratio of 5.0 mCi 90Y/1.0 mg IgG by methods that 90 veltuzumab is certainly different from that of the Y- have been previously reported in detail (18). The product was prepared epratuzumab. Even unconjugated, these two Abs seem to have on the day of its use with yields usually of >95%. Excess diethylene- different mechanisms of action (15). triaminepentaacetic acid was added to scavenge any residual unbound 90 The unconjugated Ab used was the humanized veltuzumab, Y. Each product was analyzed by instant TLC and size-exclusion which has the epratuzumab backbone and similar hyper- high performance liquid chromatography (19). Representative radio- conjugates were also tested for immunoreactivity using an anti-idiotype variable regions to rituximab (5). As the radiolabeled Ab, we 90 selected an anti-CD22 humanized Ab, epratuzumab, because Ab (20) and were >95% reactive. The activity of Y was determined in a Capintec CRC-15R dose calibrator (Capintec). Containers and other this has been found to be an effective investigational conditions were carefully controlled to ensure accurate readings of 90Y therapeutic in patients (10). The model used was Ramos activity (21). The same conjugate was labeled with 111In, following the lymphoma cells growing s.c. in nude mice, and the mice were same procedure, except with 2.5 mCi/mg IgG. treated when the tumors were established. Because these Ab therapy and biodistribution experiments. All animal experiments tumors grow very rapidly, this is a challenging model for were approved by the institutional animal welfare committee. Male therapy. The data presented show that this type of combination BALB/c nude mice were obtained from the National Cancer Institute

Fig. 1. Therapy of s.c. Ramos tumors in nude mice with 90Y-epratuzumab. Groups of 11mice were injected with 100 or 175 ACi of radiolabeled Ab on day 22 after tumor inoculation. Tumors ranged in size from 0.2 to 1.0 cm3 (mean, 0.49 cm3).The growth rate of individual tumors is shown for control untreated mice (A), mice treated with 100 ACi (B), or mice treated with 175 ACi (C). Untreated control tumors were found to regress occasionally after reaching a size of >1.0 cm 3. D, a step graph of the same experiment, showing the time required for tumors to reach a size of 3.0 cm3. ., control; o,100ACi; !,175ACi. E, body weights of the mice for 5 wk after injection of the higher dose of radioactivity,175 ACi.

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Fig. 2. Therapy of s.c. Ramos tumors in nude mice with a combination of 90Y-epratuzumab and unconjugated veltuzumab. Tumors were measured, ranked by size, and then the mice bearing tumors at least 0.1cm3 were distributed sequentially into5groupsof13to15mice.To p l e f t , the time course of Ab injections. Unconjugated veltuzumab (1.0 mg) was injected on day 0, and 90Y-epratuzumab was injected on day 1, into the appropriate groups. All mice injected with the unlabeled veltuzumab also were administered three additional doses of the same Ab at weekly intervals, at half of the initial dose. Each line shows results of an individual mouse. A, control mice not injected with Abs; B, mice injected only with veltuzumab; C, mice injected only with 175 ACi 90Y-ep ra tu zumab ; D, mice injected with both Abs; E, mice injected with a control, nonreactive Ab, hMN-14, labeled with 90Yat the same dose as the 90Y-epratuzumab. Tumors sizes were measured for 168 d after Ab injection. Only one tumor grew after the time interval shown in the graphs (100 d): this was one of the mice treated with both Abs, and the tumor first appeared at 140 d, after which it grew rapidly.

Animal Resources. Typically, groups of 60, 4- to 6-wk-old mice were Results injected s.c. on the back with 5 to 10 Â 106 tumor cells (usually 5 Â 106). Tumors grew in 80% to 95% of the mice, appearing Single agent therapy. Nude mice bearing visible Ramos consistently in f19 to 26 d. The tumors grew rapidly once they tumors were injected with either 100 ACi or 175 ACi of 90Y- appeared, with a doubling time of f3 d. Because the time of tumor epratuzumab. The mean tumor size at the time of Ab injection appearance varied over an interval of a week, groups of mice were was 0.49 cm3 (range, 0.2-1.0 cm3). As shown in Fig. 1C, all usually used in two batches, 1 at approximately day 20, and the other of the tumors, large and small, treated with 175 ACi 90Y- f5 d later. On the day of injection, animals bearing tumors >0.1 cm3 and <1.5 cm3 (caliper measured perpendicular L Â W Â D) were epratuzumab regressed by day 14, with most having no selected and distributed into groups to ensure that the mean tumor size evidence of tumor at this time. Two to 5 weeks after treatment, would be approximately the same in all groups. the tumors regrew rapidly. In contrast, tumors in untreated All Abs were injected iv into the tail vein. In most experiments, animals continued to progress. Most of the tumors in animals veltuzumab was injected on day 0, the day on which tumors were first receiving 100 ACi of 90Y-epratuzumab had a slight delay in measured, at doses of up to 1.0 mg, as indicated below. 90Y- tumor growth, but only 2 regressed, and only 1 of these A epratuzumab was injected the following day, together with 208 g regressed to 0 cm3. Occasionally, untreated tumors regressed (0.1 mL of ascites) of an irrelevant mouse IgG2a Ab (PK136) to block spontaneously after reaching a size of >1.0 cm3 (Fig. 1A). The rapid blood clearance, presumably mediated by the high affinity FcgR frequency of such regressions never exceeded 20% and, in some CD64 (22). Three additional doses of veltuzumab were injected at weekly intervals, using half the amount given in the initial dose. The experiments, did not occur at all. The tumors that regressed first studies used 175 ACi of 90Y-epratuzumab, but this dose, although spontaneously in control mice never regrew. The reason for the sometimes effective, resulted in excessive loss of body weight in a regressions is unknown. fraction of the animals 2 to 3 wk after injection, in later experiments. Figure 1D is a step graph of the same data, showing the time Therefore, the dose in subsequent experiments was reduced to 160 ACi. required for tumors to reach a size of 3.0 cm3. Using the log- The particular dose used in each experiment is given under Results. rank statistical test, the difference between the control group Tumor growth was measured once to twice per week. At each time (.) and the 175 ACi group (!) was not statistically significant point, mice were observed for signs of morbidity and body weight was because 2 tumors in the control group regressed, but the determined. Mice with >20% loss of body weight or other signs of A A morbidity were euthanized. The study was terminated when tumors difference between the 175 Ci group and the 100 Ci group grew to 3.0 cm3. Ab biodistribution using 111In-epratuzumab was done (o) was significant (P < 0.025). Using Fisher’s exact test, at day as described previously (23). 27, the difference between the control group and the 175-ACi

ClinCancerRes2008;14(19)October1,2008 6156 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2008 American Association for Cancer Research. Therapy with 90Y-Anti-CD22 and Unlabeled Anti-CD20 Abs group was significant (P < 0.001). The body weights of the mice of most or all of the mice treated with both Abs (data not treated with 175 ACi are shown in Fig. 1E. There was a transient shown). This experiment also included a group treated only loss of body weight (10.7% at day 7), but the mice had largely with 90Y-epratuzumab, and in this group, all of the tumors recovered by day 15. In later studies, some mice injected with regrew after initial regression, very similarly to the results 175 ACi had severe, >20%, loss in body weight, and thus, we shown above. We conclude that a total dose of 250 Ag (100 Ag elected to reduce the 90Y-epratuzumab dose to 160 ACi for +3Â 50 Ag weekly) veltuzumab is sufficient to produce the subsequent studies. In some experiments, the time course of maximum therapeutic effect. tumor regression was examined by measuring tumors twice The effect of veltuzumab on the tumor uptake of 111In- weekly (data not shown). After the injection of the radiolabeled epratuzumab. One possible mechanism of action of the Ab, the tumors continued to grow for at least 4 days, and veltuzumab is to increase the uptake of 90Y-epratuzumab in sometimes as long as 6 days. By day 11, tumors had become the tumor, which might be by inducing a higher level of much smaller, and reached a nadir by day 14 to 15 after Ab expression of the CD22 antigen, or by some other mechanism. injection. Thus, the time of dramatic tumor shrinkage occurred In fact, previous in vitro studies suggested that veltuzumab between day 6 to 11. binding induced an increase in CD22 expression (5). To Varying doses of up to 3.0 mg of unconjugated veltuzumab investigate this possibility, an Ab biodistribution experiment were injected when tumors reached a size of 0.1 to 0.5 cm3. was done, in which the distribution of 111In-epratuzumab was The majority of treated tumors grew at the same rate as control determined with and without injection of 1.0 mg veltuzumab, tumors, with no indication of tumor stabilization or regression. under exactly the same conditions as used in the therapy Although a subpopulation of tumors regressed after Ab experiments. 111In was used as a surrogate for 90Y because injection, this effect was not statistically significant, partly of the simpler quantification of the 111In, and because both because of the occasional regression of control tumors, as label the same 1,4,7,10-tetra-azacyclododecane–Ab conjugate. described above (data not shown; but results of a similar Results shown in Table 1 show that veltuzumab had no effect experiment are included in Fig. 2). on the uptake of the 111In-epratuzumab in either the tumor Combination therapy. Our initial studies were designed to or any of the normal organs tested. Results are shown only for examine a predose of the anti-CD20 IgG before the radio- day 3 after 111In injections, but essentially very similar results labeled anti-CD22 in established tumors. Because these tumors were obtained at day 5. The sizes of the tumors in both groups progressed rapidly, the unconjugated veltuzumab was admin- were very similar, as indicated. istered on day 0, with 90Y-epratuzumab given the next day. Three additional doses of anti-CD20 IgG were given, each at Discussion half the initial dose, at weekly intervals. In the untreated group, in this experiment (Fig. 2A), 12 of 15 tumors progressed to The results presented show that, in our experimental model, 90 >3.0 cm3 within 20 days of Ab injection. Three animals had the combination of Y-anti-CD22 Ab and unconjugated anti- complete regression of their established tumors with no CD20 IgG provided a markedly enhanced therapeutic effect, 90 evidence of regrowth over the 168-day monitoring period, compared with that of either of the Abs alone. Y-epratuzumab but 1 of these tumors had reached a size of 2.09 cm3 before alone did have a substantial effect, with essentially all of the 3 regressing. Those animals given veltuzumab (hA20) alone tumors regressing to a small size, many to 0 cm , but all of (Fig. 2B) or the same dose of an irrelevant 90Y-labeled IgG these tumors regrew a few weeks later. In contrast, veltuzumab (hMN-14 anti–carcinoembryonic antigen; Fig. 2E) failed to alone did not have a substantial effect: there were some control growth more than that seen in the untreated group, indications of therapy in a subpopulation of the mice treated with 3 in the veltuzumab-alone group and none in the 90Y-hMN-14 IgG group being tumor-free at 168 days. Consis- tent with the previous experiments, 90Y-epratuzumab alone produced a marked tumor regression in all mice, but the tumors regrew a few weeks later (Fig. 2C). When unconjugated veltuzumab was given with the 90Y-epratuzumab, 12 of 15 (80%) animals were cured (i.e., no visible tumor) over the 168-day observation period (Fig. 2D). The three tumors that developed grew very slowly. This is shown for 2 of the tumors in Fig. 2D, and the third tumor first appeared at day 140 after the first Ab injection, which is outside the scale of the graph shown. Figure 3 is a step graph of the data, showing the time at which the tumors reached a size of 3.0 cm3. Statistical analysis by the log-rank test showed that the combination treatment significantly improved survival (<3.0 cm3) compared with the other groups (P < 0.001). With the combination effect clearly established, a subsequent study was done to determine if lower doses of veltuzumab would be as effective. The dose of veltuzumab used in the initial injection was 1.0 (as above), 0.3, and 0.1 mg. In all cases, three Fig. 3. A step graph of the data shown in Fig. 2, showing the time at which the tumor size reached 3.0 cm3. .,control;o, 90Y-ep ratu zumab only ; 5, veltuzumab additional weekly injections at half the initial dose also were only; D, both Abs; n, 90Y-MN-14 (nonreactiveAb control). All remaining mice at day administered. All three doses were equally effective, with cure 168 had no detectable tumors.

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Given the effect of irrelevant mouse IgG2a on the blood Table 1. Effect of veltuzumab injection on tumor 111 clearance rate of the radiolabeled Abs, it is necessary to consider uptake of In-epratuzumab whether the effect of the unlabeled Abs in these experiments

Tissue Control With veltuzumab may be due to a similar nonspecific effect on the Ab blood clearance rate. The unlabeled Ab used was in fact a humanized Ramos tumor 14.75 F 3.60*12.76 F 3.80 IgG1, which is homologous to mouse IgG2a and binds to the F F Blood 10.31 1.77 9.93 2.02 g Liver 3.21 F 1.25 4.06 F 1.29 same high-affinity Fc receptor, CD64 (22). Although we have Spleen 3.05 F 0.73 3.34 F 0.54 not directly tested the effect of a nonreactive control human Kidney 3.21 F 0.38 3.86 F 0.70 IgG1 Ab in this study, there is strong evidence that the effect of Lung 4.50 F 0.96 4.31 F 1.25 the unlabeled Ab is not nonspecific, as follows: (a) As noted, F F Muscle 0.72 0.20 0.99 0.21 we are already injecting an irrelevant mouse IgG2a to block Fcg Tumor size (g) 0.63 F 0.39 gc 0.68 F 0.45 g receptors. The amount injected, 208 Ag, is more than twice as much as the dose required to effectively block uptake for at * % injected dose/g, on day 3 after Ab injection. Values shown are least 3 days (the latest time point tested) in nude mice (31), means F SDs of groups of 7 mice. cMass of tumors collected on day 3. which provides sufficient time for the radiolabeled Ab to localize to the tumor. Later time points are probably of little significance because most of the 90Y will have decayed. (b) Consistent with this statement, the experiment described in with veltuzumab, but most of the tumors continued growing, Table 1 shows that the injection of the unlabeled Ab had no unaffected by the Ab injection. This may seem unexpected effect on tumor uptake of the radiolableled Ab at day 3 or 5, because veltuzumab was very effective in a Daudi lymphoma which indicates that the amount of mouse IgG2a injected was model of disseminated disease (5) and has shown high sufficient to block the high-affinity Fcg receptor for at least therapeutic activity at relatively low doses in patients with 5 days. (c) In parallel experiments, we have recently shown that non–Hodgkin’s lymphoma (24). Evidently, therapeutic effects 90Y-veltuzumab, like 90Y-epratuzumab, induces temporary depend on the experimental model used and on factors such tumor regression.3 In this case, four weekly doses of unlabeled as the size of the tumor at the time of therapy and the dose veltuzumab again enhanced the therapeutic effect, but only schedule. Furthermore, it is always uncertain how well an if the first injection was administered 7 days after the experimental model mimics the human situation. radiolabeled Ab, rather than one day before (because injection The impressive effect of the 90Y-epratuzumab alone warrants of the same Ab one day before would directly compete with the some discussion. Despite the fact that this same conjugate has radiolabeled Ab). Most importantly, in the current context, been widely used in clinical trials (10, 11), there was no unlabeled epratuzumab was also tested, and was unable to previous description of efficacy of this particular conjugate in a enhance the therapeutic effect, demonstrating that the effect of mouse xenograft model, although there were reports of effective the unlabeled veltuzumab was antigen-specific (because both lymphoma therapy using other radiolabeled anti-CD22 Abs of these Abs are humanized IgG1). (25). It is interesting to note that the same is true for the There were previous reports of effective therapy with radiolabeled Abs to CD20 that were developed into Zevalin and unconjugated anti-CD20 Abs in mouse models, but most of Bexxar, with remarkably little published evidence of effective the models used are fundamentally different from that used therapy with radiolabeled anti-CD20 in mouse model systems here. The earlier studies generally used nude mice that were (26–28). 90Y-epratuzumab was shown to localize specifically further immunosuppressed by sublethal irradiation (30, 32). to tumor xenografts, although at relatively low levels (29), and The tumors did not grow as well if the mice were not irradiated, the same is true of anti-CD20 Abs (23). In the case of anti- which implies that there was some natural defense mechanisms CD20 Abs, the strong therapeutic effect of the unconjugated Ab that tended to prevent tumor growth. Whatever this mechanism in the model used by Buchsbaum et al. (30) may have obscured might be, which has not been established, it is likely that it any effect of the radiolabel. The lack of effective therapy in recovers in the weeks after irradiation, and that the action of mouse models did not prevent application of these Abs to the the Abs may be to enhance this natural defense mechanism. In clinic because the preclinical models that have been used to the current model, no sublethal irradiation or other type of evaluate therapy with unconjugated or radiolabeled Abs to immunosuppression was required. In many other more recent CD20, CD22, or other B-lineage antigens have not been studies (5, 33), therapy was tested on microscopic tumors, in predictive of clinical utility, usually underestimating clinical contrast to the established, macroscopic tumors used here. responses. In particular, radiolabeled Abs have seemed to be Typically, mice were treated within a few days after tumor cell less active in mouse models than has been shown clinically inoculation. The data obtained from such models may not be (27), perhaps partly because of the long path length of the applicable to macroscopic tumors, in which the Ab must leave h-particles used relative to the size of the mouse. In any case, it blood vessels and penetrate tumors, and in which effector is interesting to consider why 90Y-epratuzumab had a better mechanisms must function within the tumor environment. therapeutic effect in this study than in previous investigations. Isolated tumor cells in the blood, or in interstitial fluid, seem The only known difference is that we coinjected an unreactive likely to have sufficient effector cells close by to participate in mouse IgG2a Ab to block Ab binding to Fc receptors. Such Ab-dependent cellular cytotoxicity, but this is less likely to be binding occurs because of the very low levels of endogenous the case in a solid tumor, where there may not be sufficient IgG in nude mice and has major effects on Ab blood clearance and tumor uptake (22). However, there may also be other unknown variables in the experimental conditions. 3 Unpublished data.

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effector cells within the tumor and close to the tumor cells to (35, 36), and there are many reports of drug combinations with attack every cell. radiolabeled or unlabeled Abs (4). It is likely that optimal tumor The mechanism of action of this type of combined therapy is therapy will require a combination of multiple agents, and unknown, and currently under investigation, but some identifying the most effective protocols will require a large speculation is warranted. As tumors regress, due to irradiation amount of development work because there are many variables, from 90Y, the tumors must contain large number of macro- such as the timing and the doses. Although mouse models are phages that are phagocytizing and catabolizing the dead tumor not entirely applicable to the clinical situation, it seems possible cells. The presence of these cells in abundance would seem that the basic mechanisms that functioned in our model also will ideal for an attack via Ab-dependent cellular cytotoxicity on the occur in humans, and application of this combination therapy to few remaining viable cells that persist in the tumor. However, the clinic seems warranted. Finally, there is another factor that one factor that must be considered is that myelosuppression does not operate in our animal models but would be a major induced by the radiation used may inhibit the function of factor in humans: veltuzumab, rituximab, and other anti-CD20 effector cells. Another possibility is that the unconjugated anti- Abs deplete circulating normal B cells in humans (37). This effect CD20 Ab induced a higher level of expression of the CD22 in itself would be expected to increase the uptake of radiolabeled antigen, as was suggested by previous in vitro studies (5), but anti-CD22 Ab by the tumor because there would be less our Ab localization studies did not support this possibility. The competition from other antigen-expressing cells. specificity of epratuzumab (hLL2) uptake in Ramos tumors was established previously (29). The mechanism of action of Disclosure of Potential Conflicts of Interest unconjugated anti-CD20 IgG alone has been investigated in many experimental models (1, 2, 13, 33, 34), and seems to D.M. Goldenberg is employed by, is a director of, and has an ownership interest in require one or more accessory factors, such as various effector Immunomedics, Inc. cells (acting via Ab-dependent cellular cytotoxicity), comple- ment, and/or the production of chemokines by the Ab-treated Acknowledgments tumor (13). An advantage of combining radiolabeled Abs with unconju- We thank Rosana B. Michel, TomJackson, Dion Yeldell, Angelica Rosario, and gated Abs against different antigens was described previously Louis Osorio for technical assistance.

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M. Jules Mattes, Robert M. Sharkey, Habibe Karacay, et al.

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