Therapy of Advanced B-Lymphoma Xenografts with a Combination of 90Y-Anti-CD22 Igg (Epratuzumab) and Unlabeled Anti-CD20 Igg (Veltuzumab) M

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 ClinCancerRes2008;14(19)October1,2008 6154 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 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. www.aacrjournals.org 6155 Clin Cancer Res 2008;14(19) October 1,2008 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical 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.

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