A Humanized Monoclonal Antibody to Carcinoembryonic Antigen

Molecular Cancer Therapeutics 1559

A humanized monoclonal antibody to carcinoembryonic antigen, labetuzumab, inhibits tumor growth and sensitizes human medullary thyroid cancer xenografts to dacarbazine chemotherapy

Rhona Stein and David M. Goldenberg for MTC management and possibly other CEA-expressing neoplasms. [Mol Cancer Ther 2004;3(12):1559–64] Garden State Cancer Center, Center for Molecular Medicine and Immunology, Belleville, New Jersey Introduction Although medullary thyroid cancer (MTC) confined to the Abstract thyroid gland is potentially curable by total thyroidectomy f A variety of observations have shown that carcinoem- and central lymph node dissection, disease recurs in 30% bryonic antigen (CEA) is associated with growth and to 50% of these patients. The 5-year survival rate for all metastasis of cancers, including correlation of CEA serum types of MTC is between 78% and 91%, and the 10-year levels with poor clinical outcome, mediation of cell-cell survival rate is between 61% and 75% (1). These facts, as adhesion by CEA, and involvement of CEA in the immune well as the limited value shown by chemotherapy and recognition of tumors and apoptotic pathways. The radiation therapy in disseminated disease, have led to the purpose of this study was to investigate the effect that search for novel treatment modalities. Alternative strate- an anti-CEA monoclonal antibody (MAb) may have on the gies have been explored in recent years including gene growth of medullary thyroid cancer (MTC), a CEA- therapy approaches (2), dendritic cell vaccination (3), and expressing tumor, alone and in combination with chemo- monoclonal antibody (MAb) therapies (4–6). therapy. Antitumor effects were evaluated in a nude The expression of carcinoembryonic antigen (CEA) in mouse-human MTC xenograft model. Using the TT MTC MTC has been well documented for over 25 years and this cell line grown s.c., we compared tumor growth in has led to the exploitation of radiolabeled anti-CEA MAbs untreated mice with that of mice given the humanized for therapy of this disease. Numerous reports on the anti-CEA MAb labetuzumab or an isotype-matched control association of CEA with MTC have been published, MAb. The effects of time of administration post-tumor including detection of CEA in serum, immunohistologic injection, MAb dose response, specificity of response, and detection in paraffin sections of MTC biopsies, and combination with dacarbazine (DTIC) chemotherapy were scintigraphic detection of MTC lesions using radiolabeled studied. The humanized anti-CEA MAb, labetuzumab, has anti-CEA antibodies (7–10). The availability of a human direct, specific, antitumor effects in this model, without MTC cell line, TT (11), has allowed examination of conjugation to a cytotoxic agent. In addition, labetuzumab treatment options in a preclinical setting. TT cells express 131 90 sensitizes these tumor cells to chemotherapy with an a high level of CEA, and I- and Y-labeled anti-CEA effective drug in this model, DTIC, without increased MAbs specifically target TT tumors and cause significant toxicity. Significant delays in tumor growth were caused antitumor effects in nude mice bearing these xenografts (4). by the MAb therapy or chemotherapy alone; however, the In addition, the combination of radioimmunotherapy and combination of these agents was significantly more chemotherapy augments the antitumor effects of either effective than either agent given as a monotherapy or treatment alone in the xenograft model, without a use of an irrelevant MAb in this model. The superiority of significant increase in toxicity (12, 13). Clinically, excellent the combined modality treatment argues for the integra- targeting of MTC has been found with radiolabeled anti- tion of CEA MAb therapy into chemotherapeutic regimens CEA antibodies (5, 14), and antitumor effects have been achieved with 131I-labeled anti-CEA antibodies (6). Based on these observations and the large body of literature indicating that CEA is associated with growth and metastasis of cancers (15–17), we investigated the Received 7/29/04; revised 9/30/04; accepted 10/7/04. effect that an unlabeled anti-CEA MAb may have on the Grant support: Immunomedics, Inc., Morris Plains, New Jersey. growth of a CEA-expressing MTC tumor alone and in The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked combination with chemotherapy with dacarbazine (DTIC). advertisement in accordance with 18 U.S.C. Section 1734 solely to Here, we show that the unlabeled humanized anti-CEA indicate this fact. MAb, labetuzumab, has antitumor efficacy in MTC, Requests for reprints: Rhona Stein, Garden State Cancer Center, Center for without conjugation to a cytotoxic agent. However, Molecular Medicine and Immunology, 520 Belleville Avenue, Belleville, NJ 07109. Phone: 973-844-7012; Fax: 943-844-7020. combined therapy of the anti-CEA MAb with DTIC E-mail: [email protected] augments the antitumor effects of antibody or chemother- Copyright C 2004 American Association for Cancer Research. apy alone, without increased toxicity to the host.

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Materials and Methods Monoclonal Antibodies, Cell Lines, and Chemotherapy TT, a human MTC cell line, was purchased from the American Type Culture Collection (Rockville, MD). The cells were grown as monolayers in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum, penicillin (100 units/mL), streptomycin (100 Ag/mL), and L-glutamine (2 mmol/L). The cells were routinely passaged after detachment with trypsin- 0.2% EDTA. MN-14 is a class III anti-CEA (CD66e, CEACAM-5) MAb, reacting with CEA and unreactive with the normal cross- reactive antigen, biliary antigen, and meconium antigen (16). The construction and characterization of the humanized forms of MN-14 (labetuzumab) and LL2 (epratuzumab), the anti-CD22 MAb used here as a negative isotype-matched control, have been described previously (15, 18). Both antibodies were provided by Immunomedics, Inc. (Morris Plains, NJ). The antibodies were purified by protein A chromatography. The choice of DTIC as the chemotherapeutic agent for these studies and the dose schedule used were based on results of our earlier work (12). DTIC was purchased from Florida Infusion, Inc. (Palm Harbor, FL). In our previous study, the maximum given dose of DTIC was selected based on the dose of the drug given clinically to humans on a mg/m2 basis (300 mg/m2, days 0, 1, and 2; equivalent to 1.08 mg per dose in a 0.0036 m2 mouse). The present study was designed using submaximal doses of the drug, so that any putative advantage due to the combina- Figure 1. Labetuzumab treatment of nude mice bearing TT xenografts. Animals were given s.c. injections of TT cells and then (A) given an i.v. tion of agents would not be masked by the general injection of 0.5 mg labetuzumab 1 or 11 days later (y, untreated; n, day 1 sensitivity of this tumor model to DTIC. DTIC was given treated; E, day 11 treated) or (B) given weekly i.v. injections (0.25 mg at 75 Ag per dose, on days 2 to 4 post-tumor cell injection, labetuzumab/dose) initiated either 1, 3, or 7 days after inoculation of TT cells (y, untreated; ., day 1 treated; E, day 3 treated; n, day 7 treated). by i.p. injection. Points, means of respective treatment groups; bars, SE. In vivo Studies Tumors were propagated in female nu/nu mice (Taconic Farms, Germantown, NY) at 6 to 8 weeks of age by s.c. 11 days post-tumor cell injection or as weekly i.v. injections injection of 2 108 washed TT cells, which had been (0.25 mg labetuzumab per dose) initiated either 1, 3, or 7 propagated in tissue culture. Antibodies were injected i.v., days after inoculation of TT cells. Figure 1A shows the via the lateral tail vein, into the tumor-bearing animals. tumor growth curves of animals treated with a single Details on the quantities of antibodies injected and the time 0.5 mg dose of labetuzumab per mouse compared with of administration are indicated in the Results section for untreated controls. The untreated group contained 16 each study. Results are given as the mean F SE. Tumor animals; the two treatment groups contained 10 animals size was monitored by weekly measurements of the each. A significant growth delay was observed between length, width, and depth of the tumor using a caliper. the untreated group and the group treated 1 day post- Tumor volume was calculated as the product of the three tumor injection. Significant differences in the mean tumor measurements. Statistical comparisons were made using sizes (P < 0.05) were observed from days 32 to 93. Between Student’s t test to compare tumor volumes and area under days 32 and 60, there was a 64% to 70% inhibition of tumor the growth curves. Toxicity was evaluated by weekly mea- size in the labetuzumab-treated group compared with the surement of body weights. Animal studies were done untreated animals. There were no significant differences under protocols approved by the Institutional Animal between the mean tumor sizes in the day 11 group and Care and Use Committee. untreated animals. Significant delay in tumor growth was also seen by t test analysis of the area under the growth curves: P < 0.05 for the untreated group compared with Results the group treated 1 day following tumor injection but not To study the effect of labetuzumab on the growth of TT for the group treated 11 days following tumor injection. tumors in nude mice, the MAb was given either as a single Similar results were observed when labetuzumab was i.v. injection (0.5 mg labetuzumab per dose) given 1 or given as 0.25 mg weekly doses, as seen in Fig. 1B. Groups

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of seven to eight animals were studied. Significant differences in mean tumor sizes (P < 0.05) between the untreated group and all three treatment groups were observed. However, the difference in mean tumor size between the untreated mice and the day 7 treatment group was only significant at one time point, day 28. Day 1– treated mice yielded significant differences from 21 to 77 days, and day 3–treated mice yielded significant differences from 21 to 70 days. t Test analysis of the area under the growth curves indicated significant growth inhibition for the groups treated with labetuzumab either 1 or 3 days after TT cell administration compared with the untreated group. However, this analysis did not reach the 95% confidence limit for a difference between the untreated group and the group treated on day 7 (P = 0.057 at 5 weeks). No significant differences in body weights were caused by any of the treatments. Figure 3. Effect of labetuzumab dose. Animals were given i.v. injections Figure 2 summarizes the results of a study on the of increasing doses of labetuzumab 1 day after s.c. injection of TT cells. Points, means of respective treatment groups (y, untreated; ., 0.125 mg; specificity of the antitumor response. The effect of o, 0.25 mg; n, 0.50 mg; , 1.0 mg; E, 2.0 mg); bars, SE for the un- unlabeled labetuzumab on the growth of TT tumors in treated group and the group that received 0.50 mg labetuzumab per nude mice was compared with that of an isotype-matched mouse. control humanized MAb, epratuzumab (anti-CD22 IgG1), and the murine anti-CEA MAb MN-14. MAbs (0.5 mg per mouse) were given i.v. 1 day after inoculation of the TT cells followed by three additional weekly doses of 0.5 mg (insignificant) increase in growth rate. For example, at per mouse. Groups of 15 animals were studied. The growth day 37, 87% of the tumors treated with labetuzumab inhibition, without effect on body weight, shown in the were <0.5 cm3 compared with 40% of the untreated group experiment summarized in Fig. 1 was confirmed in this and 29% of the epratuzumab-treated group. t Test analysis study. Significant differences in mean tumor sizes (P < of the area under the growth curves showed significant 0.05) between the labetuzumab and the untreated groups differences (P < 0.05) between the untreated group and the were observed starting at day 23. At day 37, the mean groups treated with either labetuzumab or murine MN-14 tumor volume in the group treated with labetuzumab was but not the group treated with epratuzumab. In addition, 42.7% of the untreated control animals. Treatment with the labetuzumab group was significantly different from the murine MN-14 yielded results similar to the labetuzumab. epratuzumab group but not from the murine MN-14- Treatment with epratuzumab (anti-CD22 humanized MAb) treated animals. did not slow tumor growth; instead, there was a small The effect of dose of labetuzumab on the growth of TT tumors in nude mice was also evaluated. Antibody doses were given 1 day after the TT cells, then weekly until the termination of the study. Weekly doses ranged from 0.125 to 2.0 mg labetuzumab per mouse in groups of six mice. Significant differences in mean tumor sizes and area under the growth curves between the untreated group and all treatment groups were observed (Fig. 3), again without significant changes in body weight. For example, between days 21 and 49, mean tumor volume in the two lowest labetuzumab treatment groups were 27% to 40% of the size of tumors in the untreated animals. Treatment with the lower doses, 0.125 and 0.25 mg, seemed to be more effective than treatment with the higher doses, although the difference did not reach statistical significance. Thus, dose-dependent results were not observed in this study. Indeed, dose-dependent results have not been commonly observed in antibody treatment studies, even clinically, so this should not be unexpected. Combining naked anti-CEA MAb therapy and chemo- Figure 2. Specificity of treatment. Animals were given s.c. injections of therapy was evaluated by comparing the growth of TT in TT cells and either left untreated or given an i.v. injection of MAb (0.5 mg) 1 day later. Points, means of respective treatment groups (y, untreated; untreated mice with those treated with DTIC alone, n, labetuzumab; E, murine MN-14; , human LL2); bars, SE. labetuzumab alone, or a combined treatment of DTIC with

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labetuzumab. Animals were given i.p. injections of labetu- Discussion zumab at 100 Ag per dose on days 2 to 5, 7 to 11, and 15 and CEA, a member of the immunoglobulin supergene family then every 7 days. Groups of 10 animals were studied. (19), was first described as a tumor marker by Gold and DTIC was given at 75 Ag per dose on days 2 to 4. In the Freedman (20). CEA was originally thought to be a tumor- combined modality group, treatment with the anti-CEA specific antigen of colorectal cancer. However, it was later MAb was initiated on the same day as the first dose of found to be present in a diverse number of carcinomas, DTIC. Growth curves of TT tumors in mice given these benign tumors, and diseased tissues as well as in the treatment regimens are shown in Fig. 4. All of the treat- normal human colon (21). CEA has been shown to mediate ment groups had significant improvement in efficacy cell-cell adhesion through homotypic and heterotypic compared with the untreated animals, without increased interactions, which in turn have implicated a role for toxicity. At 7 weeks, P values for t test of area under CEA in various aspects of tumorigenesis (22). In one report, curve are 0.0017, 0.0374, and 0.0002 for DTIC-only, CEA levels have been indicated to have prognostic labetuzumab-only, and DTIC + labetuzumab, respectively, significance with a more favorable prognosis in CEA- compared with untreated animals. Combined therapy of positive tumors compared with that of CEA-negative the naked anti-CEA MAb with DTIC augments the undifferentiated tumors (23). However, other articles report antitumor effects of either antibody or chemotherapy the opposite, a more aggressive phenotype with increased alone. At 7 weeks, P values for t test of area under curve metastases was associated with high levels of CEA in of the combined treatment were 0.0026 and 0.0002 preclinical models (24, 25). The role of CEA in hepatic compared with the DTIC-only and labetuzumab-only metastasis may be based on the ability of circulating CEA to treatment groups, respectively. Eight of 10 mice treated induce Kupffer cells to release cytokines. These cytokines in with DTIC plus labetuzumab exhibited no palpable tumor turn may activate liver endothelial cells, enabling them to at 7 weeks post-treatment compared with only 1 of 10 in the bind to circulating CEA-positive tumor cells (26). CEA has DTIC-only group and none in the labetuzumab-only and also been implicated in the immune recognition of tumors, untreated groups. Mean tumor volumes at 7 weeks were possibly by interfering in natural killer cell-tumor cell F F F 0.018 0.012, 0.284 0.062, 0.899 0.172, and 1.578 adhesion (27), as well as having an effect on cellular F 3 0.303 cm for the DTIC plus labetuzumab, DTIC, differentiation, probably by interfering with normal inter- labetuzumab, and untreated groups, respectively. No cellular adhesion forces (28). significant differences in body weights were caused by The many roles attributed to CEA in neoplasia suggest any of the treatments. Thus, labetuzumab yields antitumor that targeting CEA with an anti-CEA MAb may affect efficacy in MTC, without conjugation to a cytotoxic the growth of CEA-expressing tumors by interfering with agent. However, combined therapy of the naked anti- its function(s). Indeed, antibody binding to receptor mol- CEA MAb with DTIC augments the antitumor effects of ecules on tumor cells has been shown to affect the behav- antibody or chemotherapy alone, without increased ior and proliferation of these tumor cells. For example, host toxicity. antitumor efficacy of unlabeled MAbs reacting with tumor-associated antigens has been reported in non- Hodgkin’s lymphoma with anti-CD20 (rituximab; ref. 29) and anti-CD22 (epratuzumab; ref. 30) MAbs as well as in colon cancer using the anti-epithelial glycoprotein-2 MAb, 17-1A (31), and A33 (32), in breast cancer using anti-HER-2 (33), and in colonic and other tumors with anti–epidermal growth factor receptor (34) MAbs. These unconjugated MAbs may inhibit tumor growth by blocking intracellular signaling or other biological activities of their respective antigens or by stimulating natural immunologic functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-mediated lysis. It is also interest- ing that most of these antibodies seem to have their greatest effect in combination with anticancer drugs (32–35). Common mechanisms of action among the various antibodies given with drugs include cell cycle arrest, potentiation of apoptosis, and inhibition of angiogenesis, resulting in augmentation of the antitumor effects of chemotherapy Figure 4. Effect of combination treatment, DTIC + labetuzumab. Animals were given s.c. injections of TT cells and either left untreated and, in some cases, radiation therapy (34). In the case of (y) or given an i.p. injection of labetuzumab at 100 Ag per dose on days CEA antibodies, such as labetuzumab studied here, a con- 2 – 5, 7 – 11, and 15 and then every 7 days (o); DTIC given at 75 Ag per tribution in terms of apoptosis enhancement needs to be dose on days 2 – 4 (E); or combined modality treatment consisting of the considered because of the observation that CEA plays a same labetuzumab and DTIC schedules with the labetuzumab treatment initiated on the same day as the first dose of DTIC (4). Points, means of regulatory role in apoptosis (36). Using a CEA-targeted respective treatment groups; bars, SE. ribozyme in human colon cancer cells to regulate CEA

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levels, it has been reported that CEA does not affect cell However, similar to anti-HER-2 and anti–epidermal cycle or proliferation but does protect the cells from growth factor receptor MAbs (37, 38), labetuzumab was undergoing apoptosis under various conditions, including shown to inhibit tumor cell proliferation by ADCC (17). The confluent growth, UV light, IFN therapy, and treatment lack of complement-mediated cytotoxicity against tumor with 5-fluorouracil (36). cells in vitro may be explained by the high levels of In this study, we show that labetuzumab, an antibody complement regulatory factors, CD55 and CD59, in these specific to CEA, has antitumor efficacy in MTC without colon cancer cell lines (17), which are known to inhibit conjugation to a cytotoxic agent and can sensitize these complement-mediated cytotoxicity (39, 40). Thus, the work tumor cells to chemotherapy with an effective drug in this of Blumenthal et al. (17) suggests that labetuzumab induces model. The current experiments show that CEA-binding effector-cell function against CEA-positive human colonic antibodies have direct antitumor effects and can also tumor cells as shown by the in vitro ADCC results and chemosensitize human tumor cells in vivo. In nude mice the importance of WBCs for in vivo activity. bearing human MTC xenografts, labetuzumab was shown In contrast, other anti-CEA MAbs have been reported to to significantly delay tumor growth. Differences in mean induce complement-mediated cytotoxicity as well as ADCC tumor sizes between the anti-CEA MAb–treated and the activity against a CEA-expressing gastric cancer cell line untreated groups were observed beginning at 3 weeks (41). However, CD55 and CD59 levels were not measured and lasting at least 2 months. The effect of delaying the in this cell line. These authors also found that their fully treatment with naked anti-CEA MAb was studied up to human anti-CEA MAb inhibited tumor growth in vivo in 11 days after tumor cell implantation. The antibody human gastric cancer xenografts grown in severe combined yielded statistically significant inhibition of tumor growth immunodeficient mice (41), thus agreeing with the results when given up to 3 days post-tumor cell inoculation but reported here and by Blumenthal et al. (17). not when given 7 or 11 days after the tumor cells were An important role of CEA in tumorigenic potential was transplanted. Treatment with an isotype-matched non– also shown by transfection of human colon cancer cells CEA-binding control MAb, epratuzumab (anti-CD22 hu- with a CEA antisense-expressing vector (42). Parental cells manized MAb), did not slow tumor growth, demonstrating with high CEA expression were highly tumorigenic, that the effect of labetuzumab on tumor growth is antigen whereas clones with decreased CEA expression showed specific. The observation that the effect is greatest when considerably lower tumorigenicity. It seems likely, there- treatment is given at an early time point after injection of fore, that in addition to functioning by stimulating natural tumor suggests that the antibody is most active during immunologic functions (ADCC and/or complement-medi- early spread of the tumor cells. Although this remains to ated cytotoxicity), anti-CEA MAbs may have antitumori- be proven, we find this possibility intriguing and of genic effects because they reduce or block CEA and thus possible significance in terms of blocking the establish- may overcome the putative role of CEA in protecting tumor ment of new metastatic sites. In addition, the anti-CEA cells from apoptotic stimuli, as experienced with antisense MAb was able to enhance the effect of chemotherapy; the oligonucleotides (42). It is possible that the chemosensitiza- combination of chemotherapy with DTIC and anti-CEA tion we observed for the anti-CEA antibody may also be MAb treatment was significantly more effective than either caused by overcoming inhibition of apoptosis by CEA, treatment alone. Of the mice given the combined modality including apoptosis induced by certain cytotoxic drugs. treatment, 80% had complete responses compared with Thus, the observations of the antiproliferative effects of 10% in the chemotherapy-only group and none in the CEA MAb and its potentiation of chemotherapy has been untreated and MAb-only groups. Thus, unlabeled labetu- shown in MTC as well as in other cancer model systems zumab has shown antitumor efficacy in MTC without with various levels of CEA expression and with anticancer conjugation to a cytotoxic agent, and combined therapy of drugs with diverse mechanisms of action. These effects on the naked anti-CEA MAb with DTIC augments the antitumor growth and metastasis are supportive of the general tumor effects of antibody or chemotherapy alone, without view of the role of CEA in tumor biology, despite the need increased host toxicity. for additional studies to fully understand the mechanisms These studies corroborate a recent report of Blumenthal underlying these observations. The superiority of the com- et al. (17), in which in vivo antiproliferative and antimeta- bined modality treatment argues for the integration of static effects of labetuzumab were observed in CEA- CEA MAb therapy into chemotherapeutic regimens for expressing human colonic carcinoma models. Significant MTC management. Clinical trials are needed to assess these survival increases were obtained when the animals were principles in patients with MTC and other CEA-expressing given granulocyte-macrophage colony-stimulating factor to cancers. increase their peripheral WBC counts or when the tumors were treated with IFN to up-regulate CEA expression. In Acknowledgments addition, labetuzumab potentiated the effect of two com- We thank Susan Chen for her excellent technical assistance. mon drugs used in colorectal cancer, 5-fluorouracil and CPT-11. Blumenthal et al. (17) did not observe induction References of apoptosis or complement-mediated cytotoxicity by 1. Randolph GW, Maniar D. Medullary carcinoma of the thyroid. Cancer labetuzumab in in vitro studies in colon cancer cell lines. Control 2000;7:253 – 61.

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Mol Cancer Ther 2004;3(12). December 2004

Downloaded from mct.aacrjournals.org on September 24, 2021. © 2004 American Association for Cancer Research. A humanized monoclonal antibody to carcinoembryonic antigen, labetuzumab, inhibits tumor growth and sensitizes human medullary thyroid cancer xenografts to dacarbazine chemotherapy

Rhona Stein and David M. Goldenberg

Mol Cancer Ther 2004;3:1559-1564.

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