Combined modality radioimmunotherapy
Promise and peril
Article first published online: 12 FEB 2002
Copyright © 2002 American Cancer Society
Supplement: Eighth Conference on Radioimmunodetection and Radioimmunotherapy of Cancer
Volume 94, Issue Supplement 4, pages 1320–1331, 15 February 2002
How to Cite
Burke, P. A., DeNardo, S. J., Miers, L. A., Kukis, D. L. and DeNardo, G. L. (2002), Combined modality radioimmunotherapy. Cancer, 94: 1320–1331. doi: 10.1002/cncr.10303
- Issue published online: 12 FEB 2002
- Article first published online: 12 FEB 2002
- Manuscript Accepted: 14 NOV 2001
- Manuscript Received: 31 OCT 2001
- National Cancer Institute. Grant Numbers: UO1 CA61641, PO1 CA-47829
- RGD peptide;
- bcl-2 antisense;
- breast carcinoma
Single-agent radioimmunotherapy (RIT), although potentially useful for slowing solid tumor growth, has not been effective in curing aggressive tumors, such as breast cancer. These cancers typically have p53 mutations and are less susceptible to apoptosis, the apparent mechanism of cell death from low dose-rate radiation. Thus, synergistic or combined modality radioimmunotherapy (CMRIT) agents are needed to increase radiosensitivity for therapeutic enhancement without additive toxicity.
To assess synergy in CMRIT in a breast cancer xenograft model, we evaluated RGD peptide EMD 121974, an inhibitor of αvβ3 integrin; paclitaxel, an antimicrotubule; IMC-C225, a monoclonal antibody to epidermal growth factor receptor (EGFR); and bcl-2 antisense oligonucleotide G3139. Groups of mice received 90Y-DOTA-ChL6 in combination with each agent. Tumor size, survival, and blood counts were monitored for efficacy and toxicity. Immunopathologic evaluation of apoptosis was performed at selected time points after RIT and RIT + RGD CMRIT.
CMRIT with RGD peptide increased apoptosis and resulted in 57% cures, compared with 0% cures with RIT alone. CMRIT with paclitaxel after RIT increased cures to 88%, compared with 25% cures with RIT before paclitaxel administration. CMRIT with IMC-C225 resulted in up to 20% cures if given before RIT. A time-dependent increase in toxicity was observed with IMC-C225 after RIT. CMRIT with bcl-2 antisense G3139 resulted in no cures and an increased rate of regrowth compared with RIT alone.
Some combined modality therapies resulted in higher numbers of cures, while others decreased cures and responses and increased toxicity compared with RIT alone. These results support the potential for CMRIT but illustrate the complexity of predicting the efficacy and toxicity and the importance of the relationship between dose and sequence of administration. Cancer 2002;94:1320–31. © 2002 American Cancer Society.
Systemic tumor-targeted radioimmunotherapy (RIT) has the potential to deliver radiation therapy to metastatic cells throughout the body while maintaining a high therapeutic index.1 However, single-agent RIT has not proven highly effective in patients with solid tumors. Thus, new RIT strategies to increase efficacy yet limit toxicity are currently being investigated.2 Combined modality radioimmunotherapy (CMRIT) is designed to enhance the cascade of molecular events required for apoptotic tumor cell death resulting from the continuous low dose-rate radiation from RIT.3
In normal cells, DNA damage by radiation leads to increased p53 expression, which may result in cell cycle arrest or apoptosis.4 Wild-type p53 thus acts as a proapoptotic protein. However, p53 is mutated at a frequency of 29% in primary breast carcinoma5 and expression of mutant p53 is associated with high tumor proliferation rate and poor prognosis.6 Furthermore, p53 binds to a negative response element in the bcl-2 gene, leading to decreased bcl-2 expression.7 Increased expression of bcl-2, which may prevent apoptotic cell death,8 has also been implicated in the etiology of many tumors and the resistance of cells to cancer therapy.9–13 Bcl-2 is highly expressed in up to 70% of low-grade breast cancers and in 50% of breast cancers overall.14, 15 Abnormal p53 or bcl-2 expression by tumor cells may result in decreased response to radiation or chemotherapy in many cancers.
Agents that affect the cell cycle, such that tumors are sensitized to the effects of radiation in spite of these abnormalities, are thus of particular interest for use in CMRIT. Several recent studies illustrate the potential for synergy when RIT is combined appropriately with another agent16–20 (O'Donnell, personal communication). However, not all agents demonstrating potential as single agents can be combined with RIT for increased efficacy. When an agent is part of a combination in CMRIT, both the type of agent and the timing of delivery become important in determining the outcome. We have previously presented data showing the importance of the timing of delivery of both paclitaxel and the monoclonal antibody (MAb) IMC-C225 against the epidermal growth factor receptor (EGFR) when combined with RIT.21, 22 In this study, we further illustrate both potential (increased efficacy) and problems (increased toxicity and decreased responsiveness to RIT) in investigations of specific agents and their sequence and timing in CMRIT. In our CMRIT studies, we used the RGD peptide EMD 121974 and the bcl-2 antisense oligonucleotide G3139, and compared these with key results from our paclitaxel and IMC-C225 studies. These studies utilized 90Y-DOTA-ChL6 in mice bearing breast cancer HBT 3477 xenografts in CMRIT with agents possessing properties indicating that they could render tumor tissue more sensitive to radiation-induced apoptosis.
Recent therapy studies in which chemotherapy agents have been successfully combined with nontoxic antiangiogenic agents to produce synergy23, 24 have suggested the potential of antiangiogenic agents for CMRIT. We reported our results demonstrating increased uptake of radiolabeled antibodies in xenograft tumors following 1-hour pretreatment with a cyclic RGD peptide (cRGDf-ACHA, EMD 270179),25 which binds αvβ3 integrin receptors expressed on neovasculature.26–28 Blockade of the αvβ3 receptor reportedly induces apoptosis in endothelial cells of newly developing vasculature, and has been associated with inhibition of tumor growth on chick chorioallantoic membrane and in mouse xenografts.28, 29 EMD 121974 may potentiate the effects of RIT by both increasing initial uptake of RIT by tumor and inducing apoptosis in the tumor vasculature.
Paclitaxel causes microtubular dysfunction, resulting in interference with spindle apparatus formation during cell division and interference with cell shape, anchorage, and signaling between the surface receptors and the nucleus. As a result of the microtubular dysfunction, cells are blocked in the G2/M phase of the cell cycle, which may result in increased sensitivity to radiation.31 Paclitaxel also induces bcl-2 hyperphosphorylation and functional inactivation, resulting in facilitated apoptotic death, particularly in malignancies with p53 mutations.10
Enhanced expression of EGFR has been found in most human carcinomas and in drug-resistant cancer cell lines.18, 31 Ionizing radiation may activate the EGFR tyrosine kinase and release transforming growth factor–α (TGF-α), a ligand of EGFR, leading to further activation of this receptor.18, 32, 33 Activation of EGFR could act as a mechanism for cancer cells to escape radiation-induced apoptosis, and thus could potentially inhibit RIT-induced apoptosis. Blocking EGFR may potentiate the effect of radiation34 and may also inhibit angiogenesis.35 MAb IMC-C225, a mouse-human chimeric antibody that blocks EGFR, has been shown to enhance the effect of external beam radiation on tumors in mice and thus is considered a promising agent for CMRIT.18, 34, 35
Down-regulation of bcl-2 protein expression with antisense oligonucleotides may modulate expression of bcl-2, inhibit tumor growth, and potentially resensitize cells to chemotherapy or radiation.36 The antisense oligonucleotide G3139, when used as a single agent, has resulted in complete remission in nude mice inoculated with human follicular lymphoma37 and strong inhibition of Merkel cell tumor growth in SCID mice associated with demonstrable down-regulation of bcl-2 expression and increased apoptosis.36
Each of these agents has qualities indicating potential for increased efficacy when combined with RIT. In practice, however, synergy does not always occur or is less than optimal. The CMRIT studies presented herein provide insights into the various pitfalls of CMRIT development and the timing, sequence, and dose dependency likely to achieve successful synergy.
Materials and Methods
Carrier-free yttrium-90 (90Y) (Pacific Northwest National Laboratory, Richland, WA, or New England Nuclear, Boston, MA) was purchased as chloride in 0.05 M HCl. Chimeric L6 (ChL6), a human-mouse antibody chimera (Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA), reacts with an integral membrane glycoprotein highly expressed on human breast, colon, ovary, and lung carcinomas.38–41 Cyclic RGD (cyclo-[Arg-Gly-Asp-D-Phe-(N-me)-Val]) (EMD 121974) is a selective αvβ3 antagonist with IC50 values in the lower nanomolar range. Peptide synthesis and characterization were performed as previously described.27 Paclitaxel (Taxol, Bristol-Myers Squibb, Princeton, NJ), a natural product from the taxane group with antitumor activity and novel antimicrotubule properties,30, 42 was obtained as a nonaqueous solution and diluted in 0.9% sodium chloride. IMC-C225 (a gift from ImClone Systems Incorporated, Somerville, NJ) is a chimeric anti-EGFR human-mouse MAb (immunoglobulin IgG1) with a half-life of 72 hours,43 which may alter the cell cycle and activate apoptotic pathways in cells with functional EGFR by blocking EGF or TGF-α from binding.44 Bcl-2 antisense oligonucleotide phosphorothioate G3139, with sequence 5′-TCTCCCAGCGTGCGCCAT-3′, was a gift from Genta, Incorporated (Berkeley Heights, NJ). It was reconstituted from lyophilized powder in 20 mM Tris-HCl (tris[hydroxymethyl]aminomethane) buffer, pH 7.0, which was further diluted in saline. G3139 is an 18-mer full-phosphorothioate oligonucleotide with sequence antisense to the first six codons of the open reading frame of bcl-2, which has shown efficacy in several preclinical studies.36, 45 G3139 has been shown to rapidly distribute to all tissues and concentrate in the liver, kidneys, and bone marrow after slow infusion.45 Dose levels for all agents combined with RIT were chosen to create synergy without undue toxicity, based upon studies as single agents in other tumor models.
HBT 3477, a human breast adenocarcinoma cell line, was obtained from Bristol-Myers Squibb Pharmaceutical Research Institute (Seattle, WA). More than 70% of HBT 3477 cells stained intensely with L6.46, 47 In HBT 3477 cells, p53 has been shown to be mutant, with a deleted region detecting double-stranded DNA breaks. Bcl-2 is expressed by HBT 3744 and is down-regulated by RIT within 6 hours.47
ChL6 was conjugated to 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) and radiolabeled with 90Y as previously described1, 48, 49 to prepare 90Y-DOTA-ChL6 (90Y-DOTA-peptide-000-ChL6). 90Y-DOTA-ChL6 was given as a single dose of 260 μCi 90Y-DOTA-ChL6. We chose 260 μCi because previous studies in our laboratory have demonstrated efficacy in the HBT3477 tumor model, typically with 100% response of tumors with no cures and no deaths; thus, this is the practical MTD.1
Female (age 7–10 weeks) athymic Balb/c nu/nu mice (Harlan Sprague Dawley, Inc., Frederick, MD), were maintained according to University of California animal care guidelines. HBT 3477 cells (3.0 × 106) harvested in log phase were injected subcutaneously into one or both sides of the abdomen. Injection of RIT was by tail vein in all studies, with RIT injection day designated as “Day 0” of each study. All other agents (RGD peptide, paclitaxel, IMC-C225, and bcl-2 antisense) were delivered by intraperitoneal (i.p.) injection.
RGD study treatment groups
Groups consisted of untreated (5 mice); RIT only (260 μCi 90Y-DOTA-ChL6) (5 mice); RGD only, with 250 μg RGD peptide EMD 121974 given as 6 doses on Days 0, 2, 4, 6, 8, and 10 (13 mice); and RIT + RGD, with 250 μg RGD peptide EMD 121974 given on Day 0 starting 1 hour prior to RIT (260 μCi 90Y-DOTA-ChL6), followed by 5 more doses of RGD peptide on Days 2, 4, 6, 8, and 10 (13 mice). The RGD peptide dose of 250 μg was chosen based on data showing a biologic effect with no toxicity at a similar dose delivered over 24 hours when RGD peptide was administered as a single agent to mice.50
RGD cellular apoptosis groups
Groups consisted of untreated (4 mice); RGD only, given a single dose of RGD peptide EMD 121974 (250 μg) and sacrificed 24 hours after peptide injection (2 mice); RIT only, given 260 μCi 90Y-DOTA-ChL6 and sacrificed 24 hours after RIT (2 mice); and RIT + RGD, with RGD peptide EMD 121974 (250 μg) given 1 hour prior to 260 μCi 90Y-DOTA-ChL6, followed by sacrifice at 24 hours (2 mice). After sacrifice, the tumors were removed, cut in half, frozen in O.C.T. medium (Tissue-Tek, Miles, Inc., Elkhart, IN), and stored at −70 °C until sectioning.
Paclitaxel study treatment groups
Groups consisted of untreated (10 mice); RIT only (260 μCi 90Y-DOTA-ChL6) (10 mice); paclitaxel only (600 μg i.p.) (5 mice); and 4 combination groups of RIT + paclitaxel, with paclitaxel (i.p., 600 μg) given 1 time only at 48 hours before (3 mice), 24 hours before (5 mice), 24 hours after (10 mice), or 48 hours after (4 mice) RIT (260 μCi 90Y-DOTA-ChL6, 315 μg). The dose of 600 μg paclitaxel was selected to sensitize with RIT at a conservative level equivalent to 84 mg/m2 in humans.21, 51 Doses of 100–250 mg/m2 are used in the treatment of women with metastatic breast cancer for single-agent and multimodality regimens.52, 53
IMC-C225 study treatment groups
Groups consisted of untreated (10 mice); RIT only (260 μCi 90Y-DOTA-ChL6) (10 mice); unlabeled IMC-C225 (350 μg i.p.) (9 mice); and 3 combination groups of RIT + IMC-C225, with IMC-C225 (350 μg or 150 μg i.p) given 1 time only at 24 hrs before (5 mice and 10 mice, respectively) or 24 hrs after (5 mice, 350 μg) RIT (260 μCi 90Y-DOTA-ChL6). IMC-C225 (250 μg) has been shown to suppress outgrowth of melanoma tumor in mice.55 Higher doses have also been used in mice without toxic effect.55
Bcl-2 study treatment groups
Groups consisted of untreated (4 mice); RIT only (260 μCi 90Y-DOTA-ChL6) (5 mice); bcl-2 antisense only (12.5 μg/g, i.p. for 14 days) (5 mice); and RIT + bcl-2, with bcl-2 antisense oligonucleotide given daily i.p. for 14 days, beginning 2 days before delivery of 260 μCi 90Y-DOTA-ChL6 (10 mice). G3139 has been shown to result in suppression of lymphoma when given to mice at a dose of 100 μg/day.56 Doses of 10 mg/kg are well tolerated in primates, while deaths and high toxicity in mice have been observed with doses of 50 mg/kg (1000 μg/day in a 20-g mouse).57 The dose chosen for synergy was 250 μg/day for a 20-g mouse, or approximately 50% of a theoretic maximum tolerated dose.
All mouse models
Weights and blood counts were measured 2–3 times per week for 12 weeks postinjection or until death. Tumors were measured with calipers in 3 orthogonal diameters 3 times per week. Tumor volume was calculated using the formula for hemiellipsoids.58 Blood samples were collected from tail veins using 2-μL microcapillary pipets. Samples from mice within a dose group were pooled and diluted 1:200 in phosphate-buffered saline (0.9% saline/10 mM sodium phosphate, pH 7.6) for red blood cell counts, 1:100 in 1% (weight per volume) ammonium oxalate for platelet counts, or 1:20 in 3% (weight per volume) acetic acid for white blood cell counts.59
Initial tumor volume was defined as the volume on the day before treatment. Mean tumor volume was calculated for each group on each day of measurement. Tumors that had completely regressed were considered to have a volume of zero. Tumor responses were categorized as follows: cure, tumor disappeared and did not regrow by the end of the study (≥ 84 days); complete regression, tumor disappeared for at least 7 days but later regrew; partial regression, tumor volume decreased by 50% or more for at least 7 days but then regrew.
TUNEL analysis of total and endothelial apoptosis
Tumors were cut into 10-μm sections onto Fisher superplus slides (Fisher, Pittsburgh, PA), air-dried for 1 hour, and frozen at −70 °C until terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis with an ApopTag Red kit (rhodamine used as label; Intergen, Purchase, NY) following the manufacturer's instructions. After labeling, the slides were rinsed and incubated overnight at 4 °C with a rat antimouse MAb against CD31 at 1:100 dilution (Pharmingen, San Diego, CA) to identify endothelial cells. Slides were rinsed and incubated for 1 hour with an antirat antibody linked to FITC (1:50 dilution) (Pharmingen). Slides were rinsed, dipped briefly in DAPI (4,6-diamidino-2-phenylindole, 0.2 μg/mL) for background stain, rinsed again, and mounted, followed by storage in the dark at 4 °C until quantitation.
Quantitation of total apoptosis and endothelial apoptosis
An Olympus microscope equipped with a Chroma Pinkle Filter Set (Chroma, Brattleboro, VT), with excitation filters for UV, FITC, and rhodamine and dual/triple bandpass filters to allow simultaneous viewing of multiple wavelengths, was used to quantify 6 randomly chosen ×600 fields (150,000 μm2/field) in nonnecrotic regions of each section. Fields were chosen to cover the entire viewing area using the DAPI label. Total apoptosis (rhodamine label on TUNEL-positive nuclei) was determined by the number of positive nuclei per field, while endothelial apoptosis was determined in the same fields using a dual bandpass filter to count cells labeled with both FITC (CD31) and rhodamine (TUNEL) (Fig. 1).
Comparisons of response among HBT-paclitaxel and IMC-C225 groups were done by the method of Cockran-Mantel-Haensel as previously described.60 RGD apoptosis and RGD and bcl-2 final tumor volumes were analyzed by the student t test or analysis of variance (ANOVA) (Fisher PLSD), using STATview software as appropriate, with P < 0.05 considered significant.
Mortality of Mice with HBT Tumors
The mortality of mice with HBT tumors within 7 weeks of tumor implant or within 30 days of the beginning of the studies was 10% (10 mice) for untreated mice. Mice that received unlabeled ChL6 antibody demonstrated a similar mortality rate of 11% (18 mice).23 Likewise, mice that received only IMC-C225 had a mortality rate of 10.5% (19 mice) and mice that received only paclitaxel had an 11% mortality rate (9 mice).22 Mice that received only RGD peptide or only bcl-2 antisense had 0% mortality (18 mice). Thus overall, mice not treated with RIT or CMRIT experienced a mortality rate of 8% (74 mice).
Tumoricidal Effect of RGD Peptide with Radioimmunotherapy
Tumors in untreated mice and mice that received RGD peptide alone grew without interruption. No effect of RGD peptide on tumor volume was apparent. The final tumor volumes (mm3 ± standard error) for these two groups (not including tumor size in animals that died of toxicity within 30 days) were 1204 ± 357 (no treatment) and 1301 ± 100 (RGD only). Both groups that received RIT had decreased tumor growth by 5 days after RIT, but at 37 days after RIT the tumors of mice that received RIT alone resumed a rapid growth rate, while tumors on animals that received RIT+RGD maintained a low growth rate through study termination. This resulted in final tumor volumes at the end of the 84-day trial of 78 ± 43 mm3 (RIT+RGD) compared with 1021 ± 358 mm3 (RIT), which were significantly different (ANOVA). Although 100% of mice in both the RIT and the RIT+RGD treatment groups responded to treatment, the number of cures increased from 0% for RIT-only mice in this study, and from 7% for RIT-only mice in all 4 studies, to 57% for RIT+RGD mice (Table 1). Although a preliminary study, these results suggest synergy of RGD peptide combined with RIT.
|Agent||HBT 3477 treatment groups||Time CMRIT||Tumors/mice||Response rate (%)b||cCure (%)|
|RIT onlya||260 μCi 90Y-DOTA-ChL6||30/17||87||7|
|EMD||250 μg RGD peptide (6 doses on alternate days)||13/13||0||0|
|260 μCi 90Y-DOTA-ChL6+250 μg RGD peptide (6 doses on alternate days)||−1 hr first dose||7/7||100||57|
|Paclitaxel||Paclitaxel 600 μg||6/5||0||0|
|260 μCi 90Y-DOTA-ChL6+paclitaxel 600 μg||−48 hrs||4/3||75||25|
|260 μCi 60Y-DOTA-ChL6+paclitaxel 600 μg||−24 hrs||9/5||100||22|
|260 μCi 90Y-DOTA-ChL6+paclitaxel 600 μg||+24 hrs||18/10||100||50|
|260 μCi 99Y-DOTA-ChL6+paclitaxel 600 μg||+48 hrs||8/4||100||88|
|IMC-C225||IMC-C225 350 μg||14/9||36||0|
|260 μCi 90Y-DOTA-ChL6+IMC-C225 350 μg||−24 hrs||9/5||78||11|
|260 μCi 90Y-DOTA-ChL6+IMC-C225 150 μg||−24 hrs||15/10||100||20|
|260 μCi 90Y-DOTA-ChL6+IMC-C225 350 μg||+24 hrs||7/5||29||0|
|G3139||bcl-2 antisense 12.5 μg/g daily for 14 days||10/5||0||0|
|260 μCi 90Y-DOTA-ChL6+bcl-2 antisense||−48 hrs||18/9||95||0|
|125 μg/g daily for 14 days||First dose|
Apoptosis in RGD-Treated Tumors
Using the TUNEL method to assess apoptosis, quantitation of total positive cells and positive endothelial cells indicated that the combination of RIT + RGD resulted in the greatest increase in apoptosis of both tumor and endothelial cells at 24 hours following RIT compared with untreated, RIT alone, or RGD alone (Fig. 1). Statistically significant differences in apoptosis were found in total cells and endothelial cells when CMRIT and RIT were compared and RIT and untreated were compared, as determined by ANOVA, and these differences in apoptosis were associated with differences in maximal tumor growth. We also found that RGD alone significantly increased apoptosis of both tumor and endothelial cells 24 hours following the administration of RGD peptide compared with untreated mice, but the increased apoptosis was not associated with alteration of tumor growth rate over the course of the study.
Toxicity of RGD Peptide
RGD peptide alone (250 μg, 6 doses over 10 days) did not increase mortality over untreated mice with tumors. RGD peptide–treated mice and untreated mice also displayed similar levels of RBCs, WBCs, and platelets. The average weights of RGD peptide–treated and untreated mice remained at the same level throughout the 84-day trial. Mice that received RIT and mice that received RIT + RGD demonstrated decreased RBCs, WBCs, and platelets, but RIT + RGD did not depress these values beyond those observed in RIT mice during the first 28 days following RIT. Both RIT and RIT + RGD groups experienced similar mortality, not related to the RIT + RGD combination.
Tumoricidal Effect of Paclitaxel with Radioimmunotherapy
HBT tumors in mice treated with paclitaxel alone (600 μg), unmodified ChL6 with paclitaxel, or untreated grew without interruption (Fig. 2A). There were increased percentages of partial responses, complete responses, and cures in CMRIT compared with RIT alone when paclitaxel was administered 24 or 48 hours after RIT. CMRIT with paclitaxel increased the cure rate from 0% with RIT or paclitaxel alone to 50% when paclitaxel was administered 24 hours after RIT and to 88% when paclitaxel was administered 48 hours after RIT (Table 1). However, when paclitaxel was administered prior to RIT, cure rates only reached 22% and 25% (24 and 48 hours before RIT, respectively).
Toxicity of Paclitaxel with Radioimmunotherapy
Paclitaxel (600 μg) administered before or after RIT did not result in increased mortality compared with RIT alone. All groups of mice receiving 260 μCi 90Y-DOTA-ChL6 and paclitaxel demonstrated weight loss (3–16% of their initial body weight) and subsequent weight gain. Platelet, RBC, and WBC counts reached nadirs at 12–22 days but returned to normal by 42 days. Paclitaxel following RIT was associated with less severe and less prolonged myelosuppression than paclitaxel administered prior to RIT and was similar to that induced by RIT alone.
Tumoricidal Effect of IMC-C225 with Radioimmunotherapy
Tumor response to RIT was enhanced in mice receiving IMC-C225 antibody against EGFR 24 hours prior to RIT (78% response rate with 11% cures) compared with tumors of mice receiving IMC-C225 24 hours after RIT (29% response rate with 0% cures). Mice that received RIT alone had 0% cures and a 79% response rate, while mice that received unlabeled IMC-C225 antibody had a 0% cure rate and 36% response rate.
Toxicity of IMC-C225 with Radioimmunotherapy
Even more striking than the difference in cure rates in the two groups of mice that received RIT with IMC-C225 24 hours before and 24 hours after was the difference in mortality observed, with a 20% mortality rate when IMC-C225 (350 μg) was given 24 hours before RIT, which was not statistically different than the rates for untreated animals (10%) or animals that received IMC-C225 alone (350 μg, 11% mortality). However, an 80% mortality rate was seen when IMC-C225 was given 24 hours after RIT. Although mice treated 24 hours after RIT with 350 μg of IMC-C225 had a peak weight loss that was greater than mice treated 24 hours before RIT (23.5 ± 11.7% vs. 18.0 ± 13.3%, respectively), the differences were not statistically significant, and neither group experienced weight loss statistically different from the weight loss observed after RIT alone (ANOVA, P < 0.05). RBC, WBC, and platelets decreased similarly with IMC-C225 + RIT regardless of timing of IMC-C225 dose.
Tumoricidal Effect of Bcl-2 with Radioimmunotherapy
The growth rate of most tumors in mice that received bcl-2 antisense alone did not appear different from the growth rate of untreated tumors, and no partial or complete responses were seen in these groups. Mice that received RIT alone had a 25% cure rate and a 100% response rate, while mice that received the combination therapy had a 0% cure rate and a 95% response rate. Tumors in mice that received the combined therapy resumed a rapid growth rate at a much earlier time point (21 days after RIT, Fig. 2B) than did tumors in mice that received RIT alone (49 days after RIT), leading to increased maximal tumor volumes by the end of the study (309 ± 121 mm3 for RIT alone vs. 603 ± 142 mm3 for RIT + bcl-2 antisense), although these differences were not statistically significant. Due to the increased rate of tumor regrowth in the combined therapy group, overall survival was lower in this group (20%) than in the control group that received RIT alone (80%) by the end of 84 days.
Toxicity of Bcl-2 with Radioimmunotherapy
No mice died from toxicity in the control group that received bcl-2 antisense alone. However, mice in this group experienced a slight decrease in body weight, which was restored by Day 14. Decreased RBC counts were measured beginning 1 week after the start of antisense therapy. Platelet counts in mice that received only antisense decreased during the first week of treatment reached a plateau and increased to 70% of their starting levels by Day 21 of treatment. One mouse each from the RIT only and the RIT + bcl-2 antisense treatment groups died prior to 28 days from causes other than tumor burden, resulting in similar mortality rates for these groups (20% RIT only with 5 mice in group and 10% antisense bcl-2 [12.5 μg/g daily for 14 days] with 10 mice in group). Compared with mice that received RIT alone, mice treated with RIT + bcl-2 experienced more prolonged and deeper nadirs in RBC and platelet counts, although WBC counts recovered to above-normal levels faster than RIT-only mice. Mice that received RIT + bcl-2 antisense also demonstrated increased loss of weight following RIT compared with mice that received RIT alone. Average body weight in mice that received RIT alone began increasing 7 days after RIT, while average body weights for mice on combination therapy did not begin to increase until 14 days following RIT.
In this study, we highlight the potential for agents to synergize with RIT to increase therapeutic efficacy without increasing toxicity, and also illustrate the complexity of CMRIT by describing results in which therapeutic efficacy was decreased and/or toxicity to normal tissue was increased in CMRIT. In order to optimize CMRIT, an effective agent must be chosen and the timing of its delivery and effect must be coordinated with the effect of RIT on the tumor and on normal tissue. RIT potentially can target primary tumor and metastatic cancer cells, leading to the activation of molecular pathways to programmed cell death (apoptosis).61 Cell cycle agents that overcome apoptotic blocking mechanisms, such as mutations of p53 or overexpression of bcl-2, may enhance the apoptotic effect of RIT. In addition, alternative agents, such as antiangiogenic agents, can also enhance the effect of RIT. Timing of delivery of the CMRIT agent appears to be critical to optimize effect. Most MAbs localize in tumor within 24–48 hours after administration, and the majority of the radiation dose to the tumor is delivered some 48–336 hours after delivery of the radiopharmaceutical. The majority of the radiation dose to normal tissues occurs at an earlier time due to clearance of unbound 90Y-DOTA-ChL6 from these normal, nontargeted tissues, thereby creating a window of time for CMRIT synergy when tumor radiation is high but normal tissue radiation is low.3, 61
Recent combination studies of chemotherapeutic agents with antiangiogenic agents have been promising, though their value as combined therapy agents in RIT is unknown.22, 23 The use of one agent in particular for combined modality RIT therapy, RGD peptide EMD 121974, was particularly appealing because of its reported lack of toxicity and its ability to target genetically normal endothelial cells in tumor neovasculature.29 The αvβ3integrin is highly expressed in most growing tumor vasculature but has very low expression in normal vasculature and most other normal or benign human tissue.62 When the activity of αvβ3 receptors was blocked by MAb LM609 (anti-αvβ3 antibody), rapid initiation of apoptosis in neovascular cells was seen.62 Cyclic peptides with high specific inhibition of the αvβ3 integrin were developed and cyclic peptide EMD 121974 was chosen for these studies because of its αvβ3 receptor antagonist effects at nanomolar levels and because it is currently in use in clinical trials.26 Although decreased tumor volume by blockade of integrin αvβ3 with multiple doses of RGD peptide or MAb LM609 has been reported in some tumor models,28, 63 in the HBT 3477 tumor model we found no effect of RGD peptide on tumor volumes without the addition of RIT. The growth of HBT tumors in mice that received 6 doses of RGD peptide was not different from untreated control mice. However, although only a preliminary study, inhibition of tumor growth in mice that received RGD + RIT was sustained, leading to an increased cure rate (57% for RIT + RGD vs. 0% for RIT alone within this study or 7% for RIT alone over all 4 studies). These data, and the increased apoptosis correlation observed in immunopathology (Fig. 1), are consistent with synergy of RIT and RGD peptide.
Paclitaxel administered in combination with RIT also demonstrated synergy, with the optimal strategy dependent on the sequence and timing of the delivery of CMRIT. Delivery of paclitaxel 24 or 48 hours after RIT was associated with an increased number of cures compared with delivery prior to RIT, with the highest number of cures occurring when paclitaxel was given 48 hours after RIT. Because paclitaxel is a relatively small molecule (mw 854 Da) compared with antibodies used in RIT (mw 150 kDa), it reaches peak concentrations in the tumor tissue shortly after administration, while the larger radiolabeled antibody reaches peak tumor concentrations at 24–48 hours.3, 22 Paclitaxel may be particularly effective in CMRIT, as it impairs microtubule activity,42 which leads to decreased activity of bcl-2 by phosphorylation10 and arrest of cells at the G2/M phase of the cell cycle, where they are more radiosensitive.30, 64 It also may target tumor vasculature as an antiangiogenic agent in addition to targeting tumor cells.65, 66
In contrast to the synergistic responses with RGD peptide or paclitaxel combined with RIT, other agents, when given in certain time sequences with RIT, appeared to magnify toxicity to normal tissues (IMC-C225 after 24 hrs RIT) or appeared to inhibit tumor response to radiation (bcl-2 antisense). In vitro and preclinical results demonstrate inhibition of breast cancer cells in vitro and in mouse xenografts by IMC-C225, an IgG1 against EGFR.44 Exposure of cells in vitro to IMC-C225 has been shown to decrease bcl-2/Bax ratio and to increase sensitivity to radiation.34 Our results indicate that when MAb (IMC-C225) against EGFR (350 μg) is combined with RIT 24 hours after RIT (260 μCi 90Y-DOTA-ChL6), toxicity is increased above both RIT alone or RIT with IMC-C225 given 24 hours before. Anti-EGFR IMC-C225 mortality was 80% and the response rate was 29% when provided after RIT, compared with a 20% mortality and 78% response rate when provided before RIT. These effects are consistent with the time required for a macromolecular MAb to be deposited in tumor tissue. IMC-C225 is a macromolecule; thus, when given 24 hours after RIT, its biodistribution, tumor uptake, and ability to effect changes through tumor receptors likely occur too late to “sensitize” tumor cells before the majority of the radiation energy has been deposited in the cells. However, when IMC-C225 is provided 24 hours prior to RIT, it could sensitize tumor tissue by causing G1 arrest, making the cells more radiosensitive34 as well as by decreased EGF and TGF-α growth factor stimulation from receptor blockade.18 The apparent sensitization of normal tissue, most likely bowel and bone marrow, to the effects of RIT evidenced by the high level of toxicity when IMC-C225 was given after RIT may indicate that EGFR activation creates a short but potent window of radiosensitization in these tissues. Despite our observations in mice in combination with RIT, it should be emphasized that in a clinical context, IMC-C225 is well tolerated (as a single agent or in non-RIT combined therapies). No severe adverse events have been seen even at elevated doses, either in preclinical studies in primates or during clinical trials in humans, both representing species with which IMC-C225 reacts strongly (Dr. S. Matzku, personal communication).
The potential complexity of CMRIT is also illustrated by our results with the bcl-2 antisense oligonucleotide G3139. As many cancers overexpress bcl-2 and thus are more resistant to apoptosis,9, 67–69 blocking bcl-2 overexpression of mutant genes using antisense oligonucleotides would hypothetically allow more cells to be responsive to the low-dose-rate radiation from RIT. However, in a preliminary experiment, when bcl-2 antisense oligonucleotides were administered (12.5 μg/g daily) i.p. starting 48 hours before RIT and continued daily for 12 days subsequent to RIT, the number of cures was actually decreased compared with RIT alone. This suggests that bcl-2 antisense given in this manner protected tumor tissue from radiation effect, possibly by nonspecific mechanisms. Antisense oligonucleotides can induce effects due to their ability to bind to proteins rather than through sequence-specific inhibition of mRNA activity.37 It is thus possible that G3139 may have blocked a component key to RIT-induced apoptosis. In addition to decreased efficacy, bcl-2–treated CMRIT mice had deeper and more prolonged nadirs in RBC and platelet counts for approximately the first month following RIT, suggesting that bcl-2 antisense treatment sensitized normal marrow to radiation effects.
In conclusion, we have presented data for four very different agents, all considered candidates for synergy with RIT. It should be emphasized that the effects of these agents were only tested on one breast cancer model (HBT 3477) and broad conclusions to other breast cancer models have not been confirmed. Mice bearing HBT tumors have approximately a 1 in 10 chance of dying in the first 30 days without treatment to shrink the tumors. With combined therapy there is evidence of increased tumor response as well as the risk of increased toxicity, with the end result dependent on timing. We chose these agents based on their potential to enhance the apoptotic effect, considered the major mechanism of antitumor effect in RIT. The complexity and peril of combining agents in CMRIT were apparent in our studies with IMC-C225 and bcl-2 antisense, where sequence-related toxicity and even tumor protection were suggested. However, the relatively nontoxic paclitaxel, which sensitizes cells to radiation by several mechanisms, and RGD peptide, which blocks integrin function on nonresistant endothelial cells, synergized with RIT to produce cures without increased toxicity. The synergy of these agents, when delivered in an appropriate sequence to RIT, is particularly impressive in light of the complete lack of response of the aggressive HBT tumor to these agents when used alone. These studies thus illustrate the promise and potential pitfalls of CMRIT.
The authors thank ImClone Systems Incorporated for providing IMC-C225; Genta, Incorporated for providing G3139; and Siegfried Matzku, Ph.D, and Simon L. Goodman, Ph.D, of Merck KgaA for their scientific input.
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