Volume 116, Issue S4 p. 1059-1066
Supplement
Free Access

Improved efficacy of α-particle–targeted radiation therapy

Dual targeting of human epidermal growth factor receptor–2 and tumor-associated glycoprotein 72

Diane E. Milenic MS

Corresponding Author

Diane E. Milenic MS

Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Fax: (301) 402-1923

Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Institutes of Health, 10 Center Drive, MSC-1002, Room B3B69, Bethesda, MD 20892===Search for more papers by this author
Erik D. Brady PhD

Erik D. Brady PhD

Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

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Kayhan Garmestani PhD

Kayhan Garmestani PhD

Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

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Paul S. Albert PhD

Paul S. Albert PhD

Biometric Research Branch, National Cancer Institute, Bethesda, Maryland

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Alia Abdulla MD

Alia Abdulla MD

Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

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Martin W. Brechbiel PhD

Martin W. Brechbiel PhD

Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

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First published: 02 February 2010
Citations: 44

The articles in this supplement were presented at the “12th Conference on Cancer Therapy with Antibodies and Immunoconjugates,” Parsippany, New Jersey, October 16-18, 2008.

This article is US Government work and, as such, is in the public domain in the United States of America.

Abstract

BACKGROUND:

Human epidermal growth factor receptor–2 (HER-2) and tumor-associated glycoprotein 72 (TAG-72) have proven to be excellent molecular targets for cancer imaging and therapy. Trastuzumab, which binds to HER-2, is effective in the treatment of disseminated intraperitoneal disease when labeled with 213Bi or 212Pb. 213Bi-humanized CC49 monoclonal antibody (HuCC49ΔCH2), which binds to TAG-72, inhibits the growth of subcutaneous xenografts. A next logical step to improve therapeutic benefit would be to target tumors with both molecules simultaneously.

METHODS:

Athymic mice bearing intraperitoneal human colon carcinoma xenografts were treated with a combination of trastuzumab and HuCC49ΔCH2 labeled with 213Bi administered through an intraperitoneal route. The sequence of administration also was examined.

RESULTS:

Before combining the 2 monoclonal antibodies, the effective doses of 213Bi-CC49ΔCH2 and 213Bi-trastuzumab for the treatment of peritoneal disease were determined to be 500 μCi for each labeled antibody. Treatment with 213Bi-HuCC49ΔCH2 resulted in a median survival of 45 days and was comparable to the median survival achieved with 213Bi-trastuzumab. Each combination provided greater therapeutic efficacy than either of the agents given alone. However, the greatest therapeutic benefit was achieved when 213Bi-HuCC49ΔCH2 and 213Bi-trastuzumab were coinjected, and a median survival of 147 days was obtained.

CONCLUSIONS:

Dual targeting of 2 distinct molecules in tumors such as TAG-72 and HER-2 with α-particle radiation resulted in an enhanced, additive, therapeutic benefit. The authors also observed that this radioimmunotherapeutic strategy was well tolerated. Cancer 2010;116(4 suppl):1059–66. Published 2010 by the American Cancer Society.

Targeted α-particle radiation therapy is proving to be an appropriate therapy for disseminated disease and micrometastatic disease and for the eradication of single, malignant cells, as evidenced by the increasing reports from preclinical and clinical studies.1-5 Improving monoclonal antibody (MoAb) targeting and therapeutic efficacy and reducing toxicity to normal tissues, however, remain objectives for investigators in the field of radioimmunotherapy. The strategies for doing so include 1) the administration of radiosensitizers, 2) combining MoAb monotherapy with radiolabeled MoAb, 3) altering the density of the target molecule, 4) administering multiple doses of the radiolabeled MoAb, and 5) targeting multiple antigens using a “cocktail” of radiolabeled MoAbs.6-16

The rationale for the administration of MoAb mixtures is to overcome the heterogeneous nature of tumors and to deliver what should be a more homogeneous distribution of radiation throughout the tumor if complementary molecules are targeted. Blumenthal et al6 were able to achieve greater therapeutic efficacy targeting carcinoembryonic antigen (CEA) and colon-specific antigen-p (CSAp) with a mixture of 131I-labeled MoAbs specific for these antigens. Patients with metastatic colorectal cancer received a combination of 131I-labeled MoAb against CEA and tumor-associated glycoprotein 72 (TAG-72) as well as α-interferon.11 When these patients were compared with historic controls, it appeared that there was an increase in radiation doses at tumor sites along with an increase in localization intensity as measured by γ-scintigraphy. Unfortunately, the radiation dose delivered to tumor lesions still was insufficient to result in tumor regression.

Previous studies from this laboratory have demonstrated the efficacy of high linear energy transfer radiation delivered by MoAb targeting of TAG-72 and human epidermal growth factor–2 (HER-2) to treat colon carcinoma xenografts.3-5 TAG-72 is a mucin-like glycoprotein that is overexpressed on the majority of ovarian, colorectal, gastric, pancreatic, endometrial, nonsmall cell lung, and breast carcinomas.17, 18 The genetically engineered MoAb, domain-deleted, humanized CC49 (HuCC49ΔCH2), was effective in either delaying and/or causing tumor regression in a dose-dependent manner of subcutaneous LS-174T xenografts when radiolabeled with 213Bi.4, 19 HER-2, a member of the epidermal growth factor receptor family, also is overexpressed in a wide range of carcinomas of epithelial origin, which includes from 25% to 30% of breast and ovarian cancers, from 35% to 45% of pancreatic cancers, and up to 90% of colorectal cancers.20, 21 Trastuzumab, a well recognized MoAb that reacts with HER-2, has been highly effective in increasing the survival of athymic mice bearing intraperitoneal LS-174T tumors when radiolabeled with 213Bi or 212Pb.3, 5

On the basis of the success in this laboratory with each of these MoAbs used for α-particle–targeted therapy, the next logical step was to evaluate both MoAbs in a dual-targeted radioimmunotherapy regimen with α-emitting radiation. This report presents the results from that study.

MATERIALS AND METHODS

Cell Lines

All therapy studies were conducted using LS-174T, a human colon carcinoma cell line; SKOV-3, a human ovarian carcinoma cell line that expresses ∼1 × 106 HER-2 molecules per cell, was used for in vitro analyses.13 LS-174T cells were grown in supplemented Dulbecco minimum essential medium as described previously.22 SKOV-3 cells were maintained in McCoy 5a medium supplemented with 10% fetal bovine serum and 1 mM nonessential amino acids. Media and supplements were obtained from Quality Biologicals (Gaithersburg, Md), Invitrogen (Carlsbad, Calif), or Lonza (Walkersville, Md).

Monoclonal Antibodies

The development and characterization of MoAb HuCC49ΔCH2 have been described previously.19, 23 MoAb HuC49ΔCH2 was purified from bioreactor supernatant at the Monoclonal Antibody/Recombinant Protein Production Facility (Frederick, Md), as detailed elsewhere.24 Trastuzumab (Herceptin; Genetech, South San Francisco, Calif) was obtained through the Veterinary Resources Program (National Institutes of Health, Bethesda, Md).

Chelate Synthesis and MoAb Conjugation

The synthesis, characterization, and purification of the bifunctional ligand, cyclohexyl-A″ diethylenetriaminopentaacetate (CHX-A″ DTPA), have been described previously.25 Conjugation of trastuzumab and HuCC49ΔCH2 with CHX-A″ DTPA has been previously described.3, 24, 26, 27 The final concentrations of the MoAb were quantified according to the method described by Lowry et al.28 The number of CHX-A″ DTPA molecules linked to the MoAb was determined using a spectrophotometric assays based on the titration of yttrium Arsenazo(III) complex.29

Radiolabeling

Radiolabeling of the immunoconjugate with 213Bi obtained from actinium-225 (Oak Ridge National Laboratories, Oak Ridge, Tenn or Actinium Pharmaceuticals, Inc., Florham, NJ) was conducted as previously described.4 Elution of the 213Bi and radiolabeling of HuCC49ΔCH2-CHX-A″ and trastuzumab-CHX-A′ was performed as previously described.4 Human immunoglobulin G (HuIgG) (ICN, Irvine, Calif) was conjugated similarly with CHX-A″ DTPA and radiolabeled with 213Bi, as described above, to serve as a negative control.

Immunoassays

Immunoreactivity of the radiolabeled HuCC49ΔCH2 preparations was assessed in a solid-phase radioimmunoassay as detailed previously.30 Bovine submaxillary mucin (1 μg in phosphate-buffered saline [PBS]) was adsorbed overnight to the wells of a microtiter plate at 4°C. After the wells were blocked with 1% bovine serum albumen (BSA) in PBS, serial dilutions of radiolabeled HuCC49ΔCH2 in 1% BSA in PBS were added to the wells in duplicate and incubated for 2 hours at 37°C. The plates were washed, and the radioactivity was measured in a γ-counter (Packard, Downers Grove, Ill). The percentage binding was calculated for each serial dilution. Wells coated with porcine submaxillary mucin served as a negative control.

Immunoreactivity of the radiolabeled trastuzumab preparations was assessed in a radioimmunoassay using methanol-fixed SKOV-3 cells.13 Briefly, SKOV-3 cells were trypsinized, pelleted, and resuspended in PBS, pH 7.2, that contained 1% BSA. Serial dilutions of radiolabeled trastuzumab were added in duplicate to cells (1 × 106) in 50 μL of 1% BSA in PBS. The cells were washed with 4 mL of 1% BSA in PBS after a 2-hour incubation at room temperature, pelleted at ×1000g for 5 minutes, the supernatant decanted, and the 213Bi was measured in a γ-counter (WizardOne; PerkinElmer, Shelton, Conn). The binding percentage was calculated for each dilution. The specificity of the radiolabeled trastuzumab was confirmed by incubating 1 set of cells with ∼200,000 counts per minute of radiolabeled trastuzumab with an excess (5 μg) of unlabeled trastuzumab.

Therapy Studies

Therapy studies were performed using female athymic (nu/nu) mice (Charles River Laboratories, Wilmington, Mass) that weighed between 19 g and 21 g. The mice were injected intraperitoneally with 1 × 108 LS-174T cells in 1 mL of medium or PBS as reported previously.3, 7 The initial experiment performed in the series reported here was designed to evaluate the efficacy of 213Bi-HuCC49ΔCH2 in treating disseminated peritoneal tumors and to determine the effective operating dose. On the third day, 7 groups of mice (n = 8-11) bearing intraperitoneal LS-174T tumors were given intraperitoneal injections of 213Bi-HuCC49ΔCH2 ranging from 125 μCi to 2 mCi. An additional group of mice received 1 mCi of 213Bi-HuIgG, and another group was left untreated. From the results of these initial experiments, 500 μCi was chosen as the effective operating dose for 213Bi-HuCC49ΔCH2. Previous studies had demonstrated that treatment of a tumor burden >3 days with 213Bi-trastuzumab was not effective.5 In fact, at 3 days, the average total tumor burden in the peritoneum is 25.6 ± 35.0 mg and is comprised of multiple, small nodules that range in weight from 3 mg to 177 mg (unpublished data).

In subsequent experiments, trastuzumab and HuCC49ΔCH2 labeled with 213Bi (500 μCi) were administered to the mice (n = 8-10) 3 days postimplantation of tumor in 0.5 mL PBS. HuIgG labeled with 213Bi served as a nonspecific control. Specific treatment schedules along with the results obtained are outlined below. In all of the experiments outlined below, radiation therapy doses were administered 3 days after tumor implantation. Mice were weighed before receiving radioimmunotherapy (RIT) and then twice weekly for 3 to 4 weeks.

Progression of disease was defined as a visually perceptible extension of the abdomen, development of ascites, or weight loss. Disease progression also often was evident with noticeable, palpable nodules in the abdomen. Mice were monitored and euthanized if they were in distress, moribund, or cachectic. Mice also were euthanized when 10% to 20% weight loss occurred or if bloating or tumor nodules were apparent. All animal protocols were approved by the National Cancer Institute Animal Care and Use Committee.

Statistical Analyses

A Cox proportional hazards model was used to test for a dose response relation between the dose of 213Bi-HuCC49ΔCH2 and survival (the time to euthanasia or natural death). The dose level was treated as a linear covariate in the Cox model and tested whether the corresponding regression parameter was zero using a likelihood ratio test.

For animal weight data, the maximum percentage reduction from baseline was estimated for each mouse. This was calculated as the ratio of the maximum reduction in weight from baseline during the initial 3 to 4 week period divided by the baseline weight of the mouse. Box plots were constructed for each dose level to illustrate the median, upper, and lower quartiles and to identify outliers. Differences between dose groups were tested using a Kruskal-Wallis test (nonparametric analysis of variance) for comparison of multiple groups, and the Wilcoxon rank-sum test was applied when comparing 2 groups. All reported P values correspond to 2-sided tests.

RESULTS

The first experiment that was performed in the series reported here was designed to evaluate the efficacy of 213Bi-HuCC49ΔCH2 in treating disseminated peritoneal tumors and to determine the effective operating dose. Figure 1A illustrates that, as the dose of 213Bi-HuCC49ΔCH2 increased from 125 μCi to 500 μCi, a dose response was observed; greater therapeutic benefit was derived with the increasing dose (P < .001). In this series, the median survival of mice that received 125-mCi, 250-mCi, and 500-μCi doses was 17 days, 19 days, and 25 days, respectively, compared with 15 days for the untreated mice. There appeared to be no further benefit from using doses >500 μCi: The median survival of mice that received 750 μCi was 26 days, and it was 28 days for mice that received 1-mCi, 1.5-mCi, and 2-mCi doses of 213Bi-HuCC49ΔCH2. Animal weights were monitored for 21 to 28 days after RIT as a measure of toxicity. The maximum percentage reduction in weight relating to the 7 dose levels studied is depicted in Figure 1B. Overall, differences reflective of the survival data were apparent across the dose levels (P = .002). The greatest effect on animal weight was observed at the 2-mCi dose of 213Bi-HuCC49ΔCH2. Meanwhile, the affect of the 500-μCi 213Bi-HuCC49ΔCH2 dose on animal weights was moderate. This information, in conjunction with median survival data, provided sufficient evidence for selecting 500 μCi as the effective operating dose for 213Bi-labeled HuCC49ΔCH2.

Details are in the caption following the image

Increasing doses (μCi) of 213Bi-humanized CC49 monoclonal antibody (213Bi-HuCC49ΔCH2) were administered intraperitoneally to mice bearing 3-day LS-174T intraperitoneal xenografts. Kaplan-Meier survival curves are shown. (A) The 213Bi-HuCC49ΔCH2 was injected at doses of 125 μCi (open circles), 250 μCi (solid downward triangles), 500 μCi (open upward triangles), 750 μCi (solid squares), 1 mCi (open squares), 1.5 mCi (solid diamonds), and 2 mCi (open diamonds). One set of mice was left untreated (solid circles), and 1 set received 1 mCi of 213Bi-human immunoglobulin G (HuIgG) (solid upward triangles) as a nonspecific control antibody. (B) The weights of the animals were used as indicators of toxicity after treatment with 213Bi-HuCC49ΔCH2 radioimmunotherapy. These data were plotted as the maximum relative weight reduction for each of the treatment groups and are presented as box plots. Light line indicates the median; upper region, third quartile; lower region, first quartile; brackets delineate 1.5 times the interquartile range; lines outside the brackets, outlying observations.

Then, the efficacy of 213Bi-HuCC49ΔCH2 was compared directly with that of 213Bi-trastuzumab. Groups of mice (n = 8-10) were injected with 500 μCi of 213Bi-HuCC49ΔCH2, 213Bi-trastuzumab, or 213Bi-HuIgG or were left untreated (Fig. 2). For this experiment, a median survival of 45 days was obtained for the groups of mice that were injected with either 213Bi-HuCC49ΔCH2 or 213Bi-trastuzumab compared with 17 days for the mice that were not treated. Mice that were injected with 500 μCi of 213Bi-HuIgG, the nonspecific control, experienced a median survival of 28 days.

Details are in the caption following the image

Kaplan-Meier survival curves are shown. Mice bearing 3-day LS-174T intraperitoneal xenografts either were left untreated (solid circles) or were injected with 500 μCi of 213Bi/humanized CC49 monoclonal antibody (213Bi-HuCC49ΔCH2) (open triangles), 213Bi-trastuzumab (solid squares), or 500 μCi of 213Bi-human immunoglobulin G (HuIgG) (open diamonds) as a nonspecific control.

Next, a therapy study was conducted in which the 2 targeting vehicles (500 μCi each) were administered (intraperitoneally) alone and concurrently to mice that had a 3-day intraperitoneal tumor burden. The administration sequence of these MoAbs also was explored. Groups of mice were treated with either 213Bi-HuCC49ΔCH2 or 213Bi-trastuzumab on Day 3 and were treated with the other 213Bi-labeled MoAb on Day 4; another group was coinjected with both 213Bi-labeled MoAbs on Day 3. Sequential administration of the MoAbs, 213Bi-trastuzumab followed by 213Bi-HuCC49ΔCH2 or vice versa, resulted in greater therapeutic efficacy than either MoAb administered alone and produced a median survival of 36 days and 40 days, respectively (Table 1) (Fig. 3A). This translates to a therapeutic index of 2.4 and 2.7 compared with the untreated group of tumor-bearing mice, for which the median survival was 15 days. The greatest therapeutic benefit, however, was realized in the group that was coinjected with 213Bi-HuCC49ΔCH2 and 213Bi-trastuzumab (500 μCi each). The median survival of this group was 147 days (P < .001), which translates to a therapeutic index of 9.8. This affect was additive, no interaction between trastuzumab or HuCC49ΔCH2 was indicated (P = .17). A comparison of the 3 groups corresponding to the different combinations of 213Bi-HuCC49ΔCH2 and 213Bi-trastuzumab was highly significant (P = .006); again, the concurrent administration provided the greatest increase in median survival.

Table 1. The Median Survival of Athymic Mice Bearing Intraperitoneal Tumor Xenografts Treated With Sequential or Concurrent 213Bi-Labeled Monoclonal Antibodies Targeting Tumor-Associated Glycoprotein 72 and Human Epidermal Growth Factor Receptor 2
Treatment Day Administered Median Survival, da Therapeutic Indexb
Untreated 15 1.0
Trastuzumab 3 31 2.1
HuCC49ΔCH2 3 34 2.3
HuIgG 3 21 1.4
Sequential
 Trastuzumab 3
 HuCC49ΔCH2 4 36 2.4
 HuCC49ΔCH2 3
 Trastuzumab 4 40 2.7
Concurrent 3 147 9.8
  • HuCC49ΔCH2 indicates humanized CC49 monoclonal antibody; HuIgG, human immunoglobulin G.
  • a The values presented are the median survival shown in days. Mice bearing 3-day tumor (intraperitoneal) burden were injected intraperitoneally with 500 μCi 213Bi-trastuzumab and 213Bi-HuCC49ΔCH2, alone, in sequence, or concurrently.
  • b The therapeutic index is derived from the median survival of the treatment group divided by the median survival of the untreated group.
Details are in the caption following the image

Kaplan-Meier survival curves are shown. (A) Mice bearing intraperitoneal LS-174T 3-day xenografts were injected with 500 μCi of 213Bi-labeled trastuzumab (open circles). with a humanized CC49 monoclonal antibody (HuCC49ΔCH2) (solid triangles). or with human immunoglobulin G (HuIgG) (open triangles). A combination of the 2 213Bi-labeled monoclonal antibodies (MoAbs) (500 μCi of each) was given sequentially, either with 213Bi-HuCC49ΔCH2 given on Day 3 and 213Bi-trastuzumab given on Day 4 (open squares), or the reverse sequence of 213Bi-trastuzumab given on Day 3 and 213Bi-HuCC49ΔCH2 given on Day 4 (solid squares). Another group of mice received concurrent injections of 500 μCi each of 213Bi-labeled trastuzumab and HuCC49ΔCH2 (solid diamonds). (B) The weights of the animals were used as indicators of toxicity after treatment with the 213Bi-labeled MoAbs. Data are presented as described for Figure 1.

A comparison of the maximum percentage reduction in animal weight as a measure of toxicity (Fig. 3B) indicated that the groups injected with each of the 213Bi-labeled MoAbs alone experienced weight loss as did the untreated mice and the mice that were injected first with 213Bi-HuCC49ΔCH2 and then 213Bi-trastuzumab on the following day. It is interesting to note that the animals with the longest survival, those that received the coinjection of 213Bi-HuCC49ΔCH2 and 213Bi-trastuzumab, experienced no weight loss during the follow-up period.

DISCUSSION

Low expression of an antigen does not present the same obstacle for successful targeted radiation therapy as it does for other MoAb-based therapies. Radiation may overcome the heterogeneous nature of tumors by virtue of omnidirectional particle decay; therefore, not all cells need to be targeted by a radioimmunoconjugate. Neighboring cells may receive sufficient cytotoxic doses as a result of this bystander effect. Using α-particle radiation as the cytotoxic agent has the additional advantage of requiring only a few molecules localized at the tumor cell. Three to 6 traversals of a cell nucleus by an α-particle effects cell killing, and the estimated dose rate is as low as 1 centigrays per hour.31, 32 In fact, targeting tumor burdens with low or moderate expression of a molecule may be preferred, because high expression may function as a sink and inhibit targeting of a greater number of cells. The heterogeneous nature of a tumor, however, does present the challenge of delivering a therapeutic dose of radiation throughout a tumor lesion.

The studies reported herein detail the first in vivo radioimmunotherapy study that simultaneously targets 2 molecules with α-particle radiation. To date, the concept of multiple-targeted α-particle therapy has been evaluated only in vitro against human prostate cancer (CaP) cell lines.33 In that study, 4 antibodies were radiolabeled with 213Bi, then combined, and their cytotoxic effect was evaluated against 3 CaP cell lines. The investigators reported that the 4 combined radioimmunoconjugates required only 15 to 27 μCi/mL compared with 272 to 281 μCi/mL of a control cocktail to achieve 37% cell survival. However, they also reported that a single radioimmunoconjugate, albeit not the same 1 for each of the 3 cell lines, was able to achieve the same level of cell killing. The studies were extended to the treatment of multicellular spheroids with 3 213Bi-labeled MoAbs.34 Efficacy of therapy was demonstrated against 2 sizes of spheroids (80-100 μm and 180-200 mm) of 2 human CaP cell lines. In that instance, the single radioimmunoconjugates were not evaluated for comparison.

HER-2 and TAG-72 were chosen as the target molecules based on previous RIT studies using trastuzumab and HuCC49ΔCH2 to treat human colon carcinoma xenografts with 213Bi in this laboratory.3-5 Previous studies with HuCC49ΔCH2 involved intravenous and intraperitoneal injections of radiolabeled HuCC49ΔCH2 for targeting subcutaneous tumors. Therefore, before a study evaluating the efficacy of a MoAb “cocktail,” the objective of the initial study in the current series was to establish the effective operating dose of 213Bi-HuCC49ΔCH2 for the treatment of disseminated peritoneal disease. HuCC49ΔCH2 was radiolabeled with 213Bi and administered to tumor-bearing mice in increasing doses from 125 μCi to 2 mCi. A dose-dependent therapeutic response was observed with doses up to 500 μCi. Doses >500 μCi did not provide any further increase in the median survival of the tumor-bearing mice. It is noteworthy that, with doses as high as 2 mCi per mouse, a maximum tolerated dose was not reached. A direct comparison of 213Bi-HuCC49ΔCH2 with 213Bi-trastuzumab at a dose of 500 μCi each indicated that both MoAbs were comparable in their effectiveness for treating peritoneal disease, and a median survival of 45 days was obtained by each.

Next, the 2 MoAbs were combined and assessed for their potential for the treatment of disseminated peritoneal disease in an aggressive tumor (LS-174T) model. The coinjection of 213Bi-HuCC49ΔCH2 with 213Bi-trastuzumab resulted in a median survival of 147 days, which was 4.3-fold greater and 4.7-fold greater, respectively, than either agent alone. Considering each of the agents as a drug, the sequence of their administration also was evaluated. Modest, significant increases in median survival were observed when either drug was administered first, followed the next day by the other. However, once again, the concurrent administration of the 2 213Bi-labeled MoAbs proved to be the most effective and also was additive.

The rationale for combining MoAbs to detect multiple antigens in a tumor is not without precedent. Early in vivo studies demonstrated the potential of administering 2 radiolabeled antibodies for radioimmunodetection of human colon carcinoma xenografts in athymic mice.35-37 Subsequently, the therapeutic efficacy of 2 131I-labeled MoAbs was demonstrated.6 Immunohistochemical studies demonstrated that staining biopsies with multiple MoAbs results in detecting a greater number of carcinoma cells.38 More recently, Chauhan et al39 presented a study in which microarray slides of neoplastic and benign ovarian tissue samples were stained for 1, 2, and 3 antigens (TAG-72, carbohydrate antigen 125 [CA 125] and mucin 1 [MUC1]). Positive staining for the single antigens ranged from 73% to 89.5% for the neoplastic samples, with 30% to 40% of cells stained in each sample. A combination of TAG-72 and MUC1 resulted in 96% positive cells, whereas a pairing of CA 125 and MUC1 yielded 94% positive cells. A pairing of TAG-72 and MUC1 was not evaluated. When the samples were processed for all 3 antigens, 98% of the samples were positive, with >95% of the examined cells stained in each sample. Such studies clearly suggest that targeting more than 2 antigens is a feasible approach. These immunohistochemical studies also provide an argument for selecting “cocktails” that are appropriate for the individual patient based on an analysis of their antigen profile.

Efforts are ongoing in this laboratory to identify other MoAbs with potential for targeted radiation therapy. Cetuximab and panitumumab have been assessed as radioimmunoconjugates and have demonstrated excellent tumor targeting by γ-scintigraphy and quantitative tissue analysis and currently are being evaluated as therapeutic agents.40 Future studies will include combinations of 1 of these 2 MoAbs targeting epidermal growth factor receptor with either trastuzumab or HuCC49ΔCH2.

CONFLICT OF INTEREST DISCLOSURES

The articles in this supplement represent proceedings of the “12th Conference on Cancer Therapy with Antibodies and Immunoconjugates,” held in Parsippany, New Jersey, October 16-18, 2008. Unrestricted grant support for the conference was provided by Actinium Pharmaceuticals, Inc; Bayer Schering Pharma; Center for Molecular Medicine and Immunology; ImClone Systems Corporation; MDS Nordion; National Cancer Institute; National Institutes of Health; New Jersey Commission on Cancer Research; and PerkinElmer Life & Analytical Sciences. The supplement was supported by an unrestricted educational grant from ImClone Systems Corporation, a wholly-owned subsidiary of Eli Lilly and Company, and by page charges to the authors. This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.