TARGETED ANTISENSE RADIOTHERAPY OF NON-HODGKIN'S LYMPHOMA

• Sponsoring Institution: Cooperating Schools of Veterinary Medicine
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0228426
• Project Start Date: Jan 1, 2012
• Project End Date: Dec 31, 2013
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211

Performing Department
Veterinary Medicine & Surgery

Non Technical Summary
There were 65,540 new cases of non-Hodgkin's Lymphoma (NHL) diagnosed in 2010 in the US and 20,210 patients died. NHL is one of three cancers for which mortality has increased substantially during the past decade. Multiple studies have shown that overexpression of the bcl-2 gene correlates strongly with resistance to radiation and chemotherapy and therefore becomes the only molecular feature with an independent effect on disease-free survival. Currently, bcl-2 status is not being used in routine staging of and treatment planning for NHL, because the conventional treatments are not capable of overcoming the resistance to chemotherapy associated with this gene. However, with the discovery of drugs that target this proto-oncogene, the role of bcl-2 in disease progression and treatment response or failure will be better understood, becoming more important clinically. Bcl-2-positive patients might respond better to alternative treatments, such as targeted immunotherapy, radioimmunotherapy (RIT), or antisense (mRNA inhibitor) therapy, all of which act through mechanisms that down-regulate bcl-2. The overall goal of the proposed project is to evaluate a radioactive anti-bcl-2-PNA (peptide nucleic acid) as a targeted antisense radiotherapy agent for treatment of NHL. The effector molecule DOTA-anti-bcl-2-PNA-Tyr-3-octreotate will be labeled with the radionuclide Lu-177 and evaluated in mice carrying human NHL tumors with positive bcl-2 expression. The hypotheses to be addressed in this proposal are: 1) the combination of internal radiation and antisense effects on bcl-2-mediated resistance to cell death could allow for additive therapeutic effectiveness in human lymphoma, and 2) dose fractionation of the radiolabeled PNA compound may maximize biologic and radiologic effects, therefore providing better overall effectiveness and lower toxicity than a single high dose. If successful, the proposed studies will lead to future evaluation of the Lu-177 agent for targeted antisense radiotherapy in both veterinary and human clinical trials.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3840	1040	50%
305	3840	1180	50%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1040 - Molecular biology; 1180 - Pharmacology;

Keywords

antisense therapy

radiopharmaceutical

Goals / Objectives
The overall goal of the proposed project is to evaluate Lu-177-labeled anti-bcl-2-PNA-Tyr-3-octreotate as an antisense radiotherapy agent for treatment of NHL. The effector molecule DOTA-anti-bcl-2-PNA-Tyr-3-octreotate will be labeled with the radionuclide Lu-177 and evaluated in SCID mice bearing Mec-1 xenografts. The hypotheses to be addressed in this proposal are: 1) the combination of internal cytotoxic radiation and antisense effects on bcl-2-mediated resistance to apoptosis could allow for additive therapeutic efficacy in a mouse model of human lymphoma, and 2) dose fractionation of the radiopharmaceutical may maximize biologic and radiologic effects, therefore providing better overall effectiveness and less toxicity than a single high dose. Our approach for molecular targeting of bcl-2 expression in NHL offers advantages over conventional DNA antisense technology by using a biologically stable targeting system combined with an intracellular cargo delivery system.

Project Methods
We will accomplish our objectives by 1) performing maximum tolerated dose (MTD) studies, and 2) performing single- and fractionated-dose therapy studies using Lu-177-DOTA-anti-bcl-2-PNA-Tyr-3-octreotate in SCID mice carrying human lymphoma xenografts. In the MTD study, non-tumor-bearing SCID mice (n = 5 per dose level) will be injected with doses of Lu-177-DOTA-anti-bcl-2-PNA-Tyr-3-octreotate predicted to be at or near the MTD. Control animals (n = 5) will receive normal saline only. Toxicity will be monitored by weighing and examining the animals daily signs of overt toxicity (e.g., weight loss more than 15 percent, lethargy, scruffy coat, and diarrhea) each day throughout the study. Based on prior experience, we use descriptive statistics to analyze the results of toxicity studies. If the MTD of the Lu-177 radiopharmaceutical is established, approx. 90 percent of that dose will be administered to tumor-bearing SCID mice. If the highest dose administered does not result in unacceptable toxicity, that dose will be evaluated for therapy in SCID mice bearing Mec-1 tumors. In single- and fractional-dose therapy studies, experiments will be carried out in SCID mice bearing Mec-1 tumors at 4-5 weeks post-inoculation. Control groups will include mice receiving normal saline only. Animals will be weighed and examined for any change in gross physical appearance on a daily basis. Tumor volumes will be measured with Vernier calipers using the three-dimensional formula v = (l x w x h) x 3.14/6. Endpoint tumor burdens will be assessed by post-mortem dissection. Animals will be sacrificed after surviving 8 months or when signs of overt systemic illness, described above, are observed. Multiple dosing intervals of Lu-177-DOTA-anti-bcl-2-PNA-Tyr-3-octreotate will be based on 70-80% bone marrow recovery, as determined during the course of the MTD studies, as well as the intervals of tumor regrowth observed in the single high-dose therapy study. A schedule of 4 doses at the optimum time interval of Lu-177-labeled PNA-peptide conjugate will be evaluated. Response to therapy will be assessed based on the number of days required for tumor volume to reach 1 g, by one-way ANOVA using statistical software SPSS 12.0.1. The survival fraction of each treatment group will be evaluated by Kaplan-Meier density analysis using SPSS 12.0.1, with a confidence interval of 95 percent (p less than 0.05).

Progress 01/01/12 to 12/31/13

Outputs
OUTPUTS: This project tested the hypothesis that dose fractionation of targeted antisense radiotherapy is less toxic and more effective than a single high dose. Our strategy of targeted antisense radiotherapy involves the use of a radioactive peptide nucleic acid (PNA) to decrease expression of the BCL2 cancer survival gene in tumors, while simultaneously delivering cytotoxic radiation. Because cellular internalization of PNA is very poor, this compound was conjugated to a receptor-binding peptide for intracellular delivery and targeting BCL2 at the mRNA level. One major goal of the research was to determine the maximum tolerated dose (MTD) of the PNA-peptide labeled with the therapeutic radiometal lutetium-177 in SCID mice. The second major goal of the project was to determine the efficacy of fractionating the MTD versus giving it in one dose, by evaluating mice bearing Mec-1 human lymphoma tumors. In the laboratory, the majority of this project was executed successfully by Dr. Dijie Liu and in part resulted in her promotion to Research Assistant Professor. The results of these studies have been or will be presented at several meetings and conferences. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
In order to find a safe and meaningful dose of our Lu-177-labeled PNA-peptide in a SCID mouse model of non-Hodgkin's lymphoma (NHL), the MTD was first determined. It was found to be 500 microcuries. Single doses of 125, 250, and 500 microcuries resulted in no signs of systemic toxicity, but doses of 750 and 1000 microcuries led to dehydration and weight loss, necessitating euthanasia. Mice were weighed daily and examined for signs of systemic toxicity, such as weight loss, decrease in body condition score, scruffy coat, lethargy, abdominal distention, cyanosis, and diarrhea. None of these signs were detected in any mouse at a dose less than or equal to 500 microcuries. Thus, at the MTD, no overt signs of toxicity were observed, making it unlikely that dose fractionation would be less toxic. Then, to test the hypothesis that dose fractionation is more effective than a single high dose, a study in Mec-1 NHL-bearing mice compared a single dose of 500 microcuries with a fractionated regimen of 4 doses of 125 microcuries, given on a weekly basis. The results showed that the median time to progression to a 1-g tumor in the dose fractionation group (46 days post-injection) was significantly greater (p of 0.04) than in the single dose group (30 days post-injection). Importantly, the dose fractionation regimen also prevented the formation of metastases. In the single dose group, 3 of 3 mice examined showed locoregional spread to the spinal cord, as well as metastases to the kidney and adrenal glands. In the fractionated group, no lesions were detected in any of these organs for 3 of 3 mice evaluated. No evidence of radiation damage was found in any organ. In conclusion, dose fractionation was not confirmed to be less toxic than the MTD; therefore this part of the hypothesis could not be accepted. However, dose fractionation of Lu-177-PNA-peptide upheld the part of the hypothesis that it would be more effective than a single injection of the MTD. Our future goal is to develop a targeted antisense agent with greater potency for more effective therapy.

Publications

• No publications reported this period