Anti-CD45 Pretargeted Radioimmunotherapy Prior to Bone Marrow Transplantation without Total Body Irradiation Facilitates Engraftment From Haploidentical Donors and Prolongs Survival in a Disseminated Murine Leukemia Model

s / Biol Blood Marrow Transplant 19 (2013) S211eS232 S228 chemotherapy was HIDAC (1-3 grams/m2 for 6-8 doses)/ Etoposide(15-40mg/kg) in 16 patients and growth factor alone in one patient. Median time from diagnosis to ASCT was 4.2 (range 3.6-7) months. Preparative regimen for ASCT was Busulfan (3.2mg/kg x 4)/Etoposide (60 mg/kg) in 12 patients and high dose melphalan in 5 patients. The median CD34 cells infused was 4.9 x 10e6/kg (range 2.8 to 15.9).All patients engrafted with a median time to neutrophil engraftment of 11 (range10-12) days. The median time to platelet engraftment was 20 (range15-40) days. The median length of inpatient stay during the ASCT admission was 14 (range 10-25) days. One patient died of progressive disease 14 months post ASCT. Two patients died in remission on day 53 (sepsis) and day 836 (unknown cause) post ASCT. Fourteen patients (82%) are currently alive in complete remission. at a median follow-up of 20 (range 140) months post ASCT. Conclusion: Consolidation of good risk AML patients with ASCT following induction of complete remission is safe and effective in preventing relapse in good risk AML patients

Evaluation of a Wet Chemistry Method for Isolation of Cyclotron Produced [211At]Astatine

A “wet chemistry” approach for isolation of 211At from an irradiated bismuth target is described. The approach involves five steps: (1) dissolution of bismuth target in conc. HNO3; (2) removal of the HNO3 by distillation; (3) dissolution of residue in 8 M HCl; (4) extraction of 211At from 8 M HCl into DIPE; and (5) extraction of 211At from DIPE into NaOH. Results from 55 “optimized” 211At isolation runs gave recovery yields of approximately 78% after decay and attenuation corrections. An attenuation-corrected average of 26 ± 3 mCi in the target provided isolated (actual) yields of 16 ± 3 mCi of 211At. A sixth step, used for purification of 211At from trace metals, was evaluated in seven runs. In those runs, isolated 211At was distilled under reductive conditions to provide an average 71 ± 8% recovery. RadioHPLC analyses of the isolated 211At solutions, both initial and after distillation, were obtained to examine the 211At species present. The primary species of 211At present was astatide, but astatate and unidentified species were also observed. Studies to determine the effect of bismuth attenuation on 211At were conducted to estimate an attenuation factor (~1.33) for adjustment of 211At readings in the bismuth target

Targeted 177 Lu antisense radiotherapy of B-cell non-Hodgkin's lymphoma

Title from PDF of title page (University of Missouri--Columbia, viewed on May 22, 2012).The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Dissertation advisor: Dr. Michael R. LewisVita.Includes bibliographical references."July 2011"[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] The B-cell lymphoma/leukemia-2 (bcl-2) proto-oncogene in non-Hodgkin's lymphoma (NHL) is a dominant inhibitor of apoptosis. The research goal was to develop a [superscript 177]Lu-labeled bcl-2 antisense peptide nucleic acid (PNA)-peptide conjugate designed for dual modality NHL therapy, consisting of a radiopharmaceutical capable of simultaneously down-regulating apoptotic resistance and delivering cytotoxic internally emitted radiation. In vitro results demonstrated [superscript 177]Lu-DOTA-anti-bcl-2-Tyr[superscript 3]-octreotate uptake in Mec-1 NHL cells. An in vitro dosimetry model was generated with the resulting data. Proliferation and viability assays of mass and absorbed dose indicated a mass-dose dependence and that [superscript 177]Lu-bcl-2 antisense PNA-Tyr[superscript 3]-octreotate acted additively in effecting decreased cell viability with increasing radiation doses. In vivo receptor saturation studies determined the mass of the compound necessary to saturate tumors, providing maximal compound uptake and antisense potential. Biodistribution data showed specific tumor targeting of the radiolabeled PNA-peptide in Mec-1 xenografts, which was compared to the radiolabeled peptide [superscript 177]Lu-DOTA-Tyr[superscript 3]-octreotate. In vivo dosimetry modeling calculated normalized tumor absorbed doses that would be below the 2 Gy bone marrow margin of safety. Therapy studies showed modest tumor growth delay for treatment with the [superscript 177]Lu-labeled PNA peptide. A comparison of the efficacy of a pulse/chase injection versus a single injection of the compound is also discussed

Targeted antisense radiotherapy [abstract]

Cancer is the one of the leading causes of death worldwide. While huge strides have been made in the treatment of various cancers, there is still a critical need for effective therapeutics that only target and kill the cancer cells while not affecting other cells in the body. The current invention developed by researchers at the University of Missouri is a novel agent for simultaneous, targeted gene and radiation therapy of blood cancers. The agent binds selectively to non-Hodgkin's lymphoma (NHL) cells and contains an antisense module that reduces the activity of a cancer gene to sensitize tumors to radiation. Simultaneously, the agent delivers radiation that is highly efficient at killing these sensitized cancer cells. Our invention is superior to conventional targeted radiotherapy because its two simultaneous mechanisms of action work together to ensure better cancer cell killing, potentially giving it more efficacy than existing approaches. Potential Areas of Applications: * Treatment of blood cancer * Can be modified to treat other cancers Patent Status: Provisional patent application cover sheet on file - 10UMC069prov Inventor(s): Michael Lewis, Ethan Balkin Contact Info: Harriet F. Francis, MS; J.D. [email protected] 573-884-0374 Per Stromhaug, Ph.D., MBA [email protected] 573-884-355

Evaluation of a Wet Chemistry Method for Isolation of Cyclotron Produced [ 211 At]Astatine

applied science

Anti-CD45 radioimmunotherapy with 90Y but not 177Lu is effective treatment in a syngeneic murine leukemia model.

Radioimmunotherapy (RIT) for treatment of hematologic malignancies has primarily employed monoclonal antibodies (Ab) labeled with 131I or 90Y which have limitations, and alternative radionuclides are needed to facilitate wider adoption of RIT. We therefore compared the relative therapeutic efficacy and toxicity of anti-CD45 RIT employing 90Y and 177Lu in a syngeneic, disseminated murine myeloid leukemia (B6SJLF1/J) model. Biodistribution studies showed that both 90Y- and 177Lu-anti-murine CD45 Ab conjugates (DOTA-30F11) targeted hematologic tissues, as at 24 hours 48.8 ± 21.2 and 156 ± 14.6% injected dose per gram of tissue (% ID/g) of 90Y-DOTA-30F11 and 54.2 ± 9.5 and 199 ± 11.7% ID/g of 177Lu-DOTA-30F11 accumulated in bone marrow (BM) and spleen, respectively. However, 90Y-DOTA-30F11 RIT demonstrated a dose-dependent survival benefit: 60% of mice treated with 300 µCi 90Y-DOTA-30F11 lived over 180 days after therapy, and mice treated with 100 µCi 90Y-DOTA-30F11 had a median survival 66 days. 90Y-anti-CD45 RIT was associated with transient, mild myelotoxicity without hepatic or renal toxicity. Conversely, 177Lu- anti-CD45 RIT yielded no long-term survivors. Thus, 90Y was more effective than 177Lu for anti-CD45 RIT of AML in this murine leukemia model

Anti-CD45 Radioimmunotherapy Facilitates Donor Engraftment and Prolongs Survival in the Absence of TBI Prior to Haploidentical Bone Marrow Transplantation in a Disseminated Murine Leukemia Model

Abstract 4101 Background: Despite the curative promise of hematopoietic cell transplantation (HCT), many patients with hematologic malignancies relapse and others may not proceed to HCT due to the unavailability of a matched donor. Toxicities remain high with HCT, due in part to the administration of non-specific therapies such as total body irradiation (TBI) as part of preparative regimens. We aim to overcome these limitations by replacing TBI with anti-CD45 radioimmunotherapy (RIT) for haploidentical HCT to deliver radiation directly to leukemic cells while sparing normal organs and minimizing non-specific toxicities. Methods: We established an initial TBI HCT regimen in B6SJLF1/J mice (H-2D b haplotype) conditioned with fludarabine (FLU, days -6 to -2), followed by TBI (250, 500, 750 cGy; day -1). The mice then received 15 million donor (CB6F1/J, H-2D d ) BM cells (day 0), followed by cyclophosphamide (CY) for graft-versus-host disease (GvHD) prophylaxis (day +2). Subsequent RIT HCT studies involved B6SJLF1/J mice conditioned with and without fludarabine (FLU) and escalating doses (200–400 μCi) of 90 Y-anti-CD45 Ab (30F11) RIT without TBI, followed by infusion of haploidentical BM cells from CB6F1/J mice and a single dose of cyclophosphamide (CY) 2 days after HCT. Chimerism studies were performed using flow cytometric analysis to assay for engraftment of donor CD8 + cells. Therapeutic studies were performed in B6SJLF1/J mice given 10 5 syngeneic leukemia cells via tail vein (day -5), followed by 200 or 400 μCi 90 Y-30F11 (day -3), and 1.5 × 10 7 BM donor cells (day 0) and two doses of CY (days -2 and +2) without FLU. Results: Using this model we have demonstrated that mixed chimerism was established in mice transplanted with TBI or escalating doses (200–400 μCi) of 90 Y-30F11 RIT followed by injection of haploidentical BM donor rescue cells. TBI-based HCT showed that chimerism as determined by flow cytometric analysis for donor CD8 + cells was TBI dose-dependent; mice receiving ≥500 cGy were fully chimeric 4 weeks post-HCT, and persisted ≥12 months. RIT-based HCT also revealed mice with mixed chimerism, with up to 89% of donor CD8 + cells 1 month after HCT. Elimination of FLU from the conditioning regimen did not significantly decrease chimerism, as mice transplanted without FLU showed up to 70% donor CD8 + cells 1 month after HCT. Subsequent RIT experiments in B6SJLF1/J mice harboring AML were treated with escalating doses of 90 Y-30F11 prior to HCT without FLU. Mice treated with anti-CD45 RIT using 200 μCi and 400 μCi of 90 Y-30F11 had a median overall survival (OS) of 73 (p 90 Y-30F11 group were euthanized on day 3 for excessive weight loss, without gross histology abnormality in kidneys or liver. Conclusion: These studies suggest that anti-CD45 RIT in the absence of TBI and FLU prior to haploidentical HCT can lead to establishment of mixed chimerism. Moreover, this anti-CD45 RIT in combination with haploidentical HCT can lead to improvement in survival for mice with AML. These results suggest that clinical studies with anti-CD45 RIT in lieu of TBI and FLU in a haploidentical HCT regimen should be considered for further investigation. Disclosures: No relevant conflicts of interest to declare

211At-Anti-CD45 Radioimmunotherapy Can Replace TBI Prior to Haploidentical Bone Marrow Transplantation and Yield Long-Term Hematopoietic Engraftment

Abstract Haploidentical bone marrow transplantation (haplo-BMT) is both clinically effective and widely available because related-donors can be identified for nearly all recipients. Despite the curative promise of this approach, many patients with hematologic malignancies will relapse after haplo-BMT and more effective preparative regimens are necessary. We have shown that anti-CD45 radioimmunotherapy (RIT) delivers high-doses of radiation to hematolymphoid organs while minimizing the radiation exposure to non-targeted tissues. The efficacy of beta-emitting radionuclides may be limited by their relatively low decay energies (0.66 – 2.3 MeV). We have thus investigated the higher energy alpha-emitter astatine-211 (211At) (average decay energy of 6.8 MeV), for targeted anti-CD45 radioimmunotherapy (RIT) in lieu of total body irradiation (TBI) prior to haploidentical BMT in a murine leukemia model to decrease relapse rates. Groups of five B6SJLF1/J mice (allotype H2-Db) received escalated activities (20, 30 or 40 μCi) of 211At-anti-CD45 antibody [100 μg (0.67 nmol) of B10-30F11] given by tail vein injections on day -2 in place of TBI prior to BMT. Animals received cyclophosphamide (CY; 200 mg/kg/day) on days –3, –2, or –1, and +2 for graft-versus-host disease prophylaxis, either alone, or with fludarabine (FLU; 100 mg/kg/day) for 4 days starting day -6. Transplanted mice received 1.5 × 107 haploidentical bone marrow cells from CB6F1/J mice (allotype H2-Dd) on day 0. Peripheral blood from recipient mice was then assayed monthly by flow cytometry to measure chimerism as the percentage of donor (H2-Dd) circulating CD8+ cells. The highest activity delivered of 40 µCi 211At-anti-CD45 RIT was uniformly lethal without BMT rescue, whereas 60% of transplanted mice at this dose survived to assessment at 1 month. Mice treated with 30 µCi of 211At-anti-CD45 RIT with pre- and post-transplant CY and without TBI or FLU, had high levels of engraftment with an average of 83.7 ± 5.8% donor CD8+ cells 1 month after haploidentical BMT (Table 1). The addition of FLU to 211At-anti-CD45 RIT with CY did not significantly improve chimerism levels, with mean donor CD8+ cells in mice treated with 40 µCi 211At-anti-CD45 RIT of 64.5 ± 41.6% compared to 60.0 ± 13.9% in the absence of FLU (p=0.8668). In addition, mice that received 30 µCi 211At-anti-CD45 RIT and pre-transplant CY on either day –3, –2, or –1 showed mean donor CD8+ cells of 83.7 ± 5.8%, 49.9 ± 29.6% and 55.0 ± 46.2% 1 month after haploidentical-BMT, respectively. Importantly, chimerism levels remained stable 2 months after haploidentical BMT with mean donor CD8+ cells of 80.4 ± 16.6%, 47.0 ± 37.7% and 63.2 ± 10.7% in mice treated with 30 µCi 211At-anti-CD45 RIT and pre-transplant CY on day –3, –2, and –1, respectively. Engraftment using 40 or 30 µCi 211At-anti-CD45 RIT was comparable to using 850 or 1000 cGy TBI (mean donor CD8+ cells of 70.2 ± 18.8% and 60.0 ± 4.6%, respectively) prior to haploidentical BMT. RIT alone without any chemotherapy was insufficient to facilitate clinically relevant rates of donor engraftment, as mice treated with 30 µCi 211At-anti-CD45 RIT and no FLU, CY or TBI had 15.9 ± 7.1% mean donor CD8+ cells 1 month after haploidentical BMT. These results suggest that 211At-anti-CD45 RIT prior to haploidentical BMT with pre– and post–transplant CY can result in high levels of donor hematopoietic cell engraftment in the absence of TBI and FLU. This conditioning regimen may be less toxic and more effective at preventing relapse than TBI-based approaches due to the high linear energy transfer of the alpha emissions, or the high decay energy of targeted 211At deposited over its short effective path-length. On-going studies are assessing the efficacy and toxicity associated with 211At-anti-CD45 RIT compared to a TBI-based haploidentical BMT using a syngeneic murine leukemia model. Abstract 2417. Table 1 . Preparative Therapy and CD8+ Donor Chimerism at 1 month Group FLU (100mg/kg/d) pre-BMT CY (200mg/kg) TBI 211 At-anti-CD45 RIT post-BMT CY (200mg/kg) Donor CD8+ % 1 – day –3 – 30 µCi day +2 83.7 ± 5.8 2 – day –2 – 30 µCi day +2 49.9 ± 29.6 3 – day –1 – 30 µCi day +2 55.0 ± 46.2 4 – day –3 – 40 µCi day +2 64.5 ± 41.6 5 d –6 to –3 day –3 – 40 µCi day +2 60.0 ± 13.9 6 – – – 30 µCi day +2 15.9 ± 7.1 7 – day –3 – – day +2 4.4 ± 0.6 8 – – 1000 cGy – day +2 60.0 ± 4.6 Disclosures No relevant conflicts of interest to declare