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

Pretargeted Radioimmunotherapy Using Genetically Engineered Antibody-Streptavidin Fusion Proteins for Treatment of Non-Hodgkin Lymphoma

Pretargeted radioimmunotherapy (PRIT) using streptavidin (SAv)-biotin technology can deliver higher therapeutic doses of radioactivity to tumors than conventional RIT. However, “endogenous” biotin can interfere with the effectiveness of this approach by blocking binding of radiolabeled biotin to SAv. We engineered a series of SAv FPs that down-modulate the affinity of SAv for biotin, while retaining high avidity for divalent DOTA-bis-biotin to circumvent this problem

Pretargeted radioimmunotherapy using genetically engineered antibody-streptavidin fusion proteins for treatment of non-hodgkin lymphoma.

Purpose: Pretargeted radioimmunotherapy (PRIT) using streptavidin (SAv)-biotin technology can deliver higher therapeutic doses of radioactivity to tumors than conventional RIT. However, "endogenous" biotin can interfere with the effectiveness of this approach by blocking binding of radiolabeled biotin to SAv. We engineered a series of SAv FPs that downmodulate the affinity of SAv for biotin, while retaining high avidity for divalent DOTA-bis-biotin to circumvent this problem.Experimental Design: The single-chain variable region gene of the murine 1F5 anti-CD20 antibody was fused to the wild-type (WT) SAv gene and to mutant SAv genes, Y43A-SAv and S45A-SAv. FPs were expressed, purified, and compared in studies using athymic mice bearing Ramos lymphoma xenografts.Results: Biodistribution studies showed delivery of more radioactivity to tumors of mice pretargeted with mutant SAv FPs followed by (111)In-DOTA-bis-biotin [6.2 +/- 1.7% of the injected dose per gram (%ID/gm) of tumor 24 hours after Y43A-SAv FP and 5.6 +/- 2.2%ID/g with S45A-SAv FP] than in mice on normal diets pretargeted with WT-SAv FP (2.5 +/- 1.6%ID/g; P = 0.01). These superior biodistributions translated into superior antitumor efficacy in mice treated with mutant FPs and (90)Y-DOTA-bis-biotin [tumor volumes after 11 days: 237 +/- 66 mm(3) with Y43A-SAv, 543 +/- 320 mm(3) with S45A-SAv, 1129 +/- 322 mm(3) with WT-SAv, and 1435 +/- 212 mm(3) with control FP (P < 0.0001)].Conclusions: Genetically engineered mutant-SAv FPs and bis-biotin reagents provide an attractive alternative to current SAv-biotin PRIT methods in settings where endogenous biotin levels are high. Clin Cancer Res; 17(23); 7373-82. (C)2011 AACR

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

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

Anti-CD45 Radioimmunotherapy Using the Alpha-Emitting Radionuclide 211At Combined with Bone Marrow Transplantation Prolongs Survival in a Disseminated Murine Leukemia Model

Abstract 4096 Background: Despite aggressive hematopoietic cell transplant (HCT) strategies, many patients with acute myeloid leukemia (AML) relapse. Our group has explored radioimmunotherapy (RIT) using anti-CD45 antibodies (Ab) labeled with β-emitting radionuclides such as 131 I and 90 Y as a means to augment cytotoxicity and reduce relapse. This approach has been limited by their low energy levels (0.66–2.3 MeV) and potential non-specific toxicities due to their relatively long path lengths (0.3–2.3 mm). Conversely, α-emitting agents display higher energy levels (8 MeV) delivered over a short path-length (∼60–80 μm) that can lead to superior therapeutic ratios of absorbed radiation doses that may reduce AML relapse. For this purpose 211 At is an α-emitter with an attractive half-life (7.2 hours), energy profile (6.8 MeV averages of two alpha decays, 5.9 and 7.5 MeV) and path length (average range 55–70 μm). Therefore, we evaluated the efficacy and toxicity of anti-CD45 RIT using 211 At in a clinically relevant CD45 + disseminated murine leukemia model. Methods: SJL/J mice were given 10 5 syngeneic SJL leukemic peripheral blood mononuclear cells (PBMCs) via tail vein, followed two days later by 211 At-labeled anti-murine CD45 Ab-decaborate(2-) conjugate (30F11-B10) or 211 At-labeled negative control Ab-decaborate(2-) conjugate (rat IgG-B10) in tissue biodistribution studies. Groups of 5 mice were euthanized 6, 24 and 48 hours later and organs were harvested and analyzed in a gamma counter to yield percent of the injected dose per gram (% ID/g). To assess toxicities associated with this approach, SJL/J mice were treated with 12–24 μCi of 211 At-30F11-B10 and then evaluated weekly thereafter for impact on blood counts, as well as changes in hepatic and renal function. In HCT therapeutic studies, groups of 10 leukemic mice per dose were injected with 12–24 μCi of 211 At-30F11-B10 or 211 At-rat Ab-B10, followed two days later by rescue with 15 × 10 6 syngeneic bone marrow (BM) cells. Results: Delivery of 211 At-30F1-B10 demonstrated excellent localization to the BM and spleen at 24 hours (79 and 18% ID/g, respectively) post injection with lower kidney and lung uptake (8.4 and 8.3% ID/g, respectively) at the same time point. Anti-CD45 RIT using 211 At-30F11-B10 followed by syngeneic HCT led to a dose-dependent survival benefit in leukemic mice with a median survival (OS) of 120, 98 days, and 62 days for animals treated with 24, 20, and 12 μCi 211 At-30F11-B10, respectively, compared with untreated control mice (median OS of 36 days) and mice treated with non-specific 211 At-labeled rat Ab-B10 (median OS of 46 days) (Figure). Moreover, anti-CD45 RIT with 211 At-30F11-B10 led to minimal toxicity with mild dose dependent leukopenia as the most pronounced lab abnormality. White blood cell count nadir was between 2.5 and 4.2 k/μL two weeks after HCT for mice treated with 24 and 12 μCi 211 At-30F11-B10, respectively. Counts recovered to normal levels (6–8 K/μL) 4 weeks after HCT. Mild increases in transaminase levels were seen in mice that received 211 At-30F11-B10, yet these values remained within the normal ranges (ALT 68–75 IU/L; AST 155–170 IU/L). Renal function after 211 At-30F11-B10 did not significantly deviate from baseline (BUN 15–17 mg/dL; Cr 0.3–0.4 mg/dL). Conclusion: Taken together, these data suggest that anti-CD45 RIT using the α-emitting radionuclide 211 At in conjunction with HCT is a promising therapeutic option for AML. Excellent targeting of radiation doses to BM and spleen was demonstrated with a favorable toxicity profile. Further investigation of anti-CD45 RIT for AML using 211 At in clinical trials appears to be warranted. Disclosures: No relevant conflicts of interest to declare

Astatine-211 Conjugated To Anti-CD20 Monoclonal Antibody Eliminates B-Cell Lymphoma In a Mouse Model

Stem cell rescue after myeloablative doses of beta particle emitting radiolabeled monoclonal antibodies targeting CD20 antigen can lead to remissions in up to 95% of lymphoma patients who previously failed conventional combination chemotherapy. While encouraging, toxicities with beta particle radioimmunotherapy (RIT) are significant and ∼50% of patients ultimately relapse. Higher doses of absorbed radiation to tumors have correlated with a reduced risk of disease recurrence but dose limiting toxicities prevent escalation. A potential advantage of alpha particle emitting radionuclides is the release of large amounts of energy linearly over a few cell diameters (∼50-80 mm) resulting in irreparable double-strand DNA breaks that overwhelm cellular repair mechanisms. As a result, alpha particles confer a unique capacity to kill individual targeted cells while causing minimal radiation damage to surrounding tissues. To explore alpha-emitter conjugated anti-CD20 RIT, a closo -decaborate(2-) [B10] labeling reagent was developed as a radiolabeling platform capable of providing critical stability to alpha particle-labeled biomolecules. 211At was chosen as a therapeutic radionuclide based on its high linear energy transfer, biologically relevant t1/2 (∼7.2 hr) and absence of toxic daughter radionuclide decay byproducts. Cell binding assays using CD20-expressing Ramos tumor cells demonstrated that B10 conjugated to the anti-CD20 monoclonal antibody (mAb) 1F5 (B10-1F5) was specific and that astatination did not impair antibody binding (211At-B10-1F5 binding was equivalent to 125I-B10-1F5). Blood clearance was similar for 125I-1F5, 211At-B10-1F5 and 125I-B10-1F5 in athymic nude- Foxn1nu mice (23.87 ±5.13 % injected dose/gram [ID/g], 19.41 ±3.27 %ID/g and 19.80 ±1.44% ID/g respectively) at 17 hr post infusion. In tissue biodistribution studies using nude mice bearing Ramos flank tumor xenografts (10 x106 cells injected) radioactivity was measured in blood, tumor and nonspecific organs harvested 24 hours after injection (n=5/group). Animals received either 125I-1F5, 211At-B10-1F5 and 125I-B10-1F5 (co-injected) or isotype matched control mAb 211At- B10-HB8181 and125I-B10-HB8181(co-injected). Measured activity in tumors was three-fold higher for 125I-1F5, 125I-B10-1F5 and 211At-B10-1F5 (7.62 ± 2.09%ID/g, 7.53 ± 1.59%ID/g and 9.28 ± 1.85%ID/g respectively) than for 125I-B10-HB8181 and 211At-B10-HB8181 controls (2.87 ± 0.35%ID/g and 3.45 ± 0.58%ID/g respectively). In non-target organs no appreciable difference in measured activity was seen with either 211At- or 125I-labeled B10-1F5 and their respective controls. Subsequent therapy studies performed in nude mice bearing Ramos flank tumor xenografts demonstrated only a moderate survival advantage after 211At-B10-1F5 [data not shown]. This finding was consistent with the hypothesis that the alpha particle's short path length may not be ideally suited to models of “bulky” disease. In contrast, therapy studies using disseminated Ramos and Granta tumor cells introduced into NOD-SCID mice suggest a role for alpha particle based RIT in “non-bulky” disease models. In these studies NOD-SCID animals received 1x106 tumor cells iv 48 hours prior to 211At-B10-1F5 (10 μCi or 15 μCi) or control 211At-B10-HB8181 (n=10/group). Stem cell rescue was performed 48 hours after the RIT. Eighty days after treatment 80% of animals receiving 15 μCi of 211At-B10-1F5 and 70% of animals in the 10 μCi treatment group were alive and tumor free while no animals in the non-binding 211At-B10-HB8181 (10 μCi or 15 μCi) control groups survived beyond day 47 ([Figure][1]). Blood counts, serum creatinine and transaminase levels measured in 211At-B10-1F5 treated animals ∼180 days after RIT demonstrated no significant long-term bone marrow, renal or liver toxicity. ![Figure][2] 211At-B10-1F5 can eliminate CD20 expressing tumor cells in this mouse model and further study appears to be warranted. Disclosures: No relevant conflicts of interest to declare. [1]: #F1 [2]: pending:ye