Biodistributions, Myelosuppression and Toxicities in Mice Treated with an Anti-CD45 Antibody Labeled with the α-Emitting Radionuclides Bismuth-213 or Astatine-211

We previously investigated the potential of targeted radiotherapy using a bismuth-213- labeled anti-CD45 antibody to replace total body irradiation as conditioning for hematopoietic cell transplantation in a canine model. While this approach allowed sustained marrow engraftment, limited availability, high cost and short half-life of bismuth-213 induced us to investigate an alternative α-emitting radionuclide, astatine-211, for the same application. Biodistribution and toxicity studies were conducted with conjugates of the anti-murine CD45 antibody 30F11 with either bismuth-213 or astatine-211. Mice were injected with 2-50 μCi on 10 μg or 20 μCi on 2 or 40 μg 30F11 conjugate. Biodistribution studies showed that the spleen contained the highest concentration of radioactivity, ranging from 167±23 to 417±109 % injected dose/gram (%ID/g) after injection of the astatine-211 conjugate and 45±9 to 166±11 %ID/g after injection of the bismuth-213 conjugate. The higher concentrations observed for astatine-211- labeled 30F11 were due to its longer half-life, which permitted better localization of isotope to the spleen before decay. Astatine-211 was more effective at producing myelosuppression for the same quantity of injected radioactivity. All mice injected with 20 or 50 μCi astatine-211 but none with the same quantities of bismuth-213 had lethal myeloablation. Severe reversible acute hepatic toxicity occurred with 50 μCi bismuth-213, but not with lower doses of bismuth-213 or with any dose of astatine-211. No renal toxicity occurred with either radionuclide. The data suggest that smaller quantities of astatine-211-labeled anti-CD45 antibody are sufficient to achieve myelosuppression and myeloablation with less non-hematological toxicity compared with bismuth-213-labeled antibody

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

Anti-CD45 Pretargeted Radioimmunotherapy in a Nonhuman Primate Model.

The efficacy of radiolabeled antibody (RAb) therapy has been validated by high response rates in hematologic malignancy, yet patients treated with conventional non-myeloablative doses of radioimmunotherapy (RIT) usually relapse. Low tumor-to-normal organ ratios of absorbed radioactivity are the result of ongoing non-specific radiation exposure through the circulation; this accumulates over time and is dose limiting. Our group has documented the efficacy of pretargeted RIT in murine models, using an anti-human (h)CD45 antibody (BC8). We have engineered, expressed, and purified a recombinant tetravalent single chain antibody (Ab)-streptavidin fusion protein [(scFv) 4 -SA] directed against (h)CD45. The tetrameric fusion protein (FP) retains the full antigen binding capacity of intact Ab. Because the tumor-reactive FP is not bound to a therapeutic radionuclide, it can localize to tumor sites without subjecting the rest of the body to non-specific irradiation. After maximal accumulation of BC8-FP in the tumor, a small molecular weight radioactive moiety with high affinity for the tumor-reactive FP is administered (radio-DOTA-biotin). This second reagent penetrates tumors rapidly where it binds to the pretargeted FP. Unbound radio-DOTA-biotin molecules are small enough to be rapidly cleared from the blood and excreted in the urine within minutes. In a series of studies involving fourteen fascicularis Macaques, we have shown that this two-step pretargeting approach using BC8-FP is feasible, safe and effective. We have identified that the blood clearance kinetics of radiolabeled BC8-FP are comparable to conventionally radiolabeled intact Abs (t 1/2 = 40.6 and 50.2 hrs respectively), and have determined that the optimal time-point for radio-DOTA-biotin administration is 48 hrs after FP infusion. We found BC8-FP uptake in target tissue (lymph node and spleen) to be very specific when compared with a control anti-TAG72 (scFv) 4 -SA (CC49-FP). Measured on a gamma counter at 96 hrs post infusion, the percent injected dose per gram (%ID/g) was 0.28±0.01 for BC8-FP and 0.03±0.01 for CC49-FP in lymph nodes (LN); and 0.33±0.02 and 0.07±0.002, respectively, in spleen. Serial LN biopsies and terminal spleen tissue harvests in animals co-injected with equimolar concentrations of intact BC8 antibody and BC8-FP, revealed equivalent initial uptake in target tissue, but superior retention of BC8-FP over time. After 96 hrs, the %ID/g of 111 In-BC8-FP in lymph node was 0.93±0.02, compared to 0.046±0.013 for BC8 Ab. In spleen the %ID/g was 0.158±0.0 for BC8-FP versus 0.057±0.0 for BC8 Ab. With two-step pretargeting, LN-to-blood and spleen-to-blood ratios 48 hours after 111 In-DOTA-biotin were 10.3:1 and 13.9:1, respectively, while for conventional BC8 Ab they were 2.6:1 and 3.0:1 respectively. These studies demonstrate that multi-step pretargeting can improve tumor-to-normal organ ratios of radionuclide delivery in a nonhuman primate model and offer the promise of more durable responses in patients with hematologic malignancy. Further investigation of radiolabeled BC8 as a rational therapeutic agent for both myeloid leukemia and non-Hodgkin’s lymphoma is warranted. Additional studies are planned involving introduction of a dendrimeric N-acetyl galactosamine “clearing agent” to further improve the therapeutic index. Future patient clinical trials are anticipated

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

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

Glypican-3 targeted positron emission tomography detects sub-centimeter tumors in a xenograft model of hepatocellular carcinoma

Abstract Background Early intrahepatic recurrence is common after surgical resection of hepatocellular carcinoma (HCC) and leads to increased morbidity and mortality. Insensitive and nonspecific diagnostic imaging contributes to EIR and results in missed treatment opportunities. In addition, novel modalities are needed to identify targets amenable for targeted molecular therapy. In this study, we evaluated a zirconium-89 radiolabeled glypican-3 (GPC3) targeting antibody conjugate (89Zr-αGPC3) for use in positron emission tomography (PET) for detection of small, GPC3+ HCC in an orthotopic murine model. Athymic nu/J mice received hepG2, a GPC3+ human HCC cell line, into the hepatic subcapsular space. Tumor-bearing mice were imaged by PET/computerized tomography (CT) 4 days after tail vein injection of 89Zr-αGPC3. Livers were then excised for the tumors to be identified, measured, bisected, and then serially sectioned at 500 μm increments. Sensitivity and specificity of PET/CT for 89Zr-αGPC3-avid tumors were assessed using tumor confirmation on histologic sections as the gold standard. Results In tumor-bearing mice, 89Zr-αGPC3 avidly accumulated in the tumor within four hours of injection with ongoing accumulation over time. There was minimal off-target deposition and rapid bloodstream clearance. Thirty-eight of 43 animals had an identifiable tumor on histologic analysis. 89Zr-αGPC3 immuno-PET detected all 38 histologically confirmed tumors with a sensitivity of 100%, with the smallest tumor detected measuring 330 μm in diameter. Tumor-to-liver ratios of 89Zr-αGPC3 uptake were high, creating excellent spatial resolution for ease of tumor detection on PET/CT. Two of five tumors that were observed on PET/CT were not identified on histologic analysis, yielding a specificity of 60%. Conclusions 89Zr-αGPC3 avidly accumulated in GPC3+ tumors with minimal off-target sequestration. 89Zr-αGPC3 immuno-PET yielded a sensitivity of 100% and detected sub-millimeter tumors. This technology may improve diagnostic sensitivity of small HCC and select GPC3+ tumors for targeted therapy. Human trials are warranted to assess its impact

Anti-CD38 Pretargeted Radioimmunotherapy Eradicates Multiple Myeloma Xenografts In a Murine Model

Novel therapies, including immunomodulatory agents (thalidomide, lenalidomide and pomalidomide) and proteasome inhibitors (bortezomib, carfilzomib) have improved response rates and prolonged progression free survival for patients with multiple myeloma (MM). Despite these advances the disease remains incurable. MM persistence is presumably due to residual malignant plasma cell clones that evade or develop resistance to available therapies. The efficacy of radioimmunotherapy (RIT) in the treatment of hematologic malignancies is well established and the radiosensitivity of malignant plasma cells has been demonstrated in both preclinical and clinical settings. CD38 is a plasma cell antigen that exhibits relatively specific, stable and uniform expression (95-100%) at a high epitope density on myeloma cells, making it an attractive target for antibody based therapies, including RIT. Recently, the unmodified CD38 mAb daratumumab has demonstrated anti-MM tumor cell activity both in vitro and in mouse xenografts, validating the antigen as a desirable target. Pretargeted RIT (PRIT), using a multi-step streptavidin (SA)-biotin targeting system enhances the therapeutic index of delivered radiation. We have generated an anti-CD38 antibody (Ab)-SA chemical conjugate (OKT10-SA). The OKT10-SA construct binds with high avidity to myeloma cells while retaining full biotin-binding capability for radiolabeled DOTA-biotin. Blood, tumor and nonspecific organ uptakes of OKT10-SA directly measured in biodistribution experiments involving athymic nude mice bearing human MM xenograft tumors (Green, et al. ASH 2011) demonstrated tumor-to-normal organ ratios of absorbed radioactivity that were 8:1; 10:1; 8:1; and 6:1 respectively for blood, lung, liver and kidney in mice pretargeted with OKT10-SA; compared to 0.6:1; 0.9:1; 0.8:1 and 0.4:1 respectively, in control mice pretargeted with the IgG1 isotype matched control Ab BHV1-SA (bovine herpes virus-1). In therapy studies athymic nude mice (n=9-10/group) bearing s.c. NCI-H929 human MM xenograft tumors received 1.4 nmol (300 µg) of either OKT10-SA (anti-CD38 SA) or BHV1-SA 22 hrs prior to synthetic biotin-acetyl-galactosamine clearing agent (CA; 5.8 nmol [50 µg]) and 24 hrs prior to 90Y-DOTA-biotin (2 µg) labeled with 800 µCi per mouse. Previously, a dose range of 800 µCi to 1200 µCi was identified as optimal when the high energy beta particle emitter 90Yttrium (t1/2 = 64 hrs) was used as the therapeutic radionuclide in a MM xenograft model (Green , et al. ASH 2011). All mice in the BHV1-SA control group experienced exponential MM tumor growth and 100% of these control animals required euthanasia within 37 days. All mice pretargeted with OKT10-SA demonstrated tumor shrinkage by day 6. After 70 days, 100% of the OKT10-SA treated animals remained alive and objective remissions were observed within 6 days in 100% of the mice treated with OKT10-SA followed by 800 µCi of 90Y-DOTA-biotin, including 100% complete remissions (no detectable tumor in OKT10-SA treated mice) compared to tumors that were 2982 ± 2834% of initial tumor volume [p<0.0001, Student’s t-test] in untreated control animals by day 23 [[Figure][1]]. Hepatic and renal function was assessed 160 days after mice received OKT10-SA followed by 90Y-DOTA-biotin (800 µCi, n=5) and no significant toxicity was observed. The measured serum creatinine was <0.4 mg/dl in all animals, the average BUN, ALT and AST values were 48 + 33 mg/dl, 46.8 +40 and 120.4 + 116.7 U/L respectively. The observed anti-tumor activity in this model was not attributable to an effector response mediated by the CD38 mAb in the absence of radiolabeled DOTA-biotin. Mice bearing MM xenografts (n=10) treated with OKT10-SA administered alone (without subsequent 90Y-DOTA-biotin infusion) demonstrated no objective tumor responses and 100% required euthanasia by day 17 for tumors that were 7837 ± 3492% of their initial volume. In a matched cohort of mice (n=10) treated concurrently with identical doses of OKT10-SA followed by CA and 90Y-DOTA-biotin (800µCi), 100% of the treated animals achieved a complete remission by day 17 and 70% of these animals were alive and tumor free at day 80. Tumor responses in this MM xenograft tumor model are encouraging and results support further evaluation of both PRIT and CD38 targeted RIT in MM. Studies combining PRIT and novel agents are ongoing in both xenograft and SCID-hu MM models. ![Figure][2] ![Figure][2] Disclosures: No relevant conflicts of interest to declare. [1]: #F1 [2]: pending:ye