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

Design and synthesis of bis-biotin-containing reagents for applications utilizing monoclonal antibody-based pretargeting systems with streptavidin mutants

Previous studies have shown that pretargeting protocols, using cancer-targeting fusion proteins, composed of 4 anti-CD20 single chain Fv (scFv) fragments and streptavidin (scFv(4)-SAv), followed by a biotinylated dendrimeric N-acetyl-galactosamine blood clearing agent (CA), 1, then a radiolabeled DOTA-biotin derivative (a monobiotin), 3a, can provide effective therapy for lymphoma xenografts in mouse models. A shortcoming in this pretargeting system is that endogenous biotin may affect its efficacy in patients. To circumvent this potential problem, we investigated a pretargeting system that employs anti-CD20 scFv(4)-SAv mutant fusion proteins with radioiodinated bis-biotin derivatives. With that combination of reagents, good localization of the radiolabel to lymphoma tumor xenografts was obtained in the presence of endogenous biotin. However, the blood clearance reagents employed in the studies were ineffective, resulting in abnormally high levels of radioactivity in other tissues. Thus, in the present investigation a bis-biotin-trigalactose blood clearance reagent, 2, was designed, synthesized, and evaluated in vivo. Additionally, another DOTA-biotin derivative (a bis-biotin), 4a, was designed and synthesized, such that radiometals (e.g., (111)In, (90)Y, (177)Lu) could be used in the pretargeting protocols employing scFv(4)-SAv mutant fusion proteins. Studies in mice demonstrated that the CA 2 was more effective than CA 1 at removing [(125)I]scFv(4)-SAv-S45A mutant fusion proteins from blood. Another in vivo study compared tumor targeting and normal tissue concentrations of the new reagents (2 and [(111)In]4b) with standard reagents (1 and [(111)In]3b) used in pretargeting protocols. The study showed that lymphoma xenografts could be targeted in the presence of endogenous biotin when anti-CD20 fusion proteins containing SAv mutants (scFv(4)-SAv-S45A or scFv(4)-SAv-Y43A) were employed in combination with CA 2 and [(111)In]4b. Importantly, normal tissue concentrations of [(111)In]4b were similar to those obtained using the standard reagents (1 and [(111)In]3b), except that the blood and liver concentrations were slightly higher with the new reagents. While the reasons for the higher blood and liver concentrations are unknown, the differences in the galactose structures of the clearance agents 1 and 2 may play a role

Conventional and Pretargeted Radioimmunotherapy with Bismuth-213 to Target and Treat CD20-Expressing Non-Hodgkin Lymphoma: A Preclinical Model for Consolidation Therapy to Eradicate Minimal Residual Disease.

Abstract 2705 Poster Board II-681 Conventional radioimmunotherapy (RIT) with directly radiolabeled anti-B cell antibodies (Ab) induces remissions in 50 to 80% of patients with relapsed or refractory indolent non-Hodgkin lymphomas (NHL). Although administering RIT as consolidation after chemotherapy improves response rates and produces long-term durable remissions in treatment-naive patients, the β-emitting radionuclides used in current RIT schemes may not be ideal for irradiating the microscopic tumors and the isolated tumor cells present in the setting of minimal residual disease (MRD). RIT with α-emitting radionuclides may be advantageous in the treatment of MRD because the short path length and high energies of α-particles produce optimal cytotoxicity at small target sites while minimizing damage to the surrounding normal tissues. Our group has successfully demonstrated that pretargeted RIT (PRIT) using streptavidin (SA)-Ab and radiolabeled biotin allows rapid specific localization of radioactivity at tumor sites. PRIT using α-emitting radionuclides may be particularly attractive since the most promising α-emitting radionuclides in clinical settings, such as 213 Bi (t½ = 46 min), have short half-lives and pretargeting allows the delivery of radioactivity to tumor sites before the activity decays. We therefore performed in vivo studies to evaluate the biodistribution of 213 Bi with PRIT. Athymic mice with B-cell NHL (Ramos) xenografts received a tetravalent anti-CD20 (1F5) single-chain (scFv) 4 SA fusion protein (FP) or a CC49 (scFv) 4 SA FP (non-binding negative control) followed by an N-acetyl galactosamine clearing agent (CA) and subsequent 213 Bi-DOTA-biotin infusion. Tumors, blood, and major organs were collected to determine the percent injected dose per gram (%ID/g) at various time points within ∼3 half-lives of 213 Bi. Maximal tumor uptake for 1F5 (scFv) 4 SA FP was 16.5 ± 7.0 %ID/g at 90 minutes vs. 2.3 ± 0.9 %ID/g for the control FP (p = 0.0001). Biodistributions of 213 Bi using a conventional RIT scheme with directly labeled Ab were also evaluated. Athymic mice with Ramos xenografts received 213 Bi labeled 1F5 Ab or 213 Bi labeled HB8181 Ab (a murine isotype matched nonbinding control). Maximum tumor uptake for 1F5 Ab was 3.0 ± 0.9 %ID/g at 180 minutes vs. 2.3 ± 0.7 %ID/g for the control Ab (p = 0.171). There were no significant differences in tumor uptake and normal organ distribution between the two Ab within 180 minutes of radiolabeled Ab injection presumably due to the protracted circulating half-life of radiolabeled Abs. These results were concordant with our previous experiments using other radionuclides, showing that maximal targeting of radiolabeled Ab occurs between 20 to 24 hours; well beyond the effective half-life of 213 Bi. When the results of PRIT and RIT studies were directly compared, tumor-to-blood ratios were 58 to 426-fold higher with PRIT than with conventional RIT. Tumor-to-normal organ ratios of nearly 100:1 were observed with PRIT compared to 3:1 or less with conventional RIT. Using the most favorable PRIT schemes defined in the biodistribution experiments, the therapeutic efficacy of 213 Bi was evaluated. Mice treated with PRIT using 1F5 (scFv) 4 SA FP followed by a CA and 600 μCi 213 Bi-DOTA-biotin experienced significant delays in tumor growth. The 1F5 (scFv) 4 SA FP treated animals had a mean tumor volume of 0.01 ± 0.02 vs. 203.38 ± 83.03 mm 3 for the CC49 control group at 19 days (p = 0.0006). The median survival for 1F5 group was not reached after 90 days; whereas, the median survival was 23 days for the CC49 group (p = 0.0019) and 16 days for untreated mice (p = 0.0023). The treatment was well tolerated, with no treatment-related mortalities in any group. These data demonstrate that PRIT using 213 Bi has a favorable biodistribution profile and excellent therapeutic efficacy. This model may be particularly effective in MRD settings and further studies are ongoing. Disclosures: No relevant conflicts of interest to declare

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 Ab Pretargeted Radioimmunotherapy Using An Alpha Emitting Radionuclide (213Bi) Delivers Selective Radiation to Human Myeloid Leukemias in a Mouse Xenograft Model and Results in High Rates of Complete Remission and Long Term Survival.

Abstract 1035 Poster Board I-57 Little change has been seen over the past two decades in the progression-free survival of patients with Acute Myeloid Leukemia (AML) undergoing allogeneic hematopoietic cell transplantation (HCT). Efforts to decrease the risk of relapse after HCT have included increasing the intensity of the preparative regimen. Our group has focused on targeted radioimmunotherapy using an anti-CD45 antibody (Ab) as part of the preparative regimen prior to HCT. Despite the promise of this approach, recurrent malignancy remains a problem, particularly for patients with high-risk disease. More recently we have explored a pretargeted RIT (PRIT) strategy to augment the anti-tumor efficacy of the transplant preparative regimen while diminishing overall toxicity. These studies have employed an anti-CD45 Ab conjugated to streptavidin (SA) followed by a biotinylated, N-acetylgalactosamine-containing clearing agent (CA) to remove circulating Ab-SA conjugate from the blood and then with radiobiotin using beta-emitting radionuclides that have relatively low energy transfer characteristics and long path lengths that may result in suboptimal killing of leukemia cells and normal organ toxicity due to cross-fire from malignant cells. Alpha-emitting radionuclides exhibit very high cytotoxicity coupled with a short path length, potentially increasing the therapeutic index and making them an attractive alternative to beta-emitting radionuclides. Therefore, we have now employed PRIT using an anti-CD45 Ab-SA conjugate, CA, and biotin labeled with an alpha-emitting radionuclide (213Bi) in mice with human erythroid leukemia (HEL) xenografts. Results of biodistributions of radioactivity demonstrated excellent localization of 213Bi-biotin to tumors with minimal uptake into normal organs due to elimination of non-specific radiation exposure from blood-borne radiolabeled Ab. After 10 minutes, 4.5 ± 1.1% of the injected dose of 213Bi was delivered per gram of tumor (% ID/g). Imaging using a novel alpha camera demonstrated uniform radionuclide distribution within tumor tissue at 10 minutes after 213Bi-biotin injection. Estimated radiation-absorbed doses delivered to HEL xenografts delivered 3.4-and 2.1-fold more radiation to tumor than to liver and lungs, respectively. These target-to-non-target ratios of absorbed radiation obtained using PRIT with 213Bi were similar to those observed using a beta-emitting (90Y) radionuclide in the same animal model. Based on these encouraging results, we conducted therapy experiments in a minimal disease xenograft model using a single dose of 213Bi-biotin given 24 hours after anti-CD45 Ab-SA conjugate. In an initial attempt to compare 90Y and 213Bi, we gave equal μCi doses of each radionuclide. Eighty percent of mice treated with anti-CD45 Ab-SA conjugate followed by 800 μCi of 213Bi-biotin survived leukemia-free for >100 days with minimal toxicity. By comparison, only 20% of mice treated with PRIT anti-CD45 Ab-SA conjugate followed by 800 μCi 90Y-biotin exhibited long term leukemia-free survival. While we acknowledge that equal μCi doses may not necessarily result in equivalent doses delivered to normal tissue, these data suggest that anti-CD45 PRIT using an alpha-emitting radionuclide may be highly effective and minimally toxic for the treatment of myeloid leukemias. Disclosures: No relevant conflicts of interest to declare

Anti-CD45 radioimmunotherapy using 211At with bone marrow transplantation prolongs survival in a disseminated murine leukemia model

Despite aggressive chemotherapy combined with hematopoietic stem cell transplantation (HSCT), many patients with acute myeloid leukemia (AML) relapse. Radioimmunotherapy (RIT) using monoclonal antibodies labeled with β-emitting radionuclides has been explored to reduce relapse. β emitters are limited by lower energies and nonspecific cytotoxicity from longer path lengths compared with α emitters such as (211)At, which has a higher energy profile and shorter path length. We evaluated the efficacy and toxicity of anti-CD45 RIT using (211)At in a disseminated murine AML model. Biodistribution studies in leukemic SJL/J mice showed excellent localization of (211)At-anti-murine CD45 mAb (30F11) to marrow and spleen within 24 hours (18% and 79% injected dose per gram of tissue [ID/g], respectively), with lower kidney and lung uptake (8.4% and 14% ID/g, respectively). In syngeneic HSCT studies, (211)At-B10-30F11 RIT improved the median survival of leukemic mice in a dose-dependent fashion (123, 101, 61, and 37 days given 24, 20, 12, and 0 µCi, respectively). This approach had minimal toxicity with nadir white blood cell counts >2.7 K/µL 2 weeks after HSCT and recovery by 4 weeks. These data suggest that (211)At-anti-CD45 RIT in conjunction with HSCT may be a promising therapeutic option for AML