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

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

Anti-CD38 Pretargeted Radioimmunotherapy Demonstrates Therapeutic Efficacy In a Human Multiple Myeloma Mouse Xenograft Model

Abstract 1842 Multiple myeloma (MM) remains incurable despite improved response rates and improved progression free survival in the era of therapy with novel agents, including bortezomib, thalidomide, and lenalidomide. Disease 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. The ectoenzyme receptor 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. 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 synthetic 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 were directly measured in biodistribution experiments involving athymic nude mice bearing human MM xenograft tumors. Groups of 5 mice with s.c. L363 human MM (IgG) xenograft tumors received 1.4 nmol (300 μg) of either OKT10-SA (anti-CD38 SA) or an IgG1 isotype matched control Ab BHV1-SA (bovine herpes virus-1) 22 hrs prior to synthetic biotin-acetyl-galactosamine clearing agent (CA; 5.8 nmol [50 μg]) and 24 hrs prior to trace labeled 111 In-DOTA-biotin (1 μg). The CA removed >95% of both unbound OKT10-SA and BHV1-SA from the mouse circulation within 30 minutes of administration. Animals were euthanized and comprehensive tissue biodistributions were assessed 2, 24, 48 and 96 hrs after 111 In-DOTA-biotin injection. Tumors excised from mice pretargeted with OKT10-SA contained 13.1 ± 1.9 % of the injected dose of 111 In-DOTA-biotin per gram (% ID/g) after 2 hrs and 8.8 ± 2.8 % ID/g after 24 hrs compared to 2.4 ± 0.6 % ID/g after 2 hrs and 0.9 ± 0.4 % ID/g after 24 hrs in tumors excised from control mice pretargeted with BHV1-SA. Tumor-to-normal organ ratios of absorbed radioactivity 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 BHV1-SA. Therapy studies were then performed in athymic nude mice (n=9-10/group) bearing s.c. L363 human MM xenograft tumors. Reagent concentrations and time-points for administration of OKT10-SA, BHV1-SA and CA were identical to those reported for the biodistribution studies. The high energy beta particle emitter 90 Yttrium (t 1/2 = 64 hrs) was used as the therapeutic radionuclide. 90 Y-DOTA-biotin (2 μg) was labeled with 400 μCi, 800 μCi, or 1200 μCi per mouse in 3 OKT10-SA groups and 3 control groups (untreated control; 800 μCi or 1200 μCi 90 Y-DOTA-biotin following BHV1-SA). All mice in the untreated control and BHV1-SA control groups experienced exponential MM tumor growth and 78% of the untreated control animals required euthanasia within 17 days. All mice pretargeted with OKT10-SA demonstrated tumor shrinkage by day 6 at all dose levels (see figure). After 17 days, 90% of the OKT10-SA treated animals in the 400 μCi and 1200 μCi groups and 100% of the animals in the 800 μCi remained alive. One animal treated with 1200 μCi was euthanized on day 10 due to weight loss, however the remaining 9 animals from that group were 106 ±9% of initial body weight on day 17. Objective remissions were observed within 6 days in 100% of the mice treated with OKT10-SA followed by 1200 μCi of 90 Y-DOTA-biotin, including 100% complete remissions (no detectable tumor in OKT10-SA treated mice compared to tumors that were 5240 ± 2495% of initial tumor volume in untreated control animals) by day 17. These studies represent the first application of both PRIT and CD38 targeted radioimmunotherapy in MM. Favorable OKT10-SA biodistribution findings correlate with early evidence of therapeutic efficacy. Tumor responses in this MM xenograft tumor model are encouraging, but long term toxicity and survival results are not yet mature. Future studies combining PRIT and novel agents are planned in xenograft and SCID-hu myeloma models. Disclosures: Gopal: Millenium . Wood: BD Biosciences: Research Funding

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