Development and introduction of the Filariasis Test Strip: A new diagnostic test for the Global Program to Eliminate Lymphatic Filariasis

A key component to achieving the global goal of elimination of lymphatic filariasis (LF) is the availability of appropriate tools for disease mapping, monitoring, and surveillance. However, the development of these tools for a neglected disease such as LF can be a challenge. The lack of a commercial market and low familiarity with these diseases leave little incentive for diagnostic manufacturers to invest in this space. The Filarial Test Strip (FTS) development story provides a case study on how a multi-stakeholder, public-private partnership model facilitated the development, evaluation, and introduction of a new monitoring and surveillance tool for LF. This paper will reflect on the experience with the FTS and document the process from development of the target product profile to adoption and scale-up in country programs. Lessons learned from both the successes and challenges experienced during this process may help inform future efforts to develop and introduce new diagnostic or surveillance tools for neglected diseases

Pretargeted Radioimmunotherapy for B-Cell Lymphomas

Relapsed or treatment refractory B-cell lymphomas are currently incurable with conventional chemotherapy and radiation treatments. High-dose chemoradiotherapy and stem cell transplantation can cure some patients with relapsed or refractory lymphoma, but the majority of such patients die of progressive disease. We have investigated the potential utility of pretargeted radioimmunotherapy using monoclonal antibody-streptavidin, immunoconjugates, and fusion proteins in combination with N -acetylgalactosamine dendrimeric clearing agent and radiometal-labeled 1,4,7,10-tetraazacyclododecane- N,N ′, N ″, N ‴-tetraacetic acid biotin for treatment of lymphomas using mouse and primate models. We have targeted a variety of cell surface antigens, including CD20, CD22, CD45, and HLA-DR, using conventional and pretargeted radioimmunotherapy. These studies showed the marked superiority of pretargeted radioimmunotherapy for each of the antigenic targets in terms of superior biodistributions, more complete tumor regressions, and longer survival. We are optimistic that this novel approach will provide a meaningful prolongation of survival for patients with relapsed or refractory lymphomas

Comparison of a tetravalent single-chain antibody-streptavidin fusion protein and an antibody-streptavidin chemical conjugate for pretargeted anti-CD20 radioimmunotherapy of B-cell lymphomas

The efficacy of radioimmunotherapy (RIT) for patients with relapsed non-Hodgkin lymphoma (NHL) is limited by nonspecific delivery of radiation to normal tissues due to the long circulating half-life of radiolabeled anti-CD20 antibodies (Abs). Pretargeted RIT using a covalent conjugate of the 1F5 anti-CD20 Ab with streptavidin (SA) has been shown to augment the efficacy of RIT and decrease toxicity compared with a directly labeled 1F5 Ab. We have engineered a tetravalent singlechain 1F5 (scFv)4SA fusion protein and compared it to the 1F5-SA conjugate. Athymic mice bearing Ramos lymphoma xenografts received either the conjugate or fusion protein, followed 20 hours later by a biotin-N-acetyl-galactosamine clearing agent, followed 4 hours later by 111In-DOTA-biotin. After 24 hours, 11.4% ± 2.1% of the injected dose of radionuclide was present per gram of tumor (% ID/g) using 1F5 (scFv)4SA compared with 10.8% ± 2.5% ID/g with 1F5 Ab-SA. Superior tumor-to-normal organ ratios of radioactivity were consistently seen using the fusion protein compared with the chemical conjugate (eg, tumor-to-blood ratio > 65:1 after 48 hours with the fusion protein, but < 7:1 with the conjugate). More than 90% of lymphomabearing mice could be cured with minimal toxicity using either reagent followed by 1200 μCi (44.4 MBq) 90Y-DOTA-biotin. (Blood. 2006;108:328-336

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

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 Using an Anti-Murine CD45 Monoclonal Antibody in a Syngeneic, Disseminated Murine Leukemia Model.

Abstract The efficacy of radioimmunotherapy (RIT) for patients with hematologic malignancies is currently limited by non-specific delivery of radiation to normal tissues, due to the long circulating half-life of radiolabeled antibodies (Ab) in the bloodstream. Our group has documented the promise of pretargeted RIT (PRIT) using a covalent conjugate of BC8, an anti-human (h)CD45 Ab, with streptavidin (SA) and radiobiotin to augment the efficacy and decrease the toxicity of RIT. Although pilot mouse xenograft experiments with anti-hCD45 PRIT are encouraging, their interpretation is confounded because human target antigens (including hCD45) are confined to xenograft tumor cells, whereas murine tissues lack hCD45 and will not bind anti-hCD45 Ab. We therefore compared direct one-step and pretargeted anti-murine (m)CD45 RIT using the 30F11 Ab in a model of mouse acute myeloid leukemia (mAML) where the target antigen is present on both myeloid leukemia cells and normal hematopoietic tissues to more rigorously test anti-CD45 PRIT before proceeding to human clinical trials. The in vivo blood half-life was 9.6 hr after injection of 0.65 nmol of 125I-30F11 Ab in SJL/J mice. We inoculated SJL/J mice with 1x105 leukemic PBMC or spleen cells i.v., followed 23 days later by 0.65 nmol of 125I-30F11-SA to assess biodistributions of the radioimmunoconjugate in a tumor-bearing model. Groups of mice were euthanized at serial time points, and the percent injected dose per gram (% ID/g) was measured by gamma counting. Excellent specific localization of 125I-30F11-SA was observed in leukemic sites including bone marrow (BM) and spleen compared to non-hematopoietic organs, with maximal target organ uptake seen after 8 hrs and peak target organ-to-normal organ ratios of radioactivity seen 8-24 hrs after injection of the conjugate. After 8 hrs, 106 ± 27% and 90 ± 12% ID/g of 125I-30F11-SA was delivered to BM and spleen, respectively, compared with ≤8.9% ± 2.3% ID/g for non-hematopoietic organs. The BM-to-blood and spleen-to-blood ratios after 8 hrs were 12.1:1 and 10.4:1, respectively. The estimated dose of radioactivity delivered by 131I-30F11-SA was >100 Gy/mCi for both the BM and spleen. The % ID/g in blood fell to ≤10% of the initial concentration within 30 minutes when 5.8 nmol (50 μg) of a clearing agent (CA; synthetic biotin-N-acetyl-galactosamine) was injected to remove residual conjugate from the circulation. The maximum effect of the CA was observed when it was administered 2-4 hrs after 125I-30F11-SA injection. In PRIT experiments leukemic SJL/J mice were injected with 0.65 nM of 125I-30F11-SA followed 8 hr later by 5.8 nM of a CA and 2 hr after that with 1.2 nM of 111In-DOTA-biotin. Biodistribution results demonstrated improvement in the target organ to non-target organ ratios obtained using PRIT due to elimination of non-specific radiation exposure from blood-borne radiolabeled Ab. PRIT employing the CA resulted in a BM-to-blood ration >40:1 and a spleen-to-blood ratio >20:1. These data suggest that anti-CD45 PRIT using an anti-CD45-SA conjugate in a syngeneic murine model of disseminated leukemia is highly effective and the reagents used to achieve pretargeting do not appear to be toxic. Therapeutic studies using the Ab-SA conjugate in mAML mice are on-going

Eradication of disseminated leukemia in a syngeneic murine leukemia model using pretargeted anti-CD45 radioimmunotherapy

We describe the use of pretargeted radioimmunotherapy (PRIT) using an anti–murine CD45 antibody-streptavidin (SA) conjugate followed by radiobiotin to deliver radiation selectively to murine hematolymphoid tissues, which may potentially augment the efficacy and decrease the toxicity of radioimmunotherapy for disseminated murine leukemia. Biodistribution and therapeutic results demonstrated high target organ to nontarget organ ratios of radioactivity and significant long-term survival in leukemic mice using PRIT. These data suggest that anti-CD45 PRIT using an anti–CD45-SA conjugate in a syngeneic murine model of disseminated leukemia may be more effective and less toxic than directly labeled monoclonal antibodies

Extracorporeal Adsorption Following Infusion of Radiolabeled Anti-CD20 Antibody Clears Unbound Radioimmunoconjugate from the Circulation and Reduces Radiation Exposure of Normal Organs in the Nonhuman Primate.

The efficacy of anti-CD20 radioimmunotherapy (RIT) has been validated by high rates of response in patients with non-Hodgkin’s lymphoma; however individuals treated with conventional RIT usually relapse. While dose escalation of RIT should theoretically improve patient outcomes, the maximum deliverable dose of isotope has been limited by non-specific irradiation of normal tissues. To establish the efficacy of extracorporeal adsorption therapy (ECAT) in removing unbound radioimmunoconjugate, thereby enhancing the therapeutic index of RIT; and, to investigate the lanthanide radiometal 177 Lu as a potential therapeutic isotope for use in future human trials - three groups containing two Macaca fascicularis males each were injected with rituximab trace labeled with 111 In-DOTA-biotin (n=4) or 177 Lu-DOTA-biotin (n=2). One animal in each group underwent ECAT by circulating three blood volumes over an avidin-agarose column 24 hours following the infusion. The second Macaque served as a control in each group. Animals underwent serial serum, marrow and excisional lymph node sampling. Following euthanasia, target tissues (lymph node and spleen) and non-target tissues (lung, liver and kidney) were sampled in triplicate. The radiation activity for each organ was estimated by serial quantitative gamma camera imaging and the amount of radiation per gram of tissue was directly quantified with a multi-channel gamma counter. Corrected tissue uptake values were calculated and absorbed doses to organs determined using standard MIRD methods adjusted for organ sizes. ECAT was well tolerated by all animals and no significant side effects were observed. Serial blood counts, serum creatinine, hepatic transaminase and coagulation profiles were unaffected. When compared with controls, ECAT reduced the amount of radioimmunoconjugate in the blood by greater than 95% immediately following the procedure; this effect was sustained throughout each study. Radiation doses delivered to both target and non-target tissues were decreased in the ECAT animals. Localization of antibody in lymph nodes and spleen relative to blood was superior in animals undergoing ECAT (p=0.039 by Spearman correlation). Following ECAT, the radiation doses to critical non-target tissues (lung, liver and kidney) were reduced by 52%, 30% and 40% in the 111 In-DOTA-biotin-Rituximab group and 64%, 66% and 68% in the 177 Lu-DOTA-biotin-Rituximab group compared to controls. In target tissues (lymph node and spleen), ECAT resulted in dose reductions of 12% and 27% for 111 In-DOTA-biotin-Rituximab, respectively, and 35% and 48%, respectively, for 177 Lu-DOTA-biotin-Rituximab treated animals. These findings indicate 177 Lu to be an imageable radiometal capable of conjugation to anti-CD20 antibody. Further, ECAT is identified as a safe and feasible procedure capable of achieving a significant reduction in circulating levels of unbound radioimmunoconjugate. As a result, radiation absorbed doses to normal tissues are significantly reduced