A Genetically Engineered Anti-CD45 Single-Chain Antibody-Streptavidin Fusion Protein for Pretargeted Radioimmunotherapy of Hematologic Malignancies

Abstract

Acute myelogenous leukemia (AML) currently kills the majority of afflicted patients despite combination chemotherapy and hematopoietic cell transplantation (HCT). Our group has documented the promise of radiolabeled anti-CD45 monoclonal antibodies (Ab) administered in the setting of allogeneic HCT for AML, but toxicity remains high, and cure rates are only 25% to 30% for relapsed AML. We now show the superiority of pretargeted radioimmunotherapy (PRIT) compared with conventional radioimmunotherapy using a recombinant tetravalent single-chain Ab-streptavidin (SA) fusion protein (scFv 4 SA) directed against human CD45, administered sequentially with a dendrimeric N-acetylgalactosamine-containing clearing agent and radiolabeled 1,4,7,10-tetraazacyclododecane-N,N V ,N 00 ,N V 00 -tetraacetic (DOTA)-biotin. The scFv 4 SA construct was genetically engineered by fusing Fv fragments of the human CD45-specific BC8 Ab to a full-length genomic SA gene and was expressed as a soluble tetramer in the periplasmic space of Escherichia coli. The fusion protein was purified to >95% homogeneity at an overall yield of f50% using iminobiotin affinity chromatography. The immunoreactivity and avidity of the fusion protein were comparable with those of the intact BC8 Ab, and the scFv 4 SA construct bound an average of 3.9 biotin molecules out of four theoretically possible. Mouse lymphoma xenograft experiments showed minimal toxicity, excellent tumor-specific targeting of the fusion protein and radiolabeled DOTA-biotin in vivo, marked inhibition of tumor growth, and cured 100% of mice bearing CD45-expressing tumors. These promising results have prompted large-scale cGMP production of the BC8 fusion protein for clinical trials to be conducted in patients with hematologic malignancies. (Cancer Res 2006; 66(7): 3884-92)

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

“…Further evidence can be found in the literature where recent studies have shown that a His-tagged anti-CEA scFv was also taken up rapidly by the liver (30-40% of the injected dose) [122]. In a separate study by another group, using a tag-free anti-CD45 scFv, the uptake and binding by the liver was much lower at only < 5% of the injected dose [123]. Other studies, however, have not demonstrated an increase in liver uptake of scFvs that contain a poly-his tag [124,125].…”

A Divalent Hapten-Peptide Induces Apoptosis in Human Non–Hodgkin Lymphoma Cell Lines Targeted by Anti-CD20 × Anti-Hapten Bispecific Antibodies

“…Whether pretargeted radionuclides alone will allow for greater success in solid tumors awaits clinical testing, but there are promising therapeutic results in medullary thyroid cancer, as well as promising dosimetry in colorectal cancer (22,23). Pretargeting has also been shown in preclinical models to be a more effective therapeutic in non -Hodgkin lymphoma than directly radiolabeled anti-CD20 IgG, without having to resort to doses that would lead to serious myelosuppression (8,24,25). Bispecific antibody pretargeting methods are unique from the avidin-biotin systems because they commonly employ a bivalent hapten to enhance the binding avidity of the radiolabeled hapten-peptide locally at the tumor (26).…”

Pretargeted Radioimmunotherapy for Hematologic and Other Malignancies

“…Although CD45 is mainly focused as a target for leukemia, this antigen may also be useful for PRIT of lymphoma as suggested by studies with a tetravalent anti-CD45 scFvSA fusion protein in nude mice bearing human lymphoma xenografts. 67 Another target that has been exploited for PRIT of lymphomas and leukemias using the biotin=(strept)avidin system is CD25 (interleukin-2 receptor a chain). 90 Initial studies with a chemically linked humanized anti-Tac antibodystreptavidin conjugate and 213 Bi-DOTA-biotin found significant growth inhibition of adult T-cell leukemia cells xenografted in mice and prolongation of survival of these animals compared with conventional RIT.…”

“…5 The latter forms stable complexes with yttrium-90 ( 90 Y) 59 and is widely used as an effector in PRIT. However, alternative b-or a-emitting radionuclides can be used, as DOTA forms highly stable complexes with other isotopes such as 64 Cu, 67 Ga, 149 Pm, 166 62,63 In the second approach, biotin-conjugated antibody is used to target the tumor. Antibody biotinylation is easily accomplished; importantly, addition of a few molecules of biotin (molecular weight: 244 Da) does not alter immunoreactivity, plasma kinetics, or rates of antibody permeation and diffusion and only minimally increases the molecular weight of the targeting vehicle.…”

“…57,[65][66][67] These fusion proteins have been validated for various antibodies and can be expressed as soluble tetramers in the periplasmic space of Escherichia coli. They retain full antigen and biotin-binding capabilities of their parent molecules and have tumor targeting ability comparable to whole IgG antibodies.…”