Abstract
Tumor-associated cell surface antigens and tumor-associated vascular markers have been used as a target for cancer intervention strategies. However, both types of targets have limitations due to accessibility, low and/or heterogeneous expression, and presence of tumor-associated serum antigen. It has been previously reported that a mitochondrial/cell surface protein, p32/gC1qR, is the receptor for a tumor-homing peptide, LyP-1, which specifically recognizes an epitope in tumor cells, tumor lymphatics, and tumor-associated macrophages/myeloid cells. Using antibody phage technology, we have generated an anti-p32 human monoclonal antibody (2.15). The 2.15 antibody, expressed in single-chain fragment variable and in trimerbody format, was then characterized in vivo using mice grafted subcutaneously with MDA-MB-231 human breast cancers cells, revealing a highly selective tumor uptake. The intratumoral distribution of the antibody was consistent with the expression pattern of p32 in the surface of some clusters of cells. These results demonstrate the potential of p32 for antibody-based tumor targeting strategies and the utility of the 2.15 antibody as targeting moiety for the selective delivery of imaging and therapeutic agents to tumors.
Highlights
P32 is primarily present in the mitochondria, it has been, under certain conditions [15], detected in different cellular compartments (nucleus, cellular surface, endoplasmic reticulum (13, 16 –20)) and in different cell types (B lymphocyte [13]), platelets [21], neutrophils [22], eosinophils [23], endothelial cells [24], macrophages and dendritic cells [25, 26], or fibroblasts [27]). p32 has been recently reported in the surface of tumor cells in hypoxic/ nutrient-deprived areas as well as in the cell surface of a tumor-associated macrophage/myeloid cell subpopulation closely linked to tumor lymphatics [10]
Isolation of Human Anti-rhp32 Antibodies by Panning a single-chain Fv (scFv) Library—The Griffin.1 library was panned against affinity purified recombinant human p32 immobilized in Nunc immunotubes
To identify scFv sharing the epitope with mAb 60.11, four scFv fragments (1.6, 2.9, 2.15, and 2.25) were expressed using the nonsuppressor host E. coli HB2151 and purified from the supernatant by standard affinity chromatography procedures
Summary
Cells and Culture Conditions—All cells were from the ATCC. HEK-293 cells (human embryonic kidney epithelia; CRL-1573), and MDA-MB-231 (human breast adenocarcinoma; HTB-26) were grown in DMEM supplemented with. Phages displaying scFv fragments were purified from the culture supernatant by precipitation with 20% PEG 6000 and 2.5 M NaCl and were resuspended in sterile cold PBS with 15% glycerol for long term storage at Ϫ80 °C and for subsequent rounds of selection. Soluble Antibody Expression and Purification—Phage particles from selected clones were used to infect logarithmically growing (A600 ϭ 0.5) E. coli HB2151 (nonsuppresser strain (K12, ara, ⌬(lac-pro), thi/FЈ proAϩBϩ, lacIqZ⌬M15 [35]), and soluble scFv fragments were obtained as described [33]. Competition ELISA was performed as a standard ELISA but with a previous step of blockade using mAb; after blocking with 300 l 4% BSA in PBS at 37 °C for 1 h, wells were incubated with 100 l of a 20 g/ml solution of the appropriate reagent (mAb 60.11, mAb 74.5.2, or control mouse IgG1) for 1 h at room temperature and 30 rpm. Images were acquired using a confocal scanning inverted Leica AOBS/SP2-microscope (Leica Microsystems)
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