Research article
Biodistribution and planar gamma camera imaging of 123I- and 131I-labeled F(ab′)2 and Fab fragments of monoclonal antibody 14C5 in nude mice bearing an A549 lung tumor

https://doi.org/10.1016/j.nucmedbio.2006.12.006Get rights and content

Abstract

Detection of antigen 14C5, involved in substrate adhesion and highly expressed on the membrane of many carcinomas, including lung cancer, provides important diagnostic information that can influence patient management. The aim of this study was to evaluate the biodistribution and planar gamma camera imaging characteristics of radioiodinated F(ab′)2 and Fab fragments of monoclonal antibody (mAb) 14C5 in tumor-bearing mice.

Methods

F(ab′)2 and Fab 14C5 fragments were radioiodinated using the Iodo-Gen method. In vitro stability, binding specificity and affinity of 125I-labeled 14C5 fragments were studied in A549 lung carcinoma cells. Biodistribution, blood clearance and tumor-targeting characteristics of 131I-labeled 14C5 fragments and intact mAb 14C5 were studied in Swiss nu/nu mice bearing A549 lung carcinoma tumors. Planar gamma imaging illustrated the potential use of these 123I-labeled 14C5 fragments for radioimmunodetection (RID).

Results

Saturation binding experiments showed highest affinity for 125I-labeled F(ab′)2 fragments (Kd=0.37±0.10 nmol/L) and lowest affinity for 125I-labeled Fab fragments (Kd=2.25±0.44 nmol/L). Blood clearance studies showed that the alpha half-life (t½α) value for Fab, F(ab′)2 and mAb 14C5 was 14.9, 21 and 118 min, respectively. The beta half-life t½β value for Fab, F(ab′)2 and mAb 14C5 was 439, 627 and 4067 min, respectively. 131I-Fab fragments showed highest tumor uptake 3 h after injection (2.4±0.8 %ID/g), 131I-labeled F(ab′)2 showed highest tumor uptake 6 h after injection (4.7±0.7 %ID/g) and for 131I-labeled mAb highest tumor uptake was observed at 24 h (10.7±2.3 %ID/g). In planar gamma imaging, both labeled fragments gave better tumor-to-background contrast than 123I-mAb 14C5.

Conclusion

Fab and F(ab′)2 fragments derived from intact mAb 14C5 have significant potential for diagnostic and therapeutic applications and may provide new tools in mAb-based radiopharmaceuticals for targeting non-small cell lung cancer.

Introduction

The murine monoclonal antibody (mAb) 14C5 was obtained by immunizing BALB/c mice with the membrane fraction of SK-BR-3 human breast carcinoma cells [1]. Prior work showed that antigen (Ag) 14C5 is an interesting target for radioimmunodetection (RID) and radioimmunotherapy (RIT). It is present in high amounts on the tumor surface of in situ and invasive breast cancer tissue [2], but it is hardly expressed on normal tissue. Normal epithelial, muscle and connective tissues that did not stain with mAb 14C5 were skin, thyroid, parathyroid, colon, stomach, lung, uterine tube, ovary, ureter, urethra, lymph node, nerve, chondroid tissue, skeletal muscle, esophagus and artery [1], [2]. Sometimes, low-level staining of myo-epithelial cells was observed in biopsies of breast tissue and in tubular cells of the kidney. Fibroblasts that surround invasive carcinoma cells expressed antigen 14C5, but the antigen was absent from normal fibrous tissue and fibrous tissue surrounding noninvasive islands of malignant cells [2].

An important future clinical role of mAb 14C5 may lie in the ability of this antibody to prevent the spread of tumor cells into the patient. Moreover, the expression characteristics of the Ag 14C5 are suitable for possible use of the mAb 14C5 in RID and RIT [3]. The A549 lung cancer cells showed the highest Ag 14C5 expression, providing a possible new target for non-small cell lung cancer (NSCLC) therapy. Although breast cancer is currently second as the leading cancer causing death in women in the United States, lung cancer is the leading cause of cancer-related mortality in both men and women, with NSCLC accounting for approximately 80% of all lung cancers [4], [5]. Today, the therapy of NSCLC has reached a plateau in improving patient survival [5]. Therefore, research for new treatment strategies is warranted. We previously demonstrated that 125I-mAb 14C5 binds its antigen with high affinity (A549, Kd=0.19±0.07 nmol/L; LoVo, Kd=0.20±0.05 nmol/L) [3]. Biodistribution studies of 131I-mAb 14C5 in athymic mice bearing A549 lung tumors or LoVo colon tumors showed good tumor uptake 24 h after injection: 10.4±0.8 and 9.3±0.8 %ID/g tumor tissue, respectively. Tumors could be clearly visualized by planar gamma camera imaging, but the highest tumor-to-blood ratio (∼3.0) was obtained only 168 h after injection [3].

Many efforts of various groups have been undertaken in increasing the tumor-to-nontarget ratio and lowering human antimouse antibody response. A common approach is the development of smaller antibody fragments and genetically engineered single-chain Fv (scFv) constructs of antibody molecules [6], [7], [8]. The evaluation of the enzymatic derived Fab and F(ab′)2 fragments may be useful to evaluate the potential of 14C5 mAb-derived fragments. Indeed, enzymatic derived bivalent F(ab′)2 and monovalent Fab fragments have shown promising tumor penetration and good therapeutic results in animal [9], [10], [11], [12], [13] and clinical studies [7], [14], [15], [16], [17]. Disadvantages seen with these fragments are lower tumor uptake and high renal uptake particularly when a radiometal is used, which raises concern for renal toxicity [12]. However, Behr et al. [18] reported that a high predose of cationic amino acids could significantly reduce renal tubular reabsorption of radiometal-labeled Fab.

The objective of this study was to examine the biodistribution and elimination from the blood of 123I-labeled Fab and F(ab′)2 14C5 fragments and compare with parental mAb 14C5 for the detection and possible therapy of NSCLC. Additionally, we have explored the in vitro characteristics of 125I-F(ab′)2 and 125I-Fab fragments of mAb 14C5, i.e., their stability, specificity and affinity.

Section snippets

Monoclonal antibodies

The mAb 14C5 was produced and purified as described previously [3]. Fab and F(ab′)2 fragments were derived from murine mAb 14C5 using the Immunopure IgG1 Fab and F(ab′)2 preparation kit (Perbio) according to the manufacturer's instructions. IgG and Fc fragments were removed using a protein A sepharose column (Perbio). For concentration and exchange of buffer with PBS (pH 7.4), Centricon YM-30 (Millipore) was used. Fragments were evaluated by SDS-PAGE without reduction. When purity was <90%,

Preparation of F(ab′)2 and Fab fragments

The product of the ficin-treated mAb 14C5 (150 kDa) was evaluated by SDS-PAGE (Fig. 1B). Pure F(ab′)2 fragments (>90%, ∼100 kDa) were generated in the presence of 1 mmol/L cysteine after 20 h of incubation at 37°C. Highest production of Fab fragments (∼50 kDa) was obtained in the presence of 10 mmol/L cysteine after 15 h of incubation at 37°C. MAb 14C5 was also run in parallel under nonreducing conditions and migrated at ∼150 kDa. Fab and F(ab′)2 migrated at ∼50 and ∼100 kDa, respectively. For

Discussion

When optimizing RID and RIT, it is a challenge to improve the tumor uptake while reducing the uptake in normal tissues [6], [7], [8], [9], [17], [21]. We previously described the in vitro and in vivo targeting properties in a colon cancer and a NSCLC model of radioiodinated mAb 14C5 for the potential use as novel imaging agent [3]. Although 123I-labeled mAb 14C5 clearly showed A549 lung tumors of 0.14 g by planar gamma imaging, the use of enzymatically digested antibody fragments [Fab and F(ab′)

Conclusion

This study showed that 123I-F(ab′)2 and 123I-Fab fragments of mAb 14C5 have improved characteristics as tumor imaging agents compared to the intact mAb 14C5: the fragments accumulated more rapidly in the tumor and cleared faster from the blood and nontumor tissues. However, absolute tumor uptake remained highest for mAb 14C5. Preclinical RIT studies with 131I-mAb 14C5 and its fragments, 131I-F(ab′)2 and 131I-Fab, will demonstrate which immunoconjugate has the biggest therapeutic potential.

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