Chimeric Ab derivatives of the anti-α
vβ
5 mAb 14C5 were recombinantly produced to reduce the risk of generating a HAMA response in humans. Chimerization is the first major and the most reliable step towards a more humanized therapeutic Ab molecule [
28]. Complementarity-determining region grafting could decrease immunogenicity further but often results in reduced affinity or specificity for the target antigen. Safeguarding the desired characteristics involves several labor-intensive trial-and-error approaches [
29,
30]. Integrin α
vβ
5 expression is involved in several crucial steps of cancer progression such as cell adhesion, angiogenesis, and metastasis [
31,
32]. Overexpression of α
vβ
5 has been demonstrated not only on several tumor types, but also on non-transformed tumor-promoting cells from the tumor stroma, such as cancer-associated fibroblasts [
33] and cells from the neovasculature [
11,
14,
34]. These accessory cells are less likely to lose antigen expression, making α
vβ
5 a very attractive target for Ab-based RID and/or RIT. The murine mAb 14C5 was shown to hold great potential for these applications, given the tenfold higher affinity for α
vβ
5 of the 14C5 mAb (
K
D = 0.19 ± 0.07 nM) compared to the commercially available murine P1F6 mAb [
13,
14,
17,
35]. However, despite its promising characteristics, the use of mAb 14C5 in humans might provoke a HAMA response due to its murine origin. HAMA responses generally lead to a reduced therapeutic effectiveness and can lead to potentially life-threatening severe allergic reactions. Replacing mouse Ab constant domains with their human counterparts in chimeric constructs generates less anti-chimeric Abs at lower titers as compared to HAMA in patients given the parent murine Ab [
36]. A chimeric human IgG1 mAb 14C5 was recombinantly constructed, produced, and purified. After confirmation of its stability, the affinity of recombinantly produced chAb 14C5 for the integrin α
vβ
5 was determined by cellular ELISA assays and by saturation binding assays with radioiodinated chAb. Both experiments revealed a similar and high affinity of the chAb for its antigen (
K
D = 1.4 ± 0.11 nM and
K
D = 1.19 ± 0.19 nM, respectively). Although the dissociation constant for the murine 14C5 mAb was ten times lower [
13] compared to the chAb, this still puts the chAb 14C5 on par with the P1F6 mAb. Blood clearance studies of
131I-chAb 14C5 conducted in NMRI mice revealed negligible differences in
t
1/2α and
t
1/2β in comparison with the murine mAb 14C5 (
t
1/2α = 118 min and
t
1/2β = 4067 min) [
13], since human as well as mouse IgG binds the mouse neonatal Fc receptor (FcRn) which regulates serum half-lives of IgG in both species. However, significantly different clearance values are expected in human patients, since human FcRn binds to human but not to mouse IgG, resulting in longer retention of the chAb in the blood [
37]. The highest tumor uptake for chAb as well as for the murine mAb 14C5 [
13,
17] was seen at 24 h p.i. of approximately 10%ID/g, which corresponds to other high-affinity Abs, such as the humanized anti-α
vβ
3 mAb Abegrin™ (14%ID/g at 24 h p.i.) [
38]. In mice bearing the α
vβ
5
− Colo16 squamous tumor, an initial high uptake of chAb 14C5 (approximately 10%ID/g at 24 and 48 h p.i.) was observed which decreased more rapidly compared to the uptake in the α
vβ
5
+ A549 tumor. In contrast, a negative control chAb MabThera showed significant less uptake (<5%ID/g,
p < 0.05) in the α
vβ
5
+ A549 tumor in comparison with the uptake of chAb 14C5 in both α
vβ
5
+ and α
vβ
5
− tumor types. Additionally, staining of different human tumor tissues with mAb 14C5 demonstrated tumor cell membrane and/or tumor surrounding stroma staining [
14]. Tumor sections without tumor cell membrane staining did reveal stroma staining which was co-localized with a fibroblast-specific marker (data not shown). Consequently, expression of integrin α
vβ
5 on cancer-associated fibroblasts (CAFs) was confirmed. Therefore, the uptake of chAb 14C5 seen in α
vβ
5
− Colo16-bearing animals is attributed to binding on CAFs in the tumor stroma and/or on neo-vasculature of the tumor. These results confirm α
vβ
5 as a promising target for RID and RIT. For RID smaller fragments with good tissue penetration and a fast clearance are needed, while RIT requires a good therapeutic index without too much collateral damage. 14C5 chAb Fab and F(ab')
2 fragments were generated in order to examine their pharmacokinetic properties and find the best format for RID/RIT applications. Both the chFab and chF(ab')
2 obtained from proteolytic cleavage of the chAb 14C5 as well as the recombinantly produced chFab were correctly produced and remained stable. However, expression of the chF(ab')
2 vectors did not result in significant amounts of correctly dimerized chF(ab')
2. Analysis on SDS-PAGE suggests that the stabilizing disulfide bridges between chFab' fragments were not properly formed. Procedures to oxidize the cysteine SH groups in order to form chF(ab')
2 or the removal of peptide tags on the heavy chain did not result in increased yields of stable recombinant chF(ab')
2, leaving only the proteolytically obtained chF(ab')
2 for further experiments. In cellular ELISA, the avidity of the bivalent 14C5 chF(ab')
2 (
K
D = 0.6 ± 0.05 nM) for the α
vβ
5
+ A549 lung tumor cell line was ten times higher compared to the monovalent 14C5 chFab (
K
D = 11 ± 2.34 nM). However, saturation binding experiments with
125I-labeled compounds revealed a less pronounced difference between the affinity of both fragments, with a
K
D = 0.68 ± 0.10 nM and
K
D = 2.11 ± 0.58 nM for the
125I-chF(ab')
2 and chFab 14C5, respectively. Similar results were obtained for the 14C5 murine F(ab')
2 (
K
D = 0.37 ± 0.10 nM) and Fab (
K
D = 2.25 ± 0.44 nM) [
17], indicating that the chimerization of the fragments had no influence on the
in vitro binding characteristics.
In vivo, for the chFab 14C5 fragment, the peak tumor-to-blood ratio (0.9) was reached at 6 h p.i. After 24 h the Fab was largely cleared from the system, which makes it an interesting probe for RID, despite high non-specific kidney uptake caused by renal clearance of the small fragment (<60 kDa). Behr et al. [
39] demonstrated that administration of cationic amino acids, such as
d-lysine, reduced the renal uptake of small peptides. The
131I-chF(ab')
2 demonstrated improved tumor-to-blood ratios compared to the Fab fragment, although the molecule remained in circulation longer. The
131I-chF(ab')
2 did however clear faster than the intact chAb. The AUC value is an indication for the total amount of radiation in the tumor. The chAb 14C5 AUC value (AUC
[0 → 7d] 62.75%ID∙ d∙ g
−1) was higher than the AUC value of its fragments, suggesting a higher therapeutic efficacy. However, the long residence time of the intact Ab in the blood may augment the risk of non-specific radiation damage (e.g., bone marrow). Consequently, the faster clearing chF(ab')
2 might be an attractive alternative if dose-limiting toxicity occurs after administration of the intact Ab. The AUC values may be further improved by using radiometals such as
111In and
90Y, which have been shown to improve tumor retention times and AUC values of radiolabeled immunologicals [
40]. Internalization studies with
111In-14C5 mAb have already demonstrated extended tumor retention times compared to radioiodinated Ab 14C5 [
35].