The antibody V(H) domains of camelids tend to be soluble and to resist aggregation, in contrast to human V(H) domains. For immunotherapy, attempts have therefore been made to improve the properties of human V(H)s by camelization of a small set of framework residues. Here, we have identified through sequence comparison of well-folded llama V(H) domains an alternative set of residues (not typically camelid) for mutation. Thus, the solubility and thermal refolding efficiency of a typical human V(H), derived from the human antibody BT32/A6, were improved by introduction of two mutations in framework region (FR) 1 and 4 to generate BT32/A6.L1. Three more mutations in FR3 of BT32/A6.L1 further improved the thermal refolding efficiency while retaining solubility and cooperative melting profiles. To demonstrate practical utility, BT32/A6.L1 was used to construct a phage display library from which were isolated human V(H)s with good antigen binding activity and solubility. The engineered human V(H) domains described here may be useful for immunotherapy, due to their expected low immunogenicity, and in applications involving transient high temperatures, due to their efficient refolding after thermal denaturation.
Carcinocinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is overexpressed in a number of human malignancies, especially in pancreatic cancer. It has been demonstrated that CEACAM6 is a potential target for monoclonal antibody (mAb) therapy with a safe therapeutic index. Here, we labeled three anti-CEACAM6 antibodies of different sizes, including a single-domain antibody 2A3 (16 kDa), a heavy chain antibody 2A3-mFc (80 kDa) and a full length antibody 9A6 (150 kDa), with 64Cu to image CEACAM6 expression in a xenografted pancreatic tumor model. For positron emission tomography (PET) imaging, the tumor mice were intravenously injected with 64Cu-DOTA-antibodies and static scans were obtained at 5 min, 0.5, 1, 2, 4, 8 and 24 h post-injection (p.i.). All three antibodies showed strong CEACAM6 binding. Ex vivo immunostaining on tumor sections at 24 h after Ab injection demonstrated specific tumor targeting of both 2A3-mFc and 9A6. 64Cu-DOTA-2A3 showed fast BxPC3 tumor uptake and rapid whole-body clearance. At 24 h p.i., the tumor uptakes were 98.2 ± 6.12 %ID/g for 64Cu-DOTA-2A3-mFc and 57.8 ± 3.73 %ID/g for 64Cu-DOTA-9A6, respectively. Compared with the full length antibody 9A6, the heavy chain antibody 2A3-mFc showed higher tumor uptake, lower liver uptake and shorter circulation half-life. All the data supported that the heavy chain antibody 2A3-mFc is superior to the single domain antibody and the full-length antibody with regard to tumor detection and pharmacokinetics, which has great potential to be developed for CEACAM6-targeted pancreatic cancer imaging and therapy.
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