Selection and Characterization of YKL-40-Targeting Monoclonal Antibodies from Human Synthetic Fab Phage Display Libraries

Abstract

YKL-40, also known as chitinase-3-like 1 (CHI3L1), is a glycoprotein that is expressed and secreted by various cell types, including cancers and macrophages. Due to its implications for and upregulation in a variety of diseases, including inflammatory conditions, fibrotic disorders, and tumor growth, YKL-40 has been considered as a significant therapeutic biomarker. Here, we used a phage display to develop novel monoclonal antibodies (mAbs) targeting human YKL-40 (hYKL-40). Human synthetic antibody phage display libraries were panned against a recombinant hYKL-40 protein, yielding seven unique Fabs (Antigen-binding fragment), of which two Fabs (H1 and H2) were non-aggregating and thermally stable (75.5 °C and 76.5 °C, respectively) and had high apparent affinities (KD = 2.3 nM and 4.0 nM, respectively). Reformatting the Fabs into IgGs (Immunoglobulin Gs) increased their apparent affinities (notably, for H1 and H2, KD = 0.5 nM and 0.3 nM, respectively), presumably due to the effects of avidity, with little change to their non-aggregation property. The six anti-hYKL-40 IgGs were analyzed using a trans-well migration assay in vitro, revealing that three clones (H1, H2, and H4) were notably effective in reducing cell migration from both A549 and H460 lung cancer cell lines. The three clones were further analyzed in an in vivo animal test that assessed their anti-cancer activities, demonstrating that the tumor area and the number of tumor nodules were significantly reduced in the lung tissues treated with H1 (IgG). Given its high affinity and desirable properties, we expect that the H1 anti-hYKL-40 mAb will be a suitable candidate for developing anti-cancer therapeutics.

Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library

“…To isolate a specific SARS-CoV-2 antibody, the KFab-I library [ 42 , 43 ] was panned against a recombinant SARS-2 RBD immobilized on magnetic beads ( Figure 1 a). After five rounds of panning, the phage ELISA was performed on immobilized SARS-2 RBD surfaces, using each panning library to monitor the enrichment ( Figure 1 b).…”

“…To determine whether the apparent affinities of the anti-SARS-2 RBD IgGs were altered by reformatting the Fabs into the IgGs, the apparent affinities of the IgGs were examined using ELISA ( EC 50 , nM). As shown in Figure 3 b, the five clones (C2 (IgG), C12 (IgG), D12 (IgG), F7 (IgG), and H1 (IgG)) increased their apparent affinities approximately 100- to 1800-fold compared to their Fab formats ( Figure 3 b and Table 1 ), which might have been due to an avidity effect [ 42 , 43 ]. Next, a size-exclusion chromatography analysis was performed to assess their non-aggregation properties, revealing that the IgGs were monomeric without forming high molecular weight (HMW) aggregates ( Figure 3 d and Table 1 ).…”

“…Due to the same CDR randomization design being applied to the two libraries, we reasoned that the framework could have made a difference in the panning outcome. The human V H 3 family has been shown to have the highest stability and soluble protein yield, and its germline usage out of 51 germline genes is about 43%, which is considerably higher than that of other families of human V H [ 43 , 50 ]. Indeed, for various antibody libraries, such as the Griffiths and the HuCAL libraries, it has been shown that a considerable number of antibodies selected from the libraries belonged to the human V H 3 family (74% and 36% for the Griffiths and HuCAL libraries, respectively) [ 51 , 52 ], indicating that the human V H 3 framework may be inevitably favored in the phage display selection due to its desirable properties.…”

“…Indeed, for various antibody libraries, such as the Griffiths and the HuCAL libraries, it has been shown that a considerable number of antibodies selected from the libraries belonged to the human V H 3 family (74% and 36% for the Griffiths and HuCAL libraries, respectively) [ 51 , 52 ], indicating that the human V H 3 framework may be inevitably favored in the phage display selection due to its desirable properties. Moreover, our previous phage display panning against human YKL-40, which was also performed with the two Fab libraries, showed a similar outcome in that, unlike the KFab-I library, the KFab-II yielded no binders with desirable properties [ 43 ]. However, it is believed that a panning with a mixture of both V H 3 and V H 1 frameworks from KFab-I and KFab-II libraries might result in a different outcome from the previous panning performed separately with each Fab library.…”

Synthesis of Epitope-Targeting Antibody Based on Native Protein–Protein Interactions for Ftsz Filamentation Suppressor

“…If a mutation is introduced in the VHH B22 loop by normal random mutagenesis techniques such as error-prone PCR, the sequence space reaches 20 37 , thus indicating that we cannot easily obtain the best mutants. For example, even if an antibody showing binding ability is acquired from a vast sequence space with the use of effective techniques, such as phage display and biopanning, derived antibodies often do not have the desired therapeutic effect 22 . This result indicates that the antibody is climbing a mountain in the sequence space with a high-binding a nity.…”