De novo protein sequencing, humanization and in vitro effects of an antihuman CD34 mouse monoclonal antibody

Abstract

QBEND/10 is a mouse immunoglobulin lambda-chain monoclonal antibody with strict specificity against human hematopoietic progenitor cell antigen CD34. Our in vitro study showed that QBEND/10 impairs the tube formation of human umbilical vein endothelial cells (HUVECs), suggesting that the antibody may be of potential benefit in blocking tumor angiogenesis. We provided a de novo protein sequencing method through tandem mass spectrometry to identify the amino acid sequences in the variable heavy and light chains of QBEND/10. To reduce immunogenicity for clinical applications, QBEND/10 was further humanized using the resurfacing approach. We demonstrate that the de novo sequenced and humanized QBEND/10 retains the biological functions of the parental mouse counterpart, including the binding kinetics to CD34 and blockage of the tube formation of the HUVECs.

Keywords: Antiangiogenic therapy; CD34; De novo sequencing; Humanization; Monoclonal antibody.

Fig. 1

BPI chromatogram of QBEND/10 light (A) and heavy (B) chains obtained using trypsin digestion. Ln and Hn denote the nth peptide counted from the N-terminal of QBEND/10 light and heavy chains, respectively. Stars indicate deamidated peptides. Pyro indicates pyroglutamate at Q, and oxi denotes oxidation at M.

Fig. 2

Multiple enzyme digestion and in-gel digestion sequence alignment for QBEND/10 light (A) and heavy (B) chain variable regions.

Fig. 3

Molecular model of the QBEND/10 variable regions. The 3D structure of the mouse QBEND/10 Fv region is generated using the Web-based antibody structure prediction program PIGS. Nine amino acids (in boldface), namely four and five residues in the VH and VL frameworks, respectively, are substituted with human germline residues. The CDR loops are shown in red.

Fig. 4

Sequence alignment of mouse QBEND/10 with corresponding human germline sequence. The QBEND/10 heavy (A) and light (B) chain variable regions are sequence-aligned to the most homologous human germline genes IGHV1–3*01/J4*01 and IGLV4–69*01/J1*01, respectively. The conserved surface residues are marked with empty boxes, and the nonconserved surface residues are shown as shaded boxes. The CDRs (within brackets) are unchanged.

Fig. 5

Purification of chimeric and humanized QBEND/10 antibodies. The indicated chimeric QBEND/10 (lanes 1 and 2) and humanized QBEND/10 (lanes 3 and 4) antibodies were stably expressed in the mouse myeloma NS0 cells and purified from culture media by column chromatography. The samples were electrophoresed on a 4–12% NuPAGE Bis-Tris polyacrylamide gel with the MOPS buffer under nonreducing (lanes 1 and 3) and reducing (lanes 2 and 4) conditions. The gel was stained with Coomassie brilliant blue R-250.

Fig. 6

The proportion of CD34⁺HUVECs in different culture conditions. The proportion of CD34⁺ cells was determined by flow cytometry after 7-day culturing. (A) Cells incubated with complete medium for 24 h. (B) Cells incubated with starvation medium for 24 h. (C) Cells stimulated with VEGF₁₆₅ in starvation medium for 24 h.

Fig. 7

Effects of the humanized QBEND/10 on VEGF₁₆₅-induced tube formation of human umbilical vascular endothelial cells. (A) Representative fluorescent images showing the inhibitory effect of humanized QBEND/10 on VEGF₁₆₅-induced HUVEC tube formation. HUVECs were treated with VEGF₁₆₅ (25 ng/mL) in the presence or absence of humanized QBEND/10 (10 μg/mL) or anti-VEGF IgG (10 μg/mL). (B, C) Capillary tube formation was quantified by counting the cell-covered area and junctions per field from A. One field was examined per well, with five wells per dose per experiment. Each column represents the mean±standard error of the mean. ^##P<0.01 and ^###P<0.001 compared with the control group; *P<0.05 and **P<0.01 compared with the VEGF₁₆₅ group.