Antigen identification and characterization of lung cancer specific monoclonal antibodies produced by mAb proteomics

Abstract

A mass spectrometric (MS)-based strategy for antigen (Ag) identification and characterization of globally produced monoclonal antibodies (mAbs) is described. Mice were immunized with a mixture of native glycoproteins, isolated from the pooled plasma of patients with nonsmall cell lung cancer (NSCLC), to generate a library of IgG-secreting hybridomas. Prior to immunization, the pooled NSCLC plasma was subjected to 3-sequential steps of affinity fractionation, including high abundant plasma protein depletion, glycoprotein enrichment, and polyclonal antibody affinity chromatography normalization. In this paper, to demonstrate the high quality of the globally produced mAbs, we selected 3 mAbs of high differentiating power against a matched, pooled normal plasma sample. After production of large quantities of the mAbs from ascites fluids, Ag identification was achieved by immunoaffinity purification, SDS-PAGE, Western blotting, and MS analysis of in-gel digest products. One antigen was found to be complement factor H, and the other two were mapped to different subunits of haptoglobin (Hpt). The 2 Hpt mAbs were characterized in detail to assess the quality of the mAbs produced by the global strategy. The affinity of one of the mAbs to the Hpt native tetramer form was found to have a K(D) of roughly 10(-9) M and to be 2 orders of magnitude lower than the reduced form, demonstrating the power of the mAb proteomics technology in generating mAbs to the natural form of the proteins in blood. The binding of this mAb to the beta-chain of haptoglobin was also dependent on glycosylation on this chain. The characterization of mAbs in this work reveals that the global mAb proteomics process can generate high-quality lung cancer specific mAbs capable of recognizing proteins in their native state.

Figure 1. Antigen Identification Workflow

The affinity purified antigen mixture was separated by SDS-PAGE, and then probed with a specific mAb to locate the binding antigen. The MW region, which was reactive in Western blotting, was excised from the gel, digested with trypsin and sequenced by mass spectrometry.

Figure 2. Antigen Identification for mAb #1

Reduced SDS-PAGE of the mAb #1 affinity purified mixture from pooled lung cancer patient plasma (15 μg) and stained with Coomassie blue (A). Bands 1 and 3 are the heavy (~ 50 kDa) and light chains (~ 25 kDa) of IgG, respectively. The protein (band 2) migrating at ~ 44 kDa was recognized by the mAb #1 (B) and was subsequently identified as Hpt-β chain by MALDI-TOF-MS (C).

Figure 3. Antigen Identification for mAb #2

Reduced SDS-PAGE of an affinity purified mixture (5 μg) from IgG- and albumin-depleted pooled lung cancer plasma, stained with Coomassie blue (A). Bands 1 and 2 are albumin and Hpt-β chain, respectively. Bands 3 (~18 kDa) and 4 (~10 kDa) were both recognized by mAb #2 (B) and were subsequently identified as the Hpt-α₂ and Hpt-α₁ chain by LC-ESI-MS (C).

Figure 4. N-linked Glycosylation is Essential for the mAb-Ag Interaction

Glycosylated and deglycosylated haptoglobin were separated by SDS-PAGE and stained with either Coomassie blue for protein detection (A), Schiff's base reagent to determine the deglycosylation efficiency (B) or transferred to the nitrocellulose membrane for Western blotting (C, probed with anti-Hpt-β mAb #1).

Figure 5. Surface Plasmon Resonance Analysis of Anti-Hpt-β with Hpt

Panels A-D are sensorgrams of the interaction of anti-Hpt-β with (A) native Hpt (lung cancer); (B) native Hpt (matched control); (C) reduced Hpt-β chain (matched control); and (D) reduced and deglycosylated Hpt-β chain (matched control).

Figure 6. Sandwich ELISA Using mAb #1 and mAb #2

(A) mAb #1 was labeled with HRP for detection and mAb #2 was used as the capture Ab. (B) The standard curve was generated using Hpt standard ranging from 0.005 - 10 μg/mL. The haptoglobin concentration in M-LAC material, lung cancer and matched control plasma sample were measured using this assay.