A Therapeutic Antibody for Cancer, Derived from Single Human B Cells

Abstract

Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery.

Figure 1. Cloning of Human CFH mAbs Specific to an Antigenic Peptide

(A) Isolation of CFH-specific memory B cells that bind to the epitope of interest. PBMCs were obtained from patients positive for anti-CFH antibodies and used for sorting CFH antigen-binding memory B-cells via FACS. To minimize false-positives, streptavidin was labeled with Alexa Fluor 647 and Brilliant Violet 421. Labeling with each fluorophore was carried out on separate aliquots of streptavidin, which were then mixed together prior to interaction with biotinylated antigen peptide (CFH_1110–1124). Cells showing elevated fluorescence for both fluorophores, indicated by the outlined region (0.32% of the total), were sorted into single wells for recombinant mAb synthesis. The B cell expressing mAb7968 is designated in pink. (B) Identification of cloned mAbs that bind a CFH epitope-containing peptide by ELISA. Supernatants from HEK293 cells transiently expressing pairs of V_H and V_L chains were collected, adjusted to 1 µg/ml IgG, and equal volumes tested for binding to epitope peptide bound to neutravidin (Pep-NA) or to neutravidin alone (NA). Bound antibody was detected with anti-human IgGγ-HRP and ABTS/H₂O₂ substrate. Sample 31 is an affinity purified anti-CFH autoantibody serving as a positive control.

Figure 2. Structure of Fab7968 in Complex with CFH Peptide

(A) Fab 7968 shown with the heavy chain in green and the light chain in blue. The paratope with bound peptide in red is oriented toward the top of the figure, and the CDRs are marked and labeled. (B) CFH peptide in its antibody-bound conformation (red) contrasted in a superposition to the same region in the natively folded CFH protein (gray). Here, only the 7968 epitope is highlighted in red (antibody-bound conformation) and yellow (on the natively folded protein). See also Figure S2 and Table S2.

Figure 3. Complement-Dependent Cytotoxicity of Six Tumor Cell Lines after Treatment with Recombinant Human CFH mAb7968 or Control mAb

The A549 (NSCLC - adenocarcinoma), H226 (NSCLC - squamous cell carcinoma), H460 (NSCLC - large cell carcinoma), DMS79 (small cell lung cancer), SKBR3 (breast adenocarcinoma), and KATOIII (gastric carcinoma) cell lines were treated with CFH mAb7968, alongside a subtype-matched negative control mAb (Neg), in an LDH release assay for cytotoxicity.

Figure 4. Response to CFH mAb by Tumor Cells: C3a Release, C5a Release, and C5b-9 Deposition

(A) Release of C3a from A549 or H226 lung cancer cells. Cells were incubated in the presence of either NHS alone, NHS plus mAb7968, or NHS plus human IgG. C3a was measured in cell supernatants at 1 hr and 4 hr by ELISA. (B) Release of C5a from A549 or H226 cells. Using the same cell supernatants described in (A), C5a was measured by ELISA. (C) C5b-9 deposition on A549 or H226 cells. After incubation with NHS, NHS plus mAb7968, or NHS plus a subtype-matched negative control mAb (Neg), C5b-9 deposition on cells was measured by flow cytometry.

Fig. 5. Tumor growth in the KLN205 - DBA/2 syngeneic lung cancer model with mAb treatment

(A) Growth curves. KLN205 tumor cells were injected s.c. on day 0 and mAb7968 or mAbNctl was injected i.p. every 3 days between days 1–13, after which treatment was stopped. The mean tumor volumes +/− SEM for t=7 mice treated with each mAb are plotted. P-values for the difference in tumor volumes between mAb7968 and mAbNctl on days 39, 42, and 45 are 0.027, 0.030, and 0.077, respectively. (B) Representative H&E images of the tumors. A lymphocytic component is evident in residual mAb7968-treated tumor (white arrow). The scale bar=0.030 mm and magnification is 100X.