Combinatorial antibody libraries from cancer patients yield ligand-mimetic Arg-Gly-Asp-containing immunoglobulins that inhibit breast cancer metastasis

Abstract

Combinatorial antibody libraries have the potential to display the entire immunological record of an individual, allowing one to detect and recover any antibody ever made, irrespective of whether it is currently being produced. We have termed this the "fossil record" of an individual's antibody response. To determine whether cancer patients have ever made antibodies with disease-fighting potential, we screened combinatorial antibody libraries from cancer patients for immunoglobulins that can identify metastatic tumor cells. This strategy yielded human antibodies specific for the activated conformation of the adhesion receptor integrin alphavbeta3 that is associated with a metastatic phenotype. In a remarkable example of convergent evolution, two of these antibodies were shown to contain the Arg-Gly-Asp integrin recognition motif of the natural ligand within the third complementarity-determining region of the heavy chain. These antibodies interfered with lung colonization by human breast cancer cells in a mouse model and inhibited existing metastatic disease. Our data imply that, at least at some time, these antibodies were part of a patient's surveillance system against metastatic cells, targeting the activated conformer of integrin alphavbeta3 and disrupting its functions. The ligand-mimetic nature of these antibodies, combined with specificity for a single receptor, is unique in the integrin-ligand repertoire. The convergent evolution of critical sequences in antibodies and other ligands that bind to the same target means that the immune response has sufficient power to find a best chemical solution for the optimization of binding energy, even though antibodies evolve in real time, as compared with billions of years for the natural ligand.

Fig. 1.

Cancer patient-derived scFvs Bc-12 and Bc-15 recognize cancer cells based on their expression of activated integrin αvβ3. Shown are flow cytometric analyses with or without 1 mM Ca²⁺, 1 mM Mg²⁺, or 0.2 mM Mn²⁺.(A) scFvs Bc-12 and Bc-15 are specific for αvβ3 and require cations for binding to human tumor cells. M21 melanoma, αvβ3 plus other αv integrins; M21-LIIb transfected melanoma, αIIbβ3, no αv integrins; UCLA-P3 lung adenocarcinoma, αv integrins, no β3 integrin. (B) Effect of cations and their combination on scFv-binding to M21 melanoma cells. (C) Requirement of integrin αvβ3 activation for Bc-12 and Bc-15 binding to human breast cancer cells: MDA-MB-435 (β3_–) cells lack αvβ3 or express αvβ3 either in a nonactivated (β3_WT and ParentCo) or activated functional form (β3_D723R, Bone, and Lung), and BMS cells that were isolated from breast cancer patient blood. Several independent experiments with Bc-12 and Bc-15 on each of these cell types gave similar results.

Fig. 2.

Translation of scFv DNA sequence analyses. Consensus sequence of Bc-12 and Bc-15 (specific for activated αvβ3), compared with Bc-20 (specific for αv). (Inset) Flow cytometric analysis with BMS human breast cancer cells indicates loss of binding in mutants of Bc-12 and Bc-15 containing RGE instead of RGD in CDR-H3 (Mut-12 and Mut-15). The Mut-15 signal is equivalent to negative control.

Fig. 3.

scFvs Bc-12 and Bc-15 inhibit αvβ3-mediated adhesive breast cancer cell functions. (A) Adhesion of BMS breast cancer cells to fibrinogen (Fg), vitronectin (VN), or type I collagen (Col I) with or without 3 μM Bc-12, Bc-15, or their RGE mutants Mut-12 and Mut-15, or 200 μM linear Gly-Arg-Gly-Asp-Ser-Pro-Lys (GRGDSPK) peptide. (B) Effect of scFvs on breast cancer cell arrest during blood flow. BMS breast cancer cells were labeled with hydroethidine, suspended in human blood (anticoagulated with 50 nM PPACK), and perfused over a collagen I matrix at a venous wall shear rate of 50 s^–1. As shown earlier (4), breast cancer cells arrest by αvβ3-mediated binding to platelet thrombi. This arrest was quantified by image acquisition at 30 predefined positions during blood flow. (Left) Representative images of platelet thrombi (green fluorescence) and tumor cells (red fluorescence) at identical x,y positions. (Right) Number of arrested tumor cells in the presence of 3 μM nonfunction-blocking anti-αv scFv Bc-20 or function-blocking anti-αvβ3 scFvs Bc-12 or Bc-15. (C) Effect of scFvs on haptotactic BMS cell migration toward a fibrinogen substrate in transwell chambers with or without 2 μM Bc-12, Bc-15, or their RGE mutants Mut-12 and Mut-15, compared with 200 μM GRGDSPK peptide.

Fig. 4.

Patient-derived ligand-mimetic scFvs against activated αvβ3 are internalized and affect breast cancer cell survival. (A) scFv Bc-15 is internalized by breast cancer cells isolated from patient blood: confocal images of BMS cells incubated for 4 h with FITC-Bc-15 at 4°C (binding) vs. 37°C (allowing internalization). (B) Phase-contrast images of BMS cell cultures 4 days after seeding the cells in the presence of 2 μM RGD-expressing scFv Bc-15 vs. its RGE-mutant scFv Mut-15. (C) Effect of scFv Bc-12 on the viability of matrix-deprived BMS breast cancer cells in ultra-low-adhesion plates in the presence or absence of 3.7 μM scFv or 14.36 μM camptothecin as apoptosis-inducing control. Measurement of apoptosis was based on cytoplasmic histone-associated DNA fragments after 20 h.

Fig. 5.

scFvs Bc-12 and Bc-15 prevent hematogenous breast cancer metastasis and inhibit established metastatic disease. (A) Flow cytometric analysis of scFv binding to BMS breast cancer cells in human plasma anticoagulated with 50 nM PPACK. (B) Effect of Bc-12 and Bc-15 on lung colonization by human breast cancer cells. (Left) Lungs of female severe combined immunodeficient mice 32 days after i.v. injection with 1 × 10⁵ BMS cells. The mice were treated with either 50 μg of Bc-12 or Bc-15 (i.v.) on days 1, 2, 3, and 4. Controls received PBS. (Right) Numbers of lung-surface metastases for each animal. The horizontal line indicates the median number of metastases per group. ScFv-treated mice had significantly fewer metastases (P < 0.001 by the Kruskal–Wallis test). (C) (Left) Histological sections of the above mouse lungs stained with hematoxylin/eosin. Metastases were counted for each lung in six sets of three consecutive sections separated by 140 μm. (Right) Number of metastases counted in sections of individual lungs. The horizontal line indicates the median number of metastases per group. ScFv-treated mice had significantly fewer detectable metastases (P < 0.005 by the Kruskal–Wallis test). (D) Effect of Bc-15 treatment on established breast cancer metastasis in the lungs. Female severe combined immunodeficient mice were injected i.v. with 5 × 10⁵ DsRed2-tagged MDA-MB-435 breast cancer cells expressing constitutively activated integrin αvβ3_D723R. Mice were treated on days 7, 9, 11, 14, 16, and 18 by i.v. injections of either scFv Bc-15 or its RGE mutant, Mut-15 (40 μg per dose). Metastatic foci were enumerated by fluorescence microscopy on day 19. (Left) Images of typical tumor foci within the lung tissue in Mut-15-treated (Upper) or Bc-15-treated (Lower) mice. (Scale bar, 50 μm.) (Right) Number of metastatic foci within the lung tissue of each animal. Horizontal line, median number of metastases per group. Bc-15-treated mice had significantly fewer metastases than Mut-15-treated mice (P < 0.001 by the two-sided Mann–Whitney U test).