RESUMO
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.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/uso terapêutico , Metástase Neoplásica/prevenção & controle , Oligopeptídeos , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Anticorpos Antineoplásicos/química , Anticorpos Antineoplásicos/farmacologia , Anticorpos Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Feminino , Humanos , Fragmentos de Imunoglobulinas/farmacologia , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/imunologia , Ligantes , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Camundongos , Mimetismo Molecular , Metástase Neoplásica/tratamento farmacológico , Transplante de Neoplasias , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêuticoRESUMO
Overexpression of the cell-surface glycosphingolipid G(M3) is associated with a number of different cancers, including those of the skin, colon, breast, and lung. Antibodies against the G(M3) epitope have potential application as therapeutic agents in the treatment of these cancers. We describe the chemoenzymatic synthesis of two G(M3)-derived reagents and their use in the panning of a phage-displayed human single-chain Fv (scFv) antibody library derived from the blood of cancer patients. Three scFv-phage clones, GM3A6, GM3A8, and GM3A15, were selected for recombinant expression and were characterized using BIAcore and flow cytometry. BIAcore measurements using the purified, soluble scFvs yielded dissociation constants (K(d)) ranging from 4.2 x 10(-7) to 2.1 x 10(-5) M. Flow cytometry was used to evaluate the ability of each scFv to discriminate between normal human cells (human dermal fibroblast, HDFa), melanoma cells (HMV-1, M21, and C-8161), and breast cancer cells (BCM-1, BCM-2, and BMS). GM3A6 displayed cross-reactivity with normal cells, as well as tumor cells, and GM3A15 possessed little or no binding activity toward any of the cell lines tested. However, GM3A8 bound to five of the six tumor cell lines and showed no measurable reactivity against the HDFa cells. Hence, we have demonstrated that a synthetic G(M3) panning reagent can be used to isolate a fully human scFv that is highly specific for native G(M3) on the surface of tumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer.