PO-402 Establishment of human monoclonal anti tumour antibodies with high affinity to CD9 using an in vitro B cell affinity maturation platform

Introduction Several attempts to develop an anti-CD9 antibody for cancer treatment failed pre-clinically due to the life-threatening side effect of platelet aggregation. We currently have a patient derived anti-CD9 antibody, named AT1412, under development (see abstract Schotte et al.). This patient, diagnosed with metastatic melanoma, was treated with autologous ex vivo expanded T cells and is still in remission 10 years after treatment. The antibody derived from the patient’s B cells targets a novel epitope on CD9 with low affinity, mediates ADCC against melanoma cells in vitro and, affects growth of tumour cells in mouse models. In contrast to previously described anti-CD9 antibodies AT1412 does not aggregate platelets. To improve the anti-tumour efficacy of the antibody we generated variants using a novel affinity maturation platform. Material and methods We developed an in vitro selection technique that mimics the natural process of affinity maturation of antigen specific B cells. Bcl-6/Bcl-xL immortalised primary human B cells are B-cell receptor positive, antibody-producing germinal centre like B cells. Low frequencies of AID induced somatic hypermutations in B cell clones are detected. This allows for the selection of sub clones with altered antibody binding characteristics. Sub clones of the AT1412 B cell clone were seeded and expanded. Antibody containing supernatants were collected and tested for binding to CD9 by Surface Plasmon Resonance (SPR). Results and discussions Out of 800 B cell sub clones, 9 produced an antibody with a higher affinity to CD9 as compared to the parental clone. Purified recombinant antibodies were generated to confirm enhanced binding to CD9 expressing cells by flow cytometry and SPR. One antibody variant combining two mutations reached similar affinity levels as commercial anti-CD9 mAbs. Extensive epitope mapping suggests that all high affinity variants recognise the same epitope as the parental AT1412 antibody. These high affinity antibodies mediate much stronger ADCC against melanoma cells than the wild type antibody. Importantly, the high affinity variants still do not aggregate platelets. Conclusion We describe a highly efficient technology that allows for rapid generation of antibodies with modified affinities in both the heavy and light chains within 10 weeks. This method allows for establishment of more efficacious anti-tumour antibodies. Financial interest AIMM Therapeutics is a company that develops monoclonal antibodies for prevention and treatment of infectious diseases and cancer.