HLA DR-directed bispecific single-chain Fv antibodies for lymphoma therapy

Fc receptors are important for the clinical efficacy of therapeutic antibodies. Bispecific antibodies (BsAb) are immunoglobulin-conjugates with two different binding specifities, targeting tumor antigens and effector cell trigger molecules. BsAb, produced by chemical coupling of one antibody against a tumor cell surface antigen with another against a Fc receptor, mediate effective interactions between effector and target cells.

Here, genetically coupled bispecific single chain Fv (bsscFv) were produced – as they easily enable further modifications of the molecule – directed against one of the effector cell antigens FcαRI (CD89) or FcγRIII (CD16) and against HLA class II or Lym-2. Lym-2 represents a variant form of the HLA-DR antigen and is highly expressed on the surface of malignant B cells, but only at low levels on normal cells. HLA class II and Lym-2 are both known as effective targets for effector cell-mediated lysis of malignant human B-lymphoid cells. CD89 is an interesting trigger molecule for BsAb therapy, as it recruits neutrophils as effector cells, which have tumor cytolytic potential against a broad spectrum of tumor cells and are the most abundant circulating blood leukocytes. Antibodies against CD16 have already shown biological activity in vitro and in tumor patients by recruting NK cells. The two component scFv were fused via a flexible 20aa linker. ScFv fragments were generated by producing phage display libraries from corresponding hybridomas, and screening the libraries with antigen-positive cells. Recombinant scFv against HLA class II, Lym-2, CD89 and CD16 were thus obtained from the hybridomas F3.3, Lym-2, A77 and 3G8 respectively. Functional bsscFv were expressed and secreted by insect cells and were purified via Nickel chelate chromatography. Purified BsAb reacted with HLA class II or Lym-2-positive target cells and one of the effector cell antigens, CD89 or CD16, respectivly. In ADCC experiments all constructs mediated specific lysis of HLA class II or Lym-2-positive malignant human B-lymphoid cell lines with human MNC or PMN as effector cells. The [CD89 x HLA class II] and the [CD16 x HLA class II] bsscFv also mediated significant lysis of primary cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL). In conclusion, these recombinant bsscFv may allow the specific recruitment of effector cells for an improved therapy in B-lymphoid malignancies.