Humanization and characterization of the anti-HLA-DR antibody 1D10.

1D10 is a previously described antibody that binds to cells from a majority of B-cell malignancies. The current studies were designed to further evaluate the antigen specificity of 1D10 and its potential as an immunotherapeutic agent. Studies with transfectants and immunoprecipitation demonstrated that 1D10 recognizes some, but not all, of the human HLA-DR beta chains. Both normal and malignant B cells can express the 1D10 antigen. A humanized version of 1D10 was produced using CDR grafting. The resulting antibody has an affinity that is similar to that of the parental murine antibody. In addition, the humanized antibody is capable of inducing complement-mediated cytotoxicity, antibody-dependent cell cytotoxicity, and direct apoptosis of 1D10-expressing B cells. Based on these in vitro anti-tumor activities, we conclude humanized 1D10 deserves further evaluation as an immunotherapeutic agent.

Anti-CD3-based bispecific antibody designed for therapy of human B-cell malignancy can induce T-cell activation by antigen-dependent and antigen-independent mechanisms.

Anti-CD3 x anti-B-cell antigen bispecific monoclonal antibodies (bsAbs) can redirect T-cell-mediated lysis toward malignant B cells. Clinical trials with CD3-based bsAbs have shown toxicity in patients which is likely related to nonspecific T-cell activation and targeting. Our current studies were designed to explore the mechanisms responsible for the observed in vivo toxicity by evaluating the immunologic effects of 2 different bsAb preparations in vitro. 1D10 was used as the tumor specific arm of the bsAbs. This antibody reacts with a variant of HLA-DR found on a majority of pre-B- and B-cell malignancies, and normal B cells in some individuals. Anti-CD3 served as the T-cell specific arm. A 1D10 x anti-CD3 bispecific IgG (bsIgG) produced using the hybrid-hybridoma method was compared to a 1D10 x anti-CD3 bispecific F(ab')2 [bsF(ab')2] produced using the leucine zipper technique. In cytotoxicity assays, both bsIgG and bsF(ab')2 induced lysis by pre-activated T cells of 1D10 (+) malignant B cells. bsIgG at high concentrations also induced lysis of 1D10 (-) tumor cells, while bsF(ab')2 did not. Proliferation of T cells induced by bsIgG and bsF(ab')2 was also evaluated. Both forms of bsAbs induced T-cell proliferation in the presence of antigen (+) Raji cells, while only bsIgG did so in the presence of antigen (-) malignant B cells. bsF(ab')2 induced T-cell activation in the absence of any tumor cells when testing was performed on samples where the 1D10 target antigen was present on normal peripheral blood B cells. We conclude that non-specific T-cell activation from bsAbs can occur in an antigen-independent manner due to the Fc/Fc receptor (FcR) interaction, or in an antigen-dependent manner when antigen is expressed on normal or tumor cells. Both mechanisms may have been responsible for the toxicity observed in prior clinical studies.

The role of T cell activation in anti-CD3 x antitumor bispecific antibody therapy.

Anti-CD3 x antitumor bispecific Ab can retarget T cell mediated lysis in an MHC-independent fashion and prevent tumor growth in animal models. Two bispecific Ab preparations that differ in the presence or absence of Fc were compared in the 38C13 immunocompetent murine lymphoma model to evaluate how functional Fc and T cell activation impact on response to bispecific Ab therapy. Bispecific (bs) IgG contained functional Fc and was purified from hybrid-hybridoma Ab product. Bsf(ab')2 lacked functional Fc, and was genetically constructed using the leucine zipper technique. In vitro, bsF(ab')2 induced tumor cell lysis by activated T cells more effectively than bsIgG. However, bsF(ab')2 failed to induce T cell activation in the absence of tumor cells, and did so more slowly than bsIgG when tumor cells were present. In vivo, bsIgG induced nonspecific T cell activation whereas bsF(ab')2 did not. In therapy experiments, bsIgG inhibited tumor growth in mice although a single dose of bsF(ab')2 had minimal antitumor effect. BsF(ab')2 was capable of preventing tumor growth and improving survival when mice were also treated with T cell activators (IL-2 or staphylococcal enterotoxin B), or given repeated bsF(ab')2 doses. We conclude that therapeutic response to bispecific Ab was not dependent on functional Fc, but did require T cell activation. The use of bifunctional constructs that lack functional Fc therefore allows for separate manipulation of T cell retargeting and T cell activation and deserves further evaluation as a potential immunotherapy for malignancy.

Formation of a bispecific antibody by the use of leucine zippers.

A new method is described for the production of bispecific F(ab')2 heterodimers using leucine zippers. Two heterodimer-forming "zipper" peptides derived from the Fos and Jun proteins were respectively linked to the Fab' portions of two different mAb by gene fusion. The antibodies used were 145-2C11, which binds to murine CD3, and anti-Tac, which binds to the p55 chain of the human IL-2R. Anti-Tac Fab'-Jun and anti-CD3 Fab'-Fos were expressed individually as F(ab'-zipper)2 homodimers in the mouse myeloma cell line Sp2/0. When these homodimers were reduced at the hinge region to form monomers and then reoxidized together, the resulting end products were mostly F(ab'-zipper)2 heterodimers. Bispecific anti-CD3 x anti-Tac F(ab'-zipper)2 heterodimers produced by this method were shown to be highly effective in recruiting cytotoxic T cells to lyse IL-2R-bearing HuT-102 cells in vitro.