Treatment of Human B Cell Lymphoma Xenografts with a CD3 × CD19 Diabody and T Cells

Abstract

The use of anti-CD3 × antitumor bispecific Abs is an attractive and highly specific approach in cancer therapy. Recombinant Ab technology now provides powerful tools to enhance the potency of such immunotherapeutic constructs. We designed a heterodimeric diabody specific for human CD19 on B cells and CD3ε chain of the TCR complex. After production in Escherichia coli and purification, we analyzed its affinity, stability, and pharmacokinetics, and tested its capacity to stimulate T cell proliferation and mediate in vitro lysis of CD19+ tumor cells. The effect of the diabody on tumor growth was investigated in an in vivo model using immunodeficient mice bearing a human B cell lymphoma. The CD3 × CD19 diabody specifically interacted with both CD3- and CD19-positive cells, was able to stimulate T cell proliferation in the presence of tumor cells, and induced the lysis of CD19+ cells in the presence of activated human PBL. The lytic potential of the diabody was enhanced in the presence of an anti-CD28 mAb. In vivo experiments indicated a higher stability and longer blood retention of diabodies compared with single chain Fv fragments. Treatment of immunodeficient mice bearing B lymphoma xenografts with the diabody and preactivated human PBL efficiently inhibited tumor growth. The survival time was further prolonged by including the anti-CD28 mAb. The CD3 × CD19 diabody is a powerful tool that should facilitate the immunotherapy of minimal residual disease in patients with B cell leukemias and malignant lymphomas.

Characterization of BIS20x3, a bi-specific antibody activating and retargeting T-cells to CD20-positive B-cells

“…Another important question is if BIS20x3 is more efficient in treating lymphomas than CD19xCD3 bispecific antibodies. Several CD19xCD3 bispecific antibodies have been described recently (Daniel et al, 1998;Cochlovius et al, 2000;Löffler et al, 2000) in several antibody-formats (quadroma-derived, bi-specific singlechain and diabody respectively). The findings that CD20 seems to be higher expressed on B-cells compared to CD19 and that CD20 is not internalized after antibody binding in contrast to CD19-antibody complexes are often used as arguments for the use of CD20 antibodies (e.g.…”

Effect of tetravalent bispecific CD19×CD3 recombinant antibody construct and CD28 costimulation on lysis of malignant B cells from patients with chronic lymphocytic leukemia by autologous T cells

“…13,15,18,21 Monospecific anti-CD3 tanDb was assembled from OKT3-derived V H and V L domains 23 essentially as described for CD19ϫCD3 tanDb. 15 Gene constructions were performed according to standard DNA manipulation techniques, and the experimental details are available from the authors upon request.…”

“…19 We previously demonstrated that CD19ϫCD3 BsDb is more effective than quadroma-derived bispecific antibody at mediating T-cell cytotoxicity in vitro against tumor cells. 13,18 Although BsDb was relatively rapidly cleared from the blood through the kidneys, its antitumor activity in animal tumor models was closely similar to that of the much larger quadroma-derived bispecific antibody. 18,20 However, the BsDb is formed by heterodimerization of 2 different gene products, which must be expressed in the same cell in similar amounts.…”

“…13,18 Although BsDb was relatively rapidly cleared from the blood through the kidneys, its antitumor activity in animal tumor models was closely similar to that of the much larger quadroma-derived bispecific antibody. 18,20 However, the BsDb is formed by heterodimerization of 2 different gene products, which must be expressed in the same cell in similar amounts. The problem of quantitative heterodimer formation can be overcome by linking 4 variable domains derived from antibodies of 2 specificities into a single-chain construct, resulting in either an (scFv) 2 , 2 scFv modules composed of 2 adjacent V H and V L domains of the same specificity, 16 or a diabody-like structure, a so-called scBsDb.…”

“…8 To date, different forms of the CD19ϫCD3 bispecific antibody have been generated and used in a number of in vitro and in vivo therapeutic studies. [11][12][13][14][15][16][17][18] These bispecific antibodies have been produced using either rodent hybrid hybridomas, 12,14 chemical crosslinking of 2 MAbs 11 or recombinant antibody Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; BrdU, bromodeoxyuridine; BsDb, bispecific diabody; CD, cluster of differentiation; CLL, chronic lymphocytic leukemia; CTLA-4, cytotoxic T lymphocyte-associated antigen 4; EC 50 , half-maximal effective concentration; HS, human serum; MAb, monoclonal antibody; NHL, non-Hodgkin's lymphoma; PBL, peripheral blood lymphocyte; PBMC, peripheral blood mononuclear cell; scBsDb, single-chain BsDb; scFv, single-chain variable fragment of antibody; tanDb, tandem diabody; TCR, T-cell receptor; TNF, tumor necrosis factor; TRAIL, TNF-␣-related apoptosis-inducing ligand; V H , variable domain of the heavy chain of an antibody; V L , variable domain of the light chain of an antibody; WBC, white blood count. technology.…”