Chimerization of LL2, a Rapidly Internalizing Antibody Specific for B Cell Lymphoma

Abstract

LL2 is a murine monoclonal antibody (MAb) that has been shown to be effective for the diagnosis and treatment of patients with non-Hodgkin's B cell lymphoma. Studies have also shown that radiolabeled murine LL2 (mLL2) or mLL2 and fragments thereof coupled to Pseudomonas exotoxin (PE) can effectively target human B cell lymphoma in mice. We have obtained the DNA sequences encoding the VK and VH domains of mLL2, an IgG2a MAb, which were combined with their respective human kappa and IgG1 constant region domains and expressed in SP2/0 cells. Like its murine counterpart, the chimeric LL2 (cLL2) antibody is glycosylated in the light chain variable region. Chimerization did not interfere with the immunoreactivity of the antibody, as determined by a competitive binding assay, where either antibody shows equivalent inhibition of the binding of its counterpart to the Raji cell membrane surface antigen, CD22. Both antibodies bind and are rapidly internalized by Raji cells, whereas an irrelevant humanized antibody did not bind and was not internalized under similar conditions. The internalization rates of the bound murine or chimeric antibodies were nearly identical, with Ke values of 0.106 and 0.118 min-1 for mLL2 and cLL2, respectively. The observed close equivalence between the murine and chimeric antibodies suggests potential advantages of the latter as a less immunogenic agent. Studies are currently underway to evaluate the chimeric antibody as a potential therapeutic immunoconjugate.

Characterization of a humanized IgG4 anti-HLA-DR monoclonal antibody that lacks effector cell functions but retains direct antilymphoma activity and increases the potency of rituximab

“…The humanized L243 (IgG1/ isotype), hL243␥1, was generated as described previously. [29][30][31][32] The IgG4/ isotype of hL243␥4P (IMMU-114) was constructed by replacing the heavy chain constant region coding sequence for the human ␥1 chain with that of ␥4 chain. A point mutation, Ser241Pro (based on Kabat numbering), was introduced into the hinge region of the ␥4 sequence to avoid formation of half molecules when the antibody is expressed and produced in mammalian cell cultures.…”

Characterization of a New Humanized Anti-CD20 Monoclonal Antibody, IMMU-106, and Its Use in Combination with the Humanized Anti-CD22 Antibody, Epratuzumab, for the Therapy of Non-Hodgkin’s Lymphoma

“…Similar procedures were adopted to develop the humanized anti-CD20 MAb, designated as IMMU-106, or hA20. Briefly, the V and V H genes of the parent anti-CD20 MAb were first cloned from the hybridoma cells by reverse transcription-PCR, and the complementary determining region sequences were elucidated by DNA sequencing, as described (22). The V genes of complementary determining region-grafted (or humanized) anti-CD20 MAbs were then designed and engineered.…”

Antigen binding and stability properties of non‐covalently linked anti‐CD22 single‐chain Fv dimers

“…We recently described the generation of the highly stable scFv fragment MJ-7 (V H -15-V L ) [20] that derived from the clinically established, internalizing anti-CD22 monoclonal antibody (mAb) LL2 [21]. This construct, obtained after rational mutagenesis of three destabilizing residues within the variable region from antibody heavy chain (V H ) domain core structure, retained similar antigen binding properties as the wild-type scFv but exhibited several orders of magnitude greater stability when incubated in human serum at 37°C.…”