Engineered humanized dimeric forms of IgG are more effective antibodies.

Humanized IgG1 M195 (HuG1-M195), a complementarity determining region-grafted recombinant monoclonal antibody, is reactive with CD33, an antigen expressed on myelogenous leukemia cells. M195 is in use in trials for the therapy of acute myelogenous leukemia. Since biological activity of IgG may depend, in part, on multimeric Fab and Fc clustering, homodimeric forms of HuG1-M195 were constructed by introducing a mutation in the gamma 1 chain CH3 region gene to change a serine to a cysteine, allowing interchain disulfide bond formation at the COOH terminal of the IgG. Despite similar avidity, the homodimeric IgG showed a dramatic improvement in the ability to internalize and retain radioisotope in target leukemia cells. Moreover, homodimers were 100-fold more potent at complement-mediated leukemia cell killing and antibody-dependent cellular cytotoxicity using human effectors. Therefore, genetically engineered multimeric constructs of IgG may have advantages relative to those forms that are found naturally.

Anti-Tac-H, a humanized antibody to the interleukin 2 receptor with new features for immunotherapy in malignant and immune disorders.

The Mr 55,000 interleukin 2 receptor peptide (Tac; CD25) is not expressed by normal resting T-cells but is markedly up-regulated in adult T-cell leukemia and other malignancies, as well as on T-cells activated in normal immune, autoimmune, allograft, and graft-versus-host settings. Anti-Tac is a mouse monoclonal antibody directed against the Tac peptide. Our prior attempts to use this antibody in humans for antitumor therapy and immune regulation have been limited by weak recruitment of effector functions and neutralization by antibodies to mouse immunoglobulins. To circumvent these difficulties, we prepared several chimeric "humanized" anti-Tac antibodies by genetic engineering, including one "hyperchimeric" antibody (anti-Tac-II) in which the molecule is human except for the small hypervariable segments of the complementarity-determining regions retained from the mouse antibody. These constructs maintain high affinities for antigen and abilities to block T-cell activation and demonstrate new capabilities to perform antibody-dependent cell-mediated cytotoxicity, absent in the mouse anti-Tac. Hence, humanized antibodies have been developed to a tumor-associated antigen and activated T-cell marker with significant features that offer new therapeutic possibilities for select neoplastic and immune disorders.

Biological and immunological features of humanized M195 (anti-CD33) monoclonal antibodies.

Human-mouse chimeric immunoglobulins G1 and G3 (IgG1 and IgG3) (ChG1, ChG3) and "complementarity-determining region"-grafted, humanized IgG1 and IgG3 (HuG1, HuG3) constructs of the mouse monoclonal antibody (mAb) M195 were characterized. M195 is a murine immunoglobulin G2a (IgG2a), anti-CD33 mAb, specifically reactive with acute myelogenous leukemia cells, that is active as an antileukemia agent in humans. The new mAb constructs maintained specificity and biological function, including rapid internalization after binding to the cell surface, which has been important for delivery of therapeutic isotopes in patients. Although previously reported complementarity-determining region-grafted mAbs had reduced avidities, the HuG1 and HuG3 M195 showed up to an 8.6- and 4-fold higher binding avidity, respectively, than the original murine mAb. All constructs were effective at mediating rabbit complement-mediated cytotoxicity against HL60 targets. Fibroblasts transfected with CD33 genes and expressing high levels of CD33 antigen were also lysed in the presence of human complement, but HL60 cells or fibroblasts with lower CD33 levels were not killed. Thus, the inability of M195 and constructs to kill HL60 targets with human complement is due to the much lower antigen density on HL60 cells compared to CD33+ fibroblasts. Unlike the murine M195, the chimeric and humanized M195 demonstrated antibody-dependent cell-mediated cytotoxicity using human peripheral blood mononuclear cells as effectors. Because the chimeric and humanized M195 have improved avidities as compared to the original M195 and have, in addition, the potential to avoid human anti-mouse antibody responses and to recruit human effector functions, these new constructs may be useful therapeutically, either alone or conjugated to toxins or isotopes, in the treatment of acute myelogenous leukemia.

Genetically engineered deglycosylation of the variable domain increases the affinity of an anti-CD33 monoclonal antibody.

M195 is a murine monoclonal antibody that binds to the CD33 antigen and is being tested for the treatment of myeloid leukemia. Surprisingly, a complementarity determining region (CDR)-grafted, humanized M195 antibody displayed a several-fold higher binding affinity for the CD33 antigen than the original murine antibody. Here we show that the increase in binding affinity resulted from eliminating an N-linked glycosylation site at residue 73 in the heavy chain variable region in the course of humanization. Re-introducing the glycosylation site in the humanized antibody reduces its binding affinity to that of the murine antibody, while removing the glycosylation site from the murine M195 variable domain increases its affinity. The removal of variable region carbohydrates may provide a method for increasing the affinity of certain monoclonal antibodies with diagnostic and therapeutic potential.

Regulation of differential processing of mouse immunoglobulin mu heavy-chain mRNA.

The switch between the synthesis of membrane-bound and secreted IgM during B cell differentiation is accomplished by producing, from a single gene, two alternative forms of mu heavy-chain mRNA that differ only in their 3' termini. The precursor mu RNA is either polyadenylated at the first poly(A) site, for secreted mu mRNA, or spliced between the C4 and M1 exons, for membrane-bound mu mRNA, in a mutually exclusive manner. To elucidate the molecular mechanism of the differential processing of mouse mu mRNA, we analyzed the expression of various mouse mu gene constructs stably transfected into mouse cell lines. In B cell lines, processing of the exogenously transfected mu gene transcripts accurately reflected the developmental stage of the recipient cells: both secreted and membrane-bound mu mRNAs are produced in early-stage B cells while secreted mu mRNA is primarily produced in late-stage B cells. In fibroblast cell lines, mu mRNAs transcribed from the Moloney murine sarcoma virus LTR promoter were processed primarily to the secreted form. Thus, production of the secreted form seems to be the non-regulated processing pattern. When the splicing signal of the C4-M1 intron was mutagenized, polyadenylation at the first poly(A) site occurred efficiently regardless of the recipient cell lines. On the other hand, when the polyadenylation signal was mutagenized, the splicing occurred efficiently in early-stage B cells, but only weakly in late-stage B cells and fibroblast cells. These results suggest that the splicing of the C4-M1 intron is stimulated in early-stage B cells.

Chimeric and humanized antibodies with specificity for the CD33 antigen.

L and H chain cDNAs of M195, a murine mAb that binds to the CD33 Ag on normal and leukemic myeloid cells, were cloned. The cDNAs were used in the construction of mouse/human IgG1 and IgG3 chimeric antibodies. In addition, humanized antibodies were constructed which combined the complementarity-determining regions of the M195 antibody with human framework and constant regions. The human framework was chosen to maximize homology with the M195 V domain sequence. Moreover, a computer model of M195 was used to identify several framework amino acids that are likely to interact with the complementarity-determining regions, and these residues were also retained in the humanized antibodies. Unexpectedly, the humanized IgG1 and IgG3 M195 antibodies, which have reshaped V regions, have higher apparent binding affinity for the CD33 Ag than the chimeric or mouse antibodies.

A humanized antibody that binds to the interleukin 2 receptor.

The anti-Tac monoclonal antibody is known to bind to the p55 chain of the human interleukin 2 receptor and to inhibit proliferation of T cells by blocking interleukin 2 binding. However, use of anti-Tac as an immunosuppressant drug would be impaired by the human immune response against this murine antibody. We have therefore constructed a "humanized" antibody by combining the complementarity-determining regions (CDRs) of the anti-Tac antibody with human framework and constant regions. The human framework regions were chosen to maximize homology with the anti-Tac antibody sequence. In addition, a computer model of murine anti-Tac was used to identify several amino acids which, while outside the CDRs, are likely to interact with the CDRs or antigen. These mouse amino acids were also retained in the humanized antibody. The humanized anti-Tac antibody has an affinity for p55 of 3 x 10(9) M-1, about 1/3 that of murine anti-Tac.