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Long-term survival rates of patients with acute myelogenous leukemia treated with intensive chemotherapy are 15-20%, despite efforts to develop new treatment strategies. Murine M195 (131I-M195), an anti-CD33, immunoglobulin (Ig) G2a monoclonal antibody has reactivity restricted to early myeloid cells and myeloid leukemic blasts but not hematopoietic progenitors. Previous trials in patients with relapsed or refractory myeloid leukemia showed that 131I-M195 rapidly targeted to the bone marrow and internalized into target cells. This article describes a therapeutic dose escalation study in which 24 patients received from 50 mCi/m2 to 210 mCi/m2 of 131I-M195 in divided doses. Cytoreduction of peripheral cell counts and bone marrow blasts occurred without nonhematopoietic toxicity. Doses of 131I-M195 greater than 135 mCi/m2 were associated with marrow cytoreduction sufficient to necessitate bone marrow transplant. However, 37% of the patients developed human anti-mouse antibody, preventing retreatment. To decrease immunogenicity and improve effector function, chimeric IgG1 and IgG3, and complementarity-determining region-grafted, humanized IgG1 and IgG3 versions of mouse M195 were developed by genetic engineering techniques. The new versions maintained specificity and biologic function, and they were superior to the mouse M195 in their ability to perform antibody-dependent cellular cytotoxicity against leukemia cells. Humanized M195, but not chimeric M195, showed a 4-8.6 times higher avidity than its mouse counterpart. Because effector function of IgG depends to a large extent on Fc clustering, a homodimeric HuG1 also was developed. Homodimeric HuG1 showed an ability to cause additional dramatic improvements in effector functions, as well as an ability to internalize and retain radioisotope in target leukemia cells. Monomeric and dimeric forms of humanized M195 may be advantageous in the therapy of acute myelogenous leukemia.

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.

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.

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.