( Anti-CD 33 ) Monoclonal Antibodies Biological and Immunological Features of Humanized M 195 Updated Version

Human-mouse chimeric immunoglobulins G1 and G3 (IgG1 and IgG3) (ChG1, ChG3) and "complementarity-determining region"-grafted, humanized IgG1 and IgG3 (HuGI, 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 complementaritydetermining region-grafted mAbs had reduced avidities, the HuG1 and HuG3 M195 showed up to an 8.6and 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. I N T R O D U C T I O N Mouse monoclonal IgG2a 3 antibody M195 (mAb M195) is specifically reactive with the CD33 antigen on AML cells and early myeloid progenitor cells, but not with normal tissues or hematopoietic stem cells (1-5). This sparing of normal stem cells makes it an ideal candidate for leukemia therapy and for ex vivo bone marrow purging of AML cells (6-8). M195 reacts with the CD33 antigen, a Mr 67,000 glycoprotein, and is rapidly internalized into target cells upon binding. M195 is not intrinsically cytotoxic in vitro. Although M 195 kills target cells with guinea pig and rabbit complement, it is not effective with human complement or effector cells. However, the efficient binding and internalization of M195 have allowed the use of radiolabeled mAb in trials for AML therapy (9, 10). These trials have demonstrated that specific targeting of 131I-M195 can result in marked leukemia cytoreduction, therapeutic responses, or marrow ablation with no toxicity. Genetic engineering techniques are now available to produce new human-mouse chimeric mAbs that combine mouse variable regions with human constant regions (Fig. 1) or more fully humanized mAbs that have human framework and constant regions. These humanized mAbs have been "CDR-grafted" so that only the original mouse antigen binding sites are retained. The new constructs offer advantages over murine mAbs in being more effective in recruiting human effector functions and in avoiding neutralizing H A M A responses. Avoidance of H A M A may allow for repeated treatments without loss of effectiveness and a longer circulating time for the mAb. The added features may allow us to avoid the use of radiolabels and toxins in clinical therapeutic trials (11-16). One humanized IgG1 mAb, CAMPATH-1H, derived from CDRs from a rat mAb together with human framework regions, has been used in a clinical situation (l 5). C A M P A T H l H produced remissions in two patients with refractory non-Hodgkin's iymphoma. Mathieson et al. (16) have also used CAMPATH-1H in one patient with systemic vasculitis. A second humanized mAb, anti-Tac-H, reactive with the IL-2 receptor, has recently been constructed using computer-aided three-dimensional structural design to maintain avidity at its binding site (17). Humanized anti-Tac, as well as the chimeric version, blocks T-cell activation and has the added advantage over the original murine mAb of performing ADCC against human tumor targets (18). More recently, humanized versions of two murine mAbs against herpes simplex virus gB and gD glycoproteins have resulted in similar binding, virus neutralization, and cell protection as seen with the original murine mAb (19). This paper describes four new reconstructed M195 mAbs in comparison to the original mouse M195. The four new constructs compare favorably to the parental M 195 with respect to their specificity, immunoreactivity, and internalization, and they showed increased avidity of binding and improved immunological functions, such as ADCC. Therefore, these constructs hold potential promise for a new approach to the treatment of AML. MATERIALS AND M E T H O D S Received 3/10/92; accepted 10/7/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ~This work was supported by ROICA55349-01 and the Lucille P. Markey charitable trust. D. A. S. is a Lucille P. Markey scholar. 2 TO whom requests for reprints should be addressed. 3 The abbreviations used are: IgG, immunoglobulin G; IgM, immunoglobulin M; mAb, monoclonal antibody; AML, acute myelogenous leukemia; HAMA, human anti-mouse antibody; IL-2, interleukin 2; SDS, sodium dodecyl sulfate; PBMC, peripheral blood mononuclear cells; CMC, complement-mediated cytotoxicity; MTT, dimethylthiazol diphenyltetrazolium bromide: thiazolyl blue; ADCC, antibody-dependent cellular cytotoxicity; CDR, complementarity-determining region. MAb. Mab M195 (anti-CD33) originated in BALB/c mice immunized with leukemia cells from a patient with AML and was produced from hybridomas and purified as described previously (4, 5). Humanmouse chimeric IgG, with human-derived constant regions for IgG1 and IgG3 (ChGI, ChG3), and CDR-grafted humanized M195, retaining only the CDRs and other sterically important amino acids from the mouse IgG (HuGI, HUG3), were constructed as described (20). Sp2/0 hybridoma cell lines secreting the chimeric and humanized M 195 were grown in vitro, and the mAbs were purified on PA-Sepharose (Pharmacia, Piscataway, N J) by affinity chromatography using sequential pH step elutions (4). Purity was determined on SDS-polyacrylamide gels stained with Coomassie brilliant blue.