Elsevier

Molecular Immunology

Volume 37, Issue 17, 12 December 2000, Pages 1035-1046
Molecular Immunology

Dissection and optimization of immune effector functions of humanized anti-ganglioside GM2 monoclonal antibody

https://doi.org/10.1016/S0161-5890(01)00021-9Get rights and content

Abstract

A mouse/human chimeric monoclonal antibody (MAb) KM966, specific for the cell-surface tumor antigen ganglioside GM2, was humanized by the complementarity determining regions (CDRs) grafting method. Not only the amino acid residues in the CDRs but also several in the framework regions (FRs) were changed from the human to the murine residues. A humanized variant, huKM796H/Lm-28, containing eight and five amino acid alterations in variable light (VL) and variable heavy (VH) FRs, respectively, showed a 9-fold reduction in complement-dependent cytotoxicity (CDC) compared to the chimeric KM966, despite tight antigen binding and potent antibody-dependent cellular cytotoxicity (ADCC). Several additional variants were subsequently constructed to improve the CDC of the antibody. One of the variants, designated KM8969, which differs by three amino acids, exhibited a CDC within 3-fold of the chimeric KM966. In addition, humanized KM8969 bound GM2 antigen 1.25-fold more tightly than the chimeric KM966 and showed 5-fold higher ADCC than the chimeric KM966. These results clearly show that the humanized KM8969, having the optimized immune effector functions and theoretically minimal immunogenicity, is an ideal candidate to test the effectiveness of anti-GM2 MAb in human cancer therapy. Taken together, the results obtained here indicate that the ADCC and CDC of an antibody can be dissected independently via engineering of the antibody variable region.

Introduction

Ganglioside GM2 [GalNAcβ1–4(NeuNAcα2–3)Galβ1–4Glcβ1–1ceramide] is a major tumor-differentiation antigen in human malignant melanomas and other tumors of neuroectodermal origin (Portoukalian et al., 1979, Hakomori, 1985, Zhang et al., 1997) and has been thought of as an effective target molecule for the passive immunotherapy of cancers using MAbs (Irie and Ravindranath, 1990, Scheinberg and Chapman, 1995, Jurcic et al., 1997).

Several rodent and human IgM monoclonal antibodies (MAbs) reactive to GM2 have been developed (Natoli et al., 1986, Miyake et al., 1988, Vrionis et al., 1989, Yamaguchi et al., 1990, Nishinaka et al., 1996), and a human IgM MAb, L55, showed some clinical responses in the treatment of melanoma (Irie et al., 1989). However, a number of difficulties, including the potent human anti-mouse antibody response and rapid clearance of rodent MAb in patients during immunotherapy, have greatly limited its therapeutic potential (Morell et al., 1970, Schroff et al., 1985, Shawler et al., 1985, Khazaeli et al., 1988). Moreover, comparative studies on the anti-tumor activities of IgG and IgM MAbs have revealed that the IgG MAb has superior anti-tumor activities in vivo to the IgM MAb, despite the IgM MAb having stronger anti-tumor activities in vitro (Bernstein et al., 1980). Some of the physicochemical characteristics of IgM MAbs, such as aggregation and lability to freeze-and-thawing, have been also problematic. To overcome these obstacles, we previously reported the construction of the first mouse/human chimeric anti-GM2 IgG MAb, KM966, in which the variable light (VL) and variable heavy (VH) regions of the murine anti-GM2 IgM MAb KM696 were engineered onto the constant (C) regions of human κ light- and γ 1 heavy-chain, respectively (Nakamura et al., 1994). KM966 retained specific reactivity with GM2 and exerted potent CDC and ADCC against various human cancer cells in the presence of human effector molecules and cells, respectively. Furthermore, KM966 seemed to be therapeutically effective in inhibiting the growth of human lung cancer cells implanted subcutaneously (s.c.) into nude mice and the metastasis of human lung cancer cells inoculated intravenously (i.v.) into NK cell-depleted severe combined immunodeficient (SCID) mice (Nakamura et al., 1994, Hanibuchi et al., 1998). Chimeric MAbs, however, may still induce a substantial human anti-mouse antibody response because one-third of the molecule is still of murine origin (Brüggemann et al., 1989). To further reduce the immunogenicity of chimeric MAbs and to prolong their circulating half-life, humanized MAbs were generated by grafting CDRs of murine MAbs onto the backbone of human framework regions (FRs) and C regions (Jones et al., 1986, Riechmann et al., 1988). Humanized MAbs have about 10% murine- and 90% human-derived residues, which should theoretically reduce their immunogenicity and extend their in vivo half-lives, compared with chimeric MAbs (Hakimi et al., 1991).

In the present article, we detail the construction and characterization of a humanized version of the chimeric KM966. Several variants containing variable numbers of murine FRs residues were constructed and evaluated in terms of antigen binding, CDC and ADCC. Efficient transient expression of the humanized MAb variants in COS-7 cells and molecular modeling analyses enabled us to rapidly determine the relationship between the amino acid substitutions in the FRs and the resultant antigen binding activity.

We found that the potency of the effector functions of humanized MAbs did not correlate with their antigen binding activity. The effector functions are activated only after antibodies interact with their cognate antigens, but how antigen binding, given the flexible nature of antibody molecules, triggers the effector responses at the Fc region is unknown. Some mechanisms have been proposed. In the allosteric model, antigen binding results in a conformational change that is transmitted from the Fab to the Fc region and results in the exposure of effector ligand interaction sites in the Fc region (Metzger, 1978). In the associative or aggregation model, multiple effector ligand-Fc region interactions result in the effector activation (Metzger, 1978). Burton and Woof, on the other hand, described a model for the generation of accessibility to ligand-interaction sites in the Fc region in which the Fab and Fc regions become ‘dislocated’ with respect to each other when they simultaneously interact with antigen and effector ligand, respectively (Burton and Woof, 1992). In fact, combinations of the above models are possible. Unfortunately, specific features of each of the models are difficult to assess by physical methods. In the case of the humanized MAbs, there has been no comprehensive analysis of the relationship between antigen-binding induced conformational changes and effector functions. Our results presented here suggest that the conformational changes in the V region effects on the expression of Fc effector functions and the effects of conformational changes on complement activation and the binding of Fc region to FcγR may be dissociable.

Section snippets

Cell lines

Human small cell lung cancer (SCLC) SBC-3 cells were kindly provided by Dr S. Hiraki (Okayama University, Okayama, Japan). The immunoglobulin nonsecreting rat myeloma YB2/0 cells and the monkey kidney COS-7 cells were purchased from American Type Culture Collection. Cells were maintained in RPMI1640 (SBC-3 and YB2/0) or DMEM (COS-7) supplemented with 10% heat-inactivated FBS at 37°C in a humidified atmosphere of 5% CO2 in air.

Antibodies and gangliosides

The chimeric anti-GM2 MAb KM966 was developed in our laboratory and

Design of humanized MAbs and initial characterization of binding activities

We first identified the CDRs of the original murine MAb V regions using the sequence-based definition (Kabat et al., 1991). The consensus sequences for the human FRs of V region have been defined (Kabat et al., 1991) and used for the successful humanization of many murine MAbs (Couto et al., 1995, Presta et al., 1997). We, therefore, searched for homology between the murine MAb FRs of VL region and human FRs using the most homologous consensus sequences for the four subgroups of κ VL regions.

Discussion

Here, we have constructed a humanized version of the chimeric anti-GM2 MAb KM966 in an attempt to improve its potential clinical efficacy by reducing its immunogenicity. Several humanized MAb variants were constructed, using human FRs with a high degree of homology to the murine V region, and tested for their antigen binding and effector functions. Rapid evaluation of a series of humanized MAb variants was facilitated by molecular modeling and a transient expression system. The humanized MAb

Acknowledgements

We thank Dr K. Yano for protein sequencing, Dr K. Uchida for protein purification, Dr A. Saito for oligonucleotide synthesis and Dr M.P. Quinlan for helpful suggestions.

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