Heterodimeric bispecific antibody-derivatives against CD19 and CD16 induce effective antibody-dependent cellular cytotoxicity against B-lymphoid tumor cells
Introduction
The cell surface antigen CD19 is a 95 kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily [1], [2]. It is exclusively expressed on B-lymphoid cells in nearly all developmental stages from the early pro-B cell on, but no longer on plasma cells. The protein is abundantly expressed on the majority of B-lineage acute lymphoblastic leukemias (B-ALL) and different types of non-Hodgkin-lymphomas (NHL) [3], [4], and its presence on leukemia repopulating cells (leukemia stem cells) of certain types of B-ALL has recently been reported [5], [6], [7]. The antigen is neither shed from the surface nor lost from the tumor cells upon antibody treatment and is weakly internalized after binding of antibodies and antibody fragments [8], [9], [10]. Thus, CD19 is an attractive target for the treatment of different types of B-cell malignancies, and possibly certain chronic inflammatory diseases and autoimmune disorders with antibody-derived agents [9], [11].
Although unconjugated monoclonal antibodies (mAbs) against CD19 showed encouraging results in pre-clinical tests [11], [12], [13], they displayed only modest therapeutic effects in clinical trials [14]. Therefore, different antibody-based strategies were followed to target CD19 more efficiently, including both immunotoxins and antibodies with improved effector cell-mediated functions [15], [16]. The recruitment of immune effector cells and the induction of antibody-dependent cellular cytotoxicity (ADCC) by engagement of Fc receptors (FcRs) are key events in vitro and in vivo, as demonstrated for the CD20-specific antibody Rituximab and others [17], [18], [19], [20]. Both functions are mediated by the antibody Fc-portion, and therefore, it was anticipated that optimization of the Fc-portion of CD19 antibodies may improve their therapeutic performance. Indeed, the pre-clinical efficacy of full-length CD19 antibodies was increased by strengthening the interaction between their Fc-portion and FcRs either through glyco-engineering or through amino acid substitutions achieved by mutagenesis [10], [21].
A strong improvement of effector functions was also obtained through the development of bispecific antibodies [22]. These molecules combine one binding site for a target antigen on tumor cells with a second site for an activating trigger molecule on effector cells, such as CD3 or CD28 on T-cells and CD16 (FcγRIII) on NK cells and macrophages [22], [23], [24]. In some cases, bispecific antibodies demonstrated more favorable cytotoxic properties in vitro than the parental mAbs and offered the opportunity to selectively address a desirable effector cell population [22]. In addition, the choice of antigen-combining domains from particular mAbs specific for non Fc-binding epitopes on trigger molecules allowed to avoid competition with circulating endogenous immunoglobulin (Ig) G, a known competitor of mAbs [25].
Bispecific recombinant antibody-derivatives targeting CD19 have been designed in a variety of formats [26]. Most of them used single-chain variable fragments (scFvs) as binding domains and avoided the unspecific uptake by non-activating FcRs on various cell types due to the lack of Fc-portions. Among them are tandem bispecific scFvs (bsscFvs), diabodies and tandem diabodies addressing CD16 on NK cells or CD3 on T cells [26], [27], [28]. The most advanced bispecific molecule is a tandem bsscFv with specificities for CD19 and CD3 (Blinatumomab™), which produced promising results in a phase I clinical study with NHL patients and established clinical potency for bispecific antibody-derivatives [29], [30]. However, in some clinical settings, such as the treatment of ALL patients after stem cell transplantation, NK cells may represent a more favorable population of effector cells than T cells [31]. Therefore it is still important to establish potent strategies activating NK-cells for efficient tumor cell lysis.
Although tandem bsscFvs and some of the alternative formats mentioned above offer distinct advantages, they are associated with certain limitations that might affect their therapeutic efficacy. One critical parameter for the potency of bispecific proteins is the strength of binding to the tumor cells, which is determined by the intrinsic affinity of the individual combining site and the overall functional avidity of the molecules. In one approach, an important improvement was achieved through in vitro affinity maturation of the tumor cell-directed scFv-component, which resulted in an increased cytotoxic potential in vitro [32]. In another approach, the ADCC potential of a bispecific antibody-derivative has been enhanced by including a second binding site for the tumor antigen: a heterodimeric bispecific minibody containing two scFvs for the tumor antigen Her2/neu and one scFv for CD16 was more effective in mediating ADCC than a control protein with monovalent binding for both antigens [33].
Fab fragments, assembling a binding site from an antibody light chain (L) and the Fd-fragment of the heavy chain (H), have been used as hetero-dimerization scaffolds to engineer recombinant Fab–scFv fusion proteins [34], [35], [36]. Such molecules were constructed by genetic fusion of an scFv with a different specificity to the C-terminus of either the L- or the Fd-chain of a Fab fragment. Co-expression of the extended chain with its corresponding heterologous counterpart lead to an efficient production and efficient hetero-dimerization in eukaryotic cells mediated by the complementarity of the contact zones of the L- and Fd-components and a disulfide bridge formed between them [34], [35]. Through this assembly, the binding-site of the Fab fragment was reconstituted and bispecific proteins were formed, so-called bibodies (Fig. 1A). Both chains of Fab fragments have further been extended by fusion with scFvs to build so-called tribodies (Fig. 1A). Tribodies and bibodies have intermediate molecular masses of approximately 110 and 75 kDa, respectively, and were initially designed to achieve longer plasma retention times in vivo than the smaller bsscFv fragments [34]. BsscFvs have molecular masses of only 55–65 kDa, close to the renal exclusion limit, and molecules with masses below this limit are rapidly cleared from the blood-stream by renal filtration [37]. Finally, the use of Fab fragments as binding domains in bispecific proteins is attractive, because Fab fragments in some cases had a higher affinity and a greater stability than the corresponding scFvs derived from the same hybridoma antibody [38], [39].
Our group has previously generated a disulfide-stabilized (ds) tandem bsscFv with monovalent binding for the tumor antigen CD19 and the trigger molecule CD16, ds[19 × 16] [27]. This molecule displayed strong cytotoxicity for CD19-positive human leukemic cells in ADCC reactions with mononuclear cells (MNCs) in vitro. However, monovalent targeting of tumor cells and almost similar affinities for the target antigen and the trigger molecule were considered limiting. To overcome this limitation we have designed a CD19 × CD16 tribody with one Fab and one scFv binding site for CD19 and an scFv binding site for CD16 and, for comparison, a CD19 × CD16 bibody lacking the CD19-specific scFv (Fig. 1). The tribody had the predicted increased overall avidity for CD19 over the bibody and the bsscFv, accompanied by a corresponding gain in functional ADCC activity. These results provide strong motivation to also test the in vivo properties of this molecule in future experiments.
Section snippets
Cell lines and hybridomas
Chinese hamster ovary (CHO) cells stably expressing human CD16A were from Dr. J. van de Winkel (University Medical Centre, Utrecht, The Netherlands). The 4G7 hybridoma (CD19; IgG1) [40] was from Dr. R. Levy (Stanford University, Palo Alto, CA, USA) and the hybridoma 3G8 (FcγRIII, CD16; IgG1) [41] from the American Type Culture Collection (ATCC, Manassas, VA, USA). CHO cells, the hybridomas, and the leukemia-derived cell lines SEM [42] and CEM (ATCC) were cultured in Roswell Park Memorial
Construction of Fab–scFv fusion proteins
The Fab–scFv fusion proteins were built using the CD19-directed Fab fragment from the chimeric CD19 antibody and the ds CD19 and CD16 scFvs [21], [27]. The latter were used because they were more stable than the respective scFvs containing the wild-type sequences [27]. For the simultaneous expression of both polypeptide chains building the entire molecules, double gene expression vectors were constructed (Fig. 1B). Briefly, the coding sequence for the ds CD16 scFv was fused to the 3′ end of the
Discussion
CD19 is a tumor-antigen with highly favorable properties for the design of antibody-derived therapeutics. Its attractive features have been recognized for more than 20 years, and many investigators in academic institutions and the pharmaceutical industry have attempted to target this antigen [8], [9]. However, until recently, these efforts have not produced therapeutic molecules of comparable efficacy as the CD20 antibody Rituximab (Mabthera™) or the Her2/neu antibody Trastuzumab (Herceptin™).
Conflict of interest
The authors declare no conflicts of interests.
Acknowledgements
The authors thank Dr. R. Levy for the 4G7 hybridoma, Dr. J.G. van de Winkel for the CD16 transfected CHO cells, D. Saul and B. Bock for excellent technical assistance. Th. Lange is gratefully acknowledged for administrative assistance. This work was supported by grants from Deutsche Krebshilfe e.V. (No. 108242), the association “Kaminkehrer helfen krebskranken Kindern e.V.”, the association of Friends of the University of Erlangen Children’s Hospital (bequest of Dr. Wilhelmine Fey), the
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