Six-packed antibodies punch better

In this issue of the Journal, Oostindie et al. investigate CD37-specific monoclonal antibodies (mAb) engineered to undergo hexamerization.1 Efficient hexamer formation is induced by a single amino acid substitution, E430G, in the IgG1 constant domain previously described by the same group.2 The modification potentiates complement-dependent cytotoxicity (CDC) against chronic lymphocytic leukemia (CLL) cells in vitro. Next, the authors show that combinations of hexamerization-enhanced mAb against CD20 and CD37 provide synergistic activity. Intriguingly, the CD20- and CD37-targeting mAb formed mixed hexameric complexes on the cell surface with increased anti-tumor activity.

The anti-CD20 mAb rituximab is a critical component of treatment regimens for many B-cell malignancies.3 In combination with chemotherapy, rituximab has been shown to increase response rates, response duration, and overall survival. Single-agent rituximab is quite commonly used in follicular lymphoma and as maintenance therapy in several types of B-cell non-Hodgkin lymphoma (B-NHL), including CLL. Compared to other B-NHL, CLL cells have a relatively lower expression of CD20, and single-agent rituximab has limited activity in CLL. Few studies have investigated the combination of two mAb. The combination of rituximab with the anti-CD52 targeting mAb alemtuzumab yielded a higher rate of complete responses in CLL than had historically been seen with rituximab alone.4 However, the manufacturer withdrew alemtuzumab for the treatment of CLL.

Like CD20, the tetraspanin CD37 is an integral membrane protein abundantly expressed on B cells but not on plasma cells or hematopoietic stem cells.5 T cells, natural killer (NK) cells, granulocytes, and monocytes express low levels of CD37. Tetraspanins are central to membrane organization and play important roles in cell migration and adhesion.6 CD37 has been found to co-localize with integrin a4β1 on B cells and to contribute to cell adhesion and the transduction of survival signals.7

Several anti-CD37 antibodies are undergoing clinical investigation in B-cell malignancies.5 Otlertuzumab (also called TRU-016), a single-chain variable fragment (scFv) against CD37 linked to the IgG1 Fc fragment, induces apoptosis in CLL cells and mediates antibody-dependent cellular cytotoxicity (ADCC) but not CDC. Otlertuzumab has been shown to have single-agent activity in CLL,8 and in combination with bendamustine increased the response rate and prolonged progression-free survival over single-agent bendamustine.9 BI 836826, a chimeric mouse-human mAb with Fc modifications to increase affinity to FcγRIIIa effectively mediates ADCC and also induces apoptosis of CLL cells. In a phase I dose-escalation study, BI 836826 was well-tolerated up to doses of 400 mg and had a similar adverse event profile as other Fc-modified antibodies.10 The objective response rate was 61.5% in patients treated at doses ≥200 mg. Two antibody-drug conjugates and a radioimmunoconjugate targeting CD37 are also undergoing clinical investigation.5

Currently, 36 antibody-based cancer therapies approved by the US Food and Drug Administration (FDA), including 4 biosimilars, are on the market. The majority of these treatments are mAb in IgG1 format that mediate tumor cell killing on their own or in combination with chemotherapy. A key challenge has been the identification of suitable targets for therapeutic mAb as tumor-specific antigens are rare, and tumor-associated antigens are often expressed on essential healthy cells, lowering the therapeutic index. By contrast, lineage-specific antigens that are expressed on non-essential healthy cells have emerged as preferred targets of therapeutic mAb. A prime example is CD20, which is expressed on healthy and malignant B cells, and targeted by rituximab (FDA approved in 1997), its biosimilar rituximab-abbs (in 2018), ofatumumab (in 2009), and obinutuzumab (in 2013) for treatment of B-cell malignancies. The same applies to other cell surface antigens, such as CD19, that are restricted to the dispensable B-cell lineage of the hematopoietic system. The mechanism of action (MOA) by which mAb eradicate tumor cells include the induction of apoptosis by interfering with receptor/ligand interactions at the cell surface or by recruiting components of the innate immune system, such as plasma proteins in CDC, NK cells in ADCC, and macrophages in antibody-dependent cellular phagocytosis (ADCP).11 All three principle mechanisms of innate immune system recruitment, collectively known as effector functions, involve the Fc fragment of IgG1, a homodimer comprising the hinge and the second (C_H2) and third (C_H3) constant domains of the heavy chain. To mediate CDC, ADCC, and ADCP, the Fc fragment interacts with complement protein C1q and FcγRIIIa and FcγRIIa receptors, respectively. It also mediates prolonged circulatory half-life through neonatal Fc receptor (FcRn) recycling. All of these mechanisms can be fine tuned by subjecting the Fc fragment to protein or carbohydrate engineering.12 In fact, several of the FDA-approved mAb for cancer therapy have engineered Fc fragments.

Hexabodies constitute a new class of Fc fragment-engineered therapeutic antibodies.1413 A single amino acid substitution in C_H3, E430G, enhances the formation of IgG1 hexamers upon cell surface antigen binding (Figure 1A). As such, hexamerization, which was first discovered for membrane-bound wild-type IgG1,2 facilitates the docking of the hexavalent complement protein C1q initiating CDC. Indeed, previous studies revealed that CD20-targeting IgG1 with the E430G mutation mediate significantly enhanced CDC compared to the parental mAb.1413 The current study by Oostindie et al.1 makes the same case for a CD37-targeting IgG1. In addition, combining hexameric (E430G) CD37-targeting IgG1 with one of the FDA-approved CD20-targeting IgG1 (rituximab, ofatumumab, or obinutuzumab) had a synergistic effect in terms of malignant B-cell lysis by CDC in vitro. Intriguingly, the authors provide evidence that mixing CD20- and CD37-targeting IgG1 with E430G mutation leads to the formation of hetero-hexamers that are more potent in mediating CDC than the corresponding homo-hexamers on their own or in combination (Figure 1B). This finding is exciting as it suggests that two mAb that target two different cell surface antigens may form bispecific hetero-hexamers in the membrane, effectively leading to target clustering and an increase in the density of C1q docking sites. It also sheds a light on a possible concerted MOA of polyclonal antibodies which might form hetero-hexamers if they target different cell surface antigens or different epitopes of the same cell surface antigen. Collectively, the study makes a strong case for investigating multispecific and multiparatopic biclonal, oligoclonal, and polyclonal antibodies for enhancing CDC compared to their parental mAb. Finding co-operative target combinations, such as CD20 and CD37 in the current study, that enable hetero-hexamer formation in the presence or absence of hexamerization-enhancing mutations is a key challenge in applying this concept to other hematologic malignancies and solid tumors. In this context, hexameric monoclonal and hetero-hexameric biclonal antibodies should also be tested for enhancing other effector functions in addition to CDC. While research into bispecific antibodies has accelerated, with a huge increase in the number of related clinical trials that are now ongoing,15 polyclonal antibodies16 may well be the next wave of antibody-based cancer therapy. Hetero-hexamerization in the membrane is a possible MOA of polyclonal antibodies in IgG1 format, providing an incentive to investigate their therapeutic utility with and without hexamerization-inducing mutations.

Figure 1.Hexamerization and hetero-hexamerization of CD20- and CD37-targeting mAb on the cell surface. (A) Shown are CD20- targeting (blue) and CD37-targeting (orange) mAb in IgG1 format. Monomeric in solution, they form hexamers upon cell surface antigen binding. This natural hexamerization of wild-type IgG1 is enhanced by substituting a glutamic acid residue in the IgG1 Fc fragment with a glycine residue (E430G). Mixing CD20- and CD37-targeting mAb leads to the formation of hetero-hexamers in 3:3 (shown here), 4:2, 2:4, 5:1, or 1:5 compositions. (B) Oostindie et al.¹ show a gradual increase in complement-dependent cytotoxicity (CDC) mediated by wild-type CD37-targeting IgG1 (left) to wild-type CD20-targeting IgG1 (second from left) to CD37-targeting IgG1 with E430G mutation (center) to CD20-targeting IgG1 with E430G mutation (second from right) to mixed CD20- and CD37-targeting IgG1 with E430G mutation (right). This increase in CDC correlates with the density of C1q docking sites. For simplification, only hetero-hexamers in various 3:3 compositions are shown.

In summary, Oostindie et al.1 make a compelling case for further exploration of hexamer-forming antibodies and the combination of two, or possibly even more, targeting mAb. The advantages of antibody combinations might include not only increased cytotoxic activity, as described here, but possibly also better tumor-specific targeting and mitigation of tumor escape through antigen loss or target internalization.17 However, several aspects of this promising technology need further exploration. How effective is hexamer formation in vivo and what kind of hetero-hexamers might be formed, especially in tissue sites? The current study is limited to in vitro studies with CLL cells in suspension. It is not immediately clear how these observations will translate to in vivo settings. Furthermore, hetero-hexamers may form in different ratios, some containing equal ratios of antibodies, while in others one antibody may dominate. Will there be an optimal ratio and if so, could a desired composition be engineered into the antibody backbone? Translation of this promising approach into clinical trials may well constitute the next breakthrough in antibody therapy of B-cell malignancies. A first clinical trial with mAb engineered to facilitate hexamerization is ongoing in solid tumors (clinicaltrials.gov identifier: NCT03576131). GEN1029 (also called HexaBody-DR5/DR5) consists of a mixture of two mAb that bind to different epitopes on DR5 and activate this death receptor to induce apoptosis. Results from this and other studies of hexamerization-enhanced mAb and mAb combinations are eagerly awaited.