Enhancing Fc-mediated effector functions of monoclonal antibodies: The example of HexaBodies.

Since the approval of the CD20-targeting monoclonal antibody (mAb) rituximab for the treatment of lymphoma in 1997, mAb therapy has significantly transformed cancer treatment. With over 90 FDA-approved mAbs for the treatment of various hematological and solid cancers, modern cancer treatment relies heavily on these therapies. The overwhelming success of mAbs as cancer therapeutics is attributed to their broad applicability, high safety profile, and precise targeting of cancer-associated surface antigens. Furthermore, mAbs can induce various anti-tumor cytotoxic effector mechanisms including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), all of which are mediated via their fragment crystallizable (Fc) domain. Over the past decades, these effector mechanisms have been substantially improved through Fc domain engineering. In this review, we will outline the different approaches to enhance Fc effector functions via Fc engineering of mAbs, with a specific emphasis on the so-called "HexaBody" technology, which is designed to enhance the hexamerization of mAbs on the target cell surface, thereby inducing greater complement activation, CDC, and receptor clustering. The review will summarize the development, preclinical, and clinical testing of several HexaBodies designed for the treatment of B-cell malignancies, as well as the potential use of the HexaBody technology beyond Fc-mediated effector functions.

CD20 and CD37 antibodies synergize to activate complement by Fc-mediated clustering.

CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells ex vivo Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.

Antibodies That Efficiently Form Hexamers upon Antigen Binding Can Induce Complement-Dependent Cytotoxicity under Complement-Limiting Conditions.

Recently, we demonstrated that IgG Abs can organize into ordered hexamers after binding their cognate Ags expressed on cell surfaces. This process is dependent on Fc:Fc interactions, which promote C1q binding, the first step in classical pathway complement activation. We went on to engineer point mutations that stimulated IgG hexamer formation and complement-dependent cytotoxicity (CDC). The hexamer formation-enhanced (HexaBody) CD20 and CD38 mAbs support faster, more robust CDC than their wild-type counterparts. To further investigate the CDC potential of these mAbs, we used flow cytometry, high-resolution digital imaging, and four-color confocal microscopy to examine their activity against B cell lines and primary chronic lymphocytic leukemia cells in sera depleted of single complement components. We also examined the CDC activity of alemtuzumab (anti-CD52) and mAb W6/32 (anti-HLA), which bind at high density to cells and promote substantial complement activation. Although we observed little CDC for mAb-opsonized cells reacted with sera depleted of early complement components, we were surprised to discover that the Hexabody mAbs, as well as ALM and W6/32, were all quite effective at promoting CDC in sera depleted of individual complement components C6 to C9. However, neutralization studies conducted with an anti-C9 mAb verified that C9 is required for CDC activity against cell lines. These highly effective complement-activating mAbs efficiently focus activated complement components on the cell, including C3b and C9, and promote CDC with a very low threshold of MAC binding, thus providing additional insight into their enhanced efficacy in promoting CDC.

Dietary interventions that reduce mTOR activity rescue autistic-like behavioral deficits in mice.

Enhanced mammalian target of rapamycin (mTOR) signaling in the brain has been implicated in the pathogenesis of autism spectrum disorder (ASD). Inhibition of the mTOR pathway improves behavior and neuropathology in mouse models of ASD containing mTOR-associated single gene mutations. The current study demonstrated that the amino acids histidine, lysine, threonine inhibited mTOR signaling and IgE-mediated mast cell activation, while the amino acids leucine, isoleucine, valine had no effect on mTOR signaling in BMMCs. Based on these results, we designed an mTOR-targeting amino acid diet (Active 1 diet) and assessed the effects of dietary interventions with the amino acid diet or a multi-nutrient supplementation diet (Active 2 diet) on autistic-like behavior and mTOR signaling in food allergic mice and in inbred BTBR T+Itpr3tf/J mice. Cow's milk allergic (CMA) or BTBR male mice were fed a Control, Active 1, or Active 2 diet for 7 consecutive weeks. CMA mice showed reduced social interaction and increased self-grooming behavior. Both diets reversed behavioral impairments and inhibited the mTOR activity in the prefrontal cortex and amygdala of CMA mice. In BTBR mice, only Active 1 diet reduced repetitive self-grooming behavior and attenuated the mTOR activity in the prefrontal and somatosensory cortices. The current results suggest that activated mTOR signaling pathway in the brain may be a convergent pathway in the pathogenesis of ASD bridging genetic background and environmental triggers (food allergy) and that mTOR over-activation could serve as a potential therapeutic target for the treatment of ASD.

Potent preclinical activity of HexaBody-DR5/DR5 in relapsed and/or refractory multiple myeloma.

Apoptosis induction by death receptor (DR)-specific agonistic antibodies is a potentially effective antitumor therapy. Nonetheless, to date, all conventional DR-targeting antibodies that induce apoptosis via FcγR-dependent DR clustering failed to show clinical efficacy. HexaBody-DR5/DR5 (GEN1029) has been developed to overcome full FcγR dependence. HexaBody-DR5/DR5 is a mixture of 2 noncompeting DR5-specific immunoglobulin G1 (IgG1) antibodies, each with an E430G mutation in the Fc domain. This mutation enhances Fc-Fc interactions, resulting in antibody hexamerization, followed by FcγR-independent clustering of DR5 molecules. This unique combination of dual epitope targeting and increased IgG hexamerization resulted in potent preclinical antitumor activity in various solid cancers. In this study, we explored the preclinical activity of HexaBody-DR5/DR5 in multiple myeloma (MM), because MM cells are known to express DR5. In bone marrow samples from 48 MM patients, HexaBody-DR5/DR5 induced potent cytotoxicity of primary MM cells. Importantly, HexaBody-DR5/DR5 mediated the highest cytotoxic activity in samples from relapsed/refractory MM patients, including those who are refractory to daratumumab. This improved cytotoxic activity was observed only in patients who received their last anti-MM treatment <1 month ago, suggesting that anti-MM drugs sensitized MM cells to HexaBody-DR5/DR5. Supporting this, bortezomib combined with HexaBody-DR5/DR5 synergistically increased cytotoxicity in MM cells in 7 of 11 newly diagnosed patients. Lenalidomide also synergized with HexaBody-DR5/DR5, but only via its immunomodulatory effects, presumably by enhancing the antibody-dependent cellular cytotoxicity activity of HexaBody-DR5/DR5. Daratumumab showed additive effects when combined with HexaBody-DR5/DR5. In conclusion, the results of this preclinical study indicate a therapeutic potential for HexaBody-DR5/DR5, especially in recently treated relapsed/refractory MM patients.

Bone Marrow Mesenchymal Stromal Cell-mediated Resistance in Multiple Myeloma Against NK Cells can be Overcome by Introduction of CD38-CAR or TRAIL-variant.

We have recently shown the strong negative impact of multiple myeloma (MM)-bone marrow mesenchymal stromal cell (BMMSC) interactions to several immunotherapeutic strategies including conventional T cells, chimeric antigen receptor (CAR) T cells, and daratumumab-redirected NK cells. This BMMSC-mediated immune resistance via the upregulation of antiapoptotic proteins in MM cells was mainly observed for moderately cytotoxic modalities. Here, we set out to assess the hypothesis that this distinct mode of immune evasion can be overcome by improving the overall efficacy of immune effector cells. Using an in vitro model, we aimed to improve the cytotoxic potential of KHYG-1 NK cells toward MM cells by the introduction of a CD38-specific CAR and a DR5-specific, optimized TRAIL-variant. Similar to what have been observed for T cells and moderately lytic CAR T cells, the cytolytic efficacy of unmodified KHYG-1 cells as well as of conventional, DR5-agonistic antibodies were strongly reduced in the presence of BMMSCs. Consistent with our earlier findings, the BMMSCs protected MM cells against KHYG-1 and DR5-agonistic antibodies by inducing resistance mechanisms that were largely abrogated by the small molecule FL118, an inhibitor of multiple antiapoptotic proteins including Survivin, Mcl-1, and XIAP. Importantly, the BMMSC-mediated immune resistance was also significantly diminished by engineering KHYG-1 cells to express the CD38-CAR or the TRAIL-variant. These results emphasize the critical effects of microenvironment-mediated immune resistance on the efficacy of immunotherapy and underscores that this mode of immune escape can be tackled by inhibition of key antiapoptotic molecules or by increasing the overall efficacy of immune killer cells.

Epcoritamab induces potent anti-tumor activity against malignant B-cells from patients with DLBCL, FL and MCL, irrespective of prior CD20 monoclonal antibody treatment.

Epcoritamab (DuoBody-CD3xCD20, GEN3013) is a novel bispecific IgG1 antibody redirecting T-cells toward CD20+ tumor cells. Here, we assessed the preclinical efficacy of epcoritamab against primary tumor cells present in the lymph node biopsies from newly diagnosed (ND) and relapsed/refractory (RR) B-NHL patients. In the presence of T-cells from a healthy donor, epcoritamab demonstrated potent activity against primary tumor cells, irrespective of prior treatments, including CD20 mAbs. Median lysis of 65, 74, and 84% were achieved in diffuse large B-cell lymphoma (n = 16), follicular lymphoma (n = 15), and mantle cell lymphoma (n = 8), respectively. Furthermore, in this allogeneic setting, we discovered that the capacity of B-cell tumors to activate T-cells was heterogeneous and showed an inverse association with their surface expression levels of the immune checkpoint molecule Herpesvirus Entry Mediator (HVEM). In the autologous setting, when lymph node (LN)-residing T-cells were the only source of effector cells, the epcoritamab-dependent cytotoxicity strongly correlated with local effector cell-to-target cell ratios. Further analyses revealed that LN-residing-derived or peripheral blood-derived T-cells of B-NHL patients, as well as heathy donor T-cells equally mediated epcoritamab-dependent cytotoxicity. These results show the promise of epcoritamab for treatment of newly-diagnosed or relapsed/refractory B-NHL patients, including those who became refractory to previous CD20-directed therapies.

Fc-Engineered Antibodies with Enhanced Fc-Effector Function for the Treatment of B-Cell Malignancies.

Monoclonal antibody (mAb) therapy has rapidly changed the field of cancer therapy. In 1997, the CD20-targeting mAb rituximab was the first mAb to be approved by the U.S. Food and Drug Administration (FDA) for treatment of cancer. Within two decades, dozens of mAbs entered the clinic for treatment of several hematological cancers and solid tumors, and numerous more are under clinical investigation. The success of mAbs as cancer therapeutics lies in their ability to induce various cytotoxic machineries against specific targets. These cytotoxic machineries include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), which are all mediated via the fragment crystallizable (Fc) domain of mAbs. In this review article, we will outline the novel approaches of engineering these Fc domains of mAbs to enhance their Fc-effector function and thereby their anti-tumor potency, with specific focus to summarize their (pre-) clinical status for the treatment of B-cell malignancies, including chronic lymphocytic leukemia (CLL), B-cell non-Hodgkin lymphoma (B-NHL), and multiple myeloma (MM).

DuoHexaBody-CD37®, a novel biparatopic CD37 antibody with enhanced Fc-mediated hexamerization as a potential therapy for B-cell malignancies.

Tetraspanin CD37 has recently received renewed interest as a therapeutic target for B-cell malignancies. Although complement-dependent cytotoxicity (CDC) is a powerful Fc-mediated effector function for killing hematological cancer cells, CD37-specific antibodies are generally poor inducers of CDC. To enhance CDC, the E430G mutation was introduced into humanized CD37 monoclonal IgG1 antibodies to drive more efficient IgG hexamer formation through intermolecular Fc-Fc interactions after cell surface antigen binding. DuoHexaBody-CD37, a bispecific CD37 antibody with the E430G hexamerization-enhancing mutation targeting two non-overlapping epitopes on CD37 (biparatopic), demonstrated potent and superior CDC activity compared to other CD37 antibody variants evaluated, in particular ex vivo in patient-derived chronic lymphocytic leukemia cells. The superior CDC potency was attributed to enhanced IgG hexamerization mediated by the E430G mutation in combination with dual epitope targeting. The mechanism of action of DuoHexaBody-CD37 was shown to be multifaceted, as it was additionally capable of inducing efficient antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis in vitro. Finally, potent anti-tumor activity in vivo was observed in cell line- and patient-derived xenograft models from different B-cell malignancy subtypes. These encouraging preclinical results suggest that DuoHexaBody-CD37 (GEN3009) may serve as a potential therapeutic antibody for the treatment of human B-cell malignancies.

mTOR plays an important role in cow's milk allergy-associated behavioral and immunological deficits.

Autism spectrum disorder (ASD) is multifactorial, with both genetic as well as environmental factors working in concert to develop the autistic phenotype. Immunological disturbances in autistic individuals have been reported and a role for food allergy has been suggested in ASD. Single gene mutations in mammalian target of rapamycin (mTOR) signaling pathway are associated with the development of ASD and enhanced mTOR signaling plays a central role in directing immune responses towards allergy as well. Therefore, the mTOR pathway may be a pivotal link between the immune disturbances and behavioral deficits observed in ASD. In this study it was investigated whether the mTOR pathway plays a role in food allergy-induced behavioral and immunological deficits. Mice were orally sensitized and challenged with whey protein. Meanwhile, cow's milk allergic (CMA) mice received daily treatment of rapamycin. The validity of the CMA model was confirmed by showing increased allergic immune responses. CMA mice showed reduced social interaction and increased repetitive self-grooming behavior. Enhanced mTORC1 activity was found in the brain and ileum of CMA mice. Inhibition of mTORC1 activity by rapamycin improved the behavioral and immunological deficits of CMA mice. This effect was associated with increase of Treg associated transcription factors in the ileum of CMA mice. These findings indicate that mTOR activation may be central to both the intestinal, immunological, and psychiatric ASD-like symptoms seen in CMA mice. It remains to be investigated whether mTOR can be seen as a therapeutic target in cow's milk allergic children suffering from ASD-like symptoms.