The use of therapeutic monoclonal antibodies is constrained because single antigen targets often do not provide sufficient selectivity to distinguish diseased from healthy tissues. We present HexElect ® , an approach to enhance the functional selectivity of therapeutic antibodies by making their activity dependent on clustering after binding to two different antigens expressed on the same target cell. lmmunoglobulin G (lgG)-mediated clustering of membrane receptors naturally occurs on cell surfaces to trigger complement- or cell-mediated effector functions or to initiate intracellular signaling. We engineer the Fc domains of two different lgG antibodies to suppress their individual homo-oligomerization while promoting their pairwise hetero-oligomerization after binding co-expressed antigens. We show that recruitment of complement component C1q to these hetero-oligomers leads to clustering-dependent activation of effector functions such as complement mediated killing of target cells or activation of cell surface receptors. HexElect allows selective antibody activity on target cells expressing unique, potentially unexplored combinations of surface antigens.

Antibodies are the cardinal effector molecules of the immune system and are being leveraged with enormous success as biotherapeutic drugs. A key part of the adaptive immune response is the production of an epitope-diverse, polyclonal antibody mixture that is capable of neutralizing invading pathogens or disease-causing molecules through binding interference and by mediating humoral and cellular effector functions. Avidity — the accumulated binding strength derived from the affinities of multiple individual non-covalent interactions — is fundamental to virtually all aspects of antibody biology, including antibody–antigen binding, clonal selection and effector functions. The manipulation of antibody avidity has since emerged as an important design principle for enhancing or engineering novel properties in antibody biotherapeutics. In this Review, we describe the multiple levels of avidity interactions that trigger the overall efficacy and control of functional responses in both natural antibody biology and their therapeutic applications. Within this framework, we comprehensively review therapeutic antibody mechanisms of action, with particular emphasis on engineered optimizations and platforms. Overall, we describe how affinity and avidity tuning of engineered antibody formats are enabling a new wave of differentiated antibody drugs with tailored properties and novel functions, promising improved treatment options for a wide variety of diseases. Antibody function is dependent on avidity — the accumulated strength of multiple affinity interactions between the antibody, antigen, cell surface receptors and other antibodies. In this Review, Oostindie et al. discuss the role of avidity in eliciting antibody functional responses and review the current engineering strategies for manipulating avidity interactions in antibody-based therapies.

Lymphocytes are the central actors in adaptive immune responses. When challenged with antigen, a small number of B and T cells have a cognate receptor capable of recognising and responding to the insult. These cells proliferate, building an exponentially growing, differentiating clone army to fight off the threat, before ceasing to divide and dying over a period of weeks, leaving in their wake memory cells that are primed to rapidly respond to any repeated infection. Due to the non-linearity of lymphocyte population dynamics, mathematical models are needed to interrogate data from experimental studies. Due to lack of evidence to the contrary and appealing to arguments based on Occam’s Razor, in these models newly born progeny are typically assumed to behave independently of their predecessors. Recent experimental studies, however, challenge that assumption, making clear that there is substantial inheritance of timed fate changes from each cell by its offspring, calling for a revision to the existing mathematical modelling paradigms used for information extraction. By assessing long-term live-cell imaging of stimulated murine B and T cells in vitro, we distilled the key phenomena of these within-family inheritances and used them to develop a new mathematical model, Cyton2, that encapsulates them. We establish the model’s consistency with these newly observed fine-grained features. Two natural concerns for any model that includes familial correlations would be that it is overparameterised or computationally inefficient in data fitting, but neither is the case for Cyton2. We demonstrate Cyton2’s utility by challenging it with high-throughput flow cytometry data, which confirms the robustness of its parameter estimation as well as its ability to extract biological meaning from complex mixed stimulation experiments. Cyton2, therefore, offers an alternate mathematical model, one that is, more aligned to experimental observation, for drawing inferences on lymphocyte population dynamics.

Over the last decades, several surface antigens have been identified and validated for treatment of B-cell malignancies. CD20-targeting antibodies have emerged as an effective therapy for patients with B-cell malignancies and are now broadly used in clinical practice. Nonetheless, many patients develop resistance against CD20-targeting therapies, with little or no further treatment options.1 Tetraspanin CD37 is expressed almost exclusively on hematopoietic cells with high expression on mature B-cells, including their malignant counterparts,2,3 and is a well described and validated target for B-cell malignancies. A number of CD37 targeting agents are in (pre)clinical development, including antibody-drug conjugates (IMGN529 and AGS67E), an Fc-engineered antibody (BI836826), a homodimeric therapeutic protein (otlertuzumab/TRU-016), a radioimmunoconjugate (177Lu-lilotomab), and chimeric antigen receptor T-cells.4-6 We recently reported on the development of DuoHexaBody-CD37, a novel biparatopic CD37-bispecific immunoglobulin G1 (IgG1) antibody with an E430G hexamerization-enhancing single point mutation in the Fc-domain.7 In contrast to all other newly developed CD37 targeting agents, DuoHexaBody-CD37 mediates potent complement-dependent cytotoxicity (CDC), a powerful anti-tumor effector mechanism,8,9 in addition to its Fc gamma receptor (FcγR)-mediated tumor cell kill mechanisms including antibody-dependent cellular cytotoxicity and cellular phagocytosis. DuoHexaBody-CD37 was designed based on recent discoveries that (1) initiation of CDC is dependent on Fc-mediated hexamer-formation of IgG1 antibodies after target engagement on the cell surface,10 (2) antibody Fc–Fc interactions and hexamer formation can significantly be improved by introducing an E430G single point mutation in the Fc domain,10,11 and (3) dual-epitope targeting of CD37 can potentiate or further enhance CDC.7 Here, we investigated the ex vivo therapeutic potential of DuoHexaBody-CD37 by evaluating its unique CDC-inducing capacity in primary tumor cell samples from a large cohort of newly diagnosed (ND) and relapsed/refractory (RR) patients with a broad range of B-cell malignancies, including chronic lymphocytic leukemia (CLL) and B-cell non–Hodgkin lymphoma, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma, and marginal zone lymphoma (Supplementary Material and Methods, http://links.lww.com/HS/A121). DuoHexaBody-CD37 induced potent, dose-dependent CDC of malignant primary B-cells in 45-minute complement assays with a median maximal lysis of 86% (n = 51, range: 0%-99%) and a median half maximal effective concentration (EC50) of 0.10 μg/mL (range, 0.004-15.21 μg/mL) (Figure 1A). Strikingly, sensitivity of the patient samples to DuoHexaBody-CD37-induced CDC was highly homogeneous. Only in 8 of 51 patient samples maximal lysis was lower than 60%, of which 3 samples demonstrated responses below 20% lysis. The CDC activity of DuoHexaBody-CD37 was comparable in samples from ND patients (n = 33; median maximal lysis of 85%; range CDC 0%-98%) and RR patients (n = 18; median maximal lysis of 89%; range CDC 5%-99%). Furthermore, DuoHexaBody-CD37 was significantly more potent than the CD20-targeting antibody rituximab in samples from ND patients, that is, CD20 antibody treatment naive patients (Figure 1B), even though CD37 expression was generally lower than expression of CD20 (Supplementary Figure 1, http://links.lww.com/HS/A121).Figure 1.: DuoHexaBody-CD37 induced potent CDC in samples obtained from ND and RR patients with various B-cell malignancies. A: Dose-dependent CDC induced by DuoHexaBody-CD37 in tumor B-cells (n = 51) derived from patients with various B-cell malignancy subtypes grouped together, in the presence of 20% NHS and in comparison to IgG1-ctrl (10 μg/mL). Levels of CDC-mediated tumor cell kill (% CDC) were determined by 7AAD-positive tumor cell staining, relative to a no antibody control sample. B: Comparison of CDC activity of 10 μg/mL DuoHexaBody-CD37 in samples from ND (n = 33) and RR (n = 18) patients (ns; Mann-Whitney U test), and comparison of rituximab (10 μg/mL) and DuoHexaBody-CD37 in ND patients (****P < 0.0001; Wilcoxon matched-pairs signed rank test). RR patients include CD20 therapy-refractory patients (n = 5;•), defined by progression of the disease within 6 mo post therapy, and CD20 therapy-relapse patients (n = 6;•). C: CD37 expression levels (defined as the number of CD37 molecules on the cell surface, assessed by quantitative flow cytometry), on tumor B-cells in ND and RR patient samples (ns; Mann-Whitney U test). D: Quantified CD37 expression levels (antibody molecules per cell) on tumor B-cells stratified according to B-cell malignancy subtype (ns, nonparametric Kruskal-Wallis test). All data are shown as the median and interquartile range. E: CDC activity of DuoHexaBody-CD37 (10 μg/mL) stratified according to B-cell malignancy subtype, including CLL (n = 10), FL (n = 12), MCL (n = 7), DLBCL (n = 18), and MZL (n = 4) (*P = 0.0321, nonparametric Kruskal-Wallis test with Dunn's multiple comparisons [n])). Green symbols (•) indicatepatient samples with poor prognosis: ibrutinib-refractory CLL (n = 2) and double-hit DLBCL (n =3). AAD = xxx, CDC = complement-dependent cytotoxicity, CLL = chronic lymphocytic leukemia, DLBCL = diffuse large B-cell lymphoma, FL = follicular lymphoma, IgG1 = xxx, MCL = mantle cell lymphoma, MZL = marginal zone lymphoma, ND = newly diagnosed, NHS = normal human serum, NS = not significant, RR = relapsed/refractory.Potent cytotoxicity was also observed in samples from patients who relapsed from (Figure 1B, blue symbols; n = 7) or were refractory to treatment regimens containing CD20-targeted antibodies (defined by progression of the disease within 6 months post treatment; Figure 1B, red symbols; n = 5), with the exception of 1 CD20-refractory FL patient. CD37 expression analysis (Figure 1C) revealed that this patient lacked CD37 expression on tumor B-cells. Since it has been reported that anti-CD20 therapy may induce CD20 antigen loss,12 and CD20 colocalizes with CD37 on the cell surface,13 we evaluated the possibility that CD37 expression levels were reduced on tumor B-cells derived from patients who had been exposed to anti-CD20. However, all anti-CD20–treated patient samples in our cohort showed high CD37 expression levels and no significant differences were observed in CD37 expression levels between samples from ND and RR patients (Figure 1C). In addition, CD37 was expressed homogeneously in all B-cell malignancy subtypes (Figure 1D), which is in alignment with several other reports2,6 except for a recent study that suggested variable CD37 expression in DLBCL.14 This study-related discrepancy could be due to differences in assay-dependent detection limits in our study (flow cytometry) versus the other (immunohistochemistry) study. DuoHexaBody-CD37 induced an effective and homogeneous CDC response in all samples from CLL (n = 10; median max lysis 93%; range, 80%-99%), mantle cell lymphoma (n = 7; 91%; 69%-98%) and marginal zone lymphoma (n=4; 88%; 75%-90%), and 11 of 12 FL patient samples (n=12; 86%; 5%-98%), while a more heterogeneous response was observed in DLBCL patient samples (n = 18; 74%; 0%-96%) (Figure 1E) (Kruskal-Wallis one-way analysis of variance (ANOVA), *P = 0.0321). There were no differences in sensitivity between activated B-cell and germinal center B-cell subtypes of DLBCL (Supplementary Figure 2, http://links.lww.com/HS/A121). Of note, also samples from ibrutinib-refractory CLL (n = 2) and double-hit DLBCL (n = 3) patients with poor prognosis were highly susceptible to DuoHexaBody-CD37–induced CDC (maximum lysis >60%) (Figure 1E). Complement activation and CDC-mediated tumor cell lysis is not only dependent on antigen density but also on expression levels of complement regulatory proteins (CRPs) CD46 and CD55 that inhibit complement convertases, and CD59 that inhibits the formation of the membrane attack complex.15 Expression of CRPs was not associated with B-cell malignancy subtype (Figure 2A) or treatment status (Supplementary Figure 3, http://links.lww.com/HS/A121).Figure 2.: Low sensitivity to DuoHexaBody-CD37–mediated CDC is not associated with expression of CD37 and CRPs and can be improved by combination with CD20 mAbs. A, Quantified expression levels (antibody molecules per cell) of complement regulatory proteins CD46, CD55, and CD59 on tumor B-cells in specific B-cell malignancy subtypes; CLL (n = 10), FL (n = 12), MCL (n = 7), DLBCL (n = 18), and MZL (n = 4) (ns; nonparametric Kruskal-Wallis test). Data shown are CDC in individual patient samples, in addition to the median and interquartile range. B, CDC activity of DuoHexaBody-CD37 (10 μg/mL) correlated with the ratio of CD37/CD59 expression levels for all B-cell malignancy subtypes grouped together, for DLBCL patient samples specifically (•) and for B-cell malignancy subtypes other than DLBCL (Spearman's correlation r = 0.4423, **P = 0.0013; r = 0.2843, P = 0.2678; and r = 0.5158, **P = 0.0025, respectively). Data are shown relative to a no antibody control sample. C, Expression levels of CD37 (antibody molecules per cell) correlated with C1q binding (ΔMFI) for 6 samples with low CDC response (CDC < 60%) (•), 1 CD37-negative sample as negative control (•) and 2 high responding samples as positive control (•) (Spearman's correlation r = 0.7833, *P = 0.0172). D, CDC induced by a combination of DuoHexaBody-CD37 and rituximab or ofatumumab (10 μg/mL + 10 μg/mL) vs single antibody (10 μg/mL) (n = 10) (*P < 0.05, ***P < 0.001; Friedman test with Dunn multiple comparison test) relative to a no antibody control sample. All data are shown as the median and interquartile range. CDC = complement-dependent cytotoxicity, CLL = chronic lymphocytic leukemia, CRP = complement regulatory protein, DLBCL = diffuse large B-cell lymphoma, FL = follicular lymphoma, mAbs = monoclonal antibodies, MCL = mantle cell lymphoma, MFI = median fluorescence intensity, MZL = marginal zone lymphoma.Furthermore, no correlation was observed between DuoHexaBody-CD37–induced CDC and expression of CD37 or CRPs (Supplementary Figure 4, http://links.lww.com/HS/A121). A weak, but statistically significant correlation was observed between sensitivity to DuoHexaBody-CD37 and the ratio of CD37 and CD59 expression levels (Figure 2B). However, in DLBCL patient samples, differences in (the ratio of) CD37 and CD59 expression could not explain the heterogeneous response to DuoHexaBody-CD37 ex vivo (Figure 2B, orange symbols). We therefore conclude that there is a limited impact of CRP expression on DuoHexaBody-CD37–mediated CDC. This was in contrast to rituximab-induced CDC, which already showed a strong correlation with CD20 expression but an even stronger correlation with the ratio of CD20 and CD55 (Supplementary Figure 5, http://links.lww.com/HS/A121). The first step of complement activation is the binding of C1q to membrane-bound antibodies, that together with C1r and C1s forms the C1 complex, the first component of the classical complement pathway. Toward understanding the heterogeneity in CDC responses observed in DLBCL patient samples, we investigated the C1q binding capacity of membrane-bound DuoHexaBody-CD37 in DLBCL patient samples for which DuoHexaBody-CD37–mediated CDC levels were lower than 60% (N = 6). The C1q binding capacity in low CDC responders was comparable to that of 2 patient samples highly susceptible to DuoHexaBody-CD37–induced CDC (Figure 2C), indicating that the first step of complement activation is not impaired. Whether other steps in the complement activation pathway, such as membrane attack complex assembly and stability, or cell intrinsic mechanisms, play a role in resistance to CDC induction remains to be elucidated. Finally, we also evaluated whether combination with rituximab and/or ofatumumab could further enhance the CDC activity of DuoHexaBody-CD37, as we have previously reported enhanced CDC in CLL and B-cell non–Hodgkin lymphoma primary patient cells with combinations of CD20 and CD37 antibodies.13 Indeed, in 10 patient samples including samples with intermediate to low sensitivity to DuoHexabody-CD37–mediated CDC (<80% CDC), the combination of DuoHexaBody-CD37 with ofatumumab significantly enhanced the tumor cell kill in an additive manner (Figure 2D). In contrast to ofatumumab, rituximab as single antibody could generally not induce CDC in these samples and the CDC effects of the combination with DuoHexaBody-CD37 are less striking. In conclusion, this preclinical study indicates high therapeutic potential for DuoHexaBody-CD37 in a broad spectrum of B-cell malignancies either as single agent or in combination with CD20-targeting antibodies, and supports the recently initiated first-in-human clinical trial of DuoHexaBody-CD37 for patients with relapsed or refractory B-cell NHL (NCT04358458). Disclosures SCO, MBO, and ECWB are Genmab employees and own Genmab warrants and/or stock. HJvdH, SCO, MBO, MEDC, ECWB, and TM are inventors on Genmab patent applications. MEDC has received research support from Gilead, Genmab, and Celgene. SZ has received research support from Celgene, Janssen Pharmaceuticals and Takeda; and serves in advisory boards for Celgene, Janssen Pharmaceuticals, Takeda, Amgen, and Sanofi. TM has received research support from Janssen Pharmaceuticals, Genmab, Takeda, Onkimmune, and Gadeta. All the other authors have no conflicts of interest to disclose. Acknowledgments The authors thank Henk Lokhorst for his contribution in the early stage of the study.

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.

Carbamylation is a post-translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca-IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement-dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca-IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca-IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca-IgG to activate complement was analyzed in enzyme-linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca-IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca-IgG lost its capacity to activate complement in both solid-phase and CDC assays. Mixing ca-IgG with non-modified IgG did not result in effective inhibition of complement activation by ca-IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca-IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers.

BackgroundDuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was silenced by introduction of mutations L234F L235E D265A.MethodsT-cell activation and T-cell-mediated cytotoxicity were measured by flow cytometry following co-culture with tumour cells. Anti-tumour activity of DuoBody-CD3xCD20 was assessed in humanized mouse models in vivo. Non-clinical safety studies were performed in cynomolgus monkeys.FindingsDuoBody-CD3xCD20 induced highly potent T-cell activation and T-cell-mediated cytotoxicity towards malignant B cells in vitro. Comparison of DuoBody-CD3xCD20 to CD3 bsAb targeting alternative B-cell antigens, or to CD3xCD20 bsAb generated using alternative CD20 Ab, emphasized its exceptional potency. In vitro comparison with other CD3xCD20 bsAb in clinical development showed that DuoBody-CD3xCD20 was significantly more potent than three other bsAb with single CD3 and CD20 binding regions and equally potent as a bsAb with a single CD3 and two CD20 binding regions. DuoBody-CD3xCD20 showed promising anti-tumour activity in vivo, also in the presence of excess levels of a CD20 Ab that competes for binding. In cynomolgus monkeys, DuoBody-CD3xCD20 demonstrated profound and long-lasting B-cell depletion from peripheral blood and lymphoid organs, which was comparable after subcutaneous and intravenous administration. Peak plasma levels of DuoBody-CD3xCD20 were lower and delayed after subcutaneous administration, which was associated with a reduction in plasma cytokine levels compared to intravenous administration, while bioavailability was comparable.InterpretationBased on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies.FundingGenmab

CD37 is a tetraspanin expressed on mature B cells where it orchestrates plasma membrane organization, receptor signaling, cell migration and adhesion. As CD37 is abundantly expressed on many mature B cell-derived malignancies, it represents an attractive target for new antibody therapies. DuoHexaBody®-CD37 is a novel biparatopic bispecific CD37 antibody with an Fc domain engineered to enhance antibody hexamerization upon binding to CD37 on the plasma membrane. DuoHexaBody-CD37 was shown to induce potent complement-dependent cytotoxicity (CDC) of malignant B-cell lines and primary chronic lymphocytic leukemia and non-Hodgkin lymphoma patient samples (Oostindie et al., Blood 2018 132:4170; van der Horst et al., Blood 2018 132:4179). Here, we demonstrate that the DuoHexaBody-CD37 mechanism of action also encompasses FcγR-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Daudi and Raji cells opsonized with DuoHexaBody-CD37 induced FcγRIIa and FcγRIIIa signaling in luciferase reporter assays. In agreement with efficient FcγR engagement, opsonization with DuoHexaBody-CD37 resulted in dose-dependent ADCC and ADCP by human healthy donor peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs), respectively. In a 4-hour Chromium-51 release assay, DuoHexaBody-CD37 induced ADCC in Daudi cells with PMBCs from 11 out of 12 tested donors (average EC50 of 9.87 [±9.98] ng/mL; maximum kill 20.4 [±9.2]%), and in Raji cells with 1 out of 3 donors. Image- and flow cytometry-based assays with MDMs illustrated that in presence of DuoHexaBody-CD37, Daudi cells were efficiently engulfed by effector cells from 3 different donors (average percentage phagocytic macrophages 29.7 ± 7.6%) resulting in target cell depletion (maximum depletion 75.9 [±18.0]%). Potent anti-tumor activity of DuoHexaBody-CD37 in vivo was reported in a screening approach using B-cell lymphoma patient-derived xenograft (PDX) models with single-mouse treatment groups. Two weekly doses of 5 mg/kg DuoHexaBody-CD37 resulted in strong tumor growth inhibition (tumor stasis or tumor regression) in 3/9 models compared to untreated tumors. Follow-up cohort PDX studies with eight mice per group confirmed potent, dose-dependent anti-tumor activity of DuoHexaBody-CD37 at doses as low as 1 mg/kg. In summary, DuoHexaBody-CD37 induces efficient tumor cell kill through CDC, ADCC and ADCP in vitro and shows potent anti-tumor activity in B-cell lymphoma PDX models in vivo. These data further strengthen the rationale for exploring the safety and efficacy of DuoHexaBody-CD37 in B-cell malignancy patients. Citation Format: Laurens P. Kil, Simone C. Oostindie, Kristin Strumane, Hilma J. van der Horst, Berris van Kessel, Marije B. Overdijk, Andreas Lingnau, Marcel Brandhorst, Jeroen van den Brakel, Margaret A. Lindorfer, Ronald P. Taylor, Martine E. Chamuleau, Tuna Mutis, A. Kate Sasser, Janine Schuurman, Paul W. Parren, Frank J. Beurskens, Esther C. Breij. Multifaceted mechanism of action of DuoHexaBody-CD37 involves both complement- and Fc gamma receptor-mediated cytotoxicity in pre-clinical B-cell lymphoma models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4544.

No. P1 Complement-enhancing monoclonal antibodies as a therapeutic strategy against Streptococcus pneumoniae Leire Aguinagalde , Suzanne M. Castenmiller, Carla J.C. Gosselaar-de Haas, Piet C. Aerts, Kok P.M. van Kessel, and Suzan H.M. Rooijakkers Medical Microbiology, University Medical Centre Utrecht, The Netherlands *Presenting author Streptococcus pneumoniae is a leading cause of community-acquired pneumonia, bacteraemia and meningitis. Current vaccines against pneumococcal infections are efficient for preventing invasive disease. However, the increased serotype replacement and antibiotic resistance, reinforces the necessity of developing alternative treatment strategies against S. pneumoniae. Monoclonal antibodies that boost the host immune system are attractive candidates to fight the high rates of morbidity and mortality due to this important human pathogen. Antibody-based immune activation can be induced by antibodies that bind bacterial surface structures and subsequently activate the complement cascade. Complement activation results in deposition of complement factors onto bacterial surfaces that mediate phagocytosis and intracellular killing by phagocytic immune cells. Recent studies have shown that specific point mutations in the Fc domain can enhance the clustering of antibodies into hexameric structures that are required for complement activation. This study examines the efficacy of capsule-specific human monoclonal antibodies against S. pneumoniae serogroup-6. Using flow cytometry, we show that monoclonal antibodies harboring the hexamer-enhancing E430G or E345K mutations potently increase complement activation and phagocytosis of S. pneumoniae serotype 6B. Bacterial killing assays demonstrate the strong potency of engineered antibodies to induce neutrophil-dependent killing of S. pneumoniae. Furthermore, the cross-specificity found with pneumococcal serogroup-19 broadens the efficacy of the hexamer variants to protect against highly invasive S. pneumoniae isolates or against capsular polysaccharides that currently show unsuccessful protection in vaccines. This work represents a first systematic approach to design effective therapeutic antibodies against S. pneumoniae with increased potency to activate the human immune system.

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.

Cytomegalovirus (CMV) infection is one of the most common infectious problems following kidney transplantation. In this study we sought to investigate CMV infection in the setting of renal transplant recipients in Urmia, Iran, using polymerase chain reaction (PCR) detection.Ninety-six randomly selected renal transplant recipient were enrolled in a cross-sectional study. Blood sampling via venipuncture, yielded sera investigated for anti-CMV IgM. Seropositive as well as 14 randomly selected seronegative cases were investigated with PCR assays.Thirty-three patients (34.3%) were seropositive for anti-CMV IgM; 3 (3.1%) borderline, and 60 (62.5%) seronegative. Considering borderline anti-CMV IgM levels as seropositive, 37.5% were seropositive for anti-CMV IgM. Among the 36 seropositive cases, a CMV infection was confirmed in 19 (52.7%) using PCR. Age (P = .40), educational status (P = .77), history of pretransplantation dialysis (P = .52), prior blood transfusion (P = .52), and immunosuppressive regimen were not significantly different among positive versus negative CMV PCR recipients.The seroprevalence of CMV infection was high among renal transplant recipients of Urmia, Iran, as confirmed by PCR study.

CD37 is a tetraspanin plasma membrane protein abundantly expressed on B-cells and represents a promising therapeutic target for the treatment of B-cell malignancies. Although complement-dependent cytotoxicity (CDC) has proven to be a powerful Fc-mediated effector function for killing hematological cancer cells, CD37 antibody-based therapeutics currently in clinical development are poor inducers of CDC. Here we present DuoHexaBody-CD37, a novel humanized IgG1 bispecific antibody targeting two different CD37 epitopes, with an E430G hexamerization-enhancing mutation, for the potential treatment of B-cell malignancies.

CD37 is a tetraspanin molecule expressed on mature B-cells, but absent on normal stem cells and plasma cells. Due to this cellular distribution, which is highly similar to CD20, CD37 has gained attention as a target for B-cell lymphoma, especially for rituximab-resistant and relapsed patients.

To improve the wear resistance of a C45E4 steel substrate, a Mo coating was deposited by plasma transferred arc (PTA) process. The phase and microstructure of the coating were characterized by X-ray diffraction (XRD), optical microscope (OM), scanning electron microscopy equipped with an energy dispersive spectrometer (SEM-EDS) and transmission electron microscopy (TEM), respectively. The hardness and wear resistance were investigated by Vickers hardness and pin-on-disk wear testers. It was found that the metastable Fe63Mo37, Fe solid solution, Fe7Mo6, and a certain amount of amorphous phases co-existed in the as-deposited Mo coating. The peritectic coupled growth in Fe-Mo alloys was observed. Based on the undercooling and eutectic growth theory, the possibility of the Fe63Mo37/α-Fess peritectic coupled growth has been discussed. The microhardness was much higher than that of the C45E4 steel substrate. The results showed that the coating exhibited higher wear resistance ascribed to the high hardness phases and low-friction MoO3 film on the worn surface of the coating under dry sliding wear test against ZrO2 disc at room temperature.

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 (antiCD52) 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.

IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell-expressed antigen.

Phase and bright-field microscopy are suitable for long-term, high time resolution in vitro imaging of proliferating cells. Such image sequences can extend over a period of days or weeks, and have sufficient spatiotemporal resolution to enable automated segmentation, tracking and lineaging of the cells. Fluorescence microscopy offers a more detailed insight into the cellular state, allowing the presence of specific molecular markers to be interrogated. We have developed a combined segmentation, tracking and lineaging approach that allows the phase imaging channel to be enhanced by information from a periodic fluorescence channel. The system is implemented for a two-channel fluorescence system called FUCCI that is used to indicate the timing of cell cycle progression. In combination with a new denoising algorithm, this approach has been applied to time-lapse image sequences showing clonal development for both human lung cancer cells and mouse T-cells. The method resulted in a significant decrease in the error rate of the automated algorithms, as measured in the amount of effort required by a human observer to correct all segmentation, tracking and lineaging results.