Mechanistic and pharmacodynamic studies of DuoBody-CD3x5T4 in preclinical tumor models.

CD3 bispecific antibodies (bsAbs) show great promise as anticancer therapeutics. Here, we show in-depth mechanistic studies of a CD3 bsAb in solid cancer, using DuoBody-CD3x5T4. Cross-linking T cells with tumor cells expressing the oncofetal antigen 5T4 was required to induce cytotoxicity. Naive and memory CD4+ and CD8+ T cells were equally effective at mediating cytotoxicity, and DuoBody-CD3x5T4 induced partial differentiation of naive T-cell subsets into memory-like cells. Tumor cell kill was associated with T-cell activation, proliferation, and production of cytokines, granzyme B, and perforin. Genetic knockout of FAS or IFNGR1 in 5T4+ tumor cells abrogated tumor cell kill. In the presence of 5T4+ tumor cells, bystander kill of 5T4- but not of 5T4-IFNGR1- tumor cells was observed. In humanized xenograft models, DuoBody-CD3x5T4 antitumor activity was associated with intratumoral and peripheral blood T-cell activation. Lastly, in dissociated patient-derived tumor samples, DuoBody-CD3x5T4 activated tumor-infiltrating lymphocytes and induced tumor-cell cytotoxicity, even when most tumor-infiltrating lymphocytes expressed PD-1. These data provide an in-depth view on the mechanism of action of a CD3 bsAb in preclinical models of solid cancer.

DuoBody-CD3xCD20 induces potent T-cell-mediated killing of malignant B cells in preclinical models and provides opportunities for subcutaneous dosing.

BACKGROUND: DuoBody®-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. METHODS: T-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. FINDINGS: DuoBody-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. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies. FUNDING: Genmab.

Type I CD20 Antibodies Recruit the B Cell Receptor for Complement-Dependent Lysis of Malignant B Cells.

Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20+ B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab')2 fragments, as well as C1q-binding-deficient IgG mutants, retained an ability to induce CDC, albeit with lower efficiency than for whole or unmodified IgG. Experiments using human serum depleted of specific complement components demonstrated that the observed lytic activity, which we termed "accessory CDC," remained to be dependent on C1 and the classical pathway. We hypothesized that CD20 Ab-induced clustering of the IgM or IgG BCR was involved in accessory CDC. Indeed, accessory CDC was consistently observed in B cell lines expressing an IgM BCR and in some cell lines expressing an IgG BCR, but it was absent in BCR- B cell lines. A direct relationship between BCR expression and accessory CDC was established by transfecting the BCR into CD20+ cells: OFA-F(ab')2 fragments were able to induce CDC in the CD20+BCR+ cell population, but not in the CD20+BCR- population. Importantly, OFA-F(ab')2 fragments were able to induce CDC ex vivo in malignant B cells isolated from patients with mantle cell lymphoma and Waldenström macroglobulinemia. In summary, accessory CDC represents a novel effector mechanism that is dependent on type I CD20 Ab-induced BCR clustering. Accessory CDC may contribute to the excellent capacity of type I CD20 Abs to induce CDC, and thereby to the antitumor activity of such Abs in the clinic.

A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface.

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.

Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation.

Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.

Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy.

The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3-3 µM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 µM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs.

A quantitative flow cytometric assay for determining binding characteristics of chimeric, humanized and human antibodies in whole blood: proof of principle with rituximab and ofatumumab.

Clinical successes of antibody-based drugs has led to extensive (pre-) clinical development of human(ized) monoclonal antibodies in a great number of diseases. The high specificity of targeted therapy with antibodies makes it ideally suited for personalized medicine approaches in which treatments needs are tailored to individual patients. One aspect of patient stratification pertains to the accurate determination of target occupancy and target expression to determine individual pharmacodynamic properties as well as the therapeutic window. The availability of reliable tools to measure target occupancy and expression on diseased and normal cells is therefore essential. Here, we evaluate a novel human antibody detection assay (Human-IgG Calibrator assay), which allows the flow cytometric quantification of therapeutic antibodies bound to the surface of cells circulating in whole blood. This assay not only permits the determination of the number of specific antibody bound per cell (sABC), but, when combined with quantification of exogenously added mouse antibody, also provides information on binding kinetics and antigen modulation. Our data indicate that the calibrator assay has all properties required for a pharmacodynamic tool to quantify target occupancy of chimeric, humanized and human therapeutic antibodies during therapy, as well as to collect valuable information on both antibody and antigen kinetics.

Loss of CD20 and bound CD20 antibody from opsonized B cells occurs more rapidly because of trogocytosis mediated by Fc receptor-expressing effector cells than direct internalization by the B cells.

We previously reported that 1 h after infusion of CD20 mAb rituximab in patients with chronic lymphocytic leukemia (CLL), >80% of CD20 was removed from circulating B cells, and we replicated this finding, based on in vitro models. This reaction occurs via an endocytic process called shaving/trogocytosis, mediated by FcγR on acceptor cells including monocytes/macrophages, which remove and internalize rituximab-CD20 immune complexes from B cells. Beers et al. reported that CD20 mAb-induced antigenic modulation occurs as a result of internalization of B cell-bound mAb-CD20 complexes by the B cells themselves, with internalization of ∼40% observed after 2 h at 37°C. These findings raise fundamental questions regarding the relative importance of shaving versus internalization in promoting CD20 loss and have substantial implications for the design of mAb-based cancer therapies. Therefore, we performed direct comparisons, based on flow cytometry, to determine the relative rates and extent of shaving versus internalization. B cells, from cell lines, from patients with CLL, and from normal donors, were opsonized with CD20 mAbs rituximab or ofatumumab and incubated for varying times and then reacted with acceptor THP-1 monocytes to promote shaving. We find that shaving induces considerably greater loss of CD20 and bound mAb from opsonized B cells in much shorter time periods (75-90% in <45 min) than is observed for internalization. Both shaving/trogocytosis and internalization could contribute to CD20 loss when CLL patients receive rituximab therapy, but shaving should occur more rapidly and is most likely to be the key mechanism of CD20 loss.

Statins impair antitumor effects of rituximab by inducing conformational changes of CD20.

BACKGROUND: Rituximab is used in the treatment of CD20+ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas. METHODS AND FINDINGS: Complement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a 51Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-beta-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells. CONCLUSIONS: Statins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.