Potent Complement-dependent Cytotoxicity Research Articles

GEN3014 (HexaBody ®-CD38) in Anti-CD38 Mab-Naive Patients with Relapsed/Refractory Multiple Myeloma: Preliminary Results from a Dose-Expansion Cohort of a Phase 1/2 Trial

Background: The treatment landscape for relapsed/refractory multiple myeloma (RRMM) has changed dramatically with the approval of CD38-targeted antibodies such as daratumumab. Despite this advancement in therapy, some patients still experience progression following daratumumab regimens. Clinical outcomes may be improved by more potent CD38-targeted treatment options. GEN3014 (HexaBody ®-CD38) is a next-generation human IgG1 anti-CD38 monoclonal antibody (mAb) that contains a hexamerization-enhancing mutation, E430G, that facilitates highly efficient complement-dependent cytotoxicity (CDC). In preclinical studies, GEN3014 showed robust tumor-cell killing through highly potent CDC, with increased efficiency compared with daratumumab, as well as potent antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis (Hiemstra et al, eBioMedicine 2023). Preliminary dose-escalation data from the first-in-human phase 1/2 trial of GEN3014 in RRMM patients showed clinical activity and a tolerable safety profile, and the recommended phase 2 dose was 16 mg/kg (NCT04824794). Based on these findings, expansion was initiated. Herein we describe the preliminary results of an expansion cohort in anti-CD38 mAb-naive RRMM patients. Methods: Adult RRMM patients were eligible if they had ≥3 prior lines of therapy including a proteasome inhibitor (PI) and an immunomodulatory imide drug (IMiD), were double refractory to a PI and an IMiD, or had ≥2 prior lines of therapy with 1 combining a PI and an IMiD. Patients received GEN3014 16 mg/kg intravenously in 28-day cycles (QW, cycles 1-2; Q2W, cycles 3-6; Q4W, cycles ≥7). The primary objective was to assess the preliminary antitumor activity of GEN3014. Secondary objectives included assessment of safety, tolerability, and pharmacokinetics/pharmacodynamics. Results: As of May 5, 2023, 11 anti-CD38 mAb-naive RRMM patients were treated in this expansion cohort (median age, 66 years; range, 56-75). With a median duration of exposure of 2.4 months (range, 0.2-8.3), 4 patients (36.4%) remained on treatment. Primary reasons for discontinuation included disease progression (n=3), AEs (n=3), and death (n=1). The most common treatment-emergent AEs (TEAEs) were neutropenia (36.4%), headache (27.3%), infusion-related reactions (IRRs; 27.3%), and thrombocytopenia (27.3%). IRRs were all grade 2 and did not lead to treatment discontinuation. There were 2 grade 5 TEAEs (respiratory tract infection, n=1; cardiac arrest, n=1). Of the 11 patients treated, 7 received ≥1 cycle of GEN3014, and among them, the best overall responses were 1 complete response, 2 very good partial responses, 2 partial responses, 1 minimal response, and 1 stable disease. GEN3014 treatment was associated with a transient reduction in either complement component C2 or total complement lytic activity in all patients, suggesting CDC activity. T cells transiently decreased after administration of the first dose in all patients, and T-cell expansion (≥50% increase from baseline for ≥2 visits) was observed in 2 of 4 evaluable patients who reached cycle 3 day 1 at the time of analysis. Updated data will be presented. Conclusions: GEN3014 showed a manageable safety profile and clinical activity in anti-CD38 mAb-naive RRMM patients in this expansion cohort. The preliminary pharmacodynamic observations were in line with observations from the dose-escalation part of the study. The study is ongoing and open for enrollment.

Abstract 2959: Novel CD38xCD3 bispecific IgM T cell engager, IGM-2644, potently kills multiple myeloma cells though complement and T cell dependent mechanisms

Abstract Multiple myeloma (MM), a cancer of plasma cells, occurs in ~34,000 new patients every year in the USA. Although therapeutic regimens, including anti-CD38 monospecific IgG antibodies such as daratumumab and isatuximab in combination with chemotherapies, demonstrate clinical efficacy, most patients eventually develop resistance. Several bispecific (CD38xCD3) or trispecific (CD38xCD3xCD28) T cell engager (TCE) antibody therapies are currently in development to improve upon the efficacy of CD38 targeted therapies by leveraging T cell dependent cellular cytotoxicity (TDCC) of MM cells. However, CD38 is also expressed on some normal hematopoietic cells which could potentially lead to undesired pharmacological activity such as depleting CD38+ immune cells, including activated cytotoxic T cells (i.e., fratricide). IGM-2644 is a novel, pentameric IgM antibody engineered to have ten CD38 binding sites, and an anti-CD3ε single chain Fv domain fused to a joining chain to engage CD3 on T cells, and retains the potential for complement-dependent cytotoxicity (CDC). Here we report the functional characterization of IGM-2644 using in vitro, ex vivo and in vivo anti-tumor efficacy studies with a safety evaluation of this novel IgM TCE. In vitro, IGM-2644 demonstrated significantly improved CDC activity in comparison with daratumumab and isatuximab, with >30-fold increased potency on CD38+ MM and lymphoma cell lines. IGM-2644 induced TDCC similar to a bispecific CD38xCD3 IgG on low CD38 expressing cell lines resistant to daratumumab, while demonstrating significantly lower levels of cytokine release than the bispecific IgG. In ex vivo colony forming unit (CFU) assays, IGM-2644 was able to reduce MM CFUs using primary MM patient bone marrow samples containing autologous T cells and myeloma cells, while no effect was observed on erythroid, granulocyte and macrophage CFUs in normal bone marrow samples.​ IGM-2644 dose dependently inhibited tumor growth in humanized xenograft models of CD38+ NCI-H929 (myeloma) and Raji (lymphoma). Importantly, IGM-2644 also demonstrated significantly reduced T cell fratricide compared to bispecific IgGs both ex vivo and in vivo. Significantly reduced activity of IGM-2644 on normal CD38+ innate immune cells was observed in ex vivo studies. CD38 expression has also been reported on human RBCs and platelets. However, minimal IGM-2644 binding was observed and at levels lower than daratumumab. In summary, IGM-2644 is a novel, potent, bispecific IgM TCE that has both CDC and TDCC mechanisms of cytotoxicity with the potential to be active in daratumumab resistant tumors. The balance of potent TDCC and CDC cytotoxic activity, along with an improved preclinical safety profile compared to other CD38xCD3 bispecific IgG TCEs, supports the clinical development of IGM-2644 in MM. Citation Format: Keyu Li, Rui Yun, Min Chai, Poonam Yakkundi, Rodnie Rosete, Gene Li, Liqin Liu, Mandy Li, Daniel Santos, Kevin C. Hart, Dean Ng, Paul R. Hinton, Umesh Muchhal, Thomas Manley, Maya F. Kotturi, Stephen F. Carroll, Angus M. Sinclair, Bruce A. Keyt. Novel CD38xCD3 bispecific IgM T cell engager, IGM-2644, potently kills multiple myeloma cells though complement and T cell dependent mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2959.

Abstract 4531: Clinical translation of biomarkers for ZL-1211, an anti-CLDN18.2 antibody, in patients with advanced solid tumors from a phase I dose-escalation study

Abstract ZL-1211, a humanized monoclonal IgG1 antibody targeting CLDN18.2, induces cancer cell death through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We developed ex vivo ADCC and CDC assays to study the mechanism of actions of ZL-1211. Our preclinical data has demonstrated greater antitumor activities than the clinical leading benchmark via enhanced ADCC by mutation in Fcγ domain and potent CDC activities. We used those ex vivo assays to further profile the clinical specimens from patients collected at baseline (pre-infusion) and the following cycles (post-infusion) in our phase 1 study (NCT05065710). Preliminary results in clinical samples indicated that baseline ADCC and CDC capabilities were correlated with anti-tumor activity of ZL-1211 in patients with CLDN18.2-positive locally advanced or metastatic solid tumors, suggesting probable predictive biomarkers. Results also showed that changes in circulating ZL-1211 mediated ADCC and CDC activities were dose-dependent and in accordance with the pharmacokinetic (PK) profile. In addition, we performed differential gene expression analysis and real-time PCR to compare gene expression in ZL-1211 responder and non-responder PDX models and CCLE cell lines. Our findings reveal that several immune-related genes may serve to predict the responses to ZL-1211 treatment. Other potential predictive biomarkers such as Fcγ receptor CD16A polymorphism and baseline stromal CD8 expression were also evaluated in clinical samples. Taken together, analytical approaches developed and validated from preclinical studies were successfully translated to characterize clinical samples in the current Phase 1 trial. The exploratory analysis indicated the clinical relevance of multiple biomarkers and their potential as predictive biomarkers of ZL-1211. Citation Format: Zhen Luo, Xiao Wang, Xuehuo Zeng, Jiaqing Yi, Tegan Burns, Herman Liu, Xinyu Zhang, Wenyu Liu, Karl Hsu, Hua Gong. Clinical translation of biomarkers for ZL-1211, an anti-CLDN18.2 antibody, in patients with advanced solid tumors from a phase I dose-escalation study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4531.

An anti-HER2 biparatopic antibody that induces unique HER2 clustering and complement-dependent cytotoxicity

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

Preliminary Dose-Escalation Results from a Phase 1/2 Study of GEN3014 (HexaBody®-CD38) in Patients (pts) with Relapsed or Refractory Multiple Myeloma (RRMM)

Background: CD38-targeted antibodies (Abs) are foundational treatments (Tx) for pts with newly diagnosed or RRMM. GEN3014 (HexaBody®-CD38) is a novel human IgG1 anti-CD38 monoclonal Ab (mAb) with an E430G hexamerization-enhancing mutation that facilitates highly efficient complement-dependent cytotoxicity (CDC). GEN3014 showed preclinical tumor cell killing through highly potent CDC (more efficient than daratumumab) and Ab-dependent cellular cytotoxicity and phagocytosis. We present preliminary data from dose escalation in the first-in-human, phase 1/2 trial of GEN3014 in pts with RRMM (NCT04824794). Methods: Adults with RRMM were enrolled if they had ≥3 prior lines of Tx including a proteasome inhibitor (PI) and an immunomodulatory imide drug (IMiD), were double refractory to a PI and IMiD, or had ≥2 prior lines of Tx if 1 combined a PI and IMiD. GEN3014 was tested at 6 dose levels ranging from 0.2 to 24 mg/kg and administered intravenously in 28-d cycles (first dose split equally between D1 and D2 of cycle 1, then full doses QW through cycle 2; Q2W, cycles 3-6; Q4W, cycles ≥7). Primary objectives were to determine the recommended phase 2 dose (RP2D) and maximum tolerated dose. Other endpoints included pharmacokinetic (PK) and pharmacodynamic (PD) characterization. PD biomarker analysis included immunophenotyping and quantification of complement factors and cytokines in peripheral blood. Results: As of July 10, 2022, 24 pts with RRMM were treated (median age, 65 y; range, 45-84). Pts were heavily pretreated with a median of 7 prior lines of Tx (range, 3-13). Most pts (67%) had prior exposure to a daratumumab- or isatuximab-containing regimen, with 8 pts naive to anti-CD38 mAb. GEN3014 was well tolerated; the most common Tx-emergent AEs were infusion-related reactions (IRRs; 75.0%), neutropenia (62.5%), anemia (33.3%), diarrhea (33.3%), pyrexia (25.0%), and thrombocytopenia (25.0%). IRRs were mostly low grade (G; 62.5% G1-2, 12.5% G3, no G4), occurred mainly during the first infusion, and were manageable. There were no Tx-related deaths or dose-limiting toxicities observed at doses up to 16 mg/kg, the RP2D. Disease progression led to discontinuation in 80% of pts. Among 19 response-evaluable pts (5 naive to anti-CD38 mAb, 14 refractory to anti-CD38 mAb), preliminary antitumor activity was seen. In pts naive to anti-CD38 mAb, 2 pts achieved very good partial response (1 each at 4 and 24 mg/kg); 1 achieved minimal response (MR; 16 mg/kg). In pts refractory to anti-CD38 mAb, 2 pts achieved MR (1 each at 8 and 16 mg/kg). Five pts remained on Tx (4 naive and 1 refractory to anti-CD38 mAb). Updated data will be presented. Peak GEN3014 concentrations at the end of infusion increased roughly proportionally with dose. The interval area under the concentration-time curve showed a more than proportional increase with doses ≤8 mg/kg but a proportional or less than proportional increase at doses >8 mg/kg. These results suggest target-mediated drug disposition at lower doses and a high degree of target saturation at >8 mg/kg. Trough concentrations increased with weekly administration, most notably in pts responding to Tx, indicating target cell depletion over time. GEN3014 Tx was associated with a rapid, sustained decrease in peripheral blood natural killer cells at all doses in all pts (mean 92.4% ± 9.2% peak reduction from baseline [BL], n=21). T cells transiently decreased after administration of first doses ≥4 mg/kg, followed by expansion (≥100% increase from BL), particularly in pts naive to anti-CD38 mAb. GEN3014 induced transient reduction in complement component C2 (mean 58% ± 18% peak reduction from BL, n=18) and total complement lytic activity (CH50; mean 48% ± 24% peak reduction from BL for ≥8 mg/kg, n=11) at all evaluable doses, suggesting CDC. Complement parameters rapidly returned to BL, indicating that Tx does not exhaust complement. Plasma cytokine (IL-2, IL-6, IL-8, IL-10, IFNγ, and TNFα) levels generally remained low after Tx. Conclusions: Dose-escalation results show GEN3014 had a tolerable safety profile and clinical activity in pts with RRMM, including pts with prior anti-CD38 mAb. The PK profile is characterized by target-mediated drug disposition at lower doses and signs of saturation at doses >8 mg/kg. Biomarker analyses confirm biological activity in pts with RRMM at all evaluated doses. The expansion part of the trial evaluating the 16 mg/kg RP2D is ongoing.

Bispecific mAb2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab.

Inhibition of complement activation via the overexpression of complement-regulatory proteins (CRPs), most notably CD46, CD55 and CD59, is an efficient mechanism of disguise of cancer cells from a host immune system. This phenomenon extends to counteract the potency of therapeutic antibodies that could lyse target cells by eliciting complement cascade. The manifold functions and ubiquitous expression of CRPs preclude their systemic specific inhibition. We selected CD59-specific Fc fragments with a novel antigen binding site (Fcabs) from yeast display libraries using recombinant antigens expressed in bacterial or mammalian cells. To produce a bispecific antibody, we endowed rituximab, a clinically applied anti-CD20 antibody, used for therapy of various lymphoid malignancies, with an anti-CD59 Fcab. This bispecific antibody was able to induce more potent complement-dependent cytotoxicity for CD20 and CD59 expressing Raji cell line measured with lactate dehydrogenase-release assay, but had no effect on the cells with lower levels of the primary CD20 antigen or CD20-negative cells. Such molecules are promising candidates for future therapeutic development as they elicit a higher specific cytotoxicity at a lower concentration and hence cause a lower exhaustion of complement components.

Synthetic Antibody-Rhamnose Cluster Conjugates Show Potent Complement-Dependent Cell Killing by Recruiting Natural Antibodies.

Monoclonal antibodies (mAbs) are one of the most rapidly growing drug classes used for the treatment of cancer, infectious and autoimmune diseases. Complement-dependent cytotoxicity (CDC) is one of the effector functions for antibodies to deplete target cells. We report here an efficient chemoenzymatic synthesis of structurally well-defined conjugates of a monoclonal antibody with a rhamnose- and an αGal trisaccharide-cluster to recruit natural anti-rhamnose and anti-αGal antibodies, respectively, to enhance the CDC-dependent targeted cell killing. The synthesis was achieved by using a modular antibody Fc-glycan remodeling method that includes site-specific chemoenzymatic Fc-glycan functionalization and subsequent click conjugation of synthetic rhamnose- and αGal trisaccharide-cluster to provide the respective homogeneous antibody conjugates. Cell-based assays indicated that the antibody-rhamnose cluster conjugates could mediate potent CDC activity for targeted cancer cell killing and showed much more potent efficacy than the antibody-αGal trisaccharide cluster conjugates for CDC effects.

Preclinical Anti-Tumor Activity of Hexabody-CD38 in Patient-Derived B Cell Lymphoma and Acute Myeloid Leukemia Xenograft Models

HexaBody®-CD38 (GEN3014) is a next-generation CD38-specific IgG1 molecule with a hexamerization-enhancing mutation that leads to highly efficient induction of complement-dependent cytotoxicity (CDC) upon binding to CD38 positive tumor cells. HexaBody-CD38 is designed to induce strong anti-tumor activity in patients with CD38-expressing hematological malignancies through CDC and other Fc-mediated effector functions. Previously we have shown that HexaBody-CD38 induces highly potent CDC-mediated cell death in multiple myeloma (MM), acute myeloid leukemia (AML), and B-cell non-hodgkin lymphoma (B-NHL) cells, including tumor cells in bone marrow samples from MM patients. In addition, HexaBody-CD38 demonstrated efficient ADCC and ADCP and induction of direct cell kill after Fc-crosslinking. Moreover HexaBody-CD38 induced strong inhibition of CD38 cyclase activity, which may contribute to reduction of immune suppression in the tumor microenvironment. In this study, we show that in a panel of 23 MM, AML and B-NHL cell lines HexaBody-CD38 induced CDC, with EC50 values on average 7-fold lower than those of the CD38-targeting mAb daratumumab, which is part of the standard of care for MM. Cytotoxic activity of HexaBody-CD38 showed a positive correlation with CD38 expression levels. HexaBody-CD38 was especially more potent than daratumumab in cell lines with lower CD38 expression. Sensitivity to HexaBody-CD38-induced CDC was correlated with lower expression levels of complement regulatory proteins, including CD46, CD55 and CD59. To exclude that the enhanced potency of HexaBody-CD38 resulted in lysis of healthy cell subsets known to express low levels of CD38, CDC in healthy donor leukocytes was assessed in vitro. HexaBody-CD38 (20 μg/mL) did not induce lysis of monocytes, B cells, T cells, granulocytes or erythrocytes, and only minor cytotoxicity of NK cells (median 13% lysis, range 9.5-55.1%). NK-cell lysis by daratumumab, assessed in parallel, was in the same range (median 8%, range 4.5-21.2%). Thus, HexaBody-CD38 shows superior potency in CD38-positive tumor cells while sparing healthy donor leukocytes and erythrocytes in vitro. The anti-tumor activity of HexaBody-CD38 in vivo was evaluated in patient-derived AML and B-NHL xenograft (PDX) models in nude mice. In a screen using 9 B-NHL PDX models in a 1-3 mice-per-treatment-group design, administration of two weekly doses of 5 mg/kg HexaBody-CD38 induced a strong anti-tumor response (relative tumor growth ≤10%, i.e. tumor stasis or tumor regression) in 2 models. Two models were classified as non-responder (relative tumor growth >70%) and 5 models could not be classified as either responder or non-responder (intermediates, relative tumor growth between 10 and 70%). A dose-response experiment in one of the responding models (Ly12638, 9 mice per group), confirmed potent anti-tumor activity of HexaBody-CD38, inducing complete tumor regression at a dose of 10 mg/kg (Figure 1A). In a screen using 5 AML PDX models in a 1-3 mouse-per-treatment-group design, HexaBody-CD38 (5 mg/kg) induced a strong anti-tumor response in one model. One model was classified as non-responder and the remaining 3 models could not be classified as either responder or non-responder (intermediates). A dose-response experiment in one of the intermediate models (AML11810), demonstrated statistically significant and dose-dependent anti-tumor activity of HexaBody-CD38 in an in vivo study using a classical 9 mice per treatment group design (Figure 1B). In summary, HexaBody-CD38 induced potent CDC in MM, B-NHL and AML cell lines, with superior potency compared to benchmark antibody daratumumab. HexaBody-CD38 was able to induce CDC in tumor cell lines with lower levels of CD38 expression, while sparing healthy leukocyte subsets. HexaBody-CD38 showed potent anti-tumor activity in vivo both in B-NHL and AML PDX models in nude mice. In these models, the contribution of mouse complement to the anti-tumor activity is limited and therapeutic activity is thought to be driven mainly through FcγR-mediated effector functions. This indicates that besides highly potent CDC, FcɣR-mediated effector mechanisms contribute to the preclinical activity of HexaBody-CD38 in hematological tumor models. These preclinical data support clinical investigation of HexaBody-CD38 in CD38 positive hematologic malignancies, including MM, AML, and B cell lymphomas. Figure Disclosures Hiemstra: Genmab: Current Employment, Current equity holder in publicly-traded company. Janmaat:Genmab: Current Employment, Current equity holder in publicly-traded company. Boross:Genmab: Current Employment, Current equity holder in publicly-traded company. van den Brakel:Genmab: Current Employment, Current equity holder in publicly-traded company. ten Hagen:Genmab: Current Employment, Current equity holder in publicly-traded company. van Dooremalen:Genmab: Current Employment, Current equity holder in publicly-traded company. Bosgra:Genmab: Current Employment, Current equity holder in publicly-traded company. De Goeij:Genmab: Current Employment, Current equity holder in publicly-traded company. Andringa:Genmab: Current Employment, Current equity holder in publicly-traded company. Kil:Genmab: Current Employment, Current equity holder in publicly-traded company. Sasser:Genmab: Current Employment, Current equity holder in publicly-traded company. Ahmadi:Genmab: Current Employment, Current equity holder in publicly-traded company. Satijn:Genmab: Current Employment, Current equity holder in publicly-traded company. Breij:Genmab: Current Employment, Current equity holder in publicly-traded company.

Abstract 4544: Multifaceted mechanism of action of DuoHexaBody-CD37 involves both complement- and Fc gamma receptor-mediated cytotoxicity in pre-clinical B-cell lymphoma models

Abstract 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.

DuoHexaBody-CD37\xae, 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.

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.

Duohexabody-CD37, a Novel Bispecific Antibody with a Hexamerization-Enhancing Mutation Targeting CD37, Demonstrates Superior Complement-Dependent Cytotoxicity in Preclinical B-Cell Malignancy Models

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.The natural process of antibody hexamer formation through intermolecular Fc-Fc interactions between IgG molecules after cell surface antigen binding can be improved by introducing a single point mutation such as E430G in the IgG Fc domain, thereby facilitating more efficient C1q binding and complement activation (Diebolder et al., Science 2014; de Jong et al., PLoS Biol 2016). The hexamerization-enhancing mutation E430G was introduced into two humanized CD37 monoclonal antibodies (mAbs) that bind non-overlapping CD37 epitopes. Different antibody formats and combinations, including the single antibodies, combinations of the mAbs and bispecific mAbs were tested for their capacity to induce CDC and antibody-dependent cellular cytotoxicity (ADCC). The bispecific hexamerization-enhanced antibody variant DuoHexaBody-CD37, showed superior CDC activity compared to the single hexamerization-enhanced mAbs and the combination thereof, both in vitro over a range of different B-cell lines, and ex vivo in tumor cell samples obtained from patients with chronic lymphocytic leukemia (CLL). In a CDC assay using tumor cells obtained from a relapsed/refractory CLL patient who received prior treatment with rituximab, ibrutinib and idelalisib, DuoHexaBody-CD37 induced almost complete lysis (84% lysis at a concentration 100 µg/mL), thereby outperforming the single HexaBody molecules (15% and 23% lysis) and the combination (57%) (Figure 1). In addition to its potent CDC activity, DuoHexaBody-CD37 was also capable of inducing potent ADCC of Daudi cells (EC50 = 12.3 ± 9.5 ng/mL), as assessed using peripheral blood mononuclear cells from 8 healthy human donors in a standard chromium release assay.In assays using whole blood from 6 healthy human donors, DuoHexaBody-CD37 showed efficient B-cell binding and potent and specific depletion of the B-cell population (98% ± 1.3% depletion at 10 µg/mL, EC50 = 0.85 ± 0.284 µg/mL). Furthermore, DuoHexaBody-CD37 induced significant inhibition of tumor growth in vivo in Daudi-luc Burkitt's lymphoma and JVM-3 CLL mouse xenograft models, at doses as low as 0.1 and 1 mg/kg (p<0.05), respectively.In summary, we present a novel therapeutic antibody that, for the first time, combines proprietary DuoBody® and HexaBody® platforms. DuoHexaBody-CD37 induced highly potent CDC and efficient ADCC in preclinical models, suggesting that DuoHexaBody-CD37 may serve as a potential therapeutic mAb for the treatment of human B-cell malignancies. [Display omitted] DisclosuresOostindie:Genmab: Employment, Equity Ownership. Van Der Horst:Genmab: Research Funding. Overdijk:Genmab: Employment, Equity Ownership. Strumane:Genmab: Employment, Equity Ownership. Verploegen:Genmab: Employment, Equity Ownership. Lindorfer:Genmab: Research Funding. Cook:Genmab: Research Funding. Chamuleau:Gilead: Research Funding; BMS: Research Funding; celgene: Research Funding; Genmab: Research Funding. Mutis:Gilead: Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding; Novartis: Research Funding; OnkImmune: Research Funding. Schuurman:Genmab: Employment, Other: Warrants. Sasser:Genmab: Employment, Equity Ownership. Taylor:Genmab: Research Funding. Parren:Genmab: Equity Ownership; Lava Therapeutics: Employment. Beurskens:Genmab: Employment, Equity Ownership. Breij:Genmab: Employment, Equity Ownership.

Targeting CD37 in B-Cell Malignancies Using the Novel Therapeutic Duohexabody-CD37 Results in Efficient Killing of Tumor B-Cells Ex Vivo Via Complement-Dependent Cytotoxicity, Even in Relapsed and/or Refractory Patient Samples

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.The CD37-targeting antibody-based products that are currently being evaluated in the clinic are generally poor inducers of complement-dependent cytotoxicity (CDC) - a rapid and powerful Fc-mediated effector function. DuoHexaBody-CD37 is a bispecific IgG1 antibody targeting two non-overlapping epitopes in the large extracellular loop of CD37, with a hexamerization-enhancing mutation (E430G) in the IgG Fc domain. The E430G mutation increases the intrinsic capacity of IgG1 molecules to form hexameric antibody clusters upon binding to membrane-bound antigens, thereby facilitating binding of the hexavalent complement component C1q and subsequent CDC activity (Diebolder et al., Science 2014; de Jong et al., PLoS Biology 2016). The combination of dual epitope targeting and enhanced hexamerization of DuoHexaBody-CD37 upon binding to the cell surface significantly increases complement activation and CDC efficacy in CD37-positive B-cells.To assess its clinical relevance and potential, we tested the capacity of DuoHexaBody-CD37 to induce ex vivo CDC activity using a broad range of B-cell lymphoma and leukemia patient samples (Diffuse Large B-Cell Lymphoma, Follicular Lymphoma, Mantle Cell Lymphoma, Marginal Zone Lymphoma, Chronic Lymphocytic Leukemia, n=40) derived from both newly diagnosed (ND) (n=23) and relapsed and/or refractory (RR) patients (n=17), most of whom had received prior treatment with rituximab. The CDC activity of DuoHexaBody-CD37 was compared to the CDC activity of the CD20 antibodies rituximab, ofatumumab and obinutuzumab. Our results revealed that, independent of the B-cell malignancy subtype, DuoHexaBody-CD37 induced potent CDC activity with a median lysis of 82% (n=40, both RR and ND) at 10 µg/ml, and significantly outperformed all three tested CD20 antibodies (18%, 24% and 51% median lysis for rituximab, obinutuzumab and ofatumumab, respectively). Furthermore, CDC induced by DuoHexaBody-CD37 was shown to be less sensitive to the expression of complement regulatory proteins (CD46, CD55, CD59), compared to the tested CD20 antibodies, although the few patient samples that were resistant to DuoHexaBody-CD37 in the ex vivo CDC assays expressed significantly higher CD59 levels than the responsive samples.Importantly, while CDC induced by the tested CD20 antibodies was lower in samples derived from RR patients than from ND patients, DuoHexaBody-CD37 was equally effective in inducing CDC in samples derived from ND and RR patients that received prior treatment with rituximab. In a direct comparison to the strong CDC inducer ofatumumab, DuoHexaBody-CD37 induced significantly more potent CDC in samples from 17 RR CLL and B-cell lymphoma patients with a median lysis of 82% for DuoHexaBody-CD37 versus 29% for ofatumumab (p<0.001) (Fig. 1).In conclusion, our results indicate great therapeutic potential for DuoHexaBody-CD37 in B cell malignancies, based on its strong capacity to induce CDC, even in ex vivo samples from patients that had relapsed from or were refractory to rituximab-containing treatments.Figure1 Complement-dependent cytotoxicity induced by DuoHexaBody-CD37, rituximab, ofatumumab or obinutuzumab at 10 µg/mL in frozen CLL or lymphoma B-cells of relapsed and/ or refractory patients that received prior treatment with rituximab (n=17). Statistical analysis was performed using a non-parametric t-test (Wilcoxon). ***p < 0.001 and ****p < 0.0001. Data is shown as mean ± SD. [Display omitted] DisclosuresVan Der Horst:Genmab: Research Funding. Oostindie:Genmab: Employment, Equity Ownership. Overdijk:Genmab: Employment, Equity Ownership. Chamuleau:Gilead: Research Funding; Genmab: Research Funding; celgene: Research Funding; BMS: Research Funding. Breij:Genmab: Employment, Equity Ownership. Mutis:Celgene: Research Funding; Novartis: Research Funding; Gilead: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding.

Targeting the Glyco-Antigen CD75s with the Tetravalent, Fc-Engineered Antibody ‘Ebu-141 Tetra’ Induces Potent Killing of B Cell Lymphoma and Plasma Cell Tumors

While monoclonal antibodies (MoAb) are already well established for the treatment of B cell-derived malignancies and usually show a good safety profile, not all patients benefit and relapses may be a problem. In order to identify novel surface structures suitable for antibody-based therapies and to improve killing mechanisms, ‘EBU-141 Tetra’ was developed.The parental MoAb EBU-141 is of mouse IgMk isotype, was generated in our laboratory and recognizes the glyco-antigen CD75s (previously CDw75), which are α-2,6-sialylated lactosamines on cell surface glycoproteins and glycolipids. CD75s was found on normal mature B cells and subsets of T cells, but is also expressed on certain lymphatic and solid tumors, e.g. mature B cell lymphoma, pancreatic and prostate cancer cells. EBU-141 specifically binds to CD75s on most B cell lymphoma, including Burkitt's lymphoma, FL, DLBCL, MCL, CLL, and interestingly plasma cell tumors. In addition, EBU-141 showed reactivity on a few cases of peripheral T-cell lymphoma, whereas classical Hodgkin lymphomas were consistently negative. Previously a chimeric IgG1k antibody, chEBU-141, was derived from EBU-141. Compared to the parental IgM antibody, chEBU-141 showed strongly reduced binding avidity, but was moderately effective in triggering antibody-dependent cell-mediated cytotoxicity (ADCC) of mature B cell lymphoma and malignant plasma cells via recruitment of NK cells. However, chEBU-141 lacked the potent complement-dependent cytotoxicity (CDC) observed with the parental EBU-141 antibody. The aim of this study was to generate a tetravalent binding, Fc-engineered chEBU-141 IgG1 antibody with enhanced binding avidity for CD75s and potent effector functions for antibody-based therapy of mature B cell lymphomas and multiple myeloma.Using the variable regions of EBU-141, the chimeric IgG1κ antibody with a protein-engineered Fc and tetravalent binding properties, named ‘EBU-141 Tetra’, was generated. This MoAb and relevant controls were produced by transient transfection of 293T cells and purified from cell culture supernatants by affinity chromatography. Direct anti-tumor effects and Fc-mediated modes of action were investigated in cell proliferation assays and chromium release experiments using lymphoma and myeloma cell lines. Peripheral blood mononuclear cells and serum of healthy donors were used as source of human effector cells and complement in the cytotoxicity experiments.The ‘EBU-141 Tetra’ showed improved binding to CD75s on cell surface of mature B cell lymphoma as well as myeloma plasma cells compared to the bivalent binding chEBU-141 IgG1. The higher avidity for CD75s resulted in markedly improved ADCC activity of the ‘EBU-141 Tetra’ against Daudi Burkitt's lymphoma and U266 plasma cells with EC50 values in the picomolar range and higher maximum lysis rates. In addition, the ‘EBU-141 Tetra’ regained CDC activity of the parental EBU-141 and demonstrated efficient killing of Burkitt's lymphoma and myeloma cell lines with human serum as complement source. Thus, recruitment of immune effector cells and activation of the complement system are the main modes of action of the novel, tetravalent, chimeric, Fc-engineered antibody ‘EBU-141 Tetra’ antibody.Our findings further demonstrate that highly potent IgG-like antibodies against glycan-structures can be generated from mouse IgM antibodies and may open a new therapeutic window for therapy of patients with mature B cell lymphomas and multiple myeloma. DisclosuresKlausz:Affimed: Research Funding. Otte:Affimed: Research Funding. Klapper:HTG Molecular Diagnostics, Inc.: Research Funding; Amgen: Honoraria, Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding. Peipp:Affimed: Research Funding. Gramatzki:Affimed: Research Funding.

CD38-Specific Biparatopic Heavy Chain Antibodies Display Potent Complement-Dependent Cytotoxicity Against Multiple Myeloma Cells.

CD38 is overexpressed by multiple myeloma cells and has emerged as a target for therapeutic antibodies. Nanobodies are soluble single domain antibody fragments derived from the VHH variable domain of heavy chain antibodies naturally occurring in camelids. We previously identified distinct llama nanobodies that recognize three non-overlapping epitopes of the extracellular domain of CD38. Here, we fused these VHH domains to the hinge, CH2, and CH3 domains of human IgG1, yielding highly soluble chimeric llama/human heavy chain antibodies (hcAbs). We analyzed the capacity of these hcAbs to mediate complement-dependent cytotoxicity (CDC) to CD38-expressing human multiple myeloma and Burkitt lymphoma cell lines. Combinations of two hcAbs that recognize distinct, non-overlapping epitopes of CD38 mediated potent CDC, in contrast to the hcAb monotherapy with only weak CDC capacity. Similarly, combining daratumumab with a hcAb that recognizes a non-overlapping epitope resulted in dramatically enhanced CDC. Further, introducing the E345R HexaBody mutation into the CH3 domain strongly enhanced the CDC potency of hcAbs to CD38-expressing cells. Exploiting their high solubility, we genetically fused two distinct nanobodies into heteromeric dimers via a flexible peptide linker and then fused these nanobody dimers to the hinge, CH2 and CH3 domains of human IgG1, yielding highly soluble, biparatopic hcAbs. These biparatopic hcAbs elicited CDC toward CD38-expressing myeloma cells more effectively than daratumumab. Our results underscore the advantage of nanobodies vs. pairs of VH and VL domains for constructing bispecific antibodies. Moreover, the CD38-specific biparatopic heavy chain antibodies described here represent potential new powerful therapeutics for treatment of multiple myeloma.

An IgG1-like bispecific antibody targeting CD52 and CD20 for the treatment of B-cell malignancies

Bispecific antibodies (biAb) targeting two different antigens or two distinct epitopes on the same antigen have demonstrated broad therapeutic utility. CD52 and CD20 are co-expressed on the cell surface of malignant B cells in B-cell non-Hodgkin lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL) and increased expression of both antigens is detected on dividing or recently divided cells (“proliferative fraction”) in CLL. The CD52-targeting monoclonal antibody (mAb) alemtuzumab (atz) not only depletes malignant B cells but also healthy CD52+ B and T lymphocytes and monocytes, causing severe immunosuppression. Loss of CD20 can occur in CLL after treatment with rituximab (rtx) and other CD20-targeting mAbs. To broaden the benefit of atz and rtx, we engineered an IgG1-like biAb, atz × rtx scFv-Fc. The Fc fragment of the biAb facilitates purification by Protein A affinity chromatography and supports a longer circulatory half-life. While atz × rtx scFv-Fc retained both antigen binding specificities, it showed superior binding to CD52+CD20+ B cells compared to CD52+CD20− T cells. Moreover, atz × rtx scFv-Fc mediated potent complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in vitro and exhibited B-cell depleting but T-cell sparing activities in vivo in a CLL patient-derived xenograft model. B-cell depletion was more pronounced for cells of the proliferative fraction.

Potent Therapeutic Activity Against Peritoneal Dissemination and Malignant Ascites by the Novel Anti-Folate Receptor Alpha Antibody KHK2805

Many ovarian cancer patients often show peritoneal metastasis with malignant ascites. However, unmet medical needs remain regarding controlling these symptoms after tumors become resistant to chemotherapies. We developed KHK2805, a novel anti-folate receptor α (FOLR1) humanized antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The primary aim of the present study was to evaluate whether the anti-tumor activity of KHK2805 was sufficient for therapeutic application against peritoneal dissemination and malignant ascites of platinum-resistant ovarian cancer in preclinical models. Here, both the ADCC and CDC of KHK2805 were evaluated in ovarian cancer cell lines and patient-derived samples. The anti-tumor activity of KHK2805 was evaluated in a SCID mouse model of platinum-resistant peritoneal dissemination. As results, KHK2805 showed specific binding to FOLR1 with high affinity at a novel epitope. KHK2805 exerted potent ADCC and CDC against ovarian cancer cell lines. Furthermore, primary platinum-resistant malignant ascites cells were susceptible to autologous ADCC with KHK2805. Patient-derived sera and malignant ascites induced CDC of KHK2805. KHK2805 significantly reduced the total tumor burden and amount of ascites in SCID mice with peritoneal dissemination and significantly prolonged their survival. In addition, the parental rat antibody strongly stained serous and clear cell-type ovarian tumors by immunohistochemistry. Overall, KHK2805 showed cytotoxicity against both ovarian cancer cell lines and patient-derived cells. These translational study findings suggest that KHK2805 may be promising as a novel therapeutic agent for platinum-resistant ovarian cancer with peritoneal dissemination and malignant ascites.

Abstract 578: Potent complement-dependent cytotoxicity of tumor cells by target bound hexamerization of EGFR antibodies

Abstract Introduction: Activation of complement (C) constitutes an important effector mechanism of monoclonal antibodies in cancer therapy. Unmodified IgG1 antibodies against the epidermal growth factor receptor (EGFR), a well-validated tumor target, lack the capacity to induce complement-dependent cytotoxicity (CDC) as single agents. Recently, significantly improved CDC was demonstrated after antigen-bound IgG molecules formed hexameric structures through intermolecular Fc-Fc contacts. Single point mutations (E345K or E430G) that enhance hexamerization of IgG molecules conditional on antigen binding have been identified to represent a platform for enhanced C activation (Diebolder et al Science 343:1260, 2014, de Jong et al, PLOS Biology, in press). Alternative strategies to enhance CDC have focused on enhancing C1q binding via affinity-maturation of the C1q binding site (S239D/H268F/S324T/I332E; DFTE) or by generating IgG1/IgG3 isotype chimeras (113F). The aim of the present study was to investigate whether EGFR antibodies optimized for each of these approaches differed in terms of CDC activity. Methods: EGFR antibodies comprising engineered Fc domains that carry mutations to enhance hexamerization (E345K or E430G), or increase C1q affinity (DFTE), or that exist of IgG1/IgG3 chimeras (113F) were constructed. CDC activity was assessed by 51Cr release assays in the presence of human serum. Antibody mediated C1q binding or C4b deposition on tumor cells was analyzed by flow cytometry. Apparent C1q binding affinity was determined by C1q specific ELISA. Results: Here, we demonstrate that affinity maturation of the C1q binding site (DFTE &amp; 113F) increased C1q binding in the absence of antigen, whereas mutations that enhance Fc-Fc interactions (E345K &amp; E430G), and so enhance hexamerization after target binding, did not. However, these latter mutations strongly increased C1q avidity and CDC for EGFR antibodies bound to cells. Notably, the latter strategy translated in superior CDC activity, which was particularly evident for tumor cells with low EGFR expression levels. Furthermore, hexamerization enhanced CDC was particularly sensitive to peptides inhibiting Fc-Fc interactions, supporting their different mode of action compared to other CDC-enhancing strategies. Interestingly, also the EGFR epitope recognized by different antibodies impacted on the CDC activity. Conclusion: Enhancement of hexamer formation by EGFR antibodies conditional on antigen binding is a promising novel approach to improve their anti-tumor activity. Strategies that enhanced complement binding independent of antigen engagement by the antibody appeared less effective. Citation Format: Annalina Tammen, Frank J. Beurskens, Stefanie Derer, Ralf Schwanbeck, Paul W.H.I. Parren, Janine Schuurman, Thomas Valerius. Potent complement-dependent cytotoxicity of tumor cells by target bound hexamerization of EGFR antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 578.

A bispecific antibody directly induces lymphoma cell death by simultaneously targeting CD20 and HLA-DR.

Both CD20 and HLA-DR antigens are highly expressed on a variety of B-cell lymphomas and are therapeutic targets in antibody-based lymphoma therapy. The aim of this study was to evaluate the anti-tumor effect of a bispecific antibody CD20-HLA-DR DVD-Ig on B-cell lymphoma. The gene for bispecific antibody CD20-HLA-DR DVD-Ig was constructed and expressed in FreeStyle™293-F cells, followed by purification. Their functions were characterized for binding to CD20 and HLA-DR and for cytotoxicity against B-cell lymphoma. The bispecific antibody CD20-HLA-DR DVD-Ig was engineered using the DNA fragments for the anti-CD20 rituximab and anti-HLA-DR hL243γ1. The CD20-HLA-DR DVD-Ig bound simultaneously to both CD20 and HLA-DR, induced potent complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against B-cell lymphoma, and elicited homotypic adhesion and actin reorganization. Treatment of a mixture of human whole blood and Raji cells with CD20-HLA-DR DVD-Ig effectively depleted Raji cells and had a little toxicity against normal B cells. Our data indicated that targeting both CD20 and HLA-DR was an effective way against NHL, suggesting that CD20-HLA-DR DVD-Ig may be a promising therapeutic agent for B-cell lymphoma.

Abstract 4324: Maytansinoid conjugates of a novel type III anti-CD20 antibody with potent antitumor activity.

Abstract Previously identified anti-CD20 antibodies have been classified as either Type I antibodies with lipid raft and complement-dependent cytotoxicity (CDC) activity or Type II antibodies with strong pro-apoptotic activity, but not both. We describe here a novel anti-CD20 antibody with a unique combination of functional properties that defines a new Type III anti-CD20 antibody classification. In addition, when conjugated to the potent, microtubule-acting maytansinoids DM1 or DM4, it demonstrated enhanced cell-killing activity. Novel anti-CD20 antibodies were generated by immunizing mice with CD20-positive cells. One of these murine monoclonal anti-CD20 antibodies demonstrated very strong pro-apoptotic activity against Ramos and Raji lymphoma cells in the absence of cross-linking agents, similar to the activity of Type II antibodies. The humanized version of the antibody, termed D1302A, had a stronger pro-apoptotic effect against Ramos cells than other CD20 antibodies tested including rituximab, tositumumab (B1) and GA101-NG (non-glycoengineered GA101/afutuzumab). Strikingly, D1302A also induced CD20-redistribution to lipid rafts and showed CDC activity similar to the Type I antibody rituximab, while the Type II antibodies tositumumab and GA101-NG lacked these activities. D1302A, rituximab, and ofatumumab demonstrated comparable antibody-dependent cell mediated cytotoxicity (ADCC) activity on Ramos cells with human natural killer effector cells. In vivo, D1302A showed strong efficacy in an SU-DHL-4 xenograft model (at 1 or 10 mg/kg x3) and was more active than rituximab. D1302A was conjugated to DM1 via the non-cleavable SMCC thioether linker and to DM4 via the sulfo-SPDB disulfide linker. The resulting antibody-drug conjugates retained all the functional activities of the unconjugated antibody including binding affinity, pro-apoptotic, CDC and ADCC activities, and showed enhanced and specific in vitro cytotoxicity against Granta-519 and Farage lymphoma cells. In vivo, single-dose treatment with maytansinoid conjugates of D1302A elicited enhanced activity in Daudi and SU-DHL-4 xenograft models as compared to the antibody alone and showed comparable efficacy as standard of care treatment regimens such as rituximab or rituximab + chemotherapy (R-CHOP). D1302A is a novel Type III anti-CD20 antibody that has the functional properties of both Type I (potent CDC activity) and Type II (strong pro-apoptotic activity) antibodies, together with ADCC activity. Conjugation of D1302A with maytansinoids further increases anticancer activity in vitro and in vivo, combining the potent antibody-produced activities with a fourth cytotoxic mechanism distinct from that of other CD20 targeted agents. Therefore, D1302A-maytansinoid conjugates provide a unique combination of anti-tumor activities and are promising therapeutic candidates for the treatment of CD20+ lymphomas and leukemias. Citation Format: Jutta Deckert, Peter U. Park, Yong Yi, Sharon Chicklas, Min Li, Erin K. Maloney, Rui Wu, Leanne Lanieri, Jennifer A. Coccia, Joe Ponte, Lingyun Rui, Daniel J. Tavares, Jan Pinkas, Thomas Chittenden. Maytansinoid conjugates of a novel type III anti-CD20 antibody with potent antitumor activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4324. doi:10.1158/1538-7445.AM2013-4324