Enhanced Antibody-dependent Cellular Cytotoxicity Activity Research Articles

Abstract LB130: Identification of fully human CCR8 antibodies that demonstrate excellent ADCC function, potent blocking activity, and robust anti-tumor immune responses

Abstract Immune checkpoint inhibitors have demonstrated significant benefits in treating various types of tumors. However, a considerable number of patients do not respond to this therapy. One proposed mechanism for resistance to immune checkpoint inhibitors is the infiltration of large numbers of regulatory T cells (Tregs) into tumors, which hinders the development of effective antitumor immunity and is often linked to poor prognosis. CCR8 is a G-protein-coupled receptor (GPCR) which is predominantly expressed on Tregs infiltrating tumors. The development of CCR8-specific human antibodies to deplete CCR8+ Tregs is a promising therapeutic strategy for cancer treatment. Here, we report the development and characterization of fully human monoclonal antibodies targeting CCR8 that exhibit potent antibody-dependent cellular cytotoxicity (ADCC) activity. These antibodies were generated in CCR8 knock-out RenMab® mice, which were immunized with native CCR8 antigen prepared via a proprietary cell-free membrane protein synthesis technology. Epitope mapping of the antibodies revealed recognition of a unique epitope on the N-terminus extracellular domain of CCR8. Binding assays indicated cross-reactivity of the antibodies across multiple species; importantly, however, these antibodies did not recognize related chemokine receptors, such as CCR2, CCR4, CCR5, or CX3CR1. In vitro, CCR8 antibodies effectively inhibited calcium flux induced by CCL1 binding to the CCR8 receptor. Furthermore, introduction of DE (S239D, I332E) mutations in the Fc region enhanced ADCC activity of these antibodies. To compare the efficacy of these antibodies with anti-CCR8 benchmarks in vivo, we generated benchmark analogs in-house with the same DE mutations and established syngeneic MC38 tumor models in humanized B-hCCR8 mice. In this assay, our CCR8 antibodies demonstrated potent anti-tumor activity. Notably, one mutant clone with optimized developability demonstrated robust inhibition of tumor growth when compared to both benchmark analogs and its original clone. Taken together, our fully human CCR8 antibodies demonstrate enhanced ADCC function, strong blocking activity, and superior in vivo anti-tumor activity, which may support further development into a novel cancer immunotherapy targeting Tregs. Citation Format: Huichao Liang, Yanan Guo, Meimei Yin, Aki Kawagishi, Takaho Terada, Yoshihiko Kihara, Toru Kanke, Chaoshe Guo, W. Frank An, Yi Yang. Identification of fully human CCR8 antibodies that demonstrate excellent ADCC function, potent blocking activity, and robust anti-tumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB130.

Enhancement of effector functions of anti-CD20 monoclonal antibody by increased afucosylation in CHO cell line through cell culture medium optimization

BackgroundRecombinant therapeutic anti-CD20 monoclonal antibody (mAb) is used for the treatment of non-Hodgkin’s lymphoma, a common B cell lymphoma constituting 80% of all lymphomas. Anti-CD20 mAb contains an Fc-linked biantennary glycan. Although, anti-CD20 monoclonal antibodies are being increasingly used for immunotherapy, their efficacy is limited in a section of patients with drug resistance to immunotherapy. There is a need to improve the efficacy by increasing the effector functions, such as the antibody-dependent cellular cytotoxicity (ADCC) activity of anti-CD20 monoclonal antibodies. ResultsWe developed a simple and cost-effective approach to enhance ADCC effector activity in an in-house developed clone of anti-CD20 monoclonal antibody by increasing afucosylation in a new clone of Chinese Hamster Ovary (CHO) cells using 8X uridine, manganese, and galactose (UMG) to modulate the osmolality of the medium. The purified anti-CD20 monoclonal antibody from 8X UMG-containing medium showed a 2-fold increase in afucose content and 203% ADCC activity in comparison to control antibody. ConclusionsOur study reports enhanced ADCC activity by modulating afucosylation using osmolality by altering simple feed additives in the culture medium.

Resistance to obinutuzumab-induced antibody-dependent cellular cytotoxicity caused by abnormal Fas signaling is overcome by combination therapies

BackgroundObinutuzumab, a Type II anti-CD20 antibody, is used to treat follicular lymphoma. A major mode of action of obinutuzumab is antibody-dependent cellular cytotoxicity (ADCC). Knowledge of the mechanisms of resistance to obinutuzumab is important for the development of next-line strategies to follow obinutuzumab-containing therapy, including obinutuzumab retreatment. Unfortunately, the mechanisms by which tumor cells acquire resistance to ADCC are still poorly understood. To address this, we examined the mechanisms of resistance to obinutuzumab-induced ADCC and the combination efficacy of obinutuzumab and clinically available agents in the established resistant cells.Methods and resultsWe established cells resistant to obinutuzumab-induced ADCC using the non-Hodgkin lymphoma cell line RL and examined their mechanisms of resistance and the combination efficacy of obinutuzumab and clinically available agents. Comprehensive analysis by RNA sequencing of resistance mechanisms revealed that abnormal Fas signaling decreased sensitivity to ADCC in resistant clones. Combination treatment with prednisolone, a component of CHOP and CVP, was found to enhance ADCC sensitivity of RL cells and resistant clones and to significantly suppress tumor growth in xenograft models. Treatment with prednisolone upregulated expression of CD20 and an apoptosis-inducing protein BIM, which might augment perforin/granzyme B-mediated cell death. Furthermore, pretreatment of the effector cells with bendamustine enhanced ADCC activity, and treatment with obinutuzumab plus bendamustine showed significant antitumor efficacy in xenograft models. It was speculated that bendamustine upregulates ADCC activity by potentiating granules-mediated cell killing.ConclusionsOur study revealed a novel mechanism underlying obinutuzumab-induced ADCC resistance and indicated that ADCC resistance could be overcome by combining obinutuzumab with prednisolone or bendamustine. This study provides a scientific rationale for obinutuzumab-retreatment in combination with clinically available chemotherapeutic agents for obinutuzumab resistant follicular lymphoma.

CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity

AbstractMultiple myeloma (MM) is a plasma cell neoplasm that commonly expresses CD38. Daratumumab (DARA), a human monoclonal antibody targeting CD38, has significantly improved the outcome of patients with relapsed or refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include downregulation of CD38 expression and overexpression of complement inhibitory proteins on MM target cells as well as DARA-induced depletion of CD38high natural killer (NK) cells resulting in crippled antibody-dependent cellular cytotoxicity (ADCC). Here, we tested whether maintaining NK cell function during DARA therapy could maximize DARA-mediated ADCC against MM cells and deepen the response. We used the CRISPR/Cas9 system to delete CD38 (CD38KO) in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, showed superior persistence in immune-deficient mice pretreated with DARA, and enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells. In addition, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Finally, we evaluated the impact of exposure to all-trans retinoic acid (ATRA) on wild-type NK and CD38KO NK cell function and highlighted potential benefits and drawbacks of combining ATRA with DARA in patients with MM. Taken together, these findings provide proof of concept that adoptive immunotherapy using ex vivo expanded CD38KO NK cells has the potential to boost DARA activity in MM.

Human Placental CD34+-Derived Natural Killer Cells with High Affinity and Cleavage Resistant CD16 (CYNK-101) for ADCC Mediated Cancer Immunotherapy

CYNK-101, an allogeneic off-the-shelf human placental CD34+-derived natural killer (NK) cell product, is genetically modified to express a variant of CD16 (FcγRIII) via lentiviral vector transduction. When targeting tumors with monoclonal antibodies, NK cells are key effectors of antibody dependent cellular cytotoxicity (ADCC) following recognition of antibody Fc by CD16. CYNK-101 expresses the CD16 variant (CD16VP) which has a high-affinity due to a Valine at amino acid position 158 along with proteolytic cleavage resistance imparted by Proline at amino acid position 1971,2,3,4. We hypothesize that expressing CD16VP enhances anti-tumor activity of CYNK-101 in combination with monoclonal antibody therapy. Reported here are the in vitro and ex vivo results of evaluating CYNK-101 cytotoxicity against human epidermal growth factor receptor 2 (HER2)+ solid tumors in combination with Trastuzumab, an anti-HER2 monoclonal antibody. Human placental CD34+ cells were transduced with lentivirus expressing CD16VP, and cultured in the presence of cytokines, including TPO, SCF, Flt3L, IL-7, IL-15, and IL-2, to generate CYNK-101 cells. Lentiviral transduction with CD16VP achieved high expression efficiency in multiple placental CD34+ donors. These cells (n=7) expanded at 12041 ± 6394-fold and differentiated into CYNK-101 being >90% CD56+CD3-. CYNK-101 expressed 74.1 ± 5.6% (n=7) of CD16 at the end of cultivation. Proteolytic cleavage resistance conferred by CD16VP in CYNK-101 was evaluated by activating NK cells with PMA/ionomycin (PMAi) to induce CD16 shedding. While 4h PMAi treatment resulted in >89% CD16 cleavage on non-transduced NK cells, <11% cleavage was observed on CYNK-101 cells, demonstrating CD16 shedding resistance. The in vitro anti-tumor activity of CYNK-101 against HER2+ solid tumor cell lines was assessed in combination with Trastuzumab. At effector to target (E:T) ratio of 0.6:1, CYNK-101 (n=7 donors) showed enhanced lysis of NCI-N87 gastric cancer cells in the presence of Trastuzumab compared to that of IgG control, 41.2 ± 11.1% vs. 5.3 ± 4.7% at 4h, 91.3 ± 15.0% vs. 7.5 ± 9.3% at 24h, respectively (p<0.005). Increased production of GM-CSF, IFN-γ, and TNF-α was shown in CYNK-101 plus Trastuzumab against NCI-N87 at 24h compared to that of IgG control. Overall, the enhanced ADCC activity of CYNK-101 in combination with Trastuzumab has been demonstrated against both HER2+ gastric cancer cell lines, NCI-N87 and OE-19; and HER2+ breast cancer cell lines, AU565, BT-474, HCC-1954, SKBR-3, and ZR-75-30. To further evaluate ex vivo ADCC activity of CYNK-101, CYNK-101 cells were intravenously (IV) injected at 2x107 into busulfan-pretreated NOD-scid IL2Rγnull immunodeficient (NSG) mice at Day 0. Recombinant human IL-15 was intraperitoneally injected at Days 0, 2, 4, 6, 8, 10 and 12. At Day 13, CYNK-101 cells were isolated from mouse livers (ex vivo-CYNK-101) and assessed for phenotype and ADCC activity. Ex vivo-CYNK-101 exhibited enhanced cytotoxicity against NCI-N87 in combination with Trastuzumab compared to that of IgG control (30.9% vs. 6.6% at 4h, 90.7% vs. 9.1% at 24h) at the E:T ratio of 0.5:1, as well as increased cytokine productions of GM-CSF, IFN-γ, and TNF-α at 24h. Compared to pre-infusion CYNK-101, ex vivo-CYNK-101 showed not only a higher ADCC activity against NCI-N87, but also a more matured NK cell phenotype with increased expression of CD16, KIR, NKG2D, CD94, and CD11a. Besides, these ex vivo-CYNK-101 cells demonstrated CD16 shedding resistance post PMAi stimulation. In summary, our results demonstrated enhanced in vitro and ex vivo ADCC activities of CYNK-101 in combination with Trastuzumab against HER2+ solid tumors. CYNK-101 exhibited high resistance to CD16 shedding in vitro and ex vivo. Further maturation of CYNK-101 cells post IV infusion was accompanied by ADCC activity enhancement. Evaluation of in vivo ADCC activity of CYNK-101 in combination with Trastuzumab will include both subcutaneous and orthotopic gastric cancer models. Disclosures Kang: Celularity Inc.: Current Employment. Raitman:Celularity Inc.: Current Employment. Rotondo:Celularity Inc.: Current Employment. Gleason:Celularity Inc.: Current Employment, Current equity holder in private company. He:Celularity Inc.: Current Employment, Current equity holder in private company. Somanchi:Celularity Inc.: Current Employment, Current equity holder in private company. Rousseva:Celularity Inc.: Current Employment. Guo:Celularity Inc.: Current Employment. Rana:Celularity Inc.: Current Employment. DiFiglia:Celularity Inc.: Current Employment. Ye:Celularity Inc.: Current Employment, Patents & Royalties. Mahlakõiv:Celularity Inc.: Current Employment. Van Der Touw:Celularity Inc.: Current Employment. Hariri:Celularity Inc.: Current Employment, Current equity holder in private company. Zhang:Celularity Inc.: Current Employment, Current equity holder in private company.

A novel monovalent FGFR1 antagonist: Preclinical safety profiles in rodents and non-human primates

Blocking Fibroblast Growth Factor Receptor 1 (FGFR1) is an attractive therapeutic option for treatment of cancer subtypes with amplification and over-expression of FGFR1. Selective targeting of FGFR1 can be achieved using an antibody-based approach, as small molecule inhibitors may not discriminate between FGFR1, 2, 3 and 4 due to their highly homologous kinase domain. However, development of classical bivalent FGFR1 directed antibodies has failed due to non-tolerated body weight decreases in preclinical species. M6123 is a novel IgG-like monovalent FGFR1 specific binder with enhanced Antibody-Dependent Cellular Cytotoxicity (ADCC) effector function and inhibits tumor growth significantly in FGFR1-dependent human xenograft models without reduced body weight in tumor-bearing mice. Toxicology studies reported here characterized the safety profile of M6123 in mouse, rat, and monkey. There were significant differences among animal species under similar M6123 exposure levels. Rats showed metastatic mineralization with an imbalance in serum phosphate at low doses, while mineralization was not found in mice or monkeys, even though hyperphosphatemia was detected in mice. Subtle differences in calcium/phosphate homoeostasis feedback loops may trigger the susceptibility to mineralization among animal species; nevertheless, the exact mechanism remains unknown. Monkeys showed marked, but reversible, decreases in peripheral blood NK cells and neutrophils. The latter was associated with considerably increased neutrophilic infiltrates in the liver sinusoids and red pulp of the spleen. These effects in monkeys are likely related to the enhanced ADCC activity of M6123. Overall, M6123 showed a superior safety profile in animals compared to bivalent FGFR1 antagonists or pan-FGFR inhibitors.

CD38 Knockout Primary NK Cells to Prevent “Fratricide” and Boost Daratumumab Activity

Multiple myeloma (MM) is a plasma cell neoplasm typically characterized by high and uniform CD38 expression. Daratumumab (DARA), a humanized monoclonal antibody targeting CD38 has dramatically improved the outcome of patients with refractory MM, but relapse is inevitable in most cases. DARA eliminates MM cells through several mechanisms including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis. Mechanisms of resistance to DARA include downregulated CD38 expression on target MM cells, impaired complement directed cytotoxicity via upregulation of complement inhibitory proteins (CD55, CD59), and impaired ADCC via DARA-induced NK cell "fratricide". As it relates to "fratricide", restoring ADCC by DARA-resistant NK cells has been proposed to bolster the therapeutic effect of DARA. Towards this goal, iPSC-derived CD38 knockout (CD38KO) NK cells have shown proof-of-concept for this approach but present major challenges towards clinical translation. Here, we used Cas9-RNP to generate CD38KO primary NK cells and characterized their resistance to DARA-induced "fratricide" and restoration of ADCC, but also assessed the impact of CD38 deletion on NK cell signaling and metabolism. First, primary NK cells were enriched from peripheral blood of healthy donors and expanded on feeder cells expressing mbIL-21 and 4-1BBL (PLoS One. 2012;7(1):e30264). Cas9/gRNA complexes targeting CD38 were electroporated into NK cells (J Vis Exp. 2018 Jun 14;(136)). Flow cytometric analysis revealed CD38KO efficiency was 81.9±6.9% (mean±SD, n=5). CD38KO cells were further purified to ≥ 95% purity using magnetic cell separation system. Whole genome sequencing identified no off-target mutations. CD38KO-NK cells alone showed no conjugation in the presence of DARA (Figure 1A) and no DARA-induced fratricide (Figure 1B), in stark contrast with wild type NK (WT-NK) cells. Consistent with these results, treatment of NSG mice with DARA had no impact on persistence of CD38KO-NK cells, whereas WT-NK cells were completely eliminated (NK cell frequency in peripheral blood 7 days after inoculation; WT-NK NO DARA 12.11±5.15%, KO-NK NO Dara 17.05±0.98%, WT-NK + Dara 0.21±0.08%, KO-NK + Dara 16.85±3.61%, mean±SD, n=4, p<0.01, ANOVA) (Figure 1C). To examine the functional status of CD38KO-NK cells, we used 6 MM cell lines with various CD38 expression levels (LP-1, RPMI8226, H929, MM.1s, OPM-2, and KMS-11) as well as three CD38+CD138+ primary MM samples isolated from bone marrow of newly diagnosed or relapsed patients. CD38KO-NK cells exhibited enhanced ADCC activity compared to WT-NK cells across all cell lines and primary samples tested in 4h and 24h cytotoxicity assay (Figure 1D). This was even observed in MM cells that express low levels of CD38, suggesting their potential efficacy in patients with relapsed disease after DARA (ADCC at E/T 5:1; MM.1s 7.8±1.8%(WT) vs 21.5±0.4%(KO), KMS-11 -3.5±0.6%(WT) vs 11.1±0.4%(KO), mean±SD). The CD38KO-NK cells preserved similar expansion potency as WT-NK counter parts. Next, to reveal any effect of manipulating CD38 expression on NK cells cytotoxicity and metabolism, we performed RNA sequencing analysis, which showed higher expression of several cytotoxic genes such as IFNG and GzmB in CD38-KO NK cells, and higher expression of glucose transporter genes and GAPDH, suggesting a favorable metabolic profile of these cells. Consistent with these data, we observed that CD38KO-NK cells have elevated capacity to take up glucose as measured by 2-NBDG glucose uptake assay (p=0.02, student t test). Taken together, these findings provide the proof-of-concept that CD38KO-NK cells generated from primary NK cells augment therapeutic effect of DARA and could be promising for adoptive immune cellular therapy targeting MM. Y.N. and M.N.K. contributed equally to this study. D.A.L. and G.G. contributed equally to this study. Figure 1 Disclosures Ali: Celgene: Research Funding; Poseida: Research Funding. Lee:Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Abstract 2370: Synergistic effect of carboplatin on farletuzumab (FAR) tumor cell killing via downregulation of the humoral immunosuppressive CA125 protein and enhanced antibody dependent cellular cytotoxicity (ADCC)

Abstract Farletuzumab (FAR) is a humanized monoclonal antibody (mAb) that targets folate receptor alpha (FRA), a cell surface protein highly overexpressed on a large number of cancers. Clinical evidence suggests that FAR enhances the anti-tumor effects of carboplatin and taxane in ovarian cancer patients whose tumors express low CA125 levels. In light of the recent evidence that CA125 has a direct effect on suppressing the humoral immune mechanisms [antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC)] of FAR (1, 2) and the findings that chemotherapeutic agents can enhance the ADCC activity of certain mAbs, we tested the potential synergistic effects that carboplatin may have on FAR immune effector function. Here we show the immunosuppressive effects that CA125 has on FAR-mediated tumor cell killing in vivo and demonstrate that carboplatin is able to suppress cell surface CA125 expression leading to enhanced FAR ADCC on FRA-expressing ovarian tumor cells. CA125 has been previously found to suppress FAR-mediated tumor cell killing in vitro. To determine its effect in vivo, we tested single agent FAR against isogenic tumor cells in mouse xenografts. OVCAR-3 is a FRA positive ovarian tumor cell line that expresses high levels of CA125 on its cell surface. A CA125 shRNA knockdown line (OV-KD) has been previously generated and shown to be more susceptible to FAR ADCC than its parental counterpart. Xenograft studies using these lines found that FAR had significant single agent tumor killing in the OV-KD xenografts in contrast to OVCAR-3 xenografts. We next tested FAR in vivo killing when used in combination with carboplatin. These data showed that carboplatin had a synergistic effect when combined with FAR treatment as compared to single agent treatment alone. To determine if this mechanism involved enhanced ADCC, we conducted similar studies in vitro whereby OVCAR-3 cells were treated with a sublethal concentration of carboplatin for 72 hrs, washed and then tested for FAR ADCC killing. These data found that FAR had enhanced ADCC activity on carboplatin treated cells as compared to untreated cells. Finally, to explore the potential mechanism by which this effect may occur, we examined the steady-state levels of both the mannose-6-phosphate receptor (M6PR), reported to be upregulated by chemotherapeutic drugs leading to enhanced ADCC via higher granzyme B uptake and the CA125 immunosuppressive factor. Our data found that both M6PR and CA125 expression were significantly suppressed following carboplatin exposure. These data support a mechanism by which carboplatin may enhance the humoral immune response for FAR and potentially other anti-tumor mAbs via downregulation of the immunosuppressive CA125 protein. 1. OncoTarget 8:66747-57, 20172. 2. EJI 201847707, 2018 Citation Format: J. Bradford Kline, Keiji Furuuchi, James Fulmer, Jennifer McDonough, Luigi Grasso, Nicholas C. Nicolaides. Synergistic effect of carboplatin on farletuzumab (FAR) tumor cell killing via downregulation of the humoral immunosuppressive CA125 protein and enhanced antibody dependent cellular cytotoxicity (ADCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2370.

Improving Antibody-Based Cancer Therapeutics Through Glycan Engineering.

Antibody-based therapeutics has emerged as a major tool in cancer treatment. Guided by the superb specificity of the antibody variable domain, it allows the precise targeting of tumour markers. Recently, eliciting cellular effector functions, mediated by the Fc domain, has gained traction as a means by which to generate more potent antibody therapeutics. Extensive mutagenesis studies of the Fc protein backbone has enabled the generation of Fc variants that more optimally engage the Fcγ receptors known to mediate cellular effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and cellular phagocytosis. In addition to the protein backbone, the homodimeric Fc domain contains two opposing N-linked glycans, which represent a further point of potential immunomodulation, independent of the Fc protein backbone. For example, a lack of core fucose usually attached to the IgG Fc glycan leads to enhanced ADCC activity, whereas a high level of terminal sialylation is associated with reduced inflammation. Significant growth in knowledge of Fc glycosylation over the last decade, combined with advancement in genetic engineering, has empowered glyco-engineering to fine-tune antibody therapeutics. This has culminated in the approval of two glyco-engineered antibodies for cancer therapy: the anti-CCR4 mogamulizumab approved in 2012 and the anti-CD20 obinutuzumab in 2013. We discuss here the technological platforms for antibody glyco-engineering and review the current clinical landscape of glyco-engineered antibodies.

In vivo anti-tumor efficacy of afucosylated anti-CS1 monoclonal antibody produced in glycoengineered Pichia pastoris

Monoclonal antibody (mAb) therapy has been successfully used for the treatment of B-cell lymphomas and is currently extended for the treatment of multiple myeloma (MM). New developments in MM therapeutics have achieved significant survival gains in patients but the disease still remains incurable. Elotuzumab (HuLuc63), an anti-CS1 monoclonal IgG1 antibody, is believed to induce anti-tumor activity and MM cytotoxicity through antibody dependent cellular cytotoxicity (ADCC) and inhibition of MM cell adhesion to bone marrow stromal cells (BMSCs). Modulations of the Fc glycan composition at the N297 site by selective mutations or afucosylation have been explored as strategies to develop bio-better therapeutics with enhanced ADCC activity. Afucosylated therapeutic antibodies with enhanced ADCC activity have been reported to possess greater efficacy in tumor growth inhibition at lower doses when compared to fucosylated therapeutic antibodies. The N-linked glycosylation pathway in Pichia pastoris has been engineered to produce human-like N-linked glycosylation with uniform afucosylated complex type glycans. The purpose of this study was to compare afucosylated anti-CS1 mAb expressed in glycoengineered Pichia pastoris with fucosylated anti-CS1 mAb expressed in mammalian HEK293 cells through in vitro ADCC and in vivo tumor inhibition models. Our results indicate that Fc glycosylation is critical for in vivo efficacy and afucosylated anti-CS1 mAb expressed in glycoengineered Pichia pastoris shows a better in vivo efficacy in tumor regression when compared to fucosylated anti-CS1 mAb expressed in HEK293 cells. Glycoengineered Pichia pastoris could provide an alternative platform for generating homogeneous afucosylated recombinant antibodies where Fc mediated immune effector function is important for efficacy.

Abstract DDT01-03: Discovery and preclinical pharmacology of JNJ-61186372: A novel bispecific antibody targeting EGFR and cMET

Abstract NSCLC with activating mutations in the EGFR gene are associated with high response rates to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, but invariably acquired resistance emerges over time. A primary cause of resistance is the emergence of secondary mutations in EGFR which neutralize the effectiveness of TKIs. In addition, the cMet pathway is often activated, either through MET gene amplification, overexpression of cMet protein, or an increase in the ligand HGF, to provide a compensatory survival pathway conferring resistance to EGFR TKIs. We have designed a bispecific EGFR-cMet antibody (JNJ-61186372) with a unique set of mechanisms of action resulting in anti-tumor activity in the EGFR mutant setting, with or without cMet pathway activation. We have demonstrated three mechanisms of action that contribute to the activity of JNJ-61186372: 1) inhibition of ligand-induced phosphorylation of both EGFR and cMet, 2) receptor degradation in vivo, and 3) enhanced ADCC activity. JNJ-61186372 inhibited EGF-induced phosphorylation of EGFR in cell lines with either wild-type (WT) EGFR or activating mutations in EGFR. In the same cell lines, JNJ-61186372 inhibited HGF-induced phosphorylation of cMet. JNJ-61186372 also blocked pERK and pAkt with similar IC50 values in EGFR-WT and EGFR mutant cell lines, indicating that downstream signaling pathways were inhibited. Total protein levels of both EGFR and cMet were decreased in xenograft tumor models following treatment with JNJ-61186372 compared to tumors from mice treated with PBS control suggesting that one mechanism by which JNJ-61186372 suppresses EGFR and cMet activity in vivo is through degradation of both receptors. The third mechanism of action is directing immune cells to kill tumor cells. JNJ-61186372 is produced with low levels of fucosylation, which translates to an enhanced antibody-dependent cellular cytotoxicity (ADCC). These three mechanisms of action of JNJ-61186372 provide a distinct preclinical profile for targeting both EGFR and cMET in a single bispecific antibody. JNJ-61186372 demonstrated efficacy in multiple in vivo tumor models with EGFR mutations, including both cell line and patient-derived xenografts. Importantly, JNJ-61186372 effectively inhibited tumor growth in models with mutant EGFR and cMet activation, whereas single agent EGFR inhibitors were less effective. The preclinical data support the clinical development of JNJ-61186372 in patients with lung cancer and other malignancies associated with aberrant EGFR and cMET signaling. Citation Format: Sheri L. Moores, Mark Chiu, Barbara Bushey, Kristen Chevalier, Peter Haytko, Joost Neijssen, Paul Parren, Janine Schuurman, Mark Anderson, Ricardo Attar, Robert Kramer, Matthew V. Lorenzi. Discovery and preclinical pharmacology of JNJ-61186372: A novel bispecific antibody targeting EGFR and cMET. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr DDT01-03. doi:10.1158/1538-7445.AM2014-DDT01-03

BI-HEX®-GlymaxX® cells enable efficient production of next generation biomolecules with enhanced ADCC activity

Background Despite the succes story of therapeutic monoclonal antibodies (mAbs), a medical need remains to improve their efficacy. One possibility to achieve this is to modulate important effector functions such as the antibody dependent cellular cytotoxicity (ADCC). The advantage of highly active biotherapeutic molecules is apart from the enhanced efficacy the reduction of side effects due to lower administered doses. Furthermore, these therapeutic antibodies may enable treatment of current nonresponders, e.g. patients with low antigen bearing tumors. Enhancement of the effector functions of antibodies can be achieved either by directly mutating the antibody’s amino acid sequence or by modifying its glycosylation pattern, e.g. by using a novel host cell line able to attach a desired glycostructure to the product. The latter approach has the advantage of not impacting the antibody structure itself, thereby avoiding negative effects on the PK/PD of the molecule. During the last decade it has been shown that antibodies with a reduced level of glycan fucosylation are much more potent in mediating ADCC, a mode of action particularly relevant for cancer therapeutics. Therefore, defucosylated antibodies are of major interest for biotherapeutics developers. To produce such antibodies, Boehringer Ingelheim has inlicensed the GlymaxX system from ProBioGen, Germany. This technology utilises the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) which, when stably integrated into host cell lines, inhibits fucose de-novo biosynthesis. The enzyme deflects the fucosylation pathway by turning an intermediate (GDP-4-Keto-6-Deoxymannose) into GDP-Rhamnose, a sugar that cannot be metabolised by CHO cells. As a consequence, recombinant antibodies generated by such host cells exhibit reduced glycan fucosylation and 20100 fold higher ADCC activity. Here, we show the establishment of a new host cell line, termed BI-HEXGlymaxX which is capable of producing highly active therapeutic antibodies. We furthermore present data on the cell line properties concerning cell culture performance (e.g. titer, growth, transfection efficiency), process robustness and product quality reproducibility.

Ublituximab (TGTX-1101), A Novel Anti-CD20 Monoclonal Antibody (mAb), Demonstrates Activity in Rituximab-Sensitive and Rituximab–resistant B Non-Hodgkin Lymphoma (B-NHL) Pre-Clinical In vitro and in Vivo models.

Abstract 2756The addition of rituximab to chemotherapy regimens utilized in treating B-cell non-Hodgkin lymphoma (B-NHL) has resulted in significant improvement in treatment response and clinical outcomes. On the other hand, the use of rituximab is changing the biology and response to second-line therapy in patients with relapsed/refractory disease. Novel anti-CD20 mAbs continue to be developed that may offer additional treatment options for relapsed/refractory rituximab-pre-treated patients. Ublituximab (TGTX-1101) is a novel, chimeric mAb targeting a unique epitope on the CD20 antigen. Ublituximab has been glycoengineered to enhance affinity for all variants of FcγRIIIa receptors. To further characterize the activity of ublituximab, we evaluated its anti-tumor activity in a panel of rituximab-sensitive (RSCL), rituximab–resistant (RRCL) cell lines, primary tumor cells isolated from patients with B-NHL by negative selection using magnetic beads, and in lymphoma SCID mice xenograft models. RSCL (Raji, RL, U2932, Granta, HBL-2, Jeko-1, Mino, Rec1 and Z-138), RRCL (Raji-2R, Raji 4RH, RL-4RH, and U2932-4RH); and cytarabine-resistant (AraCR) mantle cell lymphoma cell (MCL) lines (Granta-AraCR, HBL-2-AraCR, Jeko-AraCR, Mino-AraCR and Rec1-AraCR) were labeled with 51Cr. Subsequently, cells were exposed to ublituximab, rituximab or isotope control and human serum (25%) for complement dependent cytotoxicity (CDC) assays or to effector cells isolated from healthy volunteers (effector:target ratio 40:1) for antibody dependent cellular cytotoxicity (ADCC) assays, respectively. Antibody-induced direct anti-proliferative effects and induction of apoptosis were determined by alamar blue reduction assay and Annexin-V and propidium iodide staining, respectively. Primary tumor cells (n=11) were exposed to ublituximab, rituximab or isotype control +/− pooled human serum for 48 hr. Changes in ATP content were determined using the CellTiterGlo assay. For in vivo studies, 6–8 week old SCID mice were inoculated via tail vein injection with 1×106 Raji cells on day 0 and assigned to rituximab (10mg/kg/dose), ublituximab (10mg/kg/dose) or control group. MAb was given via tail vein injection on days +3, +7, +10 and +14. Differences in survival were analyzed by Kaplan-Meier curves and p values calculated using log rank test. Ublituximab induced significantly higher ADCC when compared to rituximab in 13 out of 17 cell lines tested (including all RRCL and cytarabine resistant MCL cells): (Raji 44.4% vs. 19.8%; Raji 4RH 17.5% vs. 8.3%; Raji 2R 28.2% vs. 12%; RL 40.9% vs. 17.8%; RL-4RH 33.5% vs. 17.2%; U2932 46.9% vs. 28.8%; U2932-4RH 40.2% vs. 22.1%; HBL-2AraCR 30.7% vs. 16.6%; Jeko 34.8% vs. 18.4; Jeko-AraCR 23.8% vs. 9.6; Mino 47.4% vs. 11.6%; Mino-AraCR 32.5% vs. 15.5; Rec1 30.9% vs. 0%; p-values <0.05). There was no significant difference between ublituximab and rituximab in terms of CMC (including studies performed in primary tumor cells) or direct signaling (i.e. apoptosis or cell proliferation). While ublituximab therapy prolonged the survival of lymphoma-bearing SCID mice when compared to controls, the anti-tumor activity in vivo was similar to rituximab. Our results suggest that ublituximab exhibits higher ADCC than rituximab in vitro, including in RRCL and elicits similar CDC and direct anti-tumor effects. Despite this enhanced ADCC activity, initial in vivo experiments did not result in improved survival compared to rituximab, however additional in vivo experiments investigating the activity of ublituximab in RRCL and MCL mouse models, testing alternative dose/schedule regimens and/or in combination with other anti-lymphoma agents are planned. Updated research results will be presented at the annual meeting. A Phase I/II trial of ublituximab in patients with relapsed/refractory NHL is currently ongoing. Disclosures:No relevant conflicts of interest to declare.

A Novel Fc-Engineered Antibody to CD33 with Enhanced ADCC Activity for Treatment of AML

AbstractAbstract 1363 Introduction:Acute myeloid leukemia (AML), the most frequent form of adult acute leukemia, is a heterogeneous disease with respect to presentation and clinical outcome. Although there has been progress made in treatment for some subgroups of AML patients, there is still a high medical need to develop new anti-leukemic therapies. CD33 is a myeloid lineage-specific antigen which is expressed on the cell surface of normal myeloid cells and AML blasts. CD33 has been investigated as a target for antibody-based therapies for over a decade, leading to transient approval of Mylotarg®, an antibody-calicheamicin drug conjugate. Mylotarg®has been shown to substantially improve outcome of patients with AML in combination with chemotherapy, however is associated with additional toxicity. Here we describe a novel, unconjugated Fc-engineered antibody to CD33, mAb 33.1, which mediates potent antibody dependent cellular cytotoxicity (ADCC) against AML derived cell lines and malignant AML blasts. Methods:MAb 33.1 is a fully human, Fc-engineered, IgG1-type of antibody which has increased affinity to human Fc-gamma receptor IIIa and binds CD33 with low nanomolar affinity as determined by FACS scatchard analysis. ADCC of mAb 33.1 was determined on a panel of myeloid cell lines and primary AML cells. ADCC assays were performed using peripheral blood mononuclear cells (PBMC) from healthy donors as effector cells and cytotoxicity was determined by LDH release or FACS quantification of 7-AAD-positive cells. Internalization kinetics was assessed by FACS analysis using fluorochrome labelled antibody. Epitope mapping of mAb 33.1 was performed by direct competition experiments and hydrogen/deuterium exchange mass spectrometry (HXMS). Results:Antibody mAb 33.1 is able to mediate potent ADCC on a panel of AML derived cell lines and primary AML blasts in vitro. Direct comparison of mAb 33.1 to the humanized CD33 mAb lintuzumab (HuM195) demonstrates superior cytolytic activity on AML cell lines and primary AML cells. ADCC activity of mAb 33.1 is also evident on AML cell lines and primary AML blasts that do not respond to lintuzumab treatment in vitro. In contrast to lintuzumab, mAb 33.1 shows significantly slower internalization kinetics on HL60 cells, leading to prolonged exposure of antigen/antibody complexes on the cell surface. In direct competition experiments mAb 33.1 is competing with lintuzumab for antigen binding. HXMS experiments revealed that mAb 33.1 recognizes a novel epitope on CD33 different to that of lintuzumab. Conclusion:MAb 33.1 represents a novel, Fc-engineered antibody specific for CD33 which shows decelerated internalization compared to lintuzumab, and which recognizes a novel epitope on CD33 different to that of lintuzumab. MAb 33.1 displays strongly enhanced ADCC against leukemia cell lines and primary AML cells, resulting in significantly higher cytolysis than lintuzumab. MAb 33.1 is considered as a promising candidate for treatment of patients with CD33-positive myeloid malignancies and phase I clinical trials are currently under preparation. Disclosures:Heider:Boehringer Ingelheim: Employment. Konopitzky:Boehringer Ingelheim: Employment. Ostermann:Boehringer Ingelheim: Employment. Lamche:Boehringer Ingelheim: Employment. Kuepcue:Boehringer Ingelheim: Employment. Koessl:Boehringer Ingelheim: Employment. Adam:Boehringer Ingelheim: Employment. Borges:Boehringer Ingelheim: Employment.

Mechanism of resistance to trastuzumab and molecular sensitization via ADCC activation by exogenous expression of HER2-extracellular domain in human cancer cells.

Trastuzumab, a humanized antibody targeting HER2, exhibits remarkable therapeutic efficacy against HER2-positive breast and gastric cancers; however, acquired resistance presents a formidable obstacle to long-term tumor responses in the majority of patients. Here, we show the mechanism of resistance to trastuzumab in HER2-positive human cancer cells and explore the molecular sensitization by exogenous expression of HER2-extracellular domain (ECD) in HER2-negative or trastuzumab-resistant human cancer cells. We found that long-term exposure to trastuzumab induced resistance in HER2-positive cancer cells; HER2 expression was downregulated, and antibody-dependent cellular cytotoxicity (ADCC) activity was impaired. We next examined the hypothesis that trastuzumab-resistant cells could be re-sensitized by the transfer of non-functional HER2-ECD. Exogenous HER2-ECD expression induced by the stable transfection of a plasmid vector or infection with a replication-deficient adenovirus vector had no apparent effect on the signaling pathway, but strongly enhanced ADCC activity in low HER2-expressing or trastuzumab-resistant human cancer cells. Our data indicate that restoration of HER2-ECD expression sensitizes HER2-negative or HER2-downregulated human cancer cells to trastuzumab-mediated ADCC, an outcome that has important implications for the treatment of human cancers.

Combination Therapy of a Defucosylated Anti-HM1.24 Monoclonal Antibody Plus Lenalidomide Significantly Enhances Myeloma Cell Cytotoxicity Via Antibody-Dependent Cellular Cytotoxicity

Abstract 1833Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of neoplastic plasma cells in the bone marrow. During the last decade, treatment of MM has been improved by incorporating bortezomib, thalidomide, and lenalidomide (LEN) into conventional cytotoxic and transplantation regimens in newly diagnosed and relapsed/refractory MM patients. However, MM still remains incurable despite the implementation of these new treatment options, so more efficacious therapies are needed to further improve the prognosis of MM. Monoclonal antibody (mAb)-based immunotherapy has recently become an alternative strategy for treatment of cancers. Our previous studies have shown that HM1.24 (CD317) is selectively expressed on terminally differentiated normal and neoplastic plasma cells and, moreover, expressed on the side population of MM cells that represents MM cancer stem cells. We have previously generated a humanized mAb (AHM) specific to HM1.24 for the treatment of MM. AHM carries an Fc region derived from human IgG1-k and exhibits the ability to induce antibody-dependent cellular cytotoxicity (ADCC) against human MM cells in the presence of human effector cells. To improve the efficacy of AHM, we have developed a defucosylated mAb (YB-AHM) with a higher affinity to Fc gamma RIII. LEN is a structural analog of thalidomide with more potent immunomodulatory activities. Several studies have shown that LEN activates NK cell function and enhances NK cell-mediated lysis of both MM cell lines and patient MM cells in vitro. Here, we evaluated the efficacy of combination therapy of YB-AHM and LEN. First, we investigated whether LEN stimulates the expression of HM1.24 on MM cells. LEN alone did not affect HM1.24 expression, but in the presence of peripheral blood mononuclear cells (PBMCs) LEN augmented the expression of HM1.24 in MM cell lines and primary MM cells. In PBMCs, expression levels of CD56 increased after stimulation with LEN. These results suggest that LEN might augment the ADCC activity by enhancing HM1.24 antigen and NK activity. Next, we evaluated ADCC activity of YB-AHM against RPMI 8226 cells by using flow cytometric PKH-26 assay. When we used PBMCs from healthy donors (n=5) as effectors, ADCC activity of YB-AHM was increased in an E:T ratio-dependent manner. Importantly, YB-AHM induced significantly higher ADCC activity compared with AHM (24±6% vs 11±7%, p<0.05; mAb, 100 ng/mL; E:T ratio, 10). Treatment of PBMCs with LEN (3 micro M for 2 days) slightly enhanced ADCC activity of AHM (12±5%) and YB-AHM (30±6%). In PBMCs from MM patients (n=11), YB-AHM induced ADCC activity (36±15%) that was further enhanced by treatment with LEN (45±15%). To evaluate the efficacy of this combination therapy in a more physiological manner, we assessed the efficacy of YB-AHM using total bone marrow mononuclear cells (BMMCs) from MM patients that contained both MM cells and effector cells. BMMCs were stimulated with LEN (3 micro M) for 2 days and further incubated with YB-AHM for 24 hours. Cytotoxicity was evaluated by the number of CD38-positive MM cells in total BMMCs using flow cytometry. YB-AHM plus LEN significantly reduced the number of MM cells (10.3%) compared to YB-AHM alone (21.6%) in patient No.1. Finally, RPMI 8226 cells were co-cultured with YB-AHM and LEN-stimulated PBMCs from MM patients, and MM colony formation was examined using methylcellulose assay. Colony formation of RPMI 8226 was significantly suppressed by YB-AHM and LEN-stimulated PBMCs compared to control (14±8 vs 49±10 colonies, p<0.01), suggesting that this combination therapy can target MM cancer stem cells. Thus, these results indicate that combining defucosylated HM1.24 mAb with immunomodulatory drugs provides a novel therapeutic strategy in patients with MM. Disclosures:No relevant conflicts of interest to declare.

A Recombinant Antibody to EphA3 with Pro-Apoptotic and Enhanced ADCC Activity Against Various Hematologic Malignancies Shows Selective Inhibition of Colony Formation From Long-Term Culture-Initiating Cells (LTC-ICs) In Primary Leukemia Samples

AbstractAbstract 2897EphA3 is a receptor tyrosine kinase expressed at high level on the surface of some solid tumors and certain leukemias. KB004 is a high-affinity non-fucosylated, recombinant antibody developed by KaloBios derived from a mouse monoclonal anti-EphA3 antibody using Antibody Humaneering™ Technology. The antibody binds EphA3 from human and cynomolgus macaque with equivalent affinity (610 pM for human and 570 pM for cynomolgus as determined by surface plasmon resonance (spr) analysis) making the latter a relevant toxicology species. KB004 has a direct pro-apoptotic effect on EphA3-positive cells and a induces potent antibody-dependent cellular cytotoxicity (ADCC) activity mediated by CD16-expressing effector cells. The non-fucosylated antibody has enhanced affinity for both human and cynomolgus CD16a. The affinity of KB004 for cynomolgus CD16a (7.2 nM, determined by spr analysis) was comparable to the affinity for the high-affinity variant of CD16a from humans (6.6 nM), indicating that KB004 has similar potential for ADCC in cynomolgus macaques.We first demonstrated cell-surface expression of EphA3 by flow cytometry in approximately 50% of primary human leukemia cells. Here we extend the analysis to multiple myeloma (MM) in which 3/5 patient samples show EphA3 expression. EphA3 expression was confirmed in a panel of chronic myeloid leukemia (CML) samples [n=14] and in acute myeloid leukemia (AML) [n=40] in which EphA3+ cells were identified in each French American British (FAB) classification sub-type except M0, for which only two samples were studied. CD123-positive leukemia stem cells (CD34+ CD38− CD123+) also displayed surface expression of EphA3. KB004 induced apoptosis in EphA3+ primary cells from each of the diseases analyzed, including imatinib-sensitive and resistant CML, with no activity in EphA3-negative specimens, including normal CD34+ bone marrow cells. KB004 inhibited myeloid leukemia colony formation (CFU-L) from primary AML samples in methylcellulose colony assays without affecting normal hematopoietic colony recovery and also showed dose-dependent inhibition of long-term culture-initiating cells (LTC-IC) from AML stem cells. EphA3 expression was not detected on primary human CD34+ bone marrow cells from healthy individuals.These data support the development of an anti-EphA3 antibody for the treatment of hematologic malignancies. The selective activity against leukemic stem cells supports a novel strategy for therapeutic targeting of leukemia-initiating cells. Disclosures:No relevant conflicts of interest to declare.

Enhancement of Antibody-dependent Cellular Cytotoxicity by Tandem Fc Multimerization

Monoclonal antibodies are being used as therapeutics for a number of cancers, such as leukemia, breast and colon cancers, and a lot of monoclonal antibodies specific for tumor-related antigens have been on clinical trials. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms by which antibodies exert anti-tumor effects. ADCC occurs through interaction between the Fc domains of IgG antibodies bound to target cells and Fcgamma receptors on the surface of effector cells. In our study, a chimeric antibody, designated M-Ab, was constructed with the V regions from mouse anti-CD20 mAb 1F5 and the C regions from human IgG1 and kappa chain. Two or three Fc domains were tandemly repeated downstream of the C-terminus of the M-Ab to give D0-Ab (Fc dimer Ab without a linker), T0-Ab (Fc trimer Ab without a linker), and T3-Ab (Fc trimer Ab with a (GGGGS)(3) linker in front of the second and third hinge regions). Here, we show that Fc tandem repeat antibodies bind to all the low-affinity Fcgamma receptors with very potent avidities and have greatly enhanced ADCC activity. T3-Ab is about 100 times more potent than the parental 1F5 chimeric antibody in terms of both Fcgamma receptor binding and exerted ADCC activity at a 50-100 times less concentration as compared with the parental antibody. Thus, Fc tandem repeat antibodies are anticipated to be candidates for anti-tumor therapeutics and useful tools to elucidate the biological roles of Fcgamma receptors.

A Recombinant Human Antibody to EphA3 with Pro-Apoptotic and Enhanced ADCC Activity Shows Selective Cytotoxicity against Myeloid Leukemia Cells and CD123-Positive Leukemic Stem Cells.

Abstract 1728Poster Board I-754Members of the Eph family of receptor tyrosine kinases play important roles in embryonic development but have restricted tissue distribution and activity in adult tissues. The EphA3 receptor is an oncofetal antigen expressed at high levels on the surface of several solid tumor types and certain leukemias. KaloBios is in pre-clinical development with KB004, a high-affinity recombinant human antibody, derived from a monoclonal anti-EphA3 antibody by Antibody HumaneeringTM Technology. KB004 binds EphA3 and stimulates apoptosis in primary cells from myeloid leukemia patients. The cell-surface expression of EphA3 was analyzed by flow cytometry on primary cells from chronic myeloid leukemia (CML) [n=10], acute myeloid leukemia (AML) of various sub-types [n=29], and myelodysplastic syndromes (MDS) [n=7]. EphA3 surface expression was detected in at least 50% of patient samples from each of the diseases analyzed. CD123-positive leukemia stem cells (CD34+ CD38- CD123+) also displayed surface expression of EphA3 whereas normal bone marrow CD34+ stem/ progenitor cells lacked detectable EphA3. KB004 was shown to stimulate apoptosis in EphA3+ primary leukemia cells, including CD123+ leukemic stem cells with no activity in EphA3-negative specimens, including normal CD34+ bone marrow cells. Cross-linking of the antibody was not required for induction of apoptosis. KB004 also inhibited myeloid leukemia colony formation (CFU-L) from primary AML samples in methylcellulose colony assays without affecting normal hematopoietic colony formation. In addition to its direct pro-apoptotic effect, KB004 induced potent antibody-dependent cellular cytotoxicity (ADCC) activity against EphA3-positive cells, mediated by CD16-expressing effector cells. CD16-dependent ADCC activity was further enhanced by expression of the antibody in CHO cells that lack a1,6 fucosyl transferase, generating afucosylated antibody with higher affinity for CD16a. The data support the development of an anti-EphA3 antibody for the treatment of hematologic malignancies in addition to solid tumors. In particular, the selective activity against leukemic stem cells supports a novel strategy for therapeutic targeting of leukemia-initiating cells that merits clinical investigation. DisclosuresNo relevant conflicts of interest to declare.

A Glyco-Optimized Anti-CD20 Antibody from the Aquatic Plant Lemna: Comparison of Pharmacokinetics, B-Cell Depletion and Complement Activation with Rituxan® in Cynomolgus Monkeys

Monoclonal antibodies represent one of the largest classes of drugs in preclinical and clinical development. For many antibodies, the structure and extent of the N-glycans on the Fc region of the heavy chain plays a significant role in their therapeutic function. A glyco-optimized version of the anti-CD20 antibody (rituximab) was expressed in the clonal aquatic plant Lemna. The optimized glycosylation was accomplished by co-expressing an interfering RNA (RNAi) construct targeting the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes (Cox et al., 2006). The resulting glyco-optimized rituximab contained a single major G0 N-glycan (lacking terminal galactose) without any detectable xylose or fucose. Previous in vitro cell-based studies have shown that the glyco-optimized rituximab had similar CD20 binding affinity and apoptotic effects as Rituxan® produced in mammalian cells but with significantly enhanced (up to 100- fold) antibody-dependent cellular cytotoxicity (ADCC). Enhanced ADCC activity was found for all FcgRIIIa-158 genotypes. Reported here are the results of expanded studies comparing Rituxan® with the Lemna–derived glyco-optimized rituximab. B-cell depletion was measured in genotyped human whole blood after ex vivo treatment with both anti-CD20 antibodies. Consistent with prior in vitro ADCC studies, glyco-optimized rituximab showed a significant increase in B-cell depletion for all FcgRIIIa-158 genotypes when compared to Rituxan®. To extend these findings to an in vivo setting, a comparative monkey study was conducted to evaluate:pharmacokinetic impact of the optimized glycans,B-cell depletion, andcomplement activation.The study design included two male Cynomologus monkeys per group in a dose-escalation scheme where each group received a single administration of two dose levels of either Rituxan® or glycooptimized rituximab. Results showed no difference between the two antibodies in clinical observations and pharmacokinetic profile. Overall the rate of depletion and recovery of the monkey B-cells between Rituxan® and glyco-optimized rituximab was similar with evidence of an increase in the initial rate of depletion with the glyco-optimized rituximab. The latter finding is not unexpected due to sequence differences between FcgRIIIa in humans and monkeys. Interestingly, 50% higher complement activation (as measured by serum levels of C3a,) was observed with Rituxan®. This is consistent with our previous observation that glyco-optimized rituximab had up to a ten-fold decrease in complement dependent cytotoxicity (CDC) in Raji cells compared to Rituxan®. These studies suggest that an optimized anti-CD20 antibody therapeutic can have a similar pharmacokinetic profile with enhanced ADCC activity and decreased CDC activity compared to Rituxan®. Confirmation that these differences will translate into improved efficacy with decreased side effects associated with CDC activity (Clark and Ledbetter, 2005) will require clinical research. Cox et al (2006). Nat. Biotech. 24: 1591–15197. Clark et al (2005). Ann. Rheum Dis. 64: 77–80.