Enhanced Antibody-dependent Cell-mediated Cytotoxicity Activity Research Articles

Effects of HER Family–targeting Tyrosine Kinase Inhibitors on Antibody-dependent Cell-mediated Cytotoxicity in HER2-expressing Breast Cancer

Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC. Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array-determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry-based protocols. Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell-mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone. TKIs differentially alter tumor cell phenotype which can impact NK cell-mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.

Abstract 4532: Preclinical evaluation of JTX-1811, an anti-CCR8 antibody with enhanced ADCC activity, for preferential depletion of tumor-infiltrating regulatory T cells

Abstract Introduction: Immune checkpoint blockade (ICB) has revolutionized the treatment of many cancers, still most patients do not respond to PD-1 or CTLA-4 inhibitors. Thus, new Immuno-Oncology (IO) therapies that could potentially benefit non-responding patients are greatly needed. Jounce has generated cell type-specific gene signatures as a means of probing The Cancer Genome Atlas and other large datasets for novel IO targets. Regulatory T cells (Tregs) are one attractive cell type for targeting as they may contribute to resistance to ICB. While Tregs are critical for immune homeostasis and preventing tissue damage, they are often present in large numbers within tumors where they may suppress anti-tumor immunity. Therapeutic strategies that specifically deplete tumor-infiltrating Tregs (TITRs) while sparing peripheral and normal tissue Tregs are highly desirable. Using a Treg gene signature, we have a found a strong correlation with TITRs and CCR8 (C-C motif chemokine receptor 8) across multiple tumor types. CCR8 may be differentiated from other known Treg targets in this regard, as its expression was found to be highly selective to TITRs. Methods and Results: We first assessed CCR8 levels on TITRs across multiple tumor types and compared expression to Tregs in normal colon tissue or peripheral blood. On average, peripheral blood Tregs had nearly undetectable CCR8 expression and normal colon tissue Tregs showed 4 to 5-fold lower levels of CCR8 than TITRs. We then generated a panel of monoclonal antibodies (mAbs) that bind specifically to CCR8, but not other family members, and block CCR8 signaling induced by its ligand CCL1. The ability of these mAbs to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of target cells expressing CCR8 was tested. When target cells expressed CCR8 at levels equivalent to normal tissue Tregs no ADCC activity was observed. In contrast, when cells expressed CCR8 at levels equivalent to TITRs, robust ADCC was observed, but only using antibodies in which the human IgG1 Fc was afucosylated. Thus, afucosylated anti-CCR8 antibodies demonstrated a therapeutic window whereby TITRs but not normal tissue Tregs could be depleted. An Fc competent, mouse-specific, anti-CCR8 antibody showed single agent tumor growth inhibition across several murine tumor models - including models in which anti-PD-1 was ineffective. Anti-CCR8 was a potent combination partner with anti-PD-1 resulting in 50% complete tumor regressions in PD-1 resistant models. Conclusions: Based on these pre-clinical data JTX-1811, a high affinity CCR8-specific humanized monoclonal antibody with enhanced ADCC activity, is being developed for the selective depletion of tumor-infiltrating Tregs. JTX-1811 may be useful in PD-1 resistant settings and may restore the activity of PD-1 inhibitors in the setting of primary or acquired resistance to ICB. Citation Format: Fabien Dépis, Changyun Hu, Jessica Weaver, Lara McGrath, Boris Klebanov, Joshua Buggé, Ben Umiker, Christine Fregeau, Dhruvkumar Upadhyay, Anirudh Singh, Chang-Ai Xu, Vikki Spaulding, Michelle Priess, Masie Wong, Seema Naheed, Yan Zhang, Kristin Legendre, Edward C. Stack, Alessandro Mora, Margaret Willer, Kristan Meetze, Monica Gostissa, Michael A. Meehl, Donald R. Shaffer. Preclinical evaluation of JTX-1811, an anti-CCR8 antibody with enhanced ADCC activity, for preferential depletion of tumor-infiltrating regulatory T cells [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 4532.

Glycan engineering reveals interrelated effects of terminal galactose and core fucose on antibody-dependent cell-mediated cytotoxicity.

Antibody-dependent cell-mediated cytotoxicity (ADCC) has been identified as one of the potentially critical effector functions underlying the clinical efficacy of some therapeutic immunoglobin G1 (IgG1) antibodies. It has been well established that higher levels of afucosylated N-linked glycan structures on the Fc region enhance the IgG binding affinity to the FcγIIIa receptor and lead to increased ADCC activity. However, whether terminal galactosylation of an IgG1 impacts its ADCC activity is less understood. Here, we used a new strategy for glycan enrichment and remodeling to study the impact of terminal galactose on ADCC activity for therapeutic IgG1s. Our results indicate that the degree of influence of terminal galactose on in vitro ADCC activity depends on the presence or absence of the core fucose, which is typically linked to the first N-acetyl glucosamine residue of an N-linked glycosylation core structure. Specifically, terminal galactose on afucosylated IgG1 mAbs enhanced ADCC activity with impact coefficients (ADCC%/Gal%) more than 20, but had minimal influence on ADCC activity on fucosylated structures with impact coefficient in the range of 0.1-0.2. Knowledge gained here can be used to guide product and process development activities for biotherapeutic antibodies that require effector function for efficacy, and also highlight the complexity in modulating the immune response through N-linked glycosylation of antibodies.

Blockade of CD47-Sirpα Interactions Improved Myeloid Effector Cell Mediated Lymphoma Cell Killing By the HLA-DR Antibody Apolizumab

Background:Apolizumab (APO) is a humanized IgG1 antibody targeting a HLA-DRβ chain specific epitope, which is expressed by approx. 50 % of B cell lymphomas. The antibody's clinical development was hampered by toxicity, potentially mediated by its strong CDC activity. However, previous studies demonstrated that APO efficiently triggered tumor cell killing by myeloid cells, especially by granulocytes. Myeloid effector cell engagement is strongly regulated by CD47-SIRPα interactions, and blockade of this axis has been demonstrated to improve the efficacy of therapeutic antibodies. Hence, our goal was to characterize the influence of CD47-SIRPα blockade on myeloid cell engagement by APO. Methods: Expression of the APO epitope, CD20 and CD47 on different B lymphoma cell lines was measured by flow cytometry. The capacity of APO and Rituximab (RTX) to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) by mononuclear (MNC) or polymorphonuclear (PMN) effector cells was analysed by 51Cr release assays. Antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages was analysed by fluorescence microscopy. PMN-mediated ADCC and macrophage-mediated ADCP experiments were performed in the presence or absence of the CD47 blocking antibody 5F9-IgG2σ to analyse the impact of CD47-SIRPα blockade on APO-mediated lymphoma cell killing by myeloid cells. Results: As anticipated we observed binding of APO on approx. 50% of the tested B lymphoma cell lines, including Raji (Burkitt's lymphoma), MINO (mantle cell lymphoma) and Carnaval (double hit DLBCL). Focusing on these cell lines, APO's CDC activity was more potent (Raji) or similar (Mino) compared to RTX. Furthermore, APO significantly induced tumor cell lysis by PMN or MNC in 51Cr release assays and achieved higher lysis rates in comparison to RTX. Blocking CD47-SIRPα interactions by 5F9-IgG2σ resulted in significantly increased APO-induced ADCC by PMN against all tested B cell lymphoma lines (Mino: 28.8 ± 10.2 % vs. 41.8 ± 13.3 %; Carnaval: 37.9 ± 17.9 % vs. 55.6 ± 17.6 %; Raji: 23.6 ± 11.7 % vs. 52.4 ± 19.4 % for APO alone vs. APO + 5F9-IgG2σ, respectively, p < 0.05 by ANOVA). However, no ADCC by PMN and RTX was observed - even in the presence of CD47-SIRPα blockade. Moreover, APO mediated similar or even stronger ADCP by macrophages compared to RTX, which could be further enhanced by 5F9-IgG2σ (Figure 1). Conclusions: APO's potent myeloid cell activation could be further enhanced by disruption of CD47-SIRPα interactions. Our observations suggest to generate isotype variants of APO with lower CDC but preserved or enhanced ADCC activity. Subsequently, a combination of APO and CD47-SIRPα blocking strategies may improve B cell lymphoma immunotherapy. Disclosures No relevant conflicts of interest to declare.

Abstract 619: ALT-803 enhances antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NEO-201 against human carcinoma cells

Abstract Background: ALT-803 is a novel IL-15 superagonist complex consisting of an IL-15 mutant (IL-15N72D) bound to an IL-15 receptor α/IgG1 Fc fusion protein. It has been reported that ALT-803 exerted a significant anti-tumor activity in murine myeloma, breast, colon carcinomas, in rat bladder cancer, and in human bladder, ovarian carcinomas in vivo. ALT-803 was also found to enhance ADCC against wide range of human carcinoma cells in vitro. NEO-201 is a novel humanized IgG1 monoclonal antibody (mAb) which was found to react against a variety of cultured human carcinoma cell lines and tumor tissues. NEO-201 can mediate ADCC against human carcinoma cells. Methodology: We investigate the ability of ALT-803 to modulate the ADCC mediated by NE0-201, employing NK cells isolated from normal donors as effectors. Human NK cells were treated with ALT-803 at different concentrations for 48h prior to be used as effector cells, and human carcinoma cell lines expressing the NE0-201 antigen were utilized as targets in an in vitro non-radioactive ADCC assay. The ability of ALT-803 to affect the phenotype of NK cells and to modulate NK cells gene expression was evaluated by flow cytometry and by using the Nanostring analysis respectively. Results: The treatment with ALT-803 significantly enhanced the ADCC activity mediated by NEO-201 against NEO-201 positive carcinoma cells. The effect ALT-803 was dose-dependent and achieved statistical significance at all doses tested compared to vehicle control treatment. Treatment of NK cells with ALT-803 enhanced ADCC activity also from donors with minimal ADCC activity and lowered the effective dose of NEO-201 required to initiate the ADCC response compared to untreated NK cells. ADCC activity can be blocked by using anti-CD16 and anti-TIM3 blocking antibody. Phenotypic analysis of NK cells treated with 25 ng/ml of ALT-803 for 48h demonstrated that ALT-803 can enhance the expression of TIM3 and NKG2D and the mean fluorescence intensity (MFI) of granzyme B and CD107a in CD16/CD56 positive NK cells. Nanostring analysis of human NK cells treated with ALT-803 at different concentrations for 48h, showed that ALT-803 was able to modulate mRNA expression of 62 genes (1.6 log2 fold change compared to vehicle control was considered significant). ALT-803 up-regulated the mRNA expression of 43 genes, including NK activating receptors, factors involved in the NK cytotoxicity, cytokines and their receptors, and down-regulated the mRNA expression of 19 genes, including NK inhibiting receptors and factors involved in the activation of apoptosis. Conclusions: ALT-803 can enhance ADCC activity mediated by NEO-201 against human carcinoma cells. The enhancement of the ADCC activity may be in part due to the increase in the expression of TIM3, NKG2D, granzyme B, and CD107a positive NK cells, as well as to the modulation of transcripts that are involved in the NK activation and cytotoxicity. Citation Format: Massimo Fantini, Justin M. David, Hing C. Wong, Christina M. Annunziata, Philip M. Arlen, Kwong Yok Tsang. ALT-803 enhances antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NEO-201 against human carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 619.

Abstract 1407: FPA144, a therapeutic monoclonal antibody targeting the FGFR2b receptor, promotes antibody dependent cell-mediated cytotoxicity and stimulates sensitivity to PD-1 in the 4T1 syngeneic tumor model

Abstract Five Prime Therapeutics, Inc. has developed an FGFR2b-specific humanized monoclonal antibody, FPA144, to treat patients with cancer bearing overexpression of the FGFR2b receptor, and is currently in clinical trials as a single agent for gastric cancer (NCT02318329). FGFR2 gene amplification and FGFR2b overexpression occur in approximately 5% of gastric cancers and are associated with a poor prognosis in gastric cancer patients. In addition to blocking ligand binding and inducing FGFR2b internalization, FPA144 is glycoengineered for enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). We have shown previously that FPA144 can produce complete and durable tumor growth inhibition in FGFR2b-over-expressing and FGFR2-amplified gastric cancer xenografts in immune-compromised mice (Gemo et al., Poster 5446, AACR 2014). In order to understand the contribution of the immune system to the mechanisms of action of FPA144, we evaluated the anti-tumor effects and immune cell recruitment of FPA144 in the 4T1 model of cancer in immune-competent mice. Although this model expresses FGFR2b, it is not FGFR2 amplified. Therapeutic treatment with FPA144 in the orthotopic 4T1 model resulted in a reduction in tumor burden (33%, P<0.001) and concomitant recruitment of NK cells to the site of tumor implantation, while a modified antibody lacking Fc effector function neither inhibited tumor growth nor lead to the recruitment of NK cells. Together these data support a potential role for ADCC as a mechanism of FPA144 tumor growth inhibition. In addition, treatment with FPA144 increased PD-L1 expressing cells within the tumor microenvironment, providing a strong rationale that FPA144 may combine effectively with PD-1 blockade for additional tumor growth inhibition. PD-1 blockade by the RPM1-14 antibody did not inhibit tumor growth as a single agent in the 4T1 model. Treatment with RPM1-14 in combination with FPA144, however, inhibited tumor growth by 49% (P<0.001), demonstrating an additive benefit of combination therapy. Overall, these data suggest that the enhanced ADCC activity of FPA144 may be critical for anti-tumor efficacy in tumors that have modest expression of FGFR2b. In addition, FPA144 may reprogram the tumor micro-environment in a way that primes the tumor for additional anti-tumor activity when combined with PD-1 blockade. Citation Format: Janine Powers, Servando Palencia, Susan Foy, Barbara Sennino, Toni Rose Hidalgo, Abigael Gemo, Thomas Brennan, Kisten Pierce. FPA144, a therapeutic monoclonal antibody targeting the FGFR2b receptor, promotes antibody dependent cell-mediated cytotoxicity and stimulates sensitivity to PD-1 in the 4T1 syngeneic tumor model. [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 1407.

Structural characterization of GASDALIE Fc bound to the activating Fc receptor FcγRIIIa.

The Fc region of Immunoglobulin G (IgG) initiates inflammatory responses such as antibody-dependent cell-mediated cytotoxicity (ADCC) through binding to activating Fc receptors (FcγRI, FcγRIIa, FcγRIIIa). These receptors are expressed on the surface of immune cells including macrophages, dendritic cells, and natural killer cells. An inhibitory receptor, FcγRIIb, is expressed on macrophages and other myeloid leukocytes simultaneously with the activating receptor FcγRIIa, thereby setting a threshold for cell activation. The affinity of IgG Fc for binding activating Fc receptors depends on IgG subclass and the composition of N-linked glycans attached to a conserved asparagine in the Fc CH2 domain. For example, Fc regions with afucosylated glycans bind more tightly to FcγRIIIa than fucosylated Fc, and afucosylated Fcs exhibit enhanced ADCC activity in vivo and in vitro. Enhanced pro-inflammatory responses have also been seen for Fc regions with amino acid substitutions. GASDALIE Fc is an Fc mutant (G236A/S239D/A330L/I332E) that exhibits a higher affinity for FcγRIIIa and increased effector functions in vivo compared to wild-type Fc. To explore its altered functions, we compared the affinities of GASDALIE and wild-type Fc for activating and inhibitory FcγRs. We also determined the crystal structure of GASDALIE Fc alone and bound to FcγRIIIa. The overall structure of GASDALIE Fc alone was similar to wild-type Fc structures, however, increased electrostatic interactions in the GASDALIE Fc:FcγRIIIa interface compared with other Fc:FcγR structures suggest a mechanism for the increased affinity of GASDALIE Fc for FcγRIIIa.

Antibody-Interferon-Alpha Fusion Protein Therapy for the Treatment of B-Cell Non-Hodgkin Lymphoma: Enhanced ADCC, Inhibition of Proliferation, and In Vivo Eradication of CD20+ Human Lymphomas

Background: Interferon-alpha (IFNα) is a pleiotrophic cytokine with direct anti-tumor and immunostimulatory effects. Currently IFNα is approved for the treatment of multiple hematologic malignancies, including non-Hodgkin lymphoma (NHL). However, its clinical utility has been hindered by dose-limiting toxicitiy due to systemic activation of the interferon receptor. To overcome this limitation, we engineered anti-tumor antibody-IFNα fusion proteins to selectively increase delivery of IFN to the tumor site and reduce systemic toxicity. We previously reported that IGN002, an anti-CD20-IFNα fusion protein, exhibits enhanced complement-dependent cytotoxicity (CDC) compared to rituximab, and inhibits proliferation and induces apoptosis of human B-cell NHL (Yamada et al, ASCO 2013). We now extend these previous findings and show that IGN002 possesses enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) effector function and superior in vivo anti-tumor activity against B-cell NHL, compared to rituximab.Methods: IGN002 was evaluated against a panel of human Burkitt and diffuse large B-cell lymphoma (DLBCL) cell lines. Proliferation was measured by [3 H]-thymidine incorporation, STAT1 activation by flow cytometry, ADCC by lactate dehydrogenase release using human PBMC effectors, and IFN bioactivity by encephalomyocarditis (EMC) viral protection assay. NHL xenografts were grown in SCID mice.Results: IGN002 more potently inhibited the growth of NHL cell lines expressing CD20 than rituximab or unfused IFNα. Intrinsic IFNα activity of IGN002 was reduced in viral protection and anti-proliferation assays using cells lacking CD20 expression. STAT1 activation by IGN002 was enhanced on cells expressing the target antigen, whereas a control antibody-IFNα fusion protein showed reduced STAT activation activity compared to unfused IFNα. Together, these results indicate that fusion of IFNα to the antibody results in reduced IFN effects on cells not bearing the tumor antigen target. IGN002 exhibited enhanced ADCC activity compared to rituximab against Daudi, Ramos, and Raji NHL cells in long-term (overnight incubation) assays, demonstrating both higher potency and higher maximal cytotoxicity. This result is possibly due to activation of the effector cell populations by the fused IFNα moiety, as IFN is known to activate both NK cells and monocytes. The in vivo anti-tumor efficacy of IGN002 was compared to rituximab and a control antibody-IFNα fusion protein against 10-day established Raji NHL xenografts. IGN002 was superior to both rituximab and the control fusion protein, achieving a longer median survival and higher long-term survival rate (p = 0.0015 and < 0.0001 vs. rituximab and control fusion protein, respectively). The in vivo anti-tumor efficacy of IGN002 was also compared to rituximab at three equimolar dose levels (5 mg/kg, 1 mg/kg, and 0.2 mg/kg antibody) against 10-day established Daudi NHL xenografts. IGN002 showed superior efficacy compared to rituximab at all doses (p < 0.001), achieving tumor eradication (100% long-term survival) in all mice treated at all three dose levels, whereas rituximab only delayed tumor progression.Conclusions: IGN002 demonstrated more robust direct anti-proliferative and antibody effector functions than rituximab against human NHL cells in vitro, and also showed the ability to eradicate established NHL xenografts in vivo. Against cells expressing the CD20 target antigen, IGN002 exhibited greater anti-proliferative potency than unfused IFNα. In contrast, the anti-proliferative and anti-viral potency of IGN002 was reduced against cells lacking CD20, compared to unfused IFNα. These findings support the hypothesis that tumor antigen-targeted IFN therapeutics may possess a broader therapeutic index than unfused IFNα, inhibiting tumor growth by multiple mechanisms while reducing systemic toxicity. These results support the further development of IGN002 for the treatment of B-cell NHL, and a first-in-human phase I clinical study will begin later this year in the United States. DisclosuresTimmerman:Janssen: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Valor Biotherapeutics: Research Funding. Steward:ImmunGene, Inc.: Employment. Minning:Valor Biotherapeutics, LLC: Consultancy. Sachdev:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Gresser:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Khare:Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Morrison:ImmunGene, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

A high affinity recombinant antibody to the human EphA3 receptor with enhanced ADCC activity

EphA3is expressed in solid tumors and leukemias and is an attractive target for the therapy. We have generated a panel of Humaneered® antibodies to the ligand-binding domain using a Fab epitope-focused library that has the same specificity as monoclonal antibody mIIIA4. A high-affinity antibody was selected that competes with the mIIIA4 antibody for binding to EphA3 and has an improved affinity of ∼1 nM. In order to generate an antibody with potent cell-killing activity the variable regions were assembled with human IgG1k constant regions and expressed in a Chinese hamster ovary (CHO) cell line deficient in fucosyl transferase. Non-fucosylated antibodies have been reported to have enhanced binding affinity for the IgG receptor CD16a (FcγRIIIa). The affinity of the antibody for recombinant CD16a was enhanced approximately 10-fold. This resulted in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity against EphA3-expressing leukemic cells, providing a potent antibody for the evaluation as a therapeutic agent.

A novel llama antibody targeting Fn14 exhibits anti-metastatic activity in vivo

Expression of fibroblast growth factor (FGF)-inducible 14 (Fn14), a member of the tumor necrosis factor receptor superfamily, is typically low in healthy adult organisms, but strong Fn14 expression is induced in tissue injury and tissue remodeling. High Fn14 expression is also observed in solid tumors, which is why this receptor is under consideration as a therapeutic target in oncology. Here, we describe various novel mouse-human cross-reactive llama-derived recombinant Fn14-specific antibodies (5B6, 18D1, 4G5) harboring the human IgG1 Fc domain. In contrast to recombinant variants of the established Fn14-specific antibodies PDL192 and P4A8, all three llama-derived antibodies efficiently bound to the W42A and R56P mutants of human Fn14. 18D1 and 4G5, but not 5B6, efficiently blocked TNF-like weak inducer of apoptosis (TWEAK) binding at low concentrations (0.2–2 µg/ml). Oligomerization and Fcγ receptor (FcγR) binding converted all antibodies into strong Fn14 agonists. Variants of 18D1 with enhanced and reduced antibody-dependent cell-mediated cytotoxicity (ADCC) activity were further analyzed in vivo with respect to their effect on metastasis. In a xenogeneic model using human colon carcinoma cancer cells, both antibody variants were effective in reducing metastasis to the liver. In contrast, only the 18D1 variant with enhanced ADCC activity, but not its ADCC-defective counterpart, suppressed lung metastasis in the RENCA model. In sum, this suggests that Fn14 targeting might primarily act by triggering of antibody effector functions, but also by blockade of TWEAK-Fn14 interaction in some cases.

Enhanced ADCC Activity of Affinity Maturated and Fc-Engineered Mini-Antibodies Directed against the AML Stem Cell Antigen CD96

CD96, a cell surface antigen recently described to be preferentially expressed on acute myeloid leukemia (AML) leukemic stem cells (LSC) may represent an interesting target structure for the development of antibody-based therapeutic approaches. The v-regions from the CD96-specific hybridoma TH-111 were isolated and used to generate a CD96-specific single chain fragment of the variable regions (scFv). An affinity maturated variant resulting in 4-fold enhanced CD96-binding was generated by random mutagenesis and stringent selection using phage display. The affinity maturated scFv CD96-S32F was used to generate bivalent mini-antibodies by genetically fusing an IgG1 wild type Fc region or a variant with enhanced CD16a binding. Antibody dependent cell-mediated cytotoxicity (ADCC) experiments revealed that Fc engineering was essential to trigger significant effector cell-mediated lysis when the wild type scFv was used. The mini-antibody variant generated by fusing the affinity-maturated scFv with the optimized Fc variant demonstrated the highest ADCC activity (2.3-fold enhancement in efficacy). In conclusion, our data provide proof of concept that CD96 could serve as a target structure for effector cell-mediated lysis and demonstrate that both enhancing affinity for CD96 and for CD16a resulted in mini-antibodies with the highest cytolytic potential.

Production, characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans

The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.

Colony-stimulating factor enhancement of myeloid effector cell cytotoxicity towards neuroectodermal tumour cells.

We conducted experiments to determine the optimal conditions for colony-stimulating factor-enhanced neutrophil- and mononuclear phagocyte-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) using monoclonal antibodies to disialogangliosides expressed on neuroectodermal tumour target cells. Neutrophil ADCC was most effective at effector:target ratios of 100:1, with maximal cytotoxic responses to melanoma target cells generated by 3 h. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) were the most potent stimulators of neutrophil ADCC, and enhanced ADCC activity was inhibited in the presence of antibody to Fc receptor type II (FcRII). GM-CSF and macrophage colony-stimulating factor (M-CSF) treatment of freshly isolated monocytes inhibited antibody-independent cytotoxicity but enhanced antibody-dependent responses. After 3 d in culture with CSF, 3-10-fold enhancement of ADCC against melanoma target cells was observed at effector:target cell ratios of 10:1. Greatest stimulation of macrophage ADCC was obtained when GM-CSF, M-CSF or interleukin 3 (IL-3) were used in conjunction with a secondary stimulus. Although gamma interferon (gamma-IFN) did not augment the cytotoxic capability of GM-CSF- and IL-3-stimulated macrophages, prominent cytotoxic enhancement was seen when M-CSF-stimulated macrophages were exposed to gamma-IFN. A chimaeric mouse/human monoclonal antibody was found to be equivalent in activity to the murine antibody in neutrophil ADCC; however, in macrophage ADCC assays with submaximal effector cell stimulation, the chimaeric antibody was associated with a two-fold greater response. These studies indicate that under specific conditions, CSFs capable of increasing the number and functional activity of mature myeloid effector cells enhance antibody-dependent cytotoxicity to neuroectodermal tumour target cells.