Glyco-engineered Antibodies Research Articles

Next-Generation Therapeutic Antibodies for Cancer Treatment: Advancements, Applications, and Challenges.

The field of cancer treatment has evolved significantly over the last decade with the emergence of next-generation therapeutic antibodies. Conventional treatments like chemotherapy pose significant challenges, including adverse side effects. Monoclonal antibodies have paved the way for more targeted and effective interventions. The evolution from chimeric to humanized and fully human antibodies has led to a reduction in immunogenicity and enhanced tolerance in vivo. The advent of next-generation antibodies, including bispecific antibodies, nanobodies, antibody-drug conjugates, glyco-engineered antibodies, and antibody fragments, represents a leap forward in cancer therapy. These innovations offer increased potency, adaptability, and reduced drug resistance. Challenges such as target validation, immunogenicity, and high production costs exist. However, technological advancements in antibody engineering techniques provide optimism for addressing these issues. The future promises a paradigm shift, where ongoing research will propel these powerful antibodies to the forefront, revolutionizing the fight against cancer and creating new preventive and curative treatments. This review provides an overview of three next-generation antibody-based molecules, namely bispecific antibodies, antibody-drug conjugates, and nanobodies that have shown promising results in cancer treatment. It discusses the evolution of antibodies from conventional forms to next-generation molecules, along with their applications in cancer treatment, production methods, and associated challenges. The review aims to offer researchers insights into the evolving landscape of next-generation antibody-based cancer therapeutics and their potential to revolutionize treatment strategies.

Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain.

Interactions with cell surface receptors enhance the therapeutic properties of many important antibodies, including the low-affinity Fc γ Receptors (FcγRs). These interactions require proper processing of the immunoglobulin G Fc N-glycan, and eliminating the N-glycan abolishes binding, restricting antibody production to mammalian expression platforms. Yeasts, for example, generate extensively mannosylated N-glycans that are unsuitable for therapeutics. However, Fc with a specifically truncated N-glycan still engages receptors with considerable affinity. Here we describe the creation and applications of a novel Saccharomyces cerevisiae strain that specifically modifies the IgG1 Fc domain with an N-glycan consisting of a single N-acetylglucosamine residue. This strain displayed glycoengineered Fc on its surface for screening yeast surface display libraries and also served as an alternative platform to produce glycoengineered Rituximab. An IgG-specific endoglycosidase (EndoS2) truncates the IgG1 Fc N-glycan. EndoS2 was targeted to the yeast ER using the signal peptide from the yeast protein disulfide isomerase (PDI) and a yeast ER retention signal (HDEL). Furthermore, >99% of the yeast expressed Rituximab displayed the truncated glycoform as determined by SDS-PAGE and ESI-MS analyses. Lastly, the yeast expressed Rituximab engaged the FcγRIIIa with the expected affinity (KD = 2.0 ± 0.5 μM) and bound CD20 on Raji B cells.

Precision medicine in clinical oncology: the journey from IgG antibody to IgE.

Cancer is one of the leading causes of death and the incidence rates are constantly rising. The heterogeneity of tumors poses a big challenge for the treatment of the disease and natural antibodies additionally affect disease progression. The introduction of engineered mAbs for anticancer immunotherapies has substantially improved progression-free and overall survival of cancer patients, but little efforts have been made to exploit other antibody isotypes than IgG. In order to improve these therapies, 'next-generation antibodies' were engineered to enhance a specific feature of classical antibodies and form a group of highly effective and precise therapy compounds. Advanced antibody approaches include among others antibody-drug conjugates, glyco-engineered and Fc-engineered antibodies, antibody fragments, radioimmunotherapy compounds, bispecific antibodies and alternative (non-IgG) immunoglobulin classes, especially IgE. The current review describes solutions for the needs of next-generation antibody therapies through different approaches. Careful selection of the best-suited engineering methodology is a key factor in developing personalized, more specific and more efficient mAbs against cancer to improve the outcomes of cancer patients. We highlight here the large evidence of IgE exploiting a highly cytotoxic effector arm as potential next-generation anticancer immunotherapy.

Abstract 4593: Glycoengineered antibodies for click chemistry applications

Abstract Antibody-drug conjugates (ADC) hold considerable promise as anticancer agents. A critical determinant of the effectiveness of ADCs is the chemistry that is used to conjugate the payload. Currently used approaches include primarily conjugation to either side-chain amine or carboxylic acid groups or conjugation to thiols. Because these reactions are not site specific and not easily controlled, these chemistries can result in reduced affinity for the target antigen. Further, these conjugation reactions lack selectivity and can result in heterogeneous mixtures of products that differ in the sites and stoichiometry of modification. We investigated a glycoengineering strategy that enables the introduction of artificial azide groups in the antibody without affecting their antigen affinity. This is based on the observation that glycosyltransferases can incorporate non-natural sugars (e.g., azido mannose) at different sites on an IgG molecule. The azide groups inthese artificial sugars are then available to react with alkynes through copper-catalyzed ‘click’ chemistry or with strained alkynes such as dibenzyl cyclooctyne (DBCO) allowing for biorthogonal, copper-free ‘click’ chemistry. Because the sugars are added reproducibly and at asite that does not affect antigen binding, the glycoengineering technology would overcome problems associated with traditional conjugation strategies. Using this approach, azide groups were introduced in anti-CD133 and anti-perlecan antibodies. Further, the azide groups were available to react with various DBCO conjugates including fluorophores, drug molecules and nanoparticles. Importantly, the addition of artificial sugar and subsequent azide-alkyne reactiondid not affect the affinity of the antibody for the target antigen. Conjugation of nanoparticles to antibodies using this approach resulted in enhanced cellular uptake of the nanoparticles. Similarly, conjugation of a cytotoxin to the antibody resulted in enhanced cell kill in vitro. Weexpect that this glycoengineering strategy will prove to be a unique platform technology that will have a significant impact on antibody-based therapeutics. Citation Format: Drishti Sehgal, Stephen Kalscheuer, Tanmoy Sadhukha, Jayanth Panyam. Glycoengineered antibodies for click chemistry applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4593. doi:10.1158/1538-7445.AM2017-4593

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.

Identification of a Guinea Pig Fcγ Receptor that Exhibits Enhanced Binding to Afucosylated Human and Mouse IgG.

Glyco-engineered recombinant antibodies are currently being developed as the next generation therapeutics to treat human diseases, including cancer, autoimmunity and infection. Antibodies lacking core fucosylation show great increase in affinity for FcγRIIIA, leading to an improved receptor-mediated effector function. While afucosyl human IgG1 exhibits 50-100-fold increase in antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism underlying the anti-cancer effect of some approved therapeutic antibodies, it is not clear whether such glyco-engineered antibodies would find similar use for infectious disease. Due to the species difference, human antibodies may have different binding properties towards corresponding IgG receptors from animals used for modeling infection and intoxication. During the course of studying a recombinant human IgG1 in neutralizing diphtheria toxin (DT) in Guinea pigs (Cavia porcellus), we identified a previously uncharacterized Guinea pig protein H0VDZ8 from UNIPROT database that shows high sequence homologies to human FcγRIIIA and mouse FcγRIV. This Fcγ receptor, which we named as gpFcγRIV, also demonstrates functional similarity although not to the same extent as the human and mouse counterparts, in that it binds to afucosyl human and mouse IgG much stronger than to the wild type antibodies. Thus, Guinea pigs can be used to compare the efficacies of wild type vs. afucosyl anti-DT human IgG1 in toxin removal and animal protection. Molecular and functional characterization of human FcγRIIIA and mouse FcγRIV equivalents in other species could expand the list of preclinical animal models for testing afucosyl human antibodies in treating various human diseases.

Abstract 3631: Modulation of monocyte- and macrophage-mediated antibody-dependent cell phagocytosis and cytotoxicity (ADCP/ADCC) by Fc engineering of therapeutic antibodies

Abstract Therapeutic monoclonal antibodies contribute to elimination of tumor cells by different mechanisms including blocking of receptor signaling, induction of antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), and induction of adaptive immunity. FcγR-expressing monocytes and macrophages have recently been identified as important immune effectors contributing to the activity of monoclonal antibodies in vivo. Therefore, we analyzed the FcγR-dependent effector functions mediated by both cell subsets upon treatment with glycoengineered (GE) antibodies (having enhanced FcγRIIIa (CD16a) binding affinity), wild-type (WT), and Fc-mutated antibodies that lack binding to FcγRs (P329G LALA mutation). Trastuzumab, cetuximab, rituximab and ofatumumab were used as WT antibodies, whereas GE trastuzumab, GA201 (targeting EGFR) and GA101 (targeting CD20) were used as GE antibodies. To better reflect the plasticity of myeloid lineage cells we evaluated the activities on classical and non-classical/intermediate monocytes, M1 and M2c macrophages. We found that GE and WT antibodies displayed comparable binding and induced similar monocyte/macrophage-mediated ADCC and ADCP in absence of unspecific endogenous IgGs. Interestingly, in their presence, i.e. in a situation that more closely mimics physiological conditions, GE antibodies displayed significantly superior binding and induced higher levels of ADCC/ADCP compared to WT antibodies. Data collected by imaging further indicated that the degree of phagocytized cells is higher and faster in presence of GE antibodies and that M2c macrophages perform serial phagocytosis by engulfing 2-3 tumor cells. Fc-mutated P329G LALA variant showed no binding and no functional activity. A direct comparison of the three anti-CD20 antibodies (GA101, rituximab and ofatumumab), pointed out that GA101 mediated significantly stronger elimination of tumor cells further underscoring the relevance of combination of both glycoengineering and cell death induction for monocyte/macrophage-mediated killing of tumor cells. These data show, for the first time that, in addition to enhancing NK cell-mediated cytotoxicity (ADCC), glycoengineering also enhances monocyte and macrophage cytotoxic and phagocytic activities through enhanced binding to FcγRIIIa under conditions that more closely resemble the physiological setting. Citation Format: Herter Sylvia, Christian Klein, Martina Birk, Thomas Weber, Pablo Umana, Marina Bacac. Modulation of monocyte- and macrophage-mediated antibody-dependent cell phagocytosis and cytotoxicity (ADCP/ADCC) by Fc engineering of therapeutic antibodies. [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 3631. doi:10.1158/1538-7445.AM2014-3631

Abstract 2579: Combination with the novel tumor-targeted CEA-IL2v immunocytokine enhances the activity of ADCC-competent and glycoengineered antibodies in vitro and in vivo

Abstract We recently described a novel monomeric tumor-targeted CEA-IL2v immunocytokine. CEA-IL2v comprises a single IL-2 variant (IL2v) with abolished CD25 binding that is fused to the C-terminus of a humanized CEA antibody with a heterodimeric Fc devoid of FcγR and C1q binding. CEA-IL2v recognizes a membrane-proximal epitope of CEA. Here we show that CEA-IL-2v is able to enhance the activity of ADCC competent antibodies and glycoengineered IgG1 antibodies in vitro and in vivo. The combination of CEA-IL2v with ADCC-competent IgG1 antibodies was investigated in vitro in ADCC assays and in vivo in xenografts in hCD16a transgenic SCID mice that express hFcγRIIIα on NK cells and the homologous muFcγRIV on macrophages/monocytes. Antibodies tested include trastuzumab, cetuximab and the ADCC-enhanced glycoengineered EGFR antibody GA201. In the spontaneous genetically engineered BALB-neuT breast cancer mouse model a surrogate tumor stroma Tenascin C A2 (TNCA2)-targeted IL2v immunocytokine was used in combination with a ratHER2 antibody. PBMCs stimulated by CEA-IL2v in co-cultures with tumor cells showed dose-dependent elimination of tumor cells and activation of NK cells. Addition of trastuzumab or cetuximab resulted in induction of activation markers and enhanced killing of tumor cells. As a single agent, CEA-IL2v showed minor tumor growth inhibition in orthotopic i.m.f.p. KPL4 BC and s.c. N87 GC xenografts. The combination of CEA-IL2v (1 mg/kg, q7d×3) with trastuzumab (10 mg/kg in KPL4 and 25 mg/kg in N87, q7d×3) resulted in superior inhibition of tumor growth compared to the respective single-agent treatments, combination therapy induced 6/9 complete tumor remissions in N87 and 3/5 in KPL4. In the i.v. A549 lung cancer and i.s. LS174T CRC xenografts, single-agent CEA-IL2v showed a moderate effect on median survival, whereas the combination of CEA-IL2v (1 mg/kg, q7d×3) with cetuximab (25 mg/kg, q7d×3) increased median/overall survival. Similarly, in the i.v. A549 NSCLC and i.s. LS174T CRC xenografts, the combination of CEA-IL2v (1 mg/kg for A549 and 2 mg/kg for LS174T, q7d×3) with GA201 (25 mg/kg, q7d×3) resulted in superior median survival compared to the respective single-agent treatments with long term survival of 10/10 animals in the A549 model and survival of 2/8 animals in the LS174T model. Finally in the spontaneous GEM BALB-neuT breast cancer model the combination of TNCA2-IL2v with a ratHER2 IgG1 antibody resulted in a strong and significant reduction of tumor formation in 6/7 animals as compared to the respective single agents. Combining CEA-IL2v or TNCA2-IL2v with ADCC competent/enhanced IgG1 antibodies results in enhanced anti-tumor efficacy in xenografts and in a spontaneous immunocompetent genetically engineered BALB-neuT mouse model. These data support the clinical investigation of CEA-IL2v with ADCC competent/enhanced IgG1 antibodies. Citation Format: Valeria Nicolini, Inja Waldhauer, Anne Freimoser, Sara Colombetti, Federica Cavallo, Marina Bacac, Christian Gerdes, Pablo Umana, Christian Klein. Combination with the novel tumor-targeted CEA-IL2v immunocytokine enhances the activity of ADCC-competent and glycoengineered antibodies in vitro and in vivo. [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 2579. doi:10.1158/1538-7445.AM2014-2579

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGenTM) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling® platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.

Obinutuzumab: First Global Approval

Obinutuzumab (Gazyva™) is an intravenously administered, humanized and glycoengineered, type II anti-CD20 monoclonal antibody for the treatment of B-cell malignancies. It is approved in the US for use in combination with chlorambucil for the first-line treatment of chronic lymphocytic leukaemia (CLL), and has been filed for approval in the EU in this indication. The antibody is based on GlycArt Biotechnology's (later Roche Glycart AG) proprietary GlycoMAb® technology, which uses glycoengineered antibodies that specifically increase antibody-dependent cellular cytotoxicity and thereby increase immune-mediated target cell death. Obinutuzumab is a type II anti-CD20 antibody that induces enhanced direct cell death. The monoclonal antibody is in worldwide phase III development with Roche and its subsidiaries, Genentech and Chugai Pharmaceutical, as well as Biogen Idec, for diffuse large B-cell lymphoma and non-Hodgkin's lymphoma generally, and is also in phase III development in countries outside of the US and EU for CLL.

Negative Impact Of KIR/HLA Interactions On NK Cell Activity Can Be Overcome By The Glycoengineered Antibody GA101 (Obinutuzumab)

Antibody dependent cellular cytotoxicity (ADCC) is one of the mechanisms by which therapeutic antibodies mediate tumor cell killing. The anti-CD20 antibody rituximab is the current standard of care in the treatment of B-cell lymphomas. GA101, a novel anti-CD20 antibody, contains a glycoengineered Fc-portion allowing approximately 10-fold greater affinity to FcgR3A, the Fc-IgG receptor expressed on the majority of natural killer (NK) cells.NK cell function is also regulated by inhibitory killer-cell immunoglobulin-like receptors (KIR), which interact with HLA class I antigens (2DL1-HLA-C2; 2DL2/3-HLA-C1, 3DL1-HLA-Bw4). The KIR/HLA interaction during NK cell development leads to the acquisition of full effector function in the “licensing” process, but also provides one of the main mechanisms of NK cell tolerance. The present study analyzed how KIR/HLA interactions influence ADCC, and whether there are differences between conventional and glycoengineered antibodies. We analyzed the activation (in terms of the degranulation measured by the CD107a expression) and killing capacity of KIR-positive NK cells induced by rituximab, GA101, and the parental non-Fc modified (wild-type) GA101wt. Target cells included HLA-negative B-cell lymphoma lines or B-cell lines expressing one or more HLA molecules.We confirmed previous observations that the licensing status affects the potential for rituximab-induced ADCC (degranulation against HLA-deficient 721.221 in licensed cells 35 ± 4% versus 19 ± 3% of unlicensed cells, p<0.01); and that KIR/HLA interactions strongly and selectively inhibit the response to targets expressing cognate HLA ligands (e.g. CD107a expression in KIR3DL1+ NK cells 17 ± 3% against 721.221-Bw4 cells, compared to 32 ± 4% against 721.221, p<0.01). Next, we analyzed rituximab-induced NK cell activation in donors expressing one, two, or three KIR ligands after co-incubation with target B-cell lines expressing corresponding HLA molecules. These experiments showed that the inhibitory effect during target cell encounter dominates over the activating effect of NK cell licensing, which leads to unlicensed NK cells being the strongest effectors of ADCC with rituximab (Figure, Panel A).We next compared the effect of the KIR/HLA interaction on rituximab-, GA101wt- and GA101-induced ADCC. GA101 largely compensated the hyporesponsiveness of unlicensed cells and NK cell activation was independent of the presence of HLA KIR ligands on target cells (Figure, Panel A). Finally, we addressed the question of how levels of NK cell degranulation correspond to target cell elimination. Correlation between CD107a expression and target cell elimination was excellent for all antibodies (Figure Panel B). GA101 induced the highest level of activation and the most effective target elimination. In contrast to rituximab and GA101wt, no negative impact of KIR/HLA interaction on degranulation or target cell elimination could be detected for GA101. [Display omitted] In summary, we show that KIR/HLA interactions are relevant for ADCC with rituximab, with the negative impact during target cell encounter dominating over the positive effect of licensing. In contrast, the novel glycoengineered GA101 antibody overrides the negative signals derived from the KIR/HLA interaction and activates all NK cell subsets. These data suggest that the Fc-modification to enhance ADCC can be an effective strategy to augment the efficacy of therapeutic monoclonal antibodies by recruiting NK cells irrespective of their inhibitory KIR expression. Disclosures:Terszowski:Roche: Research Funding. Klein:Roche Glycart AG: Employment. Stern:Roche: Research Funding.

Glycoengineering of Therapeutic Antibodies

Antibody dependent cellular cytotoxicity (ADCC) is the process of lysis of (tumor) target cells by immune effector cells. It requires the simultaneous binding of an antibody to a cell surface receptor on the target cell and via the Fc region of the antibody to the FcgRIIIa receptor on immune effector cells such as NK cells or macrophages/monocytes. Binding of the antibody to target and immune effector cells results in the cross-linking of FcgRIIIa with subsequent activation of FcgRIIIa signaling and release of proteins mediating the killing of the target cells such as perforin and of proteases such as granzymes. ADCC has been established as an important mode of action of therapeutic antibodies such as rituximab/ MabThera (targeting CD20), cetuximab/Erbitux (targeting EGFR) and trastuzumab/Herceptin (targeting HER2) which are approved for the treatment of various cancers. Translational research on rituximab and cetuximab has established that the response to therapeutic antibodies in patients can be influenced by the FcgRIIIa-158V/F polymorphism. This polymorphism results in the expression of a high affinity FcgRIIIa receptor (158 V) and a low affinity FcgRIIIa receptor (158F) on immune effector cells and impacts the capability of antibodies to mediate ADCC [1–7]. We have recently developed a so-called GlycoMab technology that enhances the affinity of therapeutic antibodies for both the high and low affinity FcgRIIIa receptors through the introduction of a bisecting N-acetyl-glucosamine residue in the carbohydrate chain of the Fc region of the antibodies. The introduction of this bisecting N-acetyl-glucosamine moiety in the carbohydrate chain results in a steric interference with core fucosylation of the carbohydrate. This process and other approaches resulting in the afucosylation of therapeutic antibodies are known as “antibody glycoengineering”. We and others have demonstrated that glycoengineering and lack of the core fucose residue results in an up to 50-fold enhanced affinity of human IgG1 antibodies for the human FcgRIIIa receptors, and this subsequently results in an up to 100-fold enhanced ADCC induction (potency). Recently, we have demonstrated the structural basis of the enhanced affinity of afucosylated Fc regions for FcgRIIIa by X-ray crystallography. These studies provide a structural explanation for the high affinity and FcgRIIIa specificity of glycoengineering as opposed to other technologies used for Fc-engineering such as the introduction ofmutations in the Fc region that also affect binding to other FcgRs [8–15]. Roche is currently investigating three therapeutic glycoengineered antibodies with optimized immune effector function targeting CD20, EGFR and HER3 in different phases of clinical trials. Obinutuzumab (GA101, RG7159) is a type II, anti-CD20 GlycoMab monoclonal antibody that was designed to mediate enhanced direct and immune effector cell-mediated cell killing as compared to rituximab. In addition to enhanced ADCC, due to its type II characteristics, GA101 exhibits enhanced direct cell death induction with a concomitant reduction of CDC. In NHL animal models GA101 demonstrated a superior efficacy in terms of tumor growth inhibition and survival compared to type I CD20 antibodies such as rituximab and ofatumumab, including the induction of complete tumor remission. In non-human primates GA101 induces superior and profound B cell depletion in lymphoid tissues compared to rituximab. In Phase I/II clinical trials GA101 showed promising activity in heavily pretreated NHL patients. GA101 is currently being investigated in pivotal clinical trials in B-CLL, iNHL and DLBCL in combination with chemotherapy, head-to-head with rituximab-based regimens and in Rituximab-refractory iNHL as well as in various Phase II clinical trials. GA101 was the first glycoand Fc-engineered antibody to enter clinical trials and is now the most advanced glycoand Fc-engineered antibody in clinical trials in the world [16–24]. GA201 (RG7160) is a second anti-EGFR GlycoMab monoclonal antibody that is currently being investigated in Phase II clinical trials. It was designed to combine potent EGFR signaling inhibition with enhanced ADCC induction. In xenograft models GA201 demonstrated superior activity compared to cetuximab and panitumumab. Hence, GA201 has the potential to show clinical activity in patients with solid tumors. Phase I clinical testing in heavily pretreated patients with advanced solid tumors demonstrated a manageable toxicity profile. Evidence of anti-tumor activity was also seen, especially in patients with colorectal cancer, including a number of patients who had previously received cetuximab and/or panitumumab (1 CR, 2 PR). Interestingly, one of the partial responses was reported in a tumor-harboring a k-ras mutation. GA201 is currently undergoing Phase II clinical trials in CRC and NSCLC in combination with chemotherapy as well as a neo-adjuvant biomarker study in HNSCC [25].

With or Without Sugar? (A)glycosylation of Therapeutic Antibodies

Antibodies and antibody-based drugs are currently the fastest-growing class of therapeutics. Over the last three decades, more than 30 therapeutic monoclonal antibodies and derivatives thereof have been approved for and successfully applied in diverse indication areas including cancer, organ transplants, autoimmune/inflammatory disorders, and cardiovascular disease. The isotype of choice for antibody therapeutics is human IgG, whose Fc region contains a ubiquitous asparagine residue (N297) that acts as an acceptor site for N-linked glycans. The nature of these glycans can decisively influence the therapeutic performance of a recombinant antibody, and their absence or modification can lead to the loss of Fc effector functions, greater immunogenicity, and unfavorable pharmacokinetic profiles. However, recent studies have shown that aglycosylated antibodies can be genetically engineered to display novel or enhanced effector functions and that favorable pharmacokinetic properties can be preserved. Furthermore, the ability to produce aglycosylated antibodies in lower eukaryotes and bacteria offers the potential to broaden and simplify the production platforms and avoid the problem of antibody heterogeneity, which occurs when mammalian cells are used for production. In this review, we discuss the importance of Fc glycosylation focusing on the use of aglycosylated and glyco-engineered antibodies as therapeutic proteins.

The Carbohydrate at FcγRIIIa Asn-162: AN ELEMENT REQUIRED FOR HIGH AFFINITY BINDING TO NON-FUCOSYLATED IgG GLYCOFORMS

FcgammaRIIIa plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies. Non-fucosylated bisected IgGs bind this receptor with increased affinity and trigger FcgammaRIII-mediated effector functions more efficiently than native, fucosylated antibodies. In this study the contribution of the carbohydrates of both binding partners to the strength of the complex was analyzed. Glycoengineering of the antibody increased affinity for two polymorphic forms of soluble human FcgammaRIIIa (by up to 50-fold) but did not affect binding to the inhibitory FcgammaRIIb receptor. While the absence of carbohydrate at FcgammaRIIIa's Asn-162 increased affinity for native IgG, presumably due to the removal of steric hindrance caused by the bulky sugars, it unexpectedly reduced affinity for glycoengineered (GE) antibodies by over one order of magnitude, bringing the affinity down to the same level as for native IgG. We conclude that the high affinity between GE antibodies and FcgammaRIII is mediated by productive interactions formed between the receptor carbohydrate attached at Asn-162 and regions of the Fc that are only accessible when it is nonfucosylated. As FcgammaRIIIa and FcgammaRIIIb are the only human Fcgamma receptors glycosylated at this position, the proposed interactions explain the observed selective affinity increase of GE antibodies for only these receptors. Furthermore, we predict from our structural model that only one of the two Fc-fucose residues needs to be absent for increased binding affinity toward FcgammaRIII. This information can be exploited for the design of new antibodies with altered Fc receptor binding affinity and enhanced therapeutic potential.