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Glycosylation-dependent antitumor therapeutic monoclonal antibodies
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Glycosylation-dependent antitumor therapeutic monoclonal antibodies Yiran Zhanga,b, Chun Fanc, Lijuan Zhanga,*, Xuexiao Mab,* a
Systems Biology and Medicine Center for Complex Diseases, Affiliated Hospital of Qingdao University, Qingdao, China b Department of Orthopedics, Affiliated Hospital of Qingdao University, Qingdao, China c Department of Stomatology, Affiliated Hospital of Qingdao University, Qingdao, China *Corresponding authors: e-mail address: [email protected]; [email protected]
Contents 1. Structures of immunoglobulin 2. Glycosylation of IgG and its significance 2.1 Core fucose 2.2 N-acetylglucosamine 2.3 Sialic acid 2.4 Galactose 3. Production of therapeutic monoclonal antibodies 4. FDA approved antitumor therapeutic monoclonal antibodies 5. Conclusions and perspectives Acknowledgments Conflict of interest References
2 3 5 5 6 7 7 8 10 11 11 11
Abstract The therapeutic market for monoclonal antibodies (MAbs) has grown exponentially since 2000. It is expected that the world-wide market for MAbs could reach $125 billion in 2020. For cancer treatment alone, more than 30 MAbs have been approved by the US Food and Drug Administration since 1997. Unlike structure-defined small moleculebased anti-cancer drugs, the expensive MAb is a mixture of heterogeneously glycosylated proteins. All MAbs typically have a single N-glycosylation site on each of the Fc region. The clinical efficacy of the MAbs depends on the N-glycan structures. Loss of N-glycosylation on the MAbs leads to the loss of the ability to activate complement, to bind to Fc receptors, and to induce antibody-dependent cellular cytotoxicity (ADCC). Moreover, antigen-antibody complexes produced from N-glycan-deficient MAbs are failed to be eliminated rapidly from the blood circulation. Even in certain cases, the N-glycan heterogeneity does not significantly influence pharmacokinetics or half-life of MAbs, reduced terminal galactosylation decreases complement-dependent
Progress in Molecular Biology and Translational Science ISSN 1877-1173 https://doi.org/10.1016/bs.pmbts.2019.03.004
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2019 Elsevier Inc. All rights reserved.
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cytotoxicity, the absence of core fucosylation enhances ADCC due to the increased affinities for the FcγRIIIа receptor, and high sialylation levels reduce ADCC activity and impact inflammatory responses. Furthermore, only mammalian cell lines that make human-like N-glycan structures can be used for MAbs production since certain mammalian cell lines can produce non-human glycan epitopes such as galactose-α-1,3galactose and N-glycolylneuraminic acid (NGNA), which can trigger unwanted immune response. Therefore, mastering the knowledge of N-glycan structures and glycobiology is the key to produce and provide patients with reliable MAbs with consistent glycosylation profile and expected clinical efficacy.
1. Structures of immunoglobulin Immunoglobulin, also known as antibody, is mainly produced by plasma B cells. Antibodies are a key component of the adaptive immune response and play a central role in both in the recognition of foreign antigens and the stimulation of an immune response. In mammals, there are five isotypes of antibodies known as IgA, IgD, IgE, IgG, and IgM.1 The content and distribution of the five isotypes of antibodies in human body are obviously different (Table 1). At present, almost all commercially available recombinant monoclonal antibody drugs are IgG class.2 During the past 30 years, IgG has been proved to be the most promising antibody isotype for tumor immunotherapy due to its potent antibody-dependent cellmediated cytotoxicity (ADCC) and the complement dependent cytotoxicity (CDC).3,4 IgG can be divided into four subclasses based on disulfide bonds and structural differences in heavy chain region: IgG1, IgG2, IgG3 and IgG4, which respectively account for approximately 60%, 25%, 10% and 5% of Table 1 Isotypes and characteristics of immunoglobulins in human. Characteristics IgA IgD IgE IgG
IgM
Serum content (mg/dL)
200
3
0.04
1200
120
Relative amount (%)
10–15
0.05
0.03
75–85
5–10
Synthesis rate [mg/(kg*d)]
24
0.4
0.02
33
7
Decomposition rate (%/d)
25
37
71
7
8
Half-life (d)
6
3
2
23
5
Intravascular distribution (%)
50
75
50
50
80
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serum IgG.5,6 The four subclasses have highly homologous constant region sequences, however, antigen-binding capacity, formation of immune complexes, complement activation, triggering effector cells, half-life and placental transport characteristics are very distinct.7 X-ray crystallographic study showed the similar spatial structure in IgG1, IgG2, IgG3 and IgG4, which is composed of two identical heavy chains (50–70 kDa) and two light chains (23 kDa) to form Y-shaped molecular.8 Two identical heavy chains and two identical light chains connect via disulfide bonds formed by cysteine. The variable region is near N-terminal of IgG light chain (VL), and C-terminal of light chain is constant region (CL). Meanwhile, there are one variable region (VH) and three constant regions (CH) in heavy chain of IgG.1 In consideration of function, IgG can be divided into two parts: two antigen-binding fragments (Fab), which are responsible for antigen binding, and one crystallizable fragment (Fc), which interacts with host receptors. The two moieties are connected through a flexible linker (the hinge region) (Fig. 1). Ligands of Fc include three structurally homologous cellular Fc receptor types (FcγRI, FcγRII and FcγRIII), the C1q component of complement and the neonatal Fc receptor (FcRn).9–12 By binding to specific ligand, IgG mediates different physiological effects including recognition of opsonized particles (binding to FcγR), lysis of cells (binding to complement), and degranulation of mast cells, basophils, and eosinophils (binding to FcRn).13
2. Glycosylation of IgG and its significance As the most abundant glycoproteins in human serum, correct glycosylation modification is essential for IgG to exert its physiological function. Studies have revealed that each IgG monomer contains an average of 2–3 N-glycosylation sites, of which the N-glycosylation site is conserved on the 297 asparagine (Asn).14–18 Loss of N-glycosylation on the MAbs either by treating the cells with tunicamycin19 or site-directed mutagenesis20 leads to the loss of the ability to activate complement, to bind to Fc receptors, and to induce antibody-dependent cellular cytotoxicity (ADCC). Moreover, antigen-antibody complexes produced from N-glycan-deficient MAbs are failed to be eliminated rapidly from the blood circulation.19 Besides that, the hinge region of IgG3 may contain 4 or 6 O-glycosylation sites, which is very specific in IgG subclasses and the significance of O-glycosylation in IgG3 has not been elucidated.4
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Fig. 1 See figure legend on opposite page.
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Although the N-glycan chains on Fc and Fab are predominantly biantennary complex, their glycoforms are quite different. The former contains a higher proportion of core fucose, while the latter contains a higher proportion of galactose, sialic acid and N-acetylglucosamine (GlcNAc).18 Due to non-template driven biosynthesis, the glycan structures are affected by a series of glycosyltransferases, nutrition status, and environmental factors.21–29 The decrease of galactosylation on IgG is caused by the downregulation of galactosyltransferase activity or expression in plasma cells, instead of removal by galactosidase in serum.30,31 β-galactoside alpha-2,6sialyltransferase 1 (ST6Gal1) in serum can regulate the degree of terminal sialylation of IgG molecule, and inflammatory factors such as IL-1β, IL-6 and TNF-α can influence the expression of alpha 2–6 sialytransferase, as well as fucosyltransferase VI.32,33 Common N-glycan structures detected in the recombinant monoclonal antibodies (MAbs) are shown in Fig. 2. Studies have shown that different N-glycoforms of MAbs has certain effects on their stability, clearance, immunogenicity, ADCC and CDC.34,35
2.1 Core fucose Core fucose refers to the fucose residue connected to the N-acetylglucosamine (GlcNAc) directly linked to Asn. It is estimated that about 94% Fc fragment of human IgG contains core fucose modification.18 Meanwhile, studies have proven that lack of core fucose on human IgG1 N-glycan improves binding to human Fcγ RIIIa, and enhances the ADCC activity.36–38 Additionally, it was reported that core fucosylation could increase the CDC activity.39
2.2 N-acetylglucosamine N-acetylglucosamine (GlcNAc) is the principal framework of N-glycan. The role of GlcNAc is essential for half-life of MAbs in plasma. Receptors like mannose receptor (MR) and asialoglycoprotein (ASGPR) receptor can Fig. 1 Subtypes and the structural domains of IgG. Adapted from references Wada R, Matsui M, Kawasaki N. Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms. mAbs. 2019;11(2):350–372; Cong Y, Hu L, Zhang Z, et al. Analysis of therapeutic monoclonal antibody glycoforms by mass spectrometry for pharmacokinetics study. Talanta. 2017; 165:664–670; Ucakturk E. Analysis of glycoforms on the glycosylation site and the glycans in monoclonal antibody biopharmaceuticals. J Sep Sci. 2012;35(3):341–350; Ivarsson M, Villiger TK, Morbidelli M, Soos M. Evaluating the impact of cell culture process parameters on monoclonal antibody N-glycosylation. J Biotechnol. 2014;188:88–96.
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Fig. 2 (A) N-glycoforms detected in MAbs. The common glycoforms detected include MAN5, G0, G1, G2, G0F, G1F, G2F, and G2FS. (B) Different N-glycoforms on antibodydependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Even in certain cases, the N-glycan heterogeneity does not significantly influence pharmacokinetics or half-life of MAbs, reduced terminal galactosylation decreases complement-dependent cytotoxicity, the absence of core fucosylation enhances ADCC due to the increased affinities for the FcγRIIIа receptor, and high sialylation levels reduce ADCC activity and impact inflammatory responses. Panel A: Adapted from Lan Y, Hao C, Zeng X, et al. Serum glycoprotein-derived N- and O-linked glycans as cancer biomarkers. Am J Cancer Res. 2016;6(11):2390. Panel B: Adapted from Ivarsson M, Villiger TK, Morbidelli M, Soos M. Evaluating the impact of cell culture process parameters on monoclonal antibody N-glycosylation. J Biotechnol. 2014;188:88–96; Gerngross TU. Advances in the production of human therapeutic proteins in yeasts and filamentous fungi. Nat Biotechnol. 2004;22(11):1409–1414.
specifically bind to the GlcNAc residues in glycoproteins. GlcNAc is able to bind to MR and then be removed, which reduces the half-life of glycoprotein in the blood circulation.40–43 Furthermore, IgG-complement system will be activated after terminal GlcNAc residues binding to MR.44
2.3 Sialic acid The role of sialic acid residue as a modulator of the anti-inflammatory activity of IgG has been proven.45,46 Its potential mechanism could be: 1. terminal sialic acid residue can up-regulate activation threshold of innate immunocytes, like macrophage, through inducing the expression of inhibitory Fcγ R II B receptors, and make IgG has anti-inflammatory activity.47 The terminal sialic acid residue on Fc fragment hinders the binding of
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antibody to complement C1q, which reduces the deposition of C3b on the cell surface, and results in the down-regulation of the CDC effect of the antibody.48
2.4 Galactose The N-glycans on Fc fragment of human IgG are mainly of biantennary complex type. So, there could be 0, 1 or 2 galactose residues in the N-glycans (Fig. 2). In Hodoniczky group’s study, they found that the terminal galactose on the FC fragment had high affinity to complement C1q.49 Therefore, a higher contents of terminal galactose residues would result in an enhanced CDC potency of IgG antibody.
3. Production of therapeutic monoclonal antibodies At early stage, antibodies are only tools for scientific research and play a role in immunological research, such as immunoblotting, ELISA, IHC, and immunoprecipitation. The concept of using antibodies as therapeutics was firstly proposed in 1890 by von Behring and Kitasato.50 However, the real boosting of therapeutic antibodies was the development of monoclonal hybridoma technology by Kohler and Milstein in 1975.51 Due to this technology, the first therapeutic monoclonal antibody was approved in 1986 for human use (muromonab-CD3).52 While hybridoma technology is highly efficient and productive, but it did not solve the problem of immunogenicity until the emergence of antibody display technology.53–55 Antibody display (e.g., phage display, yeast display, and ribosome display) utilizes a library of carrier to generate a humanized antigen recognition domains (complementarity determining regions, CDR), which results in a significantly reduced immunogenicity. Historically, the mammalian Chinese hamster ovary (CHO) cell line has played a dominant role, and today 70% of all recombinant therapeutic proteins are produced in CHO cells. The CHO line is selected for human therapeutic production because its glycosylation is relatively simple and similar to that in humans.56 However, immunogenicity is not the only concern in antibody pharmaceutics. Studies on the quality control of antibodies have shown that only proper expression and assembly of each fragment does not guarantee the quantity and quality of the antibody.57 A particular focus is glycosylation, as it has been shown that biological activities vary between different natural glycoforms, and consequently, biological engineering has been performed to make proper MAb-producing cell lines (CHO, NS0, Sp2/0
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cell lines, and so on).58 Currently, CHO cells, including the DUXB11, DG44, and CHO-K1 lineages, are the most heavily used for production of MAbs due to following features: (1) stable expression over long incubation time; (2) suitable for scale-up; (3) able to perform the necessary posttranslational modifications; (4) human safety.57,59 The main glycoform produced by CHO, NS0, Sp2/0 cell lines is the G0F glycoform, which indicates the proportion of galactosylated IgG Fc is low relative to normal IgG Fc.60 A particular concern in recombinant MAbs produced by engineered cells is the addition of galactose in α(1–3) linkage to galactose linked β(1–4) to the GlcNAc residues, whose structure has not been observed in human and higher primates.61 Similarly, CHO, NS0 and Sp2/0 cells can add an α(2–3)-linked N-glycylneuraminic acid that is not present in humans either and that might also be immunogenic.62 To produce MAbs with appropriately glycosylated IgG Fc and IgG Fab sites is a challenge to pharmaceutical enterprises. So, some companies try to address this problem by engineering out VH or VL glycosylation motifs when present in candidate MAbs.63
4. FDA approved antitumor therapeutic monoclonal antibodies Antitumor monoclonal antibody therapy is an important branch of tumor immunotherapy. The development of biotechnology has promoted the improvement of therapeutic MAbs. According to the format, it can be divided into murine MAbs, chimeric MAbs, humanized MAbs, and human MAbs.64–67 Since the first antitumor therapeutic MAb (Rituximab) was approved to treat non-Hodgkin lymphoma by targeting at CD20 on B cells in 1997, so far there has been nearly 30 antitumor MAbs approved by FDA (Table 2).4,35,68 As targeted drugs, antitumor therapeutic MAbs usually target at highly expressed surface antigens of tumor cells, like CD20, EGFR, and PD-L1, which could play a role in tumorigenesis, angiogenesis, and immunosuppression.69–71 The clinical trials have proved the efficacy of MAbs in alleviating the progression of disease in cancer patients. In NSCLC clinical trial of PD-1 and PD-L1 MAbs, data showed that patients received both MAbs treatment combined with chemotherapy exhibit the highest objective response rate (ORR, 71%).72 Furthermore, the median overall survival (OS) is 12.7 months with pembrolizumab (PD-1) 10 mg/kg, and 8.5 months with docetaxel 75 mg/m2.73 Similarly, a 11-years’ follow-up study of
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Antitumor therapeutic monoclonal antibodies
Table 2 The antitumor therapeutic MAbs approved by FDA from 1997 to 2017. Year of Antibody Target Indication Format approval
Rituximab
CD20
non-Hodgkin lymphoma
Chimeric IgG1
1997
Trastuzumab
HER2
Breast cancer
Humanized IgG1
1998
Alemtuzumab
CD52
Chronic lymphocytic leukemia
Humanized IgG1
2001
Ibritumomab
CD20
B cell non-Hodgkin’s lymphoma
Murin IgG1
2002
Tositumomab
CD20
non-Hodgkin lymphoma
Murine IgG2a
2003
Bevacizumab
VEGF
Colorectal cancer
Humanized IgG1
2004
Cetuximab
EGFR
Colorectal cancer
Chimeric IgG1
2004
Panitumumab
EGFR
Colorectal cancer
Human IgG2 2006
Ofatumumab
CD20
Chronic lymphocytic leukemia
Human IgG1 2009
Ipilimumab
CTLA-4
Metastatic melanoma
Human IgG1 2011
Brentuximab
CD30
Hodgkin lymphoma
Chimeric IgG1
2011
Pertuzumab
HER2
Breast cancer
Humanized IgG1
2012
Obinutuzumab
CD20
Chronic lymphocytic leukemia
Humanized IgG1
2013
Trastuzumab
HER2
Breast cancer
Human IgG1 2013
Denosumab
RANKL
Giant cell tumor of bone
Human IgG1 2013
Ramucirumab
VEGR2
Gastric cancer
Human IgG1 2014
Pembrolizumab PD-1
Melanoma
Humanized IgG4
2014
Blinatumomab
Acute lymphoblastic leukemia
Bi-specific antibody
2014
CD19, CD3
Continued
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Table 2 The antitumor therapeutic MAbs approved by FDA from 1997 to 2017.—cont’d Year of Antibody Target Indication Format approval
Nivolumab
PD-1
Metastatic melanoma
Human IgG4 2014
Nivolumab
PD-1
Lung cancer
Human IgG1 2015
Daratumumab
CD38
Multiple myeloma
Human IgG1 2015
Elotuzumab
SLAMF7
Multiple myeloma
Humanized IgG1
2015
Dinutuximab
GD2
Neuroblastoma
Chimeric IgG1
2015
Necitumumab
EGFR
NSCLC
Human IgG1 2015
Olaratumab
PDGFR-α Soft tissue sarcoma
Human IgG1 2016
Atezolizumab
PD-L1
Bladder cancer
Humanized IgG1
Avelumab
PD-L1
NSCLC
Human IgG1 2017
Durvalumab
PD-L1
Metastatic urothelial carcinoma
Human IgG1 2017
Inotuzumab ozogamicin
CD22
Acute lymphoblastic leukemia
Humanized IgG1
2016
2017
HER2-positive breast cancer treated with trastuzumab revealed that 1 year of adjuvant trastuzumab after chemotherapy for patients with HER2positive could significantly improve long-term disease-free survival (HR 076, 95% CI 068-086) compared with observation group.74 Due to the high efficacy, research and development of antitumor MAbs have competed fiercely in pharmaceutical enterprises. To discover novel targets, and to explore new functions of existing targets and interaction between multiple targets are the major tasks of biotechnology and bioengineering in the future.
5. Conclusions and perspectives Understanding the significance of the heterogeneity of MAb glycoforms has been a major challenge for the manufacturer to produce bioactive antibody products. Unlike protein and nucleic acid, the synthesis of
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glycans is a non-template driven process, the glycan structures are determined by both genes and environment. At the same time, minor changes in the number, structure and the glycosylation site may greatly alter the structure, thermal stability, solubility, secretion and biological functions of antibodies. With the development of mass spectrometry, nuclear magnetic resonance and lectin chip technologies, the analysis of N has made great progress. According to the final analyzed object, the analysis method of N-glycan chain mainly includes three kinds: (1) carbohydrate chain or monosaccharide; (2) glycopeptide; (3) glycoprotein.75–78 Based on structure knowledge, it opens up opportunities for tailoring antibody therapeutics to maximize their efficacy. Increase in ADCC activity observed for non-fucosylated rituximab and trastuzumab in in vitro whole-blood assays suggests that they may be similarly efficacious in vivo.79,80 Though higher efficacy obtained by such modification, it could have a downside in that increased and/or additional unwanted side effects may occur for example, release of inflammatory mediators and cytokines.81 Therefore, the in-depth study of glycosylation modification of IgG can better understand the immune status of the body, and promote the development of antitumor therapeutic monoclonal antibody-based drugs.
Acknowledgments This research was supported by the Natural Science Foundation of China (Grant 81,672,585), Key Technology Fund of Shandong Province (Grant 2016ZDJS07A07), the Taishan Scholar Fellowship, and the “Double First Class fund” of Shandong Province in China to L.Z.
Conflict of interest The authors declare no conflict of interest.
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47. Bruhns P, Samuelsson A, Pollard JW, Ravetch JV. Colony-stimulating factor-1dependent macrophages are responsible for IVIG protection in antibody-induced autoimmune disease. Immunity. 2003;18(4):573–581. 48. Quast I, Keller CW, Maurer MA, et al. Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity. J Clin Invest. 2015;125(11):4160–4170. 49. Hodoniczky J, Zheng YZ, James DC. Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog. 2005;21(6):1644–1652. 50. von Behring E, Kitasato S. The mechanism of diphtheria immunity and tetanus immunity in animals. 1890. Mol Immunol. 1991;28(12):1317. 1319–1320. 51. Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256(5517):495–497. 52. Goldstein G. Overview of the development of orthoclone OKT3: monoclonal antibody for therapeutic use in transplantation. Transplant Proc. 1987;19(2 suppl 1):1–6. 53. Hammers CM, Stanley JR. Antibody phage display: technique and applications. J Invest Dermatol. 2014;134(2):1–5. 54. Boder ET, Raeeszadeh-Sarmazdeh M, Price JV. Engineering antibodies by yeast display. Arch Biochem Biophys. 2012;526(2):99–106. 55. Schaffitzel C, Hanes J, Jermutus L, Pluckthun A. Ribosome display: an in vitro method for selection and evolution of antibodies from libraries. J Immunol Methods. 1999;231(1–2):119–135. 56. Esko JD, Stanley P. Glycosylation Mutants of Cultured Mammalian Cells. vol Chapter 49, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2017. 57. Rita Costa A, Elisa Rodrigues M, Henriques M, Azeredo J, Oliveira R. Guidelines to cell engineering for monoclonal antibody production. Eur J Pharm Biopharm. 2010;74(2):127–138. 58. Gaughan CL. The present state of the art in expression, production and characterization of monoclonal antibodies. Mol Divers. 2016;20(1):255–270. 59. Malphettes L, Freyvert Y, Chang J, et al. Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies. Biotechnol Bioeng. 2010;106(5):774–783. 60. Mimura Y, Church S, Ghirlando R, et al. The influence of glycosylation on the thermal stability and effector function expression of human IgG1-Fc: properties of a series of truncated glycoforms. Mol Immunol. 2000;37(12 13):697–706. 61. Macher BA, Galili U. The Galalpha1,3Galbeta1,4GlcNAc-R (alpha-Gal) epitope: a carbohydrate of unique evolution and clinical relevance. Biochim Biophys Acta. 2008;1780(2):75–88. 62. Padler-Karavani V, Yu H, Cao H, et al. Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans: potential implications for disease. Glycobiology. 2008;18(10):818–830. 63. Carter P, Presta L, Gorman CM, et al. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA. 1992;89(10):4285–4289. 64. Lonberg N, Huszar D. Human antibodies from transgenic mice. Int Rev Immunol. 1995;13(1):65–93. 65. Chadd HE, Chamow SM. Therapeutic antibody expression technology. Curr Opin Biotechnol. 2001;12(2):188–194. 66. Schmitz U, Versmold A, Kaufmann P, Frank HG. Phage display: a molecular tool for the generation of antibodies—a review. Placenta. 2000;21(suppl A):S106–S112. 67. Hanack K, Messerschmidt K, Listek M. Antibodies and selection of monoclonal antibodies. Adv Exp Med Biol. 2016;917:11–22. 68. Saini KS, Azim Jr HA, Cocorocchio E, et al. Rituximab in Hodgkin lymphoma: is the target always a hit? Cancer Treat Rev. 2011;37(5):385–390.
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69. Liu F, Hon GC, Villa GR, et al. EGFR mutation promotes glioblastoma through epigenome and transcription factor network remodeling. Mol Cell. 2015;60(2):307–318. 70. Keller S, Schmidt MHH. EGFR and EGFRvIII promote angiogenesis and cell invasion in glioblastoma: combination therapies for an effective treatment. Int J Mol Sci. 2017;18(6):E1295. 71. Wang Y, Wu L, Tian C, Zhang Y. PD-1-PD-L1 immune-checkpoint blockade in malignant lymphomas. Ann Hematol. 2018;97(2):229–237. 72. Hellmann MD, Rizvi NA, Goldman JW, et al. Nivolumab plus ipilimumab as first-line treatment for advanced non-small-cell lung cancer (CheckMate 012): results of an openlabel, phase 1, multicohort study. Lancet Oncol. 2017;18(1):31–41. 73. Herbst RS, Baas P, Kim DW, et al. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. Lancet. 2016;387(10027):1540–1550. 74. Cameron D, Piccart-Gebhart MJ, Gelber RD, et al. 11 years’ follow-up of trastuzumab after adjuvant chemotherapy in HER2-positive early breast cancer: final analysis of the HERceptin adjuvant (HERA) trial. Lancet. 2017;389(10075):1195–1205. 75. Zhang H, Li XJ, Martin DB, Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol. 2003;21(6):660–666. 76. Song X, Ju H, Lasanajak Y, Kudelka MR, Smith DF, Cummings RD. Oxidative release of natural glycans for functional glycomics. Nat Methods. 2016;13(6):528–534. 77. Liu M, Zhang Y, Chen Y, et al. Efficient and accurate glycopeptide identification pipeline for high-throughput site-specific N-glycosylation analysis. J Proteome Res. 2014;13 (6):3121–3129. 78. Hirabayashi J, Yamada M, Kuno A, Tateno H. Lectin microarrays: concept, principle and applications. Chem Soc Rev. 2013;42(10):4443–4458. 79. Iida S, Kuni-Kamochi R, Mori K, et al. Two mechanisms of the enhanced antibodydependent cellular cytotoxicity (ADCC) efficacy of non-fucosylated therapeutic antibodies in human blood. BMC Cancer. 2009;9:58. 80. Mori K, Iida S, Yamane-Ohnuki N, et al. Non-fucosylated therapeutic antibodies: the next generation of therapeutic antibodies. Cytotechnology. 2007;55(2–3):109–114. 81. Peipp M, van Bueren JJL, Schneider-Merck T, et al. Antibody fucosylation differentially impacts cytotoxicity mediated by NK and PMN effector cells. Blood. 2008;112(6): 2390–2399.
Glycosylation-dependent antitumor therapeutic monoclonal antibodies Yiran Zhanga,b, Chun Fanc, Lijuan Zhanga,*, Xuexiao Mab,* a
Systems Biology and Medicine Center for Complex Diseases, Affiliated Hospital of Qingdao University, Qingdao, China b Department of Orthopedics, Affiliated Hospital of Qingdao University, Qingdao, China c Department of Stomatology, Affiliated Hospital of Qingdao University, Qingdao, China *Corresponding authors: e-mail address: [email protected]; [email protected]
Contents 1. Structures of immunoglobulin 2. Glycosylation of IgG and its significance 2.1 Core fucose 2.2 N-acetylglucosamine 2.3 Sialic acid 2.4 Galactose 3. Production of therapeutic monoclonal antibodies 4. FDA approved antitumor therapeutic monoclonal antibodies 5. Conclusions and perspectives Acknowledgments Conflict of interest References
2 3 5 5 6 7 7 8 10 11 11 11
Abstract The therapeutic market for monoclonal antibodies (MAbs) has grown exponentially since 2000. It is expected that the world-wide market for MAbs could reach $125 billion in 2020. For cancer treatment alone, more than 30 MAbs have been approved by the US Food and Drug Administration since 1997. Unlike structure-defined small moleculebased anti-cancer drugs, the expensive MAb is a mixture of heterogeneously glycosylated proteins. All MAbs typically have a single N-glycosylation site on each of the Fc region. The clinical efficacy of the MAbs depends on the N-glycan structures. Loss of N-glycosylation on the MAbs leads to the loss of the ability to activate complement, to bind to Fc receptors, and to induce antibody-dependent cellular cytotoxicity (ADCC). Moreover, antigen-antibody complexes produced from N-glycan-deficient MAbs are failed to be eliminated rapidly from the blood circulation. Even in certain cases, the N-glycan heterogeneity does not significantly influence pharmacokinetics or half-life of MAbs, reduced terminal galactosylation decreases complement-dependent
Progress in Molecular Biology and Translational Science ISSN 1877-1173 https://doi.org/10.1016/bs.pmbts.2019.03.004
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2019 Elsevier Inc. All rights reserved.
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cytotoxicity, the absence of core fucosylation enhances ADCC due to the increased affinities for the FcγRIIIа receptor, and high sialylation levels reduce ADCC activity and impact inflammatory responses. Furthermore, only mammalian cell lines that make human-like N-glycan structures can be used for MAbs production since certain mammalian cell lines can produce non-human glycan epitopes such as galactose-α-1,3galactose and N-glycolylneuraminic acid (NGNA), which can trigger unwanted immune response. Therefore, mastering the knowledge of N-glycan structures and glycobiology is the key to produce and provide patients with reliable MAbs with consistent glycosylation profile and expected clinical efficacy.
1. Structures of immunoglobulin Immunoglobulin, also known as antibody, is mainly produced by plasma B cells. Antibodies are a key component of the adaptive immune response and play a central role in both in the recognition of foreign antigens and the stimulation of an immune response. In mammals, there are five isotypes of antibodies known as IgA, IgD, IgE, IgG, and IgM.1 The content and distribution of the five isotypes of antibodies in human body are obviously different (Table 1). At present, almost all commercially available recombinant monoclonal antibody drugs are IgG class.2 During the past 30 years, IgG has been proved to be the most promising antibody isotype for tumor immunotherapy due to its potent antibody-dependent cellmediated cytotoxicity (ADCC) and the complement dependent cytotoxicity (CDC).3,4 IgG can be divided into four subclasses based on disulfide bonds and structural differences in heavy chain region: IgG1, IgG2, IgG3 and IgG4, which respectively account for approximately 60%, 25%, 10% and 5% of Table 1 Isotypes and characteristics of immunoglobulins in human. Characteristics IgA IgD IgE IgG
IgM
Serum content (mg/dL)
200
3
0.04
1200
120
Relative amount (%)
10–15
0.05
0.03
75–85
5–10
Synthesis rate [mg/(kg*d)]
24
0.4
0.02
33
7
Decomposition rate (%/d)
25
37
71
7
8
Half-life (d)
6
3
2
23
5
Intravascular distribution (%)
50
75
50
50
80
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serum IgG.5,6 The four subclasses have highly homologous constant region sequences, however, antigen-binding capacity, formation of immune complexes, complement activation, triggering effector cells, half-life and placental transport characteristics are very distinct.7 X-ray crystallographic study showed the similar spatial structure in IgG1, IgG2, IgG3 and IgG4, which is composed of two identical heavy chains (50–70 kDa) and two light chains (23 kDa) to form Y-shaped molecular.8 Two identical heavy chains and two identical light chains connect via disulfide bonds formed by cysteine. The variable region is near N-terminal of IgG light chain (VL), and C-terminal of light chain is constant region (CL). Meanwhile, there are one variable region (VH) and three constant regions (CH) in heavy chain of IgG.1 In consideration of function, IgG can be divided into two parts: two antigen-binding fragments (Fab), which are responsible for antigen binding, and one crystallizable fragment (Fc), which interacts with host receptors. The two moieties are connected through a flexible linker (the hinge region) (Fig. 1). Ligands of Fc include three structurally homologous cellular Fc receptor types (FcγRI, FcγRII and FcγRIII), the C1q component of complement and the neonatal Fc receptor (FcRn).9–12 By binding to specific ligand, IgG mediates different physiological effects including recognition of opsonized particles (binding to FcγR), lysis of cells (binding to complement), and degranulation of mast cells, basophils, and eosinophils (binding to FcRn).13
2. Glycosylation of IgG and its significance As the most abundant glycoproteins in human serum, correct glycosylation modification is essential for IgG to exert its physiological function. Studies have revealed that each IgG monomer contains an average of 2–3 N-glycosylation sites, of which the N-glycosylation site is conserved on the 297 asparagine (Asn).14–18 Loss of N-glycosylation on the MAbs either by treating the cells with tunicamycin19 or site-directed mutagenesis20 leads to the loss of the ability to activate complement, to bind to Fc receptors, and to induce antibody-dependent cellular cytotoxicity (ADCC). Moreover, antigen-antibody complexes produced from N-glycan-deficient MAbs are failed to be eliminated rapidly from the blood circulation.19 Besides that, the hinge region of IgG3 may contain 4 or 6 O-glycosylation sites, which is very specific in IgG subclasses and the significance of O-glycosylation in IgG3 has not been elucidated.4
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Fig. 1 See figure legend on opposite page.
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Although the N-glycan chains on Fc and Fab are predominantly biantennary complex, their glycoforms are quite different. The former contains a higher proportion of core fucose, while the latter contains a higher proportion of galactose, sialic acid and N-acetylglucosamine (GlcNAc).18 Due to non-template driven biosynthesis, the glycan structures are affected by a series of glycosyltransferases, nutrition status, and environmental factors.21–29 The decrease of galactosylation on IgG is caused by the downregulation of galactosyltransferase activity or expression in plasma cells, instead of removal by galactosidase in serum.30,31 β-galactoside alpha-2,6sialyltransferase 1 (ST6Gal1) in serum can regulate the degree of terminal sialylation of IgG molecule, and inflammatory factors such as IL-1β, IL-6 and TNF-α can influence the expression of alpha 2–6 sialytransferase, as well as fucosyltransferase VI.32,33 Common N-glycan structures detected in the recombinant monoclonal antibodies (MAbs) are shown in Fig. 2. Studies have shown that different N-glycoforms of MAbs has certain effects on their stability, clearance, immunogenicity, ADCC and CDC.34,35
2.1 Core fucose Core fucose refers to the fucose residue connected to the N-acetylglucosamine (GlcNAc) directly linked to Asn. It is estimated that about 94% Fc fragment of human IgG contains core fucose modification.18 Meanwhile, studies have proven that lack of core fucose on human IgG1 N-glycan improves binding to human Fcγ RIIIa, and enhances the ADCC activity.36–38 Additionally, it was reported that core fucosylation could increase the CDC activity.39
2.2 N-acetylglucosamine N-acetylglucosamine (GlcNAc) is the principal framework of N-glycan. The role of GlcNAc is essential for half-life of MAbs in plasma. Receptors like mannose receptor (MR) and asialoglycoprotein (ASGPR) receptor can Fig. 1 Subtypes and the structural domains of IgG. Adapted from references Wada R, Matsui M, Kawasaki N. Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms. mAbs. 2019;11(2):350–372; Cong Y, Hu L, Zhang Z, et al. Analysis of therapeutic monoclonal antibody glycoforms by mass spectrometry for pharmacokinetics study. Talanta. 2017; 165:664–670; Ucakturk E. Analysis of glycoforms on the glycosylation site and the glycans in monoclonal antibody biopharmaceuticals. J Sep Sci. 2012;35(3):341–350; Ivarsson M, Villiger TK, Morbidelli M, Soos M. Evaluating the impact of cell culture process parameters on monoclonal antibody N-glycosylation. J Biotechnol. 2014;188:88–96.
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Fig. 2 (A) N-glycoforms detected in MAbs. The common glycoforms detected include MAN5, G0, G1, G2, G0F, G1F, G2F, and G2FS. (B) Different N-glycoforms on antibodydependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Even in certain cases, the N-glycan heterogeneity does not significantly influence pharmacokinetics or half-life of MAbs, reduced terminal galactosylation decreases complement-dependent cytotoxicity, the absence of core fucosylation enhances ADCC due to the increased affinities for the FcγRIIIа receptor, and high sialylation levels reduce ADCC activity and impact inflammatory responses. Panel A: Adapted from Lan Y, Hao C, Zeng X, et al. Serum glycoprotein-derived N- and O-linked glycans as cancer biomarkers. Am J Cancer Res. 2016;6(11):2390. Panel B: Adapted from Ivarsson M, Villiger TK, Morbidelli M, Soos M. Evaluating the impact of cell culture process parameters on monoclonal antibody N-glycosylation. J Biotechnol. 2014;188:88–96; Gerngross TU. Advances in the production of human therapeutic proteins in yeasts and filamentous fungi. Nat Biotechnol. 2004;22(11):1409–1414.
specifically bind to the GlcNAc residues in glycoproteins. GlcNAc is able to bind to MR and then be removed, which reduces the half-life of glycoprotein in the blood circulation.40–43 Furthermore, IgG-complement system will be activated after terminal GlcNAc residues binding to MR.44
2.3 Sialic acid The role of sialic acid residue as a modulator of the anti-inflammatory activity of IgG has been proven.45,46 Its potential mechanism could be: 1. terminal sialic acid residue can up-regulate activation threshold of innate immunocytes, like macrophage, through inducing the expression of inhibitory Fcγ R II B receptors, and make IgG has anti-inflammatory activity.47 The terminal sialic acid residue on Fc fragment hinders the binding of
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antibody to complement C1q, which reduces the deposition of C3b on the cell surface, and results in the down-regulation of the CDC effect of the antibody.48
2.4 Galactose The N-glycans on Fc fragment of human IgG are mainly of biantennary complex type. So, there could be 0, 1 or 2 galactose residues in the N-glycans (Fig. 2). In Hodoniczky group’s study, they found that the terminal galactose on the FC fragment had high affinity to complement C1q.49 Therefore, a higher contents of terminal galactose residues would result in an enhanced CDC potency of IgG antibody.
3. Production of therapeutic monoclonal antibodies At early stage, antibodies are only tools for scientific research and play a role in immunological research, such as immunoblotting, ELISA, IHC, and immunoprecipitation. The concept of using antibodies as therapeutics was firstly proposed in 1890 by von Behring and Kitasato.50 However, the real boosting of therapeutic antibodies was the development of monoclonal hybridoma technology by Kohler and Milstein in 1975.51 Due to this technology, the first therapeutic monoclonal antibody was approved in 1986 for human use (muromonab-CD3).52 While hybridoma technology is highly efficient and productive, but it did not solve the problem of immunogenicity until the emergence of antibody display technology.53–55 Antibody display (e.g., phage display, yeast display, and ribosome display) utilizes a library of carrier to generate a humanized antigen recognition domains (complementarity determining regions, CDR), which results in a significantly reduced immunogenicity. Historically, the mammalian Chinese hamster ovary (CHO) cell line has played a dominant role, and today 70% of all recombinant therapeutic proteins are produced in CHO cells. The CHO line is selected for human therapeutic production because its glycosylation is relatively simple and similar to that in humans.56 However, immunogenicity is not the only concern in antibody pharmaceutics. Studies on the quality control of antibodies have shown that only proper expression and assembly of each fragment does not guarantee the quantity and quality of the antibody.57 A particular focus is glycosylation, as it has been shown that biological activities vary between different natural glycoforms, and consequently, biological engineering has been performed to make proper MAb-producing cell lines (CHO, NS0, Sp2/0
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cell lines, and so on).58 Currently, CHO cells, including the DUXB11, DG44, and CHO-K1 lineages, are the most heavily used for production of MAbs due to following features: (1) stable expression over long incubation time; (2) suitable for scale-up; (3) able to perform the necessary posttranslational modifications; (4) human safety.57,59 The main glycoform produced by CHO, NS0, Sp2/0 cell lines is the G0F glycoform, which indicates the proportion of galactosylated IgG Fc is low relative to normal IgG Fc.60 A particular concern in recombinant MAbs produced by engineered cells is the addition of galactose in α(1–3) linkage to galactose linked β(1–4) to the GlcNAc residues, whose structure has not been observed in human and higher primates.61 Similarly, CHO, NS0 and Sp2/0 cells can add an α(2–3)-linked N-glycylneuraminic acid that is not present in humans either and that might also be immunogenic.62 To produce MAbs with appropriately glycosylated IgG Fc and IgG Fab sites is a challenge to pharmaceutical enterprises. So, some companies try to address this problem by engineering out VH or VL glycosylation motifs when present in candidate MAbs.63
4. FDA approved antitumor therapeutic monoclonal antibodies Antitumor monoclonal antibody therapy is an important branch of tumor immunotherapy. The development of biotechnology has promoted the improvement of therapeutic MAbs. According to the format, it can be divided into murine MAbs, chimeric MAbs, humanized MAbs, and human MAbs.64–67 Since the first antitumor therapeutic MAb (Rituximab) was approved to treat non-Hodgkin lymphoma by targeting at CD20 on B cells in 1997, so far there has been nearly 30 antitumor MAbs approved by FDA (Table 2).4,35,68 As targeted drugs, antitumor therapeutic MAbs usually target at highly expressed surface antigens of tumor cells, like CD20, EGFR, and PD-L1, which could play a role in tumorigenesis, angiogenesis, and immunosuppression.69–71 The clinical trials have proved the efficacy of MAbs in alleviating the progression of disease in cancer patients. In NSCLC clinical trial of PD-1 and PD-L1 MAbs, data showed that patients received both MAbs treatment combined with chemotherapy exhibit the highest objective response rate (ORR, 71%).72 Furthermore, the median overall survival (OS) is 12.7 months with pembrolizumab (PD-1) 10 mg/kg, and 8.5 months with docetaxel 75 mg/m2.73 Similarly, a 11-years’ follow-up study of
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Antitumor therapeutic monoclonal antibodies
Table 2 The antitumor therapeutic MAbs approved by FDA from 1997 to 2017. Year of Antibody Target Indication Format approval
Rituximab
CD20
non-Hodgkin lymphoma
Chimeric IgG1
1997
Trastuzumab
HER2
Breast cancer
Humanized IgG1
1998
Alemtuzumab
CD52
Chronic lymphocytic leukemia
Humanized IgG1
2001
Ibritumomab
CD20
B cell non-Hodgkin’s lymphoma
Murin IgG1
2002
Tositumomab
CD20
non-Hodgkin lymphoma
Murine IgG2a
2003
Bevacizumab
VEGF
Colorectal cancer
Humanized IgG1
2004
Cetuximab
EGFR
Colorectal cancer
Chimeric IgG1
2004
Panitumumab
EGFR
Colorectal cancer
Human IgG2 2006
Ofatumumab
CD20
Chronic lymphocytic leukemia
Human IgG1 2009
Ipilimumab
CTLA-4
Metastatic melanoma
Human IgG1 2011
Brentuximab
CD30
Hodgkin lymphoma
Chimeric IgG1
2011
Pertuzumab
HER2
Breast cancer
Humanized IgG1
2012
Obinutuzumab
CD20
Chronic lymphocytic leukemia
Humanized IgG1
2013
Trastuzumab
HER2
Breast cancer
Human IgG1 2013
Denosumab
RANKL
Giant cell tumor of bone
Human IgG1 2013
Ramucirumab
VEGR2
Gastric cancer
Human IgG1 2014
Pembrolizumab PD-1
Melanoma
Humanized IgG4
2014
Blinatumomab
Acute lymphoblastic leukemia
Bi-specific antibody
2014
CD19, CD3
Continued
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Table 2 The antitumor therapeutic MAbs approved by FDA from 1997 to 2017.—cont’d Year of Antibody Target Indication Format approval
Nivolumab
PD-1
Metastatic melanoma
Human IgG4 2014
Nivolumab
PD-1
Lung cancer
Human IgG1 2015
Daratumumab
CD38
Multiple myeloma
Human IgG1 2015
Elotuzumab
SLAMF7
Multiple myeloma
Humanized IgG1
2015
Dinutuximab
GD2
Neuroblastoma
Chimeric IgG1
2015
Necitumumab
EGFR
NSCLC
Human IgG1 2015
Olaratumab
PDGFR-α Soft tissue sarcoma
Human IgG1 2016
Atezolizumab
PD-L1
Bladder cancer
Humanized IgG1
Avelumab
PD-L1
NSCLC
Human IgG1 2017
Durvalumab
PD-L1
Metastatic urothelial carcinoma
Human IgG1 2017
Inotuzumab ozogamicin
CD22
Acute lymphoblastic leukemia
Humanized IgG1
2016
2017
HER2-positive breast cancer treated with trastuzumab revealed that 1 year of adjuvant trastuzumab after chemotherapy for patients with HER2positive could significantly improve long-term disease-free survival (HR 076, 95% CI 068-086) compared with observation group.74 Due to the high efficacy, research and development of antitumor MAbs have competed fiercely in pharmaceutical enterprises. To discover novel targets, and to explore new functions of existing targets and interaction between multiple targets are the major tasks of biotechnology and bioengineering in the future.
5. Conclusions and perspectives Understanding the significance of the heterogeneity of MAb glycoforms has been a major challenge for the manufacturer to produce bioactive antibody products. Unlike protein and nucleic acid, the synthesis of
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glycans is a non-template driven process, the glycan structures are determined by both genes and environment. At the same time, minor changes in the number, structure and the glycosylation site may greatly alter the structure, thermal stability, solubility, secretion and biological functions of antibodies. With the development of mass spectrometry, nuclear magnetic resonance and lectin chip technologies, the analysis of N has made great progress. According to the final analyzed object, the analysis method of N-glycan chain mainly includes three kinds: (1) carbohydrate chain or monosaccharide; (2) glycopeptide; (3) glycoprotein.75–78 Based on structure knowledge, it opens up opportunities for tailoring antibody therapeutics to maximize their efficacy. Increase in ADCC activity observed for non-fucosylated rituximab and trastuzumab in in vitro whole-blood assays suggests that they may be similarly efficacious in vivo.79,80 Though higher efficacy obtained by such modification, it could have a downside in that increased and/or additional unwanted side effects may occur for example, release of inflammatory mediators and cytokines.81 Therefore, the in-depth study of glycosylation modification of IgG can better understand the immune status of the body, and promote the development of antitumor therapeutic monoclonal antibody-based drugs.
Acknowledgments This research was supported by the Natural Science Foundation of China (Grant 81,672,585), Key Technology Fund of Shandong Province (Grant 2016ZDJS07A07), the Taishan Scholar Fellowship, and the “Double First Class fund” of Shandong Province in China to L.Z.
Conflict of interest The authors declare no conflict of interest.
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