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Attempts to synthesize homogeneous glycan-conjugated antibody-drug conjugates
2020 年 2 巻 3 号 p. 84-89
詳細
2020 年 2 巻 3 号 p. 84-89
Antibody-drug conjugates (ADCs) are considered as next-generation antibody medicine. At present, heterogeneous ADCs are available; however, their safety and efficacy are suboptimal. Homogeneous ADCs offer increased efficacy, reproducibility, and predictability of their pharmacokinetic and pharmacodynamic effects. The presence of N-glycan in the fragment crystallizable (Fc) region affects antibody function and understanding the mechanisms underlying this effect is critical for improvement of antibody therapy. In this mini-review, we described recent progress towards creation of homogeneous glycan-conjugated ADCs. The glycan-conjugated ADCs possess homogeneity in their drug-antibody ratio and structure. In addition, homogeneous glycan-conjugated ADCs exhibit potent cytotoxicity in vitro.
· A homogeneous antibody-drug conjugate preparation is highly required.
· Glycan affects antibody functions.
· Glycan-conjugation by combination of ENGases and ENGase mutants is one method of creating homogeneous ADCs.
Antibody-drug conjugates (ADCs) are next-generation antibody medicine [1]. Currently, seven ADCs including brentuximab vedotin, trastuzumab emtansine, polatuzumab vedotin, gemtuzumab ozogamicin, inotuzumab ozogamicin, enfortumab vedotin, and trastuzumab deruxtecan are available. Furthermore, there are more than 80 ADCs in clinical trials.
For preparing ADCs, a potent cytotoxic agent is conjugated to an antibody via a linker; the antibody binds to a specific antigen on the surface of a cancer cell. After internalization of ADC into cancer cells, the cytotoxic agents are cleaved to damage the cells. Thus, ADCs are expected to expand the narrow therapeutic index of cytotoxic agents.
Drug payloads are generally connected via amino group of Lys residues or sulfhydryl group of Cys residues. Typically, an antibody contains 90 accessible Lys residues with hydrophilic amino group located on the antibody surface. The conjugation reaction between Lys residues and drug payloads proceeds without forcing conditions. Since the drug-antibody ratios (DARs) of ADCs typically range from 2–4, up to 106 distinct species are generated in Lys-conjugated ADCs. Furthermore, addition of hydrophobic payloads to hydrophilic amino groups leads to alteration of the properties of antibodies. At present, the sulfhydryl group of Cys is the preferred conjugation site. Four disulfide bonds are cleaved under reductive conditions, yielding eight SH groups. Cys residues are less abundant than Lys residues, but this methodology still generates ~15 distinct ADCs. Heterogeneous ADCs may differ in their DAR and conjugation-site. Several studies have reported that difference in conjugation-site causes difference in stability and clearance rates of the generated ADCs [2, 3]. Therefore, each species may have different properties, which may result in a wide range of in vivo pharmacokinetic properties and activities. This can result in a narrow therapeutic index with major pharmacokinetic implications. In addition, batch-to-batch consistency in ADC production can be challenging and may require diligent manufacturing capabilities. To expand the therapeutic windows, ADC synthesis must be reproducible and regulated [4]. Although the FDA has approved certain heterogeneous ADCs, this non-selective approach could be considered suboptimal. For these reasons, the development of homogeneous ADCs is highly required.
IgG carries an N-glycan linked to each Asn297 in the fragment crystallizable (Fc) region. To prepare homogeneous ADCs, we focused on N-glycan as a conjugation site for cytotoxic agents. N-glycan is suitable as a payload conjugation site because it is far from the variable region. Additionally, the structure of glycan structure affects antibody function. For instance, absence of core-fucose enhances antibody-dependent cellular cytotoxicity (ADCC), and the addition of non-human type sugars may increase immunogenicity [5, 6]. In addition, N-glycan in IgG is heterogeneous because of its complex biosynthetic pathway. From these aspects, N-glycan on antibodies could also be a target of regulation. In this mini-review, the preparation of homogeneous ADCs through glycan-remodeling is described.
Sialic acid and fucose, a carbohydrate unit of N-glycan, have 1,2-cis diol structures and 1,2-cis diol can be oxidatively cleaved to generate a carbonyl group. Because the target antibody does not contain any carbonyl groups, it is possible to attach a payload to a generated carbonyl group through addition of an aminooxy or hydrazine group (Fig. 1(1)). The oxidation reaction was monitored through electrospray ionization-mass spectrometry (ESI-MS) and it typically involved the addition of a mass unit of 14 Da. An aminooxy group carrying the payload was conjugated to the carbonyl group [7, 8]. The acyclic diol moiety of sialic acid is flexible. In addition, sialic acid is located in non-reducing termini. Therefore, the sialic acid diol is oxidized under mild conditions. Since normally IgG contains low-levels (<20%) of sialic acid, the amount of sialic acid was increased using sialyl transferase and cytidine monophosphate sialic acid [9]. However, this strategy cannot reduce the heterogeneity of N-glycans. Additionally, careful control of reaction conditions is required, because overoxidation of aldehyde to carboxylic acid and oxidation of Met and Trp may occur as side-reactions.
Existing strategies for preparation of homogeneous antibody-drug conjugate (ADC). (1) Oxidative cleavage of 1,2-cis diol strategy, (2) monosaccharyl transferase strategy, (3) metabolic modification strategy.
An alternative approach for preparation of glycan-conjugated ADCs is use of monosaccharyl transferase (Fig. 1(2)). Galactosyl transferase and sialyl transferase, and their mutants are used for incorporation of handle for conjugation-site, because they can tolerate modified galactose and sialic acid as substrates. Although the wild-type enzyme has poor catalytic efficiency for substrates larger than galactose, mutant galactosyl transferase β (1,4) Gal-T1-Y289L could transfer C2-keto-galactose [10,11,12,13,14]. In addition to C2-keto-galactose, azide-carrying N-acetylgalactosamine (GalNAz derivative) was also transferred. Because the azide group does not exist in biomolecules and it is reactive to alkyne, this group is often employed as the payload conjugation site. Once monosaccharyl transferases transferred the azide-carrying monosaccharide, azido group modified N-glycan. Then, the bioorthogonal reaction between azide and the payload carrying alkyne led to the formation of an ADC, with the payload linked to the glycan.
Sialyl transferases can tolerate modifications at positions C5 and C9 of sialic acid. A sialic acid molecule with an azide group was incorporated to non-reducing termini of glycan and the payload was transferred by sialyl transferase I and 9-azide-functionalized cytidine-5′-monophospho-sialic acid [15]. Although the payload was specifically conjugated to N-glycan using monosaccharyl transferase, heterogeneity of N-glycan remained.
Recently, remodeling of N-glycan of antibody using a combination of endo-β-N-acetylglucosaminidase (ENGase) and its mutant has been reported [16]. In addition to monosaccharyl transferase, ENGase mutants can be used for glycan remodeling and payload conjugation. ENGase is a glycosidic hydrolase that acts on the β-1,4-glycosidic linkage within the N,N’-diacetylchitobiose core of N-glycans. The hydrolytic activity of ENGase mutants is suppressed, yet they have en block glycan transfer ability. Several ENGase mutants have been used for glycan remodeling. The scope and limitation of ENGase mutants has become clear. For instance, Endo M- and Endo CC-derived mutants do not transfer glycan to core-fucose-containing antibodies [17]. In contrast, EndoS- and EndoS2-derived mutants transfer glycan to core-fucose-containing antibodies.
II-2. Our strategy for creating a homogeneous ADC by ENGaseOur strategy of preparing homogeneous ADC though glycan-remodeling is as follows (Fig. 2):
Our strategy for creating a homogeneous antibody-drug conjugate (ADC) by ENGase
In this step, one N-acetylglucosamine (GlcNAc) at reducing end is still attached to Asn297. The heterogeneous and immunogenic moiety is removed in this step.
2) Preparation of azide group carrying N-glycan donor:Human-type oligosaccharide from sialylglycopeptide isolated from egg yolk was used. The azide group was introduced to a carboxyl acid group in sialic acid via amido bond formation. The azido group is the functional group required for the bioorthogonal reaction in the payload conjugation step. To transfer glycan, an oxazoline donor was prepared. During hydrolysis, 2-acetamide groups reacted with the substrate, leading to the formation of oxazolidium ions. Thus, the transition analogue glycan-oxazoline works as a donor for the ENGase mutant. Although sialylglycopeptide can also be used as donor for ENGase mutants, its efficacy is normally low.
3) Transfer of azide-carrying glycan to antibody:The azide-carrying glycan oxazoline was attached to the glycan-truncated antibody via an ENGase mutant. After glycan transfer, the azide group was only present at Asn297.
4) Preparation of ADCs:Bioorthogonal addition of azide and strained alkyne with payload led to the formation of homogeneous ADCs. Finally, structurally and functionally homogenous ADCs possessed a DAR of 4.
We synthesized homogeneous ADCs based on trastuzumab using EndoS as ENGase, and EndoS D233Q as ENGase mutant using the above strategy [18]. Homogeneity of glycan-trimming and glycan-addition reactions was confirmed through hydrophilic interaction chromatography (HILIC). Finally, monomethyl auristatin E (MMAE) with cathepsin B cleavable linker was conjugated between azide and strained alkyne. The synthesized ADCs were fully characterized via ultra-performance liquid chromatography (UPLC), peptide mapping, and ESI-MS to show homogeneity. The ADCs were highly toxic to N-87, OE-19, and SK-BR-3 cells, and to cells expressing HER2 at high levels. Conversely, ADCs were not toxic to MKN-45 and MCF-7 cells, and to cells expressing HER2 at low levels. These results indicate that homogeneous ADCs can be prepared through glycan-conjugation and the generated ADCs expand the therapeutic index. Similar results have been reported for this method [19,20,21].
Careful control of the reaction conditions is required when glycan-oxazoline is used for glycan addition, as the highly reactive oxazoline reacts with the amino group of Lys residues under basic conditions. Slightly acidic conditions and portion-wise addition of oxazoline suppress the side reactions [21, 22].
Furthermore, to avoid the formation of highly active oxazoline donor, one-pot glycan transfer reaction through combination of stable sialylglycopeptide with Endo M, EndoS D233Q/Q303L, and EndoS D233Q/E351Q was developed [23]. Oxazoline generated from sialylglycopeptide by Endo M via hydrolysis was transferred in situ to glycan-truncated antibody in the presence of EndoS D233Q/Q303L and EndoS D 233Q/E350Q. Side-reactions were suppressed by diluting the concentration of oxazoline.
II-3. Metabolic modification strategyAnother approach for glycan-conjugated ADC preparation is metabolic modification of glycan [24]. Unnatural sugars are metabolically incorporated into antibody glycan during preparation of antibody in CHO cells (Fig. 1(3)). Although fucosyl transferase tolerates modified fucose as a donor, sulfhydryl-, ethyl-, thioacetyl-, chloro-, or fluoro-carrying fucose was incorporated into core-fucose, but alkyne-carrying or 2-deoxy-2-fluoro fucose was not incorporated. Sulfhydryl-functionalized acetylated fucose was added outside the cells, and subsequently, the acetyl group was hydrolyzed in the cytosol and converted to guanosine 5′-diphospho-β-L-fucose through the fucose salvage pathway. Unfortunately, the sulfhydryl group in the core-fucose formed a disulfide bond with Cys outside and full-reduction and oxidation processes were utilized for creating a sulfhydryl group only in the core-fucose. However, the other sulfhydryl group on the Cys residues again formed a disulfide bond. Therefore, a maleimide-carrying payload was conjugated to a sulfhydryl group at core-fucose. ADCs prepared through this method had improved homogeneity compared to those prepared via conventional hinge disulfide reduction-maleimide conjugation method.
The strategy we employed for N-glycan conjugation is ideal for the preparation of homogeneous ADCs and homogenizes N-glycan. Other methods for the preparation of homogeneous ADCs have been reported. For instance, in vitro protein synthesis using unnatural amino acids to incorporate azide or carbonyl group, and peptide sequence recognition and conjugation using enzymes such as sortase and formylglycine [25,26,27]. However, the glycan conjugation strategy has the most advantages and it can be used to control the N-glycan structure.
There are many issues left to be addressed regarding antibody-drug conjugation. Analysis of ADCs has been difficult, especially in the case of a mixture of heterogeneous ADCs. However, the analytical methodology used to determine homogeneity has been improved. For instance, UPLC is now commonly used for structural analysis. HIC and HILIC mode column chromatography are being utilized for DAR and identification of conjugation-site. Additionally, ESI-MS analysis and rapid antibody digestion methods have been developed.
In addition, choice of bioorthogonal reactions should be considered. Often, aminooxy and hydrazine groups are added to generated carbonyl groups for N-glycan conjugation. However, the generated oxime or hydrazone linkage is susceptible to hydrolysis under physiological conditions. In order to overcome this problem, mercaptoethylpyrazolone or aminobenzamidoxime group are used to enhance stability [28].
In addition to preparing homogeneous ADCs, a mechanism for cleaving the cytotoxic drug payload is important. Linker technology for release of cytotoxic agents at the appropriate location, on appropriate time, and at appropriate dosage is highly required from the viewpoint of organic chemistry.
The author has no conflicts to interest directly relevant to the content of this article.
Our results were supported by Grant-in-Aid for Scientific Research (B) (Grant No. 19H03357) from the Japan Society for Promotion of Science. The author thanks Dr. Yoshiki Yamaguchi (Tohoku Medical and Pharmaceutical University), Dr. Minoru Suda (Fushimi Pharmaceutical Co., Ltd.), Dr. Takashi Kinoshita (Fushimi Pharmaceutical Co., Ltd.), Dr. Kenji Hirose (Nihon Waters, KK), and Dr. Yasuhiro Matsumura for cooperation with glycan-conjugated ADC project.
