Immunogenicity assessment of PEGylated proteins, Lonquex, a PEGylated G-CSF case study

6 Immunogenicity assessment of PEGylated proteins, Lonquex, a PEGylated G-CSF case study Linglong Zou, Steffen Nock BIOLOGIC R&D, TEV A P HARMACEUT ICALS , R EDWOO D CI TY, C A, UNITED STATE S

1. Introduction PEGylation refers to the process of conjugating polyethylene glycol (PEG) polymer chains to molecules, such as small molecule drugs, peptides, or therapeutic proteins, not only via covalent conjugation but also noncovalent strategies have been investigated. In pharmaceutical development, PEGylation remains an established approach to extending the circulation half-life of therapeutic proteins and this technology is expected to continue playing an important role for future therapeutic protein development. The first PEGylated biopharmaceutical product, PEGylated adenosine deaminase, was approved by the US Food and Drug Administration (FDA) approximately 30 years ago for the treatment of severe combined immunodeficiency disease [1]. Additional PEGylated products have subsequently received FDA approval. Some of those products include PEGylated interferon-alpha 2b (PegIntron), PEGylated interferon-alpha 2a (Pegasys), PEGylated granulocyte colony-stimulating factor (G-CSF) (Neulasta), PEGylated epoetinbeta (Mircera), PEG-asparaginase (Oncaspar) and PEGeuricase (Krystexxa) [2,3]. Longterm treatment of patients with these PEGylated products has demonstrated their acceptable safety and efficacy, and there are dozens of PEGylated molecules currently in clinical trials [4,5]. PEG is a chemically inert, highly hydrophilic, and nonbiodegradable polymer, which can be attached to proteins to alter physicochemical and pharmaceutical properties. A variety of PEG molecules are available for use and their molecular masses range from several thousands to tens of thousands of Daltons. Structurally, PEG molecules can be linear or branched [6]. The availability of various PEGs allows for optimized use of the best suited polymer for a given pharmaceutic. Because of its ability to render PEGylated molecules inert from their biological environment, PEGs are frequently referred to as stealth polymers. There are molecular and physical characteristics that underline the

Polymer-Protein Conjugates. https://doi.org/10.1016/B978-0-444-64081-9.00006-1 Copyright © 2020 Elsevier B.V. All rights reserved.

125

126

Polymer-Protein Conjugates

stealth properties of PEG. PEG is extremely hydrophilic, which increases solubility of PEGylated proteins. Additionally, PEG conjugation generates a hydration shell around the conjugated peptide or protein which protects them from enzymatic degradation by proteases [7]. Since its invention, PEGylation has been considered as a major breakthrough in the development of therapeutic proteins [8]. Over the last 25 years, PEGylation technology has evolved to improve specificity and other properties. Three generations of PEGylation have been consecutively introduced [9]. With the first-generation PEGylation technology, linear PEG is conjugated to a biomolecule randomly, producing a mixture of conjugates where the number of polymer chains per protein unit and the sites of attachments are highly variable. Purification and separation of desired conjugate from nondesired species is frequently required during manufacture of PEGylated products. The second-generation technology improved the PEGylation specificity by utilizing activated PEG molecules, which allows for site-specific PEGylation. With the secondgeneration technology, the amount of impurities or side products of PEGylation is dramatically reduced during synthesis of PEGylated products. In addition, higher molecular weight and branched PEG polymers are used in conjugation, prolonging the half-lives of PEGylated products compared to the first-generation technology, which is mostly based on the incorporation of linear PEG molecules. While it increases half-lives, the use of branched PEGylation can reduce the biological activity of biomolecules because of steric hindrance. The arguable third-generation PEGylation technology introduced customized linkers for conjugation to minimize the loss of a drug’s bioactivity while extending the half-life of a PEGylated product. For example, a PEGylated prodrug can be created by introducing a cleavable linker between the drug of interest and the PEG. When such molecule is delivered in vivo, the drug can be released from the conjugate through cleavage of the linker [10]. The most pronounced benefit of protein PEGylation is the extension of the circulating half-lives of PEGylated molecules. PEGylation extends their half-lives through increasing their molecular size and hydrodynamic radius, thereby reducing renal clearance [8]. Half-life extension, however, is not the only benefit that PEGylation technology can offer. Other benefits can be achieved with such a structural modification. For example, an improved solubility profile was observed for drugs formulated with a PEGylated liposomal solution [11]. PEGylation is also reported to increase the stability of proteins as the structural modification often leads to resistance to proteolysis with or without improved thermal and mechanical stability [12]. The resistance to proteolysis is often acquired, thanks to the steric hindrance of the polymer chains, shielding the protein backbone. In addition to improved solubility and stability, PEGylated molecules often show a reduction of immunogenicity versus un-PEGylated proteins, and this effect of protein PEGylation on immunogenicity has been experimentally confirmed in animals with various PEGylated model proteins [13,14]. The reduced immunogenicity can be due to an indirect effect of improved stability and reduced aggregation of the protein and/or a direct effect through shielding of immunogenic epitopes of a protein from the host

Chapter 6
Immunogenicity assessment of PEGylated proteins 127

immune system by PEG [15]. However, reduced immunogenicity of a PEGylated protein in humans cannot be taken for granted. Given the three-dimensional structure of the PEGylated protein and depending on conjugation site, PEGylation may or may not effectively block immunogenic epitopes from the immune system. In addition, PEG can illicit immune response against itself or the modified site which contains the PEG, as described in next section.

2. Immunogenicity of PEG With the introduction of PEGylation technology, the pharmacological properties of many therapeutic proteins have been improved. However, PEG-associated immunogenicity remains a concern. The immune response against PEG was reported more than 30 years ago in animals following intramuscular or subcutaneous injections of various PEGmodified proteins in Complete Freund’s Adjuvant [16]. Subsequently, anti-PEG antibodies were documented in human subjects after administration of various PEGylated proteins. High prevalence of preexisting antibodies against PEG was also reported in humans. In several studies, anti-PEG IgM antibodies were detected in 5%e40% of healthy individuals tested [17e19]. There appears to be a trend of increasing prevalence over time, as low prevalence was reported in early studies [17] and higher prevalence in more recent studies [20]. This is possibly because of improved assay sensitivity for the detection of anti-PEG antibodies and/or the increased use of PEG-containing products. PEG is proposed to be a hapten and therefore should only trigger an immune response when conjugated to a larger carrier such as a protein. Yang and Lai suggested that the epitope the anti-PEG antibodies bind to could be smaller than six to seven PEG subunits in length and that this epitope most likely resides at the interface between the PEG and its protein moiety [20]. Nowadays PEG-containing surfactants or even PEG itself is widely used in household and hygiene products (e.g., soap, shampoo, toothpaste, lotion, detergent). Frequent exposure to these PEG-containing products could naturally lead to the formation of anti-PEG antibodies, especially when PEG exposure occurs along with microbe exposure, forming a PEG conjugate as an immunogen. In animals, anti-PEG immune responses are predominantly IgM responses, independent of T-helper cell stimulation [21]. On the contrary, the anti-PEG immune responses in humans are a mix of IgM and IgG responses, indicating both T and B cell involvement. For example, Armstrong observed that 19%, 5%, and 3% of the individuals tested possess IgG only, IgM only, and both IgM and IgG preexisting anti-PEG antibodies, respectively [19]. The consequence of both preexisting and induced anti-PEG antibodies on patients treated with PEGylated proteins is not clear. It may vary from one case to another. A number of publications have shown that induced anti-PEG antibodies are linked to enhanced blood clearance and reduced efficacy of the products [22]. Preexisting anti-PEG antibodies have also been correlated with the loss of therapeutic efficacy in two PEGylated products, PEG-asparaginase (PEG-ASNase) and

128

Polymer-Protein Conjugates

PEG-urate oxidase (PEG-uricase, pegloticase), due to increased clearance [20]. In addition, anti-PEG antibodies are linked to an increase in adverse effects in several clinical studies examining different PEGylated therapeutics [23]. As the presence of preexisting anti-PEG antibodies and drug-induced immunogenicity of PEG could have many implications for the development of PEGylated therapeutic proteins, immunogenicity of the PEGylated product will have to be evaluated thoroughly. This includes the evaluation of preexisting anti-PEG antibodies and treatment-induced anti-PEG antibodies. In addition, as PEGylated products are multiple domain products, regulatory agencies, such as the FDA, expect that the immunogenicity of PEGylated proteins be evaluated with assays assessing antibody specificity to each component [24]. To understand the effect of PEGylation on the immunogenicity profile of the conjugated protein in clinical trials, an appropriate assessment strategy has to be in place. This includes establishment and implementation of adequate immunogenicity assays such as screening, confirmation, and titer assays. An example of such an immunogenicity assessment strategy is given below based on case study using Lonquex, a PEGylated G-CSF.

3. Lonquex Lonquex (drug name: lipegfilgrastim) is a PEGylated recombinant human G-CSF. Human G-CSF promotes neutrophil production, maturation, survival, and activity [25]. Recombinant G-CSFs, such as filgrastim, are commonly used for the prevention and treatment of neutropenia in patients receiving myelosuppressive chemotherapy [26]. However, recombinant G-CSFs, without structural modification, are short-acting agents because of their short half-life (3e4 h), warranting a daily administration regimen [27]. PEGylated G-CSFs were therefore developed to extend the circulating half-life of G-CSF. Lonquex was developed by Teva Pharmaceuticals Ltd. as a long-acting G-CSF and offers an alternative to pegfilgrastim. Based on a randomized, double-blind, active-controlled, phase 3 trial evaluating the efficacy and safety of lipegfilgrastim, the noninferiority of lipegfilgrastim to pegfilgrastim in the treatment of severe neutropenia was demonstrated in 202 chemotherapy-naive patients with breast cancer [28]. Lonquex was approved in the European Union in 2013 as once-per-cycle, fixed-dose prophylaxis for severe neutropenia with the following indication: reduction in the duration of neutropenia and the incidence of febrile neutropenia in adult patients treated with cytotoxic chemotherapy for malignancy (with the exception of chronic myeloid leukemia and myelodysplastic syndromes).

4. Structure and production process of Lonquex Lonquex is a conjugate of recombinant N-methionyl human G-CSF (r-metHuG-CSF) and a single mPEG molecule of 20 kDa. The conjugation is mediated through glyco-PEGylation

Chapter 6
Immunogenicity assessment of PEGylated proteins 129

and occurs at the natural O-glycosylation site at threonine134 of the r-metHuG-CSF. The glycolinker is composed of a sialic acid (SA) and an N-acetylgalactosamine (GalNAc) component. Lonquex structure, with the amino acid sequence of G-CSF and PEGylation site, is schematically shown in Fig. 6.1. The r-metHuG-CSF is produced by recombinant DNA technology in Escherichia coli. Because of its E. coli origin, there is a methionine residue added to the N-terminus, resulting in a total of 175 amino acids. The theoretical molecular mass of r-metHuG-CSF is 18,798.9 Da. After conjugation, the molecular mass of the Lonquex is approximately 39,000 Da. The structure of the linker is shown in Fig. 6.2. Cytidine monophosphate (CMP) SA-PEG is used in chemical synthesis of Lonquex and as depicted in Fig. 6.3, contains CMP, glycyl SA, and a 20-kDa methoxy PEG. CMP is not present in Lonquex as it is cleaved from CMP-SA-PEG during the Lonquex synthesis. This site-specific glycoPEGylation is achieved through sequential reactions involving two recombinant glycosyltransferases with activated sugar nucleotide donor substrates (Fig. 6.3). The first reaction is mediated by N-acetylgalactosaminyl-transferase 2 (GalNAc-T2), which catalyzes the transfer of GalNAc from UDP-GalNAc to the hydroxyl group of threonine-134 of r-metHuG-CSF. UDP is cleaved in this reaction. The second reaction is mediated with alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase 1 (ST6-GalNAc1), which catalyzes the transfer of SA and PEG from CMP-SA-PEG, resulting in glycol-PEGylated r-metHuG-CSF. CMP is cleaved in this step. HOOC- P Q A L H R L V R Y S V E L F S Q L H S 170 160 A V 10 1 L P L S S A P G L P T M -NH2 Q S SH 20 F L L K C L E Q V R K

65

18

37

30 I

Q G D G A A L Q E K L C A

S S

AB-Loop

P C S S L P A W P I G L S H G L L V L E E P H C L Q S 43 60 50 A S L 70 S Q BC-Loop 80 90 L A G C L S Q L H S G L F L Y Q G L L Q A L E G I S P

75 A F D A V D L Q L T D L T P T T I

110 120

130

W Q Q M E E L G M A P A L Q

V G 150 G T A Y R K R Q F A S A E F L 140 A G P M A O NH Q G P

CH2 mPEG 20K methyoxy polyethylene glycol

G

CD-Loop

Glycyl linker Sialic acid (SA) GalNAc

G mPEG

FIGURE 6.1 Amino acid sequence and schematic structure of Lonquex.

130

Polymer-Protein Conjugates

FIGURE 6.2 Structure of the Lonquex glycolinker.

FIGURE 6.3 Lonquex synthesis scheme.

5. Immunogenicity assessment strategy for PEGylated proteins Immunogenicity is a potential concern for any biological product and its assessment is one of the most critical elements for the development of such therapeutics. Antidrug antibody (ADA) formation, as an unwanted immune response due to product immunogenicity, may lead to serious safety consequences that manifest as a hypersensitivity reaction such as anaphylaxis and the development of cross-reactive neutralizing antibodies (NAb) to endogenous proteins [24]. A tiered analysis approach composed of ADA screening, confirmation, tittering, and NAb determination is a standard requirement for

Chapter 6
Immunogenicity assessment of PEGylated proteins 131

characterizing ADA response to a typical protein therapeutic [29,30]. PEGylated proteins are multidomain molecules for which characterization of ADA-binding specificity toward each domain is generally required [31]. For PEGylated therapeutic protein products, FDA requires that the ADA assay is able to detect antibodies against both the protein component and the PEG component [29].

6. Immunogenicity assays of Lonquex As for other PEGylated proteins, the immunogenicity of Lonquex was assessed using threetiered assays for ADA detection and characterization. Characterization assays included binding specificity determination toward protein domain (recombinant human G-CSF) and PEG component (Fig. 6.4). All assays were based on a bridging immunoassay format.

FIGURE 6.4 Sequential approach to assessing immunogenicity.

132

Polymer-Protein Conjugates

6.1

Screening assay

The screening assay was a ligand-binding assay in an electrochemiluminescent bridging format [32]. Briefly, in this assay, ADA in the samples binds to biotin-labeled Lonquex (capture reagent) and ruthenium-labeled Lonquex (detection reaction). This sandwich complex was captured on a streptavidin plate where an electrical current caused the captured ruthenium to emit measureable light. Signal was measured using a Sector Imager 6000 Analyzer (Meso Scale Discovery). The screening assay performance was monitored using the purified rabbit anti-Lonquex polyclonal antibodies as the positive control reagent. Samples with electrochemiluminescence (ECL) counts above or equal to the screening cut-point were defined as screened positive, while samples with ECL count below the screening cut-point were defined as negative samples.

6.2

Confirmatory assay

The specificity of the screened positive samples was confirmed by a competition assay using unlabeled Lonquex. Each sample was analyzed in the presence versus the absence of Lonquex and signal inhibition in the presence of Lonquex was considered an assay response. The confirmatory assay cut-point with a false-positive rate set at 1% was determined using commercially available serum samples from treatment-naive cancer patients. Samples with signal inhibition above or equal to the confirmatory cut-point were defined as confirmed positive samples and then subjected to titer determination, binding specificity characterization, and NAb assays. The samples with signal inhibitions being below the confirmatory cut-point were negative samples and required no further analysis.

6.3

Titer assay

For ADA titer determination, each sample was serially diluted and analyzed to determine the relative amount of anti-Lonquex antibodies. A titer value was reported as the log-transformed dilution factor interpolated at the titer cut-point. The titer assay was only performed on the confirmed positive ADA samples.

6.4

Binding specificity characterization assays

The confirmed positive samples were analyzed to determine if the ADA bounds to the GCSF protein moiety r-metHuG-CSF (also called filgrastim) or the PEG moiety of Lonquex via competition with filgrastim (recombinant nonglycosylated human G-CSF) and CMP-SA-PEG (cPEG), respectively. Availability of competitors for different structural components of Lonquex was critical to characterize binding specificity and dissect epitopes. cPEG was used as the competitor in the cPEG characterization assay to determine if the specificity of anti-Lonquex antibodies was toward PEG and SA components. Although UDP-GalNAc was used in the synthesis of Lonquex, it is a chemically activated compound and is not an appropriate competitor for characterizing antiLonquex antibodies toward the small glycol linker component GalNAc. In addition, UDP is not present in Lonquex. Therefore, only r-metHuG-CSF (filgrastim) and cPEG were used.

Chapter 6
Immunogenicity assessment of PEGylated proteins 133

In addition to analyzing the domain specificity of the ADA response, the cross reactivity of ADA toward the native G-CSF was also determined. This was done using Granocyte (lenograstim, manufactured by Chugai Pharmaceuticals) as assay competitor. Granocyte is a recombinant human G-CSF produced in Chinese hamster ovary cells. Unlike G-CSF moiety in Lonquex, Granocyte is a glycosylated G-CSF that mimics the native (endogenous) G-CSF [33]. In Granocyte, glycosylation occurs at threonine133, the site in endogenous G-CSF that corresponds to the amino acid threonine134 of the r-metHuG-CSF for Lonquex.

6.5

NAb assays

A cell-based assay was used to evaluate neutralizing activity of the ADA according to industry recommendations [34,35]. In this assay, an NFS-60 cell line (mouse myelogenous leukemia cell line adapted to respond to recombinant G-CSF, provided by St. Jude Children’s’ Research Hospital, US) was used with cell proliferation as the functional endpoint. Cell growth is stimulated by Lonquex or Granocyte each at their EC50 level, the concentration of the drug that gave rise to a half-maximal response. Human serum samples were added to the assay medium, and antibodies with neutralizing activity that reduced cell proliferation were measured with the WST-1 reagent [2-(4-iodophenyl)-3(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] as the chromogen. Statistically determined assay cut-points were used at the screening step to determine positivity. The assay initially screened for the presence of neutralizing antibodies against Lonquex or native G-CSF. Each human serum sample was assayed under three conditions (i.e., in the presence of inducer Lonquex, in the presence of inducer Granocyte, and with no inducer). As the cell line is also reacting to murine interleukin-3 (mIL-3), the screened positive samples underwent a mIL-3 confirmatory analysis to determine whether the observed inhibition was truly neutralizing G-CSF rather than a nonspecific inhibition, a practice recommended by FDA guidance [29]. To be considered positive for neutralizing antibodies, a sample should neutralize the activity of either Lonquex or Granocyte, but not caused cell growth in absence of an inducer, and should neutralize the activity of mIL-3. On the other hand, to be considered negative for neutralizing antibodies, a sample should not neutralize the activity of Lonquex or Granocyte and should not cause cell growth in absence of an inducer.

7. Immunogenicity analysis results of Lonquex in patients with breast cancer Immunogenicity assessments were performed by determining ADA incidence, ADA-binding specificity and neutralizing activity, and ADA impact on clinical outcomes in two independent clinical studies. These studies included both Lonquex- and Neulasta-treated patients for comparison. Full immunogenicity results of these studies

134

Polymer-Protein Conjugates

were previously reported [32], and the abbreviated results with a focus on Lonquex immunogenicity are illustrated and discussed here. A total of 208 breast cancer patients undergoing myelosuppressive chemotherapy (intravenous doxorubicin 60 mg/m2 and docetaxel 75 mg/m2) were enrolled in the first study, a phase 2, double-blind, randomized, dose-optimization study evaluating the efficacy, safety, pharmacokinetics, and immunogenicity of Lonquex in comparison to Neulasta. Patients were assigned 1:1:1:1 to receive Lonquex (3.0, 4.5, or 6.0 mg administered via subcutaneous [SC] injection) or Neulasta (6.0 mg SC) once per cycle [36]. A total of 202 breast cancer patients undergoing myelosuppressive chemotherapy were enrolled in the second study, a phase 3, double-blind, randomized, noninferiority study in which patients received either Lonquex (6.0 mg SC) or Neulasta (6.0 mg SC) once per cycle after the chemotherapy regimen [28]. In both studies, patients received the chemotherapy on day 1 of each of four 21-day cycles. All patients were Caucasian and all but three were female. Immunogenicity samples were collected at multiple time points, including baseline, before each chemotherapy cycle, the end of treatment (day 85), and on days 180 and 360 during follow-up period. ADA results from these patients are summarized in Table 6.1. In the phase 2 study, 2 of the 154 Lonquex-treated patients (Table 6.1, patients 1 and 2) had treatment-emergent ADA, representing an incidence of 1.3%. These patients had ADA-positive samples at a single time point (day 85 or day 360) after Lonquex treatment, indicating a transient ADA response. There were seven patients with preexisting ADA, including three with positive samples at both baseline and postdose time points (Table 6.1, patients 3e5) and 4 with Table 6.1

Lonquex-treated patients with ADA-Positive samples in the phase 2 study. Visit

Patient

BL

C2D1

C3D1

C4D1

D85

D180

D360

ET

1a 2a 3 4 5 6 7 8 9

Neg NA Pos Pos Pos Pos Pos Pos Pos

Neg NA Pos Pos Pos NA NA NA NA

Neg NA Pos Pos NA NA NA NA NA

Neg NA Pos Pos NA NA NA NA NA

Pos NA Pos Pos NA NA NA NA NA

NA NA Pos Pos Pos NA NA NA NA

NA Pos Pos Neg Pos NA NA Neg NA

NA NA NA NA Neg NA NA NA NA

a Patient with treatment-emergent ADA. Abbreviations include ADA: antidrug antibody; BL: baseline; C2D1: cycle 2 day 1; C3D1: cycle 3 day 1; C4D1: cycle 4 day 1; D85: day 85; D180: day 180; D360: day 360; NA: screened negative sample; Neg: confirmednegative sample; Pos: confirmed-positive sample.Note: Table includes only patients with ADA-positive samples. Gray background indicates patient with treatment-emergent ADA. ADA, antidrug antibody; BL, baseline; C2D1, cycle 2 day 1; C3D1, cycle 3 day 1; C4D1, cycle 4 day 1; D85, day 85; D180, day 180; D360, day 360; NA, screened negative sample; Neg, confirmed-negative sample; Pos, confirmed-positive sample. Reproduced from Table 1 in Zou L, Buchner A, Field F, Barash S, Liu PM. Immunogenicity assessment of tbo-filgrastim in cancer patients receiving chemotherapy. Bioanalysis 2018;10 1221e28.

Chapter 6
Immunogenicity assessment of PEGylated proteins 135

Table 6.2

Lonquex-treated patients with ADA-Positive samples in phase 3 study. Visit

Patient

Treatment group

S

BL

C2D1

C3D1

C4D1

D85

D180

D360

13a 14 15 16

Lipegfilgrastim Lipegfilgrastim Lipegfilgrastim Lipegfilgrastim

NA NA NA NA

NA Pos Pos Pos

NA Pos Pos NA

NA Pos Neg NA

NA Pos Neg NA

NA Neg Neg NA

Pos Neg Neg NA

Pos Neg Neg NA

a Patient with treatment-emergent ADA. Abbreviations include ADA: antidrug antibody; BL: baseline; C2D1: cycle 2 day 1; C3D1: cycle 3 day 1; C4D1: cycle 4 day 1; D85: day 85; D180: day 180; D360: day 360; NA: screened negative sample; Neg: confirmednegative sample; Pos: confirmed-positive sample; S: screening (predose) time point.Note: Table includes only patients with ADApositive samples. Gray background indicates patient with treatment-emergent ADA. ADA, antidrug antibody; BL, baseline; C2D1, cycle 2 day 1; C3D1, cycle 3 day 1; C4D1, cycle 4 day 1; D85, day 85; D180, day 180; D360, day 360; NA, screened negative sample; Neg, confirmed-negative sample; Pos, confirmed-positive sample; S, screening (predose) time point. Reproduced from Table 1 in Zou L, Buchner A, Field F, Barash S, Liu PM. Immunogenicity assessment of tbo-filgrastim in cancer patients receiving chemotherapy. Bioanalysis 2018;10 1221e28.

positive samples at baseline only (Table 6.1, patients 6e9). In the phase 3 study, 1 of the 101 Lonquex-treated patients (Table 6.2, patient 13) had treatment-emergent ADA, with positive samples at days 180 and 360. This reflects an ADA incidence of 1.0%. There were three patients with preexisting ADA, including one who had a positive sample at baseline only (Table 6.2, patient 16) and two who had positive samples at both baseline and a postdose time point (Table 6.2, patients 14 and 15). The binding specificity and titer of ADA were determined and results are summarized in Table 6.3, with filgrastim representing G-CSF domain, cPEG representing PEG domain, and glycoG-CSF representing native human G-CSF. Among the two patients who exhibited treatment-emergent ADA in the phase 2 study, one had a positive sample on day 85 with an antibody titer of 0.6 against cPEG only. The other patient had a positive sample on day 360. The antibody titer was undetectable in this sample and showed no recognition of filgrastim, glycoG-CSF, or cPEG. Five of the seven remaining patients showed preexisting ADA; two of these five had positive postdose ADA samples as well. One of these patients had low-titer ADA recognizing filgrastim and glycoG-CSF throughout duration of the study. The other patient had ADA recognizing filgrastim and cPEG whose titer diminished over time from 0.9 to 0.3. The remaining three of these five patients had preexisting ADA recognizing filgrastim and cPEG, but no detectable postdose ADA titer. From the phase 3 study, nine confirmed-positive samples from four patients underwent the same characterization and titer assays. One Lonquex-treated patient with a confirmed-positive sample had possible treatment-emergent ADA, recognizing filgrastim and glycoG-CSF, but not cPEG, on both day 180 and day 360 (Table 6.3). The sample also showed ADA titers of 1.2 and 2.1 for days 180 and 360, respectively. The remaining three patients had ADA-positive samples at baseline (i.e., preexisting ADA). One of these patients had ADA recognizing cPEG at baseline with a titer of 0.6 that increased to 1.2 in cycle 2 but diminished to an undetectable level by day 85. Another patient had ADA recognizing filgrastim and glycoG-CSF at baseline, with a

136

Polymer-Protein Conjugates

Table 6.3 ADA titer and binding specificity of ADA-Positive samples from Lonquextreated patients. Time point Patient

Competitor/Titer

BL

C2D1

C3D1

C4D1

D85

D180

D360

NA NA NA NA NA NA NA NA Pos Pos Neg 0 Pos Pos Pos 0.6 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Neg Neg Pos 0.6 NA NA NA NA Pos Pos Neg 0 Neg Pos Pos 0.6 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA Pos Pos Neg 0 Pos Pos Pos 0.3 Neg Neg Pos 0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA Neg Neg Neg 0 Pos Pos Neg 0 NA NA NA NA Neg Neg Pos 0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Phase 2 study 1a

Filgrastim glycoG-CSF cPEG Titer Filgrastim glycoG-CSF cPEG Titer Filgrastim glycoG-CSF cPEG Titer Filgrastim glycoG-CSF cPEG Titer Filgrastim glycoG-CSF cPEG Titer Filgrastim glycoG-CSF cPEG Titer Filgrastim glycoG-CSF cPEG Titer Filgrastim glycoG-CSF cPEG Titer Filgrastim glycoG-CSF cPEG Titer

2a

3

4

5

6

7

8

9

NA NA NA NA NA NA NA NA Pos Pos Neg 0 Pos Neg Pos 0.3 NSQ NSQ NSQ NSQ Neg Neg Neg 0.6 Pos Neg Pos 1.5 Pos Neg Pos 0.3 Pos Neg Pos 0

NA NA NA NA NA NA NA NA Pos Pos Neg 0 Pos Neg Pos 0.9 Neg Neg Pos 0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA Pos Pos Neg 0 Pos Neg Pos 0.6 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Phase 3 study a

13

Filgrastim glycoG-CSF cPEG Titer

NA NA NA NA

NA NA NA NA

NA NA NA NA

NA NA NA NA

NA NA NA NA

Pos Pos Neg 1.2

Pos Pos Neg 2.1

Chapter 6
Immunogenicity assessment of PEGylated proteins 137

Table 6.3 ADA titer and binding specificity of ADA-Positive samples from Lonquextreated patients.dcont’d Phase 3 study 14

15

16

Filgrastim glycoG-CSF cPEG Titer Filgrastim glycoG-CSF cPEG Titer Filgrastim glycoG-CSF cPEG Titer

Neg Neg Pos 0.6 Pos Pos Neg 2.1 Pos Neg Pos 0.9

Neg Neg Pos 1.2 Neg Pos Neg 1.8 NA NA NA NA

Neg Neg Pos 0.9 NA NA NA NA NA NA NA NA

Neg Neg Pos 0.6 NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA NA NA NA

a Patient with treatment-emergent ADA. Abbreviations include ADA: antidrug antibody; BL: baseline; C2D1: cycle 2 day 1; C3D1: cycle 3 day 1; C4D1: cycle 4 day 1; cPEG: PEG portion of lipegfilgrastim; D85: day 85; D180: day 180; D360: day 360; glycoG-CSF: glycosylated granulocyte-colony stimulating factor, i.e., Granocyte; NA: screened negative sample or confirmed negative (not analyzed in characterization assay); Neg: confirmed-negative sample; NSQ: insufficient sample quantity for analysis; Pos: confirmedpositive sample.Gray background indicates patient with treatment-emergent ADA. ADA, antidrug antibody; BL, baseline; C2D1, cycle 2 day 1; C3D1, cycle 3 day 1; C4D1, cycle 4 day 1; cPEG, PEG portion of lipegfilgrastim; D85, day 85; D180, day 180; D360, day 360; glycoG-CSF, glycosylated granulocyte-colony stimulating factor, i.e., Granocyte; NA, screened negative sample or confirmed negative (not analyzed in characterization assay); Neg, confirmed-negative sample; NSQ, insufficient sample quantity for analysis; Pos, confirmed-positive sample. Reproduced from Table 2 in Zou L, Buchner A, Field F, Barash S, Liu PM. Immunogenicity assessment of tbo-filgrastim in cancer patients receiving chemotherapy. Bioanalysis 2018;10 1221e28.

titer of 2.1; the ADA for glycoG-CSF persisted to cycle 2, with a titer of 1.8. All titer values were considered low. The third patient had ADA against filgrastim and cPEG with a titer of 0.9 and no detectable postdose ADA. Among patients identified as having treatment-emergent ADA, no postdose sample from any treatment group in each study tested positive for NAb activity against Lonquex or Granocyte in the cell-based neutralizing antibody assay. Clinical measures were examined for all patients with confirmed-positive ADA samples to Lonquex for a possible correlation between the presence of ADA and the potential clinical impact of immunogenicity. The pharmacodynamics measure was absolute neutrophil count (ANC), while the clinical efficacy measure was the duration of severe neutropenia (DSN), defined as the number of days with grade 4 neutropenia (ANC < 0.5  109/L) in each treatment cycle. The results were presented in a previous publication [32]. Neither ANC nor DSN values were changed in these patients after initiation of chemotherapy. No patient experienced febrile neutropenia, indicating lack of ADA impact on efficacy or pharmacodynamic parameters. Furthermore, there were no drug-related adverse events such as hypersensitivity and anaphylactic reactions in those ADA-positive patients, demonstrating no ADA impact on relevant safety parameters. The effect of ADA on the pharmacokinetics of Lonquex was investigated in a pooled analysis

138

Polymer-Protein Conjugates

of data from patients with breast cancer and patients with nonesmall cell lung cancer in a phase 3 study [32]. Only two patients for whom pharmacokinetic data were available tested positive for ADA; and there was no decrease in exposure to Lonquex, as indicated by predicted area under the curve data. A pharmacodynamic analysis with a CD34þ endpoint conducted with adult patients for all Lonquex doses found only two patients with positive ADA response. The CD34þ values for these two patients were similar to those from ADA-negative patients, indicating no impact of ADA on this pharmacodynamic parameter as well. As previously published [32], these results demonstrate a low incidence of treatmentemergent ADA (phase 2: 1.3%; phase 3: 1.0%) in Lonquex-treated patients with breast cancer and treated with doxorubicin and docetaxel. The incidences are very similar to those observed with Neulasta treatment in the same studies (phase 2: 1.9%; phase 3: 1.0%). As Neulasta is conjugated with PEG at N-terminus, these results indicate that the location of the PEG attachment does not contribute significantly to the immunogenicity of PEGylated G-CSF. As reported recently [37], immunogenicity incidence of nonPEGylated G-CSF, tbo-filgrastim, is 1.6%. The immunogenicity incidences of PEGylated G-CSF versus non-PEGylated G-CSF are thus very close, suggesting that PEGylation does not increase the immunogenicity of G-CSF. There were a significant number of patients showing preexisting ADA. In the phase 2 Lonquex study, 7 out of 154 enrolled patients (4.5%) had predose positive ADA samples. In the phase 3 study, 3 out of 101 enrolled patients have predose positive samples, a 3.0% preexisting ADA rate. The relatively high rates of preexisting ADA are in agreement with immunogenicity data of another PEGylated G-CSF product Neulasta, in which preexisting ADAs are found in approximately 6% (51/849) of patients. Preexisting antibodies against G-CSF have been previously reported for cancer patients undergoing G-CSF treatment [38]. One possible reason for the high preexisting ADA rates is that these patients may have received other G-CSF products before enrollment. Binding specificity characterization results indicate that preexisting ADA in 2 patients were solely against PEG moiety and in 5 patients were against both G-CSF and PEG moieties, so 7 out of 10 (70%) patients with preexisting ADA are partially or fully against PEG moiety. Given the high frequencies (5%e40%) of anti-PEG antibodies existed in healthy subjects [17e19], this is not surprising. Another interesting observation from the binding specificity characterization results is that patients (e.g., patients #5 and 14 in Table 6.3) with preexisting anti-PEG antibodies did not develop antibodies against G-CSF moiety after Lonquex treatment. And vice versa, the patients (e.g., patients #3 and 15 in Table 6.3) with preexisting anti-G-CSF antibodies did not develop antibodies against PEG moiety. So the immunogenic epitopes in each moiety did not spread into the other moiety after Lonquex treatment. To develop safe and potent biologic drugs, it is important to understand the potential for an immunological response in patients. As exemplified above, a through strategy for immunogenicity assessment, including assays for screening, confirmation, and titer, has to be developed and validated. Our data clearly showed the favorable immunogenicity profile of Lonquex in patients.

Chapter 6
Immunogenicity assessment of PEGylated proteins 139

References [1] Levy Y, Hershfield MS, Fernandez-Mejia C, Polmar SH, Scudiery D, Berger M, Sorensen RU. Adenosine deaminase deficiency with late onset of recurrent infections: response to treatment with polyethylene glycol-modified adenosine deaminase. J Pediatr 1988;113:312e7. [2] Szlachcic A, Zakrzewska M, Otlewski J. Longer action means better drug: tuning up protein therapeutics. Biotechnol Adv 2011;29:436e41. [3] Grigoletto A, et al. Drug and protein delivery by polymer conjugation. J Drug Deliv Sci Technol 2016; 32:132e41. https://doi.org/10.1016/j.jddst.2015.08.006. [4] Swierczewska M, Lee KC, Lee S. What is the future of PEGylated therapies? Expert Opin Emerg Drugs 2015;20:531e6. [5] Ivens IA, Achanzar W, Baumann A, Bra¨ndli-Baiocco A, Cavagnaro J, Dempster M, et al. PEGylated biopharmaceuticals: current experience and considerations for nonclinical development. Toxicol Pathol 2015;43(7):959e83. [6] Jevsevar S, Kunstelj M, Porekar VG. PEGylation of therapeutic proteins. Biotechnol J 2010;5:113e28. [7] Needham D, McIntosh TJ, Lasic DD. Repulsive interactions and mechanical stability of polymergrafted lipid membranes. Biochim Biophys Acta 1992;1108:40e8. [8] Veronese FM, Mero A. The impact of PEGylation on biological therapies. BioDrugs 2008;22:315e29. [9] Turecek PL, Bossard MJ, Schoetens F, Ivens IA. PEGylation of biopharmaceuticals: a review of chemistry and nonclinical safety information of approved drugs. J Pharm Sci 2016;105:460e75. [10] Veronese FM, Pasut G. PEGylation: posttranslational bioengineering of protein bio therapeutics. Drug Discov Today Technol 2008;5:57e64. [11] Khadka P, Ro J, Kim H, Kim I, Kim JT, Kim H, Cho JM, Yun G, Lee J. Pharmaceutical particle technologies: an approach to improve drug solubility, dissolution and bioavailability. Asian J Pharm Sci 2014;9:304e16. [12] Fishburn CS. The pharmacology of PEGylation: balancing PD with PK to generatenovel therapeutics. J Pharm Sci 2008;97:4167e83. [13] Marshall D, Pedley RB, Boden JA, Boden R, Melton RG, Begent RH. Polyethylene glycol modification of a galactosylated streptavidin clearing agent: effects on immunogenicity and clearance of a biotinylated anti-tumour antibody. Br. J. Cancer. 1996;73:565e72. [14] Katre NV. Immunogenicity of recombinant IL-2 modified by covalent attachment of polyethylene glycol. J Immunol 1990;144:209e13. [15] Harris JM, Martin NE, Modi M. Pegylation: a novel process for modifying pharmacokinetics. Clin Pharmacokinet 2001;40:539e51. [16] Richter AW, Akerblom E. Antibodies against polyethylene glycol produced in animals by immunization with monomethoxy polyethylene glycol modified proteins. Int Arch Allergy Appl Immunol 1983;70:124e31. [17] Richter AW, Akerblom E. Polyethylene glycol reactive antibodies in man: titer distribution in allergic patients treated with monomethoxy polyethylene glycol modified allergens or placebo, and in healthy blood donors. Int Arch Allergy Appl Immunol 1984;74:36e9. [18] Garratty G. Modulating the red cell membrane to produce universal/stealth donor red cells suitable for transfusion. Vox Sanguinis 2008;94:87e95. [19] Armstrong JK. The occurrence, induction, specificity and potential effect of antibodies against poly(ethylene glycol). In: Veronese FM, editor. PEGylated protein drugs: basic science and clinical applications. Basel: Birkha¨user; 2009. p. 147e68. [20] Yang Q, Lai SK. Anti-PEG immunity: emergence, characteristics, and unaddressed questions, vol. 7. Wiley Interdiscip Rev Nanomed Nanobiotechnol.; 2015. p. 655e77.

140

Polymer-Protein Conjugates

[21] Mima Y, Hashimoto Y, Shimizu T, Kiwada H, Ishida T. Anti-peg IgM is a major contributor to the accelerated blood clearance of polyethylene glycol-conjugated protein. Mol Pharm 2015;12: 2429e35. [22] Schellekens H, Hennink WE, Brinks V. The immunogenicity of polyethylene glycol: facts and fiction. Pharm Res 2013;30:1729e34. [23] Zhang P, Sun F, Liu S, Jiang S. Anti-PEG antibodies in the clinic: current issues and beyond PEGylation. J Contr Release 2016;28:184e93. [24] FDA. Guidance for industry on immunogenicity assessment for therapeutic protein products. 2014. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ UCM338856.pdf. [25] Roberts AW. G-CSF: a key regulator of neutrophil production, but that’s not all! Growth Factors, vol. 23; 2005. p. 33e41. [26] Cooper KL, Madan J, Whyte S, Stevenson MD, Akehurst RL. Granulocyte colony-stimulating factors for febrile neutropenia prophylaxis following chemotherapy: systematic review and meta-analysis. BMC Cancer 2011;11:404. [27] Granix [package insert]. North Wales PA: Teva Pharmaceuticals USA; 2014. https://www.accessdata. fda.gov/drugsatfda_docs/label/2014/125294s035lbl.pdf. [28] Bondarenko I, Gladkov OA, Elsaesser R, Buchner A, Bias P. Efficacy and safety of lipegfilgrastim versus pegfilgrastim: a randomized, multicenter, active-control phase 3 trial in patients with breast cancer receiving doxorubicin/docetaxel chemotherapy. BMC Cancer 2013;13:386. [29] FDA. Guidance for industry on assay development and validation for immunogenicity testing of therapeutic protein products. 2016. https://www.fda.gov/downloads/Drugs/Guidances/UCM192750. pdf. [30] EMA Guideline on Immunogenicity assessment of therapeutic proteins. 2017. http://www.ema. europa.eu/docs/en_GB/document_library/Scientific_guideline/2017/06/WC500228861.pdf. [31] Gorovits B, Wakshull E, Pillutla R, Xu Y, Manning MS, Goyal J. Recommendations for the characterization of immunogenicity response to multiple domain biotherapeutics. J Immunol Methods 2014;408:1e12. [32] Zou L, Buchner A, Roberge M, Liu PM. Immunogenicity assessment of lipegfilgrastim in patients with breast cancer receiving chemotherapy. J Immunol Res. 2016;23:9248061. [33] Ho¨glund M. Glycosylated and non-glycosylated recombinant human granulocyte colonystimulating factor (rhG-CSF)dwhat is the difference? Med Oncol 1998;15(4):229e33. [34] Gupta S, Indelicato SR, Jethwa V, Kawabata T, Kelley M, Mire-Sluis AR, Richards SM, Rup B, Shores E, Swanson SJ, Wakshull E. Recommendations for the design, optimization, and qualification of cell-based assays used for the detection of neutralizing antibody responses elicited to biological therapeutics. J Immunol Methods 2007;321:1e18. [35] Gupta S, Devanarayan V, Finco D, Gunn GR, Kirshner S, Richards S, Rup B, Song A, Subramanyam M. Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics. J Pharm Biomed Anal 2011;55:878e88. [36] Buchner A, Elsasser R, Bias P. A randomized, double-blind, active control, multicenter, dose-finding study of lipegfilgrastim (XM22) in breast cancer patients receiving myelosuppressive therapy. Breast Cancer Res Treat. 2014;148:107e16. [37] Zou L, Buchner A, Field F, Barash S, Liu PM. Immunogenicity assessment of tbo-filgrastim in cancer patients receiving chemotherapy. Bioanalysis 2018;10:1221e8. [38] Neulasta [package insert]. Thousand Oaks CA: Amgen Inc.; 2015. https://www.accessdata.fda.gov/ drugsatfda_docs/label/2015/125031s180lbl.pdf.