Pegfilgrastim (PEG-G-CSF) Induces Anti-polyethylene Glycol (PEG) IgM via a T Cell-Dependent Mechanism

Abstract

Protein-based therapeutics are beginning to be widely used in various clinical settings. Conjugation of polyethylene glycol (PEGylation) to protein therapeutics improves their circulation half-lives in the body. However, we and other groups observed that the initial dose of some PEGylated protein-based therapeutics may induce anti-PEG antibodies (primarily immunoglobulin M (IgM)), resulting in the accelerated clearance of a second dose. The mechanism behind the induction of anti-PEG IgM by PEGylated protein-based therapeutics is still unclear. In this study, we found that Pegfilgrastim (PEG-G-CSF, the PEGylated form of the recombinant human granulocyte colony-stimulating factor) induced anti-PEG IgM in mice when administered via either intravenous or subcutaneous administration. However, the anti-PEG IgM induction is diminished both in athymic nude mice lacking T cells and in splenectomized mice. In addition, anti-PEG IgM production was significantly diminished in the cyclophosphamide-treated mice depleted of B-cells. These results indicate that anti-PEG IgM production by Pegfilgrastim occurs in spleen in a T cell-dependent manner, which differs from anti-PEG IgM induced by PEGylated liposomes. However, B cells, both marginal zone and follicular, are essential for anti-PEG IgM production in both PEGylated preparations.

INTRODUCTION

Protein-based therapeutics have found more and more applications in recent years.¹⁾ Their high activity and selectivity give them advantages in treating disease, however, they also have disadvantages including physicochemical instability, low solubility, proteolytic degradation, relatively short circulating half-life and immunogenicity. PEGylation, the covalent attachment of the hydrophilic polymer polyethylene glycol (PEG), to protein-based therapeutics has been helpful in ameliorating some of these limitations.¹⁾ In particular, PEGylation prolongs the circulation half-lives of protein therapeutics, and may in some instances increase their biocompatibility and reduce their humoral and cellular immunogenicity. To date, over 20 PEGylated protein-based therapeutics have been approved by the U.S. Food and Drug Administration (FDA) and dozens are in clinical trials.^2,3)

Nevertheless, PEG, despite of its advantages, also has disadvantages. Some reports have claimed that PEGylated protein-based therapeutics, and liposomal drugs can elicit anti-PEG antibody responses that can adversely affect their circulation half-lives and reduce their therapeutic effects in rodents^2,4,5) and in patients.^2,6,7) Furthermore, the potential antigenicity of PEG itself has been observed in healthy individuals who have never received PEGylated therapeutics, but have anti-PEG antibodies, likely derived from PEG-containing food and cosmetics.⁸⁾ There is growing evidence showing that anti-PEG antibodies could be linked to hypersensitivity reactions and non-responsiveness to PEGylated protein-based therapeutics in some clinical cases.²⁾ Accordingly, the FDA has recommended, in its industrial guidelines for protein therapeutics in 2014, screening for anti-PEG antibodies, in addition to anti-protein antibodies, when PEGylated protein-based therapeutics undergo clinical trials.⁹⁾

In the last two decades, many studies have helped to explain the mechanism(s) underlying anti-PEG antibody induction and the accelerated blood clearance (ABC) phenomenon to PEGylated nanoparticles such as liposomes.^10–13) However, the mechanism(s) underlying anti-PEG immunity elicited by PEGylated protein-based therapeutics, are still not well understood, although anti-PEG immunity against PEGylated protein-based therapeutics can reduce the efficacy of PEGylated protein-based therapeutics. In this study, therefore, we investigated some of the mechanism(s) underlying anti-PEG immunoglobulin M (IgM) induction of a FDA-approved PEGylated protein-based therapeutic, pegfilgrastim, a PEGylated formulation of the recombinant human granulocyte colony-stimulating factor (G-CSF) analog filgrastim that stimulates the production of white blood cells.¹⁴⁾

MATERIALS AND METHODS

Materials

PEG-G-CSF (pegfilgrastim, G-Lasta^®) was purchased from Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan). Cyclophosphamide (Endoxan^®) was purchased from Shionogi & Co., Ltd. (Osaka, Japan). Cholesterol was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Hydrogenated egg phosphatidylcholine (HEPC) and 1, 2-distearoyl-sn-glycero-3 phosphoethanolamine-n-methoxy (polyethylene glycol)-2000] (mPEG₂₀₀₀-DSPE) were purchased from NOF (Tokyo, Japan). Alexa Fluor^® 647 anti-mouse CD23 IgG, APC anti-mouse IgM IgG, anti-mouse CD21 IgG-fluorescein isothiocyanate (FITC) and PE anti-mouse CD3 IgG were purchased from BioLegend (CA, U.S.A.). All other reagents were of analytical grade.

Animals

BALB/c and BALB/c nu/nu male mice, 5 weeks old, were purchased from Japan SLC (Shizuoka, Japan). The experimental animals were housed under controlled environmental conditions (constant temperature, humidity and 12 h dark–light cycle). All animal experiments were evaluated and approved by the Animal and Ethics Review Committee of Tokushima University (T2019-47).

Detection of Anti-PEG IgM in the Serum Samples

Mice (BALB/c or BALB/c nu/nu male mice) received either intravenous (i.v.) or subcutaneous (s.c.) injections of PEG-G-CSF (6 mg/kg). Serum samples were collected at day 5 following the i.v. injections and at day 7 after the s.c. injections. Sera collected from non-treated mice served as controls. An enzyme-linked immunosorbent assay (ELISA) we have previously developed¹⁵⁾ was employed to detect anti-PEG IgM in the serum samples. Absorbance was measured using a microplate reader (λ = 492 nm, InfiniteM200, TECAN Japan, Kanagawa, Japan). The absorbance detected in the control sera was subtracted from the absorbance in the serum samples. `

Effect of Splenectomy on Anti-PEG IgM Production by PEG-G-CSF

BALB/c mice were anesthetized with Isoflurane. Incisions were made in the left flanks, and the spleens were isolated and removed using a cautery instrument through ligation and cauterization of the splenic blood vessels. This procedure ensured that the spleen was removed in total and that no remaining splenic fragments were left behind. One day post splenectomy, PEG-G-CSF (6 mg/kg) was injected i.v. or s.c. into the mice. At day 5 following the injection, serum samples were collected and anti-PEG IgM in the samples was detected by ELISA, as described above.

Effect of Depletion of MZ-B Cells on Anti-PEG IgM Production by PEG-G-CSF

Cyclophosphamide treatment is known to deplete MZ-B cells in spleen.¹⁶⁾ Mice received an intraperitoneal injection of cyclophosphamide (400 mg/kg).¹⁷⁾ One day later, depletion of MZ-B cells in the spleen was detected using flow cytometry after staining with specific CD markers. Briefly, spleens were collected from mice treated with cyclophosphamide. Spleen cell suspensions were prepared as described previously.¹³⁾ Antibodies against various CD markers, diluted with 1% bovine serum albumin/phosphate buffered saline (BSA/PBS), were each added at the recommended concentrations to the target cells and the cells were analyzed by flow cytometry (Gallios, Beckman Coulter, NJ, U.S.A.). Anti-mouse CD21 IgG-FITC was detected at FL1 (Excitation 488 nm, Emission 525 nm). Alexa Fluor^® 647 anti-mouse CD23 IgG and APC anti-mouse IgM IgG were detected at FL6 (Excitation 638 nm, Emission 660 nm). PE anti-mouse CD3 IgG was detected at FL2 (Excitation 488 nm, Emission 575 nm). The data were analyzed using Kaluza software (Beckman Coulter).

Statistical Analysis

All values are expressed as mean ± standard deviation (S.D.). Statistical analysis was evaluated by unpaired t-test using Graphpad Prism 6.01 software (GraphPad Software Inc., CA, U.S.A.). The level of significance was set at p < 0.05.

RESULTS

Effect of Splenectomy on Anti-PEG IgM Production

PEG-G-CSF induced anti-PEG IgM production, with the i.v. route inducing significantly higher levels than the s.c. route (Fig. 1). This is consistent with our recent observations.¹⁸⁾ PEG-G-CSF did not elicit IgM antibodies against G-CSF itself (data not shown). We have previously shown that spleen plays an important role in the induction of anti-PEG IgM by PEGylated liposomes, and splenectomy suppresses the anti-PEG IgM response in both mice and rats.^10,15) In the current study, with PEG-G-CSF, anti-PEG IgM production in splenectomized mice was also investigated. In the splenectomized mice, the anti-PEG IgM production was completely eliminated irrespective of the administration route of PEG-G-CSF. These results indicate that the spleen is a major organ inducing the production of anti-PEG IgM, which is consistent with our observations with PEGylated liposomes.^10,15)

Fig. 1. Effect of Splenectomy on Anti-PEG IgM Production

Mice were divided into two groups, a splenectomized one and a non-splenectomized one. The former received splenectomy one day before the administration of PEG-G-CSF. The latter received a sham-operation one day before the administration of PEG-G-CSF. The mice were then injected with PEG-G-CSF at a dose of 6 mg/kg either i.v. or s.c. At day 5 after the injection, serum samples were collected. Anti-PEG IgM in the samples was determined with ELISA. Each value represents the mean ± S.D. (n = 3). ** p < 0.01 and *** p < 0.01 vs. non-splenectomized mice.

Induction of Anti-PEG IgM in Nude Mice

The production of anti-PEG IgM by i.v. administration of PEG-G-CSF was investigated in either immunodeficient athymic BALB/c mice (BALB/c nu/nu) or wild type BALB/c mice (Fig. 2). In the BALB/c nu/nu mice, anti-PEG IgM production was significantly diminished. This observation is consistent with observations for PEG-ovalbumin (OVA),¹⁹⁾ but is inconsistent with observations for PEGylated liposomes.²⁰⁾ This result indicates that the anti-PEG IgM response against PEG-G-CSF is elicited in a T cell-dependent manner.

Fig. 2. Anti-PEG IgM Induction by I.v. Injection of PEG-G-CSF in Nude Mice

BALB/c and BALB/c nu/nu mice received an i.v. injection of PEG-G-CSF at a dose of 6 mg/kg. On Day 5, serum samples were collected. Anti-PEG IgM in the samples was determined with ELISA. Each value represents the mean ± S.D. (n = 4). *** p < 0.001.

Effect of Cyclophosphamide Treatment on Anti-PEG IgM Production

To confirm that cyclophosphamide treatment depletes splenic MZ-B cells,¹⁶⁾ the effect of cyclophosphamide treatment on populations of lymphocytes in spleen was investigated (Fig. 3A). The treatment decreased the number of splenic B cells (IgM⁺ cells) and the numbers of CD21^low, CD23⁺ follicular B (FO-B) cells. MZ-B (CD21^high, CD23⁻) cells were also decreased, but the treatment did not change the number of splenic T-cells (CD3⁺). I.v. administration of PEG-G-CSF induced anti-PEG IgM production in normal, control BALB/c mice, but barely induced antibodies in the cyclophosphamide-treated mice (Fig. 3B). These results indicate that splenic B cells are responsible for production of anti-PEG IgM against administered PEG-G-CSF.

Fig. 3. Effect of Cyclophosphamide Treatment on Anti-PEG IgM Production

(A) Mice received an intraperitoneal (i.p) injection of cyclophosphamide (400 mg/kg). One day later, mice were euthanized, and spleens were collected for preparation of spleen cell suspensions. Spleens from untreated mice served as controls. Flow cytometry analysis was performed for spleen lymphocytes stained with fluorescent-labeled surface markers: IgM for total B cells, CD21 and CD23 for MZ-B cells and FO-B cells and CD3 for T cells. Each value represents the mean ± S.D. (n = 4). n.s. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control. (B) Mice received an i.p. injection of cyclophosphamide (400 mg/kg). One day later, they received i.v. injections of PEG-G-CSF at dose of 6 mg/kg. On Day 5 after the i.v. injection, serum samples were collected. Mice having no prior treatment with cyclophosphamide served as controls. Anti-PEG IgM in the serum samples was determined. Each value represents the mean ± S.D. (n = 3). *** p < 0.001 vs. control.

DISCUSSION

PEGylated protein-based therapeutics is widely applied for the treatment of a range of indications and new ones continue to be developed. Therefore, the observation of anti-PEG immunity and its negative consequences on the therapeutic efficacy/safety of PEGylated protein-based therapeutics could impact their clinical use. PEG-related immunological responses are received increasing attention.^5,21,22) Although the mechanism(s) underlying anti-PEG IgM induction have been reported for PEGylated nanoparticles such as liposomes,^12,23,24) data regarding the mechanism underlying the immunogenicity of PEG-conjugated protein-based therapeutics is still scarce. In the current study, we showed in control mice that PEG-G-CSF (pegfilgrastim, G-Lasta^®) induced the production of anti-PEG IgM in spleen in a T cell-dependent manner (Fig. 2) and that the spleen was the only source of the anti-PEG IgM production (Fig. 1), and that B cells (FO-B cells and MZ-B cells) were essential for the antibody production (Fig. 3). To the best of our knowledge, this is the first report to suggest the involvement of the splenic B cells (FO-B cells and MZ-B cells) and T cells in the induction of anti-PEG IgM by an FDA-approved PEGylated protein-based therapeutic, Pegfilgrastim (PEG-G-CSF).

In the present study, we demonstrated that i.v. injection of PEG-G-CSF induced stronger anti-PEG IgM response than the s.c. one (Fig. 1). It is known that route of administration affects distribution kinetics of injected particles. After s.c. administration, only a small fraction of the administered dose of PEG-G-CSF reaches spleen, due to slow, incomplete absorption from the site of injection. By contrary, following i.v. administration, the whole dose would be available into the blood circulation and a large fraction of PEG-G-CSF would rapidly be distributed to spleen. The preferential accumulation of PEG-G-CSF in the spleen following i.v. administration, compared to that following s.c. administration, can explain the difference in anti-PEG IgM response.

The T cell-dependent mechanism observed in this study was similar to that for PEG-OVA,¹⁹⁾ but was not the same as that for PEGylated liposomes, which induces anti-PEG IgM production in a T cell-independent manner.²⁰⁾ In our earlier studies, we hypothesized the mechanism behind anti-PEG IgM induction by PEGylated liposomes is as follows: once an initial dose of PEGylated liposomes reaches the spleen, the liposomes bind and crosslink to surface IgM on reactive MZ-B cells in the spleen and consequently trigger the production of an anti-PEG IgM that is independent of T cell help.^5,23,24) It is known that infectious pathogens are complex and composed of both T cell-dependent and T cell-independent epitopes. PEG in the PEGylated liposomes is a T cell-independent antigen,¹⁸⁾ while the protein in PEGylated protein-based therapeutics is a T cell-dependent antigen¹⁹⁾ (Fig. 1). MZ-B cells are likely to be heterogeneous and multi-reactive, reacting with a wide variety of pathogen-associated antigens. In addition to their ability to mount a local antibody response against T cell-independent antigens, MZ-B cells can participate in T cell-dependent immune responses through the capture and import of blood-borne antigens to follicular areas of the spleen.²⁵⁾ In the present study, treatment with cyclophosphamide decreased the number of splenic MZ-B cells (Fig. 3A) as previously reported^26–28) and reduced anti-PEG IgM production by PEG-G-CSF (Fig. 3B). Therefore, MZ-B cells in the splenic B cells may play a crucial role in the anti-PEG IgM production against, not only PEGylated liposomes, but also PEGylated protein-based therapeutics.

T lymphocyte is composed of different cell subsets; T-helper (Th)1, Th2, Th17 and Treg lymphocytes. These T lymphocyte subsets form a complex immune network that participates to both cellular and humoral immune responses via secreting a variety of cytokines. Among them, Th2 lymphocytes are known to be responsible for humoral immunity against extracellular pathogens by cooperation with B lymphocytes secreting antibodies through the release of cytokines, such as interleukin 4 (IL-4), which support B-cell development and production of antibodies. In the current study, Th2 cells are most likely to be the contributing cells to anti-PEG IgM production. However, the exact contribution of Th2 cells to the anti-PEG IgM production against PEGylated protein-based therapeutics is still uncertain. The investigation is still continued in our laboratory.

Since cyclophosphamide treatment also diminished the number of FO-B cell in spleen (Fig. 3A), the contribution of FO-B cells to anti-PEG IgM production cannot be negligible. Splenic FO-B cells are known to differentiate into long-lived plasma cells that are capable of secreting the IgG isotype of antibodies for several months in the absence of any cell division or re-exposure to antigen.^29,30) The MZ-B cells have been shown to be the major producers of IgM antibodies.^31,32) The anti-PEG antibodies induced by PEG-G-CSF were mainly of the IgM type (Fig. 1) and no anti-PEG IgG antibodies was produced by PEG-G-CSF up to day 14 (data not shown). This suggests that FO-B cells have little or no contribution, relative to MZ-B cells, to the production of anti-PEG IgM against PEG-G-CSF.

The logic of the hapten-carrier system would account for anti-PEG antibody production against different PEGylated products.^33,34) In this study, high dose of PEG-G-CSF could trigger anti-PEG IgM production in normal BALB/c mice (Figs. 1, 2, 3) without eliciting any anti-G-CSF IgM (data not shown). These results support the concept that the production of antibodies occurs only against PEG on the conjugates where PEG acts as a hapten. A hapten is defined a small molecule that cannot elicit an immune response unless it is covalently attached to a larger molecule (proteins, peptides, or nanoparticles), which then makes it reactive to the immune system.³⁵⁾ This haptenic characteristic of PEG is likely dependent on the molecular weight and the nature/immunogenicity of the carrier and/or the presence of adjuvants.^36–38) Previous reports have shown that proteins conjugated with a hapten perform as TD antigens,³⁹⁾ while non protein carriers, such as liposome and polysaccharide, conjugated with a hapten act as TI antigens.⁴⁰⁾ This might explain the controversy surrounding the mechanism of antibody production against PEG-G-CSF and PEGylated liposome in various developmental and clinical situations.

In animal studies, the incidence of anti-PEG immunogenicity and the ABC phenomenon against PEGylated therapeutics has been found to be varied depending on animal species used. Such variation could be attributed, at least in part, to the difference in sensitivities of animals to the immune responses as well as the differences in pharmacokinetics of drugs. Splenic MZ-B cells have been reported to play a crucial role in the production of anti-PEG antibodies by PEGylated liposomes in rodents and pigs.^23,24) However, there could be a difference in the mechanism of anti-PEG antibodies production between human and animals, especially that the anti-PEG antibody production in human is more skewed toward the IgG isotype.⁴¹⁾ Therefore, difference between animal species should be considered an important issue that must be addressed by pre-clinical study of the PEGylated formulation.

CONCLUSION

Pegfilgrastim (PEG-G-CSF) induces anti-PEG IgM production with the help of T cells and B cells (FO-B cells and MZ-B cells) in the spleen. Our study may provide a step forward towards uncovering the mechanism underlying anti-PEG IgM production by PEGylated protein-based therapeutics.

Acknowledgments

The authors are grateful to Professor Emerita Theresa M. Allen, University of Alberta for her helpful advice in developing the English manuscript. This study was supported by the Nagai Foundation, Tokyo, the Japan Society for the Promotion of Science, a Grant-in-Aid for Fund for the Promotion of Joint International Research (Fostering Joint International Research (B) (19KK0279)) and a research program for the development of an intelligent Tokushima artificial exosome (iTEX) from Tokushima University.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Harris JM, Chess RB. Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discov., 2, 214–221 (2003).
• 2) Elsadek NE, Abu Lila AS, Ishida T. 5 - Immunological responses to PEGylated proteins: anti-PEG antibodies. Polymer–Protein Conjugates. (Pasut G, Zalipsky S eds.) Elsevier, the Netherlands, pp. 103–123 (2020).
• 3) Swierczewska M, Lee KC, Lee S. What is the future of PEGylated therapies? Expert Opin. Emerg. Drugs, 20, 531–536 (2015).
• 4) Armstrong JK, Hempel G, Koling S, Chan LS, Fisher T, Meiselman HJ, Garratty G. Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients. Cancer, 110, 103–111 (2007).
• 5) Mohamed M, Abu Lila AS, Shimizu T, Alaaeldin E, Hussein A, Sarhan HA, Szebeni J, Ishida T. PEGylated liposomes: immunological responses. Sci. Technol. Adv. Mater., 20, 710–724 (2019).
• 6) White JT, Newsome SD, Kieseier BC, Bermel RA, Cui Y, Seddighzadeh A, Hung S, Crossman M, Subramanyam M. Incidence, characterization, and clinical impact analysis of peginterferon beta1a immunogenicity in patients with multiple sclerosis in the ADVANCE trial. Ther. Adv. Neurol. Disord., 9, 239–249 (2016).
• 7) Kloos R, van der Sluis IM, Mastrobattista E, Hennink W, Pieters R, Verhoef J-J. Acute lymphoblastic leukaemia patients treated with PEGasparaginase develop antibodies to PEG and the succinate linker. Br. J. Haematol., 189, 442–451 (2020).
• 8) Chen BM, Su YC, Chang CJ, Burnouf PA, Chuang KH, Chen CH, Cheng TL, Chen YT, Wu JY, Roffler SR. Measurement of pre-existing IgG and IgM antibodies against polyethylene glycol in healthy individuals. Anal. Chem., 88, 10661–10666 (2016).
• 9) Parenky A, Myler H, Amaravadi L, Bechtold-Peters K, Rosenberg A, Kirshner S, Quarmby V. New FDA draft guidance on immunogenicity. AAPS J., 16, 499–503 (2014).
• 10) Ishida T, Ichihara M, Wang X, Kiwada H. Spleen plays an important role in the induction of accelerated blood clearance of PEGylated liposomes. J. Control. Release, 115, 243–250 (2006).
• 11) Wang L, Su Y, Wang X, Liang K, Liu M, Tang W, Song Y, Liu X, Deng Y. Effects of complement inhibition on the ABC phenomenon in rats. Asian J. Pharm. Sci., 12, 250–258 (2017).
• 12) Ishida T, Kashima S, Kiwada H. The contribution of phagocytic activity of liver macrophages to the accelerated blood clearance (ABC) phenomenon of PEGylated liposomes in rats. J. Control. Release, 126, 162–165 (2008).
• 13) Shimizu T, Ishida T, Kiwada H. Transport of PEGylated liposomes from the splenic marginal zone to the follicle in the induction phase of the accelerated blood clearance phenomenon. Immunobiology, 218, 725–732 (2013).
• 14) Yamao K, Takenaka M, Yoshikawa T, Ishikawa R, Okamoto A, Yamazaki T, Nakai A, Omoto S, Kamata K, Minaga K, Hagiwara S, Sakurai T, Nishida N, Chiba Y, Watanabe T, Kudo M. Clinical safety and efficacy of secondary prophylactic pegylated G-CSF in advanced pancreatic cancer patients treated with mFOLFIRINOX: a single-center retrospective study. Intern. Med., 58, 1993–2002 (2019).
• 15) Ichihara M, Shimizu T, Imoto A, Hashiguchi Y, Uehara Y, Ishida T, Kiwada H. Anti-PEG IgM response against PEGylated liposomes in mice and rats. Pharmaceutics, 3, 1–11 (2010).
• 16) Humphrey JH. Splenic macrophages: antigen presenting cells for T1–2 antigens. Immunol. Lett., 11, 149–152 (1985).
• 17) Stockman GD, Heim LR, South MA, Trentin JJ. Differential effects of cyclophosphamide on the B and T cell compartments of adult mice. J. Immunol., 110, 277–282 (1973).
• 18) Elsadek NE, Lila ASA, Emam SE, Shimizu T, Takata H, Ando H, Ishima Y, Ishida T. Pegfilgrastim (PEG-G-CSF) induces anti-PEG IgM in a dose dependent manner and causes the accelerated blood clearance (ABC) phenomenon upon repeated administration in mice. Eur. J. Pharm. Biopharm., 152, 56–62 (2020).
• 19) 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., 12, 2429–2435 (2015).
• 20) Ishida T, Wang X, Shimizu T, Nawata K, Kiwada H. PEGylated liposomes elicit an anti-PEG IgM response in a T cell-independent manner. J. Control. Release, 122, 349–355 (2007).
• 21) McSweeney MD, Versfeld ZC, Carpenter DM, Lai SK. Physician awareness of immune responses to polyethylene glycol–drug conjugates. Clin. Transl. Sci., 11, 162–165 (2018).
• 22) Moreno A, Pitoc GA, Ganson NJ, Layzer JM, Hershfield MS, Tarantal AF, Sullenger BA. Anti-PEG antibodies inhibit the anticoagulant activity of PEGylated aptamers. Cell Chem. Biol., 26, 634–644.e3 (2019).
• 23) Shimizu T, Mima Y, Hashimoto Y, Ukawa M, Ando H, Kiwada H, Ishida T. Anti-PEG IgM and complement system are required for the association of second doses of PEGylated liposomes with splenic marginal zone B cells. Immunobiology, 220, 1151–1160 (2015).
• 24) Kozma GT, Mészáros T, Vashegyi I, Fülöp T, Örfi E, Dézsi L, Rosivall L, Bavli Y, Urbanics R, Mollnes TE, Barenholz Y, Szebeni J. Pseudo-anaphylaxis to polyethylene glycol (PEG)-coated liposomes: roles of anti-PEG IgM and complement activation in a porcine model of human infusion reactions. ACS Nano, 13, 9315–9324 (2019).
• 25) Zouali M, Richard Y. Marginal zone B-cells, a gatekeeper of innate immunity. Front. Immunol., 2, 63 (2011).
• 26) Kumararatne DS, MacLennan IC. Cells of the marginal zone of the spleen are lymphocytes derived from recirculating precursors. Eur. J. Immunol., 11, 865–869 (1981).
• 27) Zandvoort A, Lodewijk ME, Klok PA, Dammers PM, Kroese FG, Timens W. Slow recovery of follicular B cells and marginal zone B cells after chemotherapy: implications for humoral immunity. Clin. Exp. Immunol., 124, 172–179 (2001).
• 28) Pillai S, Cariappa A, Moran ST. MARGINAL ZONE B CELLS. Annu. Rev. Immunol., 23, 161–196 (2005).
• 29) Hoffman W, Lakkis FG, Chalasani G. B Cells, Antibodies, and More. Clin. J. Am. Soc. Nephrol., 11, 137–154 (2016).
• 30) Song H, Cerny J. Functional heterogeneity of marginal zone B cells revealed by their ability to generate both early antibody-forming cells and germinal centers with hypermutation and memory in response to a T-dependent antigen. J. Exp. Med., 198, 1923–1935 (2003).
• 31) Appelgren D, Eriksson P, Ernerudh J, Segelmark M. Marginal-zone B-cells are main producers of IgM in humans, and are reduced in patients with autoimmune vasculitis. Front. Immunol., 9, 2242 (2018).
• 32) Attanavanich K, Kearney J. Marginal zone, but not follicular B cells, are potent activators of naive CD4 T cells. J. Immunol., 172, 803–811 (2004).
• 33) Richter AW, Akerblom E. Antibodies against polyethylene glycol produced in animals by immunization with monomethoxy polyethylene glycol modified proteins. Int. Arch. Allergy Appl. Immunol., 70, 124–131 (1983).
• 34) Shimizu T, Ichihara M, Yoshioka Y, Ishida T, Nakagawa S, Kiwada H. Intravenous administration of polyethylene glycol-coated (PEGylated) proteins and PEGylated adenovirus elicits an anti-PEG immunoglobulin M response. Biol. Pharm. Bull., 35, 1336–1342 (2012).
• 35) Erlanger BF. The preparation of antigenic hapten–carrier conjugates: a survey. Methods Enzymol., 70 (A), 85–104 (1980).
• 36) Caliceti P, Schiavon O, Veronese FM. Immunological properties of uricase conjugated to neutral soluble polymers. Bioconjug. Chem., 12, 515–522 (2001).
• 37) Hu X, Olivier K, Polack E, Crossman M, Zokowski K, Gronke RS, Parker S, Li Z, Nestorov I, Baker DP, Clarke J, Subramanyam M. In vivo pharmacology and toxicology evaluation of polyethylene glycol-conjugated interferon beta-1a. J. Pharmacol. Exp. Ther., 338, 984–996 (2011).
• 38) Moghimi SM, Gray T. A single dose of intravenously injected poloxamine-coated long-circulating particles triggers macrophage clearance of subsequent doses in rats. Clin. Sci. (Lond), 93, 371–379 (1997).
• 39) Braley-Mullen H. Antigen requirements for induction of B-memory cells: studies with dinitrophenyl coupled to T-dependent and T-independent carriers. J. Exp. Med., 147, 1824–1831 (1978).
• 40) Mizuguchi J, Kakiuchi T, Nariuchi H, Matuhasi T. Different effect of LPS-induced macrophage factor on antibody responses to TI-1 and TI-2 antigens. Immunology, 47, 183–192 (1982).
• 41) Yang Q, Lai SK. Anti-PEG immunity: emergence, characteristics, and unaddressed questions. Nanomed. Nanobiotechnol., 7, 655–677 (2015).