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Modulation of bioavailability of proinflammatory cytokines produced by myeloid cells
Seminars in Arthritis and Rheumatism 49 (2019) S39 S42
Contents lists available at ScienceDirect
Seminars in Arthritis and Rheumatism journal homepage: www.elsevier.com/locate/semarthrit
Modulation of bioavailability of proinflammatory cytokines produced by myeloid cells Maxim A. Nosenkoa,b, Kamar-Sulu N. Atretkhanya,b, Vladislav V. Mokhonova,c, Ekaterina A. Vasilenkoc, Andrei A. Kruglovb,d, Sergei V. Tillibe, Marina S. Drutskayaa, Sergei A. Nedospasova,b,* a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia Lomonosov Moscow State University, Moscow 119234, Russia c Lobachevsky University of Nizhni Novgorod, Nizhni Novgorod 603950, Russia d German Rheumatism Research Center (DRFZ), Leibniz Institute, Berlin 10117, Germany e Institute of Gene Biology, Russian Academy of Sciences, Moscow 119991, Russia b
A R T I C L E
I N F O
Keywords: TNF IL-6 F4/80 CD11b VHH Bispecific antibodies
A B S T R A C T
In spite of successful therapeutic neutralization of proinflammatory cytokines in several autoimmune diseases, such therapy is not entirely free of side effects. The main reason relates to the fact that cytokine signaling may have protective components that need to be spared. Several approaches to achieve a less damaging cytokine inhibition are being explored. In our experimental studies we are using bispecific reagents based on VHH-modules from the heavy-chain-only antibodies to limit bioavailability of TNF and IL-6 produced by myeloid cells. After evaluation of their properties in vitro and in vivo we argue that these types of reagents may have an advantage over systemic blockers. © 2019 Elsevier Inc. All rights reserved.
Introduction At present time, there is no effective cure for most of autoimmune diseases caused by pathogenic self-reactive lymphocyte clones. The existing anti-cytokine therapies may improve the course of disease and relieve suffering, but they do not target the root of the problem and may also affect systemic immunoregulatory mechanisms. Once the patients are off the therapy, the disease is most likely to return. Another unresolved problem related to unwanted side effects of anti-cytokine therapies stems out from the fact that evolutionarily selected non-redundant functions of a given cytokine are more likely to be protective than pathogenic. Thus, pathogenic manifestations of cytokine signaling may not be dissociable from other functions, especially, when systemic inhibition is considered. With these ideas in mind we and others have set to explore a possibility of more selective anti-cytokine neutralization that may spare cytokine protective functions. Examples of such approaches for the two proinflammatory cytokines, TNF and IL-6, include selective interference with only one of the two TNF receptors [1 3,23] or interference with only one
*Corresponding author at: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov str., Moscow 119991, Russia. E-mail address: [email protected] (S.A. Nedospasov). https://doi.org/10.1016/j.semarthrit.2019.09.012 0049-0172/© 2019 Elsevier Inc. All rights reserved.
mode of IL-6 signaling based on administration of a soluble chimeric protein sgp130Fc [4,5], respectively. In studies utilizing a panel of conditional TNF knockouts, the evidence for pathogenic effects of TNF produced by myeloid cells was found in experimental arthritis, experimental autoimmune encephalomyelitis and in LPS-D/Gal-induced acute hepatotoxicity [6,7]. On the contrary, TNF produced by T cells demonstrated non-redundant protective functions in collagen-induced arthritis (manuscript in preparation) and in M. tuberculosis infection [8]. Similarly, IL-6 produced by dendritic cells was shown to promote EAE development [9] and our preliminary studies utilizing mice with IL-6 overexpression in myeloid cells reveal a pathogenic inflammatory phenotype causing postnatal lethality [22]. These results led to the idea of selective pharmacological neutralization of a proinflammatory cytokine produced by myeloid cells [10,11]. We decided to employ bispecific antibodies targeting myeloid cell surface markers that are able to capture secreted cytokine and cause its retention or internalization. We argue that in vivo such mode of retention will selectively limit bioavailability of a cytokine produced by myeloid cells only and not by other cell types. In this study, we evaluated and compared two targets on myeloid cells, F4/80 and CD11b, which served as anchors for cytokine binding moieties. Using VHH-based reagents specific for these targets [10,12] and anti-human-TNF VHH [13] we have generated several biologics, that we called myeloid-specific TNF inhibitors or MYSTIs, that were expected to interfere with
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human TNF secretion and bioavailability due to their binding to one of the two markers on the surface of myeloid cells (Suppl. Fig. 1).
MYSTI-2 and MYSTI-3 bind to the surface of macrophages and retain exogenously added human TNF We first compared bispecific antibodies in vitro for their ability to bind to macrophages (BMDM) and to human TNF (hTNF). For this purpose, we labeled MYSTI-2, MYSTI-3 and the control reagent, fMYSTI, with FITC to allow for fluorescent detection. Both MYSTI-2 and MYSTI-3, but not fMYSTI, effectively stained >98% of murine BMDM (Fig. 1(A)). Moreover, both biologics retained exogenously added hTNF via anti-TNF moiety as revealed by flow cytometry (Fig. 1(B)) or confocal microscopy (Fig. 1(C)). Control reagent did not bind to BMDM surface and, thus, did not produce the double staining despite its functional anti-TNF module. Thus, MYSTI-2 and MYSTI-3 show comparable abilities to decorate the macrophage surface and simultaneously retain hTNF.
MYSTI-2 has a longer half-life on the surface of macrophages compared to MYSTI-3 We have previously demonstrated that MYSTI-2 can be stored on the surface of macrophages for at least 24 h in vitro [11]. Interestingly, CD11b-based MYSTI-3 showed significantly shorter lifetime on BMDM surface, being almost undetectable already after 3 h of incubation, compared to MYSTI-2, which still was detectable after 24 h (Fig. 2). Both MYSTI-2 and MYSTI-3 were internalized by BMDM shortly after administration. However, the two biologics differed in their intracellular localization with MYSTI-2 being mainly located in large vesicles while MYSTI-3 was mostly detected in small granules, implicating possible proteolytic degradation in phagosomes.
MYSTI-2 and MYSTI-3 limit bioavailability of human TNF in vitro To functionally compare the retention of hTNF by both MYSTIs in vitro, BMDM isolated from hTNF KI mice [14] were preincubated with different doses of bispecific Abs, followed by
Fig. 1. MYSTI-2 and MYSTI-3 can simultaneously bind to macrophage surface and hTNF. A Flow cytometry staining of BMDM with FITC-labeled MYSTI-2 and MYSTI-3 compared to FITC-labeled control reagent (fMYSTI); B,C BMDM were consequently incubated with FITC-labeled bispecifics (MYSTI-2, MYSTI-3 or fMYSTI), recombinant hTNF, followed by anti-hTNF staining and analyzed by flow cytometry (B) or confocal microscopy (C). Staining without addition of hTNF was performed as control. Scale bar 50 mm.
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inhibit hTNF release compared to MYSTI-2 (Fig. 3). Thus we expect MYSTI-3 to have reduced therapeutic efficacy in vivo compared to MYSTI-2. Previously, MYSTI-2 was demonstrated to prevent lethality in the murine model of LPS/D-Gal-induced hepatotoxicity [10]. Our preliminary data in this model confirms inferior therapeutic potential of MYSTI-3 since it does not protect mice from lethality at the same dose, as MYSTI-2 does (data not shown). Thus we concluded that at the moment MYSTI-2 remains the best candidate reagent for further evaluation in various disease models in vivo. Conclusion
Fig. 2. MYSTI-3 has shorter life time on macrophage surface compared to MYSTI-2. BMDM were placed on glass-bottom dishes and incubated with FITC-labeled MYSTI-2, MYSTI-3 or fMYSTI for 15 min, then washed and cultivated in cell culture medium at 37 °C. At indicated timepoints cells were fixed with 4%PFA, counterstained with DAPI and analyzed using confocal microscopy. White arrows indicate internalized MYSTI-2 and MYSTI-3. Scale bar 50 mm.
In spite of its efficacy, systemic anti-cytokine therapy is not curative and is not free of unwanted side effects. Many laboratories are working to improve the specificity of proinflammatory cytokine inhibition employing different strategies. For example, selective inhibitors blocking only the soluble form of TNF [15,16], or just one of its two receptors have been developed [17,18]. In the case of IL-6, selective inhibition may target trans-signaling cascade, leaving intact at least some of the beneficial or protective IL-6 functions [4]. Alternative strategy targets bi-specific reagent with cytokine-neutralizing capacity to a damaged tissue [19 21], or to a pathogenic cell type. The fact that TNF produced by myeloid cells is pathogenic in several experimental disease models in mice led to the idea of developing a myeloid-specific TNF inhibitor, such as MYSTI [10]. Our results suggest that the properties of a surface molecule used for retention may be important and that other myeloid surface markers are worth exploring for targeting anti-cytokine reagents. We believe that bispecific cytokine blockers, such as described here, may reduce the collateral damage during anti-cytokine therapy and should be further evaluated in preclinical and clinical studies. Acknowledgments
activation with LPS. Supernatants from these cell cultures were then analyzed using ELISA (Fig. 3). Both MYSTI-2 and MYSTI-3, but not fMYSTI, limited secretion of hTNF by macrophages into culture medium in a dose-dependent manner. However, effective concentrations of two bispecifics were strikingly different with MYSTI-3 requiring almost 100 times higher concentration to fully
We thank G.A. Efimov for valuable input, M. Rashidian and H. Ploegh for providing anti-CD11b VHH and A.S. Zhdanova for help with ELISA. This work is supported by the Russian Science Foundation (grant # 19-75-30032), and confocal microscopy experiments were performed on Leica TCS SP5 and supported by grant # 14-50-00060. V.V. Mokhonov is supported by State assignment of Russian Ministry of Science and Education: 20.6445.2017/9.10.
Fig. 3. MYSTI-2 has higher TNF-inhibiting potency than MYSTI-3. MYSTI-2 and MYSTI-3 limit bioavailability of hTNF in vitro. BMDM, generated from hTNF KI mice, were incubated with indicated concentrations of MYSTI-2 (green), MYSTI-3 (blue) or fMYSTI as control (black), followed by washing and activation with LPS for 4 h. Then supernatants were collected and bioactive hTNF was measured using ELISA. Dashed bar indicates result from LPS-activated cells without incubation with any bispecific antibody. ND concentration is below detection limit (15 pg/ml). *p < 0.05; **p < 0.01; ****p < 0.0001; ns not significant (two-way ANOVA). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j.semarthrit.2019.09.012.
References [1] McCann FE, Perocheau DP, Ruspi G, Blazek K, Davies ML, Feldmann M, Dean JLE, Stoop AA, Williams RO. Selective tumor necrosis factor receptor i blockade is antiinflammatory and reveals immunoregulatory role of tumor necrosis factor receptor ii in collagen-induced arthritis. Arthritis Rheumatol 2014;66:2728–38. doi: 10.1002/art.38755. ge L, Vandenbroucke RE, Van Hauwermeiren F, Haustraete J, [2] Steeland S, Puime Devoogdt N, Hulpiau P, Leroux-Roels G, Laukens D, Meuleman P, De Vos M, Libert C. Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1. J Biol Chem 2015;290:4022–37. doi: 10.1074/jbc.M114.617787. [3] Zalevsky J, Secher T, Ezhevsky SA, Janot L, Steed PM, O’Brien C, Eivazi A, Kung J, Nguyen D-HT, Doberstein SK, Erard F, Ryffel B, Szymkowski DE. Dominant-negative inhibitors of soluble tnf attenuate experimental arthritis without suppressing innate immunity to infection. J Immunol 2007;179:1872–83. [4] Atreya R, Mudter J, Finotto S, Mullberg J, Jostock T, Wirtz S, Schutz M, Bartsch B, Holtmann M, Becker C, Strand D, Czaja J, Schlaak JF, Lehr HA, Autschbach F, Schurmann G, Nishimoto N, Yoshizaki K, Ito H, Kishimoto T, Galle PR, Rose-John S, Neurath MF. Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo. Nat Med 2000;6:583–8. doi: 10.1038/75068. [5] Garbers C, Aparicio-Siegmund S, Rose-John S. The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition. Curr Opin Immunol 2015;34:75–82. doi: 10.1016/j.coi.2015.02.008. [6] Kruglov AA, Lampropoulou V, Fillatreau S, Nedospasov SA. Pathogenic and protective functions of tnf in neuroinflammation are defined by its expression in t lymphocytes and myeloid cells. J Immunol 2011;187:5660–70. doi: 10.4049/jimmunol.1100663. [7] Grivennikov SI, Tumanov AV, Liepinsh DJ, Kruglov AA, Marakusha BI, Shakhov AN, € rster I, Clausen BE, Tessarollo L, Ryffel B, Kuprash Murakami T, Drutskaya LN, Fo DV, Nedospasov SA. Distinct and nonredundant in vivo functions of tnf produced by t cells and macrophages/neutrophils: protective and deleterious effects. Immunity 2005;22:93–104. doi: 10.1016/j.immuni.2004.11.016. [8] Allie N, Grivennikov SI, Keeton R, Hsu N-J, Bourigault M-L, Court N, Fremond C, Yeremeev V, Shebzukhov Y, Ryffel B, Nedospasov SA, Quesniaux VFJ, Jacobs M. Prominent role for t cell-derived tumour necrosis factor for sustained control of mycobacterium tuberculosis infection. Sci Rep 2013;3. doi: 10.1038/srep01809. [9] Heink S, Yogev N, Garbers C, Herwerth M, Aly L, Gasperi C, Husterer V, Croxford AL, Moller-Hackbarth K, Bartsch HS, Sotlar K, Krebs S, Regen T, Blum H, Hemmer B, Misgeld T, Wunderlich TF, Hidalgo J, Oukka M, Rose-John S, Schmidt-Supprian M, Waisman A, Korn T. Trans-presentation of IL-6 by dendritic cells is required for the priming of pathogenic TH17 cells. Nat Immunol 2017;18:74–85. doi: 10.1038/ni.3632. [10] Efimov GA, Kruglov AA, Khlopchatnikova ZV, Rozov FN, Mokhonov VV, Rose-John S, Scheller J, Gordon S, Stacey M, Drutskaya MS, Tillib SV, Nedospasov SA. Celltype-restricted anti-cytokine therapy: tnf inhibition from one pathogenic source. Proc Natl Acad Sci 2016;113:3006–11. doi: 10.1073/pnas.1520175113.
[11] Nosenko MA, Atretkhany K-SN, Mokhonov VV, Efimov GA, Kruglov AA, Tillib SV, Drutskaya MS, Nedospasov SA. VHH-Based bispecific antibodies targeting cytokine production. Front Immunol 2017;8:1073. doi: 10.3389/fimmu.2017.01073. [12] Rashidian M, Keliher EJJ, Bilate AMM, Duarte JNN, Wojtkiewicz GRR, Jacobsen JTT, Cragnolini J, Swee LKK, Victora GDD, Weissleder R, Ploegh HLL. Noninvasive imaging of immune responses. Proc Natl Acad Sci USA 2015;112:6146–51. doi: 10.1073/pnas.1502609112. [13] Coppieters K, Dreier T, Silence K, De Haard H, Lauwereys M, Casteels P, Beirnaert E, Jonckheere H, Van De Wiele C, Staelens L, Hostens J, Revets H, Remaut E, Elewaut D, Rottiers P. Formatted anti-tumor necrosis factor a vhh proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis. Arthritis Rheum 2006;54:1856–66. doi: 10.1002/art.21827. [14] Olleros ML, Chavez-Galan L, Segueni N, Bourigault ML, Vesin D, Kruglov AA, Drutskaya MS, Bisig R, Ehlers S, Aly S, Walter K, Kuprash DV, Chouchkova M, Kozlov SV, Erard F, Ryffel B, Quesniaux VFJ, Nedospasov SA, Garcia I. Control of mycobacterial infections in mice expressing human tumor necrosis factor (TNF) but not mouse tnf. Infect Immun 2015;83:3612–23. doi: 10.1128/IAI.00743-15. [15] Shibata H, Yoshioka Y, Ohkawa A, Minowa K, Mukai Y, Abe Y, Taniai M, Nomura T, Kayamuro H, Nabeshi H, Sugita T, Imai S, Nagano K, Yoshikawa T, Fujita T, Nakagawa S, Yamamoto A, Ohta T, Hayakawa T, Mayumi T, Vandenabeele P, Aggarwal BB, Nakamura T, Yamagata Y, Tsunoda S, Kamada H, Tsutsumi Y. Creation and Xray structure analysis of the tumor necrosis factor receptor-1-selective mutant of a tumor necrosis factor-alpha antagonist. J Biol Chem 2008;283:998–1007. doi: 10.1074/jbc.M707933200. [16] Kitagaki M, Isoda K, Kamada H, Kobayashi T, Tsunoda S, Tsutsumi Y, Niida T, Kujiraoka T, Ishigami N, Ishihara M, Matsubara O, Ohsuzu F, Kikuchi M. Novel TNFalpha receptor 1 antagonist treatment attenuates arterial inflammation and intimal hyperplasia in mice. J Atheroscler Thromb 2012;19:36–46. [17] Torrey H, Butterworth J, Mera T, Okubo Y, Wang L, Baum D, Defusco A, Plager S, Warden S, Huang D, Vanamee E, Foster R, Faustman DL. Targeting TNFR2 with antagonistic antibodies inhibits proliferation of ovarian cancer cells and tumorassociated tregs. Sci Signal 2017;10. doi: 10.1126/scisignal.aaf8608. [18] Kalliolias GD, Ivashkiv LB. TNF biology, pathogenic mechanisms and emerging therapeutic strategies. Nat Rev Rheumatol 2016;12:49–62. doi: 10.1038/nrrheum.2015.169. [19] Miller M-C, Manning HB, Jain A, Troeberg L, Dudhia J, Essex D, Sandison A, Seiki M, Nanchahal J, Nagase H, Itoh Y. Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritis. Arthritis Rheum 2009;60:686–97. doi: 10.1002/art.24331. [20] Kaneko K, Williams RO, Dransfield DT, Nixon AE, Sandison A, Itoh Y. Selective inhibition of membrane type 1 matrix metalloproteinase abrogates progression of experimental inflammatory arthritis: synergy with tumor necrosis factor blockade. Arthritis Rheumatol (Hoboken, NJ) 2016;68:521–31. doi: 10.1002/art.39414. [21] Hughes C, Faurholm B, Dell’Accio F, Manzo A, Seed M, Eltawil N, Marrelli A, Gould D, Subang C, Al-Kashi A, De Bari C, Winyard P, Chernajovsky Y, Nissim A. Human single-chain variable fragment that specifically targets arthritic cartilage. Arthritis Rheum 2010;62:1007–16. doi: 10.1002/art.27346. [22] Zvartsev RV, Korshunova DS, Gorshkova EA, Nosenko MA, Korneev KV, Maksimenko OG, et al. Neonatal lethality and inflammatory phenotype of the new transgenic mice with overexpression of human interleukin-6 in myeloid cells. Dokl Biochem Biophys 2018;483(1):344–7. doi: 10.1134/S1607672918060157. [23] Atretkhany K-SN, Mufazalov IA, Dunst J, Kuchmiy A, Gogoleva VS, Andruszewski D, et al. Intrinsic TNFR2 signaling in T regulatory cells provides protection in CNS autoimmunity. Proc Natl Acad Sci USA 2018;115(51):13051–6. doi: 10.1073/ pnas.1807499115.
Contents lists available at ScienceDirect
Seminars in Arthritis and Rheumatism journal homepage: www.elsevier.com/locate/semarthrit
Modulation of bioavailability of proinflammatory cytokines produced by myeloid cells Maxim A. Nosenkoa,b, Kamar-Sulu N. Atretkhanya,b, Vladislav V. Mokhonova,c, Ekaterina A. Vasilenkoc, Andrei A. Kruglovb,d, Sergei V. Tillibe, Marina S. Drutskayaa, Sergei A. Nedospasova,b,* a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia Lomonosov Moscow State University, Moscow 119234, Russia c Lobachevsky University of Nizhni Novgorod, Nizhni Novgorod 603950, Russia d German Rheumatism Research Center (DRFZ), Leibniz Institute, Berlin 10117, Germany e Institute of Gene Biology, Russian Academy of Sciences, Moscow 119991, Russia b
A R T I C L E
I N F O
Keywords: TNF IL-6 F4/80 CD11b VHH Bispecific antibodies
A B S T R A C T
In spite of successful therapeutic neutralization of proinflammatory cytokines in several autoimmune diseases, such therapy is not entirely free of side effects. The main reason relates to the fact that cytokine signaling may have protective components that need to be spared. Several approaches to achieve a less damaging cytokine inhibition are being explored. In our experimental studies we are using bispecific reagents based on VHH-modules from the heavy-chain-only antibodies to limit bioavailability of TNF and IL-6 produced by myeloid cells. After evaluation of their properties in vitro and in vivo we argue that these types of reagents may have an advantage over systemic blockers. © 2019 Elsevier Inc. All rights reserved.
Introduction At present time, there is no effective cure for most of autoimmune diseases caused by pathogenic self-reactive lymphocyte clones. The existing anti-cytokine therapies may improve the course of disease and relieve suffering, but they do not target the root of the problem and may also affect systemic immunoregulatory mechanisms. Once the patients are off the therapy, the disease is most likely to return. Another unresolved problem related to unwanted side effects of anti-cytokine therapies stems out from the fact that evolutionarily selected non-redundant functions of a given cytokine are more likely to be protective than pathogenic. Thus, pathogenic manifestations of cytokine signaling may not be dissociable from other functions, especially, when systemic inhibition is considered. With these ideas in mind we and others have set to explore a possibility of more selective anti-cytokine neutralization that may spare cytokine protective functions. Examples of such approaches for the two proinflammatory cytokines, TNF and IL-6, include selective interference with only one of the two TNF receptors [1 3,23] or interference with only one
*Corresponding author at: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov str., Moscow 119991, Russia. E-mail address: [email protected] (S.A. Nedospasov). https://doi.org/10.1016/j.semarthrit.2019.09.012 0049-0172/© 2019 Elsevier Inc. All rights reserved.
mode of IL-6 signaling based on administration of a soluble chimeric protein sgp130Fc [4,5], respectively. In studies utilizing a panel of conditional TNF knockouts, the evidence for pathogenic effects of TNF produced by myeloid cells was found in experimental arthritis, experimental autoimmune encephalomyelitis and in LPS-D/Gal-induced acute hepatotoxicity [6,7]. On the contrary, TNF produced by T cells demonstrated non-redundant protective functions in collagen-induced arthritis (manuscript in preparation) and in M. tuberculosis infection [8]. Similarly, IL-6 produced by dendritic cells was shown to promote EAE development [9] and our preliminary studies utilizing mice with IL-6 overexpression in myeloid cells reveal a pathogenic inflammatory phenotype causing postnatal lethality [22]. These results led to the idea of selective pharmacological neutralization of a proinflammatory cytokine produced by myeloid cells [10,11]. We decided to employ bispecific antibodies targeting myeloid cell surface markers that are able to capture secreted cytokine and cause its retention or internalization. We argue that in vivo such mode of retention will selectively limit bioavailability of a cytokine produced by myeloid cells only and not by other cell types. In this study, we evaluated and compared two targets on myeloid cells, F4/80 and CD11b, which served as anchors for cytokine binding moieties. Using VHH-based reagents specific for these targets [10,12] and anti-human-TNF VHH [13] we have generated several biologics, that we called myeloid-specific TNF inhibitors or MYSTIs, that were expected to interfere with
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human TNF secretion and bioavailability due to their binding to one of the two markers on the surface of myeloid cells (Suppl. Fig. 1).
MYSTI-2 and MYSTI-3 bind to the surface of macrophages and retain exogenously added human TNF We first compared bispecific antibodies in vitro for their ability to bind to macrophages (BMDM) and to human TNF (hTNF). For this purpose, we labeled MYSTI-2, MYSTI-3 and the control reagent, fMYSTI, with FITC to allow for fluorescent detection. Both MYSTI-2 and MYSTI-3, but not fMYSTI, effectively stained >98% of murine BMDM (Fig. 1(A)). Moreover, both biologics retained exogenously added hTNF via anti-TNF moiety as revealed by flow cytometry (Fig. 1(B)) or confocal microscopy (Fig. 1(C)). Control reagent did not bind to BMDM surface and, thus, did not produce the double staining despite its functional anti-TNF module. Thus, MYSTI-2 and MYSTI-3 show comparable abilities to decorate the macrophage surface and simultaneously retain hTNF.
MYSTI-2 has a longer half-life on the surface of macrophages compared to MYSTI-3 We have previously demonstrated that MYSTI-2 can be stored on the surface of macrophages for at least 24 h in vitro [11]. Interestingly, CD11b-based MYSTI-3 showed significantly shorter lifetime on BMDM surface, being almost undetectable already after 3 h of incubation, compared to MYSTI-2, which still was detectable after 24 h (Fig. 2). Both MYSTI-2 and MYSTI-3 were internalized by BMDM shortly after administration. However, the two biologics differed in their intracellular localization with MYSTI-2 being mainly located in large vesicles while MYSTI-3 was mostly detected in small granules, implicating possible proteolytic degradation in phagosomes.
MYSTI-2 and MYSTI-3 limit bioavailability of human TNF in vitro To functionally compare the retention of hTNF by both MYSTIs in vitro, BMDM isolated from hTNF KI mice [14] were preincubated with different doses of bispecific Abs, followed by
Fig. 1. MYSTI-2 and MYSTI-3 can simultaneously bind to macrophage surface and hTNF. A Flow cytometry staining of BMDM with FITC-labeled MYSTI-2 and MYSTI-3 compared to FITC-labeled control reagent (fMYSTI); B,C BMDM were consequently incubated with FITC-labeled bispecifics (MYSTI-2, MYSTI-3 or fMYSTI), recombinant hTNF, followed by anti-hTNF staining and analyzed by flow cytometry (B) or confocal microscopy (C). Staining without addition of hTNF was performed as control. Scale bar 50 mm.
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inhibit hTNF release compared to MYSTI-2 (Fig. 3). Thus we expect MYSTI-3 to have reduced therapeutic efficacy in vivo compared to MYSTI-2. Previously, MYSTI-2 was demonstrated to prevent lethality in the murine model of LPS/D-Gal-induced hepatotoxicity [10]. Our preliminary data in this model confirms inferior therapeutic potential of MYSTI-3 since it does not protect mice from lethality at the same dose, as MYSTI-2 does (data not shown). Thus we concluded that at the moment MYSTI-2 remains the best candidate reagent for further evaluation in various disease models in vivo. Conclusion
Fig. 2. MYSTI-3 has shorter life time on macrophage surface compared to MYSTI-2. BMDM were placed on glass-bottom dishes and incubated with FITC-labeled MYSTI-2, MYSTI-3 or fMYSTI for 15 min, then washed and cultivated in cell culture medium at 37 °C. At indicated timepoints cells were fixed with 4%PFA, counterstained with DAPI and analyzed using confocal microscopy. White arrows indicate internalized MYSTI-2 and MYSTI-3. Scale bar 50 mm.
In spite of its efficacy, systemic anti-cytokine therapy is not curative and is not free of unwanted side effects. Many laboratories are working to improve the specificity of proinflammatory cytokine inhibition employing different strategies. For example, selective inhibitors blocking only the soluble form of TNF [15,16], or just one of its two receptors have been developed [17,18]. In the case of IL-6, selective inhibition may target trans-signaling cascade, leaving intact at least some of the beneficial or protective IL-6 functions [4]. Alternative strategy targets bi-specific reagent with cytokine-neutralizing capacity to a damaged tissue [19 21], or to a pathogenic cell type. The fact that TNF produced by myeloid cells is pathogenic in several experimental disease models in mice led to the idea of developing a myeloid-specific TNF inhibitor, such as MYSTI [10]. Our results suggest that the properties of a surface molecule used for retention may be important and that other myeloid surface markers are worth exploring for targeting anti-cytokine reagents. We believe that bispecific cytokine blockers, such as described here, may reduce the collateral damage during anti-cytokine therapy and should be further evaluated in preclinical and clinical studies. Acknowledgments
activation with LPS. Supernatants from these cell cultures were then analyzed using ELISA (Fig. 3). Both MYSTI-2 and MYSTI-3, but not fMYSTI, limited secretion of hTNF by macrophages into culture medium in a dose-dependent manner. However, effective concentrations of two bispecifics were strikingly different with MYSTI-3 requiring almost 100 times higher concentration to fully
We thank G.A. Efimov for valuable input, M. Rashidian and H. Ploegh for providing anti-CD11b VHH and A.S. Zhdanova for help with ELISA. This work is supported by the Russian Science Foundation (grant # 19-75-30032), and confocal microscopy experiments were performed on Leica TCS SP5 and supported by grant # 14-50-00060. V.V. Mokhonov is supported by State assignment of Russian Ministry of Science and Education: 20.6445.2017/9.10.
Fig. 3. MYSTI-2 has higher TNF-inhibiting potency than MYSTI-3. MYSTI-2 and MYSTI-3 limit bioavailability of hTNF in vitro. BMDM, generated from hTNF KI mice, were incubated with indicated concentrations of MYSTI-2 (green), MYSTI-3 (blue) or fMYSTI as control (black), followed by washing and activation with LPS for 4 h. Then supernatants were collected and bioactive hTNF was measured using ELISA. Dashed bar indicates result from LPS-activated cells without incubation with any bispecific antibody. ND concentration is below detection limit (15 pg/ml). *p < 0.05; **p < 0.01; ****p < 0.0001; ns not significant (two-way ANOVA). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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M.A. Nosenko et al. / Seminars in Arthritis and Rheumatism 49 (2019) S39 S42
Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j.semarthrit.2019.09.012.
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