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An inhibitory role of complement in monoclonal antibody therapy of lymphoma
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Abstracts / Molecular Immunology 47 (2010) 2198–2294
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Upstream inhibition of key innate immune pathways in vivo in sepsis
An inhibitory role of complement in monoclonal antibody therapy of lymphoma
Ebbe Billmann Thorgersen a , Bernt Christian Hellerud a , Erik Waage Nielsen b , Andreas Barratt-Due a , Hilde Fure c , Julie Katrine Lindstad a , Anne Pharo a , Erik Fosse d , Tor Inge Tønnessen e , Harald Thidemann Johansen f , Albert Castellheim a , Tom Eirik Mollnes a
David C. Fritzinger a , Carl-Wilhelm Vogel a , Bassam B. Damaj b , Paul W. Finnegan c
a
Institute of Immunology, Rikshospitalet University Hospital and University of Oslo, Oslo, Norway b Department of Anesthesiology, Nordland Hospital, and Institute of Clinical Medicine, University of Tromsø, Norway c Department of Laboratory Medicine, Nordland Hospital, Bodø, Norway d The Interventional Centre, Rikshospitalet University Hospital, and University of Oslo, Oslo, Norway e The Interventional Centre, and Department of Anesthesiology, Rikshospitalet University Hospital, and University of Oslo, Oslo, Norway f Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway Sepsis is a severe infection-induced systemic inflammatory syndrome. Inhibition of downstream inflammatory mediators of sepsis, e.g. TNF-␣, has failed in clinical trials. We hypothesize that a better approach would be inhibition of upstream inducers instead of downstream mediators of inflammation. Complement and CD14 are two such key upstream innate immunity inducers of inflammation. Combined inhibition of the two has shown great promise in several human and porcine in vitro studies. A pig in vivo Gram-negative sepsis model has recently been developed to mimic human sepsis. The aim of the present study was to investigate the effects of inhibiting CD14, on the early inflammatory and hemostatic responses in this pig in vivo model of Gram-negative sepsis. The study comprised two arms, whole live Escherichia coli bacteria and E. coli-lipopolysaccharide (LPS) (n = 25 and n = 9 animals, respectively). The animals were allocated into treatment (anti-CD14) and control (IgG isotype or saline) groups. Inflammatory, hemostatic, physiological and microbiological parameters were measured. The proinflammatory cytokines TNF-␣, IL-1, IL6 and IL-8, but not the anti-inflammatory cytokine IL-10, were efficiently inhibited by anti-CD14. Furthermore, anti-CD14 preserved the leukocyte count and significantly reduced granulocyte enzyme matrix metalloproteinase-9 release and expression of the granulocyte membrane activation molecule wCD11R3 (pig CD11b). The hemostatic markers thrombin-antithrombin III complexes and plasminogen activator inhibitor-1 were significantly attenuated. Anti-CD14 did not affect LPS or E. coli DNA levels. This study documents that CD14-inhibition efficiently attenuates the proinflammatory cytokine response and granulocyte activation, reverse the procoagulant state, but does not interfere with LPS levels or bacterial counts in E. coli-induced sepsis. Combined inhibition of complement and CD14 will be investigated in the present model. doi:10.1016/j.molimm.2010.05.209
a
Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, HI 96813, USA b Bio-Quant, Inc., San Diego, CA 92121, USA c Incode Biopharmaceutics, Inc., San Diego, CA 92121, USA Oncologic monoclonal antibodies are thought to exert their therapeutic effects by complement-mediated cytotoxicity (CMC), antibody-dependent cellular cytotoxicity (ADCC), and/or directly or indirectly by binding to their target cell surface antigens/receptors. Rituximab is a clinically widely used therapeutic anti-CD20 monoclonal antibody for B-cell lymphoma. Evidence for CMC is mainly from in vitro studies, whereas ADCC data come from laboratory models and, more recently, from in vivo studies. Specifically, activation of NK cells by the Fc domain of a therapeutic antibody (through CD16) and NK cell-mediated lysis of rituximab-coated tumor cells are inhibited by complement activation via C3b deposition; and we recently showed that complement depletion by cobra venom factor (CVF) or HC3-1496, a human C3 derivative with CVFlike functions (humanized CVF), resulted in a survival benefit in a syngeneic murine lymphoma model using murine anti-idiotypic antibody therapy [Wang et al., 2009. Blood 114, 5322–5330]. To further assess this in vivo effect, we conducted a study of Raji lymphoma mouse survival and tested the effects of various combination regimens of HC3-1496 and rituximab. Mice were injected with 106 Raji cells on day 0. Therapy began on day 4. Drug doses were 20 mg/kg of rituximab i.p. and 500 g/kg of HC3-1496 i.p. Living mice were dosed with various regimens over 90 days. Control and HC3-1496-only treated animals died within 24 days. In comparison, rituximab alone showed < 10% survival at day 58, whereas the combination regimens showed positive trends. The best outcome was a 25% survival between day 58 and day 115 (the end of study) from a regimen consisting of rituximab every third day combined with HC3-1496 2 h before and 48 hrs after rituximab. These data appear to support that complement activation, likely through C3b deposition, inhibits the ADCC capacity of therapeutic monoclonal antibodies, and that C3 complement depletion may increase ADCC-mediated tumor cell killing; however, in this model, CMC appears to be also important and complement depletion leads to less CMC, thus resulting in a small net positive effect. doi:10.1016/j.molimm.2010.05.210 34 Pharmacokinetics of humanized cobra venom factor in mice David C. Fritzinger a , Bassam B. Damaj b , Katina Wong a , CarlWilhelm Vogel a , Paul W. Finnegan c a
Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, HI 96813, USA b Bio-Quant, Inc., San Diego, CA 92121, USA c Incode Biopharmaceutics, Inc., San Diego, CA 92121, USA
A pharmacokinetic/pharmacodynamic analysis of complement depletion with the humanized cobra venom factor (CVF) protein HC3-1496 was performed in Raji-SCID mice. SCID mice that had previously been injected i.v. with 106 Raji lymphoma cells were injected i.p. with 10 g (500 g/kg) biotinylated HC3-1496. Serum samples were obtained prior to injection, and at 0.5, 1, 2, 4, 8, 12, and 24 h post-injection. Serum was assayed for the presence of the
Abstracts / Molecular Immunology 47 (2010) 2198–2294
32
33
Upstream inhibition of key innate immune pathways in vivo in sepsis
An inhibitory role of complement in monoclonal antibody therapy of lymphoma
Ebbe Billmann Thorgersen a , Bernt Christian Hellerud a , Erik Waage Nielsen b , Andreas Barratt-Due a , Hilde Fure c , Julie Katrine Lindstad a , Anne Pharo a , Erik Fosse d , Tor Inge Tønnessen e , Harald Thidemann Johansen f , Albert Castellheim a , Tom Eirik Mollnes a
David C. Fritzinger a , Carl-Wilhelm Vogel a , Bassam B. Damaj b , Paul W. Finnegan c
a
Institute of Immunology, Rikshospitalet University Hospital and University of Oslo, Oslo, Norway b Department of Anesthesiology, Nordland Hospital, and Institute of Clinical Medicine, University of Tromsø, Norway c Department of Laboratory Medicine, Nordland Hospital, Bodø, Norway d The Interventional Centre, Rikshospitalet University Hospital, and University of Oslo, Oslo, Norway e The Interventional Centre, and Department of Anesthesiology, Rikshospitalet University Hospital, and University of Oslo, Oslo, Norway f Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway Sepsis is a severe infection-induced systemic inflammatory syndrome. Inhibition of downstream inflammatory mediators of sepsis, e.g. TNF-␣, has failed in clinical trials. We hypothesize that a better approach would be inhibition of upstream inducers instead of downstream mediators of inflammation. Complement and CD14 are two such key upstream innate immunity inducers of inflammation. Combined inhibition of the two has shown great promise in several human and porcine in vitro studies. A pig in vivo Gram-negative sepsis model has recently been developed to mimic human sepsis. The aim of the present study was to investigate the effects of inhibiting CD14, on the early inflammatory and hemostatic responses in this pig in vivo model of Gram-negative sepsis. The study comprised two arms, whole live Escherichia coli bacteria and E. coli-lipopolysaccharide (LPS) (n = 25 and n = 9 animals, respectively). The animals were allocated into treatment (anti-CD14) and control (IgG isotype or saline) groups. Inflammatory, hemostatic, physiological and microbiological parameters were measured. The proinflammatory cytokines TNF-␣, IL-1, IL6 and IL-8, but not the anti-inflammatory cytokine IL-10, were efficiently inhibited by anti-CD14. Furthermore, anti-CD14 preserved the leukocyte count and significantly reduced granulocyte enzyme matrix metalloproteinase-9 release and expression of the granulocyte membrane activation molecule wCD11R3 (pig CD11b). The hemostatic markers thrombin-antithrombin III complexes and plasminogen activator inhibitor-1 were significantly attenuated. Anti-CD14 did not affect LPS or E. coli DNA levels. This study documents that CD14-inhibition efficiently attenuates the proinflammatory cytokine response and granulocyte activation, reverse the procoagulant state, but does not interfere with LPS levels or bacterial counts in E. coli-induced sepsis. Combined inhibition of complement and CD14 will be investigated in the present model. doi:10.1016/j.molimm.2010.05.209
a
Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, HI 96813, USA b Bio-Quant, Inc., San Diego, CA 92121, USA c Incode Biopharmaceutics, Inc., San Diego, CA 92121, USA Oncologic monoclonal antibodies are thought to exert their therapeutic effects by complement-mediated cytotoxicity (CMC), antibody-dependent cellular cytotoxicity (ADCC), and/or directly or indirectly by binding to their target cell surface antigens/receptors. Rituximab is a clinically widely used therapeutic anti-CD20 monoclonal antibody for B-cell lymphoma. Evidence for CMC is mainly from in vitro studies, whereas ADCC data come from laboratory models and, more recently, from in vivo studies. Specifically, activation of NK cells by the Fc domain of a therapeutic antibody (through CD16) and NK cell-mediated lysis of rituximab-coated tumor cells are inhibited by complement activation via C3b deposition; and we recently showed that complement depletion by cobra venom factor (CVF) or HC3-1496, a human C3 derivative with CVFlike functions (humanized CVF), resulted in a survival benefit in a syngeneic murine lymphoma model using murine anti-idiotypic antibody therapy [Wang et al., 2009. Blood 114, 5322–5330]. To further assess this in vivo effect, we conducted a study of Raji lymphoma mouse survival and tested the effects of various combination regimens of HC3-1496 and rituximab. Mice were injected with 106 Raji cells on day 0. Therapy began on day 4. Drug doses were 20 mg/kg of rituximab i.p. and 500 g/kg of HC3-1496 i.p. Living mice were dosed with various regimens over 90 days. Control and HC3-1496-only treated animals died within 24 days. In comparison, rituximab alone showed < 10% survival at day 58, whereas the combination regimens showed positive trends. The best outcome was a 25% survival between day 58 and day 115 (the end of study) from a regimen consisting of rituximab every third day combined with HC3-1496 2 h before and 48 hrs after rituximab. These data appear to support that complement activation, likely through C3b deposition, inhibits the ADCC capacity of therapeutic monoclonal antibodies, and that C3 complement depletion may increase ADCC-mediated tumor cell killing; however, in this model, CMC appears to be also important and complement depletion leads to less CMC, thus resulting in a small net positive effect. doi:10.1016/j.molimm.2010.05.210 34 Pharmacokinetics of humanized cobra venom factor in mice David C. Fritzinger a , Bassam B. Damaj b , Katina Wong a , CarlWilhelm Vogel a , Paul W. Finnegan c a
Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, HI 96813, USA b Bio-Quant, Inc., San Diego, CA 92121, USA c Incode Biopharmaceutics, Inc., San Diego, CA 92121, USA
A pharmacokinetic/pharmacodynamic analysis of complement depletion with the humanized cobra venom factor (CVF) protein HC3-1496 was performed in Raji-SCID mice. SCID mice that had previously been injected i.v. with 106 Raji lymphoma cells were injected i.p. with 10 g (500 g/kg) biotinylated HC3-1496. Serum samples were obtained prior to injection, and at 0.5, 1, 2, 4, 8, 12, and 24 h post-injection. Serum was assayed for the presence of the