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BxN mouse serum transfer model of arthritis
4112
Abstracts / Molecular Immunology 45 (2008) 4095–4182
BAC-encoded hCR2 can replace mouse CR2/CR1 in Cr2−/− mice and restore humoral immune responses to model foreign antigens, we generated hCR2+/−:Cr2−/− mice and immunized them with sheep red blood cells (SRBC). Similar to our previously published P1 phage-derived hCR2 tg mice, BAC-hCR2-tg mice demonstrated anti-SRBC antibody (Ab) levels that are initially comparable to Cr2−/− mice after a single injection of the antigen, but then showed marked increases in anti-SRBC IgM and IgG1 levels after a second immunization (9223 ± 2713 RU of IgM to 13,408 ± 4198, p = 0.06; 34985.71 ± 22475.1 RU of IgG1 to 82,825 ± 36,823, p < 0.05 for Cr2−/− and hCR2+/−:Cr2−/−, respectively). To further confirm that this improvement in anti-SRBC Ab production over Cr2−/− mice was indeed due to hCR2 expression as well as to examine the effects of treating hCR2-tg mice with an inhibitory monoclonal Ab (mAb) in vivo, we used mAb 171, an anti-hCR2 mAb that we have recently shown directly recognizes the C3d binding site on hCR2. We first found that mAb 171 completely blocks hCD21-dependent co-activation of transgenic B cells by C3d:anti-IgM complexes as measured in vitro by increased intracellular calcium influx. The IP injection of 1 mg of mAb 171 was then found to induce only partial (30%) loss of hCR2 surface expression, without modifying B cell numbers, for at least three weeks. Finally, hCR2-tg mice injected with mAb 171, as compared to control IgG injection, were significantly (p < 0.01) suppressed in the development of IgM and IgG1 anti-SRBC Ab following two injections of SRBC. The development of this model system opens for the first time the possibility to study the effects of manipulating hCR2 function in vivo with potential therapeutic compounds. doi:10.1016/j.molimm.2008.08.050 O50 Gene targeting of properdin ameliorates disease development in the K/BxN mouse serum transfer model of arthritis Yuko Kimura, Lin Zhou, David Kim, Takashi Miwa, Wen-Chao Song University of Pennsylvania, Philadelphia, USA Properdin is a positive regulator of the alternative pathway (AP) complement. It facilitates AP complement activation by stabilizing and extending the half-life of the AP C3 convertase C3bBb. Recent studies have revealed that properdin may also work as a pattern recognition molecule to initiate and direct AP complement activation. In light of the fact that many human inflammatory diseases are mediated by AP complement, the question arises as to whether properdin might play a pathogenic role, and thus constitute a therapeutic target in these disease settings. To address this question, we studied a properdin knockout mouse (P−/−) in the K/BxN serum transfer model of arthritis, a disease process known to be dependent on AP complement. Compared with wild-type (WT) littermates, P−/− mice developed significantly less severe arthritis (delta ankle thickness at day 10 after serum transfer: 1.8 ± 0.3 mm for WT, 0.8 ± 0.3 mm for P−/−; total clinical index: 39 for WT and 22 for P−/− [out of a theoretical maximum of 40], N = 10 mice per group). Additionally, P−/− mice had significantly decreased inflammatory cytokine levels in their joints. To determine the source of properdin in the pathogenesis of arthritis, we generated bone marrow (BM) chimeras between WT and P−/− mice. We found that WT mice reconstituted with P−/− BM cells were protected from the disease, whereas P−/− mice reconstituted with WT BM cells developed similar disease as WT mice. In a separate approach, we engineered a properdin gene floxed mouse (Pflox ) by gene targeting and generated a myeloid cell-specific properdin knockout mouse by crossing with lysozyme-Cre transgenic mice. Importantly, this
conditional properdin knockout mouse (Pflox-lysozyme-cre ) was also protected from arthritis development. Our data thus suggest that myeloid leukocyte-derived properdin plays a pathogenic role in AP complement-dependent tissue injury and anti-properdin therapy may represent a novel option in drug development. doi:10.1016/j.molimm.2008.08.051 O51 Human C3/cobra venom factor hybrid proteins for therapeutic complement depletion: In vivo activity and lack of toxicity in primates David Fritzinger a , Brian Hew a , June Lee a , William St. John b , Michael Scaife c , Stephen Wilson d , Carl-Wilhelm Vogel a a
Cancer Research Center of Hawaii, Honolulu, USA InCode Biopharmaceuticals Inc., San Francisco, USA c MChS Consulting Inc., Los Altos, USA d Charles River Laboratories Inc., Shrewsbury, USA b
HC3-1496 is a human C3/cobra venom factor (CVF) hybrid protein in which the C-terminal 168 residues of C3 were replaced with homologous CVF residues. We have shown previously that HC3-1496 can form a stable C3 convertase that is able to deplete complement in mammalian serum in vitro and in vivo. To test the efficacy and safety of the HC3-1496 relevant for human application, it was injected into the pulmonary artery of cynomolgus monkeys over a period of 1–5 min at 62.5, 250, or 1000 g/kg—the latter dose well above what might be used clinically. At the 250 and 1000 g/kg doses, serum complement was completely depleted within 5 min and remained depleted for at least 1 h, followed by a gradual increase to approximately 20% over the next 6 h. C3a levels showed a dose-dependent rapid spike for all doses, followed by decrease to normal or subnormal levels within 1–2 h. No significant amounts of C5a were detected at any dose level, consistent with no or only residual C5-cleaving in vitro activity of the HC3-1496, Bb convertase. No reduction in serum complement was seen with 62.5 g/kg of HC3-1496. No cardiac or pulmonary changes (systolic, diastolic, and mean arterial as well as pulmonary artery blood pressures; heart rate; respiratory rate; tidal volume; maximum, minimum, and mean airway pressures; lung resistance; dynamic lung compliance) were seen after injection of 62.5 or 250 g/kg HC3-1496, while the injection of 1000 g/kg resulted in small and transitory increases in heart rate, blood pressure, and lung resistance, as well as a slight decrease in lung compliance and tidal volume. All parameters had returned to normal within 5 min, were of minor, if any, clinical significance, and could also be due to other causes. In conclusion, intra-arterial injection of HC3-1496 is a safe and efficient means of depleting complement in primates. These results suggest that HC3-1496 is a promising reagent for therapeutic complement depletion in humans. doi:10.1016/j.molimm.2008.08.052
Abstracts / Molecular Immunology 45 (2008) 4095–4182
BAC-encoded hCR2 can replace mouse CR2/CR1 in Cr2−/− mice and restore humoral immune responses to model foreign antigens, we generated hCR2+/−:Cr2−/− mice and immunized them with sheep red blood cells (SRBC). Similar to our previously published P1 phage-derived hCR2 tg mice, BAC-hCR2-tg mice demonstrated anti-SRBC antibody (Ab) levels that are initially comparable to Cr2−/− mice after a single injection of the antigen, but then showed marked increases in anti-SRBC IgM and IgG1 levels after a second immunization (9223 ± 2713 RU of IgM to 13,408 ± 4198, p = 0.06; 34985.71 ± 22475.1 RU of IgG1 to 82,825 ± 36,823, p < 0.05 for Cr2−/− and hCR2+/−:Cr2−/−, respectively). To further confirm that this improvement in anti-SRBC Ab production over Cr2−/− mice was indeed due to hCR2 expression as well as to examine the effects of treating hCR2-tg mice with an inhibitory monoclonal Ab (mAb) in vivo, we used mAb 171, an anti-hCR2 mAb that we have recently shown directly recognizes the C3d binding site on hCR2. We first found that mAb 171 completely blocks hCD21-dependent co-activation of transgenic B cells by C3d:anti-IgM complexes as measured in vitro by increased intracellular calcium influx. The IP injection of 1 mg of mAb 171 was then found to induce only partial (30%) loss of hCR2 surface expression, without modifying B cell numbers, for at least three weeks. Finally, hCR2-tg mice injected with mAb 171, as compared to control IgG injection, were significantly (p < 0.01) suppressed in the development of IgM and IgG1 anti-SRBC Ab following two injections of SRBC. The development of this model system opens for the first time the possibility to study the effects of manipulating hCR2 function in vivo with potential therapeutic compounds. doi:10.1016/j.molimm.2008.08.050 O50 Gene targeting of properdin ameliorates disease development in the K/BxN mouse serum transfer model of arthritis Yuko Kimura, Lin Zhou, David Kim, Takashi Miwa, Wen-Chao Song University of Pennsylvania, Philadelphia, USA Properdin is a positive regulator of the alternative pathway (AP) complement. It facilitates AP complement activation by stabilizing and extending the half-life of the AP C3 convertase C3bBb. Recent studies have revealed that properdin may also work as a pattern recognition molecule to initiate and direct AP complement activation. In light of the fact that many human inflammatory diseases are mediated by AP complement, the question arises as to whether properdin might play a pathogenic role, and thus constitute a therapeutic target in these disease settings. To address this question, we studied a properdin knockout mouse (P−/−) in the K/BxN serum transfer model of arthritis, a disease process known to be dependent on AP complement. Compared with wild-type (WT) littermates, P−/− mice developed significantly less severe arthritis (delta ankle thickness at day 10 after serum transfer: 1.8 ± 0.3 mm for WT, 0.8 ± 0.3 mm for P−/−; total clinical index: 39 for WT and 22 for P−/− [out of a theoretical maximum of 40], N = 10 mice per group). Additionally, P−/− mice had significantly decreased inflammatory cytokine levels in their joints. To determine the source of properdin in the pathogenesis of arthritis, we generated bone marrow (BM) chimeras between WT and P−/− mice. We found that WT mice reconstituted with P−/− BM cells were protected from the disease, whereas P−/− mice reconstituted with WT BM cells developed similar disease as WT mice. In a separate approach, we engineered a properdin gene floxed mouse (Pflox ) by gene targeting and generated a myeloid cell-specific properdin knockout mouse by crossing with lysozyme-Cre transgenic mice. Importantly, this
conditional properdin knockout mouse (Pflox-lysozyme-cre ) was also protected from arthritis development. Our data thus suggest that myeloid leukocyte-derived properdin plays a pathogenic role in AP complement-dependent tissue injury and anti-properdin therapy may represent a novel option in drug development. doi:10.1016/j.molimm.2008.08.051 O51 Human C3/cobra venom factor hybrid proteins for therapeutic complement depletion: In vivo activity and lack of toxicity in primates David Fritzinger a , Brian Hew a , June Lee a , William St. John b , Michael Scaife c , Stephen Wilson d , Carl-Wilhelm Vogel a a
Cancer Research Center of Hawaii, Honolulu, USA InCode Biopharmaceuticals Inc., San Francisco, USA c MChS Consulting Inc., Los Altos, USA d Charles River Laboratories Inc., Shrewsbury, USA b
HC3-1496 is a human C3/cobra venom factor (CVF) hybrid protein in which the C-terminal 168 residues of C3 were replaced with homologous CVF residues. We have shown previously that HC3-1496 can form a stable C3 convertase that is able to deplete complement in mammalian serum in vitro and in vivo. To test the efficacy and safety of the HC3-1496 relevant for human application, it was injected into the pulmonary artery of cynomolgus monkeys over a period of 1–5 min at 62.5, 250, or 1000 g/kg—the latter dose well above what might be used clinically. At the 250 and 1000 g/kg doses, serum complement was completely depleted within 5 min and remained depleted for at least 1 h, followed by a gradual increase to approximately 20% over the next 6 h. C3a levels showed a dose-dependent rapid spike for all doses, followed by decrease to normal or subnormal levels within 1–2 h. No significant amounts of C5a were detected at any dose level, consistent with no or only residual C5-cleaving in vitro activity of the HC3-1496, Bb convertase. No reduction in serum complement was seen with 62.5 g/kg of HC3-1496. No cardiac or pulmonary changes (systolic, diastolic, and mean arterial as well as pulmonary artery blood pressures; heart rate; respiratory rate; tidal volume; maximum, minimum, and mean airway pressures; lung resistance; dynamic lung compliance) were seen after injection of 62.5 or 250 g/kg HC3-1496, while the injection of 1000 g/kg resulted in small and transitory increases in heart rate, blood pressure, and lung resistance, as well as a slight decrease in lung compliance and tidal volume. All parameters had returned to normal within 5 min, were of minor, if any, clinical significance, and could also be due to other causes. In conclusion, intra-arterial injection of HC3-1496 is a safe and efficient means of depleting complement in primates. These results suggest that HC3-1496 is a promising reagent for therapeutic complement depletion in humans. doi:10.1016/j.molimm.2008.08.052