Keratinocyte cytokine released in response to RSV infection reverses hyperresponsiveness to methacholine in OVA-sensitized mice.

<p>(<b>A</b>) Bronchoalveolar lavage fluid keratinocyte cytokine (KC; ng/ml) levels in unsensitized, uninfected mice (UNSENS/UNINF; <i>n</i> = 5), OVA-sensitized, uninfected mice (OVA/UNINF; <i>n</i> = 11), OVA-sensitized, uninfected mice treated with 50 µg/ml heat-inactivated recombinant murine KC (OVA/UNINF + HI-KC; <i>n</i> = 5), OVA-sensitized, uninfected mice treated with 50 µg/ml recombinant murine KC (OVA/UNINF + KC; <i>n</i> = 7), OVA-sensitized mice infected with RSV (10⁶ pfu/mouse) for 2 days (OVA/DAY 2; <i>n</i> = 6), OVA-sensitized mice “infected” with UV-inactivated RSV for 2 days (OVA/UVx DAY 2; <i>n</i> = 4), and OVA-sensitized mice infected with RSV for 8 days (OVA/DAY 8; <i>n</i> = 6). *<i>P</i><0.05, ***<i>P</i><0.0005, <i>vs</i>. UNSENS/UNINF mice. (<b>B</b>) Bronchoconstrictive responses to increasing doses of nebulized methacholine (MCH) in OVA-sensitized, RSV-infected mice following nebulization of normal rat IgG (50 µg/ml; <i>n</i> = 5), KC-neutralizing monoclonal antibody (ANTI-KC, 50 µg/ml; <i>n</i> = 5), pretreatment with pertussis toxin and IgG (PTX + IgG; <i>n</i> = 6), or pretreatment with pertussis toxin and KC neutralizing antibody (PTX + ANTI-KC; <i>n</i> = 8). ***MCH dose-response curve differs significantly (<i>P</i><0.0005) from UNSENS/UNINF mice (<i>n</i> = 16).</p

RSV infection reverses airway hyperresponsiveness to methacholine in OVA-sensitized mice.

<p>Bronchoconstrictive responses to increasing doses of nebulized methacholine (MCH) in: (<b>A</b>) unsensitized, uninfected mice analyzed on day 0 (UNSENS/UNINF; <i>n</i> = 6), OVA-sensitized, uninfected mice (OVA/UNINF; <i>n</i> = 8), and OVA-sensitized mice infected with RSV (10⁶ pfu/mouse) for 2 days (OVA/DAY 2; <i>n</i> = 16), or 8 days (OVA/DAY 8; <i>n</i> = 8); (<b>B</b>) OVA/UNINF mice and OVA-sensitized mice “infected” with UV-inactivated RSV for 2 days (OVA/UVx DAY 2; <i>n</i> = 5) or 8 days (OVA/UVx DAY 8; <i>n</i> = 9). ***MCH dose-response curve differs significantly (<i>P</i><0.0005) from UNSENS/UNINF mice.</p

Keratinocyte cytokine exposure and Gαi activation are both sufficient to reverse methacholine hyperresponsiveness in OVA-sensitized, uninfected mice.

<p>Bronchoconstrictive responses to increasing doses of nebulized methacholine (MCH) following nebulization of: (<b>A</b>) heat-inactivated recombinant murine keratinocyte cytokine (HI-rmKC, 50 µg/ml; <i>n</i> = 6) or recombinant murine keratinocyte cytokine (rmKC, 50 µg/ml; <i>n</i> = 7); and (<b>B</b>) Melittin (100 µM; <i>n</i> = 7). ***MCH dose-response curve differs significantly (<i>P</i><0.0005) from OVA/UNINF mice (<i>n</i> = 8).</p

Schematic timeline of the OVA sensitization/challenge and RSV infection protocol.

<p>Mice were sensitized by intraperitoneal (i.p.) injection of OVA in alum at days −28 and −14. From −7 to −3 days, mice were challenged daily by intranasal (i.n.) OVA instillation. Animals were infected with RSV 3 days after the last OVA challenge (day 0). Airway responsiveness to methacholine (MCH) was measured at 2–8 days post-infection (d.p.i.).</p

RSV infection reverses hyperresponsiveness to methacholine in OVA-sensitized mice via a pertussis toxin-sensitive pathway.

<p>Bronchoconstrictive response to increasing doses of nebulized methacholine (MCH) following pretreatment with saline (100 µl i.p.; <i>n</i> = 4) or pertussis toxin (PTX, 100 µg/kg in 100 µl saline i.p.; <i>n</i> = 9). ***MCH dose-response curve differs significantly (<i>P</i><0.0005) from OVA/DAY 2 mice (OVA-sensitized mice infected with 10⁶ pfu/mouse RSV A2 for 2 days; <i>n</i> = 16).</p

RSV infection reduces bronchoalveolar lavage fluid cell counts in OVA-sensitized mice.

<p>Effect of OVA sensitization on (<b>A</b>) Alveolar macrophage (AM), eosinophil (EO) and small lymphocyte (SL) counts in uninfected mice (OVA/UNINF; <i>n</i> = 8) and unsensitized, uninfected controls (UNSENS/UNINF; <i>n</i> = 8); (<b>B</b>) Total cell counts in OVA-sensitized mice after mock infection for 2 days (M2; <i>n</i> = 5), 4 days (M4; <i>n</i> = 4), or 8 days (M8; <i>n</i> = 8), and infection with RSV (10⁶ pfu/mouse) for 2 days (<i>n</i> = 16), 4 days (<i>n</i> = 10), or 8 days (<i>n</i> = 6); (<b>C</b>) AM, EO, and SL counts after mock infection for 2, 4, or 8 days and infection with RSV for 2–8 days; and (<b>D</b>) Neutrophil (PMN) counts after infection with RSV for 2–8 days. No PMNs were detected in bronchoalveolar lavage fluid from uninfected or mock-infected mice at any timepoint (not shown). *<i>P</i><0.05, **<i>P</i><0.005, ***<i>P</i><0.001. N.D.: None detected.</p

Proposed mechanism for altered airway responsiveness to methacholine in ovalbumin-sensitized mice following RSV infection.

<p>In unsensitized or OVA-sensitized, uninfected mice, methacholine binds to M₃-subtype muscarinic receptors (M₃R), resulting in release of Gαq and downstream activation of phospholipase C (PLC). PLC then activates protein kinase C (PKC) and increases intracellular Ca⁺⁺ (Ca⁺⁺_i), leading to bronchoconstriction. RSV infection of respiratory epithelial cells in OVA-sensitized mice induces release of keratinocyte cytokine (KC), which binds to epithelial CXCR2 receptors in either an autocrine or paracrine fashion. KC receptor binding can be replicated in OVA-sensitized, uninfected mice by recombinant murine KC (<u>rmKC</u>), but is blocked by a neutralizing antibody to KC (<i>anti-KC</i>). Activation of CXCR2 liberates pertussis toxin (<i>PTX</i>)-sensitive Gαi, resulting in reversal of hyperresponsiveness to methacholine. Gαi can also be directly activated by <u>melittin</u>. As in previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046660#pone.0046660-McGraw1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046660#pone.0046660-McGraw2" target="_blank">[29]</a>, reversal of airway hyperresponsiveness may be a consequence of inhibition of the phospholipase C (PLC)/protein kinase C (PKC) pathway by Gαi, although we did not formally demonstrate this in the current study. Experimental agonists are shown underlined. Experimental antagonists are shown in italics. Broken lines indicate postulated mechanisms that were not formally demonstrated in this study.</p

Table_2_Translatability of findings from cynomolgus monkey to human suggests a mechanistic role for IL-21 in promoting immunogenicity to an anti-PD-1/IL-21 mutein fusion protein.docx

AMG 256 is a bi-specific, heteroimmunoglobulin molecule with an anti-PD-1 antibody domain and a single IL-21 mutein domain on the C-terminus. Nonclinical studies in cynomolgus monkeys revealed that AMG 256 administration led to the development of immunogenicity-mediated responses and indicated that the IL-21 mutein domain of AMG 256 could enhance the anti-drug antibody response directed toward the monoclonal antibody domain. Anti-AMG 256 IgE were also observed in cynomolgus monkeys. A first-in-human (FIH) study in patients with advanced solid tumors was designed with these risks in mind. AMG 256 elicited ADA in 28 of 33 subjects (84.8%). However, ADA responses were only robust and exposure-impacting at the 2 lowest doses. At mid to high doses, ADA responses remained low magnitude and all subjects maintained exposure, despite most subjects developing ADA. Limited drug-specific IgE were also observed during the FIH study. ADA responses were not associated with any type of adverse event. The AMG 256 program represents a unique case where nonclinical studies informed on the risk of immunogenicity in humans, due to the IL-21-driven nature of the response.</p

Table_1_Translatability of findings from cynomolgus monkey to human suggests a mechanistic role for IL-21 in promoting immunogenicity to an anti-PD-1/IL-21 mutein fusion protein.docx

AMG 256 is a bi-specific, heteroimmunoglobulin molecule with an anti-PD-1 antibody domain and a single IL-21 mutein domain on the C-terminus. Nonclinical studies in cynomolgus monkeys revealed that AMG 256 administration led to the development of immunogenicity-mediated responses and indicated that the IL-21 mutein domain of AMG 256 could enhance the anti-drug antibody response directed toward the monoclonal antibody domain. Anti-AMG 256 IgE were also observed in cynomolgus monkeys. A first-in-human (FIH) study in patients with advanced solid tumors was designed with these risks in mind. AMG 256 elicited ADA in 28 of 33 subjects (84.8%). However, ADA responses were only robust and exposure-impacting at the 2 lowest doses. At mid to high doses, ADA responses remained low magnitude and all subjects maintained exposure, despite most subjects developing ADA. Limited drug-specific IgE were also observed during the FIH study. ADA responses were not associated with any type of adverse event. The AMG 256 program represents a unique case where nonclinical studies informed on the risk of immunogenicity in humans, due to the IL-21-driven nature of the response.</p