Transport of anti-IL-6 antigen binding fragments into cartilage and the effects of injury

The efficacy of biological therapeutics against cartilage degradation in osteoarthritis is restricted by the limited transport of macromolecules through the dense, avascular extracellular matrix. The availability of biologics to cell surface and matrix targets is limited by steric hindrance of the matrix, and the microstructure of matrix itself can be dramatically altered by joint injury and the subsequent inflammatory response. We studied the transport into cartilage of a 48 kDa anti-IL-6 antigen binding fragment (Fab) using an in vitro model of joint injury to quantify the transport of Fab fragments into normal and mechanically injured cartilage. The anti-IL-6 Fab was able to diffuse throughout the depth of the tissue, suggesting that Fab fragments can have the desired property of achieving local delivery to targets within cartilage, unlike full-sized antibodies which are too large to penetrate beyond the cartilage surface. Uptake of the anti-IL-6 Fab was significantly increased following mechanical injury, and an additional increase in uptake was observed in response to combined treatment with TNFα and mechanical injury, a model used to mimic the inflammatory response following joint injury. These results suggest that joint trauma leading to cartilage degradation can further alter the transport of such therapeutics and similar-sized macromolecules.National Institute of Arthritis and Musculoskeletal and Skin Diseases (U.S.) (Grant AR45779)National Institute of Arthritis and Musculoskeletal and Skin Diseases (U.S.) (Grant AR60331)Janssen Pharmaceutical Ltd. (Research and Development Grant

Surrogate Antibodies That Specifically Bind and Neutralize CCL17 But Not CCL22

The chemokines CCL17 (TARC) and CCL22 (MDC) function through the same receptor, CCR4, but have been proposed to differentially affect the immune response. To better understand the role of the individual ligands, a panel of rat anti-mouse CCL17 surrogate antibodies was generated that can be used to differentiate CCL17 and CCL22 function in vitro and in vivo. We have successfully identified a panel of neutralizing antibodies by screening hybridomas for the ability to inhibit CCL17-mediated calcium mobilization. Chemotaxis in response to CCL17 is also inhibited, providing further evidence that the antibodies in this panel are antagonistic. Using a recombinant cell line expressing human CCR4, we show that the antibodies block ?-arrestin recruitment as evidence that the antibodies are specifically blocking CCL17 signaling through CCR4. The antibodies within this panel inhibit calcium mobilization with varying potency in the calcium flux assay, having apparent IC50 ranging from approximately 1 to >400?ng/mL. Although both CCL17 and CCL22 function through CCR4, only a single antibody was identified as having detectable binding to CCL22. This panel of CCL17-specific antibodies provides tools that can be used to differentiate CCL17 and CCL22 function through CCR4 interaction in vitro and in vivo.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140164/1/mab.2012.0112.pd

Engagement of two distinct binding domains on CCL17 is required for signaling through CCR4 and establishment of localized inflammatory conditions in the lung.

CCL17 (TARC) function can be completely abolished by mAbs that block either one of two distinct sites required for CCR4 signaling. This chemokine is elevated in sera of asthma patients and is responsible for establishing inflammatory sites through CCR4-mediated recruitment of immune cells. CCL17 shares the GPCR CCR4, with CCL22 (MDC) but these two chemokines differentially affect the immune response. To better understand chemokine mediated effects through CCR4, we have generated chimeric anti-mouse CCL17 surrogate antibodies that inhibit function of this ligand in vitro and in vivo. The affinities of the surrogate antibodies for CCL17 range from 685 pM for B225 to 4.9 nM for B202. One antibody, B202, also exhibits weak binding to CCL22 (KD∼2 µM) and no binding to CCL22 is detectable with the second antibody, B225. In vitro, both antibodies inhibit CCL17-mediated calcium mobilization, β-arrestin recruitment and chemotaxis; B202 can also partially inhibit CCL22-mediated β-arrestin recruitment. Both B202 and B225 antibodies neutralize CCL17 in vivo as demonstrated by reduction of methacholine-induced airway hyperreactivity in the A. fumigatus model of asthma. That both antibodies block CCL17 function but only B202 shows any inhibition of CCL22 function suggests that they bind CCL17 at different sites. Competition binding studies confirm that these two antibodies recognize unique epitopes that are non-overlapping despite the small size of CCL17. Taking into consideration the data from both the functional and binding studies, we propose that effective engagement of CCR4 by CCL17 involves two distinct binding domains and interaction with both is required for signaling

Transport of anti-IL-6 antigen binding fragments into cartilage and the effects of injury

The efficacy of biological therapeutics against cartilage degradation in osteoarthritis is restricted by the limited transport of macromolecules through the dense, avascular extracellular matrix. The availability of biologics to cell surface and matrix targets is limited by steric hindrance of the matrix, and the microstructure of matrix itself can be dramatically altered by joint injury and the subsequent inflammatory response. We studied the transport into cartilage of a 48 kDa anti-IL-6 antigen binding fragment (Fab) using an in vitro model of joint injury to quantify the transport of Fab fragments into normal and mechanically injured cartilage. The anti-IL-6 Fab was able to diffuse throughout the depth of the tissue, suggesting that Fab fragments can have the desired property of achieving local delivery to targets within cartilage, unlike full-sized antibodies which are too large to penetrate beyond the cartilage surface. Uptake of the anti-IL-6 Fab was significantly increased following mechanical injury, and an additional increase in uptake was observed in response to combined treatment with TNFα and mechanical injury, a model used to mimic the inflammatory response following joint injury. These results suggest that joint trauma leading to cartilage degradation can further alter the transport of such therapeutics and similar-sized macromolecules

Characterization of binding properties of surrogate antibodies for CCL17 and CCL22.

<p>(A) CCL17 (<i>left</i>) but not CCL22 (<i>right</i>) can compete for CCL17 binding to B202 (<i>filled squares</i>) and B225 (<i>open squares</i>). (B) Typical Biacore sensorgrams (<i>left panels</i>) and steady-state data (<i>right panels</i>) for the interaction of two lots of B202 with murine CCL22. The interaction was analyzed in PBS pH 7.4, supplemented with 3 mM EDTA, 0.005% Tween 20 and 400 µg/mL BSA, at 25°C. In the plots, each curve (sensorgrams) and each point (steady-state plots<u>)</u> corresponds to a different concentration (in duplicate) of muCCL22 ranging from 3.9 nM to 4000 nM. The dissociation rates (k_d) and affinity (K_D) for are 0.44 and 1.4 µM; for the first lot of CCL22 (<i>shown in upper panels</i>) and the k_d is 0.35 and the K_D is 1.3 µM for the second lot of CCL22 (<i>shown in lower panels</i>).</p

Treatment with B225 anti-CCL17 antibody inhibits inflammation and goblet cell metaplasia in the lung of A. fumigatus-sensitized and challenged mice.

<p>Histology of whole lung sections from day 7 post-conidia challenged mice treated with antibodies at 200 µg/kg. Panels A-C are H/E-stained and panels D-F are PAS-stained. In panels D and F, <i>black arrows</i> highlight goblet cells and <i>red arrows</i> point to the mucus released from these cells in the airways. Samples are from groups as follows: A and D are from isotype control treated; B and E are from B225 treated; C and F are from B202 treated mice. Representative photomicrographs of lung sections from n = 5 mice per group are shown. Original magnification was 200×.</p

Antibodies B202 and B225 inhibit β-arrestin recruitment mediated by CCL17 interaction with CCR4.

<p>The human CCR4 β-arrestin reporter cell line was incubated with mouse CCL17 then assayed for the ability to mobilize β-arrestin as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081465#s2" target="_blank">materials and methods</a>. Mouse CCL17 is able to trigger β-arrestin recruitment by signaling through the human CCR4 receptor in a dose-dependent manner (A). The anti-mouse CCL17 specific antibody, B225, can inhibit mouse CCL17-induced β-arrestin recruitment (B). The dual reactive antibody, B202, strongly inhibited mCCL17 induced β-arrestin recruitment (C) while partially inhibiting mCCL22 induced β-arrestin recruitment (D). In panels C and D: B202 (<i>circles</i>); positive controls mAbs <u>(</u><i>inverted triangles</i>); isotype control (<i>open diamonds</i>); no antibody (<i>dotted line</i>); media only control (<i>solid line</i>). Data are expressed as Relative Light Units (RLU) to quantitate β-arrestin recruitment which is directly proportional to luminescent activity. Antibody dose curve starts at 1 µM with a 1∶2 dilution series (mCCL22 at 20 nM; mCCL17 at 30 nM). Antibodies were tested 3 times using CCL17 and twice using CCL22; shown here are representative data from one experiment.</p

Neutralization of CCL17 in vivo ameliorates methacholine-induced AHR.

<p>Airway resistance was determined in anesthetized and ventilated mice using a Buxco plethysmograph at day 7 after conidia. Mice received IgG control or anti-CCL17 mAb at 200 µg/kg or 1 mg/kg as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081465#s2" target="_blank">Materials and Methods</a>. Baseline and methacholine-induced airway resistance are shown. Data are mean ± SEM, n = 5 mice per group. *P<0.05 compared with methacholine-challenged control group (ANOVA and Student-Newman-Keuls Multiple Comparison post Test).</p

Each antibody binds a unique and non-overlapping epitope on CCL17.

<p>(A) Both B225 and MAB529 bind to mouse CCL17 proteins directly coated on microwells but B202 does not. (B) B202 binds to mouse CCL17 that has been captured by B225 indicating that B202 and B225 recognize different epitopes. (C) Labeled B225 is not competed by either B202 or MAB529 for binding to plate-bound CCL17. (D) Labeled MAB529 is competed by B225, but not B202 antibody for binding to plate-bound CCL17.</p

In vivo neutralization of CCL17 alters whole lung cytokine levels in A. fumigatus-sensitized and challenged mice.

<p>Cytokine and chemokine levels in homogenized whole lung samples from asthmatic mice at day 7 after conidia challenge. Bioplex analysis was used to measure cytokine and chemokine levels in these samples. Data are expressed as pg/mg for isotype control treated (<i>white bars</i>), B225 treated (<i>gray bars</i>) and B202 treated (<i>black bars</i>) Data are mean ± SEM, n = 5 mice per group. *P<0.05 compared with methacholine-challenged isotype control treated group as indicated (ANOVA and Student-Newman-Keuls Multiple Comparison post Test).</p