A humanized monoclonal antibody targeting the β7 integrin selectively blocks intestinal homing of T lymphocytes

Abstract

Background and purpose: rhuMAb Beta7 is a humanized anti-human β7 monoclonal antibody currently in phase I in inflammatory bowel disease. rhuMAb Beta7 binds the β7 subunit of the integrins α4β7 and αEβ7, blocking interaction with their ligands. These integrins play key roles in immune cell homing to and retention in mucosal sites, and are associated with chronic inflammatory diseases of the gastrointestinal tract. The goal of this study was to evaluate the mucosal specificity of rhuMAb Beta7.

Experimental approach: We assessed the effect of murine anti-Beta7 on lymphocyte homing in mouse models of autoimmune disease. We also compared the effect of rhuMAb Beta7 on circulating mucosal-homing versus peripheral-homing T cells in naïve non-human primates.

Key results: In cynomolgus monkeys, occupancy of β7 integrin receptors by rhuMAb Beta7 correlated with an increase in circulating β7(+) mucosal-homing lymphocytes, with no apparent effect on levels of circulating β7(-) peripheral-homing lymphocytes. rhuMAb Beta7 also inhibited lymphocyte homing to the inflamed colons of severe combined immunodeficient mice in CD45RB(high) CD4(+) T-cell transfer models. Consistent with a lack of effect on peripheral homing, in a mouse model of experimental autoimmune encephalomyelitis, anti-β7 treatment resulted in no amelioration of CNS inflammation.

Conclusions and implications: The results presented here suggest that rhuMAb Beta7 selectively blocks lymphocyte homing to the gastrointestinal tract without affecting lymphocyte trafficking to non-mucosal tissues. rhuMAb Beta7 provides a targeted therapeutic approach with the potential for a more attractive benefit:risk ratio than currently available inflammatory bowel disease therapies.

Figure 1

The total β7 integrin receptor expression on peripheral blood CD8⁺ (A) or CD4⁺ lymphocytes (B) and unoccupied β7 receptor expression on peripheral blood CD8⁺ (C) or CD4⁺ lymphocytes (D) following single i.v. bolus administration of 5 mg·kg⁻¹ rhuMAb Beta7 to BALB/c mice (n = 4 per time point). The data are presented as the mean (±SD) percentage for each group normalized to the percentage in the control group (Time 0). Samples were analysed by flow cytometry, and 20 000 lymphocyte events were acquired using a FSC/SSC plot. Total β7 receptor expression on lymphocytes was determined using anti-β7 (clone M293), a non-competing antibody that binds a different epitope from rhuMAb Beta7. Changes in total β7 receptor expression (down-modulation) for each mouse were expressed as per cent of control GMFI, which was determined from the average of six untreated mice. Unoccupied β7 receptor expression on lymphocytes was determined using anti-β7 (clone FIB504), which binds the same epitope as rhuMAb Beta7. Changes in unoccupied β7 receptor expression (saturation) for each mouse were expressed as % of control GMFI, which was determined from the average of six untreated mice.

Figure 2

Single dose distribution study in BALB/c mice. (A) At 30 min post injection, a strong rhuMAb Beta7 signal was observed in the spleen; signal was decreased in the presence of unlabelled (cold) rhuMAb Beta7. This is consistent with specific binding of rhuMAb Beta7 to splenic β7-expressing cells. In contrast, signal in the lung was not decreased in the presence of excess unlabelled rhuMAb Beta7, suggesting that signal in this organ was due to the presence of blood rather than specific binding of rhuMAb Beta7 to cells in the lung. (B) At 24 h post injection, a strong radioactive rhuMAb Beta7 signal was observed in the spleen, lymph node and intestine; this signal was decreased by cold rhuMAb Beta7, consistent with specificity of rhuMAb Beta7 binding in these tissues. Columns represent the mean of n = 3 mice and vertical lines show SD.

Figure 3

In vivo homing of lymphocytes to the inflamed colon of CD45RB^high reconstituted SCID mice. SCID mice were reconstituted with Cr⁵¹-labelled CD45RB^high CD4⁺ T cells. In this model of colitis, rhuMAb Beta7 significantly blocked lymphocyte recruitment and homing to the inflamed colon (as measured by gamma count of harvested organs) when compared with a control Ig (A, control IgG vs. rhuMAb Beta7, P < 0.001), but had no effect on lymphocyte homing to the spleen, a non-mucosal lymphoid organ (B). Untreated mice without colitis had essentially no homing to the colon (A, see group 1); nearly all labelled lymphocytes trafficked to the spleen (B, see group 1). Group 1 = non-colitis, no test article (n = 4 mice); group 2 = control Ig (gp120; n = 9 mice); group 3 = rhuMAb Beta7 (n = 8 mice); Ig, immunoglobulin; anti-gp120, control mouse IgG1 antibody. Values shown are group mean counts per minute (CPM) and vertical lines show SD. Asterisk indicates significant P-value.

Figure 4

Anti-Beta7 in mouse model of EAE. (A) Anti-Beta7 had no effect on lymphocyte homing to brain; while treatment with anti-murine α4 (mPS/2) MAb significantly improved the mean EAE disease severity score (P = 0.003) compared with control anti-gp120, anti-murine β7 (muFIB504) had no effect (P = 0.989) on disease severity compared with control anti-gp120. MBP-TCR mice with EAE were evaluated daily using an EAE disease severity scoring system between 0 and 5; mean scores are plotted. Statistical analysis was performed on terminal disease severity scores using Dunnett's test. (B) Histological sections from each region of brain and spinal cord were scored on a scale of 0 (no inflammation) to 4 (severe inflammation involving the majority of the histological section); mean scores (and SD) were plotted. Anti-murine α4 MAb (mPS/2) significantly improved mean CNS histological inflammation (P = 0.009) compared with control anti-gp120, whereas anti-murine β7 (muFIB504) had no effect on histological inflammation in the CNS compared with control anti-gp120. Statistical analysis was performed on terminal histological inflammation scores using Dunnett's test. Asterisk indicates significant P-value. (C) % survival of MBP-TCR transgenic mice with severe EAE. Anti-murine α4 MAb (PS/2) significantly improved survival from EAE compared with control anti-gp120, while anti-murine β7 (muFIB504) had no effect on EAE survival compared with control anti-gp120. The difference between these survival curves is significant (log–rank test, P = 0.0005). MBP-TCR, myelin basic protein T-cell receptor; anti-gp120, control mouse IgG1 antibody; mPS/2, anti-murine α4; muFIB504, anti-murine β7 (rat–mouse chimeric antibody). n = 10 mice per group.

Figure 5

Pharmacodynamic (PD) biomarker analysis of rhuMAb Beta7 treatment. Three subsets of peripheral blood CD4⁺ lymphocytes, subdivided according to their homing properties, were monitored by flow cytometry as PD markers: CD45RA^-β7^high (‘gut/mucosal-homing memory’), CD45RA^-β7^low (‘peripheral-homing memory’) and CD45RA⁺β7^intermediate (‘naïve’) CD4⁺ cells. The figure shows a FACS scatter plot of CD45RA on the x-axis and β7 on the y-axis, gated on CD4⁺ cells. The figure is representative of staining pattern seen in untreated cynomolgus monkeys.

Figure 6

Mean (±SD) absolute peripheral blood CD45RA^-β7^high CD4⁺ T cell counts following four weekly i.v. bolus doses of vehicle or 5 or 25 mg·kg⁻¹ rhuMAb Beta7 to cynomolgus monkeys. Absolute CD45RA^-β7^high CD4⁺ T cell counts are shown as a percentage of predose baseline. Groups dosed with vehicle and 25 mg·kg⁻¹ had 10 animals per group from days 0 to 28, and four animals per group after day 28. There were six cynomolgus monkeys in the 5 mg·kg⁻¹ group.

Figure 7

Mean (±SD) absolute peripheral blood CD45RA⁺β7^intermediate CD4⁺ T cell counts following four weekly i.v. bolus doses of vehicle or 5 or 25 mg·kg⁻¹ rhuMAb Beta7 to cynomolgus monkeys. Absolute CD45RA⁺β7^intermediate CD4⁺ T cell counts are shown as a percentage of predose baseline. Groups dosed with vehicle and 25 mg·kg⁻¹ had 10 animals per group from days 0 to 28, and four animals per group after day 28. There were six cynomolgus monkeys in the 5 mg·kg⁻¹ group.

Figure 8

Mean (±SD) absolute peripheral blood CD45RA^-β7^low CD4⁺ T cell counts following four weekly i.v. bolus doses of vehicle or 5 or 25 mg·kg⁻¹ rhuMAb Beta7 to cynomolgus monkeys. Absolute CD45RA^-β7^low CD4⁺ T cell counts are expressed as a percentage of predose baseline. Groups dosed with vehicle and 25 mg·kg⁻¹ had 10 animals per group from days 0 to 28, and four animals per group after day 28. There were six cynomolgus monkeys in the 5 mg·kg⁻¹ group.

Figure 9

Individual cynomolgus monkey profiles of rhuMAb Beta7 serum concentrations (left y-axis) and unoccupied peripheral blood CD45RA^-β7^high CD4⁺ T cells (right y-axis) following four weekly i.v. bolus doses of 25 mg·kg⁻¹ rhuMAb β7. Unoccupied CD45RA^-β7^high CD4⁺ T cells are shown as a percentage of predose baseline. Conc., concentration.

Figure 10

Individual cynomolgus monkey profiles of absolute peripheral blood CD45RA^-β7^high CD4⁺ T cell counts (left y-axis) and unoccupied peripheral blood CD45RA^-β7^high CD4⁺ T cells (right y-axis) following four weekly i.v. bolus doses of 25 mg·kg⁻¹ rhuMAb Beta7. Absolute CD45RA^-β7^high CD4⁺ T cell counts (total) and unoccupied CD45RA^-β7^high CD4⁺ T cells are shown as a percentage of predose baseline.