An agonist antibody that blocks autoimmunity by inducing anti-inflammatory macrophages

Abstract

We have devised a method of using intracellular combinatorial libraries to select antibodies that control cell fates. Many agonist antibodies have been selected with this method, and the process appears to be limited only by the availability of a phenotypic selection system. We demonstrate the utility of this approach to discover agonist antibodies that engage an unanticipated target and regulate macrophage polarization by selective induction of anti-inflammatory M2 macrophages. This antibody was used therapeutically to block autoimmunity in a classic mouse model of spontaneous systemic lupus erythematosus.

Keywords: CD14; CTSG; M2 macrophage; cathepsin G.

Figure 1.

Selection of an agonist antibody that induces macrophage cell differentiation. A) Phenotype selection. The selection starts with a human ScFv phage library (10⁹ members). ScFv genes were transferred to a lentiviral vector to make lentiviral intrabody libraries. Total mouse bone marrow cells were infected with the antibody library and plated on soft agar. B) After 2 wk of incubation, 7 colonies with compact morphologies had grown. C and D) These colonies were harvested, and the antibody genes were recovered by PCR. One sequence was present in all colonies and was used for further studies. Hematoxylin and eosin staining of cells from the colonies showed cells with the classic morphologies of macrophages. E) The experiment was repeated using the selected sequence incorporated into lentivirus. This antibody clone (identified as LKAb) again induced colonies with a similar morphology that contained cells with macrophage morphologies. F) After a 1 wk incubation with the purified LKAb antibody, bone marrow cells differentiated in culture into cells with morphology consistent with that of macrophages.

Figure 2.

Identification of a novel antigen recognized by LKAb. A) Both commercial anti-CTSG antibody and LKAb bound to a 28 kDa protein in Western blots of lysates from cells overexpressing CTSG. B) Lysates of mouse bone marrow cells were incubated with LKAb for immunopurification. Eluates from these immune complexes bound to the commercial anti-CTSG antibody in Western blots. C) A CTSG enzymatic assay showed that CTSG enzyme activity was present in the eluates and mouse bone marrow total lysates. D) Mouse bone marrow was incubated for 2 d with lentiviruses containing CTSG shRNA, followed by incubation with LKAb (10 μg/ml) or M-CSF (10 ng/ml) for 6 d. The cells were then stained with anti-CD11b and anti-F4/80. FACS analysis showed that the induced macrophage populations were dramatically reduced when CTSG mRNA was silenced. However, M-CSF still induced macrophages when CTSG mRNA was silenced. Right: percentage of macrophages. *P < 0.05; Student’s t test.

Figure 3.

LKAb induced anti-inflammatory M2 macrophage differentiation. A) Mouse bone marrow cells were incubated with LKAb at the indicated concentrations (1–100 µg/ml) for 6 d. The cells were then stained with anti-CD11b and -CD11c and analyzed by FACS. Red outline: positive macrophage populations. LKAb induced differentiation of CD11b⁺ macrophages, but not CD11c⁺ dendritic cells, in a dose-dependent manner. B) Mouse total bone marrow cells were separated by using CD11b-specific magnetic beads, and the isolated CD11b⁺ or CD11b⁻ populations were incubated with LKAb or M-CSF for 6 d. The cells were then stained with anti-CD11b and -F4/80 and analyzed by FACS. Macrophages differentiated mainly from the CD11b⁻ population. C) Mouse bone marrow was incubated with medium, LKAb, or M-CSF for 6 d. Cells were then stained with anti-CD16/32 and -CD86 as M1 macrophage markers, and anti-CD36, -MHCΙΙ, and -CD14 as M2 macrophage markers. Red outline: M2-type-specific populations. The macrophages induced by LKAb selectively expressed M2 type markers. D) Mouse bone marrow was induced by LKAb antibody or M-CSF for 6 d. Cells were harvested, and total RNA was extracted for qRT-PCR analysis. IDO1 was used as an M1-specific gene and ARG-1 as an M2-specific gene. qRT-PCR showed high ARG-1 mRNA expression in macrophages induced by LKAb but only low levels of IDO1. E) Macrophages induced by the LKAb in vivo expressed M2 cytokines. LKAb and PBS were injected intraperitoneally into C57BL/6 or BALB/c mice 3 times/wk for 2 wk, and IL-10 levels in the serum were measured. Mice treated with LKAb showed dramatically increased levels of IL-10, one of the major anti-inflammatory cytokines. ***P < 0.0005; Student’s t test.

Figure 4.

CTSG, CD14, and NAFAT play a key role in LKAb-dependent M2 macrophage differentiation. A) Bone marrow from wild-type, CTSG knockout, CD14 knockout mice, or NFAT-knockout mice were incubated with medium, LKAb, or M-CSF for 6 d. The cells were then stained with anti-CD14 and anti-F4/80. FACS analysis showed CD14 and F4/80 expression was increased by LKAb in normal mice. However, CD14 and F4/80 expression was significantly reduced in both the CTSG- and CD14-knockout mice. CD14 and F4/80 expression was reduced in NFATc1- but not in NFATc2:CD4^Cre-knockout mice. Bottom: percentage of M2 macrophages. *P < 0.05; Student's t test. B) Wild-type mouse bone marrow cells were incubated with LKAb, a CTSG inhibitor, or both, and CTSG activity was determined. The antibody-induced reduction of CTSG activity was comparable to that of the inhibitor. C) Critical cell surface components and their downstream signaling pathways potentially involved in macrophage differentiation mediated by LKAb (depicted as bound to CTSG).

Figure 5.

LKAb induces human bone marrow cells to differentiate into M2 macrophages. Human total bone marrow was incubated with medium, LKAb, or M-CSF for 6 d. Cells were then stained with anti-CD11b and -CD64 as M1 type markers and anti-CD14 and -CD200R as M2 markers. Red outline: M2-type specific populations. As with mice, LKAb also induced human bone marrow to differentiate into anti-inflammatory macrophages.

Figure 6.

Treatment with LKAb reduces lupus-like disease in MRL-lpr mice. Mice (6–7/group) were injected intraperitoneally with LKAb or PBS (from the age of 6 wk until termination of the experiment at 20 wk) and followed for manifestations of disease. A) IgG2a anti-chromatin autoantibody levels determined by ELISA at 12 wk of age. B) Progression of lymphadenopathy between 12 and 16 wk of age assessed by palpation of axillary and salivary lymph nodes (LNs) and scored on a 0–4 scale. C) Kidney disease determined by histologic examination for GN at 20 wk of age. Representative images for treated and control mice are shown. D) Kaplan-Meier plot representing survival rates of treated and control mice. *P < 0.05, **P < 0.005, ***P < 0.0005, LKAb-treated vs. PBS control-treated mice; Student’s t test.