Folate Receptor Beta for Macrophage Imaging in Rheumatoid Arthritis

Abstract

Non-invasive imaging modalities constitute an increasingly important tool in diagnostic and therapy response monitoring of patients with autoimmune diseases, including rheumatoid arthritis (RA). In particular, macrophage imaging with positron emission tomography (PET) using novel radiotracers based on differential expression of plasma membrane proteins and functioning of cellular processes may be suited for this. Over the past decade, selective expression of folate receptor β (FRβ), a glycosylphosphatidylinositol-anchored plasma membrane protein, on myeloid cells has emerged as an attractive target for macrophage imaging by exploiting the high binding affinity of folate-based PET tracers. This work discusses molecular, biochemical and functional properties of FRβ, describes the preclinical development of a folate-PET tracer and the evaluation of this tracer in a translational model of arthritis for diagnostics and therapy-response monitoring, and finally the first clinical application of the folate-PET tracer in RA patients with active disease. Consequently, folate-based PET tracers hold great promise for macrophage imaging in a variety of (chronic) inflammatory (autoimmune) diseases beyond RA.

Keywords: PET imaging; antigen-induced arthritis; folate receptor beta; macrophage; rheumatoid arthritis.

Figure 1

Macrophage FRβ for folate-based imaging and therapeutic targeting. (A) Folate receptor isoforms FRα, FRβ, FRγ and FRδ, their GPI-membrane anchoring (except FRγ), and cell/tissue expression. (B) Selective expression of FRβ (cyanin), a glycosylphosphatidylinositol-anchored plasma membrane protein, on myeloid cells (e.g. macrophages) constitutes a suitable target for imaging of inflammatory disease, including RA. Folate (pink ellipse) is coupled to a radioactive isotope (for PET or SPECT) or near infrared fluorescent dye (for optical imaging). Following high affinity binding to FRβ, these imaging agents may stay membrane bound or potentially internalized via endocytosis. (C) Macrophage FRβ can also be subject to therapeutic targeting to ameliorate inflammation. This can be achieved by (drug-conjugated) monoclonal antibodies, folate-conjugated drugs, CAR-T cells targeted towards FRβ and with small molecule folate antagonists. (D) Folic acid, the primary circulating plasma folate form 5-CH₃-THF, and folate antagonist therapeutic drugs MTX or PMX can bind to FRβ or one of the two other folate carriers expressed on macrophages, i.e. RFC/SLC19A1 and PCFT/SLC46A1. The binding affinity of 5-CH₃-THF, MTX and PMX varies for FRβ, RFC and PCFT, respectively, as indicated by colored arrows. For example, 5-CH₃-THF binding affinity to FRβ > MTX and PMX. MTX transport is facilitated by all three folate carriers, but with slightly higher affinities for RFC and PCFT. PMX displays the highest affinity to PCFT, moderate affinity to RFC and the lowest affinity for FRβ. (E) Chemical structures of folic acid, (6S)-5CH₃-THF, methotrexate (MTX) and pemetrexed (PMX) illustrating the shared pterin moiety which is captured in the folic acid binding cleft of FRβ. (F) Chemical structure of [¹⁸F]fluoro-PEG-Folate, the folate PET imaging agent for high-affinity binding to FRβ on macrophages and utilization for diagnostics and therapy response monitoring.

Figure 2

Antigen induced arthritis (AIA) rat model used in preclinical studies for PET imaging with [¹⁸F]fluoro-PEG-folate tracer. (A) Schematic representation of the preclinical set up of the experiment: starting at day 0 and day 7 with an immunization of the rat with an emulsion of Complete Freund’s Adjuvant, Custom Bordetella Pertussis and methylated BSA (mBSA). Immunization status is checked at day 14 with a delayed type hypersensitivity (DTH) test through injection of mBSA in the ear. Around day 18, an intra-articular (i.a.) injection is given in one of the knees with mBSA. During the whole duration of the experiment or after i.a. injection, the rat can be treated with FRβ-targeted folate antagonist such as MTX or PMX. A baseline (/pre-treatment) PET scan and post-treatment PET scan with [¹⁸F]fluoro-PEG-folate have been used to monitor the effect of antifolate therapy. (B) After 1-3 days post i.a. injection a significant swelling of the knee diameter is seen in the arthritis-affected leg (****p < 0.0001, two-way ANOVA, N = 9 rats/group, paired samples). (C) Representative image of macrophage infiltration in the synovium of the arthritic leg as detected by immunohistochemical (IHC) DAB-staining of rat knee tissue with ED-1 antibody (HM3029, Hycult Biotech) (scale bar = 50μm). (D) Illustrative image of increased [¹⁸F]fluoro-PEG-folate uptake in the arthritic knee (lower panel, arthritic leg) vs. the non-arthritic, contra-lateral knee (upper panel, control leg) of an AIA rat. Both images are scaled to the same standard uptake value based on the injected dose (in MBq/ml) of the tracer and the body weight of the animal (in g.). (E) Biodistribution of [¹⁸F]fluoro-PEG-folate in the arthritic knee, liver and spleen of non-treated and MTX-treated AIA rats. Data were corrected for blood %ID/g. (F) Biodistribution of [¹⁸F]fluoro-PEG-folate in the arthritic knee, liver and spleen of non-treated and PMX-treated AIA rats. Data were corrected for blood %ID/g. Statistics for images E-F were performed in Graph-Pad Prism version 9, ^#p < 0.01, Mann-Whitney U test for non-parametric divided data, *p < 0.05, ***p < 0.001, unpaired T-TEST for parametric divided data, N = 4 rats/group. All results described in Figure 2 were derived (and reanalyzed were indicated) from own research (58, 62, 69, 70).

Figure 3

Illustration of the clinical validation of the [¹⁸F]fluoro-PEG-folate tracer in patients with rheumatoid arthritis. (A) Upper panel showing an illustrative example of immunofluorescent staining of CD68 (left), FRβ (middle) and CD68 + FRβ merged staining in a synovial knee biopsy of an RA patient ​with active disease. Lower panel depicts marked expression of CD68- and FRβ-positive macrophages in both synovial lining and sublining (see the zoomed view of the dashed marked box in the upper panel of the RA synovial tissue. (B) Representative example of high specific uptake of [¹⁸F]fluoro-PEG-folate in the hand joints of a patient with high RA disease activity vs. marginal uptake in the hand joints of a RA patient with low disease activity in the hands. Both images are scaled to the same standard uptake value based on the injected dose (in MBq/ml) of the tracer and the body weight of the patient (in kg.). All representative images of Figure 3 were derived from own research (25, 61).