We produced an anti-rat FR-β mAb and its dsFv recombinant immunotoxin consisting of the Fv portion of an anti-FRβ mAb and PE38. Using this antibody, it was previously demonstrated that expression and distribution of FRβ-expressing macrophages in rat tissues were similar to those in murine tissues in which FRβ-expressing macrophages are rarely present [18
]. After the onset of mBSA-induced arthritis, intra-articular injections of immunotoxin reduced joint swelling during the acute phase of arthritis. Antigen-induced arthritis shows several clinical and histopathological similarities to human RA. Its maximal clinical activity, two to three days post-induction, chronic arthritis develops, characterized by synovial hyperplasia, inflammatory inflammation, and cartilage and bone destruction [24
]. Destruction of cartilage matrix results predominantly from the action of connective tissue proteinase released by RA synovial tissues, chondrocytes, and pannus tissues. Several lines of evidence in RA and animal models of arthritis support a role for osteoclasts in the pannus tissue in the pathogenesis of bone erosions. RA synovial tissues, including macrophages, fibroblasts, and T cells, produce a variety of cytokines and growth factors that may increase osteoclast formation, activity, and/or survival. Specifically, the interaction of the receptor activator of nuclear factor-kappaB ligand (RANKL) produced by fibroblasts and T cells on RANK on osteoclast precursors is critical for inducing the differentiation of cells of the monocyte/macrophage lineage into osteoclasts [25
]. We previously observed that most osteoclasts originate from FRβ-expressing macrophages in RA synovial culture and that administration of dsFv anti-FRβ-PE38 reduced the number of activated fibroblasts and macrophages in RA synovial tissue engrafted into SCID mice [8
]. Thus, removal of macrophages using the immunotoxin may reduce the number of osteoclast precursors and osteoclasts, protecting against cartilage and bone destruction in arthritis.
Findings in rat arthritis models indicate that osteoclasts are formed soon after the onset of clinical arthritis and are continuously replenished by infiltrating macrophages during disease progression [26
]. Thus, early and effective blockade of osteoclastogenesis may be accomplished by targeting infiltrated macrophages. In this study, we showed that intra-articular injection of medium- and high-dose immunotoxins reduced the number of FRβ-expressing macrophages, even at 14 days post-injection. Several studies have demonstrated that systemic administration of FR-mediated drugs is effective for reducing the activity of experimental arthritis [9
]; however, it remains unknown whether intra-articular administration of these drugs is effective for treating arthritic joints. This is the first study demonstrating that intra-articular injections targeting FRβ-expressing macrophages are efficacious as local therapy.
Intra-articular injections of corticosteroids or hyaluronate are generally available for treating mono and oligoarthritis [14
]. Although these treatments appear to be safe and beneficial, the reduction in pain and inflammation is only temporary. Consistent with these clinical outcomes, it has been reported that macrophage infiltration and proinflammatory endothelial cytokine expression remained unchanged in arthroscopic biopsies of RA synovial tissues after intra-articular administration of corticosteroids [31
]. Additionally, intra-articular administration of high-molecular weight hyaluronate promoted joint swelling, inflammation, and cartilage damage during the late chronic phase of mBSA-induced arthritis [32
]. Thus, intra-articular injections of immunotoxin appear to be effective for improving long-term effects compared to the use of corticosteroids and hyaluronate. Several studies have demonstrated that intra-articular injections of anti-TNF biologics are effective for treating RA or experimental arthritic joints, although cumulative data of intra-articular administration of available biologics to RA joints show inconsistent levels of effectiveness [17
]. Additionally, local therapy using anti-TNF biologics has not been evaluated in arthritis refractory to systemic anti-TNF therapy because many patients in successful cases were naïve to systemic anti-TNF biologics. Experimental studies have demonstrated that anti-TNF biologics are capable of binding membranous TNF on macrophages and mediate antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity [34
]. An anti-FRβ immunotoxin induces apoptosis of FRβ-expressing macrophages by PE38 to inhibit protein synthesis by elongation factor 2 [36
]. However, we did not find a significant difference of ratios of apoptotic cells detected by tunnel staining in synovial macrophages between treated and non-treated synovium on day21 (data not shown). We speculate that apoptotic cells induced by the immunotoxin may be removed during the 14 days that had passed since the last injection Thus, intra-articular injection of immunotoxin may be useful for treating arthritis refractory to systemic anti-TNF therapy. Cartilage penetration by proteins is dependent on a protein's molecular weights and charges; full-length IgG cannot penetrate cartilage [37
]. The dsFv anti-FRβ immunotoxin is of lower molecular weight (62 kDa) than full-length IgG, allowing higher penetration into synovial tissues than full-length IgG. Interestingly, single-chain Fv anti-TNFα mAb inhibited acute inflammation of the knee joint induced by intra-articular administration of recombinant human TNF-α, although this model was not directly representative of RA. Conversely, a lower molecular weight may have a lower half-life and fewer prolonged effects [38
]. In fact, we did not detect the immunotoxin in sera and synovium from treated mice on the last day (data not shown). These results are reasonable because it was reported that the half-life of DsFv-PE38 immunotoxin in sera is less than 30 minutes [39
To increase retention time in joints, intra-articular injections of immunotoxins using nanoparticles and/or liposomes may be beneficial for clinical application [40
]. Clinical studies have shown that immunotoxins based on PE38 show lower nonspecific toxicity but retain antigen-dependent toxicity towards target cells [41
]. Local injection can generally be effective at lower doses than systemic injection. Thus, intra-articular administration of immunotoxin may overcome several challenges, including non-specific cytotoxicity and immunogenicity resulting from systemic usage of the immunotoxin.