**8.4. Bortezomib**

**8.2. Rituximab**

430 Current Issues and Future Direction in Kidney Transplantation

Rituximab, a chimeric monoclonal anti-CD20 antibody directed against B cells, prevents new antibody production by depletion of B cells as precursors of mature plasma cells in the circulation and the lymphoid tissue {although, some recent reports demonstrated that depletion in secondary and tertiary lymphoid structures is far less efficient and may not affect an ongoing localized humoral immune response (Genberg et al., 2006; Thaunat et al., 2008)}, prevention of B-cell proliferation, and induction of apoptosis and lysis of B cells through complement-dependent and -independent mechanisms (Salama & Pusey, 2006). Rituximab binds CD20 at the surface of precursor and mature B cells and leads to transient B-cell depletion, with typical B-cell recovery after 6–12 months in more than 80% of patients, although the degree of depletion is highly variable and is observed for up to 24 months in some individuals (Sureshkumar et al., 2007).An additional potential mechanism of action of rituximab is the direct targeting of CD20-positive cells that infiltrate the graft (Steinmetz et al., 2007). Preliminary studies indicate that rituximab decreases the concentration of pre-existing and post-transplantation antibodies (Gloor et al., 2003; Vieira et al., 2004). Conclusions and extrapolations from these studies are limited, because rituximab is usually combined with other therapies in these small and uncontrolled trials. The risk of bacterial infection as a result of immunoglobulin deficiency is also an important consideration. Based on the pathophysio‐ logic condition of this rejection process and efficacy of rituximab in B cells and antibodymediated autoimmune diseases (Eisenberg & Albert, 2006; Levesque & St Clair, 2008), a

combination treatment with rituximab/IVIG represents a logical approach.

In a multicenter study, MMF in combination with cyclosporine resulted in significantly lower frequencies of HLA antibodies when compared with azathioprine and cyclosporine treatment (Terasaki & Ozawa, 2004). Moreover, MMF was described to be effective in inhibiting primary antigen-specific antibody responses in renal transplant patients (Rentenaar et al., 2002). Heidt et al (Heidt et al., 2008) stimulated purified human B cells devoid of T cells with CD40L expressing L cells, or by anti-CD40mAb with or without Toll-like receptor triggering, all in the presence of B-cell activating cytokines. These three protocols resulted in various degrees of Bcell stimulation. Then, they added four commonly used immunosuppressive drugs (tacroli‐ mus, cyclosporin, mycophenolic acid [MPA], and rapamycin) to these cultures and tested a variety of parameters of B-cell activity including proliferation, apoptosis induction, and both IgM and IgG production. They found that MPA was extremely potent in inhibiting both proliferation and immunoglobulin production. Moreover, these effects persisted when MPA was added to already activated B cells, implying that an ongoing B-cell response may be dampened by MPA, whereas calcineurin inhibitors are ineffective. MPA levels used are lower

In the same in vitro experiments, rapamycin, like MMF, was described to be extremely potent in inhibiting humoral responses. Rapamycin was the most effective drug tested, as it inhibited not only B-cell proliferation and immunoglobulin production, but also inhibited the number of immunoglobulin producing cells. None of the other drugs tested were capable of decreasing

**8.3. Mycophenolic acid and sirolimus**

than levels that are usually achieved physiologically.

While the B cell-depleting anti-CD20 antibody rituximab is increasingly incorporated in treatment protocols of humoral rejection (Faguer et al., 2007), this reagent is neither effective in eliminating antibody-producing plasma cells (PC) – either newly created from memory or naıïve B cells or from those that existed prior to transplant- nor does it decrease circulating antibody titers (Singh et al., 2009). For an effective blockade of alloantibody formation, a specific PC-depleting reagent would be desirable. Bortezomib (BZ), a selective inhibitor of the 26S proteasome, has been approved by FDA for the treatment of relapsed multiple myeloma. Mechanisms of BZ action include inhibition of NF-κ B and cytokine expression as well as induction of apoptosis as a result of activation of the terminal unfolded protein response (Meister et al., 2007). Susceptibility to BZ-induced apoptosis is related to the high immuno‐ globulin synthesis rate of PCs associated with accumulation of unfolded proteins/DRiPs inducing endoplasmatic reticulum stress (Meister et al., 2007). Moreover, BZ not only acted on the humoral response but also effectively inhibited the influx of MHC class II+ cells, mono‐ cytes/macrophages, CD8+ as well as CD4+ T cells. In animal models, Vogelbacher and colleagues (Vogelbacher et al., 2010) found that combination of Bortezomib and sirolimus inhibit the chronic active antibody-mediated rejection in experimental renal transplantation in the rat. In humans, data are lacking. In one case report, Bortezomib failed to treat CAMR even after treatment with rituximab and IVIG.

Perry and colleagues (Perry et al., 2009) described two sensitized patients with AMR treated in February 2007 using a combination of bortezomib and multiple plasmapheresis. Both patients had resolution of AMR and decreased serum DSA levels months after treatment. Neither developed transplant glomerulopathy. In a slightly different clinical setting, Everly and colleagues (Everly et al., 2008) used bortezomib to treat six patients who had combined AMR and cellular rejection occurring from 3 months to 7.5 years after transplant. All six patients showed resolution of AMR with a decrease in DSA levels after treatment. Unfortu‐ nately, three of the six patients developed transplant glomerulopathy. Flechner and coworkers (Flechner et al., 2010) treated 20 cases (16 kidney-only and 4 kidney-combined organ recipients) with AMR 19.8 months (range 1-71 months) posttransplant using a combined regimen of intravenous corticosteroids followed by a 2-week cycle on days 1-4-8-11 of plasmapheresis and 1.3 mg/m2 bortezomib; then 0.5 mg/kg intravenous immunoglobulin four times. They found that the bortezomib-containing regimen demonstrated activity in AMR but seems to be most effective before the onset of significant renal dysfunction (serum creatinine <3 mg/dL) or proteinuria (<1 g/day).

Compared to rituximab, Waiser and colleagues (Waiser et al., 2012) found that patients with AMR treated with bortezomib had better graft survival At 18 months after treatment (P = 0.071) and renal function at 9 months was superior in patients treated with bortezomib as compared to rituximab-treated patients (P= 0.008). Whereas these early clinical experiences with protea‐ some inhibition are encouraging, the lack of controls is a major limitation in assessing true efficacy. In addition, since even successfully treated AMR can still result in the development of chronic transplant glomerulopathy, the prevention of AMR might be a more important goal of these types of therapies.

These cell-surface markers might be useful targets to prevent the development of B cells into

Current and Future Directions in Antibody-Mediated Rejection Post Kidney Transplantation

http://dx.doi.org/10.5772/55001

433

Normal plasma cells express little or no CD20 and are therefore resistant to rituximabmediated depletion. Several cell-surface molecules that are expressed by plasma cells might be considered as drug targets — syndecan-1 (CD138), CD38, α4β1-integrin (CD49d–CD29) and CXC-chemokine receptor 4 (CXCR4) — although none of these is en‐ tirely plasma-cell specific. Plasma-cell longevity is thought to be an extrinsic phenomen‐ on that is mediated by survival signals delivered by bone-marrow stromal cells (Colvin & Smith, 2005). Because the transcription factors BLIMP1 (B-lymphocyte-induced matura‐ tion protein 1) and XBP1 (X-box-binding protein 1) (as well as the repression of PAX5, paired box gene 5) are required to maintain plasma-cell function, their inhibition might

Complement antagonists could prevent the acute pathological effects of complement activa‐ tion. For example, soluble CR1 delays antibody-mediated rejection in xenograft models but is insufficient to prevent graft rejection completely (Azimzadeh et al., 2003). Other complement antagonists, such as C5-specific antibody, which blocks activation of C5 and formation of both C5a and the MAC, are in ongoing evaluation. Transgenic expression of human complementregulatory proteins (DAF and CD59) in pigs has shown potency for preventing xenograft rejection (Menoret et al., 2004), but the relevance of these studies to allografts needs to be

Immunologic barriers once considered insurmountable are now consistently overcome to enable more patients to undergo organ transplantation. Alloantibodies are a substantial obstacle to short- and long-term graft survival. To prevent or reduce alloantibody titres, more insights are needed to improve our understanding of the regulation of B cells and the devel‐

Several important issues regarding AMR remain. First, the immunologic mechanisms respon‐ sible for the development of high levels of DSA are still unclear. The contribution of memory B cells versus the role of pre-existing PCs has important therapeutic implications since each

Whereas several new therapeutic approaches have emerged, more extensive study and followup are needed to determine if these apparent advances will improve the outcomes of AMR.

opmental and differentiation pathways of memory B cells and plasma cells.

may have a differential sensitivity to various agents.

result in the loss of plasma-cell function (Shapiro-Shelef & Calame, 2005).

plasma cells.

*8.6.2. Plasma cells*

*8.6.3. Complement antagonists*

extended and tested.

**9. Summary**
