Multi-Drug Desensitization Protocol for Heart Transplant Candidates

Transplant candidates with HLA alloantibodies are at high risk for antibody mediated rejection (AMR), graft failure, and acute rejection (1,2). In heart transplantation, these complications lead to death. The 50% calculated panel reactive antibody, CPRA, threshold is chosen from a consensus statement from the International Society of Heart and Lung Transplantation (3). This value is admittedly arbitrary but does represent a consensus of accepted opinion amongst experienced and reputable heart transplant centers.

The alloantibodies that prevent these patients from being transplanted are a result of the adaptive immune system and immunologic memory. Immunologic memory is defined as the ability of the immune system to provide a faster, stronger and more specific response to a second exposure of an antigen, when the antigen was completely eliminated from the organism after the prior exposure.

There are three major cell lines involved in immunologic memory: memory T cells, memory B cells and plasma cells, all of which can survive once antigen has been eliminated (4). B cells can mount responses to both small soluble antigens and large antigens (5). Once a B cell becomes activated, it can become a short lived plasma cell and produce immunoglobulin M, (IgM), antibodies or it can enter a germinal center where it can undergo somatic hypermutation with affinity maturation and isotype switching. The B cell can then differentiate into a long lived plasma cell and compete for a bone marrow niche or can become a memory B cell (4-8).

Memory T cells can last a lifetime and can recirculate between the secondary lymphoid organs (SLO) as central memory T cells (TCM) or in the peripheral tissues as effector memory T cells (TEM) (4,9). Upon encountering their cognate antigen, they can rapidly proliferate and differentiate to effector T cells.

Memory B cells slowly proliferate and recirculate in the SLOs, last there for decades and memory B cells to vaccinia (smallpox) have been noted to survive for more than 50 years (9). They are commonly identified by the presence of CD27 and upon a second antigenic challenge; the memory B cells can rapidly proliferate and then differentiate into new plasma cells (10). This phenomenon provides a redundant system or "array" to replenish plasma cells that produce antibody for a given antigen.

Plasma cells are highly differentiated and specific cells that can actively produce alloantibody. There are two populations of plasma cells, short-lived and long-lived (8). Long-lived plasma cells can last for life and can appear within less than 1 week of antigenic stimulation (6,7). In core biopsies of kidney transplant recipients, B cells, memory B cells, plasmablasts and plasma cells have all been identified during acute rejections (11). The plasma cells are end differentiated and therefore cannot proliferate. In the bone marrow, there are a finite number of survival niches (109) dependent on the number of stromal cells present to support them. Plasma cells that don't find sanctuary in the marrow or lose sanctuary only last a few days, probably owing to the intense metabolic demands of a cell that can produce between 10,000 to 20,000 copies of antibody per second (12). Plasma cells are currently thought as being without a negative feedback loop to suppress their antibody synthesis. Plasma cells have been demonstrated to have an FcγRIIB receptor coupled to an immunoreceptor tyrosine based inhibitory motif (ITIM) (13). The FcγRIIB receptor is a low affinity receptor and cannot bind monomeric immunoglobulin G, (IgG). The homeostasis of immunoglobulin is thought to be mainly the responsibility of the endothelial cell (14). Once an antibody is produced, endothelial cells can eliminate circulating antibody by lysosomal degradation or recycling the antibody through an Fc receptor neonatal, (FcRn) dependent mechanism back into the plasma.

A successful plan to eliminate the memory of a given antigenic encounter must address the three separate systems or "arrays." The elimination of immunologic memory such that deleterious antibodies could be removed and then new favorable antibodies created would be a significant advance in the fields of transplantation and autoimmunity. A review of the medications used in IND 110875 will elucidate why this protocol may be successful where all others have failed.

Rabbit antithymocyte globulin, RATG, has anti-T-cell properties, and in particular, activity against memory T-cell surface antigens, thereby causing complement mediated T-cell death in peripheral blood and apoptosis in the spleen and lymph nodes (15). RATG has antibodies to CD27, CD38, HLA-DR and has demonstrated anti-memory B cell properties in vitro and in vivo (15-17). The combination of rATG and rituximab decreased CD27 positive B cells from the spleen in patients in a desensitization trial that was otherwise unsuccessful was a significant observation (16).

Rituximab, an anti-CD20 monoclonal antibody, has strong activity against B-cells and depletes B cells in the circulation for 5-7 months. But as B-cells differentiate into plasma cells, the CD20 surface marker is down regulated, with concomitant loss of sensitivity to rituximab (18). Rituximab has been used in a variety of autoimmune diseases (19,20). Many antibodies are not affected while others are decreased for a period of time. Short lived plasma cells may be decreased since there is not a ready supply of B cells to replace them after their short three day half-life. In a study in systemic lupus erythematosis (SLE) using rituximab; flow cytometry and autoantibody specificity studies revealed that antibodies to Ro52 and La44 and measles were not decreased but antibodies to dsDNA and C1q did decrease (19). The overall amount of immunoglobulin did not change. Plasma cells are not affected by anti-CD20, so the antibody production from long lived plasma cells continues unabated. Memory B-cells are CD 27+ and have a variable expression of CD20. A study in desensitizing kidney transplants candidates showed that rituximab did not decrease the number of CD27+ memory B cells or plasma cells in the spleen (16). In (SLE) patients treated with rituximab the cells returning after depletion were largely naïve B cells and the plasmablasts were 2.3 times higher than at baseline (19). While memory B cells circulating in the blood were lower after rituximab therapy, the memory B cells in the spleen appear to be unaffected and can then transform in to plasmablasts which can then secrete soluble antibody. This observation explains why some antibodies will disappear, at least temporarily with rituximab while others will not. If the antibody is coming primarily from short lived plasma cells or plasmablasts, rituximab will more likely have an effect; these antibodies are produced by cells with a short half-life and are dependent upon continuous proliferation of B cells. Antibodies produced by long-lived plasma cells are not affected by rituximab. Rituximab does not affect memory B cells in the spleen, so they can rapidly reform plasmablasts and plasma cells to reconstitute the cell lines producing certain clones of antibodies. Since memory B-cells can become antibody secreting plasma cells, it is advantageous to remove them before transplantation in the highly sensitized patient.

The combination of rATG and rituximab was shown in the human to reduce memory B cells in the spleen (16). This finding has not yet been incorporated into a desensitization protocol or into a protocol for autoimmune therapies from our review of the literature. No other therapy effectively reduces memory B cells in the human and it represents a novel aspect of IND 110875.

No agents previously used in transplantation, including rATG and rituximab, have the ability to inhibit mature plasma cells once they find refuge in the bone marrow, and therefore have little effect on reducing antibody production. However, bortezomib, a proteasome inhibitor used in treatment of multiple myeloma, does have the ability to deplete plasma cells via many mechanisms.

Proteasome inhibition represents a novel treatment strategy because it provides a means for depleting plasma cells within the bone marrow (21,22). Bortezomib is approved for use in the treatment of multiple myeloma and the sensitivity of myeloma cells to proteasome inhibitors is proportional to their immunoglobulin synthesis rates (22). Plasma cells are known to have high immunoglobulin synthesis rates and treatment with bortezomib depleted both short and long-lived plasma cells by more than 60% in the spleen and 95% in the bone marrow after 48 hours of treatment in the BALB/c mouse model. Bortezomib activated the unfolded protein response (UPR) documented by increases in the expression of the chaperones BiP and Chop which are markers for UPR. The authors also concluded that the late inhibition of the anti-apoptotic transcription factor NF-κB also contributed to cell death. In a lupus nephritis mouse model (NZB/W F1 mice) treated with bortezomib, dsDNA-specific antibodies decreased to the range of non-autoimmune mice. Total serum IgG, IgG2a, IgG3, IgM, and IgA concentrations were all strongly reduced but concentrations of IgG1 and IgG2 were not altered or only slightly altered (21). Total IgG concentrations were not reduced by more than 50%. The authors noted that newly formed plasma cells could return by 48 hours after bortezomib injection. These findings support the conclusion that bortezomib can kill plasma cells, and have a salutary clinical effect, but memory B cells are not depleted and plasma cells can quickly recover and produce unwanted antibody. The ability of bortezomib to kill non-malignant plasma cells represents a major finding with potential therapeutic efficacy in transplantation and autoimmune diseases, but by itself is incapable of long lasting reductions in antibody. The clinical finding of variable reductions in antibody levels with bortezomib can be explained from these findings in the basic science literature.

Bortezomib has been used as a rescue strategy for the treatment of refractory antibody mediated rejection (23-25). An in vitro study revealed that bortezomib was able to induce apoptosis in plasma cells aspirated from renal transplant recipients whereas rATG, rituximab and IVIG all failed to cause apoptosis (25). Treatment with bortezomib at concentrations that blocked antibody production in vitro was shown to significantly decrease the 20S proteasome chymotrypsin-like peptidase activity. Two patients had bone marrow biopsies during acute humoral rejection, one week after bortezomib and one year later which revealed that the total number of plasma cell allospecificities decreased as did the total percentage of plasma cells (25). Not all antibodies were reduced by bortezomib in these two patients and the total IgG levels were unchanged, yet this study did show that bortezomib could decrease plasma cells in the bone marrow.

Bortezomib is indicated for multiple myeloma wherein the malignant plasma cells are very aggressive in producing antibody. The more productive the myeloma cell line is to making antibody, the more susceptible to proteasome inhibition (22). The above literature in non-malignant plasma cells also reveals that they are susceptible to proteasome inhibition with bortezomib, but in a number of studies, certain immunoglobulin fractions did not decrease and the overall amounts of immunoglobulin were unchanged. Perhaps plasma cells are not as metabolically active as their malignant counterparts. There is some evidence that plasma cells may be able to decrease their immunoglobulin synthesis and this in turn would make them less susceptible to proteasomal inhibition (26). A reexamination of immunoglobulin homeostasis reveals a potential therapy to increase sensitivity to bortezomib.

Immunoglobulin homeostasis is largely felt to be the result of plasma cell production and then FcRn mediated recycling in the endothelial cells. Antibody that does not combine with FcRn in the endosome of the endothelial cell is then degraded while antibody that does combine is recycled back to the interstitial space (27). The concept that IgG does not have a negative feedback loop to the plasma cell is supported by data in the experimental animal and humans (28,29). However, clinicians observe patients that "rebound" after plasmapheresis with levels of antibody that were just as high or higher than before plasmapheresis and this led to the conclusion that there was a negative feedback loop (30,31). The rebound phenomenon was explained away as the return of antibody from the periphery and increased recycling by FcRn receptors (28). The regulation of protein synthesis in the plasma cell has received new attention and is controlled by a complex system of feedback loops involving the endoplasmic reticulum stress and mTOR signaling (32).

Recent investigations into the mechanism of intravenous immunoglobulin, IVIG, function offer further insight into possible explanation for a negative feedback loop to plasma cells. IVIG in the clinical literature is thought to work by a number of pathways including anti-idiotypic antibodies, inhibition of cytokine gene activation, anti-T cell receptor activity, anti CD4 activity, stimulation of cytokine receptor antagonists, inhibition of complement activity and Fc mediated interactions with antigen presenting cells to block T cell activation (33). Recent work reveals that these mechanisms are possibly erroneous. Studies in children with ITP in 1993 revealed that infusion of Fc fragments provided the anti-inflammatory properties (34). The anti-inflammatory properties of IVIG can now be attributed to Fc sialylation of IgG (35-37). Immunoglobulins are glycoproteins and a single N-linked glycan is found at Asn297 in the Fc domain. This covalently linked complex glycan is composed of a biantennary heptapolysaccharide containing N-acetylglucosamine and mannose and two terminal sialic acid residues (35). Further modifications of this carbohydrate structure are common and over 30 different glycans have been identified at this one site and glycosylation of IgG is mandatory for FcγR binding. The total anti-inflammatory activity of IVIG depends on the sialylation of the IgG Fc fragments and this represents only 5% of the IgG pool. The small amount of sialylated IgG in IVIG explains why large doses are required for its anti-inflammatory effects while much lower doses are required to treat hypogammaglobulinemia. Plasmapheresis, by decreasing sialylated IgG, may lead to the up-regulation of antibody synthesis in plasma cells and make them more susceptible to bortezomib.

The index patient treated with a protocol similar to this one, the patient had effective deletion (< 5000MFI) of all of their antibodies detected by LABScreen, including the Class II antibodies that prior to this had been difficult to remove. Using the much more stringent criteria of < 1000 MFI, the index patient had only one remaining antibody over 1000 at the end of 3 cycles of the bortezomib treatment phase. The patient was unique amongst the case reports of therapies for antibody mediated rejection and desensitization therapy in that all of the patient's antibodies dramatically declined and the total amount of soluble antibody decreased to the point where the patient required IVIG for replacement therapy. If this result is reproducible and the protocol has sufficient safety, then these results could have important ramifications in the fields of transplantation and autoimmune disease. Autoantibody mediated diseases may now have the potential of cure if the immunologic memory of the inciting epitopes is erased.

It is impossible to study these medications in the usual one-drug-at-a-time methodology given the redundant nature of immunologic memory. The potential risk of the protocol is only acceptable because of the need to develop an effective therapy for a life-threatening situation. This protocol is in line with the criteria elucidated by the FDA in the recent New England Journal of Medicine article, "Development of Novel Combination Therapies" (38). The article mainly describes combination therapies for cancer trials; however, the thematic components of the document apply to IND 110875.