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.

Inclusion Criteria:

- 1. Voluntary signed informed consent before performance of any study-related procedure
not part of normal medical care, with the understanding that consent may be withdrawn
by the subject at any time without prejudice to future medical care.

2.Female subject is either post-menopausal or surgically sterilized, or willing to use
two acceptable methods of birth control for the duration of the study and for up to 2
months after the last dose of study medication.

3.Male subject agrees to use an acceptable method for contraception for the duration
of the study.

4.Patient is greater than or equal to 18 years of age but less than 70 years old
(inclusive).

5.Patients with a Calculated Panel Reactive Antibody (CPRA) of ≥ 50% by Luminex Single
Antigen Flow Bead (SAFB) testing (LABScreen®, Canoga Park, CA), where a Mean
Fluorescence Intensity (MFI) of 1000 is the positive threshold.

6.Patient is considered compliant and intends to be available for follow-up study
period of 1 year.

7.Patient must have no known hypersensitivity to treatment with bortezomib, boron, or
mannitol.

8.Patient must have no hypersensitivity to rituximab. 9.Patient must have no history
of allergy or anaphylaxis to rabbit proteins or to any product excipients, or have
active acute or chronic infections which contraindicate additional immunosuppression.

10.Patient must have no history of an anaphylactic or severe systemic response to
Immune Globulin (Human). Individuals with selective IgA deficiencies who have antibody
against IgA (anti-IgA antibody) should not receive IVIG since these patients may
experience severe reactions to the IgA which may be present.

11.Patients without an AICD implanted will need to consent to wear a Zoll LifeVest
Wearable Defibrillator.

Exclusion Criteria:

1. Women who are pregnant, breastfeeding, or have a positive pregnancy test on
enrollment. If the patient becomes pregnant during the study, she must be removed from
the study before receiving any additional study drug.

2. History of hepatitis C virus (HCV) positivity (by polymerase chain reaction, PCR)

3. Patients who are human immunodeficiency virus (HIV)-positive, or hepatitis B surface
antigen (HBsAg)-positive.

4. Patient is deemed likely to have a second solid organ transplant or cell transplant
(e.g. kidney or islet cell) in next 3 years.

5. Patient at risk for tuberculosis (TB):

1. Current clinical, radiographic, or laboratory evidence of active or latent TB as
determined by local standard of care

2. History of active TB:

3. Within the last 2 years, even if treated

4. Greater than 2 years ago, unless there is documentation of adequate treatment
according to locally accepted clinical practice

5. Patient at risk of reactivation of TB precludes administration of conventional
immunosuppression (as determined by investigator and based upon appropriate
evaluation)

6. Patient with active peptic ulcer disease (PUD), chronic diarrhea, or gastrointestinal
malabsorption

7. Patient with a history of hypercoaguable state

8. Patient with hemoglobin < 7 g/dL, white blood cell (WBC) count < 2000/mm3 (3 x 109/L)
or platelet count < 30,000 /mm3 prior to transplant

9. Receipt of a live vaccine within 4 weeks prior to study entry

10. Patient treated with immunosuppressive therapy (e.g. methotrexate, abatacept, etc) for
indications such as autoimmune disease, or patient with comorbidity to a degree that
treatment with such agents is likely during the trial in the opinion of the
investigator

11. Patients with current or recent severe systemic infections within 2 weeks of
medication start

12. Evidence of severe liver disease with abnormal liver profile (aspartate
aminotransferase [AST], alanine aminotransferase [ALT] or total bilirubin > 1.5 times
upper limit of normal (ULN) at screening.)

13. Patient has ≥ Grade 2 peripheral neuropathy within 14 days of medication start

14. History of malignancy within the past 5 years that is not considered to be cured, with
the exception of localized basal cell carcinoma of the skin (excised ≥ 2 years prior
to study initiation)

15. Prisoner or patient compulsorily detained (involuntarily incarcerated) for treatment
of either a psychiatric or physical (e.g. infectious disease) illness

16. Patient with a history of substance abuse (drugs or alcohol) within the past 6 months,
or psychotic disorders that are not compatible with adequate study follow-up

17. Patient with a history of amiodarone exposure within three months.

18. Patient with a previous heart or other transplant