Trial Information

Nonmyeloablative Allogeneic Stem Cell Transplantation From HLA-Matched Unrelated Donor for the Treatment of Hematologic Disorders

Allogeneic Transplantation and the Graft versus Disease Effect Over the past three decades,
the transplantation of allogeneic marrow grafts has emerged as a uniquely effective therapy
for patients with hematologic malignancies, marrow failure syndromes, and other lethal
genetic and acquired diseases of hematopoiesis.1-9 Allogeneic transplantation potentially
results in curative outcomes for patients with acute leukemia, chronic myelogenous leukemia,
aplastic anemia, and lymphoid malignancies for whom standard therapies may not be effective.
The anti-tumor effect mediated by allogeneic lymphocytes is an essential factor in
eliminating residual disease post-transplant and preventing subsequent relapse.10-14
Numerous observations in patients undergoing allogeneic bone marrow transplant for
hematological malignancies have convincingly demonstrated evidence of a graft-versus-disease
(GVD) effect mediated by lymphocytes present in the donor graft. Supportive evidence for the
importance of this phenomenon includes: 1) relapse rates of syngeneic BMT recipients are
greater than in allogeneic recipients; 2) allogeneic BMT recipients who do not develop
graft-versus-host disease (GVHD) have a significantly greater relapse rate than allogeneic
BMT-recipients who do develop GVHD; 3) complete remissions have been observed in patients
with relapsed disease after allogeneic BMT in association with flares of GVHD; 4) recipients
of T-cell depleted BMT, a method of GVHD prophylaxis, relapse more frequently than
recipients of non-T-cell depleted BMT; and 5) the use of an unrelated marrow graft in both
non-T-cell depleted and T-cell depleted allogeneic BMT is associated with a reduced
incidence of relapse.

The most direct evidence for the role of allogeneic lymphocytes in mediating an antitumor
effect has been that patients who experience relapse following allogeneic transplantation
may be successfully treated by the infusion of donor leukocytes.15-22 Summarizing the
European experience, Kolb et al reported that the over 80% of patients with CML who relapse
into the chronic phase following transplant may achieve a second complete remission after
the infusion of donor leukocytes infusion in treating relapsed CML following transplant.
Complete remissions with the use of DLI for the treatment of relapsed acute leukemias,
chronic lymphocytic leukemia, myelodysplastic syndromes, multiple myeloma, and polycythemia
vera, although lower than those seen in CML, have been well demonstrated by several groups.
Additionally, a potent graft versus disease effect associated with allogeneic
transplantation and donor leukocyte infusion has been observed in patients with
non-Hodgkin's lymphoma and multiple myeloma.23-25 Thus, allogeneic transplantation offers a
uniquely effective approach to eradicating hematological malignancy in which myeloablative
therapy results in profound tumor cytoreduction and donor lymphocytes subsequently eliminate
minimal residual disease via immunological mechanisms.

Limitations in the Application of Allogeneic Transplantation The use of allogeneic BMT has
been limited by the significant treatment related morbidity and mortality associated with
this procedure. Patients often experience significant organ dysfunction as a result of
regimen related toxicity. Moderate to severe graft versus host disease occurs in
approximately 40% of patients undergoing matched sibling conventional transplants and
increases in incidence in older patients and those receiving unrelated or mismatched
grafts.26-28 Opportunistic infections due to immune dysfunction remains a major source of
morbidity and mortality particularly in older patients. The application of allogeneic
transplantation is therefore restricted to younger patients with normal underlying organ
function. Because the median age of many hematological malignancies may exceed 60 years of
age, a majority of patients with these disorders are not considered candidates for this
procedure.

The difficulty in applying allogeneic transplantation in patients with hematological
malignancies is particularly highlighted in patients with lymphoid malignancies such as
multiple myeloma, chronic lymphocytic leukemia and low-grade lymphoma. These disorders are
incurable with standard dose chemotherapy but may follow a relatively indolent course for
several years before patients develop progressive chemoresistant disease. Allogeneic
transplantation has been associated with long-term disease free survival in these settings
but is associated with a high incidence of upfront transplant related mortality. Strategies
to limit transplant related toxicity are essential to translate the decreased incidence of
relapse into an improvement in overall survival in this patient population.

Nonmyeloablative Allogeneic Transplantation One approach to limit the toxicity of allogeneic
transplantation has been the use of nonmyeloablative regimens preceding the infusion of
allogeneic cells.29-33 With this strategy, patients receive immunosuppressive therapy that
allows for the engraftment of donor cells without the immediate eradication of patient
hematopoiesis. The primary mechanism by which the underlying disease is eradicated is not
through chemotherapy-mediated cytoreduction, but rather through the donor lymphocyte
mediated graft versus tumor effect. As a result, patients experience far less regimen
related toxicity. Therefore, the adoption of this strategy may allow for the use of
allogeneic transplantation in disease settings and patient populations for which it had not
been readily applicable in the past.

Over the past several years, the use nonmyeloablative transplant has rapidly expanded.
Several reduced intensity conditioning regimens have been developed including fludarabine
and cyclophosphamide; fludarabine and melphalan; fludarabine, anti-thymocyte globulin and
low dose busulfan; and fludarabine and low dose TBI. Investigators have demonstrated the
feasibility of this treatment approach with the majority of patients demonstrating donor
engraftment, decreased regimen related toxicity, and graft mediated regression of disease.
In some studies, patients demonstrate a period of mixed donor/host chimerism in which the
infusion of donor lymphocytes is associated with achievement of complete donor chimerism.

Although regimen related toxicity is decreased following reduced intensive conditioning
regimens, graft versus host disease and opportunistic infections remain a significant source
of morbidity and mortality following nonmyeloablative allogeneic transplantation. The impact
of nonmyeloablative transplantation on immunological reconstitution has not been fully
defined. Persistence of host antigen presenting cells in the post-transplant period may
increase the incidence of GVHD due to the presentation of alloantigens to donor T cells. In
contrast, residual host cellular immunity may provide enhanced protection against infectious
pathogens and allow for more rapid education of donor lymphocytes. Of note, CMV infections
remain common in this population but present later in the post-transplant period
characteristically following the attainment of full donor chimerism.34

Nonmyeloablative Allogeneic Transplantation in Conjunction with CAMPATH Therapy One approach
to reduce the incidence of GVHD following nonmyeloablative transplantation is the addition
of CAMPATH to the preparative regimen to deplete T cells from both the recipient and the
incoming graft.35 CAMPATH-1H (Alemtuzumab) is a recombinant DNA-derived humanized monoclonal
antibody directed against the cell surface glycoprotein, CD52; produced in mammalian cell
(Chinese hamster ovary) suspension.36-39 Campath-1H is indicated for the treatment of B-cell
chronic lymphocytic leukemia in patients who have been treated with alkylating agents and
have failed fludarabine therapy. Campath-1H has been used in patients with autoimmune
neutropenia, non-Hodgkin's lymphoma, rheumatoid arthritis, and vasculitis. It has also been
used to prevent graft-versus-host disease in patients receiving stem cell transplantation.
GVHD prophylaxis was with cyclosporin A alone.

Mackinnon et al investigated a nonmyeloablative conditioning regimen in 47 patients with
hematological malignancies receiving allogeneic bone marrow stem cells from matched,
unrelated donors.40 The majority of patients had high-risk features, including having failed
a prior transplantation (29 individuals). Twenty of the transplants were mismatched for HLA
class I and/or class II alleles. They added CAMPATH-1H to a preparative regimen of
fludarabine and melphalan and administered either GCSF mobilized peripheral blood stems or
unmanipulated bone marrow from matched unrelated donors. Primary graft failure occurred in
only 2 of 44 evaluable patients (4.5%). Chimerism studies in 34 patients indicated that the
majority (85.3%) attained initial full donor chimerism. Only 3 patients developed grade III
to IV acute GVHD, and no patients have yet developed chronic extensive GVHD. The estimated
probability of nonrelapse mortality at day 100 was 14.9% (95% confidence interval [CI],
4.7%-25.1%). With a median follow-up of 344 days (range, 79-830), overall and
progression-free survivals at 1 year were 75.5% (95% CI, 62.8%-88.2%) and 61.5% (95% CI,
46.1%-76.8%), respectively. It was subsequently reported that a significant subset of
patients demonstrated evidence of CMV reactivation.41 However, the presence of CMV did not
negatively impact survival. While the investigators noted a need for longer follow-up of
this cohort, they felt that this preparative regimen incorporating in vivo CAMPATH-1H was
associated with durable engraftment and minimal treatment related toxicity.

Immune Reconstitution Following Allogeneic Transplantation Allogeneic transplantation is
associated with a period of immune dysfunction that characteristically persists for at least
1 year post-transplant.42,43 Immune recovery is dependent on the reconstitution of elements
of humoral and cellular immunity and their reeducation in the transplant recipient. It is
particularly delayed in recipients of an unrelated or T cell depleted graft. The
post-transplant period is characterized by decreased levels of helper T cells, an associated
inversion of the C4:CD8 ratio, and the blunting of T cell responses to mitogenic stimuli and
recall antigens.44-46 Humoral immune dysfunction is associated with the loss of protective
antibody levels to bacterial and viral pathogens, decreased levels of circulating
immunoglobulins particularly of the IgG2 subtype, and the reduced complexity in the pattern
of the immunoglobulin gene rearrangement.47,48

Dendritic Cells and Immune Reconstitution The nature of the recovery of antigen presenting
cells such as dendritic cells are likely to play an essential role in the reconstitution of
posttransplant immunity and host/donor tolerance. Dendritic cells (DC) form a complex
network of antigen presenting cells that play a vital role in the induction of primary
immunity as well as the modulation of tolerance.49.50 DC are the most potent antigen
presenting cells and are uniquely able to induce primary immune responses against novel
antigens through the rich expression of costimulatory and adhesion molecules. DC generated
from nonmyeloid lineages or in an immature state may mediate immune tolerance and direct T
cell responses towards a TH2 phenotype. Distinct DC populations have been identified in the
peripheral blood that are differentiated by the presence of myeloid (CD11c) or plasmacytoid
(CD123) markers.51,52 DC2 (plasmacytoid) cells are characterized by expression of type 2
cytokines and thus initiate a Th2 response, whereas DC1 (myeloid) cells initiate Th1
cytokine response. DC isolated from patients with malignancy demonstrate functional
deficiencies that potentially contribute to lack of tumor recognition by host immunity.
Little is known about DC engraftment or phenotypic and functional characteristics after
allogeneic transplantation. The interactions of DC with effector populations is likely to
be unique following nonmyeloablative conditioning given the presence of mixed
donor/recipient chimerism that may characterize the post-transplant period.

In a murine model, Shlomchik et al demonstrated that persistence of host antigen presenting
cells post-transplant was associated with the development of GVHD.53 In this model, host DC
capable of presenting minor histocompatibility antigens to infused donor T-cells potentially
initiate T-cell activation and an associated Th-1 cytokine cascade implicated in the
pathogenesis of aGVHD. The relative levels of DC1/DC2 subsets in the hematopoietic graft
strongly correlate with the incidence of graft versus host disease and incidence of relapse.
G-CSF mediated stem mobilization is associated with increased levels of DC2 populations.54
In an animal model, the infusion of splenocytes and bone marrow from donors treated with
G-CSF was associated with emergence of a DC2 phenotype, and thus, tolerance.55 In one study
of patients undergoing allogeneic transplantation, the presence of increased numbers of DC2
cells in the hematopoietic graft was associated with a decrease in GVHD, and increase in
relapse and poorer outcomes.56 The impact of DC chimerism, patterns of reconstitution, and
presence of DC1 and DC2 populations following nonmyeloablative allogeneic transplantation
has not been well studied. Recent studies have demonstrated that CD52 is expressed by some
DC populations and suggest that CAMPATH therapy may impact antigen presentation by depletion
of DC populations from the host.57-59 Depletion of host derived DC may decrease in the
incidence of GVHD, while presence of donor DC is likely essential for the reconstitution of
cellular immunity.

In the proposed study, we intend to examine the application of a CAMPATH based preparative
regimen in patients with hematological malignancies undergoing nonmyeloablative allogeneic
stem cell transplantation from an unrelated donor. The study will involve patients who
would otherwise not be candidates for a conventional myeloablative conditioning regimen
because of age or organ dysfunction and who do not have HLA-matched sibling donors. It will
also include patients suffering from lymphoid malignancies such as low-grade lymphoma, CLL,
and multiple myeloma that have experienced unacceptably high transplant related mortality
with conventional allogeneic transplantation. Inclusion of both myeloid and lymphoid
hematological malignancies in the study cohort will optimize accrual and will not limit the
ability to the ability to pursue the study goals. Patterns of engraftment, graft versus
host disease, immune reconstitution, and donor/host chimerism are primarily determined by
the preparative regimen and transplant strategy and should not differ considerably within
the patient cohort.

For the conditioning regimen, we have chosen an intermediate dose of cyclophosphamide and
fludarabine in an attempt to minimize the incidence of graft rejection without increasing
the frequency of treatment related morbidity and mortality. Based on the work of MacKinnon
et al, we will add CAMPATH-1H to the preparative regimen to further decrease the incidence
of GVHD and promote donor stem cell engraftment. Following this regimen circulating levels
CAMPATH are present for several weeks post-transplant and may excessively deplete donor T
cells as well as eliminate DC populations from the donor graft. In an effort to improve
post-transplant immune reconstitution and limit risks of infection and relapse, we have
chosen a lower dose of CAMPATH and will follow levels at serial time points post-transplant.

The principal endpoints of the trial will include defining: 1) the treatment related
toxicity profile and post-transplant hematopoietic recovery 2) the incidence and severity of
CMV infection and acute and chronic GVHD 3) the phenotypic and functional characteristics of
DC and T cell populations post-transplant; and 4) the patterns of donor/host chimerism in DC
and T cell populations and its correlation with GVHD. As a secondary endpoint, 1 and 2 year
DFS and OS will be determined.