Hematopoietic Stem Cell Support in Vasculitis

Selection of High Dose Immunosuppressive Therapy and Autologous HSCT Strategy for this Trial

Selection of the Regimen for High-Dose Immunosuppressive Therapy Cyclophosphamide with ATG
(Cy/ATG) is a common conditioning regimen with two decades of experience in the treatment of
aplastic anemia and has been used safely without reported mortality in the treatment of
autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Cy/ATG not
only has less acute toxicity, it has less chronic side effects. Cy/ATG is not associated with
late malignancies or cataracts. Cyclophosphamide and anti-thymocyte globulin (horse or rabbit
ATG) and rituximab are potent immunosuppressive agents. Rituximab and ATG contribute
additional immunosuppression without additional cytotoxicity. Rituximab and ATG given shortly
pre-transplant will contribute to the elimination of host T lymphocytes that survive
cyclophosphamide or that contaminate the CD34+selected graft. SLE, an autoimmune disease
responsive to cyclophosphamide, responds well to a Cy/ATG/rituximab conditioning regimen. For
these reasons, Cy/ATG/rituximab will be the conditioning regimen utilized in this study.

To justify any new therapy such as HSCT, the risk of dying from the disease must be higher
than that expected from its treatment, or the morbidities associated with the disease must
justify the treatment risks. Autologous stem cell transplantation has a mortality of 1-3% in
breast cancer patients using intense conditioning regimens with multiple alkylating agents,
and up to 10-15% in patients with lymphomas and other malignancies. The latter patients
usually have been heavily treated before transplant and the accumulation of treatment
toxicities is thought to play a role in their increased transplant-related mortality.
Cyclophosphamide and ATG/rituximab have been used to transplant of extremely ill patients
with systemic lupus erythematosus and multiple organ dysfunction at Northwestern University
without mortality.

Since patients in this study are earlier in the disease course, a conditioning regimen that
has historically been associated with less toxicity will be employed. The regimen will be
Cyclophosphamide at 200 mg/kg with ATG 5.5 mg/kg and rituximab 1000 mg divided over 2 doses.
Cyclophosphamide 200mg/kg with or without ATG has been used safely and effectively in a
variety of autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis,
and aplastic anemia. Rabbit ATG will be used instead of equine ATG due to the lower incidence
of hypersensitivity and fever with rabbit ATG compared to equine.

Method of Harvesting Stem Cells Based on the experience of the pilot studies, the current
protocol will mobilize stem cells with cyclophosphamide and granulocyte-colony stimulating
factor (G-CSF), and collect stem cells by apheresis, with subsequent bone marrow harvest
performed only if needed to supplement the peripheral blood stem cells (PBSC). Based on
experience of autoimmune flares in patients receiving G-CSF alone for mobilization (56),
patients will be mobilized with cyclophosphamide 2.0 g/m2 and G-CSF 10 mcg /kg.

Regimen for Autologous HSCT

Cyclophosphamide Cyclophosphamide is an active agent in patients with a wide variety of
malignancies. It is used frequently in the therapy of lymphoid malignancies and has potent
immunosuppressive activity. It is frequently used as a cytotoxic and immunosuppressive agent
in patients undergoing marrow transplants and as a treatment for patients with autoimmune
diseases. It is an alkylating agent that requires hepatic metabolism to the active
metabolites, phosphoramide mustard and acrolein. These active metabolites react with
nucleophilic groups. It is available as an oral or intravenous preparation. Bioavailability
is 90% when given orally. The half-life of the parent compound is 5.3 hours in adults, and
the half-life of the major metabolite phosphoramide mustard is 8.5 hours. Liver or renal
dysfunction will lead to prolonged serum half-life. CY is administered intravenously at a
dosage of 50 mg/kg on each of two successive days (use adjusted ideal body weight if
patient's actual body weight is greater than 100% ideal body weight). The major dose limiting
side effect at high doses is cardiac necrosis. Hemorrhagic cystitis can occur and is mediated
by the acrolein metabolite. This can be prevented by co-administration of MESNA or bladder
irrigation. Other notable side effects include nausea, vomiting, alopecia, myelosuppression
and SIADH. Refer to institutional manuals for more information about administration, toxicity
and complications.

Rabbit-Derived Anti-Thymocyte Globulin (ATG) Rabbit-derived anti-thymocyte globulin (ATG) is
a gamma globulin preparation obtained from hyperimmune serum of rabbits immunized with human
thymocytes. ATG has been used predominately in solid organ transplant immunosuppressive
regimens. ATG is a predominantly lymphocyte-specific immunosuppressive agent. It contains
antibodies specific to the antigens commonly found on the surface of T cells. After binding
to these surface molecules, ATG promotes the depletion of T cells from the circulation
through mechanisms, which include opsonization and complement-assisted, antibody- dependent,
cell-mediated cytotoxicity. The plasma half-life ranges from 1.5-12 days. ATG is administered
intravenously at a dose of 0.5 mg/kg recipient body weight on day -6 and a dose of 1.0 mg/kg
recipient body weight on days -5, -4, -3, -2 and -1. Unlike equine ATG, rabbit ATG does not
require a pre-infusion skin test to check for hypersensitivity. Methylprednisolone 1 gram
will be given before every dose of ATG. Additional medications such as diphenhydramine may be
given at the discretion of the attending physician. Although rare, the major toxicity is
anaphylaxis. Chills, fever, pruritus or serum sickness may occur.

Regimen for allogeneic HSCT

Cyclophosphamide (Cytoxan) Cyclophosphamide (CY) is administered intravenously at a dosage of
50 mg/kg on each of four successive days (use adjusted ideal body weight if patient's actual
body weight is greater than 100% ideal body weight). The major dose limiting side effect at
high doses is cardiac necrosis. Hemorrhagic cystitis can occur and is mediated by the
acrolein metabolite. This can be prevented by co-administration of MESNA or bladder
irrigation. Other notable side effects include nausea, vomiting, alopecia, myelosuppression
and SIADH. Refer to institutional manuals for more information about administration, toxicity
and complications.

Fludarabine Fludarabine phosphate is rapidly dephosphorylated to 2-fluoro-ara-A and then
phosphorylated intracellularly by deoxycytidine kinase to the active triphosphate,
2-fluoro-ara-ATP. This metabolite appears to act by inhibiting DNA polymerase alpha,
ribonucleotide reductase and DNA primase, thus inhibiting DNA synthesis. The mechanism of
action of this antimetabolite is not completely characterized and may be multi-faceted.

Phase I studies in humans have demonstrated that fludarabine phosphate is rapidly converted
to the active metabolite, 2-fluoro-ara-A, within minutes after intravenous infusion.
Consequently, clinical pharmacology studies have focused on 2-fluoro-ara-A pharmacokinetics.
After the five daily doses of 25 mg 2-fluoro-ara-AMP/m2 to cancer patients infused over 30
minutes, 2-fluoro-ara-A concentrations show a moderate accumulation. During a 5-day treatment
schedule, 2-fluoro-ara-A plasma trough levels increased by a factor of about 2. The terminal
half-life of 2-fluoro-ara-A was estimated as approximately 20 hours. In vitro, plasma protein
binding of fludarabine ranged between 19% and 29%.

CAMPATH Campath-1H is a humanized fusion protein that is directed to CD52 antigen that is
expressed on all lymphocytes, monocytes and macrophages. It has very potent immunosuppressive
property and is effective for prevention of graft-versus-host disease. 30 mg/day of CAMPATH
will be given intravenously over 2 hours on days -4, -3 and -2. The most commonly reported
adverse reactions are infusion reactions fever, chills, hypotension, urticaria, nausea, rash,
tachycardia, dyspnea), cytopenias (neutropenia, lymphopenia, thrombocytopenia, anemia), and
infections (CMV viremia, CMV infection, other infections). In clinical trials, the frequency
of infusion reactions was highest in the first week of treatment. Other commonly reported
adverse reactions include vomiting, abdominal pain, insomnia and anxiety. The most commonly
reported serious adverse reactions are cytopenias, infusion reactions, and
immunosuppression/infections. About 30 minutes before the patient gets Campath, he/she will
be given other medications (such as acetaminophen or diphenhydramine, given orally, not IV)
to help reduce side effects.

Inclusion Criteria:

- 1. Age 16 to 60 years old at the time of pretransplant evaluation.

- 2. An established diagnosis of systemic necrotizing vasculitis (Wegener's
granulomatous, polyarteritis nodosum (PAN), allergic angiitis granulomatous (AAG, also
known as Churg Strauss syndrome), microscopic polyangiitis (MPA), or overlap
syndrome)Temporal arteritis, or mixed cryoglobulinemia or primary central nervous
system vasculitis AND failure of corticosteroids and any of the following at least 6
months of oral or IV cytoxan, rituximab, or cellcept. (Failure defined as: a) patients
with a high disease activity and involvement of internal organs as measured by
increased FFS > 2 and/or BVAS > 20, or b) patients who develop recurrent flares with
subsequent progressive organ damage.)

OR

Neurovascular Behcets with recurrent oral and/or genital lesions confirmed by culture to be
herpes negative, MRI findings consistent with CNS vasculitis, recurrent neurological
symptoms, and clinical confirmation by a Neurologist (e.g., Dr. Rama Gourimeni) AND failure
of at least 3 months of oral or IV cytoxan.

OR

Pulmonary or neurovascular Sjogrens with positive SSA/SSB confirmed by a rheumatologist and
neurologist (if CNS involved) or pulmonologist (if lungs involved) and either recurrent
neurologic attacks or progressive pulmonary compromise (dyspnea on exertion, decreased DLCO
or CT findings of active disease) despite at least 6 months of intravenous monthly pulse
cyclophosphamide.

- 3. Patient eligibility must be confirmed by two Rheumatologists. For patients with
neurovascular Behcets, eligibility need only be confirmed by a neurologist.

- 4. A minimum CD34+ cell dose of 2.0 x 10e6/kg post-selection.

Exclusion Criteria:

- 1. Significant end organ damage such as:

1. LVEF <40% or deterioration of LVEF during exercise test on MUGA or echocardiogram
unless due to active disease.

2. Untreated life-threatening arrhythmia.

3. Active ischemic heart disease or heart failure.

4. DLCO < 40% of predicted value unless due to active disease.

5. Serum creatinine > 2.5 mg/dl, unless due to active disease.

6. Liver cirrhosis, transaminases >3x of normal limits, or bilirubin >2.0 unless due
to Gilberts disease.

- 2. HIV positive.

- 3. Uncontrolled diabetes mellitus, or any other illness that in the opinion of the
investigators would jeopardize the ability of the patient to tolerate aggressive
treatment.

- 4. Prior history of malignancy except localized basal cell or squamous skin cancer.
Other malignancies for which the patient is judged to be cured by local surgical
therapy, such as (but not limited to) head and neck cancer, or stage I or II breast
cancer will be considered on an individual basis.

- 5. Positive pregnancy test, inability or unable to pursue effective means of birth
control, failure to willingly accept or comprehend irreversible sterility as a side
effect of therapy.

- 6. Psychiatric illness or mental deficiency making compliance with treatment or
informed consent impossible.

- 7. Inability to give informed consent.

- 8. Active infection, excluding asymptomatic bacteruria or vaginal candidiasis.

- 9. Active hepatitis B (HBSAg positive) or active hepatitis C (PCR positive blood
lymphocytes).