Trial Information

Most Closely HLA Matched Allogeneic Virus Specific Cytotoxic T-Lymphocytes (CTL) to Treat Persistent Reactivation Or Infection With Adenovirus, CMV and EBV After Hemopoietic Stem Cell Transplantation (HSCT)

Patients may be screened for study entry when they have persistent disease despite standard
therapy as defined in the inclusion criteria. At that stage a search will be done of the
available lines. Lines were generated from HSCT donors who consented to the use of CTLs not
required for their recipient for research or from normal donors. All donors were screened
and deemed to be eligible as transplant donors. We will also manufacture additional lines
with the goal of covering common HLA types and will consult with the NMDP to determine what
HLA types would be desirable. Additional donors will be screened by a transplant donor
center physician and must be deemed eligible before a line can be manufactured.

CTL Lines: We will use trivirus specific CTL lines generated as described previously.
Generation of trivirus-specific CTL lines requires the generation of several different
components from PBMC. The CTL line will be derived from donor peripheral blood T cells, by
multiple stimulations with antigen-presenting cells (APCs) presenting CMV, EBV and
adenovirus antigens and expansion with interleukin-2 (IL-2). The APCs used to stimulate and
expand the CMV-specific T cells will be derived from patient mononuclear cells and B
lymphocytes.

To initiate the trivirus-specific CTL line, PBMC will be transduced with an adenovirus
vector (Ad5f35-pp65) expressing the immunodominant antigen of CMV, pp65. The monocyte
fraction of PBMC expressed and presented CMV-pp65 peptide epitopes to the CMV-specific T
cell fraction of the PBMC, while the virion proteins from the adenovirus vector were
processed and presented to the adenovirus-specific T cell fraction.

To expand trivirus -specific T cells we used EBV-transformed B lymphoblastoid cell lines
(EBV-LCLs) transduced with Ad5f35-pp65. This transduction allows the EBV-LCLs to present
CMV-pp65 and adenovirus virion peptides to the T cells as well as endogenously expressed EBV
antigens.

EBV-LCLs are derived from PBMC-B lymphocytes by infection with a clinical grade, laboratory
strain of Epstein-Barr virus (EBV). About 5 x 10^6 PBMC, or 5 to 10 mLs of blood is required
to generate the EBV-LCL

At the end of the CTL culture period, the frequency of T cells specific for each virus were
determined using tetramer reagents if available. To test the functional antigen specificity
of the CTL we will use overlapping peptide libraries for pp65 and adenovirus hexon and
autologous and allogeneic LCLs in Elispot assays and we will perform cytotoxicity assays
using unmodified PHA blasts and LCLs untransduced or transduced with Adhexon and CMVpp65
pepmix OR LCL transduced with Ad5f35-null and Ad5f35-pp65. Some lines will have aliquots
sent for further characterization to the NIH where Drs Melenhorst and Barrett are developing
assays to predict alloreactivity where they examine virus antigen-specific T cells using an
extensive panel of activated T cells as antigen presenting cells (T-APC). These lines will
be labeled with component number only and will not have subject identifiers.

The CTL lines will also be checked for identity, phenotype and sterility, and cryopreserved
prior to administration according to SOP. Release criteria for administering the CTL to
patients include viability >70%, negative culture for bacteria and fungi for at least 7
days, endotoxin testing less than or equal to 5EU/ml, negative result for Mycoplasma, <2%
CD19 positive B cells, <2% CD14 positive monocytes (or <2% CD83 positive cells if Dendritic
cells were used as stimulators) and HLA identity.

No Matched CTL Line Available: If no matched line is available the patient will be
registered so that the feasibility of the approach can be assessed and the eventual outcome
will also be collected.

CTL Line Available but Patient Status Changes: Patients clinical course that changes between
screening and infusion will not be given the CTL and will be followed for eventual outcome.

Survival data will be collected by asking the transplant center to submit the routine
Transplant Essential Data form that is sent to the Stem Cell Transplant Outcomes Database at
100 days and 1 year and includes data on survival status and other outcome measures.

Criteria for Selection of CTL Line: In general the line matching at the highest number of
HLA loci will be selected. Matching at the allele level will be preferred but antigen level
will be accepted or HLA-A and HLA-B. However consideration will also be given to the type of
infection and activity of the line against that virus. For example for a patient with
adenovirus infection a line that matches at 2 loci but that has recognition of adenovirus
mediated through those antigens would be preferable to a line matched at 3 loci but with no
demonstrated activity against adenovirus. The protocol chair will discuss each case with the
principal investigators at each center to determine the optimal CTL line for each patient.
If more than one line matches and there are insufficient cells to cover additional infusions
a second CTL line will be reserved in the event that additional infusions are warranted.
Patients with a partial response are eligible to receive an additional dose.

Premedications: Patients will be premedicated with Benadryl 1mg/kg (max 50 mg) IV and
Tylenol 10 mg/kg (max 650 mg) PO.

Patients will be monitored according to institutional standards for administration of blood
products and at a minimum will be monitored according to below: • Patients should remain in
the clinic for at least one hour • Patients should remain on continuous pulse oximetry for
at least 30 minutes • Vital signs should be monitored at the end of infusion then at 30 and
60 minutes

Supportive Care: Patients will receive supportive care for acute or chronic toxicity,
including blood components or antibiotics, and other intervention as appropriate.

If a patient has a partial response they are eligible to receive up to 4 additional doses at
a minimum 2 week intervals and if they meet the eligibility criteria for subsequent lines.
These doses would come from the original infused line if sufficient vials were available but
may come from another line if there are insufficient cells in the original line.

Follow-up Assessments: The timing of follow-up visits is based on the date of CTL infusion.
If a patient has multiple CTL doses the schedule resets again at the beginning so follow up
relates to the last CTL dose.

Follow up will occur at 7 days, 14 days, 21 days, 28 days, 42 days, 90 days, 180 days, and
365 days (+/- 2 days up to Week 8, and +/- 14 days for Days 90, 120, and +/- 28 days for 6
and 12 months, post-enrollment..)

The following assessments are considered standard-of-care unless identified below by " * ":

Pre-Infusion: 1. History and physical exam including height and weight 2. Viral loads for
EBV, adenovirus, CMV 3. Biopsy disease site, if appropriate 4. Imaging studies, if
appropriate 5. Complete acute GVHD staging and grading information including assessments of
rash, diarrhea, nausea/vomiting, weight and liver function tests 6. CBC with differential,
platelet count 7. Liver function tests (bilirubin, alkaline phosphatase, AST, ALT) plus
creatinine 8. Tacrolimus/cyclosporine level 9. * Samples for laboratory studies

Post-Infusion: 1. Viral loads for CMV, EBV, adenovirus weekly at 1, 2, 3, 4 and 6 weeks and
3, 6 and 12 months. 2. Complete acute GVHD staging and grading information including
assessments of rash, diarrhea, nausea/vomiting, weight and liver function tests weekly until
Day 45 3. Chronic GVHD evaluation (if present) 3, 6, 9, and 12 months 4. CBC with
differential and platelet count at 1, 2, 3, 4, and 6 weeks. 5. Infusion related toxicities
within 24 hours and toxicity evaluation weekly until Day 30 and acute GVHD until Day 45 6.
Steroid dose weekly until Day 42, and 3, 6 and 9 months 7. * Samples for laboratory studies
on Days 0, 14, 28 and 90 8. Infections through Day 42 and at 3, 6 and 12 months

Ancillary laboratory studies will include: 1) Assessment of virus-specific immunity based on
CTL levels as measured by ELISPOT assays or tetramer assays. 2) Persistence of infused T
cells based on PCR for non-shared antigen

Reporting Patient Deaths: The Recipient Death Information must be entered into the web-based
data entry system within 24 hours of knowledge of a patient's death. If the cause of death
is unknown at that time, it need not be recorded at that time. However, once the cause of
death is determined, the form must be updated.

Donor Evaluation: - Complete history and physical examination - CBC, platelets, differential
- Total protein, albumin, total bilirubin, alkaline phosphatase, ALT, AST, - HIV-1 antibody,
HIV-2 antibody, HIV NAT, HTLV-1/2 antibodies, HBsantigen, HBc antibody, HCV NAT, CMV
antibody, RPR, West Nile virus NAT, and Chagas testing - ABO and Rh typing - When the
evaluation is complete, the Transplant Physician will note in the recipient's and donor's
medical records that the tests have been evaluated, and the donor is acceptable.