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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)

Phase 1
Open (Enrolling)
Adenovirus Infection, EBV Infection

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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

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

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.

Inclusion Criteria


Pts will be eligible following any type of allogeneic transplant if they have CMV,
adenovirus or EBV infection persistent to standard therapy (as defined below).

1. Prior myeloablative or non-myeloablative allogeneic hematopoietic stem cell
transplant using either bone marrow, peripheral blood stem cells or single or double
cord blood within 18 months.

2. CMV, adenovirus or EBV infection persistent despite standard therapy

1. For CMV infection: i.Pts with CMV disease: defined as the demonstration of CMV
by biopsy specimen from visceral sites (by culture or histology) or the
detection of CMV by culture or direct fluorescent antibody stain in
bronchoalveolar lavage fluid in the presence of new or changing pulmonary
infiltrates OR ii. Failure of antiviral therapy: defined as the continued
presence of pp65 antigenemia (>1+ cell/100,000 cells) or DNAemia (as defined by
reference lab performing PCR assay but usually >400 copies/ml) after at least 7
days of antiviral therapy OR iii. Relapse after antiviral therapy defined as
recurrence of either pp65 antigenemia or DNAemia after at least 2 weeks of
antiviral therapy iv. For CMV infection, standard therapy is defined as 7 days
therapy with Ganciclovir, Foscarnet or Cidofovir for patients with disease or
recurrence after 14 days therapy

2. For EBV infection: i. EBV infection is defined as: 1. Biopsy proven lymphoma
with EBV genomes detected in tumor cells by immunocytochemistry or in situ PCR
OR 2. Or clinical or imaging findings consistent with EBV lymphoma and elevated
EBV viral load in peripheral blood. ii. For EBV infection, standard therapy is
defined as rituximab given at 375mg/m^2 in patients for 1-4 doses with a CD20+ve
tumor iii. Failure is defined as: 1. There was an increase or less than 50%
response at sites of disease for EBV lymphoma OR 2. There was a rise or a fall
of less than 50% in EBV viral load in peripheral blood or any site of disease

3. For adenovirus infection or disease: i. Adenovirus infection is defined as the
presence of adenoviral positivity as detected by PCR, DAA or culture from ONE
site such as stool, blood, urine, or nasopharynx OR ii. Adenovirus disease will
be defined as the presence of adenoviral positivity as detected by culture from
two or more sites such as stool or blood or urine or nasopharynx iii. Standard
therapy is defined as 7 days therapy with Cidofovir (if renal function permits
this agent to be given) iv. Failure is defined as a rise or a fall of less than
50% in viral load in peripheral blood or any site of disease as measured by PCR
or any other quantitative assay)

3. Pts with multiple CMV, EBV or Adenovirus infections are eligible given that each
infection is persistent despite standard therapy as defined above. Patients with
multiple infections with one persistent infection and one controlled infection are
eligible to enroll.

4. Clinical status at enrollment to allow tapering of steroids to less than 0.5
mg/kg/day prednisone.

5. Written informed consent from patient, parent or guardian.

6. Negative pregnancy test in female patients if applicable (childbearing potential who
have received a reduced intensity conditioning regimen).

The informed consent process will begin at recognition of subject eligibility and consent
will be obtained per institutional practices before study therapy is initiated. Subjects
will initially sign a screening consent to enable a search to be made for a line. If a
line is available they will sign the treatment consent.

Up to 4 additional doses can be administered if a partial response is obtained and patient
meets eligibility criteria for subsequent infusions. The minimum interval between
subsequent infusions is 2 weeks.

Donors will be eligible if they meet eligibility criteria for blood donors on history and
exam by a transplant donor physician and have negative infectious diseases testing.


For initial CTL and subsequent infusions:

1. Patients receiving ATG, or Campath or other immunosuppressive monoclonal antibodies
within 28 days of screening for enrollment.

2. Patients with other uncontrolled infections. For bacterial infections, patients must
be receiving definitive therapy and have no signs of progressing infection for 72
hours prior to enrollment. For fungal infections patients must be receiving
definitive systemic anti-fungal therapy and have no signs of progressing infection
for 1 week prior to enrollment.

Progressing infection is defined as hemodynamic instability attributable to sepsis or
new symptoms, worsening physical signs or radiographic findings attributable to
infection. Persisting fever without other signs or symptoms will not be interpreted
as progressing infection.

3. Patients who have received donor lymphocyte infusion (DLI) within 28 days.

4. Patients with active acute GVHD grades II-IV.

5. Active and uncontrolled relapse of malignancy

Donors will be ineligible if they do not meet eligibility criteria for blood donors on the
donor questionnaire or have positive infectious diseases testing on any of the tests
outlined in the inclusion criteria.

Type of Study:


Study Design:

Endpoint Classification: Safety/Efficacy Study, Intervention Model: Single Group Assignment, Masking: Open Label, Primary Purpose: Treatment

Outcome Measure:

The primary purpose of the study is to assess the safety of administering CHM-CTLs in transplant patients with EBV, CMV, or adenovirus infection. We have elected to use a dose of 2 x 107 CHM-CTLs/m2.

Outcome Time Frame:

1 year

Safety Issue:


Principal Investigator

Helen Heslop, MD

Investigator Role:

Principal Investigator

Investigator Affiliation:

Baylor College of Medicine


United States: Food and Drug Administration

Study ID:




Start Date:

November 2008

Completion Date:

August 2013

Related Keywords:

  • Adenovirus Infection
  • EBV Infection
  • CMV
  • Adenovirus
  • EBV
  • non-myeloablative transplants
  • Prior allogeneic hematopoietic stem cell transplant
  • Adenoviridae Infections
  • Epstein-Barr Virus Infections



MD Anderson Cancer Center Houston, Texas  77030-4096
Duke University Medical Center Durham, North Carolina  27710
Dana Farber Cancer Institute Boston, Massachusetts  02115
Texas Children's Hospital Houston, Texas  
University of Miami Miami, Florida  33136
The Methodist Hospital Houston, Texas  77030
Hackensack University Hackensack, New Jersey  07601
Children's Hospital of Los Angeles Los Angeles, California  90027