Allogeneic Multivirus - Directed Cytotoxic T Lymphocytes (CTL)

Viral infections are normally controlled by T-cell immunity and so are an important cause of
morbidity and mortality during the period of immune recovery after hematopoietic stem cell
transplantation (HSCT).1 Risk for infection is impacted by the degree of tissue mismatch
between donor and recipient and the immune status of the donor, including the degree and
length of immunosuppression following transplantation. Reactivation of latent viruses such as
cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are common and often cause symptomatic
disease. Respiratory viruses such as adenovirus also frequently cause infection. Antiviral
pharmacologic agents are only effective against some of these viruses; their use is costly,
and associated with significant toxicities and the outgrowth of drug-resistant mutants. As
delay in recovery of virus-specific cellular immune response is clearly associated with viral
reactivation and disease in these patients, cellular immunotherapy to restore viral-specific
immunity is an attractive option that has already been successfully used to target some of
these viruses.

Multivirus-Specific T Cells

To broaden the specificity of single CTL lines to include the three most common viral
pathogens of stem cell recipients, investigators reactivated CMV and adenovirus-specific T
cells by using mononuclear cells transduced with a recombinant adenoviral vector encoding the
CMV antigen pp65 (Ad5f35CMVpp65). Subsequent stimulations with EBV-LCL transduced with the
same vector both reactivated EBV-specific T cells and maintained the expansion of the
activated adenovirus and CMV-specific T cells. This method reliably produced CTLs with
cytotoxic function specific for all three viruses, which investigators infused into 14 stem
cell recipients in a Phase I prophylaxis study. They observed recovery of immunity to CMV and
EBV in all patients but an increase in adenovirus-specific T cells was only seen in patients
who had evidence of adenovirus infection pre-infusion. A follow-up study in which the
frequency of adenovirus-specific T cells was increased in the infused CTLs produced similar
results, thus highlighting the importance of endogenous antigen to promote the expansion of
infused T cells in vivo. Nevertheless, all patients in both clinical trials with pre-infusion
CMV, adenovirus or EBV infection or reactivation were able to clear the infection, including
one patient with severe adenoviral pneumonia requiring ventilatory support. CTLs recognizing
multiple antigens can therefore produce clinically relevant effects against all three
viruses.

CTLs for HSCT patients with virus naïve donors

All the donor specific T cell strategies discussed so far have utilized products derived from
donors who are seropositive for the virus of interest. With the increasing use of cord blood
(CB) grafts there are appreciable numbers of patients who are recipients of virus naïve donor
grafts. CMV reactivation usually occurs from endogenous virus and seropositive recipients
with seronegative donors remain the highest risk group for developing CMV. The development of
multivirus specific T cells from recipients of cord blood grafts requires the priming of
naïve T-cells rather than the simple expansion of pre-existing memory T-cells from
seropositive donors. Using a protocol stimulating CB-derived T-cells with autologous
CB-derived dendritic cells and EBV-LCL transduced with the Ad5f35CMVpp65 vector in the
presence of IL-7, 12 and 15, multivirus specific T cells can be primed in vitro from the 20%
fraction of a cord blood unit. So far, eight patients have received CTL as prophylaxis or
treatment after CBT without toxicity. No infusion-related toxicities/GvHD have been observed
and despite receiving only 80% of the CB unit, all patients engrafted neutrophils within 30
days. Early evidence of efficacy has also been demonstrated with clearance of EBV, CMV and
adenovirus in two patients and decreasing EBV viral load in a third with the other 5 patients
remaining virus free. Therefore, these results suggest that transfer of naïve T cell
(CB)-derived virus specific T cells to patients after CBT may be safe and facilitate long
term reconstitution of virus-specific T-cells in vivo.

Inclusion Criteria:

- Recipient Inclusion Criteria at the time of CTL infusion

1. Received prior myeoloablative or non-myeloablative allogeneic hematopoietic stem
cell transplant using either bone marrow or PBSC within 12 months

2. Cells administered as;

1. Prophylaxis for patients at risk of EBV, CMV, or Adenovirus.

2. Treatment of reactivation or infection for EBV, CMV, or Adenovirus.

3. Early treatment for single or multiple infections. Multiple infections with
one reactivation and one controlled infection are eligible to enroll.

3. Steroids less than 0.5 mg/kg/day prednisone

4. Karnofsky/Lansky score of ≥ 50

5. ANC greater than 500/µL.

6. Bilirubin <2x, AST <3x, Serum creatinine <2x upper limit of normal, Hgb >8.0

7. Pulse oximetry of > 90% on room air

8. Available multivirus-specific cytotoxic T lymphocytes

9. Negative pregnancy test (if female of childbearing potential)

10. Patient or parent/guardian capable of providing informed consent.

Exclusion Criteria:

- Recipient Exclusion criteria at the time of CTL infusion

1. Patients with other uncontrolled infections (see 2.3.2 for definitions)

2. Patients who received ATG, Campath, or other T cell immunosuppressive monoclonal
antibodies in the last 28 days

3. Received donor lymphocyte infusion in last 28 days

4. Evidence of GVHD > grade 2

5. Active and uncontrolled relapse of malignancy

6. Pregnant or lactating

7. Unable to wean steroids to ≤0.5 mg/kg/day prednisone.

8. Patients with Grade 3 hyperbilirubinemia