Local Modulation of Immune Receptors to Enhance the Response to Dendritic Cell Vaccination in Metastatic Melanoma

A variety of trials of immunotherapy in metastatic melanoma have clearly demonstrated that
immune responses against melanoma can be induced, but only a few patient have responded
clinically, suggesting that new strategies to enhance anti-cancer immune responses are
needed. Most of these immunotherapy trials have focused on antigen delivery and providing
stimulatory antigen-specific signals to T cells. However, the induction of antigen-specific
T cell-mediated immune responses is regulated not only by stimulatory signals, but also by
inhibitory receptor-mediated signals. Studies have demonstrated that administering mAbs
targeting such immune-modulating receptors on T cells enhances vaccine-induced anti-tumor
immunity, suggesting that such an approach might improve the efficacy of clinical cancer
vaccines. However, systemic administration such mAbs has been associated with severe,
sometimes fatal, autoimmunity. We have developed an approach for targeted delivery of such
immune modulatory proteins and mAbs, using immune modulator RNA-transfected dendritic cells
(DC), to sites where anti-tumor T cells are induced. Our preliminary studies indicate that
this approach may eliminate the adverse effects associated with systemic administration
immune modulators, while also enhancing vaccine-induced immune responses.

Therefore, the objective of this proposed Phase I immunotherapy trial is to determine the
safety and obtain preliminary data on the efficacy of vaccination of human subjects with
melanoma tumor associated antigen (TAA) RNA-transfected mature monocyte-derived DC
coadministered with additional untransfected DC (Study Arm A), DC transfected with RNA
encoding a soluble GITR-L fusion protein (Study Arm B), DC transfected with RNA encoding the
humanized heavy and light chains of an antagonistic anti-CTLA-4 mAb (Study Arm C), or DC
transfected with combined GITR-L and anti-CTLA-4 mAb RNA (Study Arm D). All study subjects
will undergo leukapheresis for collection of peripheral blood mononuclear cells (PBMC) and
purified monocytes will be cultured with the cytokines GM-CSF and IL-4 to produce immature
DC. After the induction maturation with a standard cytokine cocktail, half of the DC will
then be transfected with RNA encoding melanoma TAAs MART, tyrosinase, and gp100, and MAGE-3,
while the remaining half of the DC will be either untransfected (Study Arm A) or will be
transfected with immune modulator RNA (Study Arm B, C, and D). DC will be cryopreserved.
Subjects will then be immunized with these DC, weekly for six intranodal injections. For
each subject, safety and toxicity will be assessed, and anti-tumor immune responses will be
measured.

Inclusion Criteria:

- Patients with confirmed metastatic melanoma

- Karnofsky performance status greater than or equal to 70%.

- Estimated life expectancy > 6 months.

- Age > 17 years.

- Adequate hematologic function with:

- WBC >= 3000 mm3

- hemoglobin >= 9 mg/dl

- platelets >= 100,000/mm3

- Adequate renal and hepatic function with:

- serum creatinine < 2.5 mg/dl

- bilirubin < 2.0 mg/dl

- AST/SGOT < 70 U/L

- ALT/SGPT < 70 U/L

- Alkaline Phosphatase ≤ 135 U/L

- Ability to understand and provide signed informed consent that fulfills Institutional
Review Board guidelines.

- Ability to return to Duke University Medical Center for adequate follow-up as
required by this protocol.

Exclusion Criteria:

- Subjects undergoing concurrent chemotherapy, radiation therapy, or immunotherapy will
be excluded.

- The subject has previously irradiated, surgically treated, or newly diagnosed central
nervous system (CNS) metastases will be excluded (Pre-enrollment head CT is not
required if not indicated by clinical signs or symptoms).

- Subjects with a history of autoimmune disease such as, but not restricted to,
inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis,
scleroderma, or multiple sclerosis will be excluded.

- Subjects with serious concurrent chronic or acute illness such as pulmonary (asthma
or COPD), cardiac (NYHA class III or IV) or hepatic disease, or other illness
considered by the principal investigator to constitute an unwarranted high risk for
investigational drug administration will be excluded.

- Subjects with medical or psychological impediment to probable compliance with the
protocol will be excluded.

- Subjects with concurrent second malignancy other than melanoma or non-melanoma skin
cancer will be excluded. In the event of prior non-melanoma malignancies treated
surgically, the subject must be considered NED (no evidence of disease) for a minimum
of 3 years prior to enrollment.

- Presence of an active acute or chronic infection, including symptomatic urinary tract
infection, HIV (as determined by ELISA and confirmed by Western Blot) or viral
hepatitis (as determined by HBsAg and Hepatitis C serology) will lead to subject
exclusion.

- Subjects receiving steroid therapy (or other immunosuppressive agents such as
azathioprine or cyclosporine A) are excluded on the basis of potential immune
suppression.

- Subjects with inadequate peripheral vein access to undergo leukapheresis will be
excluded.

- Female subjects with a positive pregnancy test, as well as those who have not
previously undergone hysterectomy and/or bilateral oophorectomy and are unwilling to
utilize a medically approved form of contraception, from the time of enrollment until
6 weeks after the final immunization, will be excluded.

- Male subjects, not previously surgically sterilized, who are unwilling to use a
condom with spermicide during any sexual activity occurring over the entire
immunization period and for the 6 weeks that immediately follow the final
immunization will be excluded.

- Subjects with a documented history of severe allergic reaction to beta-lactams, eggs
or soy products.