Autologous Dendritic Cell Therapy for Type 1 Diabetes Suppression: A Safety Study
Autologous Dendritic Cell Therapy for Type 1 Diabetes Suppression: A Safety Study
The proposed studies describe a randomized trial to evaluate the safety of a new
 diabetes-suppressive cell vaccine, consisting of autologous monocyte-derived dendritic cells
 treated ex vivo with antisense phosphorothioate-modified oligonucleotides targeting the
 primary transcripts of the CD40, CD80 and CD86 co-stimulatory molecules (immunoregulatory DC;
 iDC). The hypothesis to be tested in this study is that iDC are safe and without toxicity in
 established type 1 diabetic patients.

 Fifteen (15) individuals exhibiting fully-established, insulin-dependent type 1 diabetics,
 without any diabetes-related complications, infectious disease, or other medical anomaly,
 will be enrolled to establish safety of the approach. 7/15 volunteers will be administered
 autologous control dendritic cells and 8/15 will be administered iDC. The study is
 anticipated to be complete by twelve (12) months.

 Currently, other than a humanized anti-CD3 antibody with considerable side effects, there is
 no other means to reverse new-onset type 1 diabetes. These studies will be the first ever to
 employ autologous dendritic cell transfer to suppress an autoimmune disease and to perhaps
 reverse it early on in the clinical process.
In this study, volunteers with established type 1 diabetes that requires insulin replacement
 who do not exhibit any diabetes-related complications or any other clinical pathology will
 first undergo complete physical, biochemical and immunological evaluation. Monocyte-derived
 autologous dendritic cells will be obtained following leukapheresis. The dendritic cells will
 be treated ex vivo with the antisense oligonucleotides and cryopreserved in aliquots for
 subsequent intradermal administration into sites closest to the physical location of the
 pancreas inside the body. Physiologic, biologic and immunologic responses to the dendritic
 cell vaccine will be evaluated over the period of the trial. The first group of volunteers
 will receive autologous dendritic cells without any ex vivo treatment (7/15) and the second
 group will be administered iDC (8/15). If there is no evidence of toxicity or adverse events
 associated with the dendritic cell vaccine, and only upon FDA and IRB approval we will
 initiate a new study comparing efficacy of control DC and iDC in improving glycemia and
 reversing autoimmunity in new-onset patients.

 Currently, other than a humanized anti-CD3 antibody with considerable side effects, there is
 no other means to reverse new-onset type 1 diabetes. These studies will be the first ever to
 employ autologous dendritic cell transfer to suppress an autoimmune disease and to possibly
 reverse it early on in the clinical process.

 We propose to ascertain the safety of autologous dendritic cells treated ex vivo with
 antisense oligonucleotides targeting the primary transcripts of the CD40, CD80 and CD86
 co-stimulatory molecules (we term these cells "immunoregulatory dendritic cells; iDC) first
 in volunteers with established type 1 diabetes.

 Personality traits and emotional stability have been linked to treatment adherence in other
 medical populations (e.g. Christensen et al, 2002; Dew et al., 2001). In that adherence to
 this protocol (e.g. remaining in the study) by this small sample will impact significantly on
 results and that the treatment itself may affect mood, we will assess personality traits at
 baseline and monitor mood throughout the study period.

 2.2 Significance of the studies: We do not anticipate any safety issues with this approach
 but we must be cautious at all levels given that this is immunomodulation therapy. At the
 very least, data will be obtained on whether the success we have observed with the iDC in NOD
 mice can be eventually adapted in other protocols aimed to restore normoglycemia or improve
 glycemic control along with increased prevalence of putative immunoregulatory T cells in
 recently-diagnosed humans.

 3.0 RESEARCH DESIGN AND METHODS

 3.1 Drug/Device Information 3.1.1 Autologous Dendritic Cells (Control Dendritic Cells): These
 are cells derived from the leukapheresis product of each patient. The leukapheresis product
 is processed inside an Elutra/AastromTM cell elutriation system (Refer to appended SOP #
 CPL-0158 and CPL-0166) to obtain immature dendritic cells. The final autologous product after
 these procedures is termed Control Dendritic Cells.

 3.1.2 Autologous Immunoregulatory, Diabetes-Suppressive Dendritic Cells (iDC): We have
 submitted an IND application (appended) to the FDA to cover a diabetes-suppressive embodiment
 of Control Dendritic Cells, which we term "immunoregulatory dendritic cells; iDC. The IND
 covers the treatment of Control Dendritic Cells with phosphorothioate-modified
 oligonucleotides targeting the primary transcripts of the CD40, CD80 and CD86 co-stimulation
 molecules. The Control DC and the oligonucleotide-treated dendritic cells (iDC) are the study
 cell products in this proposal.

 3.1.3 Leukapheresis Apparatus: This will be performed with the Fenwal CS3000 Plus cell
 collector, or equivalent, using a large bore double lumen catheter as peripheral venous
 access with total processing of approximately 10-12 liters per session. The device and its
 equivalents are FDA-approved for the procedures outlined herein to obtain purified
 leukocytes. Staff experienced with leukapheresis, contracted from the Central Blood Bank of
 Pittsburgh will perform these procedures.

 3.1.4 Psychological Measures: The NEO-FFI is a 60 item paper and pencil questionnaire based
 on the five-factor theory of personality (Neuroticism, Extraversion, Openness, Agreeableness,
 and Conscientiousness). For this study we will examine the Neuroticism and Conscientiousness
 scales. The Beck Depression Inventory, 2nd edition (BDI-2) and the Beck Anxiety Inventory
 (BAI) are each 21 item paper and pencil questionnaires which have been widely used in
 psychiatric and medical populations.

 3.2 Research Design and Methods 3.2.1 Setting/location of the study Systemic, physiologic and
 immunologic monitoring of patients prior to, during treatment cycle and post-treatment will
 take place in the CTRC clinic of Montefiore University Hospital of UPMC Health System
 (MUH-CTRC) under the supervision of the co-investigator physicians of this study and the
 nurses of the CTRC. Subjects will undergo leukapheresis in an outpatient setting at the
 MUH-CTRC or the University of Pittsburgh Cancer Center (UPCI) or the Central Blood Bank of
 Pittsburgh (depending on the most convenient location for each patient); receive the study
 dendritic cells in an outpatient setting at the MUH-CTRC; and undergo the remaining tests and
 procedures at the MUH-CTRC. Subjects will have approximately 16 visits at the locations
 described above, each visit lasting about 2-4 hours depending on what procedures are being
 performed at that particular visit. All results for each subject will be presented and
 discussed in an ongoing fashion at meetings held weekly at the UPCI outpatient clinic.
 Subjects will be monitored during the dendritic cell administration cycle; once every week
 following each dendritic cell administration for a period of nine weeks, then once a month
 for the remainder of the year (Refer to schematic cartoons in the Appendix, Section 6.8).

 3.2.3 Autologous Control Dendritic Cell Vaccine Preparation Autologous DC will be generated
 from monocytes, derived from leukapheresis product. Leukapheresis will be performed at either
 of the following locations: 1) MUH-CTRC; 2) the UPCI Leukapheresis Unit; or 3) the downtown
 location of the Central Blood Bank of Pittsburgh. The product will be hand carried to the
 IMCPL located inside the Hillman Cancer Center in Shadyside by a nurse participating in the
 research study. At the time of procurement, immediately following leukapheresis, the sample
 will be coded by the nurse using the same code assigned to the patient during enrollment. At
 the IMCPL, monocytes will be separated by elutriation in a closed ELUTRA system. Monocytes
 will be cultured in the closed Aastrom Replicell System in the presence of IL-4 and GM-CSF to
 generate DC. On day 6 of culture, DC will be harvested and checked for viability and purity.
 An aliquot of freshly-generated DC will be used for the control DC vaccine preparation, while
 the remaining DC will be aliquoted into sterile cryovials and cryopreserved for preparation
 of subsequent vaccines. We have calculated that to prepare a vaccine containing 1 x 107
 viable control dendritic cells or iDC (to be described below) for each delivery (One
 treatment defined as 1 x 107 dendritic cells injected at four intradermal sites
 anatomically-proximal to the pancreas) we will need a total of ~5 x 107 immature dendritic
 cells. This is the minimal cell number estimate, as additional aliquots of DC will be needed
 for testing prior to release of the vaccine for administration. The cells will be tested for
 viability, sterility (bacterial aerobic and anaerobic cultures and Gram stain), endotoxin
 level and mycoplasma as routinely performed by the IMCPL for cellular products used for human
 therapy at the UPCI. For each vaccine, the DC will be harvested, counted, adjusted into
 aliquots of 2.5 x 106 cells/mL in PBS with each aliquot and dispensed into a 1mL tuberculin
 syringe for future intradermal administration.

 3.2.4 Oligonucleotide-treated (diabetes-suppressive) iDC Vaccine Preparation The same
 procedures as for the generation of control DC will be used to generate the iDC preparation
 with the inclusion of the antisense oligonucleotides at the same time as when GM-CSF and IL-4
 will be added to the DC progenitors. The progenitors will be treated ex vivo with each of the
 antisense oligonucleotides to a final concentration of 3.3 μM according to SOP # CPL-0170
 (Appended). On day 6 of culture, DC will be harvested and checked for viability and purity.
 After extensive washing to remove the remaining oligonucleotides from the culture medium, the
 iDC will be processed identically as above, dispensed into administration aliquots, and each
 aliquot will then be cryopreserved for future administrations.

 3.2.5 Control and iDC Vaccine safety The vaccine will be prepared and administered to
 established type 1 diabetic patients meeting the criteria outlined in Section 4.0-4.1. The
 dendritic cell vaccines will be produced in the IMCPL, a cGMP facility which is in compliance
 with FDA recommendations for cellular products generated for patient therapy. At all times,
 the samples and the cell products will be identified by the same randomly-generated code
 assigned at the time of leukapheresis.

 3.2.6. Safety evaluation: Initially, 15 patients (18-35 years of age) with established
 diabetes will be randomized to receive either the control dendritic cell vaccine (7/15
 individuals) or the iDC (8/15 individuals) at the CHP GCRC. Randomization will be carried out
 using a macro (SUGI26) developed for the SAS statistics package. SUGI26 is a modification of
 a baseline adaptive randomization procedure and permits for unequal allocation ratios among
 multiple treatment groups for categorical covariates.

 Following the establishment of the randomized patients into each treatment group, a patient
 may receive either of the two dendritic cell embodiments (control DC or iDC). These patients
 will receive 1 x 107 dendritic cells intradermally (four injections of 2.5 x 106 at four
 distinct sites anatomically-proximal to the pancreas) at the times illustrated in SECTION
 3.2.1.1 and in the schematic cartoon (refer to the Appendix, Section 6.8) for a total of four
 vaccine administrations spaced every two weeks for a period of eight weeks. Per
 administration, we designate 4 unique intradermal injection sites inside the anterior
 abdominal wall perpendicularly-above the physical location of the stomach. These four sites
 will be within a quadrant of 3-4 square-inches. The cells will be delivered by a tuberculin
 syringe attached to a 27g-1/2 needle underneath a raised "bleb" of skin at each of the 4
 individual injection sites. If there is no grade 3 or 4 toxicity observed following the first
 vaccine cycle in the DC recipients, successive vaccinations will be delivered as planned.
 Dose-limiting toxicities will be defined as equal to or greater than grade 2 with the
 following exceptions: chills, malaise and fever are not considered dose limiting. All grade 4
 toxicities will be dose limiting, with the exception of lymphocytopenia. To protect patients
 against serious adverse effects of therapy the stopping rule will be observed, as described
 in Section 3.3.2 below. Following the leukapheresis procedure, the patient will be monitored
 for normoglycemia and if necessary, insulin will be administered as required to maintain
 normoglycemic levels. Glycemia will also be monitored at the end of each DC administration
 procedure and insulin can be administered to restore normoglycemia if necessary.

 3.2.7 Dose-limiting toxicities:

 The following are considered to be dose limiting toxicities (DLT's). If these toxicities are
 felt to be related to autologous dendritic cell vaccine administration, individual subjects
 will be immediately taken off study and no further injections will be given:

 -  ≥ Grade 2 or more bronchospasm,

 -  ≥ Grade 2 or more allergic reaction or generalized urticaria,

 -  ≥ Grade 2 or more autoimmune disease other than diabetes,

 -  ≥ Grade 2 injection site reaction due to vaccine or DTH testing,

 -  ≥ Grade 3 or 4 hematologic and non-hematologic toxicities

 -  Grade 2 toxicities that do not resolve within 5 days following the dendritic cell
 vaccine administration

 -  Dosing delays > 4 weeks

 Therapy may be discontinued for the following reasons:

 -  Intercurrent illness that prevents further administration of autologous dendritic cell
 vaccine, including evidence of an infectious (bacterial/viral) disease

 -  Unacceptable adverse event(s),

 -  Patient decides to withdraw from the study, or

 -  General or specific changes in the patient's condition render the patient unacceptable
 for further treatment in the judgment of the investigator, nurse or attending
 physician(s).

 3.3 DATA COLLECTION AND STATISTICAL CONSIDERATIONS 3.3.1 General Considerations This will be
 a safety trial to evaluate the safety and feasibility of administering a diabetes-suppressive
 dendritic cell vaccine based on autologous dendritic cells treated ex vivo with
 phosphorothioate-modified antisense oligonucleotides targeting the primary transcripts of the
 CD40, CD80 and CD86 co-stimulatory molecules. The vaccine will be given intradermally and
 each vaccine will contain 1 x 107 autologous dendritic cells. A total of 4 vaccines will be
 delivered to each eligible subject. A leukaphereses will be performed (14 days prior to the
 first vaccine) to ensure sufficient numbers of DCs.

 The trial is designed to assure that the toxicity rate is acceptably low to warrant further
 study of the cell vaccine in efficacy trials.

 4.5.1 CLINICAL TRIAL MONITORING

 4.5.1.1 Reporting of Serious Adverse Events (SAE)

 ADVERSE EVENTS: In general, all adverse events will be recorded and communicated to the
 safety officer, the PI and to the DSMB within 24 hours of occurrence. The safety officer will
 determine if the DSMB should meet under emergency session. Any non-serious adverse event
 reports will be submitted to the IRB within 24 hours of the DSMB meeting. The annual and
 final reports of the DSMB will also be submitted to the IRB and to the FDA within 48 hours of
 the meeting.

 Reporting of Serious Adverse Events (SAE)

 All (SAE) will be recorded on the standard toxicity sheets and reported to the FDA, the IRB,
 the DSMB safety officer, the Principal Investigator (Dr. Trucco) and/or Co-Investigators
 (Drs. Finegold, Whiteside or Giannoukakis) within 24 hours of occurrence. All
 treatment-emergent serious adverse events and pre-existing medical conditions which
 unexpectedly worsen during therapy will be recorded and reported as described immediately
 above (DSMB, DSMB safety officer, PI, FDA, IRB). All serious adverse events or deaths which
 occur beyond thirty (30) days and which are reasonably likely to be related to control or iDC
 administration will be reported within 24 hours of the event to the safety officer, the FDA
 and the full DSMB. Any serious or adverse events or deaths which occur within thirty (30)
 days of the last control DC or iDC administration will be reported identically. The safety
 officer will convene a full meeting of the DSMB within 24 hours of the occurrence of any SAE.
 The DSMB report will then be submitted to the IRB and the FDA no later than 48 hours
 following the occurrence of the SAE.

 Serious is defined as described in 21 CFR 312.32 (a)
Inclusion Criteria:

 Patients with established type 1 diabetes mellitus who meet all inclusion criteria are
 eligible for enrollment in the study.

 -  All patients enrolled must be on insulin replacement therapy.

 -  Written informed consent conforming to the institutional guidelines obtained from the
 patient.

 -  Documented evidence of insulin-requiring type 1 diabetes of >5 years duration.

 -  Adequate immune competence, as indicated by positive reaction to one or more of the
 common DTH skin tests that are part of the Multitest CMITM test system
 (Pasteur-Merieux Connaught) and as indicated by the manufacturer. Also, proof of
 vaccination for tetanus (no more than 10 years must have elapsed between tetanus
 vaccination and the onset of this study).

 -  Age 18-35.

 -  Adequate hematologic function:

 -  Absolute neutrophil count > 1,000/mm3

 -  Absolute lymphocyte count > 1,000/mm3

 -  Hemoglobin > 9 gm/dl

 -  Platelets > 100,000/mm3

 -  Liver function tests:

 -  Bilirubin (total) < 1.7 mg/dl

 -  Alkaline phosphatase < 78 u/L (2 x ULN)

 -  SGOT < 54 u/L (2 x ULN)

 -  Lactic dehydrogenase < 180 u/L (2 x ULN)

 -  Kidney profile:

 -  Serum electrolytes

 -  Sodium 135-145 mEq/L

 -  Potassium 3.5-5.0 mEq/L

 -  Bicarbonate 21-28 mEq/L

 -  Chloride 100-108 mmol/L

 -  Serum creatinine <4.5 mg/dL (3 x ULN)

 -  BUN 8-25 mg/dL

 -  No prior history of radiation therapy, immunotherapy, or chemotherapy

 -  Evidence of prior immunization to tetanus

 -  Absence of HIV, HSV, HBV, HCV viral infections

 -  At least four weeks since any prior radiation , immunotherapy or
 chemotherapy

 Exclusion Criteria:

 -  One or more of the Eligibility Criteria are not met.

 -  A significant history or current evidence of cardiac disease including, but not
 limited to, congestive heart failure, coronary artery disease, angina pectoris,
 uncontrolled hypertension, serious arrhythmias; or myocardial infarction within the
 previous six months.

 -  Evidence of active infection requiring antibiotic therapy.

 -  History of other concurrent diseases.

 -  Pregnant or lactating women.

 -  Patients requiring systemic corticosteroids

 -  Any other immune disorder including but not limited to other autoimmune diseases like
 rheumatoid arthritis, systemic lupus erythematous, multiple sclerosis, or ankylosing
 spondylitis

 -  Pregnancy

 -  History of radiation therapy, immunotherapy, or chemotherapy

 -  Breastfeeding

 -  The following therapies cannot be administered while patients are undergoing treatment
 on this protocol:

 -  radiation therapy

 -  chemotherapy

 -  corticosteroids (except when administered in life-threatening circumstances)
 other particle or cell vaccine therapies

 At the time of screening, the patients will be tested for evidence of the following viral
 infections: HIV, HBV, HCV, herpes, CMV and EBV. In addition, female volunteers will be
 asked to provide documentation from their physician stating that they have not tested
 positive for the HPV viral infection. Only patients testing negative for ALL these viral
 infections will be further considered. Should any volunteer be excluded on grounds of
 positivity for any of these infectious agents, they will be immediately notified and
 strongly advised to consult their physician. The data and the records will be maintained
 under lock and in the study participation file of the volunteer until the volunteer
 confirms in writing that they have notified their physician. At that time, these specific
 data may be submitted to the patient's physician ONLY DIRECTLY BY THE VOLUNTEER upon
 written request to the study principal investigator or immediately destroyed.