###### **Briefing Document**

###### **Biological Response Modifiers Advisory Committee** 

###### **Meeting #36** 

###### **Allogeneic Pancreatic Islets for Type 1 Diabetes** 

###### **October 9-10, 2003** 

######## Contents

[INTRODUCTION 2](#introduction)

[MEETING GOALS 2](#meeting-goals)

[BACKGROUND 2](#background)

[BIOLOGICS LICENSE APPLICATIONS FOR ALLOGENEIC ISLET PRODUCTS
3](#biologics-license-applications-for-allogeneic-islet-products)

[Manufacturing Quality and Control
3](#manufacturing-quality-and-control)

[Ensuring Quality of Source Pancreata
4](#ensuring-quality-of-source-pancreata)

[Manufacturing of Islets 5](#manufacturing-of-islets)

[Assessing the Final Product 6](#assessing-the-final-product)

[Islet Comparability 6](#islet-comparability)

[Preclinical Considerations 7](#preclinical-considerations)

[Animal Models of Testing 7](#__RefHeading___Toc51568926)

[Preclinical Evaluation of Immunosuppressive Regimens
8](#preclinical-evaluation-of-immunosuppressive-regimens)

[Preclinical Assessment of Innovative Routes of Administration
8](#preclinical-assessment-of-innovative-routes-of-administration)

[Reproductive, Developmental and Carcinogenic Potential of the Cell
Product
8](#reproductive-developmental-and-carcinogenic-potential-of-the-cell-product)

[Potential Future Issues 8](#__RefHeading___Toc51568930)

[Clinical Data 9](#clinical-data)

[Islet Study Background 9](#islet-study-background)

[Factors Influencing Islet Graft Function
11](#factors-influencing-islet-graft-function)

[Outcome Measures in the Clinical Studies
11](#outcome-measures-in-the-clinical-studies)

[ATTACHMENTS: 15](#attachments)

[UNSUPPLIED REFERENCES: 16](#unsupplied-references)

# INTRODUCTION

Allogeneic pancreatic islet transplantation is a field of intense
clinical research as a potential therapy for patients with type 1
diabetes mellitus (DM). This research typically involves the
administration of allogeneic islets, derived from cadaveric organs, into
the recipient's portal vein. Published preliminary data suggest, in the
short term, the safety risks of administering the final product may be
reasonable considering the appearance of some clinical benefit. However,
a number of manufacturing and clinical issues remain to be addressed in
order to obtain product and clinical data sufficient to successfully
support a biologics license application (BLA).

# MEETING GOALS

This meeting of the Biological Response Modifiers Advisory Committee
(BRMAC) was organized to achieve the following goals:

-   For FDA to discuss expectations for manufacturing data and clinical
    evidence to be provided in a BLA for marketing approval of
    allogeneic islets as a treatment for type 1 diabetes mellitus.

-   To obtain perspectives and advice from members of the BRMAC
    regarding the type and quality of manufacturing, preclinical and
    clinical data to be provided within a BLA for the marketing approval
    of allogeneic pancreatic islets as biological products.

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-   To provide a public forum to obtain input from stakeholders
    interested in this therapy.

# BACKGROUND

During the 10-year period from 1990 to 2000, the Food and Drug
Administration (FDA) received a total of 10 investigational new drug
applications (INDs) to investigate the use of allogeneic islets as a
therapy for type 1 diabetes mellitus. A BRMAC meeting was held in March
2000 in order to identify, clarify, and resolve certain issues relating
to exploratory clinical studies for allogeneic islets. The issues
discussed at that meeting primarily related to information on organ
procurement practices, islet processing techniques, islet
characterization procedures, pre-clinical studies to support clinical
studies, and early clinical development plans that should be provided in
an IND.

In September 2000, FDA mailed a "Dear Colleague" letter to over 250
transplant centers in the United States reminding interested parties
that allogeneic islets are regulated as biological products and prior to
the initiation of clinical studies sponsors must submit an IND to FDA
(Attachment 1).

The input received from the members of the BRMAC, its guest advisors,
and the public proved to be timely in light of a July 2000 report in the
*New England Journal of Medicine* (NEJM) describing the treatment of
seven diabetic patients with allogeneic islets isolated from cadavers
(Attachment 2). In this study, subjects received multiple
administrations of allogeneic islets and a steroid-free
immunosuppressive regimen. The median follow-up for these seven
patients, at the time of the NEJM publication, was 11.9 months (range
4.4 to 14.9 months). The apparent success was remarkable given prior
reports of little evidence of clinical benefit from allogeneic islet
administration. For example, the International Islet Transplantation
Registry reported that, for the ten years between 1989-1999, of the 267
patients who had received allogeneic islets, only 12% were insulin
independent for ≥ 7 days (Attachment 3).

The findings reported in the July 2000 NEJM ("the Edmonton Protocol")
resulted in a large increase in the number of centers performing
clinical studies using allogeneic islets. In the nearly 4 years
(2000-03) since the BRMAC meeting, FDA has received an additional 28
INDs for allogeneic islets. In these INDs, sponsors generally seek to
replicate and/or extend the results of the Edmonton Protocol.

FDA has worked with the islet transplant community and other federal
agencies to facilitate clinical studies of allogeneic islets. These
federal agencies include the Health Resources and Services
Administration (HRSA) and the National Institutes of Health (NIH). This
collaboration has helped to identify and resolve obstacles to product
development in this research area. Other outreach efforts have included
participation in at least 10 workshops or conferences related to
allogeneic islets as well as publication of an article describing FDA's
IND review expectations for allogeneic islets (Attachment 4). We have
made substantial outreach efforts in order to help guide interested
stakeholders toward the shared goal of collecting the necessary data to
demonstrate both the safety and efficacy of these products.

# BIOLOGICS LICENSE APPLICATIONS FOR ALLOGENEIC ISLET PRODUCTS

Upon submission of adequate data to demonstrate safety and clinical
efficacy, as well as other assurances that a product "meets standards
designed to assure that the biological product continues to be safe,
pure, and potent, "[^1] a biologics license (BLA) may be issued. Thus,
clinical development programs using allogeneic islets for treatment of
type 1 DM must design their studies to obtain data demonstrating:

-   The manufacturing process and facility are sufficient to assure the
    safety, purity, and potency of the product.

-   Substantial clinical evidence of the product's safety and
    effectiveness.

# 

## Manufacturing Quality and Control

Cellular biological products are complex mixtures of multiple
components, prepared from living sources. Because these products are
difficult to characterize, the manufacturing process helps to define the
composition of the final product. Manufacturing changes can impact
product safety, identity, purity, potency, consistency and stability in
unforeseen ways. The manufacturing process must be sufficient to prevent
the transmission or introduction of infectious agents while preserving
product identity, purity and potency. The integrated system of
documented manufacturing control, in-process testing and final product
release testing must be assessed to ensure the final product is safe,
pure, and potent for patient administration.

Although most allogeneic islet manufacturing procedures are similar,
there is no standard procedure. Some of the variation in isolating
islets is a result of the variability in the organ; different organs may
require modified processing parameters to optimize the yield and quality
of islets. Some of the variation is a result of different preferences
and facilities; procedures and equipment vary from center to center.
During investigational studies (IND) a certain amount of processing
innovation is expected and encouraged. However, for a BLA it is expected
that a well-defined manufacturing protocol be established and followed
to ensure manufacturing consistency and product quality. It is expected
that the manufacturing process and the manufacturing facility will be
compliant with current good manufacturing practices (cGMP), meeting lot
release testing requirements such as safety, identity, purity, potency
(as described in 21 CFR 610--General Biological Products Standards).
Further, since islets are not amenable to terminal sterilization, the
facilities should be designed for aseptic processing, meeting at least
the minimum standards as described in 21 CFR 211.42-211.58. The flow of
the manufacturing process should be designed to minimize chances for
cross contamination and product mix-up. In-process tests should be
performed at critical points in the manufacturing process with
appropriate acceptance limits established during the validation process.

So in addition to evaluating the safety and efficacy data submitted in a
BLA, FDA will evaluate the manufacturing data to determine if there is a
well-established islet preparation process and a track record of product
manufacturing consistency; confirm that the islets are prepared in a
manufacturing facility meeting cGMP requirements; and verify compliance
with applicable lot release testing requirements.

### Ensuring Quality of Source Pancreata

Unlike most drugs or other biologics, the source material for allogeneic
islets, being cadaveric organs, cannot be controlled in a traditional
way. Thus, each donor pancreas has a unique set of characteristics
(ischemia time, organ size, age, extent of fibrosis, etc). FDA
recognizes that organ procurement and allocation is clearly outside its
jurisdiction, nevertheless it is clear that consistency of islet
manufacturing is highly dependent on the quality of the organ delivered
to the manufacturing facility. In light of this constraint and in order
to be compliant with cGMPs and biologics licensing requirements, it is
imperative that predefined acceptance criteria for assessing organ
quality be established, standardized and validated to the extent
possible to ensure that unsuitable organs are excluded.

> [CMC Question:]{.underline}

1.  A key component for ensuring control of a validated islet
    manufacturing process is the use of pre-defined acceptance criteria
    to ensure that suitable donor organs with maximal potential for
    yielding adequate numbers of islets are utilized for manufacturing
    allogeneic islets, while unsuitable organs are excluded. Acceptance
    criteria may include donor suitability determination, organ
    characteristics, harvesting conditions, and transport conditions.
    Most, if not all, of this information is currently collected by
    sponsors of islet INDs. Please discuss the use of this manufacturing
    experience data as a basis for developing pre-defined acceptance
    criteria for source organs.

### Manufacturing of Islets 

One consequence of having limited control over the source donor
pancreata used to prepare islets is the need to allow some flexibility
in the exact procedures used for a specific pancreas in order to
optimize the yield and quality of islets. FDA has reviewed various
manufacturing procedures that attempt to improve islet number and
quality, such as:

-   Modifying the organ transport media to include an oxygen-enriched
    layer of perfluorocarbon

-   Varying quantity or units of dissociation enzymes used to reflect
    specific organ characteristics such as organ size, age, extent of
    fibrosis, etc.

-   Including certain additives (DNAse, Pefabloc, etc.) during islet
    processing and isolation

-   Using short-term culture before islet administration.

FDA agrees flexibility is permissible if conducted using predefined
criteria or algorithms, based upon data from manufacturing experience,
that establish processing controls within the context of a validated
manufacturing protocol.

> [CMC Question:]{.underline}

2.  Based on a given donor organ's characteristics, investigators
    frequently "customize" an islet isolation procedure by using
    different reagents, reagent concentrations and processing methods
    such that no two islet isolation procedures are exactly alike.
    However, cGMPs and BLA requirements necessitate the use of a
    well-defined, well-controlled manufacturing process. Is it
    reasonable to expect that criteria or algorithms can be developed,
    based on data collected during IND studies, to predetermine under
    what conditions the use of a specific reagent, reagent
    concentration, or processing method is appropriate?

### Assessing the Final Product 

FDA regulations for biological products mandate that specific product
safety and quality testing be done on the final product, with results
available prior to patient administration. As mentioned above, the
product testing standards are prescribed in 21 CFR 610 and include
manufacturing safety (sterility, endotoxin, mycoplasma \[if cultured\]),
identity, purity, and potency. When feasible, additional safety testing
and other assessments are also performed throughout manufacturing
(in-process testing) in order to evaluate the manufacturing process
itself and to ensure the quality and consistency of the product lots.

Of the prescribed assays, potency presents a unique challenge. In
general, assays for product potency are intended to show the ability of
the product to effect a given result. Currently, assays for islet
product potency, such as glucose stimulated insulin release or injection
of islets under the kidney capsule of diabetic mice, are performed
retrospectively. However, prior to the approval of a BLA some reasonable
measure of product potency must be developed such that results are
available prior to patient administration.

> [CMC Question:]{.underline}

3.  Please discuss any assay or assays that are currently, or could be,
    performed on the final islet product before patient administration,
    which may be predictive of the ability of the islets to perform as
    expected after patient administration.

### Islet Comparability

As mentioned previously, the manufacturing process helps to define the
product. Manufacturing changes can impact product safety, identity,
purity, potency, consistency, and stability in unforeseen ways.
Demonstrating that critical product characteristics have not changed
even when the process has changed is referred to as comparability.
Comparability testing may include analytical assays, bioassays,
preclinical studies, and clinical studies. Until comparability can be
determined, products manufactured using different processes are
considered to be different products.

At present, it is unclear how differences in methods to prepare
allogeneic islets by various groups impact the characteristics of the
final allogeneic islet product. On an individual basis, many of these
manufacturing changes appear to be minor, such as including additives
(protease inhibitors, DNAse, etc) in the dissociation medium;
dissociating allogeneic islet using manual vs. semi-automated equipment;
using a refrigerated or non-refrigerated COBE cell processor unit for
allogeneic islet enrichment; or using fresh allogeneic islets vs. using
allogeneic islets after short term culture. However, the overall effect
of any or all of these changes is not known.

FDA foresees the potential for more than one allogeneic islet sponsor to
"pool" their clinical data to support a BLA. While this can be an
acceptable approach it is highly dependent on demonstrating
comparability. Without sufficient data to demonstrate that allogeneic
islets prepared using different methods are comparable, it may not be
possible for FDA to accept data in a license application that is
"pooled" from allogeneic islet preparations in which the manufacturing
process used was different.

> [CMC Question:]{.underline}

4.  What should be key criteria for demonstrating allogeneic islet
    product comparability? Please discuss appropriate analytical assays,
    bioassays, preclinical studies, and clinical studies that may be
    required.

## Preclinical Considerations

Preclinical (pharmacology/toxicology) considerations for licensure of
allogeneic islets are not fundamentally different from those applied to
other biological therapeutic products. The types of preclinical *in
vitro* and *in vivo* studies employing various animal models that should
be performed as product development proceeds are reflective of FDA's
science-based paradigm of biologics regulation.

When properly designed, performed, and evaluated, preclinical studies
can generate data that contribute to the design of scientifically sound
clinical development programs. The goals of preclinical safety studies
are several-fold: 1) to provide supportive data for an initial safe
starting dose and subsequent dose escalation scheme; 2) to aid in
determining a risk/benefit assessment for the proposed clinical studies;
3) to identify potential endpoints for detection of toxicity and the
clinical monitoring for those toxicities; and 4) to guide in designing
appropriate patient eligibility criteria.

### Animal Models of Testing

In order to generate scientifically valid data for use in safety, as
well as efficacy assessments, it is essential that the cellular product
(clinical product or species-specific analogous cells) being tested is
biologically active in the animal species used in the preclinical
studies. Although traditionally, many *in vivo* safety studies are
preferentially performed in healthy animals, clinical investigations of
allogeneic islets have been supported by studies in a wide range of
animal species, e.g., mouse, rat, dog, pig, monkey and baboon. Many
animal models of diabetes have also been studied, including those with
genetic mutations (i.e., inbred NOD mice), medication/toxin-induced
(i.e., streptozocin treatment, corticosteroid treatment) and surgical
(i.e., pancreatectomy) disease. Each of these models has inherent
strengths and weaknesses, thus no single model will be completely
predictive of the clinical scenario. However, the data generated from
these models provide a strong scientific rationale for the potential
efficacy of allogeneic islets, thus serving to justify clinical studies
in humans with type 1 DM \[1\]. Importantly, the collection of
preclinical safety and efficacy endpoint data can be achieved by
applying principles of toxicological study design to these already
established animal models of diabetes. These studies should be done
according to good laboratory practices (GLP) or be as GLP-compliant
(with an emphasis on data integrity and quality) as possible, given the
scientific and practical restraints that may be imposed due to the
nature of the diabetes model.

### Preclinical Evaluation of Immunosuppressive Regimens

A primary focus of the March 2000 BRMAC meeting was the performance of
additional preclinical studies for the various immunosuppressive agents
proposed for clinical use. From the presentations and ensuing
discussions, it was concluded that insufficient preclinical data existed
to justify the use of any particular immunosuppressive regimen in
diabetic patients receiving allogeneic islets. General agreement was
reached on the point that, "...justification of any given
immunosuppressive or tolerance-inducing strategy should be based on
multiple preclinical models including, but not limited to, specific
islet transplantation models and at least some nonhuman primate work"
\[1\]. In concordance with the committee recommendations, FDA advises
each IND sponsor to submit preclinical toxicology data in appropriate
animal model(s), that are intended to support the safety of the
short-term and long-term use of each individual immunosuppressive agent
used, as well as any combination of agents, prior to initiation of a
clinical trial proposing use of the respective regimen. In addition, FDA
advises each IND sponsor anticipating use of a particular
immunosuppressive regimen in late stage clinical trials (Phase 3), that
the potential for reproductive/ developmental and carcinogenic toxicity
of the respective immunosuppressive regimen needs to be addressed in
preclinical studies.

###  Preclinical Assessment of Innovative Routes of Administration

Several IND sponsors currently studying allogeneic islets have proposed
that alteration of the route of administration of the cellular product
from the widely utilized route of percutaneous transhepatic portal vein
delivery to another route (such as a transjugular approach or
intraoperative administration) may promote improved islet function, as
well as a better overall safety profile. However, as concluded in the
March 2000 BRMAC meeting, sponsors proposing innovative alternate
delivery methods for allogeneic islets need to demonstrate an adequate
safety profile in animals prior to proceeding to clinical trials \[1\].

### Reproductive, Developmental and Carcinogenic Potential of the Cell Product

Based on the current knowledge regarding cellular products, the level of
concern regarding reproductive, developmental toxicity, and carcinogenic
potential raised by the administration of allogeneic islet products that
are collected, isolated, processed, and characterized according to
current FDA-specified requirements does not appear to require
preclinical studies to assess these potential toxicities. This
conclusion is based on the existing extensive literature in the public
domain for islet transplantation in many different animal models of
varying ages. Knowledge of the biology of these cells does not provide
scientific evidence for a plausible mechanism for these products to pose
reproductive/developmental or carcinogenic risks to humans at the
present time.

### Potential Future Issues 

It is important, however, to emphasize that future alterations to the
production of the allogeneic islets that result in deviation from an
accepted/licensed product could significantly change the risks
associated with the cellular product. For example, alteration of the
islets by gene transfer, or efforts at increasing the proliferative
potential of the cells by any post-harvesting manipulation would alter
FDA's level of concern for the potential risks to humans significantly
enough to require additional preclinical pharmacology/ toxicology
testing prior to the initiation of clinical trials using the new
cellular product.

## Clinical Data 

The clinical review processes for allogeneic islet IND are the same as
those for investigational biologic or drug therapies. Consequently, the
clinical development programs for allogeneic islets are expected to
mirror the paradigm for development of biologic or drug therapies. This
regulatory paradigm has been previously summarized (see Weber, et. al).

### Islet Study Background

Human cadaveric islet transplantation has been primarily used in
patients with type 1 DM, a disease which results from a selective
autoimmune destruction of insulin-producing beta (β) cells in the
pancreas. Patients with type 1 DM require replacement therapy with
exogenous insulin in order to prevent acute and chronic complications.
The most common acute complications include various hyperglycemia and
hypoglycemic events while the most common chronic complications include
nephropathy, neuropathy, retinopathy, and vasculopathy.

In a subgroup of patients with type 1 DM, maintenance of near-to-normal
blood glucose levels by intensive exogenous administration of insulin
may result in the increased occurrence of episodes of life-threatening
hypoglycemia. Additionally, some patients receiving intensive exogenous
insulin therapy still develop the chronic complications of diabetes
mellitus.

Transplantation of insulin-producing tissues has proven, when
successful, to be capable of maintaining excellent metabolic control in
the absence of life-threatening hypoglycemia. However, whole-organ
pancreas transplantation, while allowing for long-term glucose metabolic
control, insulin independence in the absence of hypoglycemic episodes,
and reversal of diabetic complications, is often associated with
significant morbidity and mortality. Islet tissue administration, on the
other hand, presents the advantage of a relatively rapid and somewhat
less invasive procedure of implantation; islets are generally injected
into the liver by percutaneous transhepatic catheterization of the
portal vein, a procedure associated with relatively few perioperative
risks and morbidites. The procedure has, at times, been performed in
type 1 diabetic patients with end-stage renal failure, as a simultaneous
islet-kidney (SIK) or as an islet-after kidney (IAK) transplant. Islet
transplantation is also sometimes performed after whole pancreatic graft
failure, since whole-organ pancreas re-transplants are associated with
an elevated rate of graft failure. Islet autotransplantation has also
been performed in patients undergoing pancreatectomy for "benign"
disease, including insulin-requiring diabetes mellitus due to cystic
fibrosis and hemochromatosis. Islet transplantation for type 2 DM has
been attempted in very few cases.

Historically, islet transplantation has been considered an experimental
procedure characterized by marginal success, and has been regarded as a
therapeutic option primarily when combined other organ transplant e.g.,
SIK or IAK. This use was thought to justify the use of chronic
immunosuppression.

The immunosuppressive protocols utilized in past years included
monoclonal or polyclonal T-cell antibodies such as ALG, ATG, OKT3, or
anti-CD4 -based induction immunosuppression, and triple-drug maintenance
therapy based on glucocorticoids,

cyclosporine or tacrolimus, and azathioprine or mycophenolate mofetil.
Unfortunately,

most of the immunosuppressive drugs such as glucocorticoids and
calcineurin inhibitors are known to exert toxic effects on the islet β
cells or to induce insulin resistance.

The anti-rejection protocols commonly used for solid organ
transplantation may have detrimental consequences on islet graft fate.
The International Islet Transplant Registry reported that a total of 405
adult islet allogeneic transplants were performed from 1983 through 1998
(see White, et. al.). Analysis of 200 C-peptide negative type 1 diabetic
patients transplanted from 1990 through 1997 showed a cumulative 1-year
patient survival of 96%, and graft survival assessed by measurable basal
C-peptide levels above 0.5 ng/ml of 35%, with insulin independence at 1
year in only 8% of the recipients. The rate of insulin independence was
higher in patients receiving multiple-donor islet preparations (19%),
when compared to those receiving single-donor preparations (11%), most
likely reflecting the need for a conspicuous islet graft mass higher
than 6,000 islet equivalents per kilogram (IE/kg) of the recipient's
body weight.

The results reported by Shapiro et al. in the NEJM in July 2000 on the
successful series of cadaveric human islet cell transplants in type 1
diabetic patients treated with a glucocorticoid-free immunosuppression
protocol, including a short course of anti-IL2 receptor antibody, and
maintenance therapy based on rapamycin and low dose of tacrolimus, has
been thought to represent a major breakthrough in the field of islet
transplantation. In this study, all seven patients received islets
isolated from more than one donor. Two to three grafts were generally
needed in order to obtain an islet mass sufficient to achieve insulin
independence (11,547 ±1,604, mean ± SD, IE/kg of recipient's body
weight). Although infusion of islets from a single donor did not result
in insulin independence in any of the patients studied, all the patients
had improved glycemic control, reduced insulin requirements, and absence
of hypoglycemic episodes soon after receiving the first graft. Two-year
follow-up data has shown continued glycemic control in a number of these
subjects (Attachment 5). A possible explanation of the need for multiple
donors in this study is the destruction of a substantial mass of islets
immediately after transplantation. Early loss of implanted islets has
been proposed to be due to their susceptibility to microenvironment
alteration with consequent functional impairment and loss to apoptosis.

Despite the apparent successful results of the Edmonton protocol, the
need to administer islets isolated from more than one organ per
recipient to achieve insulin independence represents a major hurdle to
be solved due to the shortage of organs. The need for chronic
immunosuppression also limits the application of allogeneic islet
therapy to the small cohort of patients for whom the risks of chronic
immunosuppression are outweighed by the potential benefits of islet
transplantation.

Historically, immunosuppressive regimens have frequently been modified
according to the availability of new molecules. Several strategies are
at present under evaluation, which in general aim to improve graft
acceptance and/or induce tolerance. In more than 20 of the IND currently
in effect, the immunosuppressive regimen being used consists of
sirolimus, tacrolimus and daclizumab. The use of OKT3 γ1 (Ala-Ala),
etanercept, and a few other immunomodulatory strategies are being
pursued under other INDs.

### Factors Influencing Islet Graft Function

Several factors are known to contribute to allogeneic islet engraftment
and function. The number of islets implanted appears to be a critical
factor in obtaining complete function. This number depends upon the
condition of the organ donor, the procurement and preservation of the
pancreas, and the procedure and reagents used during isolation and
purification. All these variables influence yield and quality, purity
and viability of the graft, making it difficult to completely
standardize the procedures.

Early after transplantation, islets are denervated, not yet
vascularized, implanted into a new microenvironment in the absence of
extracellular matrix, and are exposed to nonspecific inflammatory events
occurring at the site of implant. These are all conditions which may
result in functional impairment. After engraftment, islets are exposed
not only to allorecognition and rejection, but also to recurrence of the
autoimmunity that causes type 1 DM. Furthermore, immunosuppressive drugs
might also exert some toxicity on islet β cells, impairing their
function.

### Outcome Measures in the Clinical Studies

#### Glycemic control

The extent of mean fluctuations in serum glucose concentrations,
measured as mean amplitude of glycemic variation in the 24hr period is a
useful tool in the assessment of metabolic instability. The results
obtained with the 'Edmonton protocol' showed relatively dramatic
improvement following sequential islet cell transplants, with decreased
overall mean serum glucose concentrations, mean amplitude of glycemic
variations, and lability of glycemic control in a 24hr period.

#### Glycosylated hemoglobin

Hemoglobin A1c (HbA1c) is formed from the irreversible nonenzymatic
glycation of the hemoglobin β chain, and is directly proportional to the
ambient glucose concentration. The level of HbA1c highly correlates with
blood sugar levels and lasts longer after the maximum blood sugar level
is observed, making it a more reliable long-term marker of blood sugar
level control than immediate glycemia measurement. The value of HbA1c as
a retrospective and cumulative marker of glycemic balance in diabetic
patients has been confirmed by epidemiological studies. In the case of
intensive insulin therapy, the desired end result is a glycemic level
slightly higher than normal, to prevent hypoglycemic episodes. In the
long-term, however, this might result in delay, and not prevention, of
the occurrence of chronic secondary complications of diabetes.
Successful

allogeneic islet administration has been reported to improve the glucose
metabolic control with return of HbA1c levels within the normal range.
Also, in long-term functioning whole pancreas transplants, the
achievement of improved glucose metabolic control has been associated
with normalization of HbA1c during the first 2 years after
transplantation, and normal or near-normal levels over a 6-year period,
in conjunction with small doses of exogenous insulin and in the absence
of hypoglycemic episodes. However, because of the cumulative nature of
this marker, it may not represent a good means of monitoring short-term
alteration in islet function and, therefore, it is not a valuable early
marker of graft rejection/autoimmune recurrence.

#### Insulin requirements

Insulin requirement inversely correlates with glucose metabolic control.
One of the goals of successful allogeneic islet administration is the
achievement of a status of normoglycemia and insulin independence, in
the absence of insulin resistance. In the data of the Edmonton group
presented in the NEJM, all seven subjects maintained insulin
independence at 1-year follow-up post-transplant. While inversely
correlated with the mass of functionally competent islets, insulin
requirement does not represent a viable early rejection recurrence
marker.

#### C-peptide levels

Allogeneic islet administration is considered partially successful when
C-peptide levels (≥ 0.3 ng/ml) are detectable in patients who were
C-peptide negative, pre-transplant. C-peptide and insulin are produced
in equimolar amounts from the proinsulin molecule by pancreatic β cells,
and measurement of plasma C-peptide allows monitoring of β cell function
when a patient is treated with exogenous insulin.

Persistent C-peptide secretion several years after transplant in type 1
diabetic recipients has been correlated with long-term normalization or
near-normalization of blood glucose control, and significant improvement
of HbA1c levels in the absence of severe episodes of hypoglycemia.

####  Mixed meal tolerance test

Mixed meal tolerance tests are utilized to assess islet β cell function
in response to stimuli; in these studies, basal and peak insulin
C-peptide levels are expressed as an index. The mixed meal tolerance
test (MMTT) consists of the administration of 6 kcal/kg of Sustacal 140
gr/l carbohydrates, 24 g/l fat, and 61 g/l protein; 1 kcal/ml and
calculation of the stimulation index between basal and peak C-peptide
levels.

When mixed meal tolerance tests are performed in patients with only one
kidney SIK or IAK, it is important to consider that C-peptide half-life
is prolonged by about 40%, rendering the interpretation of C-peptide as
measurement of insulin production difficult.

In patients in whom insulin independence has been achieved after islet
cell transplant, MMTT might reveal impairment of function. Although a
viable means of monitoring islet function, MMTT is not performed with
high frequency, limiting the possibility of early detection of graft
impairment.

#### Intravenous glucose tolerance test

It has been reported that first-phase insulin release in response to
intravenous glucose tolerance test (IVGTT) provides an accurate
reflection of islet β cell mass, and its use is valuable in the
long-term monitoring of the islet graft mass after transplant. It
consists of the infusion of glucose (0.3g/kg body weight), over 1 min
and collection of serial samples of arterial blood before and after
injection for C-peptide determination. A variation of the test has been
used with additional β cell stimulation with tolbutamide (300 mg) 20
minutes later and the stimulation index of insulin secretion is
calculated. Similar to the oral GTT, this assay cannot be performed
frequently and in a manner that represents a simple and viable marker of
early graft failure.

#### Monitoring of autoimmunity

Standardized markers for monitoring the autoimmune response to
transplanted islets are not available to date. Analyses of humoral
autoimmune responses, such as monitoring of autoantibodies titers, have
been proposed. A correlation between rising titers of antibodies
directed at glutamate decarboxylase, GAD 65, and tyrosine phosphatase,
IA-2, and graft loss due to recurrence of autoimmunity has been reported
in whole-pancreas transplantation. Progressive islet graft failure has
been observed earlier in autoantibody-positive than in
autoantibody-negative recipients of intrahepatic islet allografts.
Further studies are necessary to establish the predictive value of these
antibodies for delineating the recurrence of autoimmunity.

Monitoring of islet graft-specific cellular auto- and allo-reactivity in
peripheral blood has also been suggested as a valuable tool to better
understand the mechanisms influencing islet allograft survival in
diabetic patients. In vitro assaying of the proliferative response of T
lymphocytes to certain islet autoantigens such as GAD65, insulin,
insulin-secretory granules, ICA69, h38kD, r38kD, insulinoma membranes
and control stimuli bovine serum albumin, aB-cystallin, and tetanus
toxoid has been proposed for the detection of changes in the recipient
autoimmune reactivity that might correlate with graft outcome; the
predictive value of such parameters to assess cell-mediated autoimmunity
recurrence awaits further confirmation.

#### Monitoring of rejection

Early markers of rejection of islet allografts are not yet available.
Many investigators regard imaging analyses and serial tissue biopsies as
too cumbersome or uninformative. Often the diagnosis of rejection is
done at the occurrence of hyperglycemia, a time point when a conspicuous
mass of islet has already been lost. At this time point, attempts at
graft rescue through antirejection treatments may be futile.

A correlation between islet allograft failure and increased titers of
anti-donor HLA antibodies has been reported (as measured by panel
reactive antibody testing).

Considerable progress in developing a molecular-based diagnostic
approach to define early markers for rejection has recently been made,
and quantitative analysis of the genes involved in the cytolytic
machinery of cytotoxic T-lymphocytes including granzyme B, perforin, and
Fas ligand in the peripheral blood has been proposed as an approach to
detect early episodes of rejection and drive antirejection therapy.

Alloreactivity assayed by cytotoxic T lymphocyte precursors and T-helper
frequencies against all mismatched HLA antigens of islet donors might
allow for the differentiation between the occurrences of auto- or
alloreactivity. A similar approach has been reported in experimental
islet transplantation to monitor the occurrence of allorejection by
performing serial mixed lymphocyte-islet co-culture.

[Clinical Questions:]{.underline}

Multiple clinical studies of allogeneic islets are completed or
on-going. Some clinical data will be presented or discussed by sponsors
during this meeting, but none of these data have undergone FDA review
and none of these data should directly impact your responses to FDA's
questions. Instead, the presentations of these data are intended to
provide examples of the types of data currently being accumulated within
the clinical investigative field.

> In light of the steady progress in clinical development of allogeneic
> islet products, FDA is seeking objective and independent advice on
> certain aspects of clinical data that may be important in designing
> confirmatory (phase 3) clinical studies and/or in interpreting the
> meaningfulness of clinical data from other types of clinical studies.

1.  Please discuss the clinical importance and limitations of each of
    the outcomes listed below. Please discuss each outcome with respect
    to its use in providing substantial evidence of efficacy if adequate
    and well-controlled clinical studies demonstrate a robust and
    durable treatment effect on the outcome.

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a.  "Insulin independence." This outcome may be defined in many ways
    but, in addition to any other definitions you regard as important,
    please specifically comment upon the following definition:
    "cessation of insulin treatment or decrease in insulin requirement
    in the setting of sufficient glycemic control." If you regard this
    as an important endpoint, please comment upon what you regard as
    evidence of "sufficient glycemic control."

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c.  Use of hemoglobin A1C, serum C-peptide concentration and mean
    amplitude of glycemic excursions (independently or in various
    composites) as measures of glycemic control and/or islet function.

d.  Acute diabetic complications. Please identify those complications
    that you regard as important outcomes (e.g., episodes of
    hypoglycemic unawareness, hospitalizations, death).

e.  Long-term diabetic sequelae. Please identify those complications
    that you regard as important outcomes (e.g., nephropathy,
    neuropathy, etc.).

f.  Other outcomes you regard as important.

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2.  Regarding the overall clinical development program for a sponsor's
    allogeneic islets, please discuss the importance and/or
    meaningfulness of the following types of clinical data with respect
    to the ability to form a risk-benefit assessment for the product.

    a.  Certain types of safety data: Specifically, the nature and
        extent of "long-term" clinical data that must be submitted in
        order to form a reasonable risk-benefit assessment. For example,
        must clinical follow-up for a certain number of subjects extend
        over a protracted period of time (e.g., 3, 5 or more years)? If
        so, please comment upon what you regard as a reasonable period
        of time and describe the types of data that must be obtained
        during this period, both pre- and post-licensure.

    b.  Historically controlled clinical data: Specifically, the
        appropriateness of the use of historical controls in the
        development program for a product, and whether data from studies
        that use no concurrent controls could be sufficient to provide
        substantial evidence of effectiveness of the product.

    c.  Clinical data from studies that only enrolled subjects with
        certain specific baseline characteristics: Specifically, the use
        and potential generalizability of clinical data from studies
        that enrolled a small subset of subjects with type 1 DM (for
        example, only subjects with a history of certain manifestations
        of "hypoglycemic unawareness"). Additionally, please discuss
        those baseline characteristics that you regard as important for
        a sponsor to consider in the clinical development of their
        product (e.g., age, extent and nature of diabetic complications,
        etc.).

# ATTACHMENTS:

1)  FDA "Dear Colleague" Letter
    (<http://www.fda.gov/cber/ltr/allpan090800.htm>)

2)  Shapiro, A, Lakey, J, Ryan, E. Korbutt, G., Toth, E., Warnock, G.,
    Kneteman, N., Rajotte, R. Islet transplantation in seven patients
    with type 1 diabetes mellitus using a glucocorticoid-free
    immunosuppressive regimen. N Engl J Med 2000; 343:230-8.

3)  White, S., James, R., Swift, S., Kimber, R., Nicholson, M. Human
    islet cell transplantation---future prospects. Diabetic Medicine
    2001; 18:78-103.

4)  Weber, D., McFarland, R., Irony, I. Selected Food and Drug
    Administration Review Issues for Regulation of Allogeneic Islets of
    Langerhans as Somatic Cell Therapy. Transplantation 2002;
    74:1816-1820.

5)  Ryan, E, Lakey, J., Paty, B., Imes, S., Korbutt, G., Kneteman, N.,
    Bigam, D., Rajotte, R., Shapiro, A. Successful islet
    transplantation; continued insulin reserve provides long-term
    glycemic control 2002. Diabetes; 51:2148-2157.

# UNSUPPLIED REFERENCES: 

1.  BRMAC 26 minutes (<http://www.fda.gov/ohrms/dockets>).

2.  US Food and Drug Administration. Guidance for Industry: Providing
    Clinical Evidence of Effectiveness for Human Drugs and Biological
    Products. 1998 Available at
    <http://www.fda.gov/cber/gdlns/clineff.pdf>

3.  International Conference on Harmonisation of Technical Requirements
    for Registration of Pharmaceuticals for Human Use. ICH harmonised
    tripartite guidelines. Guideline for Good Clinical Practice.
    May 1996. Available at http://www.ifpma.org/pdfifpma/e6.pdf 62,
    25691--709

4.  National Cancer Institute. Common Toxicity Criteria. Available at
    <http://ctep.cancer.gov/reporting/ctc.html>

5.  International Conference on Harmonisation of Technical Requirements
    for Registration of Pharmaceuticals for Human Use. ICH harmonised
    tripartite guidelines. General Considerations for Clinical Trials.
    1997 Available at <http://www.ich.org/pdfICH/e8.pdf>

6.  US Food and Drug Administration. Guidance for Industry: Providing
    Clinical Evidence of Effectiveness for Human Drugs and Biological
    Products. 1998 Available at
    <http://www.fda.gov/cber/gdlns/clineff.pdf>

7.  Pileggi, A., Ricordi, C., Alessiani, M., Inverardi, L. Factors
    influencing Islet of Langerhans graft function and monitoring.
    Clinical Chimica Acta, 2001. 310:3-16.

8.  US Food and Drug Administration. Guidance for Industry: Guidance for
    Human Somatic Cell Therapy and Gene Therapy. March 1998. Available
    at [http://www.fda.gov/cber/gdlns/somgene.pdf]{.underline}

9.  Zeng, Y., *et al.*, The effect of prednisone on pancreatic islet
    autografts in dogs. Surgery, 1993. 113: p. 98-102.

10. Yakimets, W., *et al.*, The metabolic impact of rapamycin
    (sirolimus) in chronic canine islet graft recipients.
    Transplantation, 1996. 61: p. 1206-1210.

11. Knetemanm, N., *et al.*, Prolongation of canine pancreatic islet
    allograft survival with combined rapamycin efficacy is a blood level
    related. Transplantation, 1993. 56: p. 1293-1298.

12. Chen, H., *et al.*, Combined effect of rapamycin and FK506 in
    prolongation of small bowel graft survival in the mouse. Transplant
    Proc, 1998. 30: p. 2579-2581.

13. Vu, M., *et al.*, Tacrolimus (FK506) and sirolimus (rapamycin) in
    combination are not antagonistic but produce extended graft survival
    in cardiac transplantation in the rat. Transplantation, 1997. 64: p.
    1853-1856.

14. Davalli, A.M. et al. Vulnerability of islets in the immediate post
    transplantationperiod: dynamic changes in structure and function.
    Diabetes, 1996. 45: p. 1161-1167.

15. Menger, M.D. et al. Influence of experimental hyperglycemia on
    microvascular blood perfusion of pancreatic islet isografts. J Clin.
    Invest. 1992, 90:1361-1369.

16. Kaufman, D.B. et al. Differential roles of Mac-1+ cells, and CD4+
    and CD8+ T lymphocytres in primary non-function and classic
    rejection of islet allografts. J. Exdp. Med. 1990. 172:291-302.

17. Bottino, R. et al. Transplantation of allogeneic islets of
    Langerhans in the rat liver: effects of macrophage depletion on
    graft survival and microenvironment activation. Diabetes, 1998.
    47:316-323.

18. Guan, J. et al. Glucose turnover and insulin sensitivity in rats
    with pancreatic islet transplants. Diabetes, 1998. 47:1020-1026.

19. National Cancer Institute. Common Toxicity Criteria. Available at
    <http://ctep.cancer.gov/reporting/ctc.html>

20. International Conference on Harmonisation of Technical Requirements
    for Registration of Pharmaceuticals for Human Use. ICH harmonised
    tripartite guidelines. General Considerations for Clinical Trials.
    1997 Available at <http://www.ich.org/pdfICH/e8.pdf>

21. US Food and Drug Administration. Guidance for Industry: Providing
    Clinical Evidence of Effectiveness for Human Drugs and Biological
    Products. 1998 Available at
    <http://www.fda.gov/cber/gdlns/clineff.pdf>

22. Pileggi, A., Ricordi, C., Alessiani, M., Inverardi, L. Factors
    influencing Islet of Langerhans graft function and monitoring.
    Clinical Chimica Acta, 2001. 310:3-16.

23. US Food and Drug Administration. Guidance for Industry: Guidance for
    Human Somatic Cell Therapy and Gene Therapy. March 1998. Available
    at [http://www.fda.gov/cber/gdlns/somgene.pdf]{.underline}

24. Zeng, Y., *et al.*, The effect of prednisone on pancreatic islet
    autografts in dogs. Surgery, 1993. 113: p. 98-102.

25. Yakimets, W., *et al.*, The metabolic impact of rapamycin
    (sirolimus) in chronic canine islet graft recipients.
    Transplantation, 1996. 61: p. 1206-1210.

26. Knetemanm, N., *et al.*, Prolongation of canine pancreatic islet
    allograft survival with combined rapamycin efficacy is a blood level
    related. Transplantation, 1993. 56: p. 1293-1298.

27. Chen, H., *et al.*, Combined effect of rapamycin and FK506 in
    prolongation of small bowel graft survival in the mouse. Transplant
    Proc, 1998. 30: p. 2579-2581.

28. Vu, M., *et al.*, Tacrolimus (FK506) and sirolimus (rapamycin) in
    combination are not antagonistic but produce extended graft survival
    in cardiac transplantation in the rat. Transplantation, 1997. 64: p.
    1853-1856.

29. Davalli, A.M. et al. Vulnerability of islets in the immediate post
    transplantationperiod: dynamic changes in structure and function.
    Diabetes, 1996. 45: p. 1161-1167.

30. Menger, M.D. et al. Influence of experimental hyperglycemia on
    microvascular blodd perfusion of pancreatic islet isografts. J Clin.
    Investi. 1992, 90:1361-1369.

31. Kaufman, D.B. et al. Differential roles of Mac-1+ cells, and CD4+
    and CD8+ T lymphocytres in primary non-function and classic
    rejection of islet allografts. J. Exdp. Med. 1990. 172:291-302.

32. Bottino, R. et al. Transplantation of allogeneic islets of
    Langerhans in the rat liver: effects of macrophage depletion on
    graft surivial and microenvironment activation. Diabetes., 1998.
    47:316-323.

33. Guan, J. et al. Glucose turnover and insulin sensitivity in rats
    with pancreatic islet transplants. Diabetes, 1998. 47:1020-1026.

[^1]: ^^Public Health Service Act, Title 42, USC §262, §351.