Immunological Responses to Exogenous Insulin

S. Edwin Fineberg, Thomas T. Kawabata, Deborah Finco-Kent, Robert J. Fountaine, Gregory L. Finch and Alan S.
Krasner

Endocr. Rev. 2007 28:625-652 originally published online Sep 4, 2007; , doi: 10.1210/er.2007-0002

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Immunological Responses to Exogenous Insulin
S. Edwin Fineberg, Thomas T. Kawabata, Deborah Finco-Kent, Robert J. Fountaine, Gregory L. Finch, and
Alan S. Krasner
Indiana University School of Medicine (S.E.F.), Indianapolis, Indiana 46202; University of Alabama Birmingham School of
Medicine (S.E.F.), Birmingham, Alabama 35294; and Pfizer Global Research and Development (T.T.K., D.F.-K., R.J.F.,
G.L.F., A.S.K.), Groton, Connecticut 06320
Regardless of purity and origin, therapeutic insulins continue
to be immunogenic in humans. However, severe immunolog-
ical complications occur rarely, and less severe events affect
a small minority of patients. Insulin autoantibodies (IAAs)
may be detectable in insulin-naive individuals who have a
high likelihood of developing type 1 diabetes or in patients
who have had viral disorders, have been treated with various
drugs, or have autoimmune disorders or paraneoplastic syn-
dromes. This suggests that under certain circumstances, im-
mune tolerance to insulin can be overcome. Factors that can
lead to more or less susceptibility to humoral responses to
exogenous insulin include the recipient’s immune response
genes, age, the presence of sufficient circulating autologous
insulin, and the site of insulin delivery. Little proof exists,
however, that the development of insulin antibodies (IAs) to
exogenous insulin therapy affects integrated glucose control,
I. Introduction
II. Human Experience with Exogenous Insulin Therapy
A. Animal insulin preparations
B. Recombinant human insulins
C. Insulin analogs
D. Subcutaneous and implantable insulin pumps
E. Inhaled insulin
F. Modifying factors in the antibody response to exoge-
nous insulin
III. Predicting Human Immunogenicity with Animal Models
A. Effects of inhalation of insulin
B. Mucosal delivery of insulin and prevention of type 1
diabetes in NOD mice
IV. Characterization of Insulin Antibodies
A. Measurement of insulin antibodies
B. Immunoglobulin classes
C. Antibody affinity
D. Insulin epitope and insulin antibody idiotype analyses
E. Characterization of insulin antibody immune complexes
First Published Online September 4, 2007
Abbreviations: APC, antigen-presenting cell(s); AUC, area under the
curve; CPII, continuous peritoneal insulin infusion; CSII, continuous sc
insulin infusion; dpm, disintegrations per minute; FEV1, forced expira-
tory volume in 1 sec; HLA, histocompatibility leukocyte antigen; IA,
insulin antibody; IAA, insulin autoantibody, arising spontaneously in
patients not exposed to exogenous insulin; IAS, insulin autoimmune
syndrome; IIP, implantable insulin pump; MHC, major histocompati-
bility complex; NOD, nonobese diabetic (mouse); NPH, neutral prota-
mine Hagedorn; rDNA, recombinant DNA; RLB, radioligand binding
assay.
Endocrine Reviews is published by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
625
Endocrine Reviews 28(6):625– 652
Copyright © 2007 by The Endocrine Society
doi: 10.1210/er.2007-0002
insulin dose requirements, and incidence of hypoglycemia, or
contributes to ␤-cell failure or to long-term complications of
diabetes. Studies in which pregnant women with diabetes
were monitored for glycemic control argue against a connec-
tion between IAs and fetal risk. Although studies have shown
increased levels of immune complexes in patients with dia-
betic microangiopathic complications, these immune com-
plexes often do not contain insulin or IAs, and insulin admin-
istration does not contribute to their formation. The majority
of studies have shown no relationship between IAs and dia-
betic angiopathic complications, including nephropathy, ret-
inopathy, and neuropathy. With the advent of novel insulin
formulations and delivery systems, such as insulin pumps and
inhaled insulin, examination of these issues is increasingly
relevant. (Endocrine Reviews 28: 625– 652, 2007)
V. Clinical Significance of Insulin Antibodies
A. Hypersensitivity reactions
B. Glycemic effects of insulin antibodies
C. Pregnancy
D. Autoimmune diseases
E. Inhaled insulin antibodies and pulmonary function
F. Immune complexes
G. Buffering effect of insulin antibodies
H. Insulin antibodies and risk for diabetes
VI. Conclusion
I. Introduction
D ESPITE EXPERIENCE FROM more than three quarters
of a century, uncertainties about the significance of
the immune response to therapeutic insulins remain. Such
issues are largely the residuum of early experiences with
pancreatic extracts of animal insulins, however, and have
little connection to modern-day preparations of uncontam-
inated human insulin, which now has a time-tested safety
record. Currently, severe immunological sequelae occur only
rarely, and less severe events affect a small minority of pa-
tients. Still, the following questions persist for physicians and
researchers:
• Why do IAs occur with the use of recombinant human
insulins?
• What are the key factors that determine immunogenicity
of insulin in humans?
• What are the mechanisms that lead to a break in immu-
nological tolerance for insulin?
626 Endocrine Reviews, October 2007, 28(6):625– 652
• What do IA detection and quantification techniques tell us
about the nature of the antibodies?
• Do IAs lead to increased insulin dose requirements or
hypoglycemic events?
• If the presence of autoantibodies to insulin (IAA) predicts
the development of type 1 diabetes in patients not previ-
ously treated with exogenous insulin, what is the impact
on residual ␤-cell function of antibody responses to ex-
ogenous insulin therapy in patients with established
diabetes?
• Because antibodies cross the placenta, do IAs cause dia-
betes in a developing fetus or contribute to birth defects or
postnatal complications?
This review will evaluate these questions by examining the
development of IAs to exogenous insulin (Table 1) and by
reviewing the literature on antibody responses to different
types of insulin therapies with a focus on the clinical impact
of IAs.
II. Human Experience with Exogenous
Insulin Therapy
A. Animal insulin preparations
The first effective injectable insulins developed by Banting
and Best (1) were crude acid-ethanol extracts of abattoir
bovine or porcine pancreases. Potencies were highly vari-
able, large volumes were required to reduce blood glucose in
diabetic dogs, and injections often involved toxic reactions
and abscess formation. The subsequent discovery of an in-
sulin crystallization method by Collip in 1922 led to the first
commercial insulin preparations for diabetes therapy (2).
As purification increased, complaints of local abscesses in
trial patients decreased; however, reports emerged of ana-
phylactic, local, and systemic hypersensitivity reactions (2,
3). In one patient, Tuft (4) demonstrated immunological spec-
ificity for insulin protein with regard to urticaria and edema,
and he was able to transfer dermal reactivity to nonsensitized
individuals (Prausnitz-Kustner reaction).
Resistance to insulin therapy and the discordant appear-
ance of cutaneous reactions to insulin were reported from the
1920s onward. The immunological nature of this resistance
was shown by insulin neutralization in mice that received
sera from insulin-resistant patients (5). Clinical hypersensi-
tivity and insulin resistance were first linked to circulating
antibodies to insulin in the work of Berson et al. (6).
TABLE 1. Factors affecting IA development
Insulin-related
Purity
Molecular structure
Storage condition
Formulation
Excipients
Dimer and oxidation products
Patient-related
Age
HLA type
Endogenous insulin
Delivery route
HLA, Histocompatibility leukocyte antigen.
Fineberg et al. • Immunological Responses
The use of isoelectric precipitation by Eli Lilly and Com-
pany increased the purity of exogenous bovine and porcine
insulin by 10- to 100-fold over previous preparations (2).
However, improved analytical methods showed that crys-
tallized insulin included proinsulin and intermediates, non-
convertible insulin dimers, arginyl and ethyl insulin, deami-
dated insulin, monodesamido insulin, and various
noninsulin peptides (7–9). RIAs done in 1982 showed that
twice-crystallized Novo monocomponent insulin contained
proinsulin and its intermediates as well as trace amounts of
other peptide hormones: proinsulin-like immunoreactivity
up to 20,000 ppm, glucagon 45 ppm, pancreatic polypeptide
43 ppm, somatostatin 1.4 ppm, and vasoactive intestinal pep-
tide 0.3 ppm (10). These findings raised the possibility that
noninsulin peptides were responsible for the antigenicity of
therapeutic insulins.
Local reactions to nonchromatographed insulins occurred
in up to 55% of recipients, although systemic reactions were
rarely seen (11). These dermal and systemic allergic reactions
to insulin were demonstrated to be associated with circulat-
ing IgE and antibodies to insulin, which were directed to-
ward extracted but not native insulin (12). Patients being
treated with insulin were shown to have a broad array of IA
binding classes (see Section IV.B) (13).
The incidence of insulin resistance of an immunological
nature continued to be quite rare—less than 0.1% of recip-
ients (14, 15). From the late 1970s onward, manufacturers
took further steps in insulin purification, including column
chromatography, separation of peptides by molecular siev-
ing and charge (16), HPLC (17), and reversed phase HPLC
(18). These highly purified monocomponent insulins, which
have less than 3 ppm proinsulin-like immunoreactivity, de-
creased but did not eliminate the development of IAs (19),
dermal reactions, systemic hypersensitivity, and insulin re-
sistance (20 –22).
B. Recombinant human insulins
The first human insulins for clinical use were derived from
porcine insulin by semisynthetic conversion (23). The next
advance in insulin therapy was the development of recom-
binant DNA (rDNA) human insulin made by fermentation of
Escherichia coli or Saccharomyces cerevisiae yeast that contained
DNA for insulin A and B chains, proinsulin, or modified
proinsulin. Later steps after cell lysis include removal of
bacterial and yeast components, recombination of insulin
chains, or removal of connecting peptides (24, 25).
Clinical trials revealed that semisynthetic human insulin
was less immunogenic than porcine monocomponent insulin
in patients newly diagnosed with type 1 diabetes. Schern-
thaner et al. (26) found IAs of the IgG class in 14% of patients
receiving human insulins and in 29% of the patients receiving
nonhuman insulins. Heding et al. (27) reported that 44% of
pediatric patients with type 1 diabetes receiving human
monocomponent insulin were antibody-positive, compared
with 59% of similar patients receiving porcine monocompo-
nent insulins. (These differences may be attributable to an-
tibody measurement methodologies, which will be discussed
in Section IV.A) In a 1-yr trial, Fineberg et al. (28) compared
development of antibodies to rDNA human insulin in 100
Fineberg et al. • Immunological Responses
insulin-naive children and adults with 121 similar individ-
uals treated with purified porcine insulin. At the end of the
trial period, 44% of patients treated with rDNA human in-
sulin and 60% of patients treated with porcine insulin had
developed significant levels of IAs.
In a retrospective review, injection site reactions with
highly purified mixed bovine (70% bovine, 30% porcine),
purified porcine, and rDNA human insulin were reported to
be 12, 3.9, and 2.4%, respectively (29). Sporadic case reports
have been published regarding individuals who developed
insulin resistance and/or systemic hypersensitivity reactions
while being treated with rDNA human insulins, including
short-acting and long-acting human insulin analogs (30 –34).
C. Insulin analogs
Insulin’s tendency to self-associate in concentrated solu-
tions to form dimers and hexamers is largely the reason that
sc regular rDNA human insulin and animal insulin extracts
have nonphysiological action profiles (35). In response to this
problem, researchers developed rDNA human insulin ana-
logs that have decreased tendencies to self-associate. Cur-
rently, three rapid-acting human insulin analogs are in clin-
ical use: lispro, aspart, and glulisine. These analogs consist of
molecular modifications that result in few, if any, differences
from unmodified human insulin pertaining to antigenicity
and receptor binding of insulin (36, 37). Amino acids essen-
tial for insulin receptor binding are A1 (Gly), A2 (Ile), A3
(Val), A19 (Tyr), B6 (Leu), B12 (Val), B23 (Gly), B24 (Phe), and
B25 (Asp). In addition, IGF-I binding is affected by B10 (His)
and B26 –30 (Tyr-Thr-Pro-Lys-Ala). Insulin lispro has a re-
versal of the sequence of amino acids B28 and B29 (Lis-Pro
vs. Pro-Lis), lowering the potential for dimer formation. In-
sulin aspart substitutes Asp B28 for B28 (Pro). Both of these
rapid-acting insulins have IGF-I receptor binding equivalent
to unmodified human insulin. Insulin glulisine substitutes
Glu for B29 (Lis) and Lis for B3 (Asp), resulting in decreased
self-association but somewhat longer residence time on the
IGF-I receptor (38). The rapid-acting insulin analogs, when
given sc, result in development of humoral IAs, with no
significant differences shown between the analogs and un-
modified human insulin (39 – 42). In all the clinical trials
cited, IAs were cross-reactive, i.e., they react equally to un-
modified and analog insulins with no demonstrable effects
on insulin dose, hypoglycemic events, or glycemic control.
The rapid-acting human insulin analogs are available pre-
mixed with modifications having intermediate-duration
characteristics (e.g., insulin lispro protamine suspension).
Antibodies directed to or cross-reacting antibodies with pro-
tamine have been associated with anaphylaxis during rever-
sal of anticoagulation in patients treated with neutral pro-
tamine Hagedorn (NPH) insulin (43, 44).
Two commercially available long-acting human insulin
analogs are detemir and glargine. Insulin detemir is des B30
Thr with a B29 side chain (Lys-myristic acid [tetradecanoyl-]).
When concentrated, detemir forms hexamers in sc injection
sites and, after absorption of insulin monomers, binds re-
versibly to albumin. IA data from clinical trials of this analog
have not been presented; however, type 3 hypersensitivity
Endocrine Reviews, October 2007, 28(6):625– 652 627
reactions and severe dermal reactions have been reported
(45, 46).
Insulin glargine is modified by the addition of two argi-
nines to B30 (B31 and B32) and replacement of A21 Asp with
Gly. Treatment with glargine in patients previously treated
with insulin for at least 1 yr did not result in increased IA
levels (47).
D. Subcutaneous and implantable insulin pumps
In a small, 12-month study, 45 patients with type 1 diabetes
were randomized to conventional therapy, multiple dose, or
continuous sc insulin infusion (CSII) with highly purified
porcine insulins (48). Researchers found an increase in IAs in
the multiple dose and CSII groups but not in the conventional
group, suggesting that differences in methods of sc insulin
administration affect antibody development (the study has
not been repeated with contemporary human insulins) (49,
50).
Implantable insulin pumps (IPPs) have been associated in
various studies with increases in IA levels, leading research-
ers to suggest causative mechanisms such as the storage of
insulin in the pump reservoir or to the presence of surfactant
or polyethylene glycol (48 –50). In a 2002 study, two groups
of patients with type 1 diabetes were challenged for 6 months
with either continuous peritoneal insulin infusion (CPII) or
CSII (400 or 100 U/ml, respectively) using Hoe 21 PH, a
semisynthetic human insulin using polyethylene glycol. An-
tibodies in the CPII group increased significantly vs. the CSII
group. Although these data argue against the insulin prep-
aration itself as the culprit, they do not eliminate the possi-
bility of storage degradation or an effect of silicone oil con-
tributing to the increased antibody development associated
with CPII (see Section V.B.3.c) (51).
E. Inhaled insulin
Despite well-established, long-term benefits of good gly-
cemic control (52), many patients and physicians are reluc-
tant to initiate exogenous insulin therapy (53, 54). In response
to this reluctance, researchers have explored pulmonary de-
livery of insulin as an alternative to sc injection (55). Inhaled
human insulin [Exubera (insulin human [rDNA origin]) In-
halation Powder] is the first inhalable insulin approved by
the United States and the European Union for the treatment
of type 1 and type 2 diabetes. The Exubera inhaler delivers
dry powder human insulin contained in blister packets (56).
Diabetes trials with inhaled insulins have been reviewed
in a recent article (57), and IA data have been presented for
Exubera (Pfizer Inc., New York, NY) in patients with type 1
and 2 diabetes (58, 59), and also for the AERx iDMS system
(Novo Nordisk, Bagsvaerd, Denmark) (60). [All inhaled in-
sulins currently are used as premeal insulins because they are
absorbed in the deep lung tissues as rapidly as short-acting
insulin analogs but do not have enough duration of activity
to meet basal insulin needs (58, 61, 62).]
In the Exubera trials, IA development in patients with type
1 diabetes exceeded that in comparator regular sc human
insulin groups; median IA binding increased from 3 to 29%
in the Exubera group, compared with no change in the sc
628 Endocrine Reviews, October 2007, 28(6):625– 652
group (58). Among patients with type 2 diabetes who were
previously treated with insulin, median IA binding increased
to 6% in the Exubera group and was unchanged in the sc
group. In patients with type 2 diabetes who had not previ-
ously used insulin, no change in median levels of IA binding
was seen in either the Exubera or comparator group (treated
with an oral antihyperglycemic agent). Differences between
patients with type 1 and type 2 diabetes were maintained
throughout a 24-month extension study. The IAs were of the
IgG class (Fig. 1), and no effects on insulin efficacy or adverse
effects could be attributed to IA development (58, 59). Fur-
thermore, in another study IA levels were shown to decline
after cessation of Exubera (63).
The AERx iDMS device delivers a liquid droplet of human
insulin onto a dosage strip for inhalation (64). In a 12-wk
intensive treatment trial among patients with type 2 diabetes
being treated with insulin, there was a significant increase in
IAs in the inhaled insulin group, but no change in the com-
parator sc group (60). No clinical effects were attributed to
IAs.
Causes of the increased antibody development with in-
haled insulin compared with sc insulin therapy have not
been identified. The excipients used in the formulation of
powdered insulin are not known to incite an immune re-
sponse, nor can structural differences or alterations in bio-
activity be demonstrated (58, 59). However, differences in
site of delivery per se may be paramount, because both liquid
and powdered formulations of pulmonary insulin are more
immunogenic than sc comparators.
F. Modifying factors in the antibody response to
exogenous insulin
1. Immunological mechanism of antibody responses. The gener-
ation of a primary antibody response to foreign proteins
involves the uptake by antigen-presenting cells (APC) such
as dendritic cells (65). Dendritic APC are found throughout
the body, including all current insulin delivery sites, i.e., the
sc space, the lungs, and lymph nodes that drain the perito-
neum. The protein antigen is processed to smaller peptide
fragments in endosomal vesicles. These peptides bind to
major histocompatibility complex (MHC) class II molecules
and are transported to the surface of the APC. There are
different types of class II molecules, and each has multiple
alleles. MHC binding of antigen fragments is determined by
the peptide sequence and type of MHC molecule. Thus, not
all peptides formed associate with MHC.
The MHC-peptide complex on the APC surface binds to
FIG. 1. Findings from the Exubera trials indicate that IAs associated
with inhaled and sc insulin therapy are predominantly of the IgG
class (58). A wide range of IgG levels was observed.
Fineberg et al. • Immunological Responses
helper T cells with a corresponding T cell receptor that rec-
ognizes the peptide. In addition to MHC-peptide-T cell re-
ceptor binding, a second signal from the binding of B7 (co-
stimulatory molecule) on APCs to CD28 on T cells will lead
to T cell activation and the production of various cytokines.
Costimulatory molecules are up-regulated by proinflamma-
tory cytokines produced with tissue damage or infection.
Protein antigen binding to the Ig B-cell receptors along with
cytokines from the activated T cells leads to the proliferation
and differentiation of B cells to antibody-secreting plasma
cells.
Immunological tolerance prevents the development of an-
tibody response to self-proteins. This may involve clonal
deletion of T cells that recognize self-proteins during devel-
opment in the thymus. Without costimulation by APC, T cells
may become anergic and will not respond to antigen. Tol-
erance may also occur by means of regulatory T cells that
actively down modulate T-helper cell activity. Down-regu-
lation of immune responses to self and nonself antigens
involves a subset of regulatory T lymphocytes that express
CD4, CD25, and the transcription factor Foxp3 (66). These
regulatory T cells, called suppressor T lymphocytes in the
past, may be critical in warding off or terminating autoim-
munity (67– 69). It is likely that these regulatory cells may
also play a role in the development and levels of immune
responses to exogenous insulin. These cells are present
throughout life in humans and have a very rapid turnover
rate. The peripheral T regulatory cells are prone to apoptosis
and may require the presence of continuous concentrations
of self-antigens and/or growth factors such as TGF-␤ (70).
Such conditions may be less prevalent in autoimmune dis-
eases, such as type 1 diabetes, resulting in deficient T-reg-
ulatory cell numbers or function (71, 72). T-regulatory cell
deficiencies have been demonstrated in a mouse model of
autoimmune diabetes (73, 74). Tiittanen et al. (75) recently
reported that insulin treatment may activate insulin-specific
regulatory T cells in children with newly diagnosed type 1
diabetes.
The H2b strain of mouse does not form antibodies to
porcine insulin; however, both helper and suppressor T cells
are activated in that strain, allowing the secondary devel-
opment of antibodies to bovine insulin but suppressing the
response to porcine insulin. When T suppressor cells are
removed, in vitro development of antiporcine IAs can be
shown (76). In addition, nonresponder strains of mice im-
munized with porcine insulin have been shown to have
primed T helper cells and dominant T suppressor cells that
cross-react with autologous insulin. Thus, the balance be-
tween the effects of T suppressor and T helper cells deter-
mined the effectiveness of immune tolerance to heterologous
and autologous insulins (77).
2. Sequence differences and impurities. Differences between the
amino acid sequence of a protein and the sequence of a
self-protein will increase its inherent immunogenicity. As
shown in Table 2, the sequences of bovine and porcine in-
sulin differ from human insulin by three amino acids and one
amino acid, respectively. Therefore, the administration of
bovine insulin or bovine-porcine insulin mixtures resulted in
greater IA responses than porcine or human insulin (21,
Fineberg et al. • Immunological Responses Endocrine Reviews, October 2007, 28(6):625– 652 629

TABLE 2. Comparison of amino acid sequences in human and animal insulin

A chain B chain No. of differences

Human GIVEQCCTSICSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKT
Chimpanzee GIVEQCCTSICSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKT 0
AfrGr GIVEQCCTSICSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKT 0
Macaque GIVEQCCTSICSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKT 0
Pig GIVEQCCTSICSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKA 1
Dog GIVEQCCTSICSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKA 1
Squirrel GIVEQCCTSICSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKS 1
Rabbit GIVEQCCTSICSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKS 1
Hamster GIVDQCCTSICSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKS 2
Elephant GIVEQCCTGVCSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKT 2
Cow GIVEQCCASVCSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKA 3
Whale GIVEQCCASTCSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKA 3
Cat GIVEQCCASVCSLYQLEHYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKA 4
Mouse GIVDQCCTSICSLYQLENYCN FVKQHLCGPHLVEALYLVCGERGFFYTPKS 4
Rat GIVDQCCTSICSLYQLENYCN FVKQHLCGPHLVEALYLVCGERGFFYTPKS 4
Sheep GIVEQCCAGVCSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKA 4
Goat GIVEQCCAGVCSLYQLENYCN FVNQHLCGSHLVEALYLVCGERGFFYTPKA 4
Fish (bonito) GIHEQCCHKPCDIFQLENYCN AANPHLCGSHLVDALYLVCGDRGFFYQPK– 16
Guinea pig GIVDQCCTGTCTRHQLQSYCN FVSRHLCGSNLVETLYSVCQDDGFFYIPKD 18
The sequences of amino acids for insulin A and B chains are given for each species. Differences from human insulin are highlighted in bold
type, and the number of differences is given in the far right column. Sources: National Center for Biotechnology, GenBank, www.ncbi.nlm.
nih.gov/Genbank; National Biomedical Research Foundation Protein Identification Resource, Protein Sequence Database. Fish (bonito) se-
quence from J. F. Cutfield et al.: Eur J Biochem 158:117–123, 1986 (369). AfrGr, African green monkey.

78 – 80). Studies of insulin aspart, which differs from human
insulin by one amino acid, have shown a robust antibody
response in a subpopulation of patients (81). However, in-
sulin lispro is thought to have comparable rates of antibody
formation as regular human insulin (40).
Owing to the presence of contaminants (including gluca-
gon, pancreatic polypeptide, somatostatin, proinsulin, and
insulin complexes) in early bovine and mixed bovine-porcine
preparations, some of the antibodies generated may have
been against noninsulin proteins (see Section II.A) (30). More-
over, other contaminants may also have adjuvant effects and
thereby enhance the generation of IAs (19). This finding is
consistent with studies that have shown minimal difference
in immunogenic potential between purified porcine and hu-
man insulin (28, 78, 82).
Most commonly, IAs that develop in response to exoge-
nous bovine, porcine, or human insulins are cross-reactive
with all three species (Fig. 2 and Table 3) (83– 86). In rare
cases, however, antibodies bind differentially to insulins
from different species, correlating with clinical responses
observed when switching insulin sources (87, 88).
antibodies that bind to insulin dimers have been demon-
strated (92). Lymphocyte transformation studies suggest that
insulin aggregates, rather than monomers, are the cause of
persistent cutaneous allergy (93). Insulin aggregation and
degradation products in a glycerol-based formulation of in-
sulin for use in IPPs have been shown to be associated with
increased immunogenicity (94).
Studies in animal models also indicate that aggregates are
more immunogenic. Insulin samples taken directly from an
insulin vial (material that contained no aggregates) and in-
sulin from an ip pump reservoir (material with a high level
of aggregates) were administered to rats, and IA levels were
subsequently measured (95). The percentage binding in sera
from rats immunized with insulin from the pump reservoir
was approximately 2-fold higher than that obtained with

3. Formulation and aggregates. Insulin formulation can also

affect immunogenic potential. Soluble forms of insulin, such
as regular and semilente, are less allergenic than intermedi-
ate- or long-acting preparations, such as NPH, lente, prota-
mine zinc, and ultralente (21, 89). Acid preparations are more
immunogenic than neutral ones (21). Allergic reactions to
both protamine and zinc have been reported and require
special testing to distinguish from insulin allergy (90, 91).
FIG. 2. In 1988, Fineberg et al. (85) reported antibody-bound insulin
Aggregation can clearly affect insulin immunogenicity. in previously insulin-naive individuals with type 1 or type 2 diabetes
This is also true for other biological therapeutics and has treated with rDNA human insulin, purified porcine insulin, or mixed
resulted in strict limitation on aggregates in final drug prod- bovine-porcine insulin over 2 yr. On the left, patients with type 1
ucts. It is thought that insulin aggregates may remain for diabetes reach a peak response within 6 months, with the greatest
response to mixed bovine-porcine insulin. On the right, antibody re-
longer periods at the injection site or be more readily taken sponses for patients with type 2 diabetes are diminished for all the
up by APC. Insulin aggregates are present in the circulation insulins. The insets compare antibody response to human and porcine
of patients with diabetes being treated with insulin (92), and insulin.
630 Endocrine Reviews, October 2007, 28(6):625– 652
TABLE 3. Cross-reactivity of IAs
n
Patients with type 1 diabetes treated with EXU
Human IA 353
Bovine IA 353
Porcine IA 353
Insulin-using patients with type 2 diabetes
treated with EXU
Human IA 134
Bovine IA 134
Porcine IA 134
EXU, Inhaled human insulin. Data on file, Pfizer Inc., New York, NY.
insulin directly from the vial, suggesting that insulin aggre-
gates formed in the reservoir were slightly more immuno-
genic. This may be relevant to elevated IA levels that have
been observed in patients with implanted ip pumps (see
Sections II.D and V.B.3.c).
4. Route of delivery. Animal and human studies suggest that
the site of antigen delivery can affect antibody responses.
Insulin-specific tolerance induction by iv injection in mice is
antigen-specific and dose-dependent. The same dose of in-
sulin given ip results in an augmented rather than an inhib-
ited antibody response (96). Intraperitoneal injection of in-
sulin in mice has been found to enhance the immune
responses to multiple antigens, including insulin (76, 97, 98).
Oral, parenteral, and aerosol insulin administration in dia-
betes-prone animal models can induce immune tolerance
(see Section III.B).
Intraperitoneal insulin delivery via IPPs has been shown
to increase levels of IAs in patients with type 1 diabetes (50,
99), but not in those with type 2 (see Sections II.D and V.B.3.b)
(100). Intravenous insulin delivery via IPPs (101, 102), as well
as oral insulin delivery, does not result in detectable IAs (103,
104). Pulmonary delivery of human insulin, as discussed
above, has been shown to be associated with higher levels of
IAs than sc delivery (see Section II.E) (58, 105).
5. Dose-response effects. In humans, evidence that the devel-
opment of IAs secondary to exogenous insulin is related to
dose has not been directly tested; however, clinical trial data
show no significant relationship between insulin dose and
the development of antibodies. In a study of 46 patients
newly diagnosed with type 1 diabetes, Karjalainen et al. (106)
found that 35% of them were positive for IAAs before ther-
apy. After 3 months of therapy, the IAA-positive patients had
higher antibody levels than those who were initially IAA-
negative, but this difference in IA levels did not persist with
further duration of therapy. At no time was insulin dosage
found to be related to the presence of IAs. Additional pro-
spective treatment trials of animal and human insulins, in-
sulin analogs, and inhaled human insulin in insulin-naive
and insulin-treated patients have shown no significant rela-
tionship between insulin dose and level of IAs (see Section
V.B.2.b) (22, 41, 58).
6. Patient-related factors. Known genetic determinants influ-
ence susceptibility to antibody formation. The presence of
histocompatibility leukocyte antigen (HLA)-B15, -DR4, and
-DR7 has been shown to increase the rate of IA formation,
Fineberg et al. • Immunological Responses
Median Lower quartile Upper quartile
(␮U/ml) (␮U/ml) (␮U/ml)
29.0 13.0 44.0
23.0 12.0 37.0
25.0 12.0 42.0
5.0 1.5 15.0
4.0 1.5 14.0
4.0 1.5 11.0
whereas HLA-B8 and HLA-DR3 are associated with de-
creased formation rates (26, 107–110). However, Asplin et al.
(111) assessed the development of IAs in 54 newly treated
patients with type 1 diabetes and found no effects of HLA-
DR3, HLA-DR4, or combinations on insulin-binding anti-
body formation. Most of these studies were conducted dur-
ing the late 1970s through the 1980s and evaluated antibody
responses in patients treated with bovine or porcine insulin
preparations; however, Fineberg et al. (83) studied patients
treated with human insulin and demonstrated no difference
in IA levels based on DR4 status, whereas significant differ-
ences in IA levels were observed in DR4-positive vs. DR4-
negative patients treated with porcine and mixed bovine-
porcine insulin (Fig. 3). This is not unexpected because the
peptide sequences can determine the binding affinity to dif-
ferent alleles of MHC class II molecules. The IgG heavy-chain
gene complex has also been shown to modulate susceptibility
to IA formation (112).
The presence of IAA at baseline may predict a greater IA
response to exogenous insulin. In one study, IAA-positive
individuals developed a greater IA level than IAA-negative
patients throughout 1 yr of therapy (113). However, the
increased levels could be attributed to the sum of IAA and
IA.
The findings from studies with inhaled insulin suggest
that patients with type 1 diabetes develop greater levels of
IAs than those with type 2 diabetes (58). Studies with insulin
lispro also demonstrated that patients with type 1 diabetes
had higher IA levels than those with type 2 diabetes (41). The
mechanism for this difference in response is not clear, but it
may be due to defects in regulatory T cell function, as re-
ported in nonobese diabetic (NOD) mice—a model of type 1
autoimmune diabetes (73). This defect may lead to enhanced
immune responses to human insulin in patients with type 1
diabetes (see Sections II.F.1 and V). However, it is not clear
whether exogenous antibody formation can be attributed to
further expansion of autoreactive antiinsulin T cells and B
cells that produce IAAs. There are conflicting data on
whether the presence of IAAs predicts the IA response to
exogenous insulin (106, 113, 114).
Exogenous insulin administration may also result in an-
tibody formation in individuals who do not have diabetes.
Antibody formation induced by as few as six injections of
human insulin is comparable to that seen in chronically
treated patients with type 1 diabetes (115). IAAs are also
found more commonly in individuals who have endocrine
Fineberg et al. • Immunological Responses
FIG. 3. In a 1-yr trial by Fineberg et al. (83), insulin-naive patients
received porcine insulin, mixed bovine-porcine, or rDNA human in-
sulin. Patients who were DR4-positive had greater antibody re-
sponses, in terms of percent binding, than DR4-negative subjects to
mixed bovine-porcine and porcine insulins but not for human insulin,
suggesting an interplay among HLA type, antigen structure, and
possibly other patient-related factors affecting antibody responses.
The researchers concluded that HLA type affects the magnitude of the
immune response.
autoimmune syndromes, such as Graves’ disease, Hashimo-
to’s thyroiditis, and nonimmune thyroid disease (116).
Age may play an important role in the IA response with
exogenous insulin administration. Immunological competence
declines as an individual ages, causing decreased ability to form
high-affinity antibodies, decreased ability to generate long-
lasting memory cells, and delayed hypersensitivity responses
(117–119). The age of the individual also affects the presence of
IAAs before the development of type 1 diabetes and whether
an individual is more or less likely to develop a significant
antibody response to exogenous insulin (120).
In the studies of Fineberg et al. (41, 83, 121), the develop-
ment of significant levels of antibodies to exogenous insulin
has been shown to be inversely related to C-peptide levels
and age. The two factors—age and C-peptide level—are in-
dependently correlated, although there is more evidence for
a correlation to age than to C-peptide level. A logistic re-
gression analysis of antibody development in 744 individ-
uals who were insulin naive at the beginning of therapy
revealed a 3% decrease in the chances of antibody develop-
ment for every 1-yr increase in age (odds ratio, 0.97; 95%
confidence interval, 0.96 – 0.99; P ⬍ 0.001) (122). Additional
analysis showed a 46% decrease for every 1-nmol/liter in-
crease in C-peptide levels (odds ratio, 0.54; 95% confidence
interval, 0.38 – 0.78; P ⬍ 0.001).
Endocrine Reviews, October 2007, 28(6):625– 652 631
III. Predicting Human Immunogenicity with
Animal Models
Several animal species have been used for investigations
into the immunogenicity of insulins. Antibody responses can
be generated in rabbits, mice, rats, and guinea pigs with sc
administration of human insulin and Freund’s adjuvant;
however, given the differences in amino acid sequences of
insulin between these species and humans (Table 2), these
findings are not surprising. The immunogenic potential of
insulin lispro, biosynthetic human insulin, and purified por-
cine insulin in Freund’s adjuvant were compared in rhesus
monkeys over a 6-wk period (four per dose group) (123).
Measurement of insulin-specific IgG antibody in serum by
ELISA indicated that none of the test insulin preparations
stimulated antibody formation; thus, these data indicated
that human insulin is nonimmunogenic in monkeys. Like
other species tested, however, the monkey is a poor predictor
of immunogenicity of insulins, including inhaled insulin, in
humans. In summary, it appears that animal models are poor
predictors of the immunogenicity of exogenous insulin in
humans. This is consistent with findings for other protein
therapeutic agents. Many drugs are found to be strongly
immunogenic in monkeys but weakly immunogenic in hu-
mans. Therefore, immunogenicity data from animal studies
are usually not used to predict immunogenicity in humans
but only to interpret findings in the animals studied (e.g.,
changes in toxicity, efficacy, and pharmacokinetics due to
antidrug antibodies) (124).
However, data exist that suggest animal models may be
helpful in predicting relative differences in immunogenicity
when comparing modifications of insulin. As discussed in
Section III.B, a study was conducted in rats to compare im-
munogenic responses with different levels of insulin aggre-
gates. In another study, immunological tolerance for human
insulin was induced by creating a transgenic mouse express-
ing the human insulin gene in the ␤-cells of the mouse pan-
creas. This model has been used to evaluate the immuno-
genicity of human insulin analogs (125). Twelve analogs
having varying self-association properties were found to
have differing immunogenic potential. Substitution of single
amino acids in the A8 –10 loop resulted in antibody responses
in responder mice, whereas substitutions or deletions in B3,
B9, B27, and B28 did not produce antibody responses. This
model may help identify less immunogenic insulin analogs
in the future. Comparing antibody responses to various in-
sulin analogs in these mice to those in humans will be nec-
essary to validate this approach.
A. Effects of inhalation of insulin
IA responses were evaluated in rats and monkeys exposed
daily for either 1 or 6 months to the insulin inhalation powder
used in Exubera (126). Exposures were up to maximally
tolerated doses that were limited by hypoglycemia. IAs were
measured by both ELISA and radioligand binding (RLB)
assay in serum of rats and monkeys and bronchoalveolar
lavage fluid in monkeys. A weak antibody response was
observed in rat serum by RLB assay only, but IAs were not
detectable in monkey serum or bronchoalveolar lavage fluid.
632 Endocrine Reviews, October 2007, 28(6):625– 652
A comparable weak antibody response in rats was observed
after sc injection. There were no histopathological effects of
exposure in the respiratory tract or bronchial lymph nodes
of either species. Specifically, no evidence was found for an
exposure-related immunogenic response in the respiratory
tract.
The toxicity of a different dry powder formulation of in-
haled insulin was evaluated in beagle dogs exposed for 13 wk
(127). Although the researchers did not describe any IA mea-
surements, they found no effects of exposure on pulmonary
pathological examination.
The IA response in rats, monkeys, and dogs to insulin
inhalation is consistent with findings that inhalation of harm-
less antigens leads to immunological tolerance. Inhaled an-
tigens are likely captured by intrapulmonary dendritic cells.
Under noninflammatory conditions, dendritic cells then mi-
grate to draining lymph nodes and stimulate generation of
T regulatory cells that down-modulate the immune response
to the inhaled antigen. In this manner, immune-mediated
inflammatory responses to reexposure to harmless antigens
are avoided (128).
The effects of a nebulized solution of rDNA human insulin
were evaluated in the NOD mouse model by Harrison et al.
(129). Mice were exposed to either nebulized insulin or
ovalbumin, and IAs were measured using an immunopre-
cipitation assay. Serum IA levels from aerosol–insulin-
treated mice were significantly higher than in ovalbumin-
treated mice. Aerosol–insulin-treated mice also experienced
a significant delay in the onset of spontaneous diabetes. This
antibody response to an inhaled protein may be attributed in
part to inadequate function or numbers of regulatory T cells
in NOD mice (see Section II.F.6) (73).
B. Mucosal delivery of insulin and prevention of type 1
diabetes in NOD mice
Most studies that have evaluated the effects of exogenous
insulin on insulin-specific T cell responses have been con-
ducted to investigate prevention of diabetes in NOD mice.
Exogenous insulin administration by parenteral (130 –134),
oral (135–140), or inhalation (129) routes has been shown to
result in delay of diabetes onset and/or decreased lympho-
cytic infiltration of islets. The mechanism for this protective
effect in NOD mice appears to be immunological rather than
metabolic. An inactive insulin analog (141), insulin B chain
(142), and insulin peptide B9 –23 (143) have all been shown
to be effective in preventing diabetes with sc treatment in
NOD mice. Regulatory T cells induced by insulin adminis-
tration are believed to attenuate the Th1-mediated insulitis
and diabetes through local production of Th2 cytokines (e.g.,
IL-4, TGF-␤, IL-10) in the islet (129, 137, 144).
IV. Characterization of Insulin Antibodies
A. Measurement of insulin antibodies
Initial studies conducted to characterize IAs showed that
insulin/IA immune complexes do not precipitate and there-
fore could not be measured using standard immunoprecipi-
tation/agglutination analytical methods (6). Therefore, the
Fineberg et al. • Immunological Responses
RLB assay was used, and it remains the most common format
in the measurement of IAs and IAAs. Recently, filter plate
separation of insulin/IA complexes from unbound insulin
has been incorporated into RLB assay protocols allowing for
smaller sample volume requirements and higher throughput
(145). In general, RLB assays for total insulin-binding capac-
ity involve coincubating aliquots of serum, buffer, and trace
amounts of monoiodinated 125I-insulin. Unbound and bound
insulin in the serum is often removed before incubation by
using an acidification process followed by treatment with
dextran-activated charcoal. Nonspecific binding can be as-
sessed by incubating a duplicate aliquot of the test serum
with a high concentration of unlabeled insulin and trace
levels of 125I-labeled insulin.
Immune complexes are precipitated by the addition of
antihuman globulin, protein A/G (conjugated to sepharose
beads), polyethylene glycol, or other precipitants. The pellets
of immune complexes are separated by filtration or centrif-
ugation, washed to remove unbound radiolabeled insulin,
and measured on a gamma counter. The mean nonspecific
binding disintegrations per minute (dpm) are subtracted
from total binding dpm to determine specific binding. The
specific binding dpm are converted to microunits or nano-
grams of IA-bound insulin, based on the specific activity of
the labeled insulin. Data may be expressed as insulin-binding
capacity (nanograms or microunits of insulin bound per mil-
liliter of serum). Other laboratories express the data as per-
centage binding (counts in the pellet relative to the total
counts of 125I- insulin added).
The ELISA for IAs involves adsorbing insulin to plastic
wells of 96-well plates with subsequent addition of test sera.
Insulin-specific antibodies in the test sera bind to the insulin
on the plate, and the remaining antibody is washed away.
Anti-Ig labeled with horseradish peroxidase or alkaline
phosphatase is added and binds to the IA. Substrate for
horseradish peroxidase or alkaline phosphatase is added to
each well, and a color product is formed and monitored by
measuring light absorbance. The amount of color product
formed increases with the amount of antibody bound to the
plate. Data for ELISAs are often expressed as end point titers
(minimally detectable) or titers that produce 50% maximal
signal.
In a few studies of serum from patients with diabetes being
treated with insulin, the ELISA was found to correlate with
the RLB assay (99, 146 –148). However, other studies have
demonstrated a poor correlation between the two assays
(149 –151). Serum samples were found to have very high
antibody levels/titers by the ELISA but very low titers when
using the RLB assay and vice versa. The RLB has been shown
to be more sensitive than the ELISA for detection of IAA.
Furthermore, a positive RLB assay for IAA has been found
to be a better predictor for the development of diabetes than
a similar ELISA result (152).
The differences between the RLB and ELISA findings have
been attributed to inherent differences between assay for-
mats. The RLB is a solution-based assay in which low levels
of 125I-labeled insulin are used. Thus, high affinity IAs are
primarily measured in this assay (150, 151). In contrast, as
serum is progressively diluted, the ELISA reaches the stage
of excess antigen and thus has the capacity for measuring IAs
Fineberg et al. • Immunological Responses
of varying affinities (150, 151, 153). In addition, it is believed
that the binding of insulin to solid phase (plastic wells) in the
ELISA results in antibody-binding epitopes that are different
from those available with 125I-labeled insulin in solution
phase.
Although ELISA assays can be processed rapidly and in
larger batch sizes and do not require radiolabeled insulin,
most laboratories that measure IAs use the RLB assay for the
reasons described above. Insulin autoantibody assays are
designed with high sensitivity in mind because autoanti-
bodies are found at significantly lower concentrations than
are IAs that develop in response to exogenous insulin ther-
apy; therefore, autoantibody assays may need further mod-
ification and validation to accurately measure higher IA
concentrations.
Progress has been made toward standardizing assays for
the measurement of IAAs (154, 155), but there is no stan-
dardization for assays designed to quantify insulin-therapy-
induced IAs, particularly when IAs are present at high con-
centrations. Because of the large volume of samples
generated in the Diabetes Prevention Trial Type 1 study,
micro methods were developed to analyze combined IAA
glutamic acid decarboxylase 65 and ICA512 (splice variant of
islet antigen-2 autoantigen, also referred to as IA-2A) auto-
antibodies in less than 3 h (156). Even slight differences in the
concentration of labeled insulin used, washing conditions,
precipitating agent (polyethylene glycol or protein A/G
beads), or reagents can yield significantly different results, so
the absolute concentration of antibodies determined by one
assay cannot be assumed to have a quantitative correspon-
dence with the results of another. Comparison of assays used
by different laboratories would require bridging studies be-
tween the two methods using a set of laboratory standards
of varying antibody levels. This has been accomplished with
regard to IAA, using multiple laboratory quality controls,
and expressing insulin binding as Juvenile Diabetes Foun-
dation units (154). However, the lack of standardization for
the IA assay in patients treated with insulin hampers the
ability to correlate titers with clinical findings when IAs are
measured by different laboratories using different assay con-
ditions and sometimes different assay formats.
B. Immunoglobulin classes
IA responses consisting of virtually all Ig classes and IgG
subclasses have been reported. Insulin-specific antibodies
are primarily composed of IgG1– 4 antibodies (157), but IgM,
IgA, and IgE have been reported. Antiinsulin IgM has been
detected during early insulin treatment (158 –160), and both
Andersen (157) and Reisman et al. (160) reported the presence
of that class in patients with immunological insulin resis-
tance. However, Patterson et al. (161) failed to detect IgM in
patients with diabetes who were treated with insulin. Faulk
et al. (159) reported detectable IgA in patients, and Kniker et
al. (162) associated IgA with allergic reactions in patients
with diabetes.
Most reports regarding allergic reactions implicate IgE
alone or in combination with IgG (see Section V.A) (163–168).
However, correlations have not always been found between
patients exhibiting allergic reactions and assays for insulin-
Endocrine Reviews, October 2007, 28(6):625– 652 633
specific IgE (169), and patients with detectable antiinsulin
IgE have not always exhibited allergic reactions (166, 169 –
171). IgG antibodies have been associated with cases of se-
vere insulin resistance (164, 167); however, most studies
show no correlation between IAs and increasing insulin dose
requirements (see Section V.B.2.b). Antibodies to insulin—
both sc and inhaled—are predominantly of the IgG class (58).
In a study of patients receiving either inhaled or sc insulin,
distributions of IgG subclasses were similar for both thera-
pies (172). In general, IgG1 levels were greater than those of
IgG4, which were slightly greater than IgG2 and IgG3 levels
in both study groups. Similarly, Füchtenbusch et al. (173)
demonstrated that after 12 months of sc insulin treatment,
patients with type 1 diabetes had predominantly IgG1 and
IgG4 antibodies to insulin and that IgG2 and IgG3 were lower
in concentration. These authors also showed that IgG1 levels
tend to decline, whereas IgG4 levels rise with increased du-
ration of sc insulin treatment.
Ig antibody class distributions resulting from inhaled in-
sulin therapy have been reported in two articles describing
immunogenicity data for Exubera and AERx iDMS (58, 60).
Although a wide range of antibody levels were found in the
Exubera studies, only IgG antibodies could be identified;
IgA, IgE, and IgM antibodies were undetectable (58). In 54
patients treated with AERx-iDMS, percentage binding levels
rose substantially from a baseline of 6 to 39% after 12 wk (60).
This rise in antibodies was accounted for primarily by an
increase in IgG antibodies, with small increases in IgE anti-
bodies in four patients. These data are consistent with anti-
body development reported in studies of sc insulins and
suggest that pulmonary antibody responses result from sim-
ilar immunological mechanisms.
C. Antibody affinity
The RLB assay format has been used to assess relative
affinity of IAs. In the classic equilibrium-binding assay, mul-
tiple radioligand binding incubations are set up for a single
test serum. In this way, a fixed concentration of antibody in
the test serum is incubated with increasing concentrations of
unlabeled insulin and a fixed amount of 125I-insulin. Using
higher concentrations of unlabeled insulin allows IAs with
lower binding affinities to bind the labeled insulin. The
amount of 125I-insulin bound in each tube with different
concentrations of insulin is determined, and a Scatchard
analysis of the data has been performed in several studies by
plotting the bound/free vs. bound insulin concentration.
Using this method, Goldman et al. (84) and Brooks-Worrel et
al. (174) found curvilinear Scatchard plots when serum samples
from patients with diabetes treated with insulin were analyzed.
Two populations of IAs were identified: high-affinity antibod-
ies with low binding capacity and low-affinity antibodies with
high binding capacity. These two subpopulations were typi-
cally described by reporting the binding affinity and binding
capacity determined from the apparent linear regions of cur-
vilinear Scatchard plots. The binding capacities, association
constant (Ka), and calculated dissociation constant (Kd) of each
of these two antibody subpopulations were determined. A
mean Kd for the high-affinity and low-affinity antibody pop-
ulations were 1.9 ⫻ 10⫺10 m and 1.6 ⫻ 10⫺6 m, respectively.
634 Endocrine Reviews, October 2007, 28(6):625– 652 Fineberg et al. • Immunological Responses

Similar findings were also observed in an earlier study in which ring polyclonal IAs. This approach has been used to evaluate
a gel-filtration method was used to measure bound and un- epitopes recognized by IAAs (185), but not by IAs.
bound insulin (175). Scatchard analysis has also been exten- Phage display libraries have also been used to investigate
sively used to evaluate the relationship between antibody af- insulin epitopes. The displayed random hexapeptide
finity and hypoglycemia (see Section V.B.3) (49, 176 –180). phagotopes recognized by IAA and IA from different sources
Concerns have been raised with regard to the use of Scat- have been identified and sequenced (186 –188), and consen-
chard analysis to determine the binding affinities and bind- sus sequences have been determined and compared against
ing capacities of polyclonal antibody responses (181, 182). the amino acid sequence of human insulin. These compari-
Antibody responses to most antigens, including insulin, are sons may lead to the identification of new immune markers
polyclonal and comprise antibodies with a wide range of of diabetes disease states based on insulin epitopes recog-
affinities that recognize different epitopes on the insulin mol- nized by IAA and may be used to identify patients for type
ecule (86). Determining antibody affinity and binding ca- 1 diabetes prevention therapy. Phagotype analysis may al-
pacity of two selected subpopulations in Scatchard analyses low IAAs to be distinguished from IAs (186).
excludes information about many other potentially relevant
subpopulations of antibodies. Scatchard analyses are best E. Characterization of insulin antibody immune complexes
suited for investigations of monoclonal antibodies to deter-
It was discovered early on that IAs do not form precipi-
mine binding affinity characteristics. When evaluating poly-
table immune complexes as observed with immune com-
clonal antibody responses, Scatchard analyses are best suited
plexes against larger proteins (6). Using ultracentrifugation
to determine the variance of affinities (181).
methods to analyze the nature of the IA immune complexes,
A simpler way to describe data from the equilibrium bind-
it was determined that insulin may bind IAs and form mono-
ing assay was reported by Heise et al. (59). Insulin-binding
mers and dimers. The dimers consisted of two IgG molecules
capacities determined from the RLB assay and expressed as
bound to one or two molecules of insulin (189). Two different
microunits of insulin bound per milliliter of serum at specific
epitopes on insulin are recognized (bivalent) and act as a
insulin concentrations were reported. Binding capacities at
bridge between two IgG molecules. Larger immune com-
corresponding insulin concentrations can be compared and
plexes are not formed. It is known that small immune com-
analyzed directly. Reporting binding data in this way is not
plexes do not activate complement (C1q binding) and are not
dependent upon applying a two-site binding model to de-
rapidly cleared (190). This is consistent with the finding that
termine the binding/dissociation constants of arbitrarily se-
IA immune complexes remain in circulation and are not
lected antibody subpopulations.
readily cleared. As discussed in Section V.B.1, IA immune
Heise et al. (59) used this reporting method to evaluate the
complexes circulate and thereby act as a “sink” for insulin in
clinical significance of low-affinity and high-affinity anti-
the circulation. It was also determined that C1q binding to
bodies in patients with type 1 diabetes treated with inhaled
immune complexes of serum samples taken from patients
insulin (Exubera). The researchers employed a parallel group
with type 1 diabetes with IAs was not greater than normal
design in which 23 patients were treated with sc insulin and
controls (191). Immune complex size may also explain the
24 patients were treated with inhaled insulin for 24 wk. Mean
rarity of immune complex hypersensitivity reactions or im-
(⫾sd) IA levels for the sc and inhaled insulin groups were
mune complex diseases in patients with high levels of IAs
4.3 ⫾ 9.4 and 101.4 ⫾ 140.4 ␮U/ml, respectively. The binding
who receive daily exogenous insulin (see Sections V.A and
capacities of samples measured with 10⫺10 m (high-affinity)
V.F).
and 10⫺8 m (low-affinity) insulin were determined. For the
Exubera group, the binding capacities of lower affinity an-
tibodies ranged from 667 to 3360 ␮U/ml and from 41 to 387 V. Clinical Significance of Insulin Antibodies
␮U/ml for the higher-affinity antibodies. The greater bind-
The incidence and severity of immunological complica-
ing capacities of the low-affinity antibodies are similar to
tions of insulin therapy have dramatically decreased with the
those reported with Scatchard analysis in previous studies
use of highly purified porcine or rDNA human insulin. Al-
with sc insulin. The binding capacities of the low-affinity and
though the new preparations still produce IAs, the titers are
high-affinity antibodies were compared with postprandial
lower, and they are rarely associated with clinical events.
glucose area under the curve (AUC), duration of insulin
This section will review previous investigations into the re-
action, hypoglycemic events, and fasting plasma glucose. No
lationships of IA and the following clinical issues: hyper-
correlation was observed between these pharmacodynamic
sensitivity reactions, hyper- or hypoglycemia, pregnancy,
markers and binding capacities of high or low affinities.
autoimmune markers, pulmonary function, immune com-
plexes, glycemic variability, and diabetes risk. It should be
D. Insulin epitope and insulin antibody idiotype analyses noted that interpretation of these investigations is often lim-
ited by uncontrolled observations, small sample sizes usually
Numerous mouse monoclonal antibodies against human
consisting of individual case reports, and nonstandardized
insulin have been generated to identify potential binding
antibody measurement methods.
sites within the insulin molecule (51, 56, 59, 183, 184). These
studies have shown that insulin administration could result A. Hypersensitivity reactions
in antibodies capable of recognizing many different epitopes.
Monoclonal antibodies have been used in competition assays Before the 1980s, local hypersensitivity reactions were
to determine the major sites recognized by naturally occur- common complications of insulin therapy. Since the advent
Fineberg et al. • Immunological Responses
of highly purified insulin in clinical practice, the incidence of
allergic complications has markedly decreased (78). Local
and systemic reactions with insulin administration may be
mediated by insulin-specific IgE (type 1, immediate hyper-
sensitivity reactions) or IgG (type 3, intermediate immune–
complex-mediated reactions) antibodies. In addition, non-
antibody-mediated type 4, delayed hypersensitivity
reactions have been reported. Surveys of these published
cases and reviews reveal that the exact type of local reaction
is difficult to identify, and in-depth evaluation of such pa-
tients using skin tests and skin biopsies have not been com-
monly reported; thus, the frequency of insulin reactions as-
cribed in the literature may not be entirely accurate.
Furthermore, local allergic reactions to insulin may be of
mixed types.
1. Type 1 hypersensitivity. Local, immediate reactions are the
least common cutaneous insulin reaction, occurring in less
than 1% of patients (192). These reactions begin within min-
utes of an insulin injection, peak from 12 to 24 h later, and
can be followed by generalized anaphylaxis (170). Type 1
reactions can occur at any time with respect to the initiation
of insulin, particularly when there is a history of interrupted
insulin therapy (78).
Anaphylactic reactions are often preceded by a series of
local, immediate reactions (193). They are initiated by the
binding of antiinsulin IgE to insulin. These complexes bind
to mast cells, releasing into the circulation a variety of va-
soactive substances that mediate the syndrome.
Patients with generalized reactions have higher insulin-
specific IgE levels than do patients with only local immediate
reactions (194). Antiinsulin IgE levels can be 10-fold to 20-
fold higher in patients with allergic disease than in patients
without clinical allergies (167, 195). However, the demon-
stration of circulating antiinsulin IgE does not establish the
diagnosis of insulin allergy because IgE can be found in
patients with no apparent allergy (see Section V.B) (171, 196).
Patients with antiinsulin IgEs can have concomitant insulin-
specific IgG antibodies (88, 167).
2. Type 3 hypersensitivity. Local, intermediate reactions appear
4 to 8 h after insulin injections, peak at 12 h, and generally
subside within a day (197). These phenomena are believed to
be a vasculitic response to soluble antigen-antibody com-
plexes (Arthus reactions) (192, 194, 198). Lipoatrophy— or
loss of fat at insulin injection sites—may represent a persis-
tent, localized Arthus reaction. In biopsies from affected
sites, IgM, IgE, and C3 have been demonstrated in dermal
vessel walls (199). Lipoatrophy was found to occur in up to
20% of patients before the wide availability of highly purified
forms of insulin (197), but it has become virtually nonexistent
in patients treated with purified porcine or human insulin.
It is possible that lipoatrophy is mediated by contaminant-
specific immune responses rather than by IAs. In recent
years, the more commonly observed lipohypertrophy at in-
sulin injection sites has been linked to immune responses to
insulin as well (200).
3. Type 4 hypersensitivity. Local, delayed reactions are the
most common hypersensitivity reaction and usually occur at
the start of insulin therapy (192, 198, 201, 202). They generally
Endocrine Reviews, October 2007, 28(6):625– 652 635
begin 8 to 12 h after an insulin injection and peak at 24 to 48 h.
Most local delayed reactions are mild and self-limited. Al-
though histological analyses suggest that some of these re-
actions may be mediated by sensitized T lymphocytes (192,
194), it is not known how many of these delayed reactions are
truly type 4 reactions. Occasionally, local reactions follow a
biphasic course, with temporary improvement occurring be-
tween an immediate and a delayed reaction. Type 4 hyper-
sensitivity reactions may also be attributed to contaminants
in the older insulin preparations.
4. Allergic response to inhaled insulin. In a pooled analysis of
phase 2 and phase 3 trials of Exubera, no evidence was found
for excess adverse events of an allergic nature despite higher
IA responses in patients who switched to Exubera-based
regimens (Table 4) (58).
B. Glycemic effects of insulin antibodies
A number of cross-sectional studies involving small num-
bers of patients have suggested that IAs can be associated
with alterations in a variety of insulin pharmacokinetic pa-
rameters. Some of these observed alterations might predict
a clinical tendency to hyperglycemia or insulin resistance by
neutralizing the biological effect of circulating bioactive in-
sulin, whereas other observed alterations might predict a
predisposition to clinical hypoglycemia by prolonging du-
ration of insulin action. Most clinical trial data do not show
pharmacodynamic consequences on glucose control corre-
sponding to the pharmacokinetic observations, but case re-
ports of immunological insulin resistance or hypoglycemia
syndromes attributed to IAs continue to be published. This
section will first review the published pharmacokinetic stud-
ies, then clinical data on relationships between IAs and the
following glycemic parameters: insulin resistance, metabolic
control, insulin dose requirements, and hypoglycemia.
1. Antibodies and insulin pharmacokinetics and pharmacodynam-
ics. Insulin-IA interactions like insulin-insulin receptor in-
teractions are reversible and follow the principles of equi-
librium binding. Thus, the amount of insulin bound is
dependent on antibody affinity, insulin concentration, and
antibody concentration (insulin-binding capacity). Hypo-
thetically, the amount and time course of bioavailable insulin
in plasma (insulin pharmacokinetics) could be influenced by
IAs and may be mediated by antibody affinity and binding
TABLE 4. All-causality adverse events of an allergic nature in
controlled phase 2 and phase 3 Exubera studies
Inhaled Subcutaneous
insulin insulin
(n ⫽ 920) (n ⫽ 602)
Subjects’ months of exposure 3791 2939
Body as a whole (includes potential 40 (4.3) 33 (5.5)
injection site reactions and
allergic reactions)
Lipodystrophy 4 (0.4) 4 (0.7)
Respiratory (includes asthma) 107 (11.6) 81 (13.5)
Skin and appendages (includes 64 (7.0) 28 (4.7)
nonspecific rashes and
dermatitis)
Data represent number of adverse events (%). Reprinted with per-
mission of the Journal of Clinical Endocrinology & Metabolism (58).
636 Endocrine Reviews, October 2007, 28(6):625– 652
capacity. However, evaluating the pharmacokinetic effects of
IAs presents several challenges, not the least of which is
validation of the accurate and reproducible measurement of
insulin concentrations in the presence of IAs (203, 204). Free
insulin results may not always be true reflections of bioactive
insulin in the presence of IAs (205).
With an increase in IA binding, there is an apparent in-
crease in the volume of distribution of insulin (206, 207). By
acting as a “sink” for exogenously administered insulin, an
increase in the apparent volume of distribution of free insulin
related to IAs could in theory result in alterations in the
disposition kinetics of free insulin.
It has been reported that patients with elevated levels of
IAs or antibodies with higher binding capacity experience
reduced initial rates of increase and delayed time to peak (120
to 180 min vs. 90 min), and prolonged return to baseline of
plasma-free insulin levels after sc insulin injection in com-
parison with antibody-negative patients or patients with
lower antibody-binding capacity (207–210). Although time to
peak insulin level after administration of insulin appears to
be delayed in most reports, the absolute magnitude of the
peak does not appear to be markedly different between high
and low levels of IAs (178, 208, 210). The presence of IAs has
also been associated with lower maximal (Cmax) free insulin
concentrations and AUCs after sc injection of NPH insulin
and iv infusion of porcine insulin (178, 209). Prolonged half-
life (up to 8-fold greater), increased distribution space (up to
10-fold greater), and faster metabolic clearance rates have
been documented after iv infusion of recombinant human
and/or bovine insulin in patients with type 1 diabetes (206,
207, 211). Alterations in pharmacokinetic parameters ap-
peared to be correlated with various measures of antibody
characteristics, such as insulin-binding affinity, insulin-bind-
ing capacity, or percentage of insulin binding.
Waldhausl et al. (211) found heterogeneous total insulin
responses to iv insulin administration in subjects with more
than 25 ␮g/liter insulin-binding capacity. Their data suggest
a shift in the time course of the plasma free insulin profile but
not in the overall extent of the plasma free insulin exposure.
Measurements of the pharmacodynamic responses to in-
sulin are more clinically relevant than pharmacokinetic re-
sponses and not subject to questions regarding the validity
of insulin measurements in the presence of IAs. Higher post-
prandial plasma glucose concentrations in subjects with IAs
have been described in two pharmacodynamic studies (208,
210). In the first report (208), the effects of IAs appeared to
vary with the insulin species under study. Postprandial
plasma glucose concentrations after injection with rDNA
human insulin did not differ among patients with low (⬍10
U/liter) or moderate (⬎10 U/liter) insulin-binding capacity,
but postprandial plasma glucose concentrations were sig-
nificantly greater in the moderate binding group when bo-
vine insulin was administered.
In the second report, Van Haeften et al. (210) found no
differences in postprandial insulin or glucose when compar-
ing porcine to human insulin injections, although postpran-
dial glucose excursions in response to a standard meal did
appear correlated with the IA binding. Correspondingly,
increases in plasma free insulin levels after injections of both
insulins were negatively correlated with IA binding. The
Fineberg et al. • Immunological Responses
effect of IAs on postprandial insulin and glucose was largely
accounted for by the association constant of the high-affinity
IA binding sites (K1). Peak postprandial glucose was 237 ⫾
10 mg/dl in patients in the upper quartile, compared with
166 ⫾ 12 mg/dl in patients in the lowest association constant
quartile.
In contrast, a prospective, open-label, parallel-group trial
of 47 patients with type 1 diabetes randomized to receive
inhaled insulin (Exubera) or sc regular human insulin, was
designed to evaluate whether IA development with inhaled
insulin is associated with the loss of postprandial glucose
control (59). Mean IA levels increased from baseline after
inhaled insulin treatment but not after sc regular insulin
treatment. The researchers found no significant differences in
postprandial plasma glucose profiles between treatment
arms, and no correlation between postprandial blood glucose
exposure and antibody-binding affinity was apparent.
A body of literature suggests antibody-mediated prolon-
gation of elevated insulin levels after sc injection (206 –208,
211–213); however, pharmacodynamic consequences of this
finding have not been consistently demonstrated. Impor-
tantly, increased rates of hypoglycemia have not been con-
firmed in clinical trials (see Section V.B.3.d). Although IAs
may not cause hypoglycemia, Bolli et al. (212, 213) published
two studies that demonstrated prolonged recovery times
from experimentally induced hypoglycemia in patients with
IAs. The researchers concluded that patients with insulin-
dependent diabetes can have impaired glucose counterregu-
latory hormone reserve, which can be compounded by a
prolongation of the half-life of insulin by IAs. To explain such
an IA-mediated phenomenon mechanistically, one must pos-
tulate dissociation of free insulin from antibody bound in-
sulin complexes (see Section V.B.3.d).
IAs have not consistently been shown to have the property
of prolonging the duration of insulin action. Euglycemic
clamp studies have been conducted to explore the effect of
IAs on the pharmacodynamic response to exogenously ad-
ministered insulin. In one study, glucose infusion rates did
not vary between subjects with high and low insulin-binding
capacity but were lower than in subjects without diabetes
(lower glucose infusion rates indicate decreased insulin ac-
tion) (211).
Peters et al. (209) noted that exposure (AUC) to free insulin
and glucose infusion rates were lower in patients with
greater than 10% antibody binding compared with subjects
with less than 1.5% antibody binding. Gardner et al. (214)
found no correlation between antibody status and the onset
of action of insulin administered sc. Furthermore, the peak
effects of insulin action as well as the duration of insulin
action were similar both in patients who were antibody pos-
itive and in those who were antibody negative. Similarly,
Heise et al. (59) were not able to detect a significant difference
in duration of inhaled insulin action in subjects with or with-
out IAs. They reported no correlation between IA-binding
affinity and the duration of insulin action.
It seems possible that pharmacokinetic differences attrib-
utable to IAs may be observed, but the effects are small in the
context of the myriad of other factors that influence glycemic
response. The inconsistency between pharmacokinetic and
pharmacodynamic observations may lie in the incomplete
Fineberg et al. • Immunological Responses
understanding of the in vivo conditions that control insulin
bioactivity in the presence of IAs. This is further complicated
by the imprecise relationship between insulin pharmacoki-
netics and pharmacodynamics that exists in the absence of
IAs (215, 216).
2. IAs and hyperglycemia/interference with insulin action
a. Immunological insulin resistance. IAs are determined to be
present when binding of labeled insulin is demonstrated in
vitro. Whether in vitro binding quantitatively reflects the
binding of circulating insulin in vivo is difficult to directly
determine (see Section V.B.1). If antibody binding did occur
in vivo to significant levels, one might expect to observe
increasing insulin dose requirements and, possibly, worsen-
ing glycemic control in patients with diabetes. Although a
rare syndrome of severe insulin resistance has been de-
scribed in patients with high IA levels, a mechanism for a
causal relationship between the antibodies and the syndrome
has not been clearly established. Furthermore, IAs do not
correlate with measures of glycemic control or insulin dose
requirements in most large population studies.
Insulin resistance can be defined as a daily insulin dose
requirement that exceeds the normal daily pancreatic output
in the nondiabetic state, i.e., approximately 40 ⫾ 20 U/d
(217). Severe insulin resistance is usually defined as insulin
requirement of more than 200 U/d for at least 2 d (14, 217,
218). The incidence of insulin resistance in patients with
diabetes was estimated to be 0.1% in a study at the Joslin
Clinic between 1940 and 1960 (14). However, this incidence
was acknowledged as likely to represent an overestimate
because of referral bias, and IA measurements were not
reported.
In a subsequent case series, Davidson and DeBra (218)
characterized 35 patients with severe insulin resistance as-
sociated with the presence of high levels of circulating IAs.
Patients studied in this series had mean daily insulin re-
quirement of 550 U/d (range, 200 to 2000 U/d) and maxi-
mum insulin-binding capacity greater than 10,000 ␮U/ml. In
addition, the following underlying explanations for large
insulin requirements were ruled out: diabetic ketoacidosis,
significant infection, significant dietary indiscretion, li-
poatrophic diabetes, significant complicating endocrine dis-
ease, insulin receptor defects, antibodies to insulin receptors,
or factitious claim of insulin dose greater than 200 U/d.
At least 60% of patients diagnosed with immunological
insulin resistance had a history of diabetes with onset after
age 30 yr, suggesting that the syndrome is more commonly
observed in patients with type 2 diabetes (20, 218). The onset
and subsequent course of insulin resistance is often associ-
ated with symptomatic hyperglycemia, including episodes
of ketoacidosis and hyperosmolar coma (217, 218). The du-
ration of insulin therapy before the onset of severe insulin
resistance has been reported to range from 1 month to 15 yr
(217, 218), although 50 – 85% of patients with insulin resis-
tance received insulin for less than 1 yr, and 10 –25% received
insulin for less than 1 month before the onset of severe insulin
resistance (217, 218). Insulin allergy may coexist with im-
mune insulin resistance in 10 –35% of cases (20, 217). Some
cases of insulin resistance that occurred as insulin allergy
spontaneously subsided, prompting speculation that in-
Endocrine Reviews, October 2007, 28(6):625– 652 637
creasing concentrations of IgG can inhibit IgE-mediated clin-
ical events (20, 167, 219). Although the dominant feature of
this syndrome is decreased insulin action, episodes of hy-
poglycemia can occur. Furthermore, the frequency of hypo-
glycemia may diminish after resolution of the insulin-resis-
tant state (218).
Immunological insulin resistance is thought to occur less
commonly now than it did before the 1980s, but cases of at
least partial resistance attributed to IAs continue to be re-
ported— even in patients treated only with human insulin or
insulin analogs (30, 164, 220 –227). Severe insulin resistance
has also rarely been described in the setting of high levels of
serum insulin-binding activity associated with underlying
chronic lymphocytic leukemia, lymphoma, multiple my-
eloma, and macroglobulinemia (228). Possibly, the severe
resistance in these cases was due at least in part to the pro-
duction of monoclonal paraproteins with significant insulin-
binding activity, although monoclonal insulin binding was
demonstrated in only two cases. Insulin resistance occurring
in patients with hematological malignancy is not always
associated with an insulin-binding monoclonal protein (87).
b. Treatment of immunological insulin resistance. Because im-
mune insulin resistance tends to be self-limited (50% of pa-
tients are insulin resistant for ⬍ 6 months and 75% for ⬍ 1
yr), therapy is aimed at speeding a remission while avoiding
metabolic complications (14). The initial treatment for severe
insulin resistance has generally been to switch therapy to
alternative insulin formulations. Before the advent of human
insulin in clinical practice, such patients were successfully
treated by switching to purified porcine insulin (20, 229), fish
insulin (219, 230), and modified insulins, including sulfated
insulin (218) and desalanine-porcine insulin (231). The ben-
eficial effect on immunological insulin resistance of sulfated
insulin may have been mediated by CD8⫹ T cells (232).
In patients with type 2 diabetes, insulin cessation may be
a successful treatment for immunological insulin resistance
(221, 233). Switching insulin-resistant patients to human in-
sulin (87) or insulin lispro (220, 224) or from U-100 to U-500
insulin has also been successful (234). When switching in-
sulin formulations in patients with immune-mediated insu-
lin resistance, substantially lower doses may be necessary to
avoid hypoglycemia (217). In a case report, endogenous in-
sulin stimulated by tolbutamide was found to be effective in
a patient with type 2 diabetes who was felt to be unrespon-
sive to exogenous insulin (235).
Glucocorticoids are a second-line therapy for patients with
nonremitting immunological insulin resistance who do not
respond adequately to switching insulin formulations (14, 15,
20, 236), or in whom ketoacidosis ensues (79). Prednisone at
doses of 60 to 80 mg/d can result in lower insulin require-
ments in up to 75% of patients, often within a few days and
even before declines in antibody levels (20). Responses in
some cases can be dramatic, and severe hypoglycemia can
ensue (14, 217, 236), leading some to recommend that glu-
cocorticoid therapy be administered in a hospital or other
monitored setting (217). Prednisone doses should be tapered
when there is a clinical response. Often, 2 to 3 wk of glu-
cocorticoid therapy is adequate to achieve a sustained re-
sponse (20, 217). In rare cases, plasmapheresis (223, 237) or
638 Endocrine Reviews, October 2007, 28(6):625– 652
cytotoxic immunosuppressive agents, such as 6-mercapto-
purine (238), have been used for immune insulin resistance
not related to an underlying paraproteinemia.
Although high levels of IAs have been demonstrated in
patients with syndromes of severe insulin resistance, it is
difficult to arrive at a causal relationship between the anti-
bodies and the syndrome with available data. Insulin dose
requirements are not predicted by insulin-binding capacity
measurements, even in patients with severe insulin resis-
tance (20, 218). Furthermore, there is substantial overlap in
antibody levels between resistant and nonresistant patients
(218). An approximate maximum antibody-binding thresh-
old of 30,000 ␮U/ml, above which patients may be at risk for
this syndrome, has been suggested; however, 15% of patients
with the syndrome described in the literature have values
below this range (217, 218).
If an antibody threshold exists, one still needs to postulate
that there is an undiscovered attribute of the antibodies or
related cofactors that determines why some patients develop
insulin resistance, whereas other patients with comparable
antibody levels do not. Subpopulations of antibodies iden-
tified by insulin affinity have been correlated with clinical
resistance and response to therapy in case reports (223, 239,
240). Both high-affinity (221) and low-affinity (240) antibod-
ies have been identified as correlating best with clinical
findings.
IA levels clearly do not by themselves explain why some
patients with this syndrome require 200 U/d and others
require 2000 U/d (241). Furthermore, the mechanism by
which IAs would cause insulin resistance is not clear. IAs
could function as a circulating binding reservoir for insulin;
however, in theory, once this reservoir is saturated, normal
free insulin levels should be achievable with typical insulin
doses (20, 217, 241, 242).
Cyclical expression of important idiotypic determinants—
despite unchanged levels of total insulin binding— could
explain clinical syndromes that occur and remit spontane-
ously. Although such cyclic idiotypic expression has been
described in one report (243), it is not clear that this phe-
nomenon involves the insulin-receptor-binding epitope,
which would be necessary to explain the occurrence and
remission of immunological insulin resistance. Rarely, insu-
lin resistance is associated with antiinsulin receptor antibod-
ies (244). Most patients with insulin resistance due to insulin
receptor antibodies have evidence for systemic autoimmu-
nity to noninsulin autoantigens (244), unlike the immune
response to exogenous insulin in patients with diabetes (245).
Modern definitions of immunological insulin resistance re-
quire excluding the presence of insulin receptor antibodies
(218).
c. Overall metabolic control and insulin dose requirements.
Some pharmacodynamic studies that examined the relation-
ship between IAs and postprandial glycemia have suggested
that IAs can be associated with relative hyperglycemia after
meals (49, 208, 210) (see Section V.B.1). A prospective study
evaluating postprandial glucose tolerance during the devel-
opment of IAs with inhaled insulin therapy showed no loss
of postprandial glucose control (59).
Some investigators have reported IA levels correlated with
Fineberg et al. • Immunological Responses
higher average glucose in populations of patients (246 –248),
but most showed no correlation between IA and glucose
control— usually measured as glycated hemoglobin (19, 58,
99, 114, 241, 249 –265). Most studies published since 1980
have shown no evidence for a significant relationship be-
tween IA and average glycemia (58, 99, 114, 241, 250 –256,
259 –263, 265).
Researchers have postulated that IAs can be associated
with mild degrees of insulin resistance, which would be
detected clinically as mild to moderate increases in insulin
dose requirements. Additionally, some studies have sug-
gested that increasing insulin dose requirements correlate
with IA levels (249, 264, 266 –268); however, most studies
have showed no relationship or correlations between IAs and
decreasing dose requirements (58, 99, 114, 241, 247, 250, 252,
255, 259, 260, 262, 265, 266, 269 –273). Evidence suggests that
IA-positive patients who switch insulins to a less immuno-
genic preparation can experience reduced dose requirements
in concert with declining IA levels (27, 264, 274, 275), or that
switching insulin preparations can result in declining IA
levels without decreased insulin dose requirements (19, 251,
263, 276 –280).
3. IAs and hypoglycemia. Rare syndromes in which recurrent
or prolonged hypoglycemia is the dominant feature have
been attributed causally to IAs. Most frequently, this situa-
tion is encountered in insulin autoimmune syndrome (IAS;
also called Hirata’s disease), in which nondiabetic patients
with no history of insulin exposure spontaneously develop
IAAs and hypoglycemia. Evidence that IAs induced by ex-
ogenous insulin therapy can also cause hypoglycemia is lim-
ited to case reports. Although hypoglycemia is the most
notable feature of these syndromes, some affected patients
have also been reported to have clinical evidence of atten-
uated insulin action (including severe insulin resistance). It
remains to be determined whether antibodies associated
with pathological hypoglycemia can be distinguished in vitro
from the far more commonly occurring IAs that are not
linked to clinical hypoglycemia.
a. Hirata’s disease. IAS consists of high levels of IAs with or
without concomitant Graves’ disease associated with fasting
hypoglycemia in insulin-naive patients. The disease was first
described in 1970 by Hirata et al. (281). This disorder is
HLA-linked (282) and is the third most common cause of
hypoglycemia in Japan, but has been sporadically reported
outside Japan (283, 284).
In most patients, remission occurs within 6 months; how-
ever, life-threatening hypoglycemia may necessitate mea-
sures such as plasmapheresis (285, 286). Impairment in glu-
cose tolerance is reported for some of these patients (283).
Patients with IAS often have late postprandial hypoglyce-
mia, possibly due to late release of endogenous insulin from
the autoantibody pool (286). It is also possible that some
instances of hypoglycemia associated with IAS can be at-
tributed to the development of antiidiotypic antibodies that
have insulin agonistic properties. Among white patients in
the United States, IAS manifests primarily as postprandial
hypoglycemia; as in Japanese patients, the syndrome can be
associated with polyclonal or monoclonal IgG insulin-bind-
ing antibodies (287).
Fineberg et al. • Immunological Responses
b. Hypoglycemia and antibodies to exogenous insulin. Pub-
lished case reports have attributed unusually prolonged ep-
isodes of hypoglycemia to high levels of antibodies to ex-
ogenous animal insulin (176, 178, 288, 289). The first case,
reported in 1960 by Harwood (288), described a 44-yr-old
woman with type 1 diabetes who for 9 yr experienced pe-
riods in which she would need to discontinue insulin therapy
for up to 23 d because of prolonged hypoglycemia. She was
found to have 106,000 ␮U/ml insulin-binding capacity with
an unusually slow rate of dissociation of the antibody-anti-
gen complex.
Seven case reports have been published of hypoglycemia
occurring in patients with antibodies found in the setting of
human insulin therapy (223, 239, 290 –293). Interestingly,
these cases were all reported from Japan, where IAS is
thought to be more prevalent than in the rest of the world.
These cases also occurred predominantly in patients over 70
yr of age, as is the case with IAS.
Hypoglycemia has also been reported in up to 54% of
patients receiving a combined pancreas and kidney trans-
plant (294). Tran et al. (295) compared patients who had
repeated episodes of hypoglycemia or hypoglycemic symp-
toms after a pancreatic transplant matched with patients who
did not have hypoglycemia after pancreatic transplant. They
found a decrease in the ratio of fasting free insulin to total
insulin in the patients who had a hyperglycemic response to
a liquid meal challenge. This subgroup had a substantial
increase in total, but not free, insulin concentrations. Al-
though IAs were not directly measured, these data are con-
sistent with the hypothesis that hypoglycemia was associ-
ated with high levels of circulating IAs (295).
Although anecdotal information from these case reports
suggests that very high levels of IAs can be associated with
unusual hypoglycemia syndromes, studies of large popula-
tions have failed to establish a relationship between IAs and
hypoglycemia event rates (58, 59, 251, 259, 263, 265, 296, 297).
c. Hypoglycemia in IIP patients. Intraperitoneal insulin de-
livery via IIPs has been associated with high levels of IAs in
40% of patients with type 1 diabetes, and these antibody
levels persist for at least 3 yr (see Sections II.D, II.F.3, and
II.F.4) (50). Lassmann-Vague et al. (99) found no correlations
between IAs and hypoglycemic event rates experienced by
24 patients who had been treated for 2 yr with ip insulin
therapy. IA levels also did not correlate with glycated he-
moglobin, insulin requirements, or free insulin levels in these
patients. In a contrasting study, Jeandidier et al. (49) tracked
IA response in 62 patients with type 1 diabetes on ip pump
therapy for 2 yr. Although an association between IA status
and the number of low blood glucose values per month was
reported, severe hypoglycemic event rates dropped dramat-
ically in patients implanted with ip pumps regardless of IA
responses.
The possibility that nocturnal hypoglycemia could be re-
lated to the antibody response— despite downward titra-
tions of overnight basal insulin infusion—was first raised by
Olsen et al. (50), who described four of 25 patients using IIPs
with unexplained significant decreases in overnight basal
insulin dose requirements. All four of these patients had peak
IA levels above 200 ␮U/ml (range, 205 to 1021 ␮U/ml),
Endocrine Reviews, October 2007, 28(6):625– 652 639
although one patient remained symptomatic despite a de-
cline in antibody level to 84 ␮U/ml while still receiving ip
insulin therapy.
d. Clinical trials and limitations of literature. Although infor-
mation from case reports suggests that high levels of IAs can
be associated with prolonged periods of hypoglycemia, stud-
ies of large populations have failed to establish a relationship
between IAs and hypoglycemia event rates (58, 59, 251, 259,
263, 265, 296, 297). In a pooled analysis of phase 2 and phase
3 trials involving more than 350 patients with type 1 diabetes
and more than 400 patients with type 2 diabetes treated with
inhaled insulin (Exubera), no relationships between hypo-
glycemic event rates and IA levels were observed (58).
Published case reports describe patients with unusual hy-
poglycemia syndromes who are found to have very high
levels of IAs. Because of an inherent reporting bias, however,
it is difficult to determine from the literature how many
patients with high antibody levels do not have hypoglyce-
mia. Likewise, it is difficult to know the frequency of similar
syndromes in patients who have low IA levels.
Although both hypoglycemia and IAs are common in pa-
tients receiving insulin, a causal relationship between the
two has been difficult to demonstrate. For the presence of
antibodies to explain hypoglycemia in such patients, it must
be presumed that antibodies first bind the insulin in circu-
lation and then later dissociate from the insulin, allowing
activation of cellular insulin receptors. Despite multiple at-
tempts to characterize antibodies associated with hypogly-
cemia syndromes, it is not clear that consistent affinity/
binding capacity profiles can distinguish antibodies in
patients with and without hypoglycemia syndromes (49,
179).
However, recent studies have reported potential progress
toward identifying in vitro characteristics of IAs associated
with clinical hypoglycemia syndromes in small numbers of
patients (177, 180, 222, 223, 298). One of those studies has
suggested that insulin-receiving individuals with high anti-
body levels and recurrent hypoglycemia have a higher dis-
sociation constant for insulin measured by surface plasmon
resonance than monoclonal antibodies to human insulin
(298). It remains to be seen whether these recently proposed
assays correlate better with symptoms than with standard IA
measurements.
It is also not clear what factors may precipitate the hy-
pothesized unregulated dissociation of antibody-insulin
complexes. Because some patients are reported to have noc-
turnal hypoglycemia, researchers often presume that bind-
ing equilibrium is shifted toward antibody-insulin dissoci-
ation when free insulin concentrations decline as time
elapses overnight from evening insulin administration.
However, were this the case, a correlation between IA and
lower fasting glucose measured in the morning would be
expected, and no such relationships have been demonstrated
(58, 59, 251, 256).
Clinical trials comparing inhalation with sc delivery of
insulin in patients with type 1 or type 2 diabetes have dem-
onstrated reduced fasting plasma glucose levels by as much
as 40 mg/dl associated with inhaled insulin, the mechanism
for which is unknown (299). However, IAs were found not
640 Endocrine Reviews, October 2007, 28(6):625– 652
to correlate with these fasting glucose levels (58) (Fig. 4, from
Pfizer data on file). This is consistent with the observation
that treatment group differences in fasting plasma glucose
are fairly consistent in patients with type 1 or type 2 diabetes,
whereas IA responses to inhaled insulin are significantly
higher in patients with type 1 compared with patients with
type 2 diabetes (58).
To be clinically significant, prolonged dissociation of IA
complexes should result in a measurable prolongation of the
duration of insulin action. This hypothesis was examined in
a prospective 6-month pharmacodynamic study in which
duration of insulin action was measured by 12-h euglycemic
clamps (59). This study showed that in patients with IA
responses to inhaled insulin (Exubera), duration of insulin
action was unchanged, despite a rise in IA levels, and no
differences were found between these patients and those
treated with sc insulin. Furthermore, there were no correla-
tions between high-affinity or low-affinity insulin binding on
duration of insulin action, fasting glucose, or clinical hypo-
glycemic events (see Section V.B.1).
Clinical trial data suggest that hypoglycemia does not
correlate with IAs in large populations of patients. Never-
theless, rare patients with high antibody levels may develop
hypoglycemia syndromes owing in part to prolonged or
unregulated appearance of free insulin in the circulation
(178). Because the levels of IAs do not in themselves account
for recurrent hypoglycemia, it seems likely that clinical hy-
poglycemia in this setting would have a multifactorial eti-
ology, perhaps involving deficient glucose counterregula-
tion coupled with a prolonged half-life of free insulin (213,
300).
C. Pregnancy
Maternofetal IgG transfer begins early in the second tri-
mester, with most antibodies transferring to the fetus during
the third trimester (301). Because organogenesis occurs dur-
ing the first trimester, a lack of correlation between congen-
ital malformations and maternal IAs is not surprising (302).
Recently, it has been shown that transmission of maternal
antibodies to exogenous insulin does not affect diabetes risk
in offspring (303).
Pregnancy-related risks from IAs have not been clearly
demonstrated. Some early papers reported associations be-
tween IAs and neonatal hypoglycemia (304 –306). However,
these papers did not adequately describe maternal glycemic
FIG. 4. In a long-term phase 3 study (data on file, Pfizer
Inc.), no correlation was found between fasting plasma
glucose and IA levels in patients with type 1 diabetes
(n ⫽ 252) treated with inhaled human insulin (Exubera)
at 24 months (Spearman correlation coefficient,
⫺0.073).
Fineberg et al. • Immunological Responses
control in the patients studied. Maternal glycemic control is
important because it is known to influence many fetal and
neonatal risks, including neonatal hypoglycemia and mac-
rosomia (307). Most notably, despite the strong association
between neonatal hypoglycemia and increased birth weight
in the infant of a diabetic mother (307), no consistent linkage
was found between IAs and birth weight, even in the early
studies that linked IAs to neonatal hypoglycemia. Further-
more, hypoglycemia remains a common complication in ne-
onates born to mothers with diabetes, despite the fact that
insulin preparations of low immunogenicity are now in rou-
tine use (22, 308).
The potential for increased incidences of neonatal hypo-
glycemia, respiratory distress syndrome, and hypocalcemia
has also been suggested in small studies published from 1980
to 1990 (304, 305, 309, 310); however, these reports also lacked
adequate documentation of maternal glycemic control. Sub-
sequent studies, performed in the modern era of prenatal
care for women with diabetes, argue against a connection
between IAs and fetal morbidities (254, 311). Additionally,
multiple studies have failed to show a relationship between
maternal IAs and birth weight. A randomized trial compar-
ing human insulin with animal insulin during pregnancy
found that improved glycemic control, not IAs, influenced
infant birth weight (312). Wellik et al. (313) showed no cor-
relation between IAs and neonatal glucose level or birth
weight. Three recent studies with substantially larger sample
sizes reported no relationships between IAs and birth weight
(314 –316). The most recent report found similar birth
weights in offspring of 138 mothers with type 1 diabetes
across a range of maternal IA levels and cord blood insulin
levels (314). Islet autoantibody concentrations also were
found to have no influence on birth weight.
Two hypotheses have been put forward to explain a pos-
sible relationship between maternal IAs and neonatal hypo-
glycemia (79), both of which would predict clear correlations
between IA levels, birth weight, and neonatal hypoglycemia
risk. In the first, maternally derived antibody interference
with insulin action in the fetal circulation could result in
compensatory fetal hyperinsulinemia. In this scenario, fetal
hyperinsulinemia results in increased birth weight and neo-
natal hypoglycemia. Although neonatal cord blood C-pep-
tide levels (reflecting endogenous insulin secretion) were
found to correlate with IAs in one study (305), the finding
was not reproducible (302, 315). Recently, no relationship
Fineberg et al. • Immunological Responses
was found between cord blood insulin levels and birth
weight (314). Furthermore, multiple studies, including the
only randomized prospective study, have found no relation-
ship between IAs and fetal birth weight (312).
A second hypothesis suggests that insulin is transferred to
the fetus via IA complexes. These complexes could then
dissociate in the fetal circulation, releasing bioactive insulin.
Were this to happen during fetal life, increased birth weight
would be expected. As discussed, multiple studies have
failed to show relationships between maternal IAs and mac-
rosomia. If IA complex dissociation occurred after birth, un-
usually prolonged neonatal hypoglycemia syndromes might
be expected, given that the biological half-life of circulating
IgG is approximately 23 d (317). No distinguishing clinical
characteristics of neonatal hypoglycemia associated with
IAs, such as unusual prolongation, have been described in
babies born to mothers with IAs.
D. Autoimmune diseases
Antibody responses to exogenous insulin have not been
shown to cause generalized immune activation resulting in
autoimmune disease states. Lassmann-Vague et al. (245)
measured a panel of autoantibodies before implantation with
pumps for ip insulin delivery and then subsequently every
year in 28 patients with type 1 diabetes. At baseline, 19 of 28
patients were negative for all tested autoantibodies (antithy-
roglobulin, antithyroperoxidase, gastric parietal cell, smooth
muscle, mitochondrial, liver-kidney microsome, antinuclear,
antiendomysium, and antigliadin antibodies). During 2 yr of
ip insulin treatment, the sera of the patients negative at
baseline remained negative throughout the study, despite
the expected IA response (see Sections II.D and V.B.2.c). Nine
patients with preexisting autoantibodies had no change in
most autoantibody titers. Two of these patients had increases
in antithyroperoxidase titers, whereas three patients had de-
creases in these titers. No difference was seen in IA responses
to ip insulin delivery in the nine patients with preexisting
autoantibodies relative to those who did not have preexisting
antibodies.
E. Inhaled insulin antibodies and pulmonary function
Inhaled human insulin is associated with higher levels of
IAs compared with sc human insulin (see Section II.E). In-
haled insulin (Exubera) is also associated with small treat-
ment group differences in pulmonary function tests, which
develop early after treatment initiation, are not associated
with clinical sequelae, are nonprogressive with up to 2 yr of
therapy, and are reversible with treatment discontinuation
(63, 318). This pulmonary function test change does not,
however, appear to be mediated by the immunological re-
sponse to inhaled insulin. Teeter and Riese (63) showed that
the treatment group difference in forced expiratory volume
in 1 sec (FEV1) is manifest as early as 2 wk after inhaled
insulin initiation, at which time the mean IA response has not
yet evolved. Furthermore, Fineberg et al. (58) showed no
correlation between IA levels and FEV1 in pooled analyses of
inhaled insulin (Exubera) clinical trials.
Endocrine Reviews, October 2007, 28(6):625– 652 641
F. Immune complexes
A series of reports published from the 1960s through the
1980s yielded conflicting results regarding insulin-antiinsu-
lin immune complexes in the development of long-term di-
abetic complications in animal models (319). Studies in hu-
mans have not shown consistent links between IAs and
diabetic complications. Although some studies have shown
increased levels of immune complex formation in patients
with diabetic microangiopathic complications compared
with patients without complications (320 –322), these im-
mune complexes often do not contain insulin or IA (321–324).
Furthermore, insulin administration does not contribute to
this immune complex formation (325). The nonspecific im-
mune complexes observed in patients with diabetes may
reflect general inflammatory reactions associated with an-
giopathies (321).
Overall, no direct evidence has shown that immune com-
plexes, insulin immune complexes, or IAs are capable of
mediating vascular or glomerular damage. Associations
have been reported, but they have been largely based on the
prevalence of immune complex detectability rather than on
quantitative measurements of the amount of immune com-
plex present (326). Andersen (327) showed that high porcine
insulin-binding levels were more frequent in patients with
complications from diabetes than in patients without com-
plications but found no significant differences in mean an-
tibody levels between groups. Virella et al. (328) found cor-
relations between the presence or absence of detectable
insulin-antiinsulin immune complexes and the presence or
absence of some diabetic complications.
Although immune complexes in insulin-treated patients
have been associated with procoagulant markers in some
studies (323, 329, 330), the majority of studies have shown no
relationship between IA and diabetic angiopathic complica-
tions. Specifically, no relation has been found between IAs
and histological findings of nephropathy (331), muscle base-
ment membrane thickness (332), clinical nephropathy (114,
333, 334), clinical retinopathy (114, 273, 321–323, 332–336),
clinical autonomic neuropathy (337), clinical peripheral neu-
ropathy (338), or complement activation in patients with
diabetes (339).
The absence of a pathogenic effect may be related to the
properties of the immune complex. It has been observed that
insulin and IA immune complexes do not precipitate (at
antigen or antibody excess) and cannot be detected by stan-
dard immunodiffusion methods (340). In addition, insulin-
insulin–antibody immune complexes are monomers or
dimers, and large immune complexes are not formed. Small
immune complexes do not readily bind to C1q complement
and are, therefore, not readily cleared by the reticuloendo-
thelial system of the liver or spleen (190). Small immune
complexes have a similar half-life to circulating Ig molecules,
which explains the high levels of circulating total insulin
(antibody bound) in patients with high levels of IAs (see
Section IV.E).
G. Buffering effect of insulin antibodies
Both glycemic stability and instability have been attrib-
uted to the presence of IAs. Instability has been described
642 Endocrine Reviews, October 2007, 28(6):625– 652
only in case reports and is characterized either by both hy-
perglycemia and hypoglycemia occurring within a single
24-h interval or by periods lasting days to weeks of hyper-
glycemia alternating with periods of hypoglycemia of similar
duration (49, 176, 178, 223, 229, 239, 290, 291, 341, 342). Some
of these case reports describe patients with relative overnight
hypoglycemia and daytime hyperglycemia, although there is
no clear mechanistic explanation for such diurnal patterns
(see Section V.B.3.c). Because these glycemic instability syn-
dromes are rare and variable in nature and because in vitro
parameters are not predictive of clinical findings (see Section
V.B), it is difficult to establish cause and effect relationships
between the antibodies and such glucose variability
syndromes.
Conversely, because studies in patients being treated with
insulin—as well as those in individuals with insulin auto-
immune-hypoglycemia syndrome— have suggested that the
presence of high levels of IAs is associated with a retarded
disappearance rate of insulin, some authors have suggested
that antibodies may serve as a “buffer” to glucose variability
(see Section V.B.1) (6, 178, 223, 286, 343, 344). Limited studies
demonstrated that this slowed rate of insulin disappearance
appeared to decrease the likelihood of diabetic ketoacidosis,
contributing to stability of glycemic control (269, 345–347).
However, for the majority of patients, the levels of antibodies
seen with insulin therapy are unlikely to result in significant
effects on glycemic variability (348).
H. Insulin antibodies and risk for diabetes
1. IAs and ␤-cell loss. Although the spontaneous appearance
of IAA in nondiabetic patients is known to be predictive of
type 1 diabetes development (349, 350), there is no evidence
that IAAs or IAs themselves causally mediate ␤-cell destruc-
tion. Support for this comes from studies in which nondia-
betic patients with circulating antibodies to insulin were
followed for the onset of diabetes (351–354). For example,
Bock et al. (351) investigated whether insulin treatment of
patients without diabetes who were undergoing insulin
shock therapy for psychiatric disorders would be at in-
creased risk for the development of diabetes. In their retro-
spective analysis of 481 patients observed for an average of
22 yr, one patient developed type 1 diabetes, and 12 devel-
oped type 2 diabetes. These instances did not differ from the
background population. Only two of 27 patient samples ex-
amined were positive for IAs, and none was positive for islet
cell antibodies. The researchers concluded that exogenous
insulin used in diabetes prevention trials was safe and would
not increase the risk for diabetes.
2. Cellular immune response to exogenous insulin. Most of the
reports on insulin-specific T cell responses have focused on
the autoreactive T cells involved in the pathogenesis of type
1 diabetes. Studies using NOD mice have demonstrated the
presence of CD4⫹ and CD8⫹ cells that recognize insulin and
lead to the destruction of ␤-cells in this model of spontaneous
autoimmune diabetes (355). However, multiple interven-
tions, including parenteral, oral, or aerosolized insulin treat-
ment (see Sections III.A and III.B) have been shown to delay
the onset of diabetes in these mice.
Fineberg et al. • Immunological Responses
T cell clones have been generated from the draining pan-
creatic lymph nodes of patients with type 1 diabetes that
were found to be responsive to insulin (356). These data
indicate that insulin may be one of the initial antigens rec-
ognized by autoreactive T cells before the onset of clinical
type 1 diabetes and exogenous insulin treatment.
A few studies have evaluated insulin-specific T cell re-
sponses in patients with diabetes being treated with insulin.
The most commonly used assay to measure insulin-
responsive T cells involves coincubating peripheral blood
mononuclear cells with or without insulin for 6 to 10 d. The
proliferative response is measured by the incorporation of
radio-labeled nucleotide (357). T cell responses in patients
with type 1 diabetes treated with porcine and bovine insulin
or porcine-bovine mixtures were first reported in 1975 (358,
359). Patients with recent-onset diabetes (32%) and those
with long-standing illness (47%) were found to have positive
T cell responses to human insulin (360). Since T cell help is
required for B cell development and antibody production, IA
levels might be expected to correlate with T cell responsive-
ness. However, poor correlations between IA and/or IAA
levels and T cell responsiveness have been observed (360 –
362). Patients with high IAs or IAAs had very low T cell
responses, and patients with low IA or IAA levels had high
T cell responses. The relatively infrequent finding of a strong
cellular immune response to insulin in patients with type 1
diabetes treated with exogenous insulin may be partly ex-
plained by the activation of regulatory T cells (75).
Additional investigations suggest that T cells respond to
different regions (epitopes) of the insulin molecule. T cell
responses are greater with the B chain than the A chain of
insulin (363, 364). Theoretically, insulin-specific T cells mea-
sured in these studies may be autoreactive T cells generated
by endogenous insulin, autoreactive T cells expanded by
exogenous insulin, and/or T cells initially generated by ex-
ogenous insulin; however, based on the assays used, it is not
possible to differentiate among these possibilities. T cell re-
sponses with exogenous insulin treatment likely will be fur-
ther investigated if insulin treatment is found to prevent the
onset of type 1 diabetes in humans, as observed in NOD mice.
3. Exogenous insulin and prevention of type 1 diabetes in clinical
studies. Results from the NOD mouse studies led to the Di-
abetes Prevention Trial Type 1, which was designed to test
the ability of sc and orally administered insulin to prevent
type 1 diabetes in subjects known to be at risk based on the
presence of autoimmune markers (e.g., IAAs and islet cell
antibodies) (365). The sc insulin administration arm of the
trial showed no acceleration or delay of type 1 diabetes onset.
The same was true for the oral insulin treatment arm when
all patients were included (366). A subset analysis of the oral
insulin treatment group, however, demonstrated a signifi-
cantly lower annualized rate of diabetes onset in patients
with baseline IAA levels of at least 80 nU/ml treated with
oral insulin compared with patients treated with placebo.
Additional studies showed no effect of oral insulin on
residual ␤-cell function in patients with new onset type 1
diabetes (103, 104). More recently, 38 children at risk for type
1 diabetes were treated with intranasal insulin and showed
no evidence for accelerated loss of ␤-cell function (367). Fur-
Fineberg et al. • Immunological Responses
thermore, IA responses to intranasal insulin were demon-
strated, as were immune changes consistent with mucosal
tolerance to insulin. A small trial suggested that low-dose sc
insulin may have favorable immunomodulatory effects in
adult patients with latent autoimmune diabetes (368).
VI. Conclusion
Regardless of purity and origin, therapeutic insulins con-
tinue to be immunogenic in humans; however, severe im-
munological complications occur rarely, and less severe
events affect just a small minority of patients. Today’s human
insulins are free of noninsulin peptides and variations in
insulin structure that contributed to the antigenicity seen
with pancreatic insulins in the past; however, insulins must
be manufactured, stored, and delivered by nonphysiological
means, possibly contributing to the humoral and cellular
immune responses seen in patients treated with insulin.
IAs may be detectable in insulin-naive individuals who
have a high likelihood of developing type 1 diabetes, have
had viral disorders, have been treated with various drugs, or
have autoimmune disorders or paraneoplastic syndromes.
This suggests that under certain circumstances immune tol-
erance to insulin can be overcome. Factors that can lead to
more or less susceptibility to humoral responses to exoge-
nous insulin include the recipient’s immune response genes,
age, the presence of sufficient circulating autologous insulin,
and the site of insulin delivery. Down-regulation of immune
responses to self- and nonself-antigens involves a subset of
regulatory T lymphocytes that may be critical in warding off
or terminating autoimmunity (67– 69). These regulatory cells
may also play a role in the development and levels of im-
mune responses to exogenous insulin.
Researchers have postulated that IAs can be associated
with insulin resistance, which would be detected clinically as
increases in insulin dose requirement. However, most stud-
ies have shown no relationship between dose and IAs (58, 99,
114, 241, 247, 250, 252, 255, 259, 260, 262, 265, 266, 269 –273).
In long-term, follow-up studies of children with type 1 di-
abetes, neither the presence of IAAs nor the development of
IAs affected insulin dose requirements (114).
Case reports indicate that high levels of IAs can be asso-
ciated with prolonged or recurrent hypoglycemia, but stud-
ies of large populations have failed to establish a relationship
between IAs and hypoglycemia event rates. Reports describe
patients with hypoglycemia syndromes who are found to
have markedly high levels of IAs, but it is difficult to deter-
mine from the literature how many patients with high an-
tibody levels do not have hypoglycemia.
It is not clear what factors may precipitate the hypothe-
sized unregulated dissociation of antibody-insulin com-
plexes. Because some patients are reported to have nocturnal
hypoglycemia, researchers often presume that binding equi-
librium is shifted toward antibody-insulin dissociation when
free insulin concentrations decline. Clinical trial data suggest
that hypoglycemia does not correlate with IAs in large pop-
ulations of patients; however, it cannot be excluded that a
small number of patients with high antibody levels develop
hypoglycemia syndromes owing in part to prolonged or
Endocrine Reviews, October 2007, 28(6):625– 652 643
unregulated appearance of free insulin in the circulation
(178). Clinical hypoglycemia in this setting may be multi-
factorial in etiology, involving deficient glucose counterregu-
lation coupled with a prolonged half-life of free insulin (213,
300). Most studies published since 1980 have shown no ev-
idence for a significant relationship between IA and average
glycemia.
Some early studies suggested that IAs were associated
with increases in neonatal morbidity, including hypoglyce-
mia, respiratory distress syndrome, and hypocalcemia (304,
310). However, studies in which pregnant women with di-
abetes were monitored for glycemic control argue against a
connection between IAs and fetal risk. No distinguishing
clinical characteristics of neonatal hypoglycemia associated
with IAs, such as unusual prolongation, have been described
in babies born to mothers with IAs.
Human studies have not shown consistent links between
IAs and diabetic complications. Although studies have
shown increased levels of immune complexes in patients
with diabetic microangiopathic complications (320 –322),
these immune complexes often do not contain insulin or IAs
(321–324). Neither does insulin administration contribute to
immune complex formation (325). The majority of studies
have shown no relationship between IAs and diabetic an-
giopathic complications, including nephropathy, retinopa-
thy, and neuropathy.
The absence of a pathogenic effect may be related to the
properties of the immune complex. Researchers have ob-
served that insulin and IA-immune complexes do not pre-
cipitate (at antigen or antibody excess) and cannot be de-
tected by standard immunodiffusion methods (340). In
addition, binding data suggested that insulin binding is uni-
valent, which limits the formation of large immune com-
plexes. Small immune complexes do not readily bind to C1q
complement and are not, therefore, readily cleared by the
reticuloendothelial system of the liver or spleen (190). Small
immune complexes have a similar half-life to circulating Ig
molecules. This explains the high levels of circulating total
insulin (antibody bound) in patients with high levels of IAs.
In conclusion, humoral antibody responses to exogenous
insulin continue to be largely unavoidable. Little proof exists
that the development of antibodies to exogenous insulin
therapy affects glycemic control, insulin dose requirements,
and hypoglycemia, or contributes to ␤-cell failure or to the
long-term complications of diabetes. Current human insulin
and insulin analog therapies have resulted in decreased IA
levels when contrasted with animal insulins. Local reactions
to the most recent formulations of insulin continue to be
observed but are infrequent, and systemic reactions are rare.
Until therapeutic insulin can be delivered in a physiological
manner, we are likely to continue to observe infrequent im-
munological sequelae. The development of IAs to exogenous
insulin reflects the exquisite sensitivity of the immune sys-
tem to even minor perturbations.
Acknowledgments
Address all correspondence and requests for reprints to: S. Edwin
Fineberg, 3504 Mountain Lane, Mountain Brook, Alabama 35213. E-mail:
efineber@iupui.edu
644 Endocrine Reviews, October 2007, 28(6):625– 652
Disclosure Statement: S.E.F. serves on advisory boards for Pfizer Inc.,
Eli Lilly and Company, and MannKind Corporation. T.T.K., D.F.-K.,
R.J.F., G.L.F., and A.S.K. are employees of Pfizer Inc.
Statistical support was provided by Pamela F. Schwartz of Pfizer Inc.
Editorial support was provided by Mark Poirier, BA, of PAREXEL and
was funded by Pfizer Inc.
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