Peptides having insulin autoantibody but not insulin receptor binding capacity

A therapeutic agent that includes a purified peptide bound to a cytotoxic moiety, the peptide being specifically reactive with human insulin autoantibodies and non-reactive with human insulin cell surface receptor, is disclosed.

BACKGROUND OF THE INVENTION 
This invention is in the general field of insulin-related peptides. 
Human insulin is a two-chain peptide hormone formed in the beta cells of 
the pancreatic islets of Langerhans. In controlling the level of blood 
glucose, insulin is the major fuel-regulating hormone in humans and causes 
a variety of physiological responses. It is believed that a certain 
portion of the insulin molecule is a receptor-binding region, i.e., that 
portion of the molecule that binds to receptor molecules on the surface of 
cells to initiate physiological responses. Gammeltoft, "Insulin 
Receptors," Physiol. Rev., 64:1321,1322 (1984). More specifically, 
referring to FIGS. 1 and 2, human insulin consists of an A chain of 21 
amino acids SEQ ID NO. 1 and a B chain of 30 amino acids, SEQ ID NO. 2 
held in three-dimensional conformation by disulfide bonds. Id. at 1352. 
The portion of the insulin molecule indicated by the shaded area shown in 
FIG. 2 is believed to be the receptor-binding region. Id. at 1343. 
Diabetes mellitus Type I is a chronic and progressively degenerative 
illness in which the immune system of the patient produces antibodies 
which interfere with insulin-related function. In particular, diabetics 
may produce "auto," or spontaneously occurring, antibodies specific for 
the beta cells of the pancreatic islets, causing destruction of the cells 
and thus of the insulin-producing capacity of the patient. Darnell, et 
al., Molecular Cell Biology. Scientific American Books. New York: 1986. In 
most cases, insulin therapy, consisting of injections of insulin into the 
patient's bloodstream, can overcome the problem. However, the development 
of antibodies to insulin in the patient being treated is an accepted 
consequence of the therapy. Palmer et al., Science, 222:1337 (1983). Human 
insulin antibodies, or insulin antibodies from other animals arising in an 
immunological response to insulin injection, are believed to recognize a 
binding site on the insulin molecule that overlaps the insulin 
receptor-binding site, for a truncated insulin analogue, 
desoctapeptide-insulin (B23-30 desoctainsulin), is substantially reduced 
in both biological and immunologic activity compared to naturally occuring 
insulin. Bromer et al., Biochim. Biophys. Acta, 133:219 (1967). 
Recently, the presence of spontaneously occurring insulin antibodies (or 
insulin "auto"antibodies) has been reported in the serum of at least 18% 
of newly diagnosed Type I diabetic patients before they had received any 
insulin therapy. Palmer et al., Science, 222:1337 (1983). This result has 
been confirmed by a number of different groups. Arslanian et al., 
Diabetes, 34:926 (1985); Srikanta et al., Diabetes, 35:139 (1985); 
Soeldner et al., N.E. J. Med., 313:893 (1985); and Dean et al., 
Diabetalogia, 29:339 (1986). 
SUMMARY OF THE INVENTION 
We have discovered that, in contrast with the behavior of insulin 
antibodies elicited in an immune response to insulin therapy, 
spontaneously arising human insulin autoantibodies recognize an epitope, 
or antibody binding site, on the insulin molecule that is distinct from 
the receptor-binding region of the molecule. Thus, insulin analogues 
properly designed not to contain the receptor binding site can retain the 
ability to bind to insulin autoantibodies and yet lack receptor binding 
(and thus receptor stimulating) ability. Using such insulin analogues, we 
are able to produce therapeutic agents that can be used safely to 
inactivate insulin autoantibodies released in the patient's serum and also 
to target for destruction insulin autoantibody-producing B lymphocytes 
having insulin autoantibodies exposed on their surface, thereby decreasing 
the production of insulin antibodies and decreasing T cell responses to 
insulin. 
In one aspect, the invention features a therapeutic agent including a 
purified peptide bound to a cytotoxic moiety, the peptide being 
specifically reactive with human insulin autoantibodies and non-reactive 
with human insulin cell surface receptor. By "specifically reactive with" 
insulin autoantibodies is meant that the peptide is capable of 
specifically recognizing human insulin autoantibodies in preference to 
other antibodies and that the immunologic reactivity or binding between 
the peptide and human insulin autoantibody is at least 50% of the level of 
reactivity of insulin with human insulin autoantibodies. By "non-reactive 
with human insulin cell surface receptor" is meant that the peptide has 
less than 1% of the receptor binding activity of insulin. Therefore, the 
peptide is unable to initate the physiological responses of the cell to 
insulin. 
Preferred embodiments include the following features. The cytotoxic moiety 
is a toxic portion of a toxin molecule; the peptide is an analogue of 
human insulin including an A chain and a B chain; the A chain includes, at 
positions A12 and A13, the amino acid residues at positions A12 and A13 of 
human insulin, or conservative substitutions thereof; the B chain 
includes, at position B3 of human insulin, the amino acid residue at 
position B3 of human insulin, or a conservative substitution thereof; and 
the B chain has a C-terminus before amino acid residue B23 of human 
insulin. Alternatively, the A chain of the human insulin analogue includes 
the above-indicated amino acid residues at positions A12, A13, and B3, and 
the A and B chains further include at least one non-conservative 
substitution for an amino acid at any one of positions A1, A21, B12, B16, 
B24, B25, or B26 of the corresponding A chain or the B chain of human 
insulin. By "analogue" of human insulin is meant a peptide whose general 
tertiary structure resembles that of insulin but whose amino acid sequence 
has been modified. By "conservative substitution" is meant a substitution 
for the indicated amino acid that does not substantially affect the 
antibody binding function of the peptide. By "non-conservative 
substitution" is meant a substitution for the indicated amino acid that 
does substantially affect the antibody binding function of the peptide. 
Analogues of insulin included within the claims have the property of being 
specifically reactive with human insulin autoantibodies and non-reactive 
with human insulin cell surface receptor as defined. When the above 
description is applied to analogues in which amino acids at specific 
positions in the insulin A and B chains have been deleted from the 
corresponding positions in the analogue, the analogue still retains the 
numbering system that would permit the greatest correspondence with the 
numbering system of the A and B chains of insulin. 
In particularly preferred embodiments, the peptide is 
desoctapeptide(B23-B30)-insulin or an analogue of human insulin in which 
the A chain includes the amino acid residues at positions A11-A14 of human 
insulin and the B chain includes the residues at positions B1-B4 of human 
insulin. 
In other aspects, the invention features a method for controlling human 
insulin autoimmunity in a patient and a method for gradually inducing 
patient tolerance to insulin. The methods include the steps of providing a 
therapeutic agent that includes a purified peptide specifically reactive 
with human insulin autoantibodies and non-reactive with human insulin cell 
surface receptor, and administering to the patient a therapeutically 
effective amount of the agent in a pharmaceutically acceptable carrier 
substance. In preferred embodiments of the method for controlling human 
insulin autoimmunity, the peptide is bound to a cytoxic moiety, preferably 
the toxic portion of a toxin molecule which, most preferably, is ricin or 
diphtheria toxin. In preferred embodiments of either method, the 
therapeutic agent includes the preferred peptide described above. 
In yet another aspect, the invention features a method for detecting the 
presence of human insulin autoantibodies in a patient. The method includes 
the steps of providing a sample of serum from a patient, contacting the 
serum with a purified peptide that is specifically reactive with human 
insulin autoantibodies and non-reactive with human insulin cell surface 
receptor, and measuring the amount of binding of the peptide to the serum 
sample. In a similar method, the presence of human insulin-related 
autoantibodies in a patient can be detected by contacting a sample of 
serum from the patient with purified human proinsulin or with a purified 
peptide analogue of proinsulin that includes an A chain, comprising the 
amino acid residues at positions A12 and A13 of human proinsulin or 
conservative substitutions thereof, and a B chain, comprising the amino 
acid residue at position B3 of human proinsulin or a conservative 
substitution thereof. 
The invention provides for agents and methods of using them that will 
diagnose and destructively target insulin autoantibodies in serum, or the 
autoantibody-producing B lymphocytes, and thus help prevent the onset of 
Type I diabetes mellitus without triggering the physiological responses 
that are a consequence of insulin/receptor binding. Because insulin has 
been so thoroughly studied, the agents are easy to make by standard 
molecular or biochemical techniques. 
Other features and advantages of the invention will be apparent from the 
following description of the preferred embodiment thereof and from the 
claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The invention features peptide analogues of insulin which retain the 
ability of the insulin molecule to be immunologically reactive with 
insulin autoantibodies present in the serum of patients having pre Type I 
diabetes mellitus, while at the same time the peptides are biologically 
non-reactive with insulin receptor. Preferred analogues contain the 
epitope to which human anti-insulin autoantibodies bind and lack the 
insulin receptor binding portion of the insulin molecule. 
Such analogues can be used therapeutically, e.g., as a carrier for a toxin 
molecule to target insulin autoantibodies or insulin 
autoantibody-producing B lymphocytes, thus reducing the concentration of 
insulin autoantibodies, or decreasing their production, without triggering 
a diabetic's characteristic response to insulin stimulation. 
1. Determination of an epitope recognized by human anti-insulin 
autoantibodies that is distinct from the insulin receptor binding domain. 
Insulin autoantibodies isolated from sera of well-characterized 
first-degree relatives of patients with Type I diabetes mellitus were 
analyzed in comparative studies by competition with a series of insulins 
from different species and with insulin analogues. Insulin binding 
activity was detected using a competitive binding radioassay and expressed 
in nU of insulin precipitated per ml of sera (1 nU/ml=7.18 fmole/L). Each 
of the studied individuals consistently had insulin autoantibodies for at 
least one year. The data indicate that these Type I diabetes-associated 
autoantibodies are homogeneous. 
Using the results of the comparative studies, a recognition epitope for 
human anti-insulin autoantibodies arising in Type I diabetes was mapped 
and shown to include regions A11-A14 and B1-B4 of the insulin molecule and 
not to include regions A17 and B23-B30. Referring to FIG. 4, the included 
regions can be seen to be adjacent to one another in insulin's three 
dimensional crystallographic structure. At the same time, referring to 
FIG. 2, the autoantibody-binding region of the insulin molecule can be 
distinguished from the insulin receptor-binding domain that lies on the 
opposite surface of the molecule; specific details are given below. 
Competition of a series of insulins and insulin analogues, whose amino acid 
sequences are given in FIG. 1, with .sup.125 I-human insulin for binding 
to human insulin autoantibodies isolated from patient sera was evaluated. 
Referring to FIG. 3, human insulin consistently inhibited binding at lower 
concentrations than the other insulins, and the rank order potency of 
various insulins was similar for different sera. Guinea pig insulin failed 
to compete in concentrations 1000 times greater than that which gave 
maximal inhibition with human insulin. Fish (salmon) insulin also competed 
poorly. All other species of insulin studied gave similar maximal 
inhibition of .sup.125 I-human insulin precipitation. Two specific insulin 
analogues were studied: GluA17.fwdarw.Gln human insulin (similar to fish 
(salmon) and guinea pig insulin) and desoctapeptide(B23-B30)-insulin, an 
analogue lacking the carboxyl terminal eight amino acids of the B chain. 
Both of these analogues competed effectively for .sup.125 I-insulin 
binding to insulin autoantibodies. 
In human proinsulin the N-terminus of the insulin A-chain (GlyA1) is 
connected to the C-terminus of the B-chain (ThrB30) by a 35 amino acid 
connecting peptide. In contrast to all other insulin analogues studied, 
human proinsulin was found to be at least as potent as human insulin in 
binding to insulin autoantibodies, and for several patients only 
proinsulin autoantibodies were found. 
Although the affinities and capacities of the autoantibodies from different 
subjects varied, for all subjects the overall order of immunologic 
recognition of the insulins and related analogues was similar. Comparisons 
between the sequences of these insulins allows an autoantibody recognition 
domain to be mapped. 
The primary sequence of porcine insulin differs from human insulin by one 
residue; bovine, rat and ovine insulins each differs from human by three 
amino acids (FIG. 1). Each of these insulins exhibits high-affinity 
recognition by the human insulin autoantibodies. The affinity of chicken 
insulin, which differs from human insulin at 7 positions, is reduced 
.about.20-fold. The affinity of the human insulin autoantibodies for fish 
insulin is markedly reduced, and guinea pig insulin is essentially not 
recognized by the insulin autoantibodies. These insulins differ from human 
insulin at 15 and 18 different positions, respectively. 
In bovine and ovine insulins, residues ThrA8 and IleA10 of human insulin 
are substituted by alanine and valine, respectively; these conservative 
changes result in a minimal reduction in insulin autoantibody binding 
affinity. In contrast, the A8-A10 positions of chicken and salmon insulin 
and A9-A10 positions of guinea pig insulin, all insulins with reduced 
insulin autoantibody affinity, are substituted non-conservatively. More 
significant regions of sequence dissimilarity can be mapped to A13-A15, 
A17, B1-B3 and B27 domains of insulin. Analogues GluA17.fwdarw.Gln insulin 
and desoctapeptide(B23-B30)-insulin compete for binding effectively, 
demonstrating that the A17 and B27 positions are not important for human 
insulin autoantibody recognition. 
Insulin autoantibodies from several patients with the autoimmune syndrome 
differ dramatically from the Type I diabetes associated insulin 
autoantibodies studied here. In particular, such patients have been 
reported to have markedly higher insulin binding capacities, and for 
several patients the epitope of insulin recognized by the autoantibodies 
can distinguish porcine from human insulin. Porcine insulin differs from 
human insulin only at the B30 residue (threonine changed to alanine), a 
residue unimportant for reactivity of Type I diabetes-associated insulin 
autoantibodies. 
From these comparative studies we have mapped the recognition epitope for 
human anti-insulin autoantibodies arising in Type I diabetes mellitus to 
include regions A11-A14 and B1-B4. Referring to FIG. 4, these regions can 
be seen to be adjacent to one another in insulin's three dimensional 
crystallographic structure. 
The receptor binding region of insulin, as indicated in FIG. 2, has been 
mapped previously by comparing primary sequences and biologic potencies of 
insulins from many species and related analogues to the crystallographic 
structure of insulin. Gammeltoft, supra, p. 1349. Residues at the 
amino-(GlyA1, GlnA5) and carboxyl-terminus (TyrA19, AsnA21) of the A-chain 
and the center (VB12, TyrB16) and carboxyl-terminus (PheB24, PheB25, 
TyrB26) of the B-chain are thought to be involved directly in receptor 
recognition. This is in striking contrast to the residues contributing to 
the dominant immunogenic epitope of this study. The epitope of insulin 
that reacts with the insulin autoantibodies can therefore be distinguished 
from insulin's receptor binding domain which lies on the opposite surface 
of the three-dimensional structure of the insulin molecule. 
2. Preparation of insulin analogues that are immunologically active but 
inactive metabolically 
Given the information obtained concerning the separation of the insulin 
autoantibody and insulin receptor binding activity, specific analogues can 
be prepared that are presumed to retain autoantibody binding activity 
while at the same time to lack receptor binding activity. Some appropriate 
analogues are desoctapeptide(B23-B30)-insulin or proinsulin; 
desheptapeptide(B24-B30)-insulin or proinsulin; 
despentapeptide(A1-A5)-insulin or proinsulin; des-(A21) proinsulin; 
insulin or proinsulin modified at GlyA1, ValB12, TyrB16, PheB24, or PheB25 
with a non-conservative substitution (e.g., Arg or Glu) or chemically; or 
combinations of the above. 
The analogues can then be screened in the following manner: 
Human autoantibody binding affinity is determined by reacting 
autoantibodies obtained from new onset Type I diabetics prior to insulin 
therapy (sera from such patients is readily available worldwide) with the 
appropriate analogue and then determining competition with I.sup.125 
insulin by Scatchard analysis. Receptor binding activity is also 
determined by Scatchard analysis utilizing a series of standard insulin 
receptor assays including reactivity with whole cells and with purified 
normal human receptors. In addition, bioactivity can be determined by 
injecting an analogue-containing solution intravenously or subcutaneously 
into humans or animals and then monitoring blood glucose levels. In 
humans, a standard insulin tolerance test involves intravenously injecting 
0.1 U/kg insulin and then monitoring blood glucose levels over a 1/2 hour 
period. In a more formal procedure, a euglycemic glucose clamp can be 
employed, with the intravenous infusion of insulin, or the analogue being 
tested, and glucose; the end point is glucose utilization. Acceptable 
analogues should have greater than 50% of the affinity of human insulin 
for pre-diabetic insulin autoantibodies and less than 1% of the 
bioactivity of human insulin. 
3. Preparation of therapeutic agents 
Referring to FIG. 5, an insulin analogue/toxin conjugate is prepared 
according to the following scheme: An appropriate insulin analogue, 
selected as described above, is reversibly modified with the 
t-butyloxycarbonyl (Boc) group selectively at A1.sup..alpha. and 
B29.sup..epsilon. positions; the B1.sup..alpha. amino group is not 
modified. This material is then reacted with N-succinimidyl 
3-(2-pyridyldithio) propionate (SPDP) to form SPDP-insulin. Following 
removal of boc groups B1.sup..alpha. -SPDP-insulin is obtained in pure 
form. This is reacted with the free sulfhydryl group of Toxin A chain 
(e.g., of a peptide toxin such as ricin, diphtheria toxin, etc.) to form a 
disulfide-linked conjugate. 
Use 
Insulin analogues that dissociate receptor from immunogenic binding can be 
used in broad based therapy for the detection of incipient Type I diabetes 
mellitus and the prevention of further development of the condition. 
For detection of insulin autoimmunity heralding Type I diabetes, analogues 
having specifically designed epitopes, so as to react with a subset of 
human insulin autoantibodies, can be prepared. 
Proinsulin is an excellent agent for detecting insulin autoimmunity. As 
described earlier, human proinsulin is at least as potent as human insulin 
in binding to insulin autoantibodies, and in some patients only proinsulin 
autoantibodies are found at early stages of diabetes development. 
Radioactive biotinylated or otherwise labeled proinsulin, or insulin or 
proinsulin analogue, can be utilized in a fluid phase assay as a 
diagnostic reagent to detect pre Type I autoimmunity. The reagent, with 
invarient residues A12, A13, B1, B2 and B3, should be labeled at sites 
distinct from those residues (e.g., at TyrA14 or at TyrB26). Specifically, 
a kit for detecting pre Type I diabetes mellitus can be prepared for 
routine use in a hospital setting. Such a kit would include a container to 
receive a sample of serum from a patient; a measured amount of purified, 
labeled human proinsulin (or insulin or proinsulin analogue) to be placed 
in contact with the serum sample; and a filter or standard means of 
precipitation (e.g., 14% polyethylene glycol) for recovering the antibody 
population in the serum so as to permit measurement of the amount of 
specific binding of the labeled reagent to the serum sample. 
In a therapeutic context, analogues of insulin with minimal receptor 
binding activity but greater than 50% immunogenic activity can be 
administered intravenously or orally in doses of 0.01 mg to 10 mg/kg/day 
to induce patient tolerance to insulin. 
For a more immediate effect, an appropriate analogue can be coupled to, 
e.g., diphtheria toxin, as described above, and administered intravenously 
or subcutaneously (in doses of 0.01 mg to 10 mg/kg/day). Such a toxin 
conjugate can specifically target autoreactive B lymphocytes thereby 
decreasing the production of insulin autoantibodies, and can eliminate the 
insulin specific B lymphocyte presentation to T cells of insulin or 
proinsulin, thus reducing T cell responses to insulin. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
GlyIleValGluGlnC ysCysThrSerIleCysSerLeuTyrGlnLeu 
51015 
GluAsnTyrCysAsn 
20 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
PheValAsnGlnHisLeuCysGlySerHisLeuValGluAlaLeuTyr 
51015 
LeuValCysGly GluArgGlyPhePheTyrThrProLysThr 
202530 
Other embodiments are in the claims.