Insulin receptor

Insulin receptor is purified in accordance with this invention to a level sufficient to enable amino acid sequencing thereof. DNA encoding insulin receptor is provided, as well as methods for synthesizing insulin receptor or its mutant in heterologous host cells transformed with vectors containing such DNA. Knowledge of the amino acid sequence for insulin receptor enables the preparation of novel immunogenic conjugates and antibodies raised against such conjugates. Novel therapeutically useful forms of the insulin receptor and anti-receptor antibodies are described.

BACKGROUND INFORMATION 
This relates to the mammalian insulin receptor. In particular it relates to 
the synthesis of the insulin receptor by recombinant cells and to the 
manufacture and use of certain novel products enabled by the 
identification and cloning of DNA encoding the insulin receptor. 
The rapid matabolic effects of insulin as well as its long term growth 
promoting actions are initiated by the interaction of the hormone with 
specific, high affinity cell surface receptors.sup.1-3. The indulin 
receptor (apparent M.sub.r 350,000-400,000) is an integral membrane 
glycoprotein composed of two .alpha. subunits (apparent M.sub.r 
=120,000-130,000) and two .beta. subunits (apparent M.sub.r =90,000), 
which are linked by disulfide bonds .sup.4-8. Photoaffinity labelling of 
the receptor.sup.9,10 as well as affinity cross linking.sup.7,11 have 
shown that the .alpha. submit is predominantly labelled by radioactive 
insulin derivatives. In intact cells, insulin stimulates the 
phosphorylation of the .beta. subunit on serine and tyrosine residues, a 
phenomenon first observed in rat hepatoma cells and IM9 
lymphoblasts.sup.12,13, and then extended to a variety of cell 
types.sup.14-16. In vitro, insulin-dependent tyrosine kinase 
activity.sup.14-22 copurifies with the insulin-binding activity to 
homogeneity.sup.19,20,23. The protein kinase activity of the insulin 
receptor catalyzes both phosphorylation of the .beta. subunit as well as 
exogenous peptides and proteins.sup.14,21,24-26. Studies with model 
peptide substrates indicate that the specificity of the insulin-dependent 
protein kinase is similar to that of the epidermal growth factor (EGF) 
receptor kinase and the src family of tyrosine specific protein 
kinases.sup.24,25. In addition to being the principal substrate for 
autophosphorylation, the .beta. subunit bears an ATP binding 
site.sup.21,22. 
The .alpha. and .beta. subunits of the heterotetrameric insulin receptor 
derive from a single, glycosylated, polypeptide precursor of approximately 
190,000 daltons.sup.29-31. Disulfide bridges linking the .alpha. and 
.beta. subunit regions probably begin to form as the protein folds. After 
being transported from the endoplasmic reticulum to the Golgi apparatus, 
the precursor is further glycosylated and is then cleaved, followed by its 
transport to the plasma membrane.sup.27. Although similar in purported 
structure to the IGF-1 receptor.sup.28, the insulin receptor differs from 
the EGF receptor, which functions in the plasma membrane as a single 
polypeptide chain. 
The amino acid sequence of the human EGF receptor was recently reported 
.sup.32,33. However, although a need has existed for identifying the DNA 
encoding the insulin receptor and for expressing the DNA in recombinant 
culture, and notwithstanding reports of purified human placental insulin 
receptor in 1981 (7), no sequence information for this complex protein as 
yet has been disclosed in the literature 
SUMMARY 
By a novel final purification step we have obtained the human placental 
insulin receptor in sequenceable grade. Thereafter we determined the 
nucleotide and imputed complete amino acid sequence of the insulin 
receptor (also abbreviated herein as "IR" or "HIR" in the case of human 
insulin receptor). DNA encoding IR or its mutants is expressed in 
recombinant cells, thereby enabling the synthesis of (a) IR compositions 
having the amino acid sequence of the natural insulin receptor which are 
entirely free of other proteins of the species of origin and (b) novel 
mutant insulin receptors. This DNA or its fragments are useful in the 
hybridization diagnosis of defective IR DNA or mRNA and for obtaining DNA 
encoding IR from natural sources. IR and its mutants are therapeutically 
useful in the treatment of insulin overdose-induced nypoglycemia and in 
the study of the mechanisms of insulin-dependent cell metabolism. Known 
polypeptide fragments of IR are useful for generating antibodies in 
animals which are capable of binding IR in predetermined domains. These 
antibodies are useful, for example, as therapeutic agents and as 
components in diagnostic assays for IR and its mutants. 
The DNA species provided herein are novel. cDNA which encodes IR is 
obtained by reverse transcription of mRNA from cells. Accordingly, it 
contains no introns and is free of any flanking regions encoding other 
proteins of the organism from which the mRNA originated. Chromosomal DNA 
encoding IR is obtained by probing genomic DNA libraries with theIR cDNA 
or its fragments. Chromosomal DNA encoding IR in its entiret.y is excised 
free of the nonmal chromosomal flanking regions encoding other proteins, 
but it may contain introns. 
The isolated IR DNA is readily modified by substitution, deletion or 
insertion of nucleotides, thereby resulting in novel DNA sequences 
encoding IR or its derivatives. These modified sequences are used to 
produce mutantIR or to enhance the expression of IR species. 
In processes for the synthesis of IR, DNA which encodes IR is ligated into 
a replicable (reproducible) vector, the vector used to transform host 
cells, the host cells cultured and IR recovered from the culture. The IR 
species which are capable of synthesis herein include mature 
(amino-terminal) IR, preIR and IR derivatives including (a) fusion 
proteins wherein IR or any fragment thereof (including mature IR) is 
linked to other proteins or polypeptides by a peptide bond at the amino 
and/or carboxyl terminal amino acids of IR or its fragments, (b) IR 
fragments, including mature IR or fragments of preIR in which any. 
preprotein amino acid is the amino-terminal amino acid of the fragment, 
(c) mutants of IR or its fragments wherein one or more amino acid residues 
are substituted, inserted or deleted, and/or (d) methionyl or modified 
methionyl (such as formyl methionyl or other blocked methionyl species) 
amino-terminal addition derivatives of the foregoing proteins, fragments 
or mutants. 
Ordinarily, mammalian cells are transformed with a vector containing the 
sequence of mature IR or its fragments or mutants linked at its 5' end to 
the IR presequence or other secretory leader recognized by eukaryotic 
cells, the cell cultured, and mature IR, fragments or mutants are 
recovered from the culture. IR is secreted by expression of an IR mutant 
in mammalian cells where the transmembrane sequence has been deleted or 
substituted by a hydrophilic domain. 
Also within the scope of this invention are derivatives of IR other than 
variations in amino acid sequence or glycosylation. Such derivatives are 
characterized by covalent or aggregative association with chemical 
moieties. The derivatives generally fall into three classes: Salts, side 
chain and terminal residue covalent modifications, and adsorption 
complexes, e.g. with cell membranes. 
Antibodies against predetermined fragments of IR are raised by immunization 
of animals with conjugates of the fragments with immunogenic proteins. 
Monoclonal antibodies are prepared from cells secreting desired antibody. 
These antibodies are screened for insulin-like activity on normal or 
defective receptors. 
Recombinant IR or IR antibodies are purified and then combined for 
therapeutic use with physiologically innocuous stabilizers and excipients, 
sterile filtered and placed into dosage form as by lyophilization in 
dosage vials or storage in stabilized aqueous preparations. Within the 
scope herein are derivatives of antibodies which are not complement 
binding. 
IR compositions are administered to animals in therapeutically effective 
doses to neutralize excessive circulating insulin, as for example in 
overdose emergencies. Suitable dosages will be apparent to the artisan in 
the therapeutic context. Similarly, anti-IR compositions having 
insulin-like activity are administered as required to induce metabolism of 
blood glucose.

DETAILED DESCRIPTION 
For the purposes herein, IR is defined as a protein or polypeptide which is 
substantially homologous with the amino acid sequence depicted in FIG. 1b 
or a fragment thereof, excluding any protein or polypeptide which exhibits 
substantially the same or lesser homology to the selected FIG. 1b sequence 
than does the insulin-like growth factor receptor (IGFR), epidermal growth 
factor receptor (EGFR) or the oncogenes v-abel, v-svc, v-fes, v-fms, v-ros 
or v-erb B. Ordinarily insulin receptor polypeptides will be about from 40 
to 100 percent homologous to the FIG. 1b sequence, preferably 80 to 90 
percent homologous, and they will exhibit at least some biological 
activity in common with the insulin receptor of FIG. 1b. Biological 
activity shall include, but is not limited to, insulin binding, ATP 
binding, protein phosphorylation activity and cross-reactivity with 
anti-IR antibodies raised against IR from natural (i.e., nonrecombinant) 
sources. Homology is determined by optimizing residue matches b.y 
introducing gaps as required but without considering conservative 
substitutions as matches. This definition is intended to include natural 
allelic variations in IR sequence. 
IR includes the insulin receptors of animals other than humans, e.g. 
bovine, porcine or ovine. 
PreIR is a species of IR included within the foregoing definition. It is 
characterized by. the presence in the molecule of a signal (or leader) 
polypeptide which serves to post-translationally direct the protein to a 
site inside or outside of the cell. Generally, the signal polypeptide 
(which will not have IR activity in its own right) is proteolytically 
cleaved from a residual protein having IR activity as part of the 
secretory process in which the protein is transported into the host cell 
periplasm or culture medium. The signal peptide may be microbial or 
mammalian (including the native, 27 residue presequence), but it 
preferably is mammalian. 
Derivatives of IR included herein are amino acid sequence mutants, 
glycosylation variants and covalent or aggregative conjugates with other 
chemical moieties. Covalent derivatives are prepared by linkage of 
functionalities to groups which are found in the IR amino acid side chains 
or at the N- or C-termini, by means known in the art. These derivatives 
may, for example, include: aliphatic esters or amides of the carboxyl 
terminus or residues containing carboxyl side chains, O-acyl derivatives 
of hydroxyl group-containing residues, and N-acyl derivatives of the amino 
terminal amino acid or amino-group containing residues, e.g. lysine or 
arginine. Acyl groups are selected from the group of alkyl-moieties 
(including C3 to C18 normal alkyl), thereby forming alkanoyl aroyl 
species. 
A major group of derivatives are covalent conjugates of IR or its fragments 
with other proteins or polypeptides. These derivatives are synthesized in 
recombinant culture as N- or C-terminal fusions or by the use of 
difunctional agents known per se for use in cross-linking proteins to 
insoluble matrices through reactive side groups. Preferred IR 
derivatization sites with cross-linking agents are at cysteine and lysine 
residues. Preferred agents are M-Maleimidobenzoyl succinimide ester and 
N-hydroxysuccinimide. 
Covalent or aggregative derivatives are useful as immunogens, reagents in 
immunoassay or for affinity purification procedures of insulin or other 
binding ligands. For example, IR is insolubilized by covalent bonding to 
cyanogen bromide-activated Sepharose by methods known per se or adsorbed 
to polyolefin surfaces (with or without glutaraldehyde cross-linking) for 
use in the assay or purification of anti-IR antibodies or insulin. IR also 
is labelled with a detectable group, e.g., radioiodinated by the 
chloramine T procedure, covalently bound to rare earth chelates or 
conjugated to another fluorescent moiety for use in diagnostic assays. 
Mutant IR derivatives include the predetermined, i.e. site specific, 
mutations of IR or its fragments. Mutant IR is defined as a polypeptide 
otherwise falling within the homology definition for IR as set forth 
herein but which has an amino acid sequence different from that of IR as 
found in nature, whether by way of deletion, substitution or insertion. 
For example, the Arg Lys Arg sequence at residues 720-723, inclusive, may 
be mutated by deletion (in order to produce a single chain receptor) or by 
substitution with another proteolysis recognition sequence more compatible 
with a recombinant host cell. Similarly, the transmembrane sequence, 
believed to span residues 918-940, inclusive, is deleted or substituted 
with hydrophilic residues such as serine to facilitate secretion of the 
receptor into cell culture medium. 
While the mutation sites are predetermined, it is unnecessary that the 
mutation per se be predetermined. For example, in order to optimize the 
performance of mutants at a given residue position, random mutagenesis may 
be conducted at the target codon and the expressed IR mutants screened for 
the desired activity. Techniques for making substitution mutations at 
predetermined sites in DNA having a known sequence are well known, for 
example M13 primer mutagenesis. 
IR mutagenesis is conducted by making amino acid insertions, usually on the 
order of about from 1 to 10 amino acid residues, or deletions of about 
from 1 to 30 residues. Substitutions, deletions, insertions or any 
subcombination may be combined to arrive at a final construct. Insertions 
include amino or carboxyl-terminal fusions. Obviously, the mutations in 
the DNA must not place coding sequences out of reading frame and 
preferably will not create complementary regions that could hybridize to 
produce secondary mRNA structure such as loops or hairpins. 
Not all mutations in the DNA which encode IR will be expressed in the final 
product. For example, a major class of DNA substitution mutations are 
those in which a different secretory leader or signal has been substituted 
for the native human secretory leader, either by deletions within the 
leader sequence or by substitutions, wherein most or all of the native 
leaderis exchanged for a leader more likely to be recognized by the 
intended host. However, the human secretory leader will be recognized by 
hosts other than human cell lines, most likely in cell culture of higher 
eukaryotic cells. When the secretory leader is "recognized" by the host, 
the fusion protein consisting of IR and the leader ordinarily is cleaved 
at the leader-mature IR peptide bond in the events that lead to secretion 
of IR or its insertion into the cell membrane. Thus, even though a mutant 
preIR is synthesized as an intermediate, the resulting IR will be mature. 
Another major class of DNA mutants that are not expressed as IR derivatives 
are nucleotide substitutions made to enhance expression, primarily to 
avoid amino terminal loops in the transcribed mRNA (see EP 75,444A, 
incorporated by reference) or to provide codons that are more readily 
transcribed by the selected host, e.g. the well-known E. coli preference 
codons for E. coli expression. 
Compositions comprising IR may include such substances as the stabilizers 
and excipients described below, predetermined amounts of proteins from the 
cell or organism that served as the source of DNA encoding the IR, 
proteins from other than the IR source cells or organisms, and synthetic 
polypeptides such as poly-L-lysine. Recombinant IR which is expressed in 
allogeneic hosts of course will be expressed completely free of gene 
source proteins. For example, the expression of human IR in CHO or other 
higher mammalian cells results in a composition where the receptor is not 
only free of human proteins but the IR in the culture is not denatured, 
unlike the partially purified HIR preparations reported in the literature. 
DNA which encodes IR is obtained by chemical synthesis, by screening 
reverse transcripts of mRNA from placental cells or cell line cultures, or 
by screening genomic libraries from any cell. 
This DNA is covalently labelled with a detectable substance such as a 
fluorescent group, a radioactive atom or a chemiluminescent group by 
methods known per se. It is then used in conventional hybridization 
assays. Such assays are employed in identifying IR vectors and 
transformants as described in the Examples infra, or for in vitro 
diagnosis such as detection of the aberrant IR DNA or mRNA in tissue 
samples. 
IR is synthesized in host cells transformed with vectors containing DNA 
encoding IR. A vector is a replicable DNA construct. Vectors are used 
herein either to amplify DNA encoding IR and/or to express DNA which 
encodes IR. An expression vector is a replicable DNA construct in which a 
DNA sequence encoding IR is operably linked to suitable control sequences 
capable of effecting the expression of IR in a suitable host. The need for 
such control sequences will vary depending upon the host selected and the 
transformation method chosen. Generally, control sequences include a 
transcriptional promoter, an optional operator sequence to control 
transcription, a sequence encoding suitable mRNA ribosomal binding sites, 
and sequences which control the termination of transcription and 
translation. Amplification vectors do not require expression control 
domains. All that is needed is the ability to replicate in a host, usually 
conferred by an origin of replication, and a selection gene to facilitate 
recognition of transformants. 
Vectors comprise plasmids, viruses (including phage), and integratable DNA 
fragments (i.e., fragments integratable into the host genome by 
recombination). The vector replicates and functions independently of the 
host genome, or may, in some instances, integrate into the genome itself. 
In the present specification, "vector" is generic to "plasmid", but 
plasmids are the most commonly used form of vector at present. However, 
all other forms of vectors which serve an equivalent function and which 
are, or become, known in the art are suitable for use herein. Suitable 
vectors will contain replicon and control sequences which are derived from 
species compatible with the intended expression host. Transformed host 
cells are cells which have been transformed or transfected with IR vectors 
constructed using recombinant DNA techniques. Transformed host cells 
ordinarily express IR, but host cells transformed for purposes of cloning 
or amplifying IR DNA do not need to express IR. Expressed IR will be 
deposited in the cell membrane or secreted into the culture supernatant, 
depending upon the IR DNA selected. 
DNA regions are operably linked when they are functionally related to each 
other. For example, DNA for a presequence or secretory leader is operably 
linked to DNA for a polypeptide if it is expressed as a preprotein which 
participates in the secretion of the polypeptide; a promoter is operably 
linked to a coding sequence if it controls the transcription of the 
sequence; or a ribosome binding site is operably linked to a coding 
sequence if it is positioned so as to permit translation. Generally, 
operably linked means contiguous and, in the case of secretory leaders, 
contiguous and in reading phase. 
Suitable host cells are prokaryotes, yeast or higher eukaryotic cells. 
Prokaryotes include gram negative or gram positive organisms, for example 
E. coli or Bacilli. Higher eukaryotic cells include established cell lines 
of mammalian origin as described below. A preferred host cell is E. coli 
W3110(ATCC 27,325), although other prokaryotes such as E. coli B, E. coli 
X1776 (ATCC 31,537), E. coli 294 (ATCC 31,446), Pseudomonas species, 
Bacillus species or Serratia Marcesans are suitable. 
Prokaryotic host-vector systems are preferred for the expression of IR 
fragments that do not require extensive proteolytic and disulfide 
processing. A plethora of suitable microbial vectors are available. 
Generally, a microbial vector will contain an origin of replication 
recognized by the intended host, a promoter which will function in the 
host and a phenotypic selection gene, for example a gene encoding proteins 
conferring antibiotic resistance or supplying an autotrophic requirement. 
Similar constructs will be manufactured for other hosts. E. coli is 
typically transformed using pBR322, a plasmid derived from an E. coli 
species (Bolivar, et al., 1977, "Gene" 2: 95). pBR322 contains genes for 
ampicillin and tetracycline resistance and thus provides easy means for 
identifying transformed cells. 
Expression vectors should contain a promoter which is recognized by the 
host organism. This generally means a promoter obtained from the intended 
host. Promoters most commonly used in recombinant microbial expression 
vectors include the .beta.-lactamase (penicillinase) and lactose promoter 
systems (Chang et al., 1978, "Nature", 275: 615; and Goeddel et al., 1979, 
"Nature" 281: 544), a tryptophan (trp) promoter system (Goeddel et al., 
1980, "Nucleic Acids Res." 8: 4057 and EPO App. Publ. No. 36,776) and the 
tac promoter [H. De Boer et al., "Proc. Nat'l. Acad. Sci. U.S.A." 80: 
21-25 (1983)]. While these are the most commonly used, other known 
microbial promoters are suitable. Details concerning their nucleotide 
sequences have been published, enabling a skilled worker operably to 
ligate them to DNA encoding IR in plasmid or viral vectors (Siebenlist et 
al., 1980, "Cell" 20: 269). The promoter and Shine-Dalgarno sequence (for 
prokaryotic host expression) are operably linked to the DNA encoding the 
IR, i.e., they are positioned so as to promote transcription ofIR 
messenger from the DNA. 
Eukaryotic microbes such as yeast cultures may be transformed with 
IR-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is 
the most commonly used among lower eukaryotic host microorganisms, 
although a number of other strains are commonly available. Yeast vectors 
generally will contain an origin of replication from the 2 micron yeast 
plasmid or an autonomously replicating sequence (ARS), a promoter, DNA 
encoding IR, sequences for polyadenylation and transcription termination 
and a selection gene. An exemplary plasmid is YRp7, (Stinchcomb et al., 
1979, "Nature", 282: 39; Kingsman et al., 1979, "Gene", 7: 141; Tschemper 
et al., 1980, "Gene", 10: 157). This plasmid already contains the trp1 
gene which provides a selection marker for a mutant strain of yeast 
lacking the ability to grow in tryptophan, for example ATCC No. 44076 or 
PEP4-1 (Jones, 1977, "Genetics", 85: 12). The presence of the trp1 lesion 
in the yeast host cell genome then provides an effective environment for 
detecting transformation by growth in the absence of tryptophan. 
Suitable promoting sequences in yeast vectors include the promoters for 
metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., 1980, "J. 
Biol. Chem.", 255: 2073) or other glycolytic enzymes (Hess et al., 1968, 
"J. Adv. Enzyme Reg.", 7: 149; and Holland et al., 1978, "Biochemistry", 
17: 4900), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, 
hexokinase, pyruvate decarboxylase, phosphofructokinase, 
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, 
triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. 
Suitable vectors and promoters for use in yeast expression are further 
described in R. Hitzeman et al., EPO Publn. No. 73,657. 
Other promoters, which have the additional advantage of transcription 
controlled by growth conditions, are the promoter regions for alcohol 
denydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes 
associated with nitrogen metabolism, and the aforementioned 
metallothionein and glyceraldehyde-3-phosphate denydrogenase, as well as 
enzymes responsible for maltose and galactose utilization. In constructing 
suitable expression plasmids, the termination sequences associated with 
these genes are also ligated into the expression vector 3' of the IR 
coding sequences to provide polyadenylation and termination of the mRNA. 
Cultures of cells derived from multicellular organisms are the preferred 
hosts for recombinant IR synthesis. This is particularly so for mature IR 
or the IR .alpha. or .beta. chains as extensive host cell processing is 
required. In principal, any higher eukaryotic cell culture is workable, 
whether from vertebrate or invertebrate culture. However, mammalian cells 
are preferred. Propagation of such cells in cell culture has become a 
routine procedure in recent years [Tissue Culture, Academic Press, Kruse 
and Patterson, editors (1973)]. Examples of useful host cell lines are 
VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, 
BHK, COS-7, CV, and MDCK cell lines. Expression vectors for such cells 
ordinarily include (if necessary) an origin of replication, a promoter 
located upstream from the gene to be expressed, along with a ribosome 
binding site, RNA splice site (if intron-containing genomic DNA is used), 
a polyadenylation site, and a transcriptional termination sequence. 
The transcriptional and translational control sequences in expression 
vectors to be used in transforming vertebrate cells are often provided by 
viral sources. For example, commonly used promoters are derived from 
polyoma, Adenovirus 2, and most preferably Simian Virus 40 (SV40). The 
early and late promoters are particularly useful because both are obtained 
easily from the virus as a fragment which also contains the SV40viral 
origin of replication (Fiers et al., 1978, "Nature", 273: 113). Smaller or 
larger SV40fragments may also be used, provided the approximately 250 bp 
sequence extending from the Hind III site toward the Bg1 I site located in 
the viral origin of replication is included. Further, it is also possible, 
and often desirable, to utilize the human genomic IR promoter, control 
and/or signal sequences, provided such control sequences are compatible 
with the host cell chosen. 
An origin of replication may be provided either by construction of the 
vector to include an exogenous origin, such as may be derived from SV40or 
other viral (e.g. Polyoma, Adenovirus, VSV, or BPV) source, or may be 
provided by the host cell chromosomal replication mechanism. If the vector 
is integrated into the host cell chromosome, the latter is often 
sufficient. 
Rather than using vectors which contain viral origins of replication, one 
can transform mammalian cells by the method of cotransformation with a 
selectable marker and the IR DNA. An example of a suitable selectable 
marker is dinydrofolate reductase (DHFR) or thymadine kinase. Such markers 
are proteins, generally enzymes that enable the identification of 
transformant cells, i.e., cells which were competent to take up exogenous 
DNA. Generally, identification is by survival of transformants in culture 
medium that is toxic or from which the cells cannot obtain critical 
nutrition without having taken up the marker protein. In selecting a 
preferred host mammalian cell for transfection by vectors which comprise 
DNA sequences encoding both IR and DHFR, it is appropriate to select the 
host according to the type of DHFR protein employed. If wild type DHFR 
protein is employed, it is preferable to select a host cell which is 
deficient in DHFR thus permitting the use of the DHFR coding sequence as a 
marker for successful transfection in selective medium which lacks 
hypoxanthine, glycine, and thymidine, critical nutrients that are not 
available without DHFR. An appropriate host cell in this case is the 
Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared 
and propagated as described by Urlaub and Chasin, 1980, "Proc. Natl. Acad. 
Sci. USA" 77: 4216. This method is further described in U.S. Pat. No. 
4,399,216; the procedures therein are adapted here for use in IR synthesis 
by substitution of DNA encoding an IR species for the genomic or 
.beta.-globin DNA used in the cited patent using appropriate synthetic 
linkers as required. 
Note that if DNA encoding DHFR protein with low binding affinity for 
methotrexate (MTX) is used as the controlling sequence, it is not 
necessary to use DHFR resistant cells. Because the mutant DHFR is 
resistant to MTX, MTX containing media can be used as a means of selection 
provided that the host cells are themselves MTX sensitive. Most eukaryotic 
cells which are capable of absorbing MTX appear to be methotrexate 
sensitive. One such useful cell line is a CHO line, CHO-K1 (ATCC No. CCL 
61). 
Other methods suitable for adaptation to the synthesis of IR in recombinant 
vertebrate cell culture include those described in M-J. Gething et al., 
"Nature" 293:620-625 (1981); N. Mantei et al., "Nature" 281:40-46; A. 
Levinson et al., EP 117,060A and 117,058A. 
HIR synthesized in recombinant culture is characterized by the presence of 
non-human cell components, including proteins, in amounts and of a 
character which depend upon the purification steps taken to recover HIR 
from the culture. These components ordinarily will be of yeast, 
procaryotic or non-human higher eukaryotic origin and and preferably are 
present in innocuous contaminant quantities, on the order of less than 
about 1 percent by weight. Further, recombinant cell culture enables the 
production of IR absolutely free of homologous proteins. Homologous 
proteins are those which are normally associated with the IR as it is 
found in nature in its species of origin, e.g. in cells, cell exudates or 
body fluids. For example, a homologous protein for HIR is human serum 
albumin. Heterologous proteins are the converse, i.e. they are not 
naturally associated or found in combination with the IR in question. 
IR or anti-IR is prepared for administration by mixing IR or anti-IR having 
the desired degree of purity with physiologically acceptable carriers. 
Such carriers will be nontoxic to recipients at the dosages and 
concentrations employed. Ordinarily, the preparation of such compositions 
entails combining the IR with buffers, antioxidants such as ascorbic acid, 
low molecular weight (less than about 10 residues) polypeptides, proteins, 
amino acids, carbohydrates including glucose or dextrins, chelating agents 
such as EDTA, glutathione and other stabilizers and excipients. 
IR compositions are administered to counteract insulin overdose or to 
adsorb autoimmune anti-IR antibodies. The route of administration is 
intravenous and dose is measured by amelioration of hypoglycemia, i.e., 
increases in blood sugar or, in the case of anti-IR antibody therapy, by 
insulin efficacy. IR compositions used in the emergency therapy for 
insulin-induced hypoglycemia desirably are combined with conventional 
agents such as i.v. dextrose used to treat insulin overdose. IR produced 
by recombinant techniques also is useful in preparing receptor affinity 
columns for the purification of insulin. 
In order to simplify the Examples certain frequently occurring methods may 
be referenced by shorthand phrases. 
Plasmids are designated by a low case p preceded and/or followed by capital 
letters and/or numbers. The starting plasmids herein are commercially 
available, are publically available on an unrestricted basis, or can be 
constructed from such available plasmids in accord with published 
procedures. In addition, other equivalent plasmids are known in the art 
and will be apparent to the ordinary artisan. 
"Digestion" of DNA refers to catalytic cleavage of the DNA with an enzyme 
that acts only at certain locations in the DNA. Such enzymes are called 
restriction enzymes, and the sites for which each is specific is called a 
restriction site. "Partial" digestion refers to incomplete digestion by a 
restriction enzyme, i.e., conditions are chosen that result in cleavage of 
some but not all of the sites for a given restriction endonuclease in a 
DNA substrate. The various restriction enzymes used herein are 
commercially available and their reaction conditions, cofactors and other 
requirements as established by the enzyme suppliers were used. Restriction 
enzymes commonly are designated by abbreviations composed of a capital 
letter followed by other letters and then, generally, a number 
representing the microorganism from which each restriction enzyme 
originally was obtained. In general, and unless otherwise provided, about 
1 .mu.g of plasmid or DNA fragment is used with about 1 unit of enzyme in 
about 20 .mu.l of buffer solution. Appropriate buffers and substrate 
amounts for particular restriction enzymes are specified by the 
manufacturer. Incubation times of about 1 hour at 37 .degree. C. are 
ordinarily used, but may vary in accordance with the supplier's 
instructions. After incubation, protein is removed by extraction with 
phenol and chloroform, and the digested nucleic acid is recovered from the 
aqueous fraction by precipitation with ethanol. Digestion with a 
restriction enzyme infrequently is followed with bacterial alkaline 
phosphatase hydrolysis of the terminal 5' phosphates to prevent the two 
restriction cleaved ends of a DNA fragment from "circularizing" or forming 
a closed loop that would impede insertion of another DNA fragment at the 
restriction site. Unless otherwise stated, digestion of plasmids is not 
followed by 5' terminal dephosphorylation. Procedures and reagents for 
dephosphorylation are conventional (T. Maniatis et al., 1982, Molecular 
Cloning pp. 133-134). 
"Recovery" or "isolation" of a given fragment of DNA from a restriction 
digest means separation of the digest on polyacrylamide gel 
electrophoresis, identification of the fragment of interest by comparison 
of its mobility versus that of marker DNA fragments of known molecular 
weight, removal of the gel section containing the desired fragment, and 
separation of the gel from DNA. This procedure is known generally. For 
example, see R. Lawn et al., 1981, "Nucleic Acids Res." 9: 6103-6114, and 
D. Goeddel et al., 1980, "Nucleic Acids Res." 8: 4057. 
"Southern Analysis" is a method by which the presence of DNA sequences in a 
digest or DNA-containing composition is confirmed by hybridization to a 
known, labelled oligonucleotide or DNA fragment. For the purposes herein, 
unless otherwise provided, Southern analysis shall mean separation of 
digests on 1 percent agarose, denaturation and transfer to nitrocellulose 
by the method of E. Southern, 1975, "J. Mol. Biol." 98: 503-517, and 
hybridization as described by T. Maniatis et al., 1978, "Cell" 15: 
687-701. 
"Transformation" means introducing DNA into an organism so that the DNA is 
replicable, either as an extrachromosomal element or chromosomal 
integrant. Unless otherwise provided, the method used herein for 
transformation of E. coli is the CaCl.sub.2 method of Mandel et al., 1970, 
"J. Mol. Biol." 53: 154. 
"Ligation" refers to the process of forming phosphodiester bonds between 
two double stranded nucleic acid fragments (T. Maniatis et al., Id., p. 
146). Unless otherwise provided, ligation may be accomplished using known 
buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 
.mu.g of approximately equimolar amounts of the DNA fragments to be 
ligated. 
"Preparation" of DNA from transformants means isolating plasmid DNA from 
microbial culture. Unless otherwise provided, the alkaline/SDS method of 
Maniatis et al., Id. p. 90., may be used. 
"Oligonucleotides" are short length single or double stranded 
polydeoxynucleotides which are chemically synthesized by known methods and 
then purified on polyacrylamide gels. 
All literature citations are expressly incorporated by reference. 
EXAMPLE 1 
N-terminal Amino Acid Sequences and DNA Probe Design 
The insulin receptor was purified from human placental membrane 
preparations by chromatography on wheat germ agglutinin agarose and 
insulin agarose as described previously.sup.23, except that 
phenylmethylsulfonyl fluoride (PMSF) was used to inhibit proteolysis; 
elution from wheat germ agglutinin was performed at 4.degree. C. and the 
protein was eluted from the insulin column with 0.5 percent SDS. 
Chromatograpy on hydroxylapatite in SDS.sup.43 was used to concentrate the 
partially purified receptor as follows. The insulin agarose eluate was 
diluted to less than 0.2 percent SDS and made 0.01M in sodium phosphate, 
pH 6.4, and 1 mM in dithiothreitol and passed over a three ml column of 
hydroxylapatite at 37.degree. C.; bound protein was eluted with 0.6M 
sodium phosphate, pH 6.4, 1 mM dithiothreitol, 0.1 percent SDS. 
Purification of the subunits was obtained by preparative polyacrylamide 
gel electrophoresis after dialysis against 0.1 percent SDS. Samples were 
electrophoresed on seven percent gels with 1 mM sodium thioglycolate in 
the upper reservoir buffer, and bands were excised from the Coomassie blue 
stained gel and electroeluted as described.sup.44. Quantitation of the 
alpha and beta subunits was based on a determination of the staining 
intensity of individual bands with Coomassie blue after polyacrylamide gel 
electrophoresis. Staining was determined by laser densitometry (Model 2202 
Ultroscan, LKB) and the receptor bands were compared with known amounts of 
standard proteins (myosin, beta galactosidase, and phosphorylase B) run on 
the same gel. However, particularly in the case of the beta subunit the 
protein sequence analysis results suggest that our estimate of the amount 
of receptor is low. 
Purified protein was applied to the vapor phase protein sequencer (Model 
470A, Applied Biosystems) described by Hewick et al..sup.45, and the amino 
acid derivatives released were determined by reversed phase HPLC on a 
Microsorb C8 column (4.6.times.250 mm, Rainin) using an aqueous phosphate 
buffer with acetonitrile as the organic solvent. In the case of the alpha 
subunit, 65 pmoles of sequence was obtained from an estimated 130 pmoles 
of protein, while 400 pmoles of sequence was obtained from an estimated 
300 pmoles of the beta subunit. The following are the amino terminal amino 
acid sequences of the insulin receptor .alpha. and .beta. subunits and the 
nucleotide sequences of the .alpha. and .beta. subunit probes designed 
therefrom. Codons for (XXX) positions were chosen on the basis of 
tentative amino acid assignments. Parentheses indicate uncertainty in 
assignment. Asterisks indicate ligation points between independently 
synthesized oligonucleotides. Underlined nucleotides represent later 
determined mismatches with the natural insulin receptor cDNA complement. 
##STR1## 
The double stranded nybridization probes were prepared using an automatic 
DNA synthesizer (Biosearch). Short overlapping oligonucleotides were 
synthesized and purified by acrylamide gel electrophoresis. Approximately 
10 pmol of each oligonucleotide were phosphorylated in separate reactions 
using three-fold excess of .gamma..sup.32 P-ATP (Amersham) and T4 
polynucleotide kinase. Full-length probes were prepared by combining the 
5' end labeled oligonucleotides and ligation with 2 units of T4 DNA ligase 
at 20.degree. C. for 2 hrs. Analytical examination of the ligation result 
demonstrated that about 69-80 percent of the DNA had been ligated to probe 
monomer size. The entire ligation mixture was boiled to separate DNA 
strands and used for nybridization as described.sup.37. 
EXAMPLE 2 
Identification of cDNA Which Encodes IR 
Total polyA-containing RNA was isolated from frozen term placenta. A clone 
library (1.5.times.10.sup.6 pfu) with cDNA (&gt;500 bp) and the 
.lambda.gt10vector system was prepared as described.sup.33,34. 
Initial screening with the .alpha.-subunit probe detected 15 hybridization 
positive recombinant phage. Characterization of purified phage DNAs by 
EcoRI restriction analysis followed by Southern blot hybridization.sup.35 
showed that each clone contained two EcoRI fragments, one of which 
hybridized with the synthetic .alpha. subunit probe. Only one phage 
(.lambda.HIR-P12) contained a second EcoRI fragment that hybridized with 
the .beta. subunit probe. The entire cDNA insert of phage .lambda.HIR-P12 
measured about 5 kb (1 kb and 4 kb EcoRI fragments, FIG. 1a) and was 
therefore large enough to code for the entire human insulin receptor 
precursor, estimated to be approximately 190,000 daltons including 
carbonydrate side chains.sup.31. 
Nucleotide sequence analysis confirmed that the 1 kb EcoRI fragment 
contained an open reading frame which coded for the amino terminal 
sequence of the insulin receptor .alpha. subunit. The leucine residue 
which we had determined to be located at position two of the mature 
.alpha.-subunit was found to be preceded by a histidine. An initiation ATG 
codon was identified 27 amino acids upstream from His (1), flanked by 
nucleotides which match Kozak's.sup.36 criteria for a translation 
initiation site. The amino acid residues between the ATG codon and the 
NH.sub.2 -terminus of the mature protein are highly hydrophobic, and are 
believed to represent the signal peptide sequence necessary for transport 
of the nascent insulin receptor precursor polypeptide into the lumen of 
the endoplasmic reticulum. B Even though the remaining nucleotide sequence 
upstream from methionine -27 does not contain any in-frame stop codons, 
the presence of an adjacent, suitable signal sequence led us to propose 
that the ATG at position -27 is used for translation initiation. This 
assignment predicted the orientation of the .alpha. and .beta. subunit 
sequences within the insulin receptor precursor: the .alpha. subunit of 
the receptor was expected to be located upstream from the NH.sub.2 
-terminus of the .beta. subunit within their common precursor. This 
prediction was confirmed by determination of the complete 5,181 bp long 
nucleotide sequence of the .lambda.HIR-P12 cDNA insert (FIG. 1B). The 
longest open reading frame starting with methionine within this sequence 
codes for 1,370 amino acids including the 27 residue signal peptide. The 
coding sequence is preceded by 50 nucleotides of 5' untranslated sequence 
and is followed by a signal for translational termination (TAA) and 1,018 
nucleotides of 3' untranslated sequence. It is not certain that the 
A-stretch at the 3' end of our sequence represents part of a polyA tail or 
an internal sequence within a larger 3' untranslated region, since this 
A-stretch is preceded by an imperfect polyadenylation signal (AATATA). To 
resolve this question, three additional, independent cDNAs representing 3' 
untranslated sequences were selected from the placental library and 
compared by restriction analysis with .lambda.HIR-P12 EcoRI fragments. 
Each of them appeared to end with the same 3' terminal sequence and 
matched .lambda.HIR-P12 restriction patterns, although none of them 
extended as far upstream. 
Comparison of final cDNA-derived nucleotide sequences with our synthetic 
probes revealed that the .alpha. subunit N-terminal probe contained 9 
mismatches (86 percent match) with the longest stretch of perfect match 
being 11 bp, and the 57 bp, .beta. subunit N-terminal probe contained 12 
mismatches (79 percent match) and a 12 bp maximum uninterrupted match. No 
false positives were identified. 
Based on our cDNA sequence we calculated molecular weights of 155,000 for 
the pre-insulin-receptor-precursor and 152,000 for the mature precursor. A 
tetrapeptide (ArgLysArgArg) at position 720 of the insulin receptor 
precursor sequence directly precedes the .beta. subunit N-terminal 
sequence (FIG. 1b) and is believed to represent the cleavage site for the 
receptor precursor processing enzyme. Omitting this peptidase recognition 
sequence, the final unmodified subunit molecular weights are predicted to 
be 82,400 (.alpha.) and 69,700 (.beta.). 
Hydropathy analysis was conducted by scanning the 1370-amino-acid-long 
pre-insulin receptor precursor sequence using the computer program of Kyte 
and Doolittle.sup.46. Landmarks within the sequence are indicated 
schematically: signal sequence (a), cysteine-rich region (b), precursor 
processing site (c), transmembrane sequence (d) and tyrosine kinase domain 
(e). Hydrophobicity results in positive and hydrophilicity in negative 
values (see Kyte and Doolittle.sup.46). The results are shown in FIG. 3. 
The 719-residue-long .alpha. subunit sequence (FIG. 1b) is largely 
hydrophilic (FIG. 3) with a few short hydrophobic stretches, none of which 
are long enough to qualify as potential membrane anchor sequences. Fifteen 
consensus sequences for asparagine-linked glycosylation 
(Asn.times.Ser/Thr) are evenly distributed over the 719-residue-long 
.alpha. subunit region, which is also characterized by an unusually large 
number (37) of cysteine residues. Twenty-six of the cysteines are 
concentrated between residues 155 and 312 (FIG. 1b) and are contained 
within a rather hydrophilic domain (FIG. 3). Although in most cases there 
is no direct evidence as to which of the potential asparagine-linked 
carbohydrate attachment sites are in fact glycosylated, our failure to 
detect the asparagines at positions 16 and 7 of the alpha and beta 
subunits, respectively, during protein sequencing strongly suggests that 
these two sites are glycosylated. 
The 620-amino-acid-long .beta. subunit sequence contains only nine cysteine 
residues (1.5 percent) and can be divided into 3 domains. The 
amino-terminal 194-residue-long domain contains 4 potential 
asparagine-linked glycosylation sites and four cysteine residues. An 
adjacent stretch of 23-26 highly hydrophobic amino acids (915 or 918 - 
940) is believed to represent the single transmembrane domain that anchors 
the insulin receptor in the membrane. The transmembrane sequence is 
flanked at its C-terminal end by three basic amino acids (ArgLysArg; FIG. 
1b), which are part of the 403-residue-long carboxy terminal domain that 
contains two potential N-linked glycosylation sites as well as a typical 
number of cysteine residues. 
EXAMPLE 3 
Multiple Related mRNAs 
Northern blot analysis.sup.38 was undertaken for polyA.sup.+ RNA (5 .mu.g) 
isonated from mouse 3T3-LI fibroblasts before (a) and after 
differentiation into adipocytes.sup.40 (b) and human term placenta tissue 
(c) after separation on a formaldehyde-containing one percent agarose gel 
and transfer to nitrocellulose. Rat ribosomal RNA was used as a size 
marker. The filter was hybridized with radiolabelled insulin receptor cDNA 
fragments.sup.47 (1010 bp and 4169 bp EcoRI). Exposure was for 3 days at 
-60.degree. C. using an intensifying screen (Cronex Lightning Plus). 
Additional Northern blot experiments with placenta and IM-9 RNAs were 
carried out as mentioned in the text. The following cDNA fragments were 
used in parallel experiments: EcoRI 1-1011; EcoRI 1012-XhoI 2904; StuI 
3234-StuI 3728; StuI 2399-XhoI 3080; PstI 4341-EcoRI 5169. 
Northern blot hybridization.sup.38 analysis using polyA.sup.+ RNA from term 
placenta, as well as fetal placenta (20 week) and a human lymphoblast cell 
line (IM-9), yielded a complex pattern of five common hybridization bands, 
8.2 kb, 7.3 kb, 6.5 kb, 5.5 kb and 4.6 kb in length (FIG. 2A). A faint, 
2.9 kb band was also detected, but was present only in term placenta and 
IM-9 RNAs. The hybridization signal intensities for the bands was 
different for each band, indicating that either variable amounts of 
different length mRNAs are synthesized from the same gene, or that 
different but related genes are transcribed to yield multiply-sized mRNAs. 
To further investigate the identity of mRNAs from placenta and IM-9 cells 
which hybridize with our 5.2 kb insulin receptor cDNA probe, we used a 
variety of cDNA probe fragments as hybridization probes in Northern blot 
hybridization experiments. In all cases, at least all of the four largest 
transcripts (8.2, 7.3, 6.5 and 5.5 kb) were detected. 
After treatment with dexamethasone and isobutyl methylxanthine, mouse 
3T3-LI fibroblasts differentiate into adipocytes, in a process which leads 
to a ten to twenty fold increase in the number of insulin receptor 
molecules on their surfaces..sup.39,40 Northern blot analysis carried out 
with polyA-containing mRNA obtained from these cells before and after 
differentiation revealed that the increase in receptor molecules parallels 
an approximately tenfold increase in two insulin receptor mRNAs (FIG. 2A, 
a and b). Unlike the complex transcription pattern observed in placenta 
(FIG. 2A, C), the 3T3-LI cells synthesized only comparable amounts of a 
6.5 and an 8.2 kb mRNA, both before and after induction. These experiments 
strongly suggest that the major transcripts of 6.5 and 8.2 kb represent 
insulin receptor mRNAs. The mRNAs may differ in the lengths of their 3' or 
5' untranslated sequences, as has been described for other gene 
systems.sup.41,42 or alternatively these mRNAs could be generated by 
variable splicing of a primary transcript from the same gene. 
EXAMPLE 4 
DNA Probe Analysis of Genomic DNA 
To determine the number of insulin receptor genes present in the human 
genome, Southern blot analyses.sup.35 were carried out using an 857 bp 
PstI-EcoRI cDNA fragment derived from the 3' most terminal untranslated 
sequence as a hybridization probe. High molecular weight DNA (10 .mu.g per 
lane) isolated from placental nuclei was digested with excess amounts of 
EcoRI (a), HincII (b), and PstI (c), separated on a one percent agarose 
gel and analyzed by Southern blot hybridization.sup.35. The radiolabelled 
probe used under high stringency conditions consisted of a 857 bp 3' 
terminal PsI-EcoRI fragment. .lambda. wt phage DNA digested with EcoRI and 
HindIII was used as size marker. These sequences were chosen because they 
are the most rapidly divergent sequences within gene families and would 
therefore avoid detection of closely related genes. As can be seen in FIG. 
2B, this probe hybridized with single restriction fragments from high 
molecular weight, nuclear DNA, consistent with the presence of only one 
insulin receptor gene in the haploid human genome. 
EXAMPLE 5 
Expression of HIR in Mammalian Cells 
A gel purified SalI fragment (.about.5.2 kb) from .lambda.HIR-P12 
containing the entire HIR coding sequence was subcloned into the pUC12 
(New England Biolabs) polylinker region by digesting pUC12 with SalI and 
ligating the purified SalI fragment to the vector. Plaques were grown up 
and screened for clones having the desired XbaI-SalI-HIR-SalI-HindIII 
orientation (where the XbaI and HindIII sites originate from the pUC12 
polylinker and flank the SalI inserted HIR gene). This vector was 
designated PUC12/HIRc. pUC12/HIRc was cut with XbaI and DraI (DraI is 
located in the 3' untranslated region of HIR) and the HIR-containing 
fragment was isolated. This fragment was inserted into a mammalian 
expression vector (pCVSVEHBVE400, European Publn. No. 117060), which had 
been digested first with BamHI. The BamHI expression vector sticky ends 
were filled in with Klenow PolI and subsequently the plasmid was digested 
with XbaI. Thus, insertion of the XbaI-DraI was only possible in the 
orientation necessary for expression of the HIR mRNA. The resulting 
insulin receptor expression plasmid was designated pCVSVE-HIRc-2. It is 
subsequently transfected by standard procedures into several mammalian 
cell lines such as MDCK, CHO, Ratl (a rat fibroblast cell line) or COS and 
cultured in transient expression culture medium under conditions favoring 
transient expression as are generally known in the art. Stable 
transformants are identified by culture in medium to select for DHFR 
expression from pCVSVE-HIRc-2. Transient expression is measured after 
about 40 hrs by binding of radioiodinated insulin to transfected vs. 
nontransfected control cells. 
EXAMPLE 6 
Expression of Mutant HIR in Mammalian Cells 
This example describes the method generally usable for deletional 
mutagenesis of IR. The following embodiment describes the deletion of the 
transmembrane region (TMR) of HIR. 
The gel purified SalI fragment from Example 5 is ligated to SalI digested 
M13mp8 replicative form DNA (J. Adelman et al., 1983, "DNA" 2 (3):183-193) 
and transfected into E.coli JM103. Transformants (pSalHIR) are identified 
as described in Adelman et al., op cit. 
The synthetic oligodeoxyribonucleotides 5'GCTGCCTCTTTCTTTTTGCAATATTTG-3' 
(".DELTA.") (complementary to the HIR coding regions immediately proximal 
and distal to the TMR), 5'GAAGTCACAACACTAACCTTC3' ("loop") (complementary 
to a portion of the TMR), 5'AGAAGCGTAAAGCGGTCC3' (a sequencing primer 
complementary to the region encloding HIR amino acid residues 950-955) and 
5'GTTTTCCCAGTCACGAC3' ("LAC", regularly used as a sequencing primer on 
recombinant phage M13 DNA) were prepared by the method of Crea et al., 
"Nucl. Acids Res." 8:2331-2348 (1980). 
The oligonucleotides were phosphorylated as described in Adelman et al., op 
cit. 
Phosphorylated .DELTA. and LAC are annealed to pSalHIR and primer extended 
as described in Adelman et al., op cit. DNA is recovered as described and 
used to transform E.coli JM103. Transformant phage are identified by 
screening with "loop" and .DELTA.. A transformant having the transmembrane 
region deletion (designated pSalHIRd918-940) is identified by DNA sequence 
analysis using the sequencing primer described above in the method of 
Adelman et al., op cit. DNA is recovered from pSalHIRd918-940 and digested 
with SalI. The mutant HIR DNA is recovered and ligated to SalI digested 
pUC12 as described above in Example 5. Thereafter, Example 5 is repeated 
except that expression is measured by an HIR sandwich immunoassay of the 
cell culture supernatant using immobilized and labelled polyclonal 
antisera raised against HIR (host cell IR will be cell membrane bound and 
antisera are used in the assay which are selected for low titer 
cross-reactivity with the host cell IR). 
Other deletional, substitution and insertional mutations e.g. mutations of 
residues 720-723, are prepared in similar fashion and/or by the use of 
synthetic oligonucleotide inserts using the methods of U.S. patent 
application No. 614,617; U.K. patent application No. 2,130,219A; R. 
Wallace et al., 1981, "Nucleic Acids Research" 9 (15): 3647-3656; G. 
Winter et al., 1982, "Nature" 299: 756-758; and A. Hui et al., 1984, "The 
EMBO Journal" 3 (3): 623-629), all of which are expressly incorporated 
herein. 
EXAMPLE 7 
Preparation of Antibodies Against Predetermined Amino Acid Sequences of IR 
In this contemplated example, the peptides described in the following Table 
I are synthesized by the Merrifield solid phase synthesis technique (68) 
and conjugated to the proteins scheduled in Table I using the indicated 
bifunctional agents in accord with conventional practice. 
TABLE I 
______________________________________ 
Polypeptides 
(designated by 
HIR residues, Conjugating 
inclusive) Agent Protein 
______________________________________ 
147-158 MBS.sup.a 
keyhole limpet 
hemocyanin (KLH) 
(Calbiochem) 
158-168 and/or 320-334 
MBS KLH 
175-187 MBS KLH 
267-282 NHS.sup.b 
KLH 
166-177 and/or 275-287 
NHS KLH 
149-163 and/or 134-149 
NHS KLH 
241-252 MBS bovine serum albumin (BSA) 
Cys(345-359).sup.c 
MBS BSA 
120-133 and/or 309-325 
MBS BSA 
179-190 and/or 495-508 
NHS BSA 
197-206 and/or 433-446 
NHS BSA 
193-206 MBS soybean trypsin inhibitor 
(STI) 
2-20 and/or 524-535 
MBS STI 
704-720 and/or 724-736 
MBS STI 
954-966 and/or 1142-1154 
MBS STI 
1330-1343 MBS STI 
183-191 MBS bovine thyraglobulin (BT) 
319-322 and/or 213-224 
NHS BT 
90-112 and/or 310-324 
NHS BT 
______________________________________ 
.sup.a MBS: mMaleimidobenzoyl sulfosuccinimide ester. Conjugation is 
through cysteine residues. 
.sup.b NHS: N--hydroxysuccinimide. Conjugation is through lysine residues 
.sup.c The indicated polypeptide is synthesized with an added Nterminal 
cysteine. 
Other bifunctional cross-linking agents are known and useable in the 
preparation of immunogenic conjugates. Examples include glutaraldehyde, 
succinic anhydride, SOCl.sub.2, and R'N=C=NR. 
Other proteins than those enumerated above are useful. They preferably will 
be heterologous to the species to be immunized, otherwise the immune 
response likely will be of low titer if at all. 
Preferably, the IR polypeptide fragments chosen are selected from the 
.alpha. subunit, although fragments from the ATP binding, protein 
phosphokinase and autophosphorylation regions of the receptor are included 
within the scope hereof. 
Animals are immunized against the immunogenic conjugates by combining 1 mg 
or 1 .mu.g of conjugate (for rabbits or mice, respectively) with 3 volumes 
of Freund's complete adjuvant and injecting the solution intradermally at 
multiple sites. One month later animals are boosted with 1/5 to 1/10 the 
original amount of conjugate in Freund's complete adjuvant by subcutaneous 
injection at multiple sites. 7 to 14 days later animals are bled and the 
serum assayed for anti-HIR titer. Animals are boosted until the titer 
plateaus. Preferably, the animal is boosted with the conjugate of the same 
IR polypeptide, but conjugated to a different protein and/or through a 
different crosslinking agent. 
Monoclonal antibodies are prepared by recovering spleen cells from 
immunized animals and immortilizing the cells in conventional fashion, 
e.g. by fusion with myeloma cells or by EB virus transformation and 
screening for clones expressing the desired antibody. 
Antibodies which are capable of binding to or in the steric vicinity of the 
IR insulin binding site are identified by immobilizing rabbit anti-IR 
.beta.-subunit on goat anti-rabbit IgG-coated microtiter wells, adding 
dilutions of test sample in .sup.125 I-insulin-containing PBS to the 
coated wells, incubating overnight, aspirating the test sample from the 
wells, washing with PBS, and determining the radioactivity remaining in 
the wells by gamma counting. Test samples containing desired antibodies 
are indicated by increases in insulin displacement at higher sample 
concentrations. 
Similar assays are conducted to identify antibodies which bind to the 
ATP-binding, protein phosphokinase active site, and autophosphorylation 
domains of IR. 
Diabetes is believed to be primarily a function of inadequate insulin 
levels or defective or insufficient insulin receptors. For example, an 
insulin receptor may be defective because of an inability to bind insulin 
in a fashion that will activate the tyrosine kinase activity of the 
receptor. This may result, for example, from dominant point mutations in 
the insulin receptor binding region of IR. 
Knowledge of the amino acid sequence for IR has now made possible the 
generation of antibodies against selected regions of the receptor which 
can be methodically screened for insulin-like or insulin agonist activity. 
Such antibodies or their derivatives are useful as insulin substitutes for 
the therapeutic treatment of diabetes stemming from defective receptor 
binding of insulin. While anti-insulin receptor antibodies are known that 
induce glucose uptake by test cells.sup.50, the method herein makes it 
possible to raise such antibodies against predetermined sites on the IR 
molecule, thereby avoiding the generation of contaminant antibodies having 
undesirable side effects.sup.51,52, and for the first time disclose 
methods for stimulating defective insulin receptors. 
Suitable candidate antibodies such as those prepared above, are screened by 
preparing IR in conventional fashion from a tissue sample obtained from a 
diabetic patient found partly or wholly refractory to insulin. It is 
unnecessary to purify the IR to the sequenceable grade described elsewhere 
herein. It is satisfactory to purify the IR by known methods up to insulin 
affinity adsorption (which in any case probably would be ineffective where 
the defective receptor is incapable of binding insulin). 
The IR preparation is combined in aqueous solution with (a) 0.1-5 mg/ml of 
a protein tyrosine kinase substrate such as (Glu AlaTyr)n.sup.49 or 
histone H2B, (b) (gamma-.sup.32 P) ATP and (c) aliquots of test sample and 
varying dilutions of the test sample candidate antibody in control 
antibody solution so as to prepare a curve in which essentially the same 
antibody ccncentration is present for each data point. The assay is 
conducted generally as described previously.sup.49, except that the IR 
need not be immobilized. Normal antibody from the animal species in which 
the candidate antibody was raised was prepared in the same way as the 
candidate antibody to serve as a negative control. Afterincubation the 
proteins in the solution are precipitated and washed free of labelled ATP, 
or otherwise separated from labelled ATP as for example by gel 
electrophoresis. The radioactivity found in the protein fractions from the 
varying antibody dilutions and negative control is compared. Candidate 
antibody which induces or stimulates incorporation of .sup.32 P into 
protein is suitable for therapeutic use as an insulin substitute. 
Altenatively, a less direct assay will be the measurement of cellular 
glucose uptake rates in the presence of dilutions of the candidate 
antibody. 
It is prefered to use the Fab fragments of the selected antibody because 
such fragments, even though divalent, will not bind complement and 
therefore will not participate in potentially toxic autoimmune responses. 
Such fragments are obtained by enzymatic digestion of the antibody using 
conventional methods. 
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