Herstatin protein, human

EXAMPLE 16

Recombinant Herstatin Specifically Inhibited Human Carcinoma Cells that Over-Express HER-2

Herstatin, according to the present invention, can be used therapeutically to treat cancer, particularly cancers of the breast, ovary, lung, colon, glia and prostate. The ability of recombinant, purified herstatin to specifically inhibit human carcinoma cells that over-express HER-2 was assessed in both ovarian and breast cancer cell lines.

Methods. Production of recombinant, purified herstatin. Recombinant herstatin was produced as a histidine-tagged protein in insect S2 cells using standard insect expression systems from Invitrogen. The recombinant protein was secreted into serum-free media and was present at about 10 mg per liter. Herstatin protein was purified by nickel affinity chromatography and by conconavalin A affinity chromatography. The purified protein was judged to be greater than 90% pure based on protein staining.

Treatment of cancer cell lines with herstatin. Purified recombinant herstatin was added (to a concentration of 240 nM) to cancer cells lines at the time of plating, at 2 days and at 4 days. Growth and/or survival of the living cells was quantitated by the art-recognized MTT live cell assay (Hansen et al., Immunol. Methods 119:201-210, 1989).

Results. FIG. 24 shows that growth and/or survival of an ovarian carcinoma cell line (SKOV-3) and a breast carcinoma cell line (SKBR-3) that over-express HER-2 was inhibited by about 60% relative to non-herstatin treated cells (left-most two bars). By contrast, the normal breast cell line (HBL-100) and the MCF-7 breast carcinoma cell line that express low levels of HER-2, were not significantly inhibited by purified recombinant herstatin (right-most two bars).

Thus, the level of herstatin-mediated growth inhibition reflected the HER-2 expression level. Herstatin inhibition showed specificity toward those cancer cells that over-express HER-2 relative to normal cells or to transformed cells that express only low or normal levels of HER-2.

FIG. 25 illustrates that inhibition of growth and/or survival (optical density at 600 nM) of SKOV-3 carcinoma (left-most three bars), but not normal HBL-100 cells (right-most three bars), showed a dose response to purified recombinant herstatin.

EXAMPLE 14

Herstatin Blocked the Activated Akt-Mediated EGF Survival Signal

The protein kinase called Akt is a key regulator of cellular survival. Activation of Akt is both necessary and sufficient for survival of cells. Stimulation of activated Akt causes inappropriate cell survival, or prevents normal cell death, which has been found to occur in several human cancers. HER-2 and the EGF receptor both cause activation of the Akt survival signal whereby, according to current theory and belief, they cause oncogenic growth (Blume-Jensen & Hunter, Nature 411:355-365, 2001; Datta et al., Genes and Development 13:2905-2907, 2000; Yarden & Slikowski, Nature Reviews, Molecular Cell Biology, 2:127-137).

Methods. Measurement of activated phospho-akt (activated AKT) in EGFR3T3 cells. Measurement of activated AKT (phospho-akt) was accomplished using standard Western blotting techniques, employing a commercially available anti-phospho-akt antibody (Santa Cruz). Transfection of cells was as described herein, above.

Results. Herstatin transfected control cells that over-express the EGF receptor (EGFR3T3) were further treated with EGF (100 ng/ml in DMEM) or vehicle (control) and incubated for different times to test the affects of herstatin on EGF survival (FIG. 21). To test for activated Akt, cell extracts were reacted with an antibody (commercially available from Santa Cruz) specific for the activated form of Akt (phospho-akt).

A strong stimulation of the activated phospho-akt at 20 minutes was seen after stimulation of the EGF receptor with EGF (upper panel). Significantly, in the presence of herstatin, the activated Akt signal was eliminated. The overall level of the Akt protein was constant as shown by control lanes (lower panel of FIG. 21).

Thus, according to the present invention, diminished cell survival was not only correlated to a reduction in tyrosine phosphorylation of p185HER-2 and to interference with EGF-mediated activation of the EGF receptor (as shown in the Examples herein, above), but also to blocking the EGF-receptor-meidated cell survial signal. This result demonstrates the efficacy of herstatin in blocking the activated Akt-mediated cell survival signal generated by EGF.

EXAMPLE 1

This example provides the results from an experiment to investigate HER-2 mRNA diversity within the extracellular domain (ECD) coding sequence using polymerase chain reaction (PCR). A cDNA library from SKOV-3 cells (American Type Culture Collection (Rockville, Md.) maintained in DMEM, supplemented with 10% fetal bovine serum and 0.05% gentamycin), an ovarian carcinoma cell line in which the HER-2 gene is amplified eight times (Tyson et al., Am. J. Obstet. Gynecol. 165:640-646, 1991) was examined using a forward primer specific for exon 1 (Tal et al., Mol. Cell. Biol. 7, 2597-2601, 1987) identical to nucleotides 142-161 and a reverse primer complementary to nucleotides 1265-1286 in exon 9 (Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993). Briefly, The SKOV-3 cDNA library was provided by Origene Technologies, Inc. (Rockville, Md.), and was prepared from RNA extracted from SKOV-3 cells. RNA was extracted from SKOV-3 cells grown to 80% confluence on 15 cm plates with TriReagent (Molecular Research Center, Inc., Cincinnati, Ohio), according to the manufacturer's protocol, to obtain total RNA. RNA was resuspended in 10 mM Tris-EDTA, pH 8.0, for reverse transcription and cDNA library construction, or in RNA hybridization buffer (80% formamide, 40 mM PIPES, 4 mM NaCl, 1 mM EDTA, pH 7.5) for ribonuclease protection assay (RPA). RNA concentrations were determined spectrophotometrically at OD₂₆₀. Poly A⁺ mRNA was selected from total RNA using a mRNA extraction kit (Oligotex, Qiagen).

A product of ˜1420 bp, determined to be HER-2-specific by Southern blotting, was approximately 270 bp larger than the expected size of 1144 bp from the previously described cDNA sequence (Coussens et al., Science 230:1132-1139, 1985). Briefly, the Southern blotting procedure transferred nucleic acids from agarose gels under vacuum (Bio-Rad Model 785 Vacuum Blotter) in 0.4 M NaOH to Gene Screen Plus Hybridization Transfer Membrane (NEN Research Products, Boston, Mass.). Nucleic acids were fixed to membranes by UV crosslinking in a UV-Stratalinker (Stratagene, Inc., La Jolla, Calif.), and the membranes were blocked in hybridization buffer (50% formamide, 5×SSC, 1% SDS, 10 mg/ml herring sperm DNA) at 42° C. for 2 h. The membranes were hybridized at 42° C. for 16 h in hybridization buffer with 10⁷ cpm of a 220 bp Kpn-HincII fragment from ECDIIIa cDNA labelled with (α-³²P)dCTP (NEN Life Sciences) using a Random Prime DNA Labelling Kit (Boehringer Mannheim).

Templates were amplified in a Perkin Elmer GeneAmp PCR System 2400 (Perkin Elmer Cetus, Emeryville, Calif.) using the Expand High Fidelity PCR System (Boerhinger Mannheim) with 1× High Fidelity PCR buffer containing 2.5 mM MgCl₂, 5 μM of each primer, and 200 μM dNTPs. All primers were obtained from GIBCO BRL (Life Technologies). Numbering of nucleotide and amino acid residues is according to the HER-2 cDNA sequence reported by Coussens et al. (Coussens et al., Science 230:1132-1139, 1985). The HER-2 extracellular domain was targeted for amplification from an SKOV-3 cDNA library (Origene Technologies, Inc.) using a forward primer (A) identical to nucleotides (nt) 142-161 of HER-2 cDNA (5′-TGAGCACCATGGAGCTGGC-3′ [SEQ ID NO 3]), which spans the initiation codon (underlined) and a reverse primer (B) (5′-TCCGGCAGAAATGCCAGGCTCC-3′ [SEQ ID NO 4]), which is complementary to HER-2 exon sequence at nt 1265-1286. Cycling parameters were: 94° C., 30 sec; 58° C., 45 sec; 68° C., 3 min, for 30 cycles. The region spanning the alternative sequence (denoted ECDIIIa) from genomic DNA, was amplified using a forward primer (C) (5′-AACACAGCGGTGTGAGAAGTGC-3′ [SEQ ID NO 5]) identical to HER-2 exon-specific sequence at nt 1131-1152 and the reverse primer (B) [SEQ ID NO. 4] on DNA prepared as described (Bond et al., FEBS Letters 367:61-66, 1995) with cycling parameters: 94° C., 30 sec; 62° C., 30 sec; 72° C., 60 sec, for 25 cycles.

Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the structure of mRNA containing the ECDIIIa sequence. First strand cDNA was reverse transcribed (Bond et al., FEBS Letters 367:61-66, 1995) using 5 μg RNA primed with 0.5 μg oligo-dT. To amplify the ECDIIIa insert and adjacent 5′ HER-2 exon sequence, a forward primer (A) described above and a reverse primer (D) (5′-ATACCGGGACAGGTCAACAGC-3′ [SEQ ID NO 6]) which is complementary to the 3′ECDIIIa-specific sequence were used. Cycling parameters were: 94° C., 30 sec; 60° C., 40 sec; 68° C., 2 min, for 30 cycles.

Amplification of the ECDIIIa insert and adjacent 3′ HER-2 exon-specific sequence was with a forward primer (E) (5′-TCTGGGTACCCACTCACTGC-3′ [SEQ ID NO 7]) which is identical to the 5′ECDIIIa-specific sequence and contains a Kpn1 restriction site and a reverse primer (F) (5′-TTCACACTGGCACGTCCAGACC-3′ [SEQ ID NO 8]) which is complementary to HER-2 exon sequence at nt 3898-3919 and spans the termination codon (underlined). Cycling parameters were: 94° C., 30 sec; 60° C., 40 see; 68° C., 5 min, for 30 cycles.

The PCR product was subcloned and the nucleotide sequence was determined.

The results showed that the normal HER-2 coding sequence was present beginning with the 5′ primer sequence and continued uninterrupted through nucleotide 1171. At this position, a 274 nucleotide insertion was found, followed by the expected coding sequence, including the 3′ primer sequence. Analysis of the predicted protein product showed that the 274 nucleotide insertion encodes an extension of the known HER-2 protein, beginning at residue 340 (Coussens et al., Science 230:1132-1139, 1985), and introduces an in-frame stop codon 79 amino acids later (FIG. 1). Comparison of the inserted nucleotides and their predicted amino acid sequence with sequences in Genbank showed no homologies. Examination of the 5′ and 3′ junctions of the divergent sequence revealed consensus splice donor and acceptor sites (Sharp, and Burge, Cell 91:875-879, 1997) and include a pyrimidine tract and potential branchpoint adenine residues near the 3′end of the insert sequence (FIG. 1). Thus, the inserted sequence is likely to be an intron.

Inspection of the predicted amino acid sequence of the novel 79 amino acids [SEQ ID NO. 1] encoded by the inserted sequence shows a consensus N-linked glycosylation site and a high proline content of 19% (FIG. 1). The inserted sequence was designated ECDIIIa since it is located at the boundary between subdomains II and III in the extracellular domain of the p185HER-2 sequence (Lax et al., Mol. Cell. Biol. 8:1831-1834, 1988). The insert sequence is in-frame with the adjacent 5′ HER-2 exon sequence for 236 nt where there is a termination codon.

EXAMPLE 2

This example provides the results from experiments characterizing ECDIIIa as contiguous with HER-2 exons in the genome. To investigate the HER-2 gene structure in the region of the ECDIIIa sequence, a forward primer, identical to nucleotides 763-785, and a reverse primer, complementary to nucleotides 1265-1286 of the HER-2 cDNA, were used in the PCR on human genomic DNA. The amplification product was anticipated to span exon 5 (Tal et al., Mol. Cell. Biol. 7:2597-2601, 1987) to an exon which is immediately 3′ of the ECDIIIa sequence. Intron number and sizes were estimated based on PCR product sizes, restriction digest analysis, and partial sequence analysis of amplification products.

Next, human genomic DNA was examined using HER-2 exon-specific primers that directly flank the insert to determine the sequences immediately flanking the ECDIIIa sequence. A ˜430 bp product was amplified from normal human genomic DNA and from genomic DNA extracted from carcinoma cell lines SKOV-3, SKBR-3 and BT474, all of which have HER-2 gene amplification (Kraus et al., EMBO J. 6:605-610, 1987) and were found to express ECDIIIa in their cDNA. The identities of the PCR products as HER-2 were verified by Southern blot analysis using the procedure described in Example 1. Nucleotide sequence analysis showed that the PCR product from human genomic DNA contained the ECDIIIa insert, flanked immediately on both sides by known HER-2 coding sequence; no mutations or rearrangements were seen. These data show that the ECDIIIa sequence represents a wholly retained intron, likely intron 8 based on the size of products amplified following intron 4 and on the location of intron 8 in the homologous EGFR gene and HER-3 gene (Lee and Maihle, Oncogene 16:3243-3252, 1998).

EXAMPLE 11

This example illustrates that the sequence of intron 8 is polymorphic within that portion of intron 8 that serves as in-frame (with the extracellular domain of p185HER-2) coding sequence when intron 8 is alternatively retained in mRNA.

Intron 8 of the human HER-2 gene is alternatively retained in mRNA, and encodes a novel 79-residue domain at the C-terminus of a part of the extracellular domain of p185HER-2. The product, “herstatin,” of the alternative transcript with the retained intron functions as an autoinhibitor of the HER-2 oncogene. The intron 8 encoded domain, alone, was shown to bind with nM affinity to p185HER-2. (Doherty et al., Proc. Natl. Acad. Sci. USA 96:10, 869-10,874, 1999).

Polymorphisms in the nucleotide and deduced amino acid sequence of intron 8 in the HER-2 gene were identified by sequencing genomic DNA from 15 different individuals. FIG. 8 and SEQ ID NO:1 show the most common nucleotide and corresponding amino acid sequences, respectively, of intron 8. This region contains 10 different polymorphisms (marked by the letters W (2x), Y (3x), R, N, M, and S (2x) in SEQ ID NO:10; or marked by an “X” in FIG. 8) that result in nonconservative amino acid substitutions (see legend to TABLE 1). For example, the polymorphism (G→C) at nucleotide position 161 (FIG. 8; TABLE 1) would result in a substitution of Arginine (R) for Proline (P) at amino acid residue #54 of SEQ ID NO:1, or residue #394 of SEQ ID NO:2. The N-terminal Glycine (G), designated as position 1 in FIG. 8 or SEQ ID NO:10, corresponds to amino acid residue #341 in the “herstatin” sequence (Doherty et al., Proc. Natl. Acad. Sci. USA 96:10, 869-10,874, 1999). The nucleotide sequence shown in FIG. 1(A) is a polymorphic form that differs at amino acid residues #6 and #73 from the most commonly detected sequence shown here in FIG. 8.

This result demonstrates that in the human population there are several variations in the intron-8 encoded domain that could lead to altered biochemical and biological properties among ECDIIIa-containing protein variants. Some identified variants are summarized in Table 1:

EXAMPLE 12

This example shows (see Table III, below) five polymorphic HER-2 intron 8 polymorphisms (sequence variants 12-16) identifiable in DNA samples from African Americans.

Specifically, four polymorphic sites were identified within that portion of intron 8 that serves as in-frame (with the extracellular domain of p185HER-2) coding sequence when intron 8 is alternatively retained in mRNA (i.e., four polymorphic sites within the sequence region encompassed by SEQ ID NO:10, or within that encompassed by the sequence region of FIG. 8). Two of these polymorphic sites (variants 12 and 15) correspond in position to those (variants 3 and 10, respectively) disclosed above in Example 11, whereas the other two (variants 13 and 14) represent additional polymorphic sites (Table II).

Furthermore, (see Table II and Table III, below) an additional polymorphic site (variant 16) was identified in a region of intron 8 that remains as “non-coding” sequence when intron 8 is alternatively retained in mRNA. This “non-coding” intron 8 polymorphic site is located 3′, or downstream from that portion of intron 8 that contains the other polymorphic sites shown in this Example and Example 11, and that serves as in-frame (with the extracellular domain of p185HER-2) coding sequence when intron 8 is alternatively retained in mRNA.

Methods. Polymorphisms in the nucleotide and deduced amino acid sequence of intron 8 in the HER-2 gene were identified by sequencing genomic DNA (using blood samples) from 215 individuals corresponding to 75 African Americans (Black), 135 Caucasians (White), one Asian American (Asian) and 4 Hispanics. As for Example 11, above, the N-terminal Glycine (G or Gly) designated as position 1 in FIG. 8 or SEQ ID NO:1 or SEQ ID NO:10, corresponds to amino acid residue #341 in the “herstatin” sequence of SEQ ID NO:2 or SEQ ID NO:13.

Results. Table II designates the nucleotide substitutions and the two amino acid residue substitutions in the coding sequence of intron 8 and a third nucleotide substitution in a non coding sequence of intron 8 using numbering corresponding to the entire “herstatin” protein sequence (SEQ ID NO:2 or SEQ ID NO:13):

Table III, below, illustrates that the sequence data revealed polymorphisms at nucleotide positions #17 and #217 (also corresponding to nucleotide positions of the sequence region shown in FIG. 8 or SEQ ID NO:10). The polymorphism at position #17 (variant 12) corresponds to variant 3 of Table I (Example 11). The polymorphism at position #217 (variant 15) corresponds (at least at the protein level) to variant 10 of Table I (Example 11) (see SEQ ID NO:12 and SEQ ID NO:13).

Additionally, the sequence data (see SEQ ID NO:11) revealed (see Table III) that intron 8 contains three polymorphic sites (corresponding to variants 13, 14 and 16) in addition to those disclosed in Example 11, above. Two of these (variants 13 and 14) are located at nucleotide positions #49 and #92 of SEQ ID NO:11 (also corresponding to nucleotide positions #49 and #92 of SEQ ID NO:10 (or FIG. 8). The third (variant 16) is located at a nucleotide position #259 of SEQ ID NO:11 [also corresponding to nucleotide position #259 relative to the sequence region of SEQ ID NO:10 (or to position #264 of the sequence shown in FIG. 1, panel A)]. Thus, the polymorphism corresponding to variant 16 is located 19 nucleotide positions 3′ (downstream) from that portion of intron 8 that contains the other polymorphic sites shown in this Example and Example 11 (i.e., that portion represented by SEQ ID NO:10), and that serves as in-frame (with the extracellular domain of p185HER-2) “coding” sequence when intron 8 is alternatively retained in mRNA.

Two of these polymorphisms result in nonconservative amino acid substitutions (see Table II and Table III, and legend of Table III; also see SEQ ID NO:12 and SEQ ID NO:13). For example, the polymorphism (C→T) found at the nucleotide position corresponding to nucleotide #49 of SEQ ID NO:11 [or to position # 49 of SEQ ID NO:10 or FIG. 8] (i.e., the polymorphism at position X(49) of Table 2) would result in a substitution of Arginine (Arg) for Cysteine (Cys) at the amino acid position corresponding to amino acid residue #17 of SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:10 or SEQ ID NO:11) or to amino acid residue-#357 of SEQ ID NO:13 or SEQ ID NO:2.

SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 show the four variant amino acid positions described in this example, along with those of Example 11 that are also shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:10.

Table III designates (in addition to variants 12 and 15, which correspond to variants 3 and 10, respectively of Table I) the nucleotide substitutions and the corresponding two additional (relative to those of Table I of Example 11) amino acid residue substitutions (i.e., variants 13 and 14) in the “coding” sequence of intron 8, along with the third nucleotide substitution in the 3′ “non-coding” region of intron 8; The numbers in parenthesis after each X (polymorphic position) refer to nucleotide positions of SEQ ID NO: II [or, as in Table I, correspond to (or are relative to, in the case of X(259) the nucleotide positions in the DNA sequences shown in FIG. 8 or SEQ ID NO:10].

As for Example 11, above, the N-terminal Glycine (G or Gly) designated as position 1 in SEQ ID NO:11, FIG. 8, SEQ ID NO:1 or SEQ ID NO:10, corresponds to amino acid residue #341 in the “herstatin” sequence of SEQ ID NO:2.

SKOV3 ovarian carcinoma cells. Two additional polymorphisms were found in a cell line derived from human ovarian cancer (SKOV3). These two polymorphisms result in nonconservative amino acid substitutions. One polymorphism is a substitution (C-T) at nucleotide #17 in the intron 8 sequence and nucleotide #1037 in the “herstatin” sequence resulting in a substitution of leucine for proline at amino acid residue #6 in the intron 8 sequence and at amino acid residue #346 in the “herstatin’ sequence (i.e., of SEQ ID NO:2 or SEQ ID NO:13). The second polymorphism found in the SKOV3 ovarian carcinoma cells line is a substitution (G-A) at nucleotide #217 in the intron 8 sequence and nucleotide #1237 in the “herstatin” sequence resulting in a substitution of Asparagine for Aspartic Acid at amino acid residue #73 in the intron 8 sequence and amino acid #413 in the “herstatin” sequence (i.e., of SEQ ID NO:2 or SEQ ID NO:13).

Significantly, the five polymorphic sites identified in the sequence analysis of this Example 12 were found only in DNA samples from African Americans (Black).

Summary of Examples 11 and 12

Together, Examples 11 and 12 of the present invention disclose 13 polymorphic positions in intron 8 of the Her-2 gene. Example 12, involved a relatively large DNA sample size, and indicated that the five polymorphic sites identified (three of which are distinct from the ten polymorphic sites identified in Example 11) are unique to African Americans (Black).

Twelve of the thirteen polymorphisms (i.e., except for variant 16 of Example 12) of these two Examples are present in that portion of intron 8 that serves as in-frame (with the extracellular domain of p185HER-2) coding sequence when intron 8 is alternatively retained in mRNA.

The polymorphism corresponding to variant 16 is located in a region of intron 8 that remains as “non-coding” sequence when intron 8 is alternatively retained in mRNA. This “non-coding” intron 8 polymorphic site is located 19 nucleotide positions 3′, or downstream from that portion of intron 8 that contains the other polymorphic sites, and that serves as in-frame (with the extracellular domain of p185HER-2) coding sequence when intron 8 is alternatively retained in mRNA.

These HER-2 intron 8 polymorphisms provide for novel DNA and protein sequences, novel pharmaceutical compositions for treating solid tumors that overexpress HER-2, and monoclonal antibodies that bind to ECDIIIa variants corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:12 or SEQ ID NO:13. These HER-2 intron 8 polymorphisms also provide for prognostic and diagnostic assays for the treatment and prevention of cancer.

Example 21

Assay to Measure Modulation of Insulin Binding Affinity for INSR by Anti-INSR Antibodies

To determine the ability of the modulating antibodies to affect the binding of insulin to the insulin receptor, the affinity of unmodified insulin binding to human INSR expressed on the surface of serum starved CHOK1 cells (hINSR8-CHOK1) was measured in the presence and absence of monoclonal antibodies to INSR. A KinExA assay was developed to measure very low levels of insulin in cell culture media. This assay allowed the binding of insulin to cells expressing INSR to be measured by determining the level of insulin depletion from the cell culture media. As insulin became bound to the cells, the concentration of insulin in the cell culture media dropped. By using a titration of cells expressing INSR and measuring the percent free insulin, the affinity of the INS-INSR interaction could be estimated using KinExA software. This assay was used to measure the degree of modulation of insulin binding activity shown by various anti-INSR antibodies.

hINSR8-CHOK1 cells were serum starved overnight and then prepared for assay by pelleting cells and resuspending at a concentration of 2× the final assay concentration for the highest dilutions (between 3.5×10⁷ and 2.0×10⁷ cells/mL in assay dilution buffer of PBS (Teknova, Hollister Calif.) with 500 μg/mL BSA and 0.1% sodium azide (Sigma Aldrich, St. Louis, Mo.)). A two-fold serial dilution of cells was prepared creating a ten-point dilutions series and a no-cells control was also used. Cell suspensions were aliquoted into polypropylene assay tubes in 2 mL volume each. To these cell suspensions 1 mL of 40 ug/mL test antibody (or 100 ug/mL for Ab078) was added to each tube, gently mixed and incubated for 30-45 minutes on ice. The antibodies used were tested in comparison to the negative control human IgG2 anti-KLH antibody. 1 mL of 200 pM insulin was added to each tube to establish a final insulin concentration of 50 pM (300 pg/mL) (Sigma-Aldrich, St. Louis, Mo.). Samples were incubated overnight at 4° C. for 18 hours then centrifuged to pellet cells and supernatants were removed for testing.

KinExA 3000 analysis was performed using beads coated with an anti-insulin monoclonal antibody. 2 grams of poly(methyl methacrylate) (PMMA) beads (Sapidyne, Boise, Id.) was suspended in 9 mL of assay buffer PBS containing 65 ug/mL of clone D6C4 mouse anti-insulin monoclonal antibody (Fitzgerald Industries, Acton Mass.). Beads were rotated at room temperature for 6 hours then allowed to settle. Supernatant was replaced with PBS with 50 mg/mL BSA Fraction V (Sigma-Aldrich, St. Louis, Mo.) and rotated overnight at 4° C. Detection solution used was biotinylated mouse anti-insulin clone D3E7 (Fitzgerald Industries, Acton Mass.) at 0.15 μg/mL in assay dilution buffer with Streptavidin-PE at 1 ug/mL (Invitrogen, Carlsbad, Calif.). On the KinExA 3000 the sample was injected at 0.25 mL/minute for 240 seconds, then rinsed for 60 seconds in running buffer (PBS with 0.05% sodium azide), then 240 seconds of the detection solution was injected, followed by a final 90 second wash at 1 mL/minute. The difference in voltage from an early initial time-point and a time point near the end of the run was measured and used to calculate affinities. The INSR concentration on the cells was estimated at 2.5×10⁵ receptors/cell. Affinity was determined using the KinExA software (Sāpidyne, Boise Id.) and EC50's were calculated by non-linear fit in Prism (GraphPad Software, La Jolla Calif.).

A number of anti-INSR antibodies enhanced the affinity of insulin for the cells, as shown in Table 4 below. One of the tested antibodies decreased the affinity of insulin for the cells by approximately three-fold.

These data illustrate that the screening methods described herein produce positive modulator antibodies that strengthen the binding affinity of insulin for insulin receptor.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications, variations and combinations of embodiments may be made without deviating from the spirit and scope of the invention. Such modifications, variations and combinations are intended to be encompassed by the following claims.