©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Recombinant Antibodies in Bioactive Peptide Design (*)

(Received for publication, June 27, 1994; and in revised form, January 9, 1995)

Cristina Monfardini (1) (2) Thomas Kieber-Emmons (3)(§) Joan M. VonFeldt (1) (2)(¶) Brigid O'Malley (1) (2) Helga Rosenbaum (1) (2) A. Paul Godillot (1) (2) Kenneth Kaushansky (6)(**) Christopher B. Brown (7) Donald Voet (5) Daniel E. McCallus (1) (2) David B. Weiner (1) (2) (3)(§§) William V. Williams (1) (2) (4)

From the  (1)Department of Medicine, Rheumatology Division, (2)Institute for Biotechnology and Advanced Molecular Medicine, and (3)Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine and (4)Childrens' Hospital of Philadelphia and the (5)Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, the (6)Division of Hematology, University of Washington, Seattle, Washington 98195, and the (7)University of Calgary Health Sciences Center, Calgary, Alberta, Canada T2N 4N1

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is important in many immune and inflammatory processes. GM-CSF binds to specific cellular receptors which belong to a recently described supergene family. These receptors are potential targets for pharmacologic design, and such design depends on a molecular understanding of ligand-receptor interactions. One approach to dissecting out critical intermolecular interactions is to develop analogs of specific interaction sites of potential importance. Monoclonal antibodies have been employed for these purposes in prior studies. Here we present application of recombinant antibody technology to the development of analogs of a site on GM-CSF bound by a neutralizing anti-GM-CSF monoclonal antibody.

Polyclonal antisera with high titer neutralizing activity against human GM-CSF were developed in BALB/c mice. Purified immunoglobulins were prepared and used to immunize syngeneic mice. Anti-anti-GM-CSF was developed which demonstrated biological antagonist activity against GM-CSF-dependent cellular proliferation. RNA was extracted from spleen cells of mice with biologically active anti-anti-GM-CSF, cDNA synthesized, and polymerase chain reaction performed with primers specific for murine kappa light chain V regions. Polymerase chain reaction products were cloned into the pDAB(L) vector and an expression library developed. This was screened with anti-GM-CSF neutralizing mAb 126.213, and several binding clones isolated. One clone (23.2) which inhibited 126.213 binding to GM-CSF was sequenced revealing a murine kappa light chain of subgroup III. Comparison of the 23.2 sequence with the human GM-CSF sequence revealed only weak sequence similarity of specific complementarity determining regions (CDRs) with human GM-CSF. Structural analysis revealed potential mimicry of specific amino acids in the CDR I, CDR II and FR3 regions of 23.2 with residues on the B and C helices of GM-CSF. A synthetic peptide analog of the CDR I was bound by 126.213, specifically antagonized GM-CSF binding to cells and blocked GM-CSF bioactivity. These studies indicate the feasibility of using recombinant antibody libraries as sources of interaction site analogs.


INTRODUCTION

Development of small molecular mimics of larger, polypeptide ligands is one approach to pharmacophore design. Several strategies are available for the development of such mimics, including the use of small oligopeptide analogs derived from native sequence(1, 2, 3, 4, 5) , development of peptidic and non-peptidic analogs based on molecular structure data(6, 7) , and analysis of alternative ligands(8) . Alternative ligands that bind to the same site as the native ligand provide the opportunity to investigate structural and chemical constraints for binding in the setting of diverse backbone geometries. This has the potential to identify critical contact residues based on similar structural and chemical characteristics between the diverse ligands.

Prior studies have investigated a monoclonal antibody (mAb)(^1), 87.92.6, which mimicked a neutralizing epitope on the reovirus type 3 hemagglutinin(9, 10, 11, 12) . 87.92.6 was bound both by a reovirus type 3 neutralizing mAb and the reovirus type 3 receptor. Sequence similarity between 87.92.6 light chain second complementarity determining region (CDR II) and the reovirus type 3 hemagglutinin (13) allowed the development of synthetic peptides and peptidomimetics which bound both the neutralizing mAb and the reovirus type 3 receptor. These peptides and peptidomimetics also demonstrated biological activity on reovirus type 3 receptor bearing cells. The use of anti-receptor mAbs as a source of sequence-structural information to aid in peptide design has allowed the development of similar biologically active peptides in several systems, including the platelet fibrinogen receptor(14) , the thyroid-stimulating hormone receptor(15) , and epitopes on the human immunodeficiency virus (16) and hepatitis B surface antigen(17) .

Recombinant antibodies have been developed which are expressed in bacteria (18, 19) or on the surface of filamentous bacteriophage (20, 21, 22, 23) . The advantages of recombinant approaches to antibody development include the ability to rapidly screen thousands of clones simultaneously, the potential to detect binding moities poorly represented in the initial repertoire, and the potential to express isolated variable regions. While intact mAbs contain both light and heavy chain variable regions (V(L) and V(H), respectively), recombinant antibodies can be developed which express both V(L) and V(H), or V(L) or V(H) alone. This limits the potential interaction sites of the recombinant antibody, allowing more precise delineation of critical interaction regions.

Here we describe the development of a recombinant light chain library in Escherichia coli derived from mice immunized with polyclonal anti-GM-CSF. This library was screened with a previously described neutralizing anti-GM-CSF mAb 126.213 (24) which inhibits GM-CSF binding to HL-60 cells, neutralizes GM-CSF induced colony formation, and competes with the alpha chain of the GM-CSF receptor for GM-CSF binding(25) . Screening with radioiodinated 126.213 yielded several binding clones, including one that inhibited immunoprecipitation of GM-CSF by 126.213. Comparison of the recombinant V(L) sequence with the human GM-CSF sequence revealed only weak similarity with GM-CSF, but structural analysis suggested mimicry of residues on the B and C helices of GM-CSF by a site chiefly made up of the CDR I region of 23.2. A synthetic peptide corresponding to the CDR I was bound by the neutralizing anti-GM-CSF mAb and specifically inhibited GM-CSF binding and the growth of GM-CSF-dependent cells. These studies suggest a structural basis for recombinant antibody mimicry of a predominately helical molecule (human GM-CSF), demonstrate a bioactive peptide analog of a GM-CSF site implicated in receptor binding, and indicate the feasibility of using recombinant antibody libraries as sources of interaction site analogs.


MATERIALS AND METHODS

Bacterial Strains

E. coli DH5alpha competent cells (Life Technologies, Inc.) were used for transformation. Bacteria were grown in Luria broth containing 100 mg/ml ampicillin (LB/amp)(26, 27) . Solid media contained 1.5% agar (Difco Inc.).

Enzymes and Oligonucleotides

Restriction endonucleases and T4 DNA ligase were purchased from Life Technologies, Inc. Enzyme reaction conditions were according to those of the supplier. Oligonucleotides for PCR primers and for Southern blotting were synthesized by the DNA Synthesis Facility of the Wistar Institute. The primers were selected by analysis of immunoglobulin sequences as published by Kabat et al.(28) . The specific primers are listed in Table 1. PCR amplification employed primers 3315 (relatively specific for the murine Vkappa III family) and 5591 (near the 3` end of the Ckappa coding region). Note that primer 5591 introduces a stop codon at codon 207, resulting in a truncated light chain lacking the carboxyl-terminal 8 amino acids including the cysteine at position 214. This should result in production of light chains which are predicted to remain monomers. Primers were tested on various hybridoma cell lines in the laboratory prior to their use in library construction. In these studies amplified sequences were isolated from the gels cloned and sequenced to verify the utility of the primers and their specificity for amplification of Ig variable regions (data not shown).



Cell Lines and Proliferation Assay

CTLL cells and the proliferation assay in response to IL-2 or rat spleen concanavalin A was described previously(29) . AML 193 cells were obtained from the American Type Culture Collection (ATCC), and MO7E cells were from R. Zollner, Genetics Institute (Cambridge MA). AML 193 was grown serum free in Iscove's modified Dulbecco's medium with insulin (10 µg/ml), transferrin (5-10 units/ml), 1% OPI media additive (oxalate, pyruvate, and insulin), and GM-CSF (0.5 ng/ml). MO7E was grown in RPMI 1640 with 10% heat-inactivated fetal calf serum, Pen/strep, L-glutamine, and GM-CSF 0.5 ng/ml. For proliferation assays, 2 times 10^4 AML 193 or MO7E cells were cultured per well in 96-well round bottomed plates in the above medium along with test antisera in a final volume of 200 µl. Following a 3-5 day incubation, tritiated thymidine (1 µCi/well) was added for an additional 18 h, the cells harvested onto glass fiber filters utilizing a PhD cell harvester, and counts/minute incorporated determined in a standard liquid scintillation system.

Development of Anti-GM-CSF and Anti-anti-GM-CSF

Recombinant human GM-CSF (obtained from Bachem Biosciences, Philadelphia, PA) was used to immunize BALB/c mice as described previously(30) . Serum was obtained 1 week following each boost with antigen. Antisera from three to five animals were pooled for the assays performed. Following the third boost, significant neutralizing titers against human GM-CSF-dependent cellular proliferation were demonstrated (Fig. 1). The mice were bled after five boosts and IgG purified from serum by affinity chromatography with Sepharose-protein A. This was used to immunize syngeneic BALB/c mice (50 µg of purified IgG/immunization) and serum obtained following each boost. The sera were assayed for inhibition of GM-CSF-dependent proliferation (see below), and significant (>50%) inhibition was seen following the eighth boost against both MO7E and AML193 cells ( Fig. 1and data not shown). Mice that exhibited neutralizing activity on this assay served as spleen cell donors.


Figure 1: Biological activity of antisera. Proliferation of the human GM-CSF-dependent cell line MO7E was performed as noted under ``Materials and Methods'' in the presence of varying dilutions of murine anti-GM-CSF (following the fifth boost) and murine anti-anti-GM-CSF (following the ninth boost). Counts/min incorporated ± the standard deviation of triplicate wells is shown for various dilutions of antisera. In similar experiments, the inhibition induced by anti-GM-CSF titered out at 1:20,000 to-:100,000 dilutions.



Amplification of Anti-anti-GM-CSF Immunoglobulin Light Chain Variable Regions (V(L))

Spleenocytes were isolated from four anti-GM-CSF immunized mice who displayed neutralizing activity against GM-CSF-dependent proliferation. A cell suspension was prepared and lymphocytes isolated by Ficoll-Hypaque density gradient centrifugation. RNA was extracted with the RNAzol kit (Biotecx Laboratories Inc., Houston, TX), according to the manufacturer's instructions. Following isolation, the RNA was precipitated with isopropyl alcohol, pellets washed in 70% ethanol, and rotary evaporated. The dried pellets were resuspended in 50 µl of diethylpyrocarbonate-water and RNA quantified spectrophotometrically.

For reverse transcription, 10-20 µg of RNA in 10 µl was utilized to synthesize cDNA primed with random hexamers in the following reaction mixture: 3 µl of Maloney murine leukemia virus reverse transcriptase with 6 µl of 5 times reverse transcriptase buffer, 1.5 µl of RNase inhibitor, and 3 µl of 0.1 M dithiothreitol (all from Life Technologies, Inc.), 3 µl (100 pmol) of random hexamers (from Pharmacia LKB Biotechnol), and 1 µl of 40 mM dNTPs (10 mM in each dNTP, from Boehringer Mannheim, GmbH W., Germany). Following a 10-min preincubation at 25 °C, the reaction was carried out for 1 h at 42 °C, then 95 °C for 5 min followed by storage at -20 °C until use.

For PCR amplification, the oligonucleotide primers 3315 and 5591 listed in Table 1were employed at 0.5 nM/ml final concentrations. The relative position of these primers on Igkappa cDNA is shown in Fig. 2. The PCR mixture (100 µl) consisted of 10 µl of PCR primers, 16 µl of dNTPs (final concentration 200 µM in each dNTP), 10 µl of PCR buffer (10 times; Perkin-Elmer Cetus), 61.5 µl of dH(2)O, 2 µl cDNA, and 1.2 units of Taq polymerase (Perkin-Elmer Cetus). Amplification was carried out in a Programmable Thermal Cycler (MJ Research, Watertown, MA). The amplification program was 94 °C for 3 min followed by five cycles of 94 °C for 60 s, 52 °C for 60 s, 72 °C for 60 s; followed by 25 cycles of 94 °C for 60 s, 52 °C for 90 s, and 72 °C for 120 s. Following 30 cycles, the temperature was held at 72 °C for 7 min. Positive amplification was determined by agarose gel electrophoresis. The PCR products were cloned into the pDAB(L) plasmid, which is of utility for protein expression as has been published previously (31, 32) . PCR products and plasmid DNA were cut with the appropriate endonucleases and plasmid DNA was treated with calf intestinal phosphatase (Boehringer Mannheim), followed by ligation using 1 unit of T4 DNA ligase overnight at 16 °C. Ligation mixtures were transformed into E. coli DH5alpha competent cells as described by the manufacturer.


Figure 2: Library screening. A, first round library screening was carried out on 30 filters lifted from 30 LB/amp plates representing a total of 15,000-20,000 colonies. A representative filter is shown here. B, second round screening of one positive clone (clone 23.2) replated and probed with I-126.213. Compare with panel C, second round screening of a control clone with an irrelevant V(L) region. D, third round screening of clone 23.2. Compare with panel E, E. coli transformed with pDAB(L) alone.



Library Characterization

Competent Epicurian coli cells (the Cell Center, University of Pennsylvania) transformed with the amplified Vkappa/pDAB(L) library was plated on LB/amp plates. Control ligated pDAB(L) vector produced four to five colonies/plate, while the appropriately ligated Vkappa/pDAB(L) transformants produced 175-465 colonies/plate. Inserts were confirmed by plasmid miniprep analysis(26, 27) , which revealed appropriately sized inserts in 75% of colonies. Approximately 2,000 colonies on 10 plates were screened in this study.

Protein Expression

Bacterial clones possessing the Vkappa genes inserted into pDAB(L) were plated onto LB/amp plates. Control plates contained E. coli transformed with either pDAB(L) alone, pUC19,or pUC18. Following overnight growth, replica plating, and additional overnight growth, 0.45-m nitrocellulose filters were placed on the bacterial plate. Filters were lifted to other LB/amp plates on which 50 µl of isopropyl-beta-thio-galactopyranoside (IPTG) (25 mg/ml; Stratagene, La Jolla, CA) had been spread and were then incubated for 4 h at 37 °C. ilters were then exposed to chloroform vapor for 15 min and incubated overnight (with shaking) in lysis buffer (100 mM Tris-Cl, pH 7.8,150 mM NaCl,5 mM MgCl(2),1.5% bovine serum albumin (BSA),1 µg/ml pancreatic DNase I,and 40 µg/ml lysozyme). Filters were then blocked for 4 h with blocking buffer (5% non-fat dry milk and 0.05% BSA in phosphate-buffered saline (PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na(2)HPO(4), 1.8 mM KH(2)PO(4); pH .4)). Following blocking, filters were screened for specific variable region expression as noted below.

For some experiments, lysates were prepared of bacteria expressing the recombinant antibody fragments. Lysates of E. coli XL1 Blue cells (Stratagene, La Jolla, CA) were prepared either from unmanipulated bacteria or E. coli transformed with pDAB(L) alone, or the various V(L) regions ligated into pDAB(L). Colonies were grown overnight in LB/Amp, and 500 µl used to seed 5-l cultures grown to 0.6 A units in Superbroth (Cell Center, University of Pennsylvania), then induced with 1 mM IPTG for for 4-12 h. The cells were centrifuged (10,000 revolutions/min for 30 min) and the pellets dissolved in 2 ml of lysis buffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and aprotinin diluted 1:100 from a concentration of 2.1 mg/ml, all from Sigma). These cells were sonicated for 45 s on ice and clarified by centrifugation (11,000 times g for 15 min at 4 °C) and the supernatant (lysate) used as sources of V(L) fragments.

Library Screening

For binding of I-labeled 126.213 or control mAb ID6 (33) blocked filters were incubated for 2 h at 37 °C with I-labeled mAb (purified by staphylococcal protein A affinity chromatography), 500,000-1,000,000 cpm/ml, labeled by the chloramine T method (9) in PBS containing 1% BSA and 0.05% Tween-20 (PBS-BSA). Filters were washed extensively with PBS-BSA and autoradiographed (Kodak XRP film) for 2-24 h.

DNA Sequencing

Double-stranded DNA sequencing employed the primers listed in Table 1and followed previously published protocols (34) . Sequencing proceeded in both the 5` and 3` orientations to confirm all sequence information.

Western Blotting

Bacterial lystaes (prepared as above) or recombinant human GM-CSF were run on 15% SDS-polyacrylamide gel in reducing sample buffer (2% SDS, 50 mM Tris-HCl, pH 6.8, 10% glycerol, 0.001% bromphenol blue) with 500 µg of bacterial protein (as determined by the Bio-Rad protein assay) loaded in each well. Following electrophoresis, the gel was transferred to Immobilon P transfer membranes (Millipore) as described(35) , and the blot blocked with 1% bovine serum albumin, 5% non-fat dry milk, 0.05% Tween 20 in PBS (blocking buffer) for >1 h at 37 °C or overnight at 4 °C. The blots were then incubated with 5 ml of purified mAb 126.213 diluted to 2 µg/ml in blocking buffer for 2 h at 37 °C, and washed four times in PBS, 0.05% Tween 20 (wash buffer). Polyclonal goat anti-mouse IgG (Sigma) (radioiodinated as described(9) ) was then added (1,000 cpm/µl in blocking buffer) and the blot incubated for 2 h at 37 °C, washed extensively in wash buffer, dried, and exposed for 12-72 h to Kodak XAR film.

Immunoprecipitation

5 µg of purified 126.213 was reacted with protein G beads (Sigma) in Eppendorf tubes and rotated overnight at 4 °C. The tubes were centrifuged and the liquid aspirated. The beads were then washed three time with lysis buffer (1% Triton, 0.05% SDS, 10 mM Na(2)HPO(4)-NaH(2)PO(4), 150 mM NaCl, 5 mM EDTA, 100 µM Na(3)VO(4), and 5 µg/ml aprotinin all from Sigma) to remove unbound antibody. The beads were resuspended in 100 µl of lysis buffer, and I-GM-CSF was added to the tubes in the presence or absence of inhibitors (100 µl of total volume) and rotated at 4 °C for 1 h. The tubes were then centrifuged, the liquid discarded, and the beads washed three times. The beads were then resuspended in 2 times sample buffer (0.5 M Tris-HCl, pH 6.8, 16% glycerol, 3.2% SDS, 8% 2-mercaptoethanol, and 0.04% bromphenol blue (all from Sigma) in distilled H(2)0) and heated at 95 °C for 5 min to dissociate bonds. Samples were then loaded onto 10% SDS-polyacrylamide gel electrophoresis gels and analyzed by autoradiography as described(35, 36) .

Radioreceptor Binding Assay

This was modified from previously published protocols(37, 38) . Briefly, HL-60 cells (from ATCC) were grown in RPMI 1640 with 10% fetal calf serum and added L-glutamine. 1-2 times 10^6 HL-60 cells were washed twice in RPMI 1640 with 1% BSA and 25 mM HEPES, pH 7.4 (binding buffer), centrifuged, and incubated with inhibitors as noted in figure legends in a 25-µl volume for 1 h at room temperature. 0.5 nM of I-GM-CSF (118 µCi/µg, from DuPont NEN) was then added for 30 min at room temperature, the cells layered over 500 µl of chilled fetal calf serum, centrifuged, and the pellets counted.

Peptide Synthesis

All peptides were synthesized by solid-phase methods as described previously (9, 10, 11, 12) by the Wistar Institute Peptide Synthesis Facility or Macromolecular Resources at Colorado State University, deprotected, and released from the resin using anhydrous HF.

Enzyme-linked Immunosorbent Assay (ELISA)

ELISA was performed with polystyrene plates (Dynatech Laboratories Inc.) coated with peptide by evaporation of peptides (at the concentrations noted) in distilled water overnight at 37 °C. The wells were washed with PBS, blocked with 0.05% Tween, 2% BSA in PBS, and washed with PBS. Primary antibodies were added at varying dilutions for >1 h at 37 °C. After washing, secondary antibody, goat anti-mouse conjugated to horseradish peroxidase (Sigma) was added per well in 1% BSA in PBS for 1-2 h at 37 °C. The substrate used for color development was 3,3`,5,5`-tetramethyl-benzidine dihydrochloride (Sigma). The wells were decanted, washed extensively, and absorbance of samples was measured in a plate reader (MR 5000; Dynatech Laboratories) and expressed as A nm. Specific values were determined by subtracting the absorbance measured from uncoated wells from the absorbance to peptide coated wells(39) .


RESULTS

Construction and Screening of Anti-anti-GM-CSFV (L)C Library

Polyclonal neutralizing antibodies against human GM-CSF raised in BALB/c mice were used to develop syngeneic anti-anti-GM-CSF with neutralizing activity (Fig. 1). The PCR was used to amplify immunoglobulin Vkappa genes from these mice. Oligonucleotide primers for amplification of immunoglobulin genes were chosen based on conserved DNA sequences found in V(L) variable framework regions and from the kappa constant region domain. The 5` primer used in these experiments was relatively specific for the murine VkappaIII family. The 3` primer introduced a stop codon at position 207, eliminating 8 amino acids including the carboxyl-terminal cysteine residue, thereby eliminating the tendency for the produced light chains to dimerize. Spleen cells were isolated, RNA extracted, and cDNA synthesized. This served as a template for PCR amplification of the V(L)C regions. Bands of the expected size (680 base pairs) were observed following agarose gel electrophoresis. This amplification was specific as control cellular DNA from human T lymphocyte cell lines did not yield a PCR product (data not shown).

The PCR products were ethanol precipitated (to remove residual primer DNA) and digested with appropriate restriction endonucleases (XbaI and EcoRI). These were ligated into similarly restricted, alkaline phosphatase-treated pDAB(L). Following ligation, the reaction products were transformed into E. coli DH5alpha cells and plated onto 30 LB/amp plates. This V(L)C library was then screened after induction with IPTG with radioiodinated neutralizing mAb 126.213, which specifically neutralizes GM-CSF activity. Thirty filters containing 500-1,000 colonies each were screened in this manner. A representative filter is shown in Fig. 2A. Based on the observed binding of I-126.213 to colonies we picked 30 reactive colonies. These were expanded and replated and rescreened using fresh I-126.213 and a control mAb (ID6) specific for HIV-1 gp120(33) . Approximately 50% of the filters were bound by I-126.213 but not by I-ID6 following the second round of screening (see Fig. 2, B and C, for representative filters screened with I-126.213). Most of these were bound by I-126.213 in subsequent rounds of screening (Fig. 2, D and E). Ten colonies which were consistently bound by I-126.213 but not I-ID6 in subsequent assays were selected for further characterization.

Characterization of V(L)C Regions

Western blot analysis was performed of bacterial lysates prepared from the bacterial colonies consistently bound by 126.213. For Western analysis, lysates were prepared from E. coli transformed with the pDAB(L) plasmid alone, or containing the specific light chain inserts. The cultures were then either left uninduced or induced with IPTG, bacterial lysates prepared, separated electrophoretically, and transferred to Immobilon filters. These were probed with 126.213 followed by I-goat anti-mouse IgG and analyzed by autoradiography. A typical Western blot is shown in Fig. 3A. This compares cultures of bacteria both uninduced and following induction with IPTG. As can be seen, IPTG induces the appearance of a 21-23 kDa band for the clones containing specific V(L) regions, while only nonspecific bands are present in the cultures transformed with pDAB(L) alone. Notably, this gel was run under non-reducing conditions, suggesting that the light chains do not dimerize, instead remaining as monomers. The molecular mass of the band detected is somewhat lower than the 23 kDa predicted for the isolated light chain. This may be due to inaccuracy of the molecular weight markers used or could reflect compact folding of the V(L)C fragments.


Figure 3: Characterization of rAb V(L) regions. A, Western blot analysis of rAb fragments. E. coli transformed with various plasmids were induced or left uninduced, lysates prepared, and Western blotting performed with 126.213 as the primary antibody as noted under ``Materials and Methods.'' Lanes were as follows: 1, clone 23.2 uninduced; 2, clone 23.2 induced; 3, clone 5.1 uninduced; 4, clone 5.1 induced; 5, pDAB(L) alone uninduced; 6, pDAB(L) alone induced; 7, 300 ng of GM-CSF (positive control). Molecular weight markers are indicated. The arrow indicates the band specifically induced. B, inhibition of immunoprecipitation by 23.2. Immunoprecipitation of I-GM-CSF was performed as noted under ``Materials and Methods.'' Lysates of E. coli expressing 23.2 or control (irrelevant clone) were prepared, protein quantified, and 400 µg used to inhibit immunoprecipitation. Inhibitors were added as follows: 1, pDAB(L) alone induced; 2, 300 ng of GM-CSF; 3, clone 25.1 uninduced; 4, clone 25.1 induced; 5, clone 23.2 uninduced; 6, clone 23.2 induced; 7, ^14C molecular weight markers.



The neutralizing mAb 126.213 specifically immunoprecipitates I-GM-CSF. This assay allowed investigation of the ability of various rAb V(L)C regions to compete with I-GM-CSF binding to 126.213. Of the 10 rAb V(L)C regions screened, only one (clone 23.2) reproducibly inhibited immunoprecipitation by 126.213 (Fig. 3B). Inhibition with the lysates from bacteria transformed with 23.2 reproducibly inhibited immunoprecipitation on multiple experiments (Fig. 3B and data not shown). Inhibition was much greater for IPTG-induced cell lysates compared with uninduced lysates. Clone 23.2 was selected for further characterization.

Inhibition of GM-CSF Binding to HL-60 Cells by 23.2

GM-CSF specifically binds GM-CSF receptors present on HL60 (human myelomonocytic leukemia) cells, and this binding is inhibited by 126.213(24) . We examined the ability of rAb 23.2 to inhibit binding of I-GM-CSF to HL60 cells on a standard cellular binding assay. In this assay, HL-60 cells were preincubated with lysates from E. coli induced with IPTG following transformation with the 23.2 plasmid or an irrelevant plasmid (pUC18). The counts/minute bound are shown versus increasing amounts of lysate added in Fig. 4. 23.2 transformed bacterial lysates inhibited binding of I-GM-CSF to HL-60 cells, while control lysates had no effect. This result indicates that 23.2 competes with GM-CSF for binding to a site on HL-60 cells and may bind to the GM-CSF receptor present on these cells.


Figure 4: Inhibition of I-GM-CSF binding to HL-60 cells by 23.2. The binding assay was performed as noted under ``Materials and Methods'' using 2 times 10^6 HL-60 cells, in the presence or absence of increasing amounts of 23.2 or control (pUC18) lysates. The counts/min (cpm) bound ± standard error of replicate determinations for two lysate preparations are shown.



Sequence of Clone 23.2

As clone 23.2 was specifically bound by mAb 126.213, and competed with GM-CSF for binding to 126.213 and to HL-60 cells, the 23.2 insert was sequenced. The nucleic acid sequence and derived amino acid sequence of 23.2 is shown in Fig. 5. The 23.2 V(L) region is a member of the murine Vkappa III family as defined by Kabat et al.(28) , or the Vkappa 21 group as defined by Weigert et al. (40), with the J region derived from the Jkappa1 family(40) . Data base searching reveals that the 23.2 V/J amino acid sequence is very similar to the previously described Vkappa 21 hybridoma light chains 6684 and 7940 derived from NZB mice (40) , differing by only 6 amino acid substitutions from 6684 and 8 substitutions from 7940.


Figure 5: Nucleic acid and derived amino acid sequences of clone 23.2. Sequencing was performed by double-stranded DNA sequencing with Taq polymerase, as described previously, using both the PCR primers and primers derived from the pDAB(L) plasmid. FR, framework; codon numbering (above the sequence) is according to Kabat et al.(28) with codon one corresponding to the first amino acid residue of the FR1 region. Leader peptide sequence is not shown.



The intact 23.2 sequence and the individual CDR sequences were compared with the human GM-CSF sequence using the Bestfit, Gap, Wordsearch, and Segments programs of the Wisconsin package(41) . Several regions of weak sequence similarity were noted which involved CDR regions of 23.2. Prior studies of 126.213 used murine/human chimeric forms of GM-CSF to map interaction sites(24) . These studies suggested that residues 77-83 were critical for 126.213 binding to GM-CSF. We noted weak homology of the CDR I and CDR II with this epitope. An additional region of weak sequence similarity was also seen between amino acids 54-61 of GM-CSF and the CDR III of 23.2. Interestingly, amino acids 54-61 (on the B helix of GM-CSF) lie immediately adjacent to amino acids 77-83 (on the C helix) in the crystal structure of GM-CSF(42) . However, the weak sequence similarity seen here indicated that the mimicry of GM-CSF by 23.2 might be better accounted for on a structural level.

Structural Analysis of GM-CSF Mimicry by 23.2

Structural analysis of 23.2 was carried out following development of molecular models of the V(L) domain. A molecular model of the light chain was developed by examining sequence homologies of the 23.2 sequence with sequences of crystallographically known light chain structures. Identification of crystallographic templates for the light chain model included examining the length of the respective CDRs to match those of the template. As many light chain structures display equivalent CDR II and CDR III lengths, several alternative models were developed. Model 1 was developed using as a template the antibody 50.1 (Iggb), an antibody directed against the V3 loop of HIV gp120. This template displayed equivalent CDR lengths with the 23.2 sequence. The CDRs and framework (FR) regions of the 50.1 template were mutated to those of 23.2 using the program Insight (Biosym Technologies). The side chain angles of the substituted residues were set according to angles identified in a data base of side chains. Each CDR and FR region were changed individually, followed by 1000 cycles of energy minimization to eliminate close contacts between atoms. As in our prior studies (43, 44, 45) , the program Discover (version 2.8, Biosym Technologies) was used for energy minimization with supplied constant value force field.

Alternative models were also generated by searching the crystallographic data base for loops of the same size as the CDR I region. The spatially conserved Cartesian positions at the NH(2)- and COOH-terminal regions of CDR I were held fixed in the search procedure. A Cartesian distance matrix was constructed for combinations of the residues on the NH(2)- and COOH-terminal regions of the CDR I and compared to a precalculated Cartesian distance matrix data base of high resolution protein structures(46) . The 20 best matches were examined using the program Insight II and appropriate choices were made based upon similarities in chiralities of side chains at the junctures of the CDR I loop. The choice was spliced into the template using the program Insight II. Two alternative models were constructed using this approach. The one involved splicing a loop identified in the immunoglobulin Fc fragment 1Fc2 (Model 2). The other involved the heavy chain CDR I of 50.1 (Model 3). It is well known that light chains can adopt heavy chain conformations in the absence of heavy chain(47) . The CDR I of the heavy chain 50.1 was spliced into the template. The alternative structures were mutated to the 23.2 sequence and the structures energy minimized. These models are presented in Fig. 6.


Figure 6: Structural basis for mimicry. The structure of GM-CSF was determined from coordinates derived from the crystal structure (J. M. LaLonde, K. Swaminathan, and D. Voet, manuscript in preparation), displayed on the MacImdad program (Molecular Applications Group, Palo Alto, CA) on a Macintosh Quadra 950 computer. The 23.2 V(L) models were derived as described under ``Results.'' The GM-CSF view is directed at the B and C helices, while the 23.2 models' view is directed at the CDR I region. Specific residues implicated in mimicry are indicated. The models are further discussed in the text.



Prior studies investigating the epitope on GM-CSF recognized by 126.213 by mutagenesis (24) implicated residues 77-83, located on the C helix of GM-CSF. Peptide mapping studies of this antibody suggest recognition by the B and C helices as well as an epitope representing the first beta strand, which are all structurally adjacent. (^2)Analysis of the 23.2 models suggests a structural basis for mimicry of this site. This is shown in Fig. 6. All three models center our attention on residues Thr, Glu, and Lys63 of GM-CSF. The proximity of Lys and Glu suggest a charge-charge interaction. In all three models, these 2 residues are mimicked by Arg and Asp of 23.2. For Models 1 and 3, the mimicry suggests similar orientations of 23.2, while for Model 2 the structure is rotated 90°. The other GM-CSF residues mimicked include: Thr mimicked by Ser in Models 1 and 3, and by Thr in Model 2; Lys mimicked by Arg in Models 1 and 3, and Lys in Model 2; Thr and Ser mimicked by Ser in Models 1 and 3, and by Ser and Tyr in Model 2; Lys mimicked by Lys in Models 1 and 3, and by Ser in Model 2; and Glu mimicked by Ser and Ser in Model 1, and by Ser in Model 3. Thus, while sequence similarity between GM-CSF and 23.2 is quite low, structural similarity is suggested centered on the B and C helices of GM-CSF and the 23.2 CDR I.

Binding and Bioactivity of CDR Peptides

To further investigate the basis for mimicry by 23.2, synthetic peptides were developed based on the 23.2 CDR sequences. These are shown in Table 2. The CDR peptides were used in an ELISA assay to determine binding by 126.213 (Fig. 7). Binding to the CDR II and CDR III peptides was not higher than binding to the control peptide used, although it was higher than the isotype matched control mAb used (Fig. 7). However, the CDR I peptide was bound at higher levels than the other CDR peptides and the control peptide, and was bound by 126.213 but not the control mAb. Additional studies using a competitive ELISA indicate that this peptide blocks GM-CSF binding by 126.213 (data not shown). This suggests that the CDR I region of 23.2 is the major recognition site for 126.213.




Figure 7: Binding of 126.213 to synthetic peptides derived from the 23.2 sequence. Binding was performed by ELISA assay as described under ``Materials and Methods.'' The values shown are A nm binding to the peptides at the concentration noted minus A nm binding to BSA-coated control plates. Results are compared for 126.213 versus an isotype matched control mAb (D1.H3) specific for a peptide derived from the hamster beta-adrenergic receptor. The mean ± S.D. of triplicate wells is shown for increasing amounts of purified 126.213 added. A and B, binding to CDR I peptide. C and D, binding to the CDR II peptide. E and F, binding to the CDR III peptide. G and H, binding to the control peptide. The mAbs used were: 126.213 in A, C, E, and G and D1.H3 in B, D, F, and H.



The ability of these peptides to compete with GM-CSF for binding to HL-60 cells was examined using a radioreceptor assay. HL-60 cells were preincubated with peptides prior to the addition of I-GM-CSF and specific binding determined in the presence of excess unlabeled GM-CSF. A representative experiment is shown in Fig. 8. Increasing amounts of CDR I peptide were able to specifically inhibit GM-CSF binding in a dose-dependent manner, while CDR II and CDR III peptides did not demonstrate any specific binding inhibition. Thus, the CDR I peptide antagonizes I-GM-CSF binding to HL-60 cells, suggesting interaction of this peptide with the GM-CSFR.


Figure 8: Inhibition of I-GM-CSF binding to HL-60 cells by CDR peptides. The radioreceptor assay was performed as noted under ``Materials and Methods,'' using 10^6 HL-60 cells. The cells were preincubated with peptides at varying dilutions for 60 min at room temperature prior to the addition of I-GM-CSF. The specific proportion of cpm bound was determined by subtracting the proportion of cpm bound under identical conditions in the presence of saturating amounts (50 nM) of unlabeled GM-CSF. The standard deviation of this assay was 10% on multiple determinations. The percent inhibition of binding is shown versus increasing amounts of peptides.



The bioactivity of these peptides was assessed by their effect on GM-CSF-dependent cellular proliferation. This was compared with their effect on interleukin-2-dependent proliferation by the CTLL cell line, to control for nonspecific toxic effects. The results are shown in Fig. 9. At the concentrations used, none of the peptides were toxic to CTLL cells with the exception of the CDR III peptide at 2 mg/ml. The CDR II peptide had no inhibitory effect on either cell line. In contrast, the CDR III and CDR I peptides inhibited GM-CSF-dependent cellular proliferation. For the CDR III peptide, the IC was 1 mg/ml (approximately 400 µM), while for the CDR I peptide, it was 50 µg/ml (approximately 21 µM). These data indicate that the CDR I peptide is a specific antagonist of GM-CSF-dependent cellular proliferation in a micromolar concentration range.


Figure 9: Inhibition of GM-CSF-dependent cell proliferation by peptides. The proliferation assay was performed as noted under ``Materials and Methods'' on AML193 cells (GM-CSF-dependent) and CTLL cells (IL-2-dependent), in the presence or absence of increasing amounts of peptides as noted. Results from two experiments are combined, with the mean ± standard error percent inhibition of proliferation shown versus increasing peptide concentration. A, CDR I peptide; B, CDR II peptide; C, CDR III peptide.




DISCUSSION

GM-CSF-Receptor Interactions

GM-CSF activity is mediated by binding to specific cellular receptors (GM-CSFR) which belong to a recently described supergene family(38, 49, 50, 51, 52, 53, 54) . The high affinity GM-CSFR is comprised of an alpha chain (GM-CSFRalpha) specific for GM-CSF (38) , and a beta chain (beta(c)), which can also associate with the IL-3 and IL-5 receptor alpha chains(52) . The GM-CSFRalpha imparts specificity to the interaction with GM-CSF, and when expressed without beta(c) is able to bind GM-CSF, albeit with lower affinity than the heterodimeric receptor(55) . The high affinity receptor (GM-CSFRalpha and beta(c)) appears to be the signal transducing unit(56, 57) , with a sequential binding of GM-CSF to GM-CSFRalpha followed by binding to beta(c) postulated. The formation of a ternary complex of GM-CSF with GM-CSFRalpha and beta(c) implies that more than one site on GM-CSF is needed for receptor binding and bioactivity.

GM-CSF binding and bioactivity have been analyzed at a molecular level. Mutagenesis studies implicate the first (A) helix in binding of GM-CSF to the high affinity GM-CSFRalpha/beta(c) complex, but not to the low affinity receptor (GM-CSFRalpha alone)(55, 58, 59) . This is illustrated most strikingly by studies using mutants of residue Glu of GM-CSF, which inhibit binding of GM-CSF to the low affinity receptor, but display little activity in inhibiting binding to the high affinity receptor(58) . Based on these experiments, it has been proposed that the first alpha helix of GM-CSF is responsible for binding to beta(c)(59) . Murine and human GM-CSF display species specificity and are not cross-reactive. As the substitutions are scattered throughout the molecule, it was possible to swap regions of murine and human GM-CSF to locate sites critical for receptor interaction(37) . These studies indicated a critical role for amino acids 21-31 and 77-94 in mediating the activity of human GM-CSF, suggesting that the second site may be involved in binding to the GM-CSFRalpha. However, other potential GM-CSFRalpha interaction sites have also been suggested in mutagenesis studies(60, 61, 62) , mapping of neutralizing mAbs(24, 63, 64, 65) , and synthetic peptide studies(48, 63, 66) . Thus, in spite of considerable study, the GM-CSFRalpha interaction site(s) on GM-CSF remain incompletely characterized.

Recent studies from our group have used synthetic peptides, anti-peptide antisera, and neutralizing mAbs to map epitopes on GM-CSF critical for bioactivity.^2 The major findings were: a peptide derived from the sequence of the A helix (residues 17-31) and antibodies to this peptide inhibited GM-CSFdependent cellular proliferation; a peptide comprising portions of the B and C helices (residues 54-78) was recognized by two neutralizing monoclonal antibodies (including 126.213) and exhibited biological antagonist activity. Other peptides were also bound by 126.213 corresponding to residues 78-99 and 31-54, but were not specific antagonists of GM-CSF bioactivity. These three peptides together constitute a ``face'' on GM-CSF centered on the B and C helices and opposite the A helix. Together with the prior studies noted above, these studies suggest two binding sites on GM-CSF important in receptor binding: the A helix which likely interacts with beta(c), and the opposite face centered on the B and C helices which we propose interacts with the GM-CSFRalpha. The ability of synthetic peptides corresponding to these epitopes to specifically inhibit GM-CSF bioactivity strongly supports their role in receptor interaction.

GM-CSF Mimicry by Recombinant Antibody Light Chain

The studies described here suggest that the 23.2 rAb fragment mimics a binding site on GM-CSF involved in interaction with the GM-CSFRalpha. 23.2 was selected to bind to the antigen binding idiotopes of the neutralizing mAb 126.213, which competes with a soluble form of the GM-CSFRalpha for binding to GM-CSF. 23.2 displays several features characteristic of an ``internal image'' of the antigen including competition with GM-CSF for binding to 126.213 and to HL-60 cells ( Fig. 3and Fig. 4). The sequence similarity of 23.2 with GM-CSF noted, while weak, is spread out over residues 53-98, which comprises the B and C helices as well as the BC interhelical loop, and represent one ``face'' of the GM-CSF molecule(42) . The weak sequence similarity seen led to the development of molecular models of 23.2 to investigate a potential structural basis for the mimicry observed (Fig. 6). This suggests mimicry of specific residues on the GM-CSF B and C helices by specific residues in the 23.2 CDR I, CDR II, and FR3 regions. Synthetic peptides corresponding to the 23.2 CDR regions were developed and evaluated. This analysis led to the observation that the CDR I peptide is recognized by 126.213 (Fig. 7) and is a biological and receptor antagonist of GM-CSF ( Fig. 8and Fig. 9). The CDR I region of 23.2 contributes most of the residues implicated in the structural analysis. The activity of the CDR I peptide confirms the importance of these residues and suggests that this peptide interacts with the GM-CSFRalpha, functioning as a receptor antagonist.

In prior studies, we described the molecular basis for antibody mimicry of a viral hemagglutinin(9, 10, 11, 12) . Other groups have applied this technology to platelet fibrinogen receptor(14) , the thyroid-stimulating hormone receptor(15) , and epitopes on the hepatitis B surface antigen(17) . Monoclonal antibodies were utilized in these studies as mimics and to derive sequence information. The studies presented here are the first to suggest that recombinant antibodies can be similarly employed to develop alternative ligands. The prior studies of antibody mimicry in general described mimicry of structures either known or predicted to represent reverse turns. As antibody CDRs are generally reverse turns, the ability of antibody CDRs to mimic other reverse turn regions does not necessarily imply that CDRs can mimic amino acid residues presented by other diverse backbone geometries. The epitopes involved in this study are largely alpha helical in nature. In spite of this, molecular modeling of this epitope suggests a structural basis for mimicry as noted above. This indicates that antibody mimicry of amino acid arrays on helical regions can be understood on a molecular-structural level. The application of recombinant antibody technology to development of such mimics should broaden the applicability of alternative ligand development in the analysis of active site structures.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM-46400. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a grant by the American Cancer Society and a NIH Cancer Center grant to the Wistar Institute.

Supported by a National Research Development Award.

**
Supported by NIH Grant RO1 CA 31615.

§§
Supported by grants from the American Foundation for AIDS Research and NIH.

(^1)
The abbreviations used are: mAb, monoclonal antibodies; CDRs, complementarity determining regions; GM-CSF, granulocyte-macrophage colony-stimulating factor; PCR, polymerase chain reaction; IL, interleukin; cpm, counts/minute; IPTG, isopropyl-beta-thio-galactopyranoside; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; rAb, recombinant antibody.

(^2)
VonFeldt, J. M., Monfardini, C., Fich, S., Rosenbaum, H., Kieber-Emmons, T., Williams, R. M., Kahn, S. A., Weiner, D. B., and Williams, W. V.(1995) Pept. Res., in press.


ACKNOWLEDGEMENTS

We wish to thank L. Marie for her helpful comments and S. Fish for her most kind assistance.


REFERENCES

  1. Miele, L., Cordella-Miele, E., Facchiano, A., and Mukherjee, A. B. (1988) Nature 335, 726-730 [CrossRef][Medline] [Order article via Infotrieve]
  2. Graf, J., Ogle, R. C., Robey, F. A., Sasaki, M., Martin, G. R., Yamada, Y., and Kleinman, H. K. (1987) Biochemistry 26, 6896-6900 [Medline] [Order article via Infotrieve]
  3. Cardwell, M. C., and Rome, L. H. (1988) J. Cell Biol. 107, 1551-1559 [Abstract]
  4. Iwamoto, Y., Robey, F. A., Graf, J., Sasaki, M., Kleinman, H. K., Yamada, Y., and Martin, G. R. (1987) Science 238, 1132-1134 [Medline] [Order article via Infotrieve]
  5. Kleinman, H. K., Graf, J., Iwamoto, Y., Sasaki, M., Schasteen, C. S., Yamada, Y., Martin, G. R., and Robey, F. A. (1989) Arch. Biochem. Biophys. 272, 39-45 [Medline] [Order article via Infotrieve]
  6. Kieber-Emmons, T. (1992) in Biologically Active Peptides: Design, Synthesis and Utilization (Williams, W. V., and Weiner, D. B., eds) Vol. 1, pp. 3-34, Technomic Publishing Co., Lancaster, PA
  7. Balaji, V. N., and Ramnarayan, K. (1992) in Biologically Active Peptides: Design, Synthesis and Utilization (Williams, W. V., and Weiner, D. B., eds) Vol. 1, pp. 35-54, Technomic Publishing Co., Lancaster, PA
  8. Von Feldt, J. M., Ugen, K. E., Kieber-Emmons, T., and Williams, W. V. (1992) in Biologically Active Peptides: Design, Synthesis and Utilization (Williams, W. V., and Weiner, D. B., eds) Vol. 1, pp. 55-86, Technomic Publishing Co., Lancaster, PA
  9. Williams, W., Guy, H., Rubin, D., Robey, F., Myers, J., Kieber-Emmons, T., Weiner, D., and Greene, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6488-6492 [Abstract]
  10. Williams, W., Moss, D., Kieber-Emmons, T., Cohen, J., Myers, J., Weiner, D., and Greene, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5537-5541 [Abstract]
  11. Williams, W., Kieber-Emmons, T., Rubin, D., Greene, M., and Weiner, D. (1991) J. Biol. Chem. 266, 9241-9250 [Abstract/Free Full Text]
  12. Williams, W., Kieber-Emmons, T., VonFeldt, J., and Weiner, D. (1991) J. Biol. Chem. 266, 5182-5190 [Abstract/Free Full Text]
  13. Bruck, C., Co, M., Slaoui, M., Gaulton, G., Smith, T., Fields, B., Mullins, J., and Greene, M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6578-6582 [Abstract]
  14. Taub, R., Gould, R. J., Garsky, V. M., Ciccarone, T. M., Hoxie, J., Friedman, P. A., and Shattil, S. J. (1989) J. Biol. Chem. 264, 259-265 [Abstract/Free Full Text]
  15. Taub, R., Hsu, J. C., Garsky, V. M., Hill, B. L., Erlanger, B. F., and Kohn, L. D. (1992) J. Biol. Chem. 267, 5977-84 [Abstract/Free Full Text]
  16. Levi, M., Sallberg, M., Ruden, U., Herlyn, D., Maruyama, H., Wigzell, H., Marks, J., and Wahren, B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4374-8 [Abstract]
  17. Pride, M. W., Shi, H., Anchin, J. M., Linthicum, D. S., LoVerde, P. T., Thakur, A., and Thanavala, Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11900-11904 [Abstract]
  18. Ward, E., Güssow, D., Griffiths, A., Jones, P., and Winter, G. (1989) Nature 341, 544-546 [CrossRef][Medline] [Order article via Infotrieve]
  19. Huse, W., Sastry, L., Iverson, S., Kang, A., Alting-Mees, M., Burton, D., Benkovic, S., and Lerner, R. (1989) Science 246, 1275-1281 [Medline] [Order article via Infotrieve]
  20. Barbas, C. 3., Crowe, J. J., Cababa, D., Jones, T. M., Zebedee, S. L., Murphy, B. R., Chanock, R. M., and Burton, D. R. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10164-10168 [Abstract]
  21. Burton, D. R., Barbas, C. 3., Persson, M. A., Koenig, S., Chanock, R. M., and Lerner, R. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10134-10137 [Abstract]
  22. Barbas, C. 3., Bain, J. D., Hoekstra, D. M., and Lerner, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4457-4461 [Abstract]
  23. Barbas, C. 3., Kang, A. S., Lerner, R. A., and Benkovic, S. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7978-7982 [Abstract]
  24. Brown, C. B., Hart, C. E., Curtis, D. M., Bailey, M. C., and Kaushansky, K. (1990) J. Immunol. 144, 2184-2189 [Abstract/Free Full Text]
  25. Williams, W. V., VonFeldt, J. M., Rosenbaum, H., Ugen, K. E., and Weiner, D. B. (1994) Arthritis Rheum. 37, 1468-1478 [Medline] [Order article via Infotrieve]
  26. Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J., and Struhl, K. (eds) (1989) Current Protocols in Molecular Biology , John Wiley & Sons, New York
  27. Sambrook, J., Fritsch, E., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  28. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest , U. S. Department of Health and Human Services, Bethesda, MD
  29. Borofsky, M. A., Weiner, D. B., Zurier, R. B., and Williams, W. V. (1992) Immunol. Res. 11, 154-164 [Medline] [Order article via Infotrieve]
  30. Romano, C., Williams, W. V., Fischberg, D. J., Cocero, N., Weiner, D. B., Greene, M. I., and Molinoff, P. B. (1989) J. Neurochem. 53, 362-369 [Medline] [Order article via Infotrieve]
  31. McCallus, D. E., Ugen, K. E., Sato, A. I., Williams, W. V., and Weiner, D. B. (1992) Viral Immunol. 5, 163-172 [Medline] [Order article via Infotrieve]
  32. Williams, W. V., McCallus, D. E., Satre, M., Eldridge, D., Frank, I., O'Donnell, E. A., and Weiner, D. B. (1993) Transgene 1, 113-124
  33. Ugen, K. E., Refaeli, Y., Ziegner, U., Agadjanyan, M., Satre, M. A., Srikatan, V., Wang, B., Sato, A., Williams, W. V., and Weiner, D. B. (1993) Vaccines 1993, 215-221
  34. Wang, B., Fang, Q., Williams, W., and Weiner, D. B. (1992) BioTechniques 13, 527-530 [Medline] [Order article via Infotrieve]
  35. Weiner, D., Kokai, Y., Wada, T., Cohen, J., Williams, W., and Greene, M. (1989) Oncogene 4, 1175-1183 [Medline] [Order article via Infotrieve]
  36. Weiner, D., Liu, J., Cohen, J., Williams, W., and Greene, M. (1989) Nature 339, 230-231 [CrossRef][Medline] [Order article via Infotrieve]
  37. Kaushansky, K., Shoemaker, S., Alfaro, S., and Brown, C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1213-1217 [Abstract]
  38. Gearing, D. P., King, J. A., Gough, N. M., and Nicola, N. A. (1989) EMBO J. 8, 3667-3676 [Abstract]
  39. Ugen, K. E., Goedert, J. J., Boyer, J., Refaeli, Y., Frank, I., Williams, W. V., Willoughby, A., Landesman, S., Mendez, H., Rubinstein, A., KeiberEmmons, T., and Weiner, D. B. (1992) J. Clin. Invest. 89, 1923-1930 [Medline] [Order article via Infotrieve]
  40. Weigert, M., Gatmaitan, L., Loh, E., Schilling, J., and Hood, L. (1978) Nature 276, 785-790 [Medline] [Order article via Infotrieve]
  41. Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395 [Abstract]
  42. Diederichs, K., Boone, T., and Karplus, P. A. (1991) Science 254, 1779-1782 [Medline] [Order article via Infotrieve]
  43. Lohman, K. L., Kieber-Emmons, T., and Kennedy, R. C. (1993) Mol. Immun. 30, 1295-1306
  44. Kieber-Emmons, T., VonFeldt, J. M., Godillot, A. P., McCallus, D., Srikantan, V., Weiner, D. B., and Williams, W. V. (1994) Lupus 3, 379-392 [Medline] [Order article via Infotrieve]
  45. Karp, S. L., Kieber-Emmons, T., Sun, M. J., Wolf, G., and Neilson, E. G. (1993) J. Immunol. 150, 867-79 [Abstract/Free Full Text]
  46. Jones, T. A., and Thirup, S. (1986) EMBO J. 5, 819-22 [Abstract]
  47. Schiffer, M., Girling, R. L., Ely, K. R., and Edmundson, A. B. (1973) Biochemistry 12, 4620-4631 [Medline] [Order article via Infotrieve]
  48. Clark-Lewis, I., Lopez, A. F., To, L. B., Vadas, M. A., Schrader, J. W., Hood, L. E., and Kent, S. B. H. (1988) J. Immunol. 141, 881-889 [Abstract/Free Full Text]
  49. Chiba, S., Tojo, A., Kitamura, T., Urabe, A., Miyazono, K., and Takaku, F. (1990) Leukemia 4, 29-36 [Medline] [Order article via Infotrieve]
  50. Cannistra, S. A., Groshek, P., Garlick, R., Miller, J., and Griffin, J. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 93-97 [Abstract]
  51. DiPersio, J., Billing, P., Kaufman, S., Eghtesady, P., Williams, R. E., and Gasson, J. C. (1988) J. Biol. Chem. 263, 1834-1841 [Abstract/Free Full Text]
  52. Hayashida, K., Kitamura, T., Gorman, D. M., Arai, K.-i., Yokota, T., and Miyajima, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9655-9659 [Abstract]
  53. Park, L., Friend, D., Gillis, S., and Urdal, D. (1986) J. Biol. Chem. 261, 4177-4183 [Abstract/Free Full Text]
  54. Onetto-Pothier, N., Aumont, N., Haman, A., Bigras, C., Wong, G. G., Clark, S. C., De Lean, A., and Hoang, T. (1990) Blood 75, 59-66 [Abstract]
  55. Shanafelt, A. B., and Kastelein, R. A. (1992) J. Biol. Chem. 267, 25466-25472 [Abstract/Free Full Text]
  56. Yokota, T., Watanabe, S., Mui, A. L., Muto, A., Miyajima, A., and Arai, K. (1993) Leukemia 7, S102-S107
  57. Sakamaki, K., Miyajima, I., Kitamura, T., and Miyajima, A. (1992) EMBO J. 11, 3541-3549 [Abstract]
  58. Lopez, A. F., Shannon, M. F., Hercus, T., Nicola, N. A., Cambareri, B., Dottore, M., Layton, M. J., Eglinton, L., and Vadas, M. A. (1992) EMBO J. 11, 909-916 [Abstract]
  59. Shanafelt, A. B., Miyajima, A., Kitamura, T., and Kastelein, R. A. (1991) EMBO J. 10, 4105-4112 [Abstract]
  60. Gough, N., Grail, D., Gearing, D., and Metcalf, D. (1987) Eur. J. Biochem. 169, 353-358 [Abstract]
  61. Shanafelt, A. B., and Kastelein, R. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4872-4876 [Abstract]
  62. Altmann, S. W., Johnson, G. D., and Prystowky, M. B. (1991) J. Biol. Chem. 266, 5333-5341 [Abstract/Free Full Text]
  63. Kanakura, Y., Cannistra, S. A., Brown, C. B., Nakamura, M., Seelig, G. F., Prosise, W. W., Hawkins, J. C., Kaushansky, K., and Griffin, J. D. (1991) Blood 77, 1033-1043 [Abstract]
  64. Nice, E., Dempsey, P., Layton, J., Morstyn, G., Cui, D. F., Simpson, R., Fabri, L., and Burgess, A. (1990) Growth Factors 3, 159-169 [Medline] [Order article via Infotrieve]
  65. Seelig, G., Prosise, W., Scheffler, J., Nagabhushan, T., and Trotta, P. (1990) J. Cell. Biochem. 14, 246
  66. Greenfield, R. S., Braslawsky, G. R., Kadow, K. F., Spitalny, G. L., Chace, D., Bull, C. O., and Bursuker, I. (1993) J. Immunol. 150, 5241-51 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.