CA2000355A1 - Human fc-gamma receptor iii - Google Patents
Human fc-gamma receptor iiiInfo
- Publication number
- CA2000355A1 CA2000355A1 CA002000355A CA2000355A CA2000355A1 CA 2000355 A1 CA2000355 A1 CA 2000355A1 CA 002000355 A CA002000355 A CA 002000355A CA 2000355 A CA2000355 A CA 2000355A CA 2000355 A1 CA2000355 A1 CA 2000355A1
- Authority
- CA
- Canada
- Prior art keywords
- ser
- ala
- riii
- cells
- leu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Abstract
Abstract of the Disclosure Soluble and membrane-bound forms of human Fc.gamma.RIII are provided, together with nucleic acids capable of encoding the same. Soluble Fc.gamma.RIIIs are useful in ameliorating the serum platelet deficiency associated with immune thrombocytopenic purpura. Cells expressing membrane-bound Fc.gamma.RIII are useful components in assays for serum immune complexes.
Description
~OOV35S
Case 2510XQ
Field of the Invention The invention relates generally to therapeutic compounds, and more particularly to soluble and membrane-bound forms of a low-affinity receptor for human immunoglobulin G, nucleic acids encodinq the same, and diagnostic and therapeutic uses of such receptors.
-I BACKGROUND
Receptors for the Fc poition of immunoglobulin G (IgG~ play a central role in cellular immune ~ -defenses. Three types of such receptors have been identified: A 72 kilodalton (kD) receptor with hiqh affinity for monomeric IgG is found on monocytes and some macrophages, a 40 kD receptor with low affinity for monomeric IgG is found on monocytes, neutrophils, eosinophils, platelets and certain human tumor-cell lines, and a 50-70 kD receptor with low affinity for monomeric IgG is found on neutrophils, eosinophils, natural killer cells, and macrophages. These three types ; ~ -- of Fc7 receptor are referred to as Fc7RI, Fc~RII, and Fc~RIII, respectively: Unkeless et al., Ann. Rev.
Immunol., Yol. 6, pgs. 251-281 (1988).
It is believed that ~c~RIII-mediated removal IgG-coated platelets plays an important part in the -~
;` zoov~s pathogene~ls of immune thrombocytopenic ~urpura (I~P), a platelet-det~clency condltlon charact-rlz-d by ~xce~-lv-bleedlng: von dem Borne, pg-. 222-256, ln Immunohaematoloav, Engelfrl-t t al., d~. (El~vler, Amsterdam, 1984). Clarkson et al., New Enaland J. Med., Vol. 314, pgs. 1236-1239 (1986), report that the infu-ion of ligand-blocking anti-Fc~RIII antibody into a patient with refractory ITP resulted in a transient increase in platelet count. This observation suggests that the most deleterious manifestation of ITP could be temporarily ameliorated by the administration of agents that block or compete with Fc~RIII for binding sites on IgG-coated platelets.
In a separate area of clinical i~munology, elevated serum levels of aggregates consisting of immunoglobulin and antigen (so-called ~immune coaplexes~) have been correlated with a wide variety of disorders, particularly-autoimmune diseases, such as systemic lupus erythematosus (SLE), and rheumatoid arthritis. The level of such complexes has become an important diagnostic for presence of autoimmune disorders; e.g. Theofilopoulos et al., Am.J. Pathol., Col. 100, pgs. 531-591 (1980).
Present assays for the serum level of immune complexes include solid-phase assays which take advantage of the affinity of the complexes for certain conplement - `
or rheumatoid factor proteins, and cellular assays which ~`
take advantage of the property of Ra~i cell~ to preferentially bind immune complexes; ~heofilopoulos et --al., chapter 28, and Toth et al., chapter 29, in Rose et al., eds. Manual of Clinical Immunology, 3rd Ed.
(American Society for Microbiology, Washington, D.C., 1986). Unfortunately, like many mammalian-cell based assays, the Raji-cell assay is difficult to perform, and requires elaborate controls and standards because of the inherent variability of Ra~i-cell binding to imaune complexes.
,t .~r~`. :~ ., ~ 200Q355 In light of the ~oregoing, it would be advantageous for the medical community to hav-alternative assay methods for immune co~plex-- utllizing well characterized, widely available,` and conveniently cultured cell line~ It would also be advantageou~ i~
soluble Fc~Rs could be produced in su~icient quantity to permit practical emergency therapy ~or re~actory cases of ITP
SUMMARY OF THE INVENTION
The present invention is directed to ~oluble and membrane-bound human Fc~RIII polypeptides, their muteins, nucleic acids capable of encoding the same, and diagnostic and therapeutic uses of such polypeptides and muteins .
The invention is based on the discovery of a - ~;
cDNA encoding a human Fc7RIII A cDNA clone, pcD(SR~
containing the Fc~RIII-encoding insert (illustrated in Figure 1) is deposited in E coli ~12 strain MC1061 with ~ ~ ;
the American Type Culture Collection (ATCC), Rockville, Maryland, USA, under accession number 67707 -~
Preferred embodiments of the invention include proteins having amino acid sequences selected from the group of 2-fold substituted and l-fold deleted sequences -defined by the formulas --~
X(Arg) - X(Thr) - X(Glu) - X(Asp) - X(Leu) - X(Pro) - ---~
X(Lys) - X(Ala) - X(Val) - X(Val) - X(Phe) - X(Leu) - --X(Glu) - X~Pro) - X(Gln) - X(Trp) - X(Tyr) - X(Arg) - ~-X(Val) - X(Leu) - X(Glu) - X(Lys) - X(Asp) - X(Ser) -X(Val) - X(Thr) - X(Leu) - X(Lys) - X(Cys) - X(Gln) - -~
X(Gly) - X(Ala) - X(Tyr) - X(Ser) - X(Pro) - X(Glu) -X(Asp) - X(Asn) - X(Ser) - X(Thr) - X(Gln) - X(Trp) - -~
X(Phe) - X(His) - X(Asn) - X(Glu) - X(Asn) - X(Leu) - ~ -~
X(Ile) - X(Ser) - X(Ser) - X(Gln) - X(Alà) - X(Ser) - -' ~1)0~;~55 X(Ser) - X(Tyr) - X~Phe) - X(Tle) - X~Asp) - X(Ala) -X~Ala) - X(Thr) - X(Val) - X(A~p) - X(Asp) - X~8er) -X(Gly) - X(Glu) - X(Tyr) - X(Arg) - X(Cys) - X(Gln) -X(Thr) - X(Asn) - X~Leu) - X~Ser) - X(Thr) - X~Leu) -X~Ser) - X(Asp) - X(Pro) - X(Val) - X~Gln) - X(Leu) -X(Glu) - X(Val) - X(His) - X(Val) - X(Gly) - X(Trp) -X(Leu) - X(Leu) - X(Leu) - X(Gln) - X(Ala) - X(Pro) -X(Arg) - X(Trp) - X(Val) - X(Phe) - X(Lys) - X(Glu) -X(Glu) - X(Asp) - X(Pro) - X(Ile) - X(His) - X(Leu) -X(Arg) - X(Cy6) - X(His) - X(Ser) - X(Trp) - X(Lys) -X(Asn) - X(Thr) - X(Ala) - X(Leu) - X(His) - X(Lys) -X(Val) - X(Thr) - X(Tyr) - X(Leu) - X(Gln) - X(Asn) -X(Gly) - X(Lys) - X(Asp) - X(Arg) - X(Lys) - X(Tyr) -X(Phe) - X(~is) - X(His) - X(Asn) - X(Ser) - X(Asp) -X(Phe) - X(His) - XlIle) - X(Pro) - X(Lys) - X(Ala) -X(Thr) - X(Leu) - X(Lys) - X(Asp) - X(Ser) - X(Gly) -X(Ser) - X(Tyr) - X(Phe) - X(Cys) - X(Arg) - X(Gly) -X(Leu) - X(Val) - X(Gly) - X(Ser) - X(Lys) - X(Asn) -X(Val) - X(Ser) - X(Ser) - X(Glu) - X(Thr) - X(Val) -X(Asn) - X(Ile) - X(Thr) - X(Ile) - X(Thr) - X(Gln) -X(Gly) - X(Leu) - X(Ala) - X(Val) - X(Ser) - X(Thr) -X(Ile) - X(Ser) - X(Ser) - X(Phe) - X(Ser) - X(Pro) -X(Pro) - X(Gly) Formula 1 and X(Arg) - X(Thr) - X(Glu) - X(Asp) - X(Leu) - X(Pro) -X(Lys) - X(Ala) - X(Val) - X(Val) - X(Phe) - X(Leu) -X(Glu) - X(Pro) - X(Gln) - X(Trp) - X(Tyr) - X(Arg) -X(Val) - X(Leu) - X(Glu) - X(Lys) - X(Asp) - X(Ser) -X(Val) - X(Thr) - X(Leu) - X(Lys) - X(Cys) - X(Gln) -X(Gly) - X(Ala) - X(Tyr) - X(Ser) - X(Pro) - X(Glu) -X(A8p) - X(A6n) - X(Ser) - X(Thr) - X(Gln) - X(Trp) --,.. ..
~ ,,. -. :, :
~; ~
~)0~3~S~i :`"
X(Phe) - X~Hio) - X~A~n) - X~Glu) - X(8er) - X~Leu) -X~Ile) - X~Ser) - X~Ser) - X~Gln) - X~Ala) - X~8er) -X~Ser) - X~Tyr) - X~Phe) - X~Ile) - X~A-p) - X~Ala) -X~Ala) - X~Thr) - X~Val) - X~Asp) - X~Asp) - X~Ser) -X~Gly) - X(Glu) - X~Tyr) - X~Arg) - X~Cy~) - X~Gln) -X~Thr) - X~Asn) - X~Leu) - X~Ser) - X~Thr) - X~Leu) -X~Ser) - X~Asp) - X~Pro) - X~Val) - X~Gln) - X~Leu) -X~Glu) - X~Val) - X~His) - X~Ile) - X~G~y) - X~Trp) -X(Leu) - X~Leu) - X~Leu) - X~Gln) - X(Ala) - X(Pro) -X(Arg) - X~Trp) - X~Val) - X~Phe) - X(Lys) - X(Glu) -X~Glu) - X~Asp) - X~Pro) - X~Ile) - X~His) - X~Leu) -X~Arg) - X~Cys) - X~His) - X~Ser) - X~Trp) - X~Lys) -X~Asn) - X~Thr) - X~ala) - X~Leu) - X~His) - X~Lys) -X~Val) - X~Thr) - X~Tyr) - X(Leu) - X(Gln) - X(Asn) -X(Gly) - X(Lys) - X(Gly) - X(Arg) - X(Lys) - X(Tyr) -X(Phe) - X(His) - X(His) - X(Asn) - X(Ser) - X(Asp) -X~Phe) - X(Tyr) - X~Ile) - X~Pro) - X~Lys) - X~Ala) -X~Thr) - X~Leu) - X~Lys) - X~Asp) - X~Ser) - X~Gly) -X~Ser) - X~Tyr) - X(Phe) - X~Cys) - X~Arg) - X~Gly) -X~Leu) - X~Phe) - X~Gly) - X(Ser) - X~Lys) - X~Asn) -X~Val) - X~Ser) - X~Ser) - X(Glu) - X(Thr) - X(Val) -X(Asn) - X~Ile) - X~Thr) - X~Ile) - X~Thr) - X~Gln) -X~Gly) - X~Leu) - X~Ala) - X~Val) - X~Ser) - X~Thr) -X~Ile) - X~Ser) - X~Ser) - X~Phe) - X~Phe) - X~Pro) -X~Pro) - X~Gly) ~ "'~:-~'', ~ Formula II --~
:' :.-,':"' wherein the term X~Xaa) represents the group of synonymous L-amino~ acids to the amino acid Xaa.
~; Synonymous amino acids within a group have sufficiently similar physiochemical properties for substitution between members of the group to preserve the biological function of the molecule, Grantham, Science, Vol. 185, pgs. 862-864 ~1974); and Dayhoff et al., Atlas of Protein 1~ ' ,, . ;' :` ZOOQ355 ff_ Sequence and Structure 1972, Vol. S, W ~ 89-99. It i-clear that deletions of amino acids may al~o b- mad- ln the above-identified sequence wlthout altering biological function, particularly if only a few amino acids are deleted and amino acids that are critical to a functional conformation are not removed or displaced, e.g. some cysteine residuesi: Anfisen, ~Principles That Govern The Folding of Protein Chains~, Science, Vol. 181, pgs. 223-230 (1973). Proteins and muteins produced by deletions are within the purview of the present invention.
Whenever an amino acid residue of a protein of Formula I
or II is referred to herein by number, such a number is in reference to the N-terminus of the protein.
Preferred, and more preferred, groups of synonymous amino acids for each amino are listed in Table I. The respective qrOups for each amino acid are indicated by slashes (/). Amino acids to the left of the slash make up the most preferred group. All the amino ---acids on the same line make up the more preferred group.
As used herein, ~N-fold substituted~ in reference to Formula I or II describes a group of amino -:
acid seguences differing from the native amino acid sequences of Formula III or IV, respectively, by zero-N
substitutions, such that the replacing amino acid is selected from t~e appropriate group of amino acids synonymous to the amino acid at the location of the substitution. That is, if the X(Ala) is at.the location for substitution, then the Ala at that location can be replaced with Ser, Thr, or Gly.
~ Likewise, as used herein, the term ~N-fold deleted~ in reference to Formula I or II describesi a group of amino acid seguences differing from the native amino acid sequences of Formula III or IV, respectively, by zero-N deletions of amino-acids.
.
: 200Q;~55 Throughout, 6tandard abbrovi~tlon~ ~re u~ed to designate amino acid~, nucleotide~, r--triotlon endonucle~se~, and the li~e; e.g. Cohn, ~Nomencl~tur- ~nd Symbol~sm of a -Amino Acids,~ Method~ in EnzvmoloqY, Vol.
106, pgs. 3-17 (1984); Wood et al. BlochemistrY: A
Problems ApProach~ 2nd ed. ~Ben~amin, Menlo Park, 1981);
and Roberts, ~Directory of ~estriction Endonucleases~, Methods in EnzvmolosY, Vol. 68, pgs. 27-40 (1979).
Table I
, :
Preferred and More Preferred Groups of Synonymous Amino Acids Amino Acid SYnonYmous GrouPs Ala Ala Ser / Thr Gly -~
Arg Arg / Lys --~
Asn Asn Asp / Ser Asp I Asp Glu / Asn Cys ~ Cys Gln Gln / Glu ~--Glu Glu Asp / Gln -Gly Gly Ala / Ser ., His His / Asn Ile Ile Val / Leu Leu Leu Val / Ile --~
Lys Lys Arg / Asn Met Met / Leu ~-Phe Phe Pro Pro / Ala Ser Ser Ala Thr I Gly Asn Thr Thr Ser Ala / Lys -~
Trp Trp - '~
Tyr - Tyr Val Val Ile / Ala Leu ':
, ,' , ~, , Z~)O(.t355 . "
~rief DescriPtion of The Drawina~
Pigure 1, parts A and ~ (on two she-t- to be read side-by-side) illustrates th- nucleotide ~eguence o~
the cDNA insert of pcD(SRa)-GP5;
Figure 2 illustrates the relative location o~
the stop codon inserted to produce a soluble human Fc~RIII, secreted as an FC~RIII mutant;
Figure 3 illustrates the result of gel electrophoretic analysis of the soluble Fc7RIII produced in Example III below; and ~ Figure 4 parts ~ and B (on two sheets to be read side-by-side) illustrates the necleotide sequence o~
the cDNA insert of pcD(SRo)-NL10.
DETAILED DESCRIPTION OF THE INVENTION - -The present invention includes nucleic acids encoding polypeptides capable of binding to the Fc portion of human IgG. These polypeptides are derived from human Fc~RIII. The invention includes soluble and membrane-bound polypeptides for human Fc~RIII. These and other modified versions of the polypeptides are readily produced using standard protein engineering techniques.
once nucleic acid sequence and/or amino acid -~-sequence information is available for a native protein, a variety of techniques become available for producing virtually any mutation in the native sequence. Shortle, in Science, Vol. 229, pgs. 1193-1201 ~1985), reviews techniques for mutating nucleic acids which are applicable to the present invention. Preferably, mutants -of *he protein of the present invention are produced by site-specific oligonucleotide-directed mutagenesis, e.g.
Zoller and Smith, Methods in Enzymologv, Vol. 100, pg~.
468-500 (1983), and Mark et al., U.S. Patent 4,518,584 entitled ~Human Recombinant Interleukin-2 Muteins^, which ~
~,. ~1-.~, ~ , . . . ~ , Z00~355 g are lncorporated by reference; or by so-called ~cassette~
mutagenesis described by Wells et al. in Gene, Vol. 34, pg8. 315-323 (1985), by Estell et al. ln Scienco, Vol.
233, pqs. 659-663 (1986), and al80 essentially by Mullenbach et al. in J. 8101. Chem., Vol. 261, pgs. 719-722 (1986), and by Feretti et al. in Proc. Natl. Acad.
Sci., Vol. 83, pg5. 597-603 (1986).
Polypeptides with amino acid modifications (i.e. muteins) may be desirable in a variety of circumstances. For example, undesirable side effects might be reduced by certain muteins, particularly if the side effect is associated with a different part of the polypeptide from that of the desired activity. In some expression systems, the native polypeptide may be -~
susceptible to degradation by proteases. In such cases, selected substitutions and/or deletions of amino acids which change the susceptible sequences can significantly enhance yields, e--.g. British patent application 2173-804-A where Arg at position 275 of human tissue plasminogen activator is replaced by Gly or Glu. Muteins may also increase yields in purification procedures and/or increase shelf lives of proteins by eliminating amino acids susceptible to oxidation, acylation, alkylation, or other chemical modifications. For exa~rle, methionine readily undergoes oxidation to form a sul~oxide, which in many proteins is associated with loss of biological activity: e.g. Brot and Weissbach, Arch. Biochem.
Biophys., Vol. 223, pg. 271 (1983). Methionines can often be replaced by more inert amino acids with little or no loss of biological activity, e.g. Australian patent application AU-A-52451/86. In bacterial expression systems, yields can sometimes be increased by eliminating or replacing conformationally inessential cysteine residues, e.g. Mark et al., U.S. Patent 4,518,584.
200(~355 Preferably, soluble form~ of tho FC~RIII o~ the invention are produced by introduoing a atop oodon prlor to (i.e. ln the 5'- or ~upstream~ dlreotlon of) the codlng region for the transmembrane and lntraoellul~r portion~ of the Fc~RIII cDNA. Thls 1- convenlently done by site-speciflc mutagenesis. Transmembrane region~ are readily identified by the presence of an amlno acld segment containing from about 20-25 residues having high average bydrophobicity, e.g. Wickner, Sclence, Vol. 210, pgs. 861-868 (1980), and Greene et al., Ann. Rev.
Immunol., Vol. 4, pgs. 69-9S (1986).
Plasmid pcD(SR~)-GPS is slmllar to the pcD
shuttle vector described by Okayama and Berg, Mol. Cell.
Biol., Vol. 2, pgs. 161-170 (1983), and Vol. 3, pgs. 280-289 (1983), except that the SV40 promoter has been modified to improve expression by the downstream insertion of a portion of the long terminal repeat (LTR) from a HTLV(I)~retrovirus, described by Takebe et al., Mol. Cell. Biol., Vol. 8, pgs. 466-472 (1988). The plasmid is conveniently propagated in E. coli K12 strain MC1061, or like host.
The immunoglobulin G binding property of the Fc~RIIIs of the invention is measured by standard techniques, e.g. (1) in the case of membrane-bound Fc~RIII: the ability of cells transfected with the Fc~RIII cDNA to form rosettes in the presence of IgG-coated red blood cells (RBCs) or to preferentially bind human IgG aggregated by heat treatment; or (2) in the case of soluble Fc~RIII: the ability to preferentially remove Fc~RIII from solution by an immunoadsorbent column comprising human IgG, or to inhibit rosette formation between IgG-coated RBCs and cells known to have Fc~RIIIs. The former measurements can be made with fluorescently labeled human IgG molecules, e.g. Haugland, Handbook of Fluorescent Probes (Molecular Probes, Inc., ?355 Junction City, OR, 1985). The latter measurements can be made by constructing an IgG column ~rom lsolated hum~n IgG and a commercially available ~ctiv~ted sepharos~
column, e.g. from Bio-Rad Laboratories (Richmond, CA).
-Rosette assays are standard ln the ~rt, e.g.
Winchester et al., chapter 31, in Rose et al., ed~., Manual of Clinical Laboratory Immunology, 3rd Ed.
(American Society for Microbiology, Washington, D.C., 1986).
Once the cDNA of the invention has been cloned, a wide range of expression systems (i.e. combinations of host and expression vector) can be used to produce the proteins of the invention. Possible types of host cells include, but are not limited to, bacterial, yeast, insect, mammalian, and the like. Selecting an expression system, and optimizing protein production thereby, involves the consideration and balancing of many factors, including (1~ the nature of the protein to be expressed, e.g. the protein may be poisonous to some host organisms, it may be susceptible to degradation by host proteases, or it may be expressed in inactive conformations or in insoluble form in some hosts, (2) the nature of the messenger RNA (mRNA) corresponding to the protein of interest, e.g. the mRNA may have sequences particularly susceptible to host endonucleases, which drastic?lly -~
reduce the functional lifetime of the mRNA, or the mRNA
may form secondary structures that mask the start codon or ribosome binding site, thereby inhibiting initiation of translation in some hosts, (31 the selection, availability, and arrangement of host-compatible expression-control sequences in the 3'- and 5'-regions -~
flanking the coding region -- these include promoters, 5'- and 3'-protector sequences, ribosome binding sites, transcription terminators, enhancers, polyadenylate addition sites, cap sites, intron-splice sites, and the Z~OU3~i like, (4) whether the protein h~s a secretion-signal sequence whi¢h can be processed by th- host, or wheth-r an expre~sion-control ~-quence encodlng a signal s-quenoe endogenous to the host ~ust be splic-d onto the region encoding the mature protein, ~5) the available modes and efficiencies of tran~fection or transformatlon o~ the host, and whether transient or stable expression is desired, (6) the scale and cost of the host cuIture system desired for expressing the protein, (7) whether, and what type of, posttranslational modifications are desired, e.g. the extent and kind of glyco~ylation desired may affect the choice of host, ~8) the ease with which the expressed protein can be separated from proteins and other materials of the host cells and/or culture medium e.g. in some cases it may be desirable to express a fusion protein with a specialized signal sequence to aid in later purification steps, e.g.
Sassenfeld et al., Biotechnology, January 1984! (9) the stability and copy number of a particular vector in a selected host, e.g. Hofschneider et al., eds. Gene Cloning in Orqanisms Other than E. coli (Springer Verlag, Berlin, 1982), and (10) like factors known to those skilled in the art. -Many reviews are available which provide guidance for making choices and/or modifications of specific expression systems in light of the recited factors, e.g., de Boer and Shepard, ~Strategies for Optimizing Foreign Gene Expression in Escherichia coli pgs. 205-247, in Kroon, ed. Genes: Structure and Expression (John Wiley~ Sons, New York, 1983), review several E. coli expression systems; Xucherlapati et al., Critical Reviews in Biochemistrv, Vol. 16, Issue 4, pgs.
349-379 (1984), and Banerji et al., Genetic Engineerinq, Vol. 5, pgs. 19-31 (1983) review methods for transfecting and transforming mammalian cells; Reznikoff and Gold, eds., Maximizinq Gene Ex~res~ion ~8utterworth~, Boston, 1986) review selected topic~ in gene xpresalon ln ~. -coli, ye~st, and mammalian cells; and Thilly, Ma~malian `Cell Technoloay ~Butterworths, Boston, 1986) revlews mammalian expression systems.
Likewise, many reviews are available which describe techniques and conditions for linking and/or - manipulating specific cDNAs and expression control sequences to create and/or modify expression vectors suitable for use with the present invention: e.g.
Maniatis et al., Molecular Cloninq: A Laboratorv Manual (Cold Spring Harbor Laboratory, N.Y., lg82); Glover, DNA
Cloninq: A Practical Approach, Vol. I and II (IRL Press, Oxford, 1985), and Perbal, A Practical Guide to Molecular Cloning (John Wiley & Sons, N.Y., 1984).
Suitable expression systems for the invention include those disclosed by Itakura and Riggs, U.S. patent 4,704,362 (bacterial expression), by Clark et al., U.S.
patent 4,675,285 and by Hamer U.Sl patent 4,599,308 (mammalian expression), and by Kurjan et al., U.S. patent 4,546,082 (yeast expression). Accordingly, the above patents are incorporated by reference.
Whenever SV40-based vectors are used, e.g. pcD
vectors, a preferred host is the COS7 cell line, described by Gluzman~ Cell, Vol. 23, pgs. 175-182 (1981) -and available from the ATCC under accession number ~--CRL1651. -~
` Soluble Fc~RIIIs of the invention are administered as a pharmaceutical composition for treating ITP. Such compositions contain an effective amount of the Fc~RIII in a pharmaceutical carrier. A -~
pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivering the compositions of the -invention to a patient. General~y, compositions useful ~
for parenteral administration of such drugs are well ~A
, ', ` 200~)355 known, e.g. Reminqton's Pharmaceuti al Science, 15th Ed.
(Mack PubliEihing Company, Easton, PA 1980).
Alternatively, compositions Or the invention may be introduced into a patient's body by an implantable drug delivery system, e.g. Urquhart et al., Ann. Rev.
Pharmacol. Toxicol., Vol. 24, pgs. 199-236 (1984).
When administered parenterally, the soluble Fc7RIIIs will be formulated in a unit dosage in~ectable form (solution, suspension, emulsion) in association with a pharmaceutical carrier. Such oarriers are inherently non-toxic and non-therapeutic. Examples of such carriers are normal saline, Ringer's solution, dextrose solution, and Hank's solution. Nonagueous carriers such as fixed oils and ethyl oleate may also be used. A preferred carrier is 5% dextrose/saline. The carrier may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The soluble Fc7RIII is prefera~ly formulated in purifiéd form substantially free of -aggregates and other proteins at a concentration in the range of about 5 to 30 mg/ml, and preferably at a concentration in the range of about 10 to 20 mg/ml.
Selecting an administration regimen to deliver to a patient an amount of soluble Fc7RIIi which is effectiYe in ameliorating the thrombopenia associated with ITP depends on several factors, including the serum turnover rate of the soluble Fc~RIII, the serum level of competing endogenous Fc7RIII associated with the immune disorder, the possible immunogenicity of the soluble Fc7RIII, and the like. Preferably, an administration regimen maximizes the amount of soluble Fc7RIII delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of soluble Fc7RIII
delivered depends in part on the particular soluble Fc7RIII employed and the severity of the disease being - ;
. ' ! .' ' ' ' ~ . ' ' ` . . .
rj.~. ., ~ , .: ' :
ZO~()3s5 treated. Guidance in selecting appropriate do~e- ls found in the literature on therapeutic u~es of antlbodie~
and antibody ~ragments, whlch are polypeptid-~ of roughly the same size as the soluble Fc~RIIIs, .g. ~4ch t al., chapter 22, in Ferrone et al., eds., Handbook of Monoclonal Antibodies (Noges Publications, Park Ridge, .
NJ, 1985); and Russell, pgs. 303-357, and Smith et al., pgs. 365-389, in Haber et al., eds. Antibodies in Human Diagnosis and Therapy (Raven Press, New York, 1977).
Preferably, the dose is in the range of about 1-20 mg/kg per day, more preferably about 1-10 mg/kg per day.
.
EXAMPLES
The following examples serve to illustrate the present invention. Selection of vectors and hosts as well as the concentration of reagents, temperatures, and the values of other variables are only to exemplify application of the present invention-and are not-to be considered limitations thereof. ~
Example I. Construction of stable mammalian cell ~--transformants which exPress human Fc~RIII
; . - .
Preferably, ma D alian cell lines capable of stable expression of the Fc~RIII are produced by cotransfecting a host mammalian cell with a vector carrying a selectable marker and a vector carrying a ;
host-compatible promoter and the Fc7RIII cDNA insert.
For pcD(SR~)-GP5, suitable hosts include Chinese hamster -~
ovary cells, COS monkey cells, and mouse L cells, such as a thymidine kinase deficient mutant (tk ) L cell ~- -available from the American Type Culture Collection under accession number CCL 1.3. The selectable marker allows ~- one t~ select host cells which have a high probability of containing the Fc~RIII gene fully integrated into the host genome. Typically, the ratio of pcD(SRb)-GP5 to the marker-containing vector in the tran8~ection ~olutlon i~
about 10:1. Thus, if the marker gene i- lnt~gr~ted into the host genome, it is very likely that pcD(SR~)-GP5 will also be integrated by virtue of it~ highor concontration, The selectable marker also provides a means o~ preventing the cultures of desired transformants from being overgrown by revertant cells.
tk mouse L cells are cotransfected with pcD(SR~)-GP5 and pSV2tk, a pSV2 plasmid carrying a thymidine kinase gene under control of the SV40 early promoter. The pSV2 plasmid is described by Mulligan et al., Science, Vol. 209, pgs. 1422-1427 (1980), and by Subramani et al~, Mol. Cell. Biol., Vol. 1, pgs. 854-864 (1981), and is available from the American Type Culture Collection under accession number 37146. Both plasmids are amplified in E. coli, e.g. strain HBlOl available from the ATCC under accession number 33694, and purified by cesium chloride equilibrium centrifugation. A
suspension of abut 1 x 105 of tk L cells in 10 ml of Dulbecco's Modified Eagle Medium (DME) with 10% fetal bovine serum is placed in a Falcon 3003 dish and cultured at 37C for 20 hours in a 5% carbon dioxide gas incubator, after which the medium is replaced by 10 ml of fresh DME with 10% fetal bovine serum. The culture is incubated for an additional 4 hours. After incubation, 0.5 ml of solution A (50 mM Hepes, 280 mM NaCl, 1.5 mM
sodium phosphate buffer, pH 7.22) and 0.5 ml of solution B (2M CaCl2, 10 ~g pcD(SR~)-GP5, 1 ~g pSV2tk) are added to the culture medium, and the culture is incubated at 37C for 24 hours in a 5% C02 atmosphere, after which the cells are placed in a selective medium with HAT (e.g.
Sigma Chemical Co., St. Louis, M0). After two weeks the surviving colonies are subcloned by limiting dilution, and clones are assayed for expression of Fc~RIII.
`: Z~)OQ355 xample II. Use of stable L cell transfor~ant Qxpressinq membrane-bound Fc7RIII
to measure serum levels of lmmune complex A stably transformed mammalian cell expressing the Fc~RIII of the invention can replace the Ra~i cell line in assays for immune complexes, e.g. Theo~ilopoulos et al., chapter 28, Manual of Clinical Laboratory Immunoloqy, 3rd Ed. (American Society for Microbiology, Washington, D.C. 1986).
Antiserum to human IgG i8 prepared in rabbits, and the IgG fraction is isolated by ammonium sulfate precipitation followed by fractionation on an anion-exchange chromatography column (DEAE-cellulose 52;
Whatman Chemical Separation Ltd., Maidstone, England).
Antiserum from commercial sources may also be used. The IgG frac~ion of the antiserum is brought to 5 mg/ml, and 1 ml is labeled with 125I. After iodination and dialysis, the antiserum is diluted to 1 mg/ml with phosphate-buffered saline (PBS) to give a specific activity of about 3 x 105 cpm/~g. Cells transformed with -~
the Fc~RIII cDNA are harvested after 72 hours of culture, and portions of 2 x 1o6 cells in 200 ~l of medium are -~
placed in 1.5-ml plastic Eppendorf conical tubes (Brinkmann Instruments, Inc., Westbury, N.Y.; catalog no.
22-36-411-1). One ml. of Spinner medium (Eagle minimal ~ --medium without Ca2+ and Mg2+) is added to each tube, and the cells are centrifuged at 800 x g for 8 min. ~-~
.
Supernatant fluids are aspirated, and the cell pellets are resuspended in 50 ~l of Spinner medium. Serum to be tested is diluted fourfold in 0.15 M ~aCl (physiological sal~ne), and 25 ~l is added to the transformed cells.
After a 45-min incubation period at 37C with gentle shaking by hand every 5 to 10 min, the cells are washed 2(~()Q35S
three times with Spinner medium. Arter the ~inal wa~h, the cells, gently 6haken every 5 to 10 minute~, ~r-allowed to react for 30 min at 4C wlth 125I-l~beled rabbit anti-human IgG diluted with Spinner medium containing 10 g of human serum albumin (HSA) per liter.
After incubation, the cells are washed three times, supernatant fluids are completely aspirated, and radioactivity of the cell pellet i8 determined in a gamma counter. All assays are done in duplicate. The amount of uptake, expressed as absolute counts, percentage of the input, or micrograms of antibody, is referred to a standard curve of radioactive antibody uptake by cells incubated with normal human serum (complement source) containing various amounts of aggregated human IgG ~i-(AHG). The quantity of immune complex in serum is equated to an amount of AHG after correction for the dilution factor and is xpressed as micrograms of AHG
equivalent per milliliter of serum. The soluble AHG is formed from a solution of 6.5 mg of Cohn fraction II or isolated human IgG per ml of physiological saline, heated at 63C for 30 min, and centrifuged (1,500 x g, 15 min) to remove insoluble large aggregates. The supernatant is then diluted with buffer to yield a final concentration of approximately 1.6 mg/ml. Portions (0.5 ml) of this preparation are stored at -70C and can be used for as long as 1 month.
~ he standard curve of radioactive antibody uptake i6 constructed as follows. Fifty-microliter portions of AHG (80 ~g of protein) are serially diluted (11 twofold dilutions) in saline. Subsequently, 50 ~l of a twofold dilution of normal human serum (source of complement), freshly obtained or stored at -70C, i8 added to each dilution of AHG, mixed carefully, and incubated at 37C for 30 min. Thereafter, 25 ~l of each mixture is added to 2 x 106 cells in duplicate ~fourfold `: 200()35S
final dilutions of 6erum eont~lnlng from 20 ~g to about 20 ng of AHG); the mixture i- lnoubat-d, wa~h-d, and reaet-d wlth radiolabsled antlbody. Radioaetlvity 1 then eounted as with th- t--t -ra. A b~-- lin- of r~dioaetive antibody uptake ~baekground) by e-ll~
ineubated with 25 ~1 of a fourfold dilutlon of noraal human serum, used as a ~ouree of eomplement in the reference eurve, is also established.
Standard preparatinis of IgG aggregates and of tetanus toxoid-human anti-tetanus toxoid immun- eomplexe~
have recently been developed under the auspiees of the International Union of Immunologists and are available through the Swiss Red Cross Blood Transfusion Serviee, e.g. Nydegger et al., Clin. Exp. Immunol., Vol. 58, pgs.
502-509-(1984).
Example III. Construction of a soluble human Fc7RIII
Soluble Fc~RIII was eonstrueted using site-l specifie oligonucleotide-directed mutagenesis of the Fc~RIII cDNA insert of pcD(SR~)-GP5. The entire cDNA --insert of pcD(SR~)-GP5 was subcloned as a 2.4 kilobase Bam HI fragment into the Bluescript XS plasmid (Stratagene, San Diego, CA) and single-stranded DNA was then prepared. --A synthetie oligonucleotide (minimum size about 18 nucleotides) encoding a TAA stop eodon and a Bam NI
site (with mutations shown in boldfaee in Figure 2) was ~;
used as a primer for complementary strand syntbesis using DNA polymerase I large fragment (Amersham, Arlington Heights, IL). Figure 2 indicates the position of the stop codon and Bam HI site relative to the ma~or domains eneoded by the Fe~RIII eDNA: S represents the region eneoding the signal peptide, EC represents the region ~ -encoding the extraceliular doaain of Fe~RIII, H ; ~-~
' ' ' -: -, ~:
ZOU(~355 represents the region encod~ng tho hydrophobic, or tran~membrane, domain of Fc~RIII, and C represento th-region encoding the cytopl~m~c do~ain. Introducing the stop codon as indicated by Figure 2 produce~ a mutant Fc~RIII lacking both the cytoplasmic and tran~membrane domains; it is soluble as shown by the designation ~sFc~RIII~ beside the modified DNA sequence. After the identity of the mutant was confirmed by DNA sequence ~.
analysis, it was subcloned back into the Bam HI ~ite of the pcD(SR~) vector, amplified in E. coli, and transfected into COS 7 cells by electroporation. One day prior to transfection, approximately (1.5-2.0) x 106 COS
7 monkey cells were seeded onto individual 100 mm plates in Dulbecco's modified Eagle's medium (DME) containing 6%
fetal calf serum and 2 mM glutamine. To perform the transfection, cells were harvested by trypsinization and counted, washed twice in serum-free DME, and suspended to (1-7) x 106 cells per ml in serum-free DME. DNA was added to 25 ~g/ml and the mixture was allowed to stand at room temperature for 10 minutes, after which 0.8 ml was pulsed in a 0.4 cm sterile cuvette with a Bio Rad -(Richmond, CA) Gene Pulser at 250 volts and 960 microfarads. After pulsing, cells were allowed to stand for 10 minutes and then were plated at (1.5-2.0) x 106 per 100 mm plate in DME plus 6% fetal calf serum.
Supernatants were ~arvested and assayed for soluble Fc~RIII after 72 hours.
The soluble Fc~RIII in the COS7 culture supernatants was analyzed further by gel electrophoresis, and immunoprecipitated. The results of this are illustrated in Figure 3. Lanes 1, 3 and 6 each contain proteins from culture supernatants of COS7 cells transfected with the pcD carrying the soluble Fc~RIII
cDNA. Lane 1 proteins were immunoprecipitated with a non-specific mouse IgG2 antibody; Lane 3 proteins were :.:` 200Q355 ~mmunopreclpitated with human IgG, presumably by the binding o~ the Fc portion o~ th- ~ntibody wlth th-soluble Fc~RIII; and Lan- 6 prot-ln~ w r-immunoprecipitat-d wlth th- ~onoclon~l antlbody 3G8 which i8 specific ~or the extracellular dom~in o~ Fc~RIII.
Size marker6 ad~acent to the Control lane (lan- 1) are in kilodaltons. The soluble Fc7RIII corresponds to the broad band at about 40 ki~odaltons. Lanes 4 and 7 contain proteins from culture supernatants of COS7 cells transfected with the pcD carrying the soluble Fc~RIII -cDNA and cultured in the presence of tunicamycin.
Tunicamycin inhibits the posttranslational attachment of N-linked carbohydrates to proteins. It was applied in an attempt to determine the nature of the apparent heterogeneity of the 40 kilodalton band. As shown in Figure 2, the tunicamycin causes the appearance of two bands of lower molecular weight, which is consistent with the deglycosylation of the soluble Fc~RTII. Tunicamycin -~
was added to~the cultures as disclosed by Martens et al., ~-PNAS 84: 809-813 (1987). Lanes 2 and 5 contain proteins from culture supernatants of COS7 cells which had undergone the same manipulations as the COS cells of --~
lanes 3 and 4 and lanes 6 and 7, respectively, with the excepticn that no plasmid DNA was included in the transfectio~ protocol.
Example IY Isolation of a Variant human Fc~RIII from Natural Killer Cells.
A cDNA library was constructed from mRNA `;
extracted from a human natural killer (NX) cell line -~ -.~, . .
using the pcD(SR~) expression vector. The library was screened with a probe constructed from the cDNA inQert of ~;~ pcD(SR~)-GP5, which was radiolabeled with 31p by random-primed DNA labcling (Boehringer Mannheim, Indianapolis, : , .
,~,~,. ,. ~ .. - ~, , ~ ~ . , . :
2(~003SS
IN). A clone pcD(SR~)-NL10 was obtained that exhibited human IgG bindlng activity. The sequence of the cDNA
insert of pcD(SR~)-NL10 ls illustrated in Figure 4. It can be seen that the amino acid sequence o~ this Fc~RIII
and that encoded by pcD(SR~)-GP5 are very similar, differing in the extracellular domain by only six amino acids.
The descriptions of the foregoinq embodiments of the invention have been presented for purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching.
The embodiments are chosen and described in order to best explain the principles of the invention and its practical application to thereby enable others skilled in the art to best utilize the invention in various embodiments and with such modifications as are suited to-the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto.
Applicants have deposited pcD(SR~)-GP5 with the American Type Culture Collection Rockville, MD, USA
(ATCC), under accession number 67707. This deposit is made under the Budapest Treaty for the Deposit of Microorganisms; and also under conditions as provided under ATCC's agreement for Culture Deposit for Patent Purposes, which assures that the deposit will be made available to the US Commissioner of Patents and Trademarks pursuant to 35 U.S.C. 122 and 37 C.F.R. 1.14, and will be made available to the public upon issue of a U.S. patent, which requires that the deposit be maintained. Availability of the deposited strain is not to be contrued as a license to practice the invention in contravent-ion of the rights granted under the authority of any government in accordance with its patent laws.
Case 2510XQ
Field of the Invention The invention relates generally to therapeutic compounds, and more particularly to soluble and membrane-bound forms of a low-affinity receptor for human immunoglobulin G, nucleic acids encodinq the same, and diagnostic and therapeutic uses of such receptors.
-I BACKGROUND
Receptors for the Fc poition of immunoglobulin G (IgG~ play a central role in cellular immune ~ -defenses. Three types of such receptors have been identified: A 72 kilodalton (kD) receptor with hiqh affinity for monomeric IgG is found on monocytes and some macrophages, a 40 kD receptor with low affinity for monomeric IgG is found on monocytes, neutrophils, eosinophils, platelets and certain human tumor-cell lines, and a 50-70 kD receptor with low affinity for monomeric IgG is found on neutrophils, eosinophils, natural killer cells, and macrophages. These three types ; ~ -- of Fc7 receptor are referred to as Fc7RI, Fc~RII, and Fc~RIII, respectively: Unkeless et al., Ann. Rev.
Immunol., Yol. 6, pgs. 251-281 (1988).
It is believed that ~c~RIII-mediated removal IgG-coated platelets plays an important part in the -~
;` zoov~s pathogene~ls of immune thrombocytopenic ~urpura (I~P), a platelet-det~clency condltlon charact-rlz-d by ~xce~-lv-bleedlng: von dem Borne, pg-. 222-256, ln Immunohaematoloav, Engelfrl-t t al., d~. (El~vler, Amsterdam, 1984). Clarkson et al., New Enaland J. Med., Vol. 314, pgs. 1236-1239 (1986), report that the infu-ion of ligand-blocking anti-Fc~RIII antibody into a patient with refractory ITP resulted in a transient increase in platelet count. This observation suggests that the most deleterious manifestation of ITP could be temporarily ameliorated by the administration of agents that block or compete with Fc~RIII for binding sites on IgG-coated platelets.
In a separate area of clinical i~munology, elevated serum levels of aggregates consisting of immunoglobulin and antigen (so-called ~immune coaplexes~) have been correlated with a wide variety of disorders, particularly-autoimmune diseases, such as systemic lupus erythematosus (SLE), and rheumatoid arthritis. The level of such complexes has become an important diagnostic for presence of autoimmune disorders; e.g. Theofilopoulos et al., Am.J. Pathol., Col. 100, pgs. 531-591 (1980).
Present assays for the serum level of immune complexes include solid-phase assays which take advantage of the affinity of the complexes for certain conplement - `
or rheumatoid factor proteins, and cellular assays which ~`
take advantage of the property of Ra~i cell~ to preferentially bind immune complexes; ~heofilopoulos et --al., chapter 28, and Toth et al., chapter 29, in Rose et al., eds. Manual of Clinical Immunology, 3rd Ed.
(American Society for Microbiology, Washington, D.C., 1986). Unfortunately, like many mammalian-cell based assays, the Raji-cell assay is difficult to perform, and requires elaborate controls and standards because of the inherent variability of Ra~i-cell binding to imaune complexes.
,t .~r~`. :~ ., ~ 200Q355 In light of the ~oregoing, it would be advantageous for the medical community to hav-alternative assay methods for immune co~plex-- utllizing well characterized, widely available,` and conveniently cultured cell line~ It would also be advantageou~ i~
soluble Fc~Rs could be produced in su~icient quantity to permit practical emergency therapy ~or re~actory cases of ITP
SUMMARY OF THE INVENTION
The present invention is directed to ~oluble and membrane-bound human Fc~RIII polypeptides, their muteins, nucleic acids capable of encoding the same, and diagnostic and therapeutic uses of such polypeptides and muteins .
The invention is based on the discovery of a - ~;
cDNA encoding a human Fc7RIII A cDNA clone, pcD(SR~
containing the Fc~RIII-encoding insert (illustrated in Figure 1) is deposited in E coli ~12 strain MC1061 with ~ ~ ;
the American Type Culture Collection (ATCC), Rockville, Maryland, USA, under accession number 67707 -~
Preferred embodiments of the invention include proteins having amino acid sequences selected from the group of 2-fold substituted and l-fold deleted sequences -defined by the formulas --~
X(Arg) - X(Thr) - X(Glu) - X(Asp) - X(Leu) - X(Pro) - ---~
X(Lys) - X(Ala) - X(Val) - X(Val) - X(Phe) - X(Leu) - --X(Glu) - X~Pro) - X(Gln) - X(Trp) - X(Tyr) - X(Arg) - ~-X(Val) - X(Leu) - X(Glu) - X(Lys) - X(Asp) - X(Ser) -X(Val) - X(Thr) - X(Leu) - X(Lys) - X(Cys) - X(Gln) - -~
X(Gly) - X(Ala) - X(Tyr) - X(Ser) - X(Pro) - X(Glu) -X(Asp) - X(Asn) - X(Ser) - X(Thr) - X(Gln) - X(Trp) - -~
X(Phe) - X(His) - X(Asn) - X(Glu) - X(Asn) - X(Leu) - ~ -~
X(Ile) - X(Ser) - X(Ser) - X(Gln) - X(Alà) - X(Ser) - -' ~1)0~;~55 X(Ser) - X(Tyr) - X~Phe) - X(Tle) - X~Asp) - X(Ala) -X~Ala) - X(Thr) - X(Val) - X(A~p) - X(Asp) - X~8er) -X(Gly) - X(Glu) - X(Tyr) - X(Arg) - X(Cys) - X(Gln) -X(Thr) - X(Asn) - X~Leu) - X~Ser) - X(Thr) - X~Leu) -X~Ser) - X(Asp) - X(Pro) - X(Val) - X~Gln) - X(Leu) -X(Glu) - X(Val) - X(His) - X(Val) - X(Gly) - X(Trp) -X(Leu) - X(Leu) - X(Leu) - X(Gln) - X(Ala) - X(Pro) -X(Arg) - X(Trp) - X(Val) - X(Phe) - X(Lys) - X(Glu) -X(Glu) - X(Asp) - X(Pro) - X(Ile) - X(His) - X(Leu) -X(Arg) - X(Cy6) - X(His) - X(Ser) - X(Trp) - X(Lys) -X(Asn) - X(Thr) - X(Ala) - X(Leu) - X(His) - X(Lys) -X(Val) - X(Thr) - X(Tyr) - X(Leu) - X(Gln) - X(Asn) -X(Gly) - X(Lys) - X(Asp) - X(Arg) - X(Lys) - X(Tyr) -X(Phe) - X(~is) - X(His) - X(Asn) - X(Ser) - X(Asp) -X(Phe) - X(His) - XlIle) - X(Pro) - X(Lys) - X(Ala) -X(Thr) - X(Leu) - X(Lys) - X(Asp) - X(Ser) - X(Gly) -X(Ser) - X(Tyr) - X(Phe) - X(Cys) - X(Arg) - X(Gly) -X(Leu) - X(Val) - X(Gly) - X(Ser) - X(Lys) - X(Asn) -X(Val) - X(Ser) - X(Ser) - X(Glu) - X(Thr) - X(Val) -X(Asn) - X(Ile) - X(Thr) - X(Ile) - X(Thr) - X(Gln) -X(Gly) - X(Leu) - X(Ala) - X(Val) - X(Ser) - X(Thr) -X(Ile) - X(Ser) - X(Ser) - X(Phe) - X(Ser) - X(Pro) -X(Pro) - X(Gly) Formula 1 and X(Arg) - X(Thr) - X(Glu) - X(Asp) - X(Leu) - X(Pro) -X(Lys) - X(Ala) - X(Val) - X(Val) - X(Phe) - X(Leu) -X(Glu) - X(Pro) - X(Gln) - X(Trp) - X(Tyr) - X(Arg) -X(Val) - X(Leu) - X(Glu) - X(Lys) - X(Asp) - X(Ser) -X(Val) - X(Thr) - X(Leu) - X(Lys) - X(Cys) - X(Gln) -X(Gly) - X(Ala) - X(Tyr) - X(Ser) - X(Pro) - X(Glu) -X(A8p) - X(A6n) - X(Ser) - X(Thr) - X(Gln) - X(Trp) --,.. ..
~ ,,. -. :, :
~; ~
~)0~3~S~i :`"
X(Phe) - X~Hio) - X~A~n) - X~Glu) - X(8er) - X~Leu) -X~Ile) - X~Ser) - X~Ser) - X~Gln) - X~Ala) - X~8er) -X~Ser) - X~Tyr) - X~Phe) - X~Ile) - X~A-p) - X~Ala) -X~Ala) - X~Thr) - X~Val) - X~Asp) - X~Asp) - X~Ser) -X~Gly) - X(Glu) - X~Tyr) - X~Arg) - X~Cy~) - X~Gln) -X~Thr) - X~Asn) - X~Leu) - X~Ser) - X~Thr) - X~Leu) -X~Ser) - X~Asp) - X~Pro) - X~Val) - X~Gln) - X~Leu) -X~Glu) - X~Val) - X~His) - X~Ile) - X~G~y) - X~Trp) -X(Leu) - X~Leu) - X~Leu) - X~Gln) - X(Ala) - X(Pro) -X(Arg) - X~Trp) - X~Val) - X~Phe) - X(Lys) - X(Glu) -X~Glu) - X~Asp) - X~Pro) - X~Ile) - X~His) - X~Leu) -X~Arg) - X~Cys) - X~His) - X~Ser) - X~Trp) - X~Lys) -X~Asn) - X~Thr) - X~ala) - X~Leu) - X~His) - X~Lys) -X~Val) - X~Thr) - X~Tyr) - X(Leu) - X(Gln) - X(Asn) -X(Gly) - X(Lys) - X(Gly) - X(Arg) - X(Lys) - X(Tyr) -X(Phe) - X(His) - X(His) - X(Asn) - X(Ser) - X(Asp) -X~Phe) - X(Tyr) - X~Ile) - X~Pro) - X~Lys) - X~Ala) -X~Thr) - X~Leu) - X~Lys) - X~Asp) - X~Ser) - X~Gly) -X~Ser) - X~Tyr) - X(Phe) - X~Cys) - X~Arg) - X~Gly) -X~Leu) - X~Phe) - X~Gly) - X(Ser) - X~Lys) - X~Asn) -X~Val) - X~Ser) - X~Ser) - X(Glu) - X(Thr) - X(Val) -X(Asn) - X~Ile) - X~Thr) - X~Ile) - X~Thr) - X~Gln) -X~Gly) - X~Leu) - X~Ala) - X~Val) - X~Ser) - X~Thr) -X~Ile) - X~Ser) - X~Ser) - X~Phe) - X~Phe) - X~Pro) -X~Pro) - X~Gly) ~ "'~:-~'', ~ Formula II --~
:' :.-,':"' wherein the term X~Xaa) represents the group of synonymous L-amino~ acids to the amino acid Xaa.
~; Synonymous amino acids within a group have sufficiently similar physiochemical properties for substitution between members of the group to preserve the biological function of the molecule, Grantham, Science, Vol. 185, pgs. 862-864 ~1974); and Dayhoff et al., Atlas of Protein 1~ ' ,, . ;' :` ZOOQ355 ff_ Sequence and Structure 1972, Vol. S, W ~ 89-99. It i-clear that deletions of amino acids may al~o b- mad- ln the above-identified sequence wlthout altering biological function, particularly if only a few amino acids are deleted and amino acids that are critical to a functional conformation are not removed or displaced, e.g. some cysteine residuesi: Anfisen, ~Principles That Govern The Folding of Protein Chains~, Science, Vol. 181, pgs. 223-230 (1973). Proteins and muteins produced by deletions are within the purview of the present invention.
Whenever an amino acid residue of a protein of Formula I
or II is referred to herein by number, such a number is in reference to the N-terminus of the protein.
Preferred, and more preferred, groups of synonymous amino acids for each amino are listed in Table I. The respective qrOups for each amino acid are indicated by slashes (/). Amino acids to the left of the slash make up the most preferred group. All the amino ---acids on the same line make up the more preferred group.
As used herein, ~N-fold substituted~ in reference to Formula I or II describes a group of amino -:
acid seguences differing from the native amino acid sequences of Formula III or IV, respectively, by zero-N
substitutions, such that the replacing amino acid is selected from t~e appropriate group of amino acids synonymous to the amino acid at the location of the substitution. That is, if the X(Ala) is at.the location for substitution, then the Ala at that location can be replaced with Ser, Thr, or Gly.
~ Likewise, as used herein, the term ~N-fold deleted~ in reference to Formula I or II describesi a group of amino acid seguences differing from the native amino acid sequences of Formula III or IV, respectively, by zero-N deletions of amino-acids.
.
: 200Q;~55 Throughout, 6tandard abbrovi~tlon~ ~re u~ed to designate amino acid~, nucleotide~, r--triotlon endonucle~se~, and the li~e; e.g. Cohn, ~Nomencl~tur- ~nd Symbol~sm of a -Amino Acids,~ Method~ in EnzvmoloqY, Vol.
106, pgs. 3-17 (1984); Wood et al. BlochemistrY: A
Problems ApProach~ 2nd ed. ~Ben~amin, Menlo Park, 1981);
and Roberts, ~Directory of ~estriction Endonucleases~, Methods in EnzvmolosY, Vol. 68, pgs. 27-40 (1979).
Table I
, :
Preferred and More Preferred Groups of Synonymous Amino Acids Amino Acid SYnonYmous GrouPs Ala Ala Ser / Thr Gly -~
Arg Arg / Lys --~
Asn Asn Asp / Ser Asp I Asp Glu / Asn Cys ~ Cys Gln Gln / Glu ~--Glu Glu Asp / Gln -Gly Gly Ala / Ser ., His His / Asn Ile Ile Val / Leu Leu Leu Val / Ile --~
Lys Lys Arg / Asn Met Met / Leu ~-Phe Phe Pro Pro / Ala Ser Ser Ala Thr I Gly Asn Thr Thr Ser Ala / Lys -~
Trp Trp - '~
Tyr - Tyr Val Val Ile / Ala Leu ':
, ,' , ~, , Z~)O(.t355 . "
~rief DescriPtion of The Drawina~
Pigure 1, parts A and ~ (on two she-t- to be read side-by-side) illustrates th- nucleotide ~eguence o~
the cDNA insert of pcD(SRa)-GP5;
Figure 2 illustrates the relative location o~
the stop codon inserted to produce a soluble human Fc~RIII, secreted as an FC~RIII mutant;
Figure 3 illustrates the result of gel electrophoretic analysis of the soluble Fc7RIII produced in Example III below; and ~ Figure 4 parts ~ and B (on two sheets to be read side-by-side) illustrates the necleotide sequence o~
the cDNA insert of pcD(SRo)-NL10.
DETAILED DESCRIPTION OF THE INVENTION - -The present invention includes nucleic acids encoding polypeptides capable of binding to the Fc portion of human IgG. These polypeptides are derived from human Fc~RIII. The invention includes soluble and membrane-bound polypeptides for human Fc~RIII. These and other modified versions of the polypeptides are readily produced using standard protein engineering techniques.
once nucleic acid sequence and/or amino acid -~-sequence information is available for a native protein, a variety of techniques become available for producing virtually any mutation in the native sequence. Shortle, in Science, Vol. 229, pgs. 1193-1201 ~1985), reviews techniques for mutating nucleic acids which are applicable to the present invention. Preferably, mutants -of *he protein of the present invention are produced by site-specific oligonucleotide-directed mutagenesis, e.g.
Zoller and Smith, Methods in Enzymologv, Vol. 100, pg~.
468-500 (1983), and Mark et al., U.S. Patent 4,518,584 entitled ~Human Recombinant Interleukin-2 Muteins^, which ~
~,. ~1-.~, ~ , . . . ~ , Z00~355 g are lncorporated by reference; or by so-called ~cassette~
mutagenesis described by Wells et al. in Gene, Vol. 34, pg8. 315-323 (1985), by Estell et al. ln Scienco, Vol.
233, pqs. 659-663 (1986), and al80 essentially by Mullenbach et al. in J. 8101. Chem., Vol. 261, pgs. 719-722 (1986), and by Feretti et al. in Proc. Natl. Acad.
Sci., Vol. 83, pg5. 597-603 (1986).
Polypeptides with amino acid modifications (i.e. muteins) may be desirable in a variety of circumstances. For example, undesirable side effects might be reduced by certain muteins, particularly if the side effect is associated with a different part of the polypeptide from that of the desired activity. In some expression systems, the native polypeptide may be -~
susceptible to degradation by proteases. In such cases, selected substitutions and/or deletions of amino acids which change the susceptible sequences can significantly enhance yields, e--.g. British patent application 2173-804-A where Arg at position 275 of human tissue plasminogen activator is replaced by Gly or Glu. Muteins may also increase yields in purification procedures and/or increase shelf lives of proteins by eliminating amino acids susceptible to oxidation, acylation, alkylation, or other chemical modifications. For exa~rle, methionine readily undergoes oxidation to form a sul~oxide, which in many proteins is associated with loss of biological activity: e.g. Brot and Weissbach, Arch. Biochem.
Biophys., Vol. 223, pg. 271 (1983). Methionines can often be replaced by more inert amino acids with little or no loss of biological activity, e.g. Australian patent application AU-A-52451/86. In bacterial expression systems, yields can sometimes be increased by eliminating or replacing conformationally inessential cysteine residues, e.g. Mark et al., U.S. Patent 4,518,584.
200(~355 Preferably, soluble form~ of tho FC~RIII o~ the invention are produced by introduoing a atop oodon prlor to (i.e. ln the 5'- or ~upstream~ dlreotlon of) the codlng region for the transmembrane and lntraoellul~r portion~ of the Fc~RIII cDNA. Thls 1- convenlently done by site-speciflc mutagenesis. Transmembrane region~ are readily identified by the presence of an amlno acld segment containing from about 20-25 residues having high average bydrophobicity, e.g. Wickner, Sclence, Vol. 210, pgs. 861-868 (1980), and Greene et al., Ann. Rev.
Immunol., Vol. 4, pgs. 69-9S (1986).
Plasmid pcD(SR~)-GPS is slmllar to the pcD
shuttle vector described by Okayama and Berg, Mol. Cell.
Biol., Vol. 2, pgs. 161-170 (1983), and Vol. 3, pgs. 280-289 (1983), except that the SV40 promoter has been modified to improve expression by the downstream insertion of a portion of the long terminal repeat (LTR) from a HTLV(I)~retrovirus, described by Takebe et al., Mol. Cell. Biol., Vol. 8, pgs. 466-472 (1988). The plasmid is conveniently propagated in E. coli K12 strain MC1061, or like host.
The immunoglobulin G binding property of the Fc~RIIIs of the invention is measured by standard techniques, e.g. (1) in the case of membrane-bound Fc~RIII: the ability of cells transfected with the Fc~RIII cDNA to form rosettes in the presence of IgG-coated red blood cells (RBCs) or to preferentially bind human IgG aggregated by heat treatment; or (2) in the case of soluble Fc~RIII: the ability to preferentially remove Fc~RIII from solution by an immunoadsorbent column comprising human IgG, or to inhibit rosette formation between IgG-coated RBCs and cells known to have Fc~RIIIs. The former measurements can be made with fluorescently labeled human IgG molecules, e.g. Haugland, Handbook of Fluorescent Probes (Molecular Probes, Inc., ?355 Junction City, OR, 1985). The latter measurements can be made by constructing an IgG column ~rom lsolated hum~n IgG and a commercially available ~ctiv~ted sepharos~
column, e.g. from Bio-Rad Laboratories (Richmond, CA).
-Rosette assays are standard ln the ~rt, e.g.
Winchester et al., chapter 31, in Rose et al., ed~., Manual of Clinical Laboratory Immunology, 3rd Ed.
(American Society for Microbiology, Washington, D.C., 1986).
Once the cDNA of the invention has been cloned, a wide range of expression systems (i.e. combinations of host and expression vector) can be used to produce the proteins of the invention. Possible types of host cells include, but are not limited to, bacterial, yeast, insect, mammalian, and the like. Selecting an expression system, and optimizing protein production thereby, involves the consideration and balancing of many factors, including (1~ the nature of the protein to be expressed, e.g. the protein may be poisonous to some host organisms, it may be susceptible to degradation by host proteases, or it may be expressed in inactive conformations or in insoluble form in some hosts, (2) the nature of the messenger RNA (mRNA) corresponding to the protein of interest, e.g. the mRNA may have sequences particularly susceptible to host endonucleases, which drastic?lly -~
reduce the functional lifetime of the mRNA, or the mRNA
may form secondary structures that mask the start codon or ribosome binding site, thereby inhibiting initiation of translation in some hosts, (31 the selection, availability, and arrangement of host-compatible expression-control sequences in the 3'- and 5'-regions -~
flanking the coding region -- these include promoters, 5'- and 3'-protector sequences, ribosome binding sites, transcription terminators, enhancers, polyadenylate addition sites, cap sites, intron-splice sites, and the Z~OU3~i like, (4) whether the protein h~s a secretion-signal sequence whi¢h can be processed by th- host, or wheth-r an expre~sion-control ~-quence encodlng a signal s-quenoe endogenous to the host ~ust be splic-d onto the region encoding the mature protein, ~5) the available modes and efficiencies of tran~fection or transformatlon o~ the host, and whether transient or stable expression is desired, (6) the scale and cost of the host cuIture system desired for expressing the protein, (7) whether, and what type of, posttranslational modifications are desired, e.g. the extent and kind of glyco~ylation desired may affect the choice of host, ~8) the ease with which the expressed protein can be separated from proteins and other materials of the host cells and/or culture medium e.g. in some cases it may be desirable to express a fusion protein with a specialized signal sequence to aid in later purification steps, e.g.
Sassenfeld et al., Biotechnology, January 1984! (9) the stability and copy number of a particular vector in a selected host, e.g. Hofschneider et al., eds. Gene Cloning in Orqanisms Other than E. coli (Springer Verlag, Berlin, 1982), and (10) like factors known to those skilled in the art. -Many reviews are available which provide guidance for making choices and/or modifications of specific expression systems in light of the recited factors, e.g., de Boer and Shepard, ~Strategies for Optimizing Foreign Gene Expression in Escherichia coli pgs. 205-247, in Kroon, ed. Genes: Structure and Expression (John Wiley~ Sons, New York, 1983), review several E. coli expression systems; Xucherlapati et al., Critical Reviews in Biochemistrv, Vol. 16, Issue 4, pgs.
349-379 (1984), and Banerji et al., Genetic Engineerinq, Vol. 5, pgs. 19-31 (1983) review methods for transfecting and transforming mammalian cells; Reznikoff and Gold, eds., Maximizinq Gene Ex~res~ion ~8utterworth~, Boston, 1986) review selected topic~ in gene xpresalon ln ~. -coli, ye~st, and mammalian cells; and Thilly, Ma~malian `Cell Technoloay ~Butterworths, Boston, 1986) revlews mammalian expression systems.
Likewise, many reviews are available which describe techniques and conditions for linking and/or - manipulating specific cDNAs and expression control sequences to create and/or modify expression vectors suitable for use with the present invention: e.g.
Maniatis et al., Molecular Cloninq: A Laboratorv Manual (Cold Spring Harbor Laboratory, N.Y., lg82); Glover, DNA
Cloninq: A Practical Approach, Vol. I and II (IRL Press, Oxford, 1985), and Perbal, A Practical Guide to Molecular Cloning (John Wiley & Sons, N.Y., 1984).
Suitable expression systems for the invention include those disclosed by Itakura and Riggs, U.S. patent 4,704,362 (bacterial expression), by Clark et al., U.S.
patent 4,675,285 and by Hamer U.Sl patent 4,599,308 (mammalian expression), and by Kurjan et al., U.S. patent 4,546,082 (yeast expression). Accordingly, the above patents are incorporated by reference.
Whenever SV40-based vectors are used, e.g. pcD
vectors, a preferred host is the COS7 cell line, described by Gluzman~ Cell, Vol. 23, pgs. 175-182 (1981) -and available from the ATCC under accession number ~--CRL1651. -~
` Soluble Fc~RIIIs of the invention are administered as a pharmaceutical composition for treating ITP. Such compositions contain an effective amount of the Fc~RIII in a pharmaceutical carrier. A -~
pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivering the compositions of the -invention to a patient. General~y, compositions useful ~
for parenteral administration of such drugs are well ~A
, ', ` 200~)355 known, e.g. Reminqton's Pharmaceuti al Science, 15th Ed.
(Mack PubliEihing Company, Easton, PA 1980).
Alternatively, compositions Or the invention may be introduced into a patient's body by an implantable drug delivery system, e.g. Urquhart et al., Ann. Rev.
Pharmacol. Toxicol., Vol. 24, pgs. 199-236 (1984).
When administered parenterally, the soluble Fc7RIIIs will be formulated in a unit dosage in~ectable form (solution, suspension, emulsion) in association with a pharmaceutical carrier. Such oarriers are inherently non-toxic and non-therapeutic. Examples of such carriers are normal saline, Ringer's solution, dextrose solution, and Hank's solution. Nonagueous carriers such as fixed oils and ethyl oleate may also be used. A preferred carrier is 5% dextrose/saline. The carrier may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The soluble Fc7RIII is prefera~ly formulated in purifiéd form substantially free of -aggregates and other proteins at a concentration in the range of about 5 to 30 mg/ml, and preferably at a concentration in the range of about 10 to 20 mg/ml.
Selecting an administration regimen to deliver to a patient an amount of soluble Fc7RIIi which is effectiYe in ameliorating the thrombopenia associated with ITP depends on several factors, including the serum turnover rate of the soluble Fc~RIII, the serum level of competing endogenous Fc7RIII associated with the immune disorder, the possible immunogenicity of the soluble Fc7RIII, and the like. Preferably, an administration regimen maximizes the amount of soluble Fc7RIII delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of soluble Fc7RIII
delivered depends in part on the particular soluble Fc7RIII employed and the severity of the disease being - ;
. ' ! .' ' ' ' ~ . ' ' ` . . .
rj.~. ., ~ , .: ' :
ZO~()3s5 treated. Guidance in selecting appropriate do~e- ls found in the literature on therapeutic u~es of antlbodie~
and antibody ~ragments, whlch are polypeptid-~ of roughly the same size as the soluble Fc~RIIIs, .g. ~4ch t al., chapter 22, in Ferrone et al., eds., Handbook of Monoclonal Antibodies (Noges Publications, Park Ridge, .
NJ, 1985); and Russell, pgs. 303-357, and Smith et al., pgs. 365-389, in Haber et al., eds. Antibodies in Human Diagnosis and Therapy (Raven Press, New York, 1977).
Preferably, the dose is in the range of about 1-20 mg/kg per day, more preferably about 1-10 mg/kg per day.
.
EXAMPLES
The following examples serve to illustrate the present invention. Selection of vectors and hosts as well as the concentration of reagents, temperatures, and the values of other variables are only to exemplify application of the present invention-and are not-to be considered limitations thereof. ~
Example I. Construction of stable mammalian cell ~--transformants which exPress human Fc~RIII
; . - .
Preferably, ma D alian cell lines capable of stable expression of the Fc~RIII are produced by cotransfecting a host mammalian cell with a vector carrying a selectable marker and a vector carrying a ;
host-compatible promoter and the Fc7RIII cDNA insert.
For pcD(SR~)-GP5, suitable hosts include Chinese hamster -~
ovary cells, COS monkey cells, and mouse L cells, such as a thymidine kinase deficient mutant (tk ) L cell ~- -available from the American Type Culture Collection under accession number CCL 1.3. The selectable marker allows ~- one t~ select host cells which have a high probability of containing the Fc~RIII gene fully integrated into the host genome. Typically, the ratio of pcD(SRb)-GP5 to the marker-containing vector in the tran8~ection ~olutlon i~
about 10:1. Thus, if the marker gene i- lnt~gr~ted into the host genome, it is very likely that pcD(SR~)-GP5 will also be integrated by virtue of it~ highor concontration, The selectable marker also provides a means o~ preventing the cultures of desired transformants from being overgrown by revertant cells.
tk mouse L cells are cotransfected with pcD(SR~)-GP5 and pSV2tk, a pSV2 plasmid carrying a thymidine kinase gene under control of the SV40 early promoter. The pSV2 plasmid is described by Mulligan et al., Science, Vol. 209, pgs. 1422-1427 (1980), and by Subramani et al~, Mol. Cell. Biol., Vol. 1, pgs. 854-864 (1981), and is available from the American Type Culture Collection under accession number 37146. Both plasmids are amplified in E. coli, e.g. strain HBlOl available from the ATCC under accession number 33694, and purified by cesium chloride equilibrium centrifugation. A
suspension of abut 1 x 105 of tk L cells in 10 ml of Dulbecco's Modified Eagle Medium (DME) with 10% fetal bovine serum is placed in a Falcon 3003 dish and cultured at 37C for 20 hours in a 5% carbon dioxide gas incubator, after which the medium is replaced by 10 ml of fresh DME with 10% fetal bovine serum. The culture is incubated for an additional 4 hours. After incubation, 0.5 ml of solution A (50 mM Hepes, 280 mM NaCl, 1.5 mM
sodium phosphate buffer, pH 7.22) and 0.5 ml of solution B (2M CaCl2, 10 ~g pcD(SR~)-GP5, 1 ~g pSV2tk) are added to the culture medium, and the culture is incubated at 37C for 24 hours in a 5% C02 atmosphere, after which the cells are placed in a selective medium with HAT (e.g.
Sigma Chemical Co., St. Louis, M0). After two weeks the surviving colonies are subcloned by limiting dilution, and clones are assayed for expression of Fc~RIII.
`: Z~)OQ355 xample II. Use of stable L cell transfor~ant Qxpressinq membrane-bound Fc7RIII
to measure serum levels of lmmune complex A stably transformed mammalian cell expressing the Fc~RIII of the invention can replace the Ra~i cell line in assays for immune complexes, e.g. Theo~ilopoulos et al., chapter 28, Manual of Clinical Laboratory Immunoloqy, 3rd Ed. (American Society for Microbiology, Washington, D.C. 1986).
Antiserum to human IgG i8 prepared in rabbits, and the IgG fraction is isolated by ammonium sulfate precipitation followed by fractionation on an anion-exchange chromatography column (DEAE-cellulose 52;
Whatman Chemical Separation Ltd., Maidstone, England).
Antiserum from commercial sources may also be used. The IgG frac~ion of the antiserum is brought to 5 mg/ml, and 1 ml is labeled with 125I. After iodination and dialysis, the antiserum is diluted to 1 mg/ml with phosphate-buffered saline (PBS) to give a specific activity of about 3 x 105 cpm/~g. Cells transformed with -~
the Fc~RIII cDNA are harvested after 72 hours of culture, and portions of 2 x 1o6 cells in 200 ~l of medium are -~
placed in 1.5-ml plastic Eppendorf conical tubes (Brinkmann Instruments, Inc., Westbury, N.Y.; catalog no.
22-36-411-1). One ml. of Spinner medium (Eagle minimal ~ --medium without Ca2+ and Mg2+) is added to each tube, and the cells are centrifuged at 800 x g for 8 min. ~-~
.
Supernatant fluids are aspirated, and the cell pellets are resuspended in 50 ~l of Spinner medium. Serum to be tested is diluted fourfold in 0.15 M ~aCl (physiological sal~ne), and 25 ~l is added to the transformed cells.
After a 45-min incubation period at 37C with gentle shaking by hand every 5 to 10 min, the cells are washed 2(~()Q35S
three times with Spinner medium. Arter the ~inal wa~h, the cells, gently 6haken every 5 to 10 minute~, ~r-allowed to react for 30 min at 4C wlth 125I-l~beled rabbit anti-human IgG diluted with Spinner medium containing 10 g of human serum albumin (HSA) per liter.
After incubation, the cells are washed three times, supernatant fluids are completely aspirated, and radioactivity of the cell pellet i8 determined in a gamma counter. All assays are done in duplicate. The amount of uptake, expressed as absolute counts, percentage of the input, or micrograms of antibody, is referred to a standard curve of radioactive antibody uptake by cells incubated with normal human serum (complement source) containing various amounts of aggregated human IgG ~i-(AHG). The quantity of immune complex in serum is equated to an amount of AHG after correction for the dilution factor and is xpressed as micrograms of AHG
equivalent per milliliter of serum. The soluble AHG is formed from a solution of 6.5 mg of Cohn fraction II or isolated human IgG per ml of physiological saline, heated at 63C for 30 min, and centrifuged (1,500 x g, 15 min) to remove insoluble large aggregates. The supernatant is then diluted with buffer to yield a final concentration of approximately 1.6 mg/ml. Portions (0.5 ml) of this preparation are stored at -70C and can be used for as long as 1 month.
~ he standard curve of radioactive antibody uptake i6 constructed as follows. Fifty-microliter portions of AHG (80 ~g of protein) are serially diluted (11 twofold dilutions) in saline. Subsequently, 50 ~l of a twofold dilution of normal human serum (source of complement), freshly obtained or stored at -70C, i8 added to each dilution of AHG, mixed carefully, and incubated at 37C for 30 min. Thereafter, 25 ~l of each mixture is added to 2 x 106 cells in duplicate ~fourfold `: 200()35S
final dilutions of 6erum eont~lnlng from 20 ~g to about 20 ng of AHG); the mixture i- lnoubat-d, wa~h-d, and reaet-d wlth radiolabsled antlbody. Radioaetlvity 1 then eounted as with th- t--t -ra. A b~-- lin- of r~dioaetive antibody uptake ~baekground) by e-ll~
ineubated with 25 ~1 of a fourfold dilutlon of noraal human serum, used as a ~ouree of eomplement in the reference eurve, is also established.
Standard preparatinis of IgG aggregates and of tetanus toxoid-human anti-tetanus toxoid immun- eomplexe~
have recently been developed under the auspiees of the International Union of Immunologists and are available through the Swiss Red Cross Blood Transfusion Serviee, e.g. Nydegger et al., Clin. Exp. Immunol., Vol. 58, pgs.
502-509-(1984).
Example III. Construction of a soluble human Fc7RIII
Soluble Fc~RIII was eonstrueted using site-l specifie oligonucleotide-directed mutagenesis of the Fc~RIII cDNA insert of pcD(SR~)-GP5. The entire cDNA --insert of pcD(SR~)-GP5 was subcloned as a 2.4 kilobase Bam HI fragment into the Bluescript XS plasmid (Stratagene, San Diego, CA) and single-stranded DNA was then prepared. --A synthetie oligonucleotide (minimum size about 18 nucleotides) encoding a TAA stop eodon and a Bam NI
site (with mutations shown in boldfaee in Figure 2) was ~;
used as a primer for complementary strand syntbesis using DNA polymerase I large fragment (Amersham, Arlington Heights, IL). Figure 2 indicates the position of the stop codon and Bam HI site relative to the ma~or domains eneoded by the Fe~RIII eDNA: S represents the region eneoding the signal peptide, EC represents the region ~ -encoding the extraceliular doaain of Fe~RIII, H ; ~-~
' ' ' -: -, ~:
ZOU(~355 represents the region encod~ng tho hydrophobic, or tran~membrane, domain of Fc~RIII, and C represento th-region encoding the cytopl~m~c do~ain. Introducing the stop codon as indicated by Figure 2 produce~ a mutant Fc~RIII lacking both the cytoplasmic and tran~membrane domains; it is soluble as shown by the designation ~sFc~RIII~ beside the modified DNA sequence. After the identity of the mutant was confirmed by DNA sequence ~.
analysis, it was subcloned back into the Bam HI ~ite of the pcD(SR~) vector, amplified in E. coli, and transfected into COS 7 cells by electroporation. One day prior to transfection, approximately (1.5-2.0) x 106 COS
7 monkey cells were seeded onto individual 100 mm plates in Dulbecco's modified Eagle's medium (DME) containing 6%
fetal calf serum and 2 mM glutamine. To perform the transfection, cells were harvested by trypsinization and counted, washed twice in serum-free DME, and suspended to (1-7) x 106 cells per ml in serum-free DME. DNA was added to 25 ~g/ml and the mixture was allowed to stand at room temperature for 10 minutes, after which 0.8 ml was pulsed in a 0.4 cm sterile cuvette with a Bio Rad -(Richmond, CA) Gene Pulser at 250 volts and 960 microfarads. After pulsing, cells were allowed to stand for 10 minutes and then were plated at (1.5-2.0) x 106 per 100 mm plate in DME plus 6% fetal calf serum.
Supernatants were ~arvested and assayed for soluble Fc~RIII after 72 hours.
The soluble Fc~RIII in the COS7 culture supernatants was analyzed further by gel electrophoresis, and immunoprecipitated. The results of this are illustrated in Figure 3. Lanes 1, 3 and 6 each contain proteins from culture supernatants of COS7 cells transfected with the pcD carrying the soluble Fc~RIII
cDNA. Lane 1 proteins were immunoprecipitated with a non-specific mouse IgG2 antibody; Lane 3 proteins were :.:` 200Q355 ~mmunopreclpitated with human IgG, presumably by the binding o~ the Fc portion o~ th- ~ntibody wlth th-soluble Fc~RIII; and Lan- 6 prot-ln~ w r-immunoprecipitat-d wlth th- ~onoclon~l antlbody 3G8 which i8 specific ~or the extracellular dom~in o~ Fc~RIII.
Size marker6 ad~acent to the Control lane (lan- 1) are in kilodaltons. The soluble Fc7RIII corresponds to the broad band at about 40 ki~odaltons. Lanes 4 and 7 contain proteins from culture supernatants of COS7 cells transfected with the pcD carrying the soluble Fc~RIII -cDNA and cultured in the presence of tunicamycin.
Tunicamycin inhibits the posttranslational attachment of N-linked carbohydrates to proteins. It was applied in an attempt to determine the nature of the apparent heterogeneity of the 40 kilodalton band. As shown in Figure 2, the tunicamycin causes the appearance of two bands of lower molecular weight, which is consistent with the deglycosylation of the soluble Fc~RTII. Tunicamycin -~
was added to~the cultures as disclosed by Martens et al., ~-PNAS 84: 809-813 (1987). Lanes 2 and 5 contain proteins from culture supernatants of COS7 cells which had undergone the same manipulations as the COS cells of --~
lanes 3 and 4 and lanes 6 and 7, respectively, with the excepticn that no plasmid DNA was included in the transfectio~ protocol.
Example IY Isolation of a Variant human Fc~RIII from Natural Killer Cells.
A cDNA library was constructed from mRNA `;
extracted from a human natural killer (NX) cell line -~ -.~, . .
using the pcD(SR~) expression vector. The library was screened with a probe constructed from the cDNA inQert of ~;~ pcD(SR~)-GP5, which was radiolabeled with 31p by random-primed DNA labcling (Boehringer Mannheim, Indianapolis, : , .
,~,~,. ,. ~ .. - ~, , ~ ~ . , . :
2(~003SS
IN). A clone pcD(SR~)-NL10 was obtained that exhibited human IgG bindlng activity. The sequence of the cDNA
insert of pcD(SR~)-NL10 ls illustrated in Figure 4. It can be seen that the amino acid sequence o~ this Fc~RIII
and that encoded by pcD(SR~)-GP5 are very similar, differing in the extracellular domain by only six amino acids.
The descriptions of the foregoinq embodiments of the invention have been presented for purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching.
The embodiments are chosen and described in order to best explain the principles of the invention and its practical application to thereby enable others skilled in the art to best utilize the invention in various embodiments and with such modifications as are suited to-the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto.
Applicants have deposited pcD(SR~)-GP5 with the American Type Culture Collection Rockville, MD, USA
(ATCC), under accession number 67707. This deposit is made under the Budapest Treaty for the Deposit of Microorganisms; and also under conditions as provided under ATCC's agreement for Culture Deposit for Patent Purposes, which assures that the deposit will be made available to the US Commissioner of Patents and Trademarks pursuant to 35 U.S.C. 122 and 37 C.F.R. 1.14, and will be made available to the public upon issue of a U.S. patent, which requires that the deposit be maintained. Availability of the deposited strain is not to be contrued as a license to practice the invention in contravent-ion of the rights granted under the authority of any government in accordance with its patent laws.
Claims (14)
1. A protein consisting of soluble or membrane-bound human Fc.gamma.RIII.
2. The protein of claim 1 consisting of the extracellular domain of a human Fc.gamma.RIII.
3. The protein of claim 1 having an amino acid sequence selected from the group consisting of 2-fold substituted and 1-fold deleted sequences defined by the formula:
or wherein:
X(Ala) represents Ala, Ser, Thr or Gly;
X(Arg) represents Arg or Lys;
X(Asn) represents Asn, Asp or Ser;
X(Asp) represents Asp, Glu, or Asn;
X(Cys) is Cys;
X(Gln) represents Gln or Glu;
X(Glu) represents Glu, Asp or Gln;
X(Gly) represents Gly, Ala or Ser;
X(His) represents His or Asn;
X(Ile) represents Ile, Val, or Leu;
X(Leu) represents Leu, Val, or Ile;
X(Lys) represents Lys, Arg, or Asn;
X(Met) represents Met or Leu;
X(Phe) is Phe;
X(Pro) represents Pro or Ala;
X(Ser) represents Ser, Ala, Thr, Gly, or Asn;
X(Thr) represents Thr, Ser, Ala, or Lys;
X(Trp) is Trp;
X(Tyr) is Tyr; and X(Val) represents Val, Ile, Ala, or Leu.
or wherein:
X(Ala) represents Ala, Ser, Thr or Gly;
X(Arg) represents Arg or Lys;
X(Asn) represents Asn, Asp or Ser;
X(Asp) represents Asp, Glu, or Asn;
X(Cys) is Cys;
X(Gln) represents Gln or Glu;
X(Glu) represents Glu, Asp or Gln;
X(Gly) represents Gly, Ala or Ser;
X(His) represents His or Asn;
X(Ile) represents Ile, Val, or Leu;
X(Leu) represents Leu, Val, or Ile;
X(Lys) represents Lys, Arg, or Asn;
X(Met) represents Met or Leu;
X(Phe) is Phe;
X(Pro) represents Pro or Ala;
X(Ser) represents Ser, Ala, Thr, Gly, or Asn;
X(Thr) represents Thr, Ser, Ala, or Lys;
X(Trp) is Trp;
X(Tyr) is Tyr; and X(Val) represents Val, Ile, Ala, or Leu.
4. The protein of claim 3 wherein said group consists of 1-fold substituted sequences of said formulas.
5. The protein of claim 3 wherein:
X(Ala) represents Ala or Ser;
X(Arg) is Arg;
X(Asn) represents Asn or Asp;
X(Asp) represents Asp or Glu;
X(Cys) is Cys;
X(Gln) is Gln;
X(Glu) represents Glu or Asp;
X(Gly) represents Gly or Ala;
X(His) is His;
X(Ile) represents Ile or Val;
X(Leu) represents Leu or Val;
X(Lys) represents Lys or Arg;
X(Met) is Met;
X(Phe) is Phe;
X(Pro) is Pro;
X(Ser) represents Ser, Ala or Thr;
X(Thr) represents Thr, Ser or Ala;
X(Trp) is Trp;
X(Tyr) is Tyr; and X(Val) represents Val or Ile.
X(Ala) represents Ala or Ser;
X(Arg) is Arg;
X(Asn) represents Asn or Asp;
X(Asp) represents Asp or Glu;
X(Cys) is Cys;
X(Gln) is Gln;
X(Glu) represents Glu or Asp;
X(Gly) represents Gly or Ala;
X(His) is His;
X(Ile) represents Ile or Val;
X(Leu) represents Leu or Val;
X(Lys) represents Lys or Arg;
X(Met) is Met;
X(Phe) is Phe;
X(Pro) is Pro;
X(Ser) represents Ser, Ala or Thr;
X(Thr) represents Thr, Ser or Ala;
X(Trp) is Trp;
X(Tyr) is Tyr; and X(Val) represents Val or Ile.
6. The protein of claim 5 wherein said group consists of 1-fold substituted sequences of said formulas.
7. The protein of claim 6 having the amino acid sequence:
or
or
8. A nucleic acid capable of encoding soluble or membrane-bound human Fc.gamma.RIII.
9. The nucleic acid of claim 8 capable of encoding a polypeptide having an amino acid sequence selected from the 2-fold substituted and 1-fold deleted sequences defined by the formula:
or wherein the various amino acids represented by X(Xaa) have the meanings defined in claim 3.
or wherein the various amino acids represented by X(Xaa) have the meanings defined in claim 3.
10. The nucleic acid of claim 9 capable of encoding a polypeptide selected from and
11. A method of treating immune thrombocytopenic purpura in a person, the method comprising administering an effective amount of soluble human Fc.gamma.RIII.
12. The method of claim 11 wherein said step of administering includes intravenous delivery of an amount of said soluble human Fc.gamma.RIII in the range of about 1-20 mg/kg body weight of said person per day.
13. A method of detecting immune complexes in a sample, the method comprising the steps of:
providing cells transformed by a vector carrying a cDNA insert encoding an Fc.gamma.RIII, the Fc.gamma.RIII
being expressed on the surface of the cells;
exposing the cells to the sample containing immune complex so that the immune complex can adhere to_ the surface of the cells by way of the Fc.gamma.RIIIs;
labeling the immune complex adhering to the surfaces of the cells; and measuring the amount of labeled immune complex adhering to the cells.
providing cells transformed by a vector carrying a cDNA insert encoding an Fc.gamma.RIII, the Fc.gamma.RIII
being expressed on the surface of the cells;
exposing the cells to the sample containing immune complex so that the immune complex can adhere to_ the surface of the cells by way of the Fc.gamma.RIIIs;
labeling the immune complex adhering to the surfaces of the cells; and measuring the amount of labeled immune complex adhering to the cells.
14. The method of claim 13 wherein said cells are mouse L cells or COS monkey cells and said vector is selected from pcD(SR.alpha.)-GP5 and pcD(SR.alpha.)-NL10.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US30003989A | 1989-01-19 | 1989-01-19 | |
| US300,039 | 1989-01-19 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2000355A1 true CA2000355A1 (en) | 1990-07-19 |
Family
ID=23157428
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002000355A Abandoned CA2000355A1 (en) | 1989-01-19 | 1989-10-10 | Human fc-gamma receptor iii |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2000355A1 (en) |
-
1989
- 1989-10-10 CA CA002000355A patent/CA2000355A1/en not_active Abandoned
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