Soluble mammal-derived Fc receptor which binds at a pH ranging from about 5.5 to 6.5 and releases at a pH ranging from about 7.5 to 8.5

The present invention pertains to a new composition of matter in the form of a soluble Fc receptor (pHsFcR) is created. The pHsFcR has a pH-determinable binding capability for at least one antibody or complex thereof. The pHsFcR is produced using the cDNA for a transmembrane, pH-determinable Fc receptor found in nature which has the capability of binding to at least one antibody or complex thereof. The most preferred embodiment of the present invention is a secreted form of pHsFcR which is produced using a modified cDNA of the transmembrane Fc receptor; the modified cDNA is obtained by the creation of a new in-frame stop codon and the deletion of the DNA sequence encoding the transmembrane (and typically the cytoplasmic) domain of the transmembrane Fc receptor. This creates a new gene that encodes a completely soluble protein consisting of the extracellular domains of the transmembrane Fc receptor. The new gene is used in formation of an expression vector which is introduced into a procaryotic or eucaryotic cell, followed by selection for a procaryotic or eucaryotic cell comprising the modified cDNA of the desired secreted pHsFcR. The selected cells are grown, during which pHsFcR is secreted into the growth medium. The secreted pHsFcR can then be separated from the growth medium using an affinity column comprising an antibody to which the pHsFcR can bind.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a soluble Fc receptor and a method for its 
production. 
2. Description of the Background Art 
Immunoglobulins, also referred to as antibodies, are a major component of 
the humoral immune response of all mammals. These glycoproteins are 
divided into five major structural classes, each of which can be divided 
into subclasses. The five major classes are IgA, IgD, IgE, IgG, and IgM. 
IgG is the most common class of immunoglobulins found in the serum. 
It is the highly specific interaction between an antibody and its target 
antigen that makes an immunoglobulin an effective agent against an 
invading pathogen. The high specificity of the immunoglobulin--antigen 
interaction allows the immune system to attack the foreign antigen with 
minimal harm to the host organism.degree. In general, immunoglobulins have 
a high affinity for their target antigen with an average dissociation 
constant (Kd) for the antigen--antibody complex on the order of 10.sup.-9 
to 10.sup.-12 moles/liter. 
Immunoglobulins are composed of four polypeptide chains; two identical 
light chains, each of approximately 25 kD in molecular mass and two 
identical heavy chains, each of approximately 50-80 kD in molecular mass. 
The different classes of antibodies are distinguished by structural 
differences in their heavy chains. A schematic drawing of an IgG 10 is 
shown in FIG. 1. 
Light chains 12 and 14 have been characterized as having a single amino 
terminal variable domain (a domain is a distinct region of tertiary 
protein structure), V.sub.L and a single constant domain, C.sub.L. The 
term variable refers to the variability in amino acid sequence found in 
this type of domain between antibodies with different antigen binding 
specificities, which antibodies are of the same immunoglobulin class and 
subclass. The heavy chains 16 and 18 of an IgG contain a single amino 
terminal variable domain, VH, followed by three constant domains, C.sub.H 
1, C.sub.H 2, and C.sub.H 3, as shown in FIG. 1. 
In general, an immunoglobulin, and in particular IgG, may be characterized 
as a Y shaped molecule in which each upper arm of the Y is formed by a 
pairing of a single light chain (V.sub.L+ C.sub.L) with the two most amino 
terminal domains of a single heavy chain (V.sub.H+ C.sub.H1). It is the 
pairing of the variable domains of the light and heavy chains that forms 
the antigen binding sites 20 and 22. The constant domain of the light 
chain (C.sub.L) interacts with the first constant domain of the heavy 
chain .(C.sub.H 1). The heavy and light chains are covalently bound to 
each other by a disulfide bond 24 between the paired C.sub.L and C.sub.H 1 
domains. The two heavy chains dimerize through interactions between their 
2nd and 3rd constant domains (C.sub.H 2 and C.sub.H 3). The two heavy 
chains are also covalently bound to each other through interchain 
disulfide bonds 26. These disulfide bonds 26 connect the two heavy chains 
in a region between the C.sub.H 1 and C.sub.H 2 domains that is known as 
the hinge region. Beneath the hinge region are oligosaccharide units 
(carbohydrates) 28 and 30, which are attached to the C.sub.H 2 domains 
within the structure. The V.sub.L and C.sub.L of the light chain paired 
with V.sub.H and C.sub.H 1 of the heavy chain is called an Fab, FIG. 1 at 
32 and 34; and, the paired C.sub.H 2 and C.sub.H 3 domains of the IgG 
heavy chains is referred to as the Fc, FIG. 1 at 36, of the antibody. 
Typically, immunoglobulin molecules are soluble in aqueous solution and 
bind to compounds in a highly specific manner with a great affinity. It is 
possible to produce antibodies against virtually any organic compound 
known to humans. These characteristics of immunoglobulins have resulted in 
their widespread analytical and therapeutic use throughout the biological 
sciences. 
Fc receptor is a general term that refers to any one of several proteins 
that bind to the Fc region of an immunoglobulin. Fc receptors can be 
soluble or membrane-bound. An example of a membrane-bound Fc receptor is 
the FcRn from the intestines of neonatal rats. The FcRn was recently 
cloned and characterized by N. E. Simister and K. E. Mostov, as described 
in "An Fc Receptor Structurally Related to MHC Class I Antigens" Nature 
(London) Vol. 337, pp. 184-187 (1989). 
__________________________________________________________________________ 
Met 
Gly 
Met 
Ser 
Gln 
Pro 
Gly 
Val 
Leu 
Leu 
Ser 
Leu 
Leu 
Leu 
Val 
Leu 
-22 -20 -15 -10 
Leu 
Pro 
Gln 
Thr 
Trp 
Gly 
Ala 
Glu 
Pro 
Arg 
Leu 
Pro 
leu 
Met 
Tyr 
His 
-5 -1 +1 5 10 
Leu 
Ala 
Ala 
Val 
Ser 
Asp 
Leu 
Ser 
Thr 
Gly 
Leu 
Pro 
Ser 
Phe 
Trp 
Ala 
15 20 25 
Thr 
Gly 
Trp 
Leu 
Gly 
Ala 
Gln 
Gln 
Tyr 
Leu 
Thr 
Tyr 
Asn 
Asn 
Leu 
Arg 
30 35 40 
Gln 
Glu 
Ala 
Asp 
Pro 
Cys 
Gly 
Ala 
Trp 
Ile 
Trp 
Glu 
Asn 
Gln 
Val 
Ser 
45 50 55 
Trp 
Tyr 
Trp 
Glu 
Lys 
Glu 
Thr 
Thr 
Asp 
Leu 
Lys 
Ser 
Lys 
Glu 
Gln 
Leu 
60 65 70 
Phe 
Leu 
Glu 
Ala 
Ile 
Arg 
Thr 
Leu 
Glu 
Asn 
Gln 
Ile 
Asn 
Gly 
Thr 
Phe 
75 80 85 90 
Thr 
Leu 
Gln 
Gly 
Leu 
Leu 
Gly 
Cys 
Glu 
Leu 
Ala 
Pro 
Asp 
asn 
Ser 
Ser 
95 100 105 
Leu 
Pro 
Thr 
Ala 
Val 
Phe 
Ala 
Leu 
Asn 
Gly 
Glu 
Glu 
Phe 
Met 
Arg 
Phe 
110 115 120 
Asn 
Pro 
Arg 
Thr 
Gly 
Asn 
Trp 
Ser 
Gly 
Glu 
Trp 
Pro 
Glu 
Thr 
Asp 
Ile 
125 130 135 
Val 
Gly 
Asn 
Leu 
Trp 
Met 
Lys 
Gln 
Pro 
Glu 
Ala 
Ala 
Arg 
Lys 
Glu 
Ser 
140 145 150 
Glu 
Phe 
Leu 
Leu 
Thr 
Ser 
Cys 
Pro 
Glu 
Arg 
Leu 
Leu 
Gly 
His 
Leu 
Glu 
155 160 165 170 
Arg 
Gly 
Arg 
Gln 
Asn 
Leu 
Glu 
Trp 
Lys 
Glu 
Pro 
Pro 
Ser 
Met 
Arg 
Leu 
175 180 185 
Lys 
Ala 
Arg 
Pro 
Gly 
asn 
Ser 
Gly 
Ser 
Ser 
Val 
Leu 
Thr 
Cys 
Ala 
Ala 
190 195 200 
Phe 
Ser 
Phe 
Tyr 
Pro 
Pro 
Glu 
Leu 
Lys 
Phe 
Arg 
Phe 
Leu 
Arg 
Asn 
Gly 
205 210 215 
Leu 
Ala 
Ser 
Gly 
Ser 
Gly 
Asn 
Cys 
Ser 
Thr 
Gly 
Pro 
Asn 
Gly 
Asp 
Gly 
220 225 230 
Ser 
Phe 
His 
Ala 
Trp 
Ser 
Leu 
Leu 
Glu 
Val 
Lys 
Arg 
Gly 
Asp 
Glu 
His 
235 240 245 250 
His 
Tyr 
Gln 
Cys 
Gln 
Val 
Glu 
His 
Glu 
Gly 
Leu 
Ala 
Gln 
Pro 
Leu 
Thr 
255 260 265 
Val 
Asp 
Leu 
Asp 
Ser 
Pro 
Ala 
Arg 
Ser 
Ser 
Val 
Pro 
Val 
Val 
Gly 
Ile 
270 275 280 
Ile 
Leu 
Gly 
Leu 
Leu 
Leu 
Val 
Val 
Val 
Ala 
Ile 
Ala 
Gly 
Gly 
Val 
Leu 
285 290 295 
Leu 
Trp 
Asn 
Arg 
Met 
Arg 
Ser 
Gly 
Leu 
Pro 
Ala 
Pro 
Trp 
Leu 
Ser 
Leu 
300 305 310 
Ser 
Gly 
Asp 
Asp 
Ser 
Gly 
Asp 
Leu 
Leu 
Pro 
Gly 
Gly 
Asn 
Leu 
Pro 
Pro 
315 320 325 330 
Glu 
Ala 
Glu 
Pro 
Gln 
Gly 
Val 
Asn 
Ala 
Phe 
Pro 
Ala 
Thr 
Ser. 
335 340 344 
__________________________________________________________________________ 
This Fc receptor (FcRn) is physiologically expressed on the luminal surface 
of neonatal rat intestinal epithelial cells. The FcRn was determined to 
optimally bind to IgG at the intestinal pH of 6-6.5 and to release bound 
IgG at the serosal pH of approximately 7.5. The physiological role of FcRn 
is to bind to maternal IgG consumed by the newborn when it drinks its 
mother's milk. The FcRn is then involved in the transport of the bound IgG 
across the intestinal epithelial barrier and the release of the IgG into 
the blood of the newborn. By this means the neonatal rat can passively 
acquire some resistance to disease. This is especially important during 
the first few weeks of independent life of the rat (as well as the cat) 
because at birth these mammals are practically agammaglobulinemic (without 
antibodies). FcRn, as found on the surface of neonatal rat intestinal 
epithelial cells, is a heterodimer consisting of an FcRn heavy chain (p51) 
of approximately 45-53 kD in molecular mass and a light chain 
(.beta..sub.2 m) of approximately 14 kD in molecular mass. The FcRn heavy 
chain appears to have 3 extracellular domains, a transmembrane domain and 
a cytoplasmic tail. The three extracellular domains of the FcRn heavy 
chain have significant sequence similarity to the corresponding domains of 
Class I Major Histocompatibility Complex (MHC) molecules. This sequence 
similarity suggests that the FcRn may have a tertiary protein structure 
similar to that observed for Class I MHC molecules. The FcRn light chain 
is a .beta..sub.2 m (.beta.-2 microglobulin), a soluble single domain 
protein also found as a component of the Class I MHC molecule heterodimer. 
The sequence for rat .beta..sub.2 m was published by J. Sundelin et al. 
Scand. J. Immunol. 27, pp. 195-199 (1988) as follows: 
__________________________________________________________________________ 
Ile 
Gln 
Lys 
Thr 
Pro 
Gln 
Ile 
Gln 
Val 
Tyr 
Ser 
Arg 
His 
Pro 
Pro 
Glu 
1 5 10 15 
Asn 
Gly 
Lys 
Pro 
Asn 
Phe 
Leu 
Asn 
Cys 
Tyr 
Val 
Ser 
Gln 
Phe 
His 
Pro 
20 25 30 
Pro 
Gln 
Ile 
Glu 
Ile 
Glu 
Leu 
Leu 
Lys 
Asn 
Gly 
Lys 
Lys 
Ile 
Pro 
Asn 
35 40 45 
Ile 
Glu 
Met 
Ser 
Asp 
Leu 
Ser 
Phe 
Ser 
Lys 
asp 
Trp 
Ser 
Phe 
Tyr 
Ile 
50 55 60 
Leu 
Ala 
His 
Thr 
Glu 
Phe 
Thr 
Pro 
Thr 
Glu 
Thr 
Asp 
Val 
Tyr 
Ala 
Cys 
65 70 75 80 
Arg 
Val 
Lys 
His 
Val 
Thr 
Lys 
Leu 
Glu 
Pro 
Lys 
Thr 
Val 
Thr 
Trp 
Asp 
85 90 95 
Arg 
Asp 
Met. 
99 
__________________________________________________________________________ 
The transmembrane domain of the FcRn heavy chain anchors the FcRn 
heterodimer into the cell membrane of the intestinal epithelial cells. The 
hydrophobic nature of this transmembrane domain precludes the 
solubilization of this protein in an aqueous buffer in the absence of 
surfactants, which are often toxic, which can reduce the stability of 
proteins, and which are often difficult to remove once they have been in 
contact with the protein. 
There are numerous potential applications for an Fc receptor which is 
soluble in aqueous solutions without the use of a surfactant. In addition, 
an Fc receptor, such as the FcRn, which is capable of binding antibodies 
at one pH and releasing the antibodies at another, is one that could be 
used in the biotechnology industry. Fc receptors can be used in medicine 
for the detection of antibodies under conditions of physiological pH. In 
addition, in U.S. patent application, Ser. No. 07/819,040, of Andrew Huber 
et al., filed Jan. 10, 1992, it is disclosed that soluble FcRn receptors 
of the kind disclosed and claimed herein can be attached to a surface and 
used to concentrate and purify antibodies from mixtures comprising 
antibodies such as IgG from blood, ascites or tissue culture supernatants 
(growth medium in which cells are cultured). 
However, the usefulness of the membrane-bound FcRn is limited by the fact 
that, like other transmembrane proteins, it is not readily soluble in an 
aqueous solution in the absence of surfactants, as described above. 
Although cDNA for the membrane-bound FcRn was available from N. E. 
Simister, it was questionable whether a soluble FcRn could be produced. 
The soluble FcRn, like other proteins must be able to fold into a 
functional three dimensional structure. This folding of a protein in 
solution is a very complex process and is influenced by many factors, such 
as the presence of other proteins which assist in the folding process. 
Upon removal of the transmembrane domain from the FcRn, the resultant 
protein might not be able to fold properly in solution, rendering it 
non-functional and probably insoluble. Even if the protein were able to 
fold properly, it might not be secreted from the cell in which it is 
produced and/or it might simply be degraded within the cell. Further, 
depending on where the heavy chain of FcRn is truncated to remove the 
transmembrane domain, the truncated heavy chain might be less heat stable 
and might not associate with its intended light chain, which would affect 
its ability to bind to an antibody. 
Definitions 
The following definitions are for use in understanding the descriptions 
presented throughout the present application. 
FcRn is understood to mean rat neonatal, intestinal, pH-determinable 
membrane-bound Fc receptor(s); 
pHsFcRn is understood to mean a soluble form of FcRn produced by 
applicants; 
pHFcR is understood to mean a membrane-bound fc receptor having the ability 
to bind to an antibody monomer or complex thereof at one pH and to release 
the antibody or complex thereof at another pH; 
sFcR is understood to mean soluble, vertebrated-derived Fc receptor in 
general; 
pHsFcR is understood to mean soluble, vertebrate-derived Fc receptor having 
the ability to bind to an antibody monomer or complex thereof at one pH 
and to release the antibody or complex thereof at another pH; 
.alpha.MEM is understood to mean a commercially available growth medium 
supplied by Irvine Scientific and Gibco/BRL; 
.beta..sub.2 m is understood to mean .beta..sub.2 -microglobulin; 
cDNA is understood to mean complementary DNA prepared from mRNA. 
DAF is understood to mean decay-accelerating factor; 
DMEM is understood to mean a commercially available growth medium supplied 
by Irvine Scientific and Gibco/BRL; 
MHC is understood to mean major histocompatibility complex; 
MSX is understood to mean L-methionine sulfoximine; and 
PI-PLC is understood to mean phosphatidylinositol--specific phospholipase 
C; 
SUMMARY OF THE INVENTION 
In accordance with the present invention, a new composition of matter in 
the form of a soluble vertebrate-derived Fc receptor (pHsFcR) has been 
created. The pHsFcR has a pH-determinable binding capability for at least 
one antibody monomer or complex thereof. The pHsFcR was produced using the 
cDNA of a transmembrane, pH-determinable, vertebrate-derived Fc receptor 
found in nature that is capable of binding to at least one antibody 
monomer or complex thereof. The pHsFcR of the present invention can be 
produced by removing the amino acid residues responsible for the 
attachment of the pHFcR to the cell membrane. In the case of secreted 
pHsFcR, this is accomplished at the DNA level by eliminating the DNA that 
encodes the transmembrane domain(s) of the protein. In the case of 
lipid-linked pHFcR, a pHsFcR can be generated at the protein level by 
cleaving the protein polypeptide chain between the desired extracellular 
domains and the amino acids responsible for the attachment of the protein 
to the cell membrane. 
The secreted pHsFcR was produced from the transmembrane Fc receptor cDNA by 
the creation of a new in-frame stop codon and the deletion of the DNA 
sequence encoding the transmembrane (and typically the cytoplasmic) domain 
of the transmembrane protein. The new gene thereby created encoded a 
completely soluble protein consisting of the extracellular domains of the 
vertebrate-derived transmembrane protein. In the preferred embodiment of 
the secreted pHsFcR, the cDNA sequence encoding the transmembrane and 
cytoplasmic domains of an FcRn heavy chain was deleted from the FcRn cDNA. 
Specifically, a modified heavy chain cDNA fragment was produced by 
inserting a stop codon after the codon for amino acid 269 in the full 
length heavy chain cDNA for FcRn. The amino acid 269 position is shown in 
FIG. 2A for FcRn heavy chain (p51 WT). The full amino acid sequence for 
FeRn heavy chain, including the FcRn cDNA, is provided in the N. E. 
Simister and K. E. Mostov paper referenced previously (Nature, 337, pp. 
184-187, 1989). The position selected for insertion of the stop codon 
(after the codon for amino acid 269) was based on similarities between the 
heavy chain subunit, p51, of FcRn and the corresponding domains of the 
Class I MHC proteins, wherein the last residue of the Class I .alpha.3 
domain is residue 274. The FcRn heavy chain cDNA fragment described above 
was used to produce a truncated heavy chain of the FcRn, which associated 
with a .beta.2m light chain to produce a soluble FcRn (pHsFcRn). The full 
amino acid sequence for .beta..sub.2 m light chain is provided in a paper 
by J. Sundelin et al., "The Complete Amino Acid Sequence of Rat 
.beta..sub.2 -microglobulin", Scand. J. Immunol., Vol. 27, pp. 195-199 
(1988). 
The FcRn heavy chain amino acid 269 position is probably not the only 
position at which truncation is possible. Because the histidine amino 
acids of the alpha 3 domain are probably involved in the binding of the Fc 
of IgG, the position of truncation has to preserve these amino acids as 
part of the alpha 3 domain. In the alpha 3 domain, there are 4 histidine 
residues which are located at sequence positions 237, 250, 251 and 258. 
Studies in the laboratory, are in progress to establish the exact role of 
these residues in the Fc binding property of pHsFCRn. However, applicants 
believe that a truncation within the amino acid range of about 260 to 280 
will produce a suitable truncated FcRn heavy chain. 
Because the alpha 3 domain of mHC Class I molecules interacts principally 
with .beta..sub.2 m, and FcRn appears to be structurally analogous to 
Class I MHC molecules, the alpha 3 domain of FcRn probably binds to 
.beta..sub.2 m also. The functionality of FcRn heavy chain appears to be 
dependent upon the presence of an associated .beta..sub.2 m. Thus, the 
functionality of the Fc receptor may be lost if the alpha 3 domain is 
deleted. 
The pHsFcRn was tested and found to have maintained its ability to bind to 
antibodies. In addition, as described in U.S. patent application, Ser. No. 
07/819,040 which was filed simultaneously with the present patent 
application, it is possible to attach the above-described pHsFcRn of the 
present invention to a surface and to use the attached, immobilized 
pHsFcRn to concentrate, isolate or purify antibodies from a mixture 
comprising such antibodies. 
In the specific embodiments of the present invention, the cDNAs for the 
heavy and light chains of membrane-bound FcRn were used as the starting 
materials to produce the pHsFcRn. However, one skilled in the art, in view 
of the present disclosure, should be able to produce a soluble form of a 
different, membrane-bound Fc receptor, providing it has adequate 
similarity in the heavy chain domains. For example, the present method can 
be used to produce pH dependent, soluble Fc receptors from vertebrate (and 
particularly mammalian) transmembrane Fc receptors homologous to FcRn. 
In the specific embodiments of the present invention the expression 
vectors, as illustrated in FIG. 2B, contained the glutamine synthetase 
gene which confers resistance to L-methionine sulfoximine. (The CellTech 
glutamine synthetase-based selection/amplification gene was used to 
produce the desired expression vectors.) However, other 
selection/amplification genes can be expected to perform well also, such 
as the DHFR (dihydrofolate reductase) gene, which confers resistance to 
methotrexate. This latter gene is commonly used for protein expression 
(selection & amplification). 
In the specific embodiments of the present invention, the expression of the 
lipid-linked forms of FcRn and the secreted sFcRn was carried out using 
CHO cells (eukaryotic cells). However, since it has presently been 
discovered that the carbohydrate component of the Fc receptor is not 
involved in Fc binding, the bacterial production (prokaryotic cells) of 
lipid-linked pHFcR and secreted pHsFcR is expected to be feasible. 
Applicants believe expression of the lipid-linked pHFcR and secreted pHsFcR 
of the present invention can be carried out in Eukaryotic cells such as 
CHO cells, Rat2 cells, and 3T3 (mouse cell line), for example and not by 
way of limitation. 
When the sFcR is to be used to concentrate or purify antibodies from 
mixtures comprising the antibodies, it is desirable that the technique for 
concentration or purification be a gentle one. In the case of a 
physiological pHsFcRn, the receptor binds to the Fc portion of antibodies 
at a pH ranging from about 6.0 to about 6.5, and releases the antibodies 
at a pH ranging from about 7.5 to about 8.0. A process operable over this 
pH range is most preferred, since it does not affect the antibody molecule 
in a manner which affects its normal performance as an antibody. The 
preferred pHsFcR of the present invention are those which can bind to the 
Fc portion of at least one kind of immunoglobulin (antibody) at a pH 
ranging from about 5.5 to about 6.5 and can release the Fc portion of the 
immunoglobulin at a pH ranging from about 7.5 to about 8.5. In general, 
the pHsFcR of the present invention are those which can both bind to and 
release from the Fc portion of the antibody over a pH ranging from about 4 
to about 9. The pHsFcR may bind to the antibody at a given pH and release 
at a higher pH or may bind at a given pH and release at a lower pH. 
A method of producing the vertebrate-derived pHsFcR of the present 
invention comprises the following steps: 
(a) creating a gene fragment for a truncated heavy chain of the desired 
vertebrate-derived pHsFcR, said fragment excluding the transmembrane 
domain of said heavy chain; 
(b) forming an expression vector comprising a gene useful as a selectable 
marker and optionally a means of gene amplification, the pHsFcR heavy 
chain gene fragment from step (a), and a gene fragment for the light chain 
compatible with the pHsFcR heavy chain gene fragment of step (a); 
(c) introducing the expression vector of step (b) into a procaryotic or 
eucaryotic cell; 
(d) selecting for a procaryotic or eucaryotic cell comprising the modified 
and optionally amplified cDNAs of the desired vertebrate-derived pHsFcR; 
(e) growing the cell of step (d), from which the desired pHsFcR is 
harvested. 
The pHsFcR is typically secreted from the cell of step (d) into the growth 
medium during the growing process of step (e); however, when secretion 
does not occur, it may be necessary to carry out an additional step (f): 
lysing the cells grown in step (e), whereby the pHsFcR is released from 
the cells. 
An alternative method of producing the vertebrate-derived pHsFcR receptor 
of the present invention pertains to production of a lipid-linked pHFcR, 
wherein the lipid linkage is cleaved, thereby releasing a pHsFcR; this 
method comprises the following steps: 
(a) creating a gene fragment comprising a vertebrate-derived Fc receptor 
truncated heavy chain, the fragment excluding the transmembrane domain of 
the heavy chain but including a signal specifying the attachment of a 
lipid; 
(b) forming an expression vector comprising a gene useful as a selectable 
marker and optionally a means of gene amplification, the step (a) heavy 
chain gene fragment including the lipid attachment signal, and a gene 
fragment for the light chain compatible with the pHFcR heavy chain gene 
fragment of step (a); 
(c) introducing the expression vector of step (b) into a procaryotic or 
eucaryotic cell; 
(d) selecting a procaryotic or eucaryotic cell comprising the modified and 
optionally amplified cDNA of the desired vertebrate-derived pHFcR; 
(e) growing the cell of step (d) from which the desired pHFcR is obtained; 
(f) cleaving the pHFcR lipid linkage of the step (d) cell membrane to 
produce a pHsFcR. 
The above-described method for production of a pHsFcR from a lipid-linked 
pHFcR can also be carried out wherein the signal for attachment of a lipid 
is included in the gene fragment of the light chain and wherein the gene 
fragment for the truncated heavy chain has been constructed to delete the 
DNA sequence encoding the transmembrane domain of the transmembrane 
protein from which it was derived. 
An additional step will typically be used with the three above-described 
methods, wherein: 
separation of the pHsFcR from a growth medium or other solution containing 
contaminating proteins is achieved using an affinity column comprising an 
antibody to which the pHsFcR can bind. 
Typically the compatible light chain used in combination with the truncated 
heavy chain derived from the membrane-bound Fc receptor is a .beta..sub.2 
microglobulin (.beta..sub.2 m). 
The gene fragment for the heavy chain of the desired pHsFcR or pHFcR can be 
introduced into the procaryotic or eucaryotic cell for amplification in 
the same expression vector with the gene fragment for the light chain 
compatible with the heavy chain, as described above. It is also possible 
to introduce the gene fragment for the heavy chain and the gene fragment 
for the compatible light chain into the procaryotic or eucaryotic cell in 
separate expression vectors, so long as the selection and optionally, 
amplification process within the cell is not materially affected.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
The present invention pertains to a composition of matter comprising a 
soluble, vertebrate-derived Fc receptor having a pH-determinable 
capability for binding to the Fc portion of an antibody monomer or a 
complex thereof, this composition has been named a pHsFcR herein. The 
invention also pertains to a method of producing a pHsFcR, since, in the 
known art, Fc receptors which have a pH-determinable capability for 
binding to the Fc portion of an antibody monomer or a complex thereof are 
membrane-bound (transmembrane proteins). The ability of the pHsFcR of the 
present invention to bind to antibodies in a pH-determinable 
(pH-dependent) manner is not affected upon rendering the membrane-bound Fc 
receptor soluble by the method of the present invention. 
The pHsFcR of the present invention can be produced by removing the amino 
acid residues responsible for the attachment of the pHFcR to the cell 
membrane. In the case of secreted pHsFcR this is accomplished at the DNA 
level by eliminating the DNA that encodes the transmembrane domain(s) of 
the protein. In the case of lipid-linked pHFcR, a pHsFcR can be generated 
at the protein level by cleaving the protein polypeptide chain between the 
desired extracellular domains and the amino acids responsible for the 
attachment of the protein to the cell membrane. 
Due to the complexity of macromolecules and their interactions in general, 
it was necessary for the applicants to use the cDNA of an existing, 
pH-determinable, membrane-bound Fc receptor molecule as the starting 
material in the method of producing the soluble Fc receptor of the present 
invention. However, a strategy for secretion of a normally-membrane-bound 
heterodimeric glycoprotein by deletion of the transmembrane region has not 
always been successful. 
Applicants decided to use the cDNA from rat Fc receptor, FcRn, as the 
starting material of the preferred embodiment. Deletion of the 
transmembrane region of the FcRn was attempted through introduction of an 
in-phase translation stop codon. Due to the uncertainty involved, prior to 
construction of a secreted pHsFcRn, two different lipid-linked forms of 
FcRn (pHFcR) were constructed which were likely to be solubilized by 
treatment with PI-PLC to generate pHsFcRn. Subsequently, a cell line that 
secreted a pHsFcR was developed. 
The first form of lipid-linked FcRn was produced by constructing a modified 
FcRn heavy chain in which amino acid residue 269 was followed by the 
lipid-anchoring signal from DAF. The lipid-anchoring signal from DAF is 
shown in FIG. 2A attached to the C-terminus of the truncated p 51 at 212 
and attached to the C-tenninus of.beta..sub.2 -m at 222. This portion of 
the FcRn was expressed together with rat .beta..sub.2 m in CHO cells using 
a glutamine synthetase-based amplifiable expression system. (Residue 269 
is the counterpart of the human MHC-Class I residue 274, which is the last 
residue of the class I .alpha.3 domain.) 
The second lipid-linked form of FcRn was produced by constructing a 
modified rat .beta..sub.2 m in which its C Terminal residue was followed 
by the lipid-anchoring signal from DAF. This portion of the FcRn was 
expressed together with a heavy chain truncated after residue 269. 
Both lipid-linked forms of FcRn were determined to bind labeled rat Fc at 
pH 6.5, but not at pH 8.0, as shown in FIG. 12 at D1-D4. This reproduces 
the physiological pH dependence of Fc binding, confirming that the 
presence of the PI anchor on one of the two chains of the modified FcRn 
heterodimer does not interfere with proper heterodimer formation or Fc 
binding. Diminished binding of rat Fc to lipid-linked heavy chains 
transfected in the absence of rat .beta..sub.2 m, as shown in FIG. 12 at 
D4, suggests that the FcRn molecule is not fully functional in the absence 
of rat .beta..sub.2 m, even when a hamster or bovine .beta..sub.2 m light 
chain is provided for potential association. Thus, in the most preferred 
embodiment of the present invention, the .beta..sub.2 m associated with 
the sFcRn heavy chain is the corresponding wild type .beta..sub.2 m. 
Because the truncated p51 (FcRn heavy chain) was capable of being 
transported to the surface of the cell, and because little or no exchange 
of rat .beta..sub.2 m with bovine or hamster .beta..sub.2 m present in the 
process medium occurred, applicants next attempted to make a secreted, 
soluble form of FcRn. 
As previously disclosed, the starting material described herein for the 
method of producing the more preferred pHsFcR embodiment, secreted pHsFcR, 
was the cDNA for FcRn (the membrane-bound Fc receptor on the intestinal 
epithelial cells of newborn rats). It is understood that the present 
method for producing pHsFcRn can be applied to other membrane-bound 
vertebrate Fc receptors as well. 
The FcRn, found in nature attached to cell membranes is limited in its 
functional uses. The pHsFcRn of the present invention, freed from the cell 
membrane, is available for attachment to any compatible, functional 
surface. 
METHOD FOR PRODUCING SOLUBLE Fc RECEPTOR 
Reagents 
Rat IgG, fluorescein-conjugated rat Fc, and phycoerythrin-conjugated 
F(ab').sub.2 fragments of goat anti-rabbit IgG were from Jackson 
ImmunoResearch. Fluorescein-conjugated goat anti-mouse was from Cappel 
Products. Goat anti-rabbit IgG-peroxidase conjugate and purified rabbit 
anti-human .beta..sub.2 m IgG for Western blots were from Boehringer 
Mannheim. CNBr-activated Sepharose 4B was from Pharmacia. Endoglycosidase 
F/N-glycosidase F was from Boehringer Mannheim. Methionine sulfoximine 
(MSX), phospholipase C, and lentil lectin-Sepharose 4B were from Sigma. 
Dulbecco's modified Eagle's medium (DMEM), e minimum essential medium 
(eMEM), and dialyzed fetal bovine serum were from Irvine Scientific and 
GIBCO/BRL. Anti-p51, a rabbit polyclonal antiserum against the FcRn heavy 
chain was supplied by Neil E. Simister of Brandeis University, Waltham, 
Mass. 2B10C11, a mouse monoclonal antibody against rat .beta..sub.2 m, was 
supplied by Lennart Logdgerg of Sandoz Pharmacological Corporation. The 
pBJ1 and pBJ5 plasmid vectors were obtained from the Mark Davis Laboratory 
at Stanford University. 
It should be mentioned here that the pHsFcR of the present invention, if 
conjugated with a reporter group such as phycoerythrin, fluorescein or 
peroxidase, can be used in place of some of the reagents listed above; eg. 
in place of fluorescein-conjugated goat anti-mouse and goat anti-rabbit 
IgG-peroxidase conjugate. 
EXAMPLE 1 
Method of Producing a Vector for Expression of the cDNA for a Lipid-Linked 
Truncated FcRn Heavy Chain and a Wild-Type .beta..sub.2 m 
Molecular biological experiments were performed by standard methods, as 
described by J. Sambrook, et al. in "Molecular Cloning: A Laboratory 
Manual" (Cold Spring Harbor Lab, Cold Spring Harbor N.Y.) 2nd Ed. (1989). 
The p51-DAF chimera was constructed by methods similar to those used for 
the expression of a lipid-linked form of the T-cell antigen receptor, as 
described by A. Y. Lin et al. in Science, Vol. 249, pp. 677-679 (1990). 
The chimeric protein consisted of the phosphatidyl inositol (PI)-anchoring 
signal of decay-accelerating factor (DAF; residues 311-347; as described 
by I. W. Caras et al. in Science, Vol. 238, pp. 1280-1283 (1987)) fused 
C-terminal to amino acid 269 of the FcRn heavy chain. The sequence for 
this PI-anchoring signal (as given in FIG.2A) is as follows: 
__________________________________________________________________________ 
Pro 
Asn 
Lys 
Gly 
Ser 
Gly 
Thr 
Thr 
Ser 
Gly 
Thr 
Thr 
Arg 
Leu 
Leu 
Ser 
Gly 
His 
Thr 
Cys 
Phe 
Thr 
Leu 
Thr 
Gly 
Leu 
Leu 
Gly 
Thr 
Leu 
Val 
Thr 
Met 
Gly 
Leu 
Leu 
Thr. 
__________________________________________________________________________ 
pBS/p51-DAF, not shown in the FIGS., was constructed as follows: 
(a) The cDNA encoding the DAF PI-anchoring signal, residues 311-347, as 
described by I. W. Caras et al., referenced above, was inserted into the 
polylinker of pBluescript SK(-) at the plasmid's unique EcoR V restriction 
site, using techniques known in the art, to generate the plasmid pBS/DAF. 
Bluescript SK(-) is available from Stratagene. 
(b) A Bam HI fragment comprising the full length FcRn heavy chain cDNA was 
obtained from N. E. Simister of Brandeis University. This cDNA can be 
produced using techniques known in the art. 
(c) pBS/DAF was cleaved using the restriction enzyme Bam HI to generate a 
linearized pBS/DAF. 
(d) The Bam HI DNA fragment from step (b) was ligated to the linearized 
pBS/DAF of step (c) to produce pBS/DAF/p51. 
(e) Oligonucleotide-directed in vitro deletional mutagenesis (of the kind 
described by T. A. Kunkel et al., in Methods Enzymol, Vol. 154, pp. 
367-382 (1987)) was used on pBS/DAF/p51 of step (d) to fuse the DNA 
sequence encoding the PI anchoring signal of DAF to the 3'end of the DNA 
codon encoding amino acid 269 of the FcRn heavychain, producing the 
plasmid pBS/p51-DAF. 
The pBJ5-GS expression vector (schematic shown in FIG. 3) is a derivative 
of pBJ-5, (schematic shown in FIG. 4). pBJ-5 was derived from pBJ-1 
(schematic shown in FIG. 5) by the elimination of the SAL I restriction 
site located between the ampicillin restriction gene and the poly A 
addition signal, using techniques known in the art. pBJ-1 was derived from 
pcDL-SR.alpha.296 (not shown in the FIGS.) in the same manner that 
pBJ1-Neo was derived (described by A. Y. Lin et al., Science, Vol. 249, 
pp. 677-679 (1990)), with the single exception that a neomycin gene was 
not inserted during construction of the PBJ1 plasmid. The pBJ-1 and pBJ-5 
plasmid vectors were obtained from the Mark Davis laboratory at Stanford 
University. 
pBJ5-GS, shown in FIG. 3, was constructed as follows: 
(a) The CellTech vector pSVLGS.1, (schematic shown in FIG. 6) was cleaved 
using the restriction enzymes Aat II and BAM HI to generate a DNA fragment 
comprising the GS minigene. pSVLGS.1 is available from Celltech Limited, 
Berkshire, U.K. 
(b) The single-stranded DNA tails on the ends of the GS minigene fragment 
described above were treated using 4-dNTP (dATP, dGTP, dTTP and dCTP 
combined) and T.sub.4 DNA ligase to form blunt ends, using techniques 
known in the art. 
(c) pBluescript SK(-) was cleaved with Xho I and this linearized plasmid 
was treated with 4-dNTP and T.sub.4 ligase to form blunt ends, using 
techniques known in the art. 
(d) The blunt-ended GS minigene fragment from step (b) was ligated to the 
blunt-ended pBluescript SK(-) from step (c) to produce a circular plasmid 
pBS/GS (not shown in the FIGS.), using techniques known in the art. 
(e) The pBS/GS plasmid from step (d) was cleaved using the restriction 
enzymes Sal I and Xho I to generate a DNA fragment comprising the GS 
minigene, using techniques known in the art. 
(f) The pBJ5 vector described above (schematic shown in FIG.4) was 
linearized by cutting it with the Sal I restriction enzyme. 
(g) The GS minigene fragment from step (e) was ligated to the linearized 
pBJ5 vector from step (f) to produce the circular expression vector 
pBJ5-GS shown in FIG. 3. pBJ5-GS employs the glutamine synthetase gene as 
a selectable marker and means of gene amplification in the presence of the 
drug MSX, a system developed by C. R. Bebbington et al. of Celltech and 
described in DNA Cloning, ed. D. M. Glover IRL, Oxford, Vol. 3chapter 8, 
p. 163 (1987). 
pBJ5-GS/p51-DAF (schematic shown in FIG. 7) was constructed as follows: 
(a) The p51-DAF cDNA was cleaved from the pBS/p51-DAF described above using 
Xho I and Not I restriction enzymes, using techniques known in the art. 
(b) The circular pBJ5-GS, expression vector shown in FIG. 3, was linearized 
using Xho I and Not I restriction enzymes, using techniques known in the 
art. 
(c) The p51-DAF cDNA of step (a) was then ligated into the linearized 
pBJ5-GS from step (b), using techniques known in the art, to produce the 
expression vector pBJ5-GS/p51-DAF shown in FIG. 7. 
PBJ1/.beta..sub.2 m (schematic shown in FIG. 8) was constructed as follows: 
(a) A Bam HI fragment containing the full length rat .beta..sub.2 m cDNA 
was obtained from N. E. Simister of Brandeis University. This cDNA can be 
produced using techniques known in the art. 
(b) pBluescript SK(-) was cleaved using BAM HI, using techniques known in 
the art. 
(c) The BAMHI fragment described in (a) was ligated to the linearized 
pBluescript SK(-) of step (b) to produce pBS.beta..sub.2 m (not shown in 
the FIGS), using techniques known in the art. 
(d) pBS/.beta..sub.2 m was cleaved using the restriction enzymes Not I and 
Xho I to generate a DNA fragment comprising the full length rat 
.beta..sub.2 m cDNA, using techniques known in the art. 
(e) pBJ1 (schematic shown in FIG. 5) was cleaved using restriction enzymes 
Xho I and Not I, using techniques known in the art. 
(f) The DNA fragment from step (d) was ligated to the linearized pBJ1 of 
step (e) to produce pBJ1/.beta..sub.2 m, shown in FIG. 8, using techniques 
known in the art. 
The expression vector pBJ5-GS/p51-DAF/.beta..sub.2 m (schematic shown in 
FIG. 2B, wherein cDNAs p51-DAF and .beta..sub.2 m are inserted) was 
constructed as follows: 
(a) The pBJ5-GS/p51-DAF, shown in FIG. 7, was cleaved using the restriction 
enzyme Sal I, using techniques known in the art. 
(b) The pBJ1/.beta..sub.2 m, shown in FIG. 8, was cleaved using the 
restriction enzyme Sal I to generate a DNA fragment comprising the full 
length rat .beta..sub.2 m cDNA, the SRe promoter, and the poly A addition 
signal, using techniques known in the art. 
(c) The DNA fragment from step (b) was ligated to the linearized 
pBJ5-GS/p51-DAF from step (a) to produce circular expression vector 
pBJ5-GS/p51-DAF/.beta..sub.2 m, using techniques known in the art. 
EXAMPLE 2 
Method of Producing a Vector for Expression of the eDNA for a Lipid-Linked 
.beta..sub.2 m and a Truncated FcRn Heavy Chain 
Molecular biological experiments were performed by standard methods, as 
described by J. Sambrook, et al. in "Molecular Cloning: A Laboratory 
Manual" (Cold Spring Harbor Lab, Cold Spring Harbor N.Y.) 2nd Ed. (1989). 
The .beta..sub.2 m-DAF chimera was constructed by methods similar to those 
used for the expression of a lipid-linked form of the T-cell antigen 
receptor, as described by A. Y. Lin et al. in Science, Vol. 249, pp. 
677-679 (1990). The chimeric protein consisted of the phosphatidyl 
inositol (PI)-anchoring signal of decay-accelerating factor (DAF; residues 
311-347; as described by I. W. Caras et al. in Science, Vol. 238, pp. 
1280-1283 (1987)) fused C-terminal to the final amino acid 99 of 
.beta..sub.2 m. 
pBS/.beta..sub.2 m-DAF, not shown in the FIGS., was constructed as follows: 
(a) A Bam HI fragment comprising the full length .beta..sub.2 m light chain 
cDNA was obtained from N. E. Simister of Brandeis University. This cDNA 
can be produced using techniques known in the art. 
(b) pBS/DAF produced as described in EXAMPLE 1, was cleaved using the 
restriction enzyme Bam HI to generate a linearized pBS/DAF. 
(c) The Bam HI DNA fragment from step (a) was ligated to the linearized 
pBS/DAF of step (b) to produce pBS/DAF/.beta..sub.2 m. 
(d) Oligonucleotide-directed in vitro deletional mutagenesis (of the kind 
described by T. A. Kunkel et al., in Methods Enzymol, Vol. 154, pp. 
367-382 (1987)) was used on pBS/DAF/.beta..sub.2 m of step (d) to fuse the 
DNA sequence encoding the PI anchoring signal of DAF to the 3'end of the 
DNA codon encoding amino acid 269 of the .beta..sub.2 m light chain, 
producing the plasmid pBS/.beta..sub.2 m-DAF. 
pBJ5-GS/.beta..sub.2 m-DAF (schematic shown in FIG. 9), was constructed as 
follows: 
(a) The .beta..sub.2 m-DAF cDNA was cleaved from pBs/.beta..sub.2 m-DAF 
using Xho I and Not I restriction enzymes, using techniques known in the 
art. 
(b) The circular pBJ5-GS expression vector, shown in FIG. 3, was linearized 
using Xho I and Not I restriction enzymes, using techniques known in the 
art. 
(c) The .beta..sub.2 m-DAF cDNA of step (a) was then ligated into the 
linearized pBJ5-GS of step (b), using techniques known in the art, to 
produce the pBJ5-GS/.beta..sub.2 m-DAF shown in FIG. 9. 
pBS/p51-stop (not shown in FIGS.) was constructed as follows: 
(a) A Bam HI fragment comprising the full length FcRn heavy chain cDNA was 
obtained as previously described. 
(b) pBluescript SK(-) was cleaved using the restriction enzyme Bam HI to 
generate a linearized pBluescript SK(-). 
(c) The Bam HI DNA fragment of step (a) was ligated to the linearized 
pBluescript SK(-) of step (b), using techniques known in the art, to 
produce pBS/p51 (not shown in the FIGS.). 
(d) Oligonucleotide-directed in vitro deletional mutagenesis (of the kind 
described by T. A. Kunkel et al.) was used on pBS/p51 of step (c) to 
insert a translational stop codon immediately 3' of the codon encoding 
amino acid 269 of the FcRn heavy chain, producing the plasmid pBS/p51-stop 
(comprising the modified FcRn heavy chain cDNA, p51-stop). 
pBJ1/p51-stop, shown in FIG. 10, was constructed as follows: 
(a) The pBS/p51-stop described above was cleaved using the restriction 
enzymes Not I and Xho I to generate a DNA fragment comprising the p51-stop 
cDNA, using techniques known in the art. 
(b) pBJ1, shown in FIG. 5, was cleaved using restriction enzymes Xho I and 
Not I using techniques known in the art. 
(c) The DNA fragment from step (a) was ligated to the linearized pBJ1 of 
step (b) to produce pBJ1/p51-stop, shown in FIG. 10, using techniques 
known in the art. 
The expression vector .pBJ5-GS/p51-stop/.beta..sub.2 m-DAF (shown in FIG. 
2B, wherein cDNAs .beta..sub.2 m-DAF and p51-stop are inserted) was 
constructed as follows: 
(a) The pBJ5-GS/.beta..sub.2 m-DAF, shown in FIG. 9, was cleaved using the 
restriction enzyme Sal I, using techniques known in the art. 
(b) The pBJ1/p51-stop, shown in FIG. 10, was cleaved using the restriction 
enzyme Sal I to generate a DNA fragment comprising the p51-stop cDNA, the 
SRa promoter, and the poly A addition signal, using techniques known in 
the art. 
(c) The DNA fragment from step (b) was ligated to the linearized 
pBJ5-GS/.beta..sub.2 m-DAF from step (a), using techniques known in the 
art, to produce circular expression vector pBJ5-GS/p51-stop/.beta..sub.2 
m-DAF. 
EXAMPLE 3 
Method of Producing a Vector for Expression of the cDNA for a Truncated 
FcRn Heavy Chain and a Wild-Type .beta..sub.2 m 
Molecular biological experiments were performed by standard methods, as 
described by J. Sambrook, et al. in "Molecular Cloning: A Laboratory 
Manual" (Cold Spring Harbor Lab, Cold Spring Harbor N.Y.) 2nd Ed. (1989). 
pBJ5-GS/p51-stop (schematic shown in FIG. 11), was constructed as follows: 
(a) pBS/p51-stop prepared as described in Example 2 above, was cleaved 
using the restriction enzymes Xho I and Not X to generate a DNA fragment 
comprising the p51-stop cDNA, using techniques known in the art. 
(b) The pBJ5-GS prepared as described in Example 1 above and shown in FIG. 
3, was linearized using Xho I and Not I restriction enzymes, using 
techniques known in the art. 
(c) The DNA fragment from step (a) was ligated to the linearized pBJ5-GS 
from step (b) to produce pBJ5-GS/p51-stop as shown in FIG. 11, using 
techniques known in the art. 
pBJ5-GS/p51-stop/.beta..sub.2 m (schematic shown in FIG. 2B, wherein cDNAs 
p51-stop and .beta..sub.2 m are inserted) was constructed as follows: 
(a) pBJ1/.beta..sub.2 m produced as described in Example 1 and shown in 
FIG. 8 was cleaved using the Sal I restriction enzyme to generate a DNA 
fragment comprising the full length .beta..sub.2 m eDNA, the SRe promoter 
and the poly A addition signal, using techniques known in the art. 
(b) pBJ5-GS/p51-stop produced as described above and shown in FIG. 11 was 
cleaved using the Sal I restriction enzyme, using techniques known in the 
art. 
(c) The DNA fragment from step (a) was ligated to the linearized 
pBJ5-GS/p51-stop from step (b), using techniques known in the art, to 
produce the circular expression vector pBJ5-GS/p51-stop/.beta..sub.2 m. 
EXAMPLE 4 
Production of the Three FcRn Forms in CHO Cells 
Each of the three expression vectors described in Examples 1-3 was 
individually transfected into CHO (Chinese Hamster Ovary) cells by the 
following calcium phosphate procedure (Stratagens). The CHO cell line was 
CHO-K1, obtained from ATCC (American Type Culture Collection), catalog 
number CCL61. A calcium phosphate/DNA precipitate containing 30 .mu.g of 
pure DNA, or no DNA as a mock control, was added to CHO cells in 10 ml of 
fresh DMEM with serum. Selection and amplification of the glutamine 
synthetase gene were carried out according to the following protocol. The 
next day, the cells were washed three times with eMEM without serum and 
then incubated in a .alpha.MEM with 10% dialyzed fetal bovine serum and 25 
.mu.M MSX. Viable CHO cells containing the transfected expression vectors 
described above were visible after two weeks and were isolated and grown 
(using standard techniques known in the art for CHO cell growth) in 24 
well plates in eMEM with a 10% dialyzed fetal bovine serum and 25 .mu.M 
MSX. The isolation and growth of viable cells was repeated to produce 
several distinct homogeneous populations of transfected cells (clones of 
cells). 
Clones transfected with the two lipid-linked forms of FcRn were tested for 
expression of both protein subunits by immunofluorescence with anti-p51 
and 2B10C11. Clones expressing the desired lipid-linked form of FcRn were 
put in six-well plates and submitted to increasing MSX concentrations from 
50 to 500 .mu.M, to select for cells in which the transfected genes had 
been amplified. 
Clones expressing the desired lipid-linked form of FcRn were also tested 
for their ability to differentially bind rat Fc at pH 6.5 as follows: 106 
cells were incubated in suspension for 1 hour at room temperature in 500 
.mu.l of phosphate-buffered saline (pH 6.5 or pH 8.0) with 
fluorescein-conjugated rat Fc (0.5 .mu.M) and then washed twice. The cell 
pellet was resuspended in 1 ml of buffer at the appropriate pH and 
analyzed by flow cytometry using an Ortho cell sorter available from Ortho 
Pharmaceutical Company of Raritan, New Jersey (U.S.A.). 
Supernatants from CHO clones transfected by pBJ5-GS/p51-stop/.beta..sub.2 m 
(the secretable, soluble form of FcRn) were tested for secreted FcRn 
heterodimer by Western blotting with anti-p51 and a rabbit anti-human 
.beta..sub.2 m antiserum (crossreactive with rat .beta..sub.2 m). Clones 
expressing the secreted, soluble form of FcRn were put in six-well plates 
and submitted to increasing MSX concentrations from 50 to 500 .mu.M, to 
select for cells in which the transfected genes had been amplified. 
Phospholipase C Treatment of Clones Expressing the Lipid-Linked Form of 
FcRn 
Cleavage of lipid-linked proteins was done as described by A. Y. Lin et 
al., referenced above, but using a stock of phospholipase C containing 
PI-PLC (Sigma P6135; 1 mg/ml), which was added to 107 cells at a 100-fold 
dilution. Cells were incubated for 2 hours at 37.degree. C. in an 
atmosphere having controlled humidity and carbon dioxide content, within 
the standard technology ranges. The above cleavage reaction produced a 
soluble pHsFcRn, derived from each lipid-linked form of FcRn, which 
soluble pHsFcR could be harvested via the purification technique described 
below. 
Purification of pHsFcRn by Affinity Chromatography and Biochemical Analysis 
Rat IgG (70 mg) was covalently linked to 7 ml of CNBr-activated Sepharose 
(10 mg/ml) according to the manufacturer's directions. The pH of 100-500 
ml of either supernatant from a CHO clone secreting sFcRnheterodimer 
growing in 100 .mu.M MSX or cleavage reaction solution containing pHsFcR 
was decreased to pH 6.5 and put on the column at a constant flow rate of 
20 ml/hr at 4.degree. C., and the column was washed with 300 ml of 50 mM 
sodium phosphate, pH 6.5/0.05% NaN.sub.3. Elution of pHsFcRn was initiated 
with 50 mM sodium phosphate, pH 8.0/0.05% NaN.sub.3. Fractions of 2 ml 
were collected and their optical density at 280 nm measured. Typically, 
.apprxeq.10 mg of secreted pHsFcRn heterodimer [quantified by 
bicinchoninic acid (BCA) assay; Pierce] was eluted in six fractions from 
250 ml of supernatant harvested from 10.sup.8 -10.sup.9 cells. The protein 
was concentrated by Centricon 10000 concentration devices available from 
Amicon Corp. 
pHsFcR derived from lipid-linked FcRn was also obtained using the 
above-described purification technique and was evaluated along with the 
secreted pHsFcR as described below. 
EXAMPLE 5 
ANALYTICAL DATA CONFIRMING THE PRODUCTION OF SOLUBLE FcRn (pHsFcRn) WAS 
OBTAINED AS PRESENTED BELOW 
N-Terminal Sequencing. 
N-terminal sequencing was performed on 20-40 .mu.g of purified secreted 
pHsFcRn in a phosphate buffer dried on a poly(vinylidene difluoride) 
membrane (in the manner described by P. Matsudaira, J. Biol. Chem. Vol. 
262, pp. 10035-10038 (1987) and inserted into an Applied Biosystems model 
4778 sequencer reaction cartridge. CD Spectra. 
A Jasko J-600 spectropolarimeter was used in the wavelength range of 190 to 
260 nm with a 0.1 mm cell. Purified HLA-B40 (A Class I MHC molecule) 
(supplied by Don Wiley and Anastasia Haykov, of Harvard University, 
Cambridge, Mass.,) and secreted pHsFcRn heterodimer were concentrated to 
0.1-0.5 mg/ml in 20 mM sodium phosphate (pH 8.0). The percent helix, 
.beta.-strand, and disordered structure (random) was estimated by fitting 
the spectra to reference data (from N. Greenfield et al., Biochemistry, 
Vol. 8, pp. 4108-4115 (1969) with a nonrestrained least-squares algorithm. 
Crystallization of Secreted pHsFcRn. 
Crystals were grown by vapor diffusion from protein solutions (20 mg/ml) in 
10 mM Pipes, pH 6.5/0.05% NAN.sub.3, in 2-.mu.l droplets with 24.0% 
(wt/vol) polyethylene glycol 3350, 2.0% (wt/vol) saturated ammonium 
sulfate, and 100 mM Pipes (pH 6.5). 
SUMMARY OF RESULTS FROM EXAMPLES 1 THROUGH 5 
Expression of Two Lipid-Linked (membrane-bound) Forms of FcRn. 
An expression vector, as illustrated in FIG. 2B, containing the hamster 
glutamine synthetase gene 252 (for selection and amplification), the gene 
encoding the extracellular portion of the FcRn heavy chain fused in-frame 
to the cDNA from DAF encoding its lipid attachment signal (p51-DAF), and 
the rat .beta..sub.2 m gene was transfected into CHO cells. Stable lines 
were generated and the transfected gene was amplified by selection with 
MSX. (Cell surface expression of FcRn heavy and light chains were detected 
by immunostaining with anti-p51 and anti-rat .beta..sub.2 m antibodies as 
shown in FIG. 12 at B1 and B2; and the amount of rat .beta..sub.2 m 
detected was reduced after treatment with PI-PLC, as illustrated in FIG. 
12 at B2 (- - -). CHO cells expressing these lipid-linked FcRn 
heterodimers bound rat Fc. The physiological pH dependence of Fc binding 
observed in nature was reproduced, in that binding was observed at pH 6.5 
but not at pH 8.0., as illustrated in FIG. 3 at D2 and D3, wherein-- 
represents pH 6.5, and - - - - represents pH 8. Binding of labeled Fc was 
inhibited by addition of unlabeled rat IgG or rat Fc at 100 .mu.g/ml, but 
not by the equivalent amount of an unrelated protein. Expression of the 
lipid-linked FcRn heavy chain in the absence of rat .beta..sub.2 m was at 
a level comparable to the expression in the presence of rat .beta..sub.2 m 
(data not shown), but the binding of fluorescein-conjugated rat Fc was 
diminished, as illustrated in FIG. 12 at D4. 
The second lipid-linked form of FcRn was expressed by transfecting a 
plasmid containing the DNA encoding the DAF PI-attachment signal fused to 
the rat .beta..sub.2 m gene, together with the gene encoding the FcRn 
heavy chain truncated with an in-frame stop codon after amino acid 269 
(p51-stop), and the glutamine synthetase gene, as illustrated in FIG. 2B. 
Stable CHO lines expressing this lipidlinked form of FcRn were generated 
by selection with MSX and stained with the anti-p51 and anti-rat 
.beta..sub.2 m antibodies as shown in FIG. 12 at C1 and C2; and the amount 
of rat .beta..sub.2 m detected was reduced after treatment with PI-PLC, as 
illustrated in FIG. 12 at C2 (- - -). The FcRn/.beta..sub.2 m heterodimers 
also showed the expected pH dependence of Fc binding, as illustrated in 
FIG. 3 at D3. Expression levels of the two forms of lipid-linked FcRn 
heterodimers appeared comparable, as illustrated by comparison of FIG. 3 
at B1 and C1. 
Expression of Secreted sFcRn 
An expression vector (FIG. 2B) containing the glutamine synthetase gene, 
the truncated FcRn heavy chain gene, and the rat .beta..sub.2 m gene was 
transfected into CHO cells. Media collected from individual clones were 
assayed for secretion of pHsFcRn heterodimers by Western blotting aliquots 
of supernatants with antibodies specific for the heavy and light chains. 
Positive clones were amplified with increasing amounts of MSX. Medium (250 
ml) from a high-expressing clone growing in 100 .mu.M MSX was adjusted to 
pH 6.5 and passed over a rat IgG affinity column. The column was 
extensively washed, and pHsFcRn heterodimers were eluted by changing the 
pH to 8.0, yielding .apprxeq.10 mg of purified protein. The 
high-expressing CHO clone was grown in a Cell Pharm II hollow-fiber 
bioreactor device (Unisyn Fibertec) in the presence of 100 .mu.M MSX. The 
yield of soluble pHsFcRn was typically 7-10 mg per daily harvest. 
Purified secreted pHsFcRn was analyzed by SDS/17.5% PAGE, as illustrated in 
FIG. 13. FIG. 13 shows SDS/17.5% PAGE comparison of purified secreted 
pHFcRn before (lanes a and c) and after (lanes b and d) treatment with 
endoglycosidase F/N-glycosidase F. Samples were run under nonreducing 
(lanes a and b) or reducing (lanes c and d) conditions. Under either 
reducing or nonreducing conditions, two bands were detected: a sharp band 
of apparent molecular mass 13 kDa, corresponding to .beta..sub.2 m, and a 
broad diffuse band at .apprxeq.43 kDa, corresponding to the truncated FcRn 
heavy chain. Treatment of purified pHsFcRn with a mixture of 
endoglycosidase F and Nglycosidase F had no effect on the apparent 
molecular mass of .beta..sub.2 m, but the majority of the heavy chain 
shifted its position of migration to 30 kDa, in close agreement with the 
predicted molecular mass of the unmodified truncated heavy chain (30,274 
Daltons). These data suggest that .apprxeq.13 kDa of the extra molecular 
mass of the truncated FcRn heavy chain was due to N-linked glycosides, a 
figure that is not inconsistent with the utilization of all four potential 
N-linked glycosylation sites in the FcRn heavy chain sequence. 
Deglycosylated pHsFcRn retained its ability to bind to the Fc affinity 
column (data not shown), suggesting that the pHsFcRn carbohydrate moieties 
were not involved in the interaction between the Fc Receptor and Fc. This 
functionality in the absence of the heavy chain carbohydrates suggests 
that it could be possible to produce most of (if not all of) normal 
functional pHFcR in procaryotic and eukaryotic cell lines that fail to add 
carbohydrate to newly synthesized proteins. 
Purified pHsFcRn was subjected to N-terminal sequence analysis to verify 
the origin of the .beta..sub.2 m species associated with the pHsFcRn heavy 
chain, and to look for possible peptides associated with the FcRn heavy 
chain. The first 16 residues of bovine .beta..sub.2 m (as described by M. 
L. Groves et al., J. Biol. Chem., Vol. 257, pp. 2619-2626 (1982) and rat 
.beta..sub.2 m (as described by J. Sundelin et al., Scand. J. Immunol., 
Vol. 27, pp. 195-199 (1988) differ at amino acid residues 3, 4 and 6 (of 
the mature protein), and the sequences of hamster (as determined by 
applicants) and rat .beta..sub.2 m differ at residues 3, 4, 7 and 11. Two 
N-terminal sequences in equimolar amounts were identified in the soluble 
pHsFcRn sample: the sequence AEPRLPLMYHLAAVSD(SEQ. I.D. NO. 1), 
corresponding to the first 16 amino acids of the mature FcRn heavy chain 
as described by N. E. Simister et al., Cold Springs Harbor Symp. Quant. 
Biol. referenced above, and the sequence IQKTPQIQVYSRHPPE(SEQ. I.D. NO. 
2), corresponding to the sequence of the first 16 residues of mature rat 
.beta..sub.2 m (J. Sundelin et al. as referenced above). No evidence of 
sequences corresponding to bound peptides was seen. 
CD Spectral Comparison of pHsFcRn and MHC Class I Proteins. 
Analysis of CD spectra of HLA-B40 (a human Class I MHC molecule) and 
soluble pHsFcRn (as illustrated in FIG. 14) by a nonrestrained least 
squares fitting procedure indicated a dominant .beta.-strand component 
with a minor helical contribution, similar to that reported previously for 
HLA-B7 and HLA-A2, although lower amounts of .beta.-structure were 
predicted for other class I MHC molecules from CD spectral analyses. The 
percent helix estimated from our analysis of the spectrum of HLA-B40 
agrees well with the amount helix found in Class I MHC crystal structures 
(.apprxeq.20% helix); however, our CD spectral data predicts higher .beta. 
structure content (.congruent.60% compared with previous estimates of 
.congruent.42%). 
FIG. 14 shows Far-UV CD spectra of secreted pHsFcRn and HLA-B40 expressed 
as ellipticity per mean residue. Comparison of the data shows % .alpha. 
pHsFcRn=15, with % .alpha. HLA-B40=20; % .beta. of pHsFcRn=85, with % 
.beta.HLA-B40=70; and % random of pHsFcRn=0, with % random of HLA-B40=10. 
The correlation coefficient for secreted pHsFcRn=0.988 and the correlation 
coefficient for HLAB-40=1.0. It is important to mention that the CD 
spectral information presented herein is consistent within itself but may 
not be consistent with information generated by others skilled in the art 
due to subjectivity in the methods of analyzing data. 
Crystals of Secreted pHsFcRn. 
Soluble, secreted pHsFcRn protein formed crystals of approximate dimensions 
0.3 mm.times.0.1 mm.times.0.1 mm in space group C222.sub.1. The unit cell 
dimensions were a=126.4 .ANG., b=191.7 .ANG., and c=149.6 .ANG.. The 
asymmetric unit of the crystal was estimated to contain two to four 
molecules based on average volume to mass ratios (V.sub.m) of protein 
crystals (as described by B. W. Mathews, J. Mol. Biol. Vol. 33, pp. 
491-497 (1968)), representing solvent contents between 73% (if two 
molecules per asymmetric unit) to 32% (if four molecules per asymmetric 
unit). The crystals diffracted to 3.5 .ANG.resolution using 
nickel-filtered CuK.alpha. radiation from a rotating-anode x-ray 
generator. Single crystals of a complex of a complex between pHsFcRn and 
rat Fc have also been obtained by A. H. Huber and the applicants. 
Additional General Information 
As previously discussed, pertaining to production of secreted pHsFcRn, The 
gene encoding the truncated FcRn heavy chain was transfected together with 
the rat .beta..sub.2 m gene, using the Celltech glutamine synthetase-based 
amplifiable expression system. With this expression system, a high level 
of expression can be obtained after an initial selection, and gene 
amplification is rapidly achieved. Clones secreting soluble FcRn 
heterodimer were evaluated for relative expression levels by analysis of 
supernatant samples on Western blots, and a high-expressing clone 
secreting FcRn at 40 mg/liter was selected at 100 .mu.M MSX. FcRn was 
purified on a rat IgG affinity column by passing supernatants over the 
column at pH 6.5 and eluting protein at pH 8.0. This purification scheme 
is gentle, taking advantage of the physiological pH dependence of Fc 
binding, thereby avoiding the harsh elution conditions typically necessary 
for elution from immunoaffinity columns. 
SDS/PAGE analysis of purified, secreted pHsFcRn showed two bands 
corresponding to truncated heavy chain and .beta..sub.2 m. No evidence of 
covalent dimerization of the pHsFcRn heavy chain mediated by a disulfide 
bond was seen by comparison of the mobility under reducing and nonreducing 
conditions, and purified pHsFcRn was eluted from a gel filtration column 
at the position expected for a complex of a single heavy and light chain. 
Microsequencing of pHsFcRn heterodimer revealed the expected N-terminal 
residues of the heavy chain and rat .beta..sub.2 m in equimolar amounts, 
suggesting that .beta..sub.2 m exchange with bovine .beta..sub.2 m in the 
medium or hamster .beta..sub.2 m inside the CHO cells was either minimal 
or did not occur. 
Because the structurally related Class I MHC molecules are transported to 
the cell surface with peptides derived from intracellular proteins and 
appear to depend upon the presence of bound peptide for structural 
stability, it was of interest whether secreted pHsFcRn heterodimers showed 
evidence of bound peptide. No other sequences were detectable, suggesting 
that the pHsFcRn heterodimer was not complexed with a unique peptide. A 
mixture of peptides would probably not have been detected and cannot be 
ruled out as a possibility. 
Samples of secreted pHsFcRn and the human Class I MHC molecule HLA-B40 were 
analyzed by CD spectroscopy (FIG. 14). Analysis of CD spectra of the Class 
I molecule was reasonably consistent with the known Class I x-ray 
structures and with previously reported CD spectra, as described by D. 
Lancet et al. in Proc. Natl. Acad. Sci. U.S.A., Vol.76, pp.3844-3848, 
(1979), and by J. C. Gorga, et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 
86, pp. 2321-2325 (1989), suggesting that the molecule is primarily 
composed of .beta.-structure with a minor .alpha.-helical contribution. 
The CD spectrum of secreted pHsFcRn appears similar, which together with 
the primary sequence similarity further suggests that the two types of 
molecules may adopt similar tertiary structures. 
The above-described preferred embodiments of the present invention are not 
intended to limit the scope of the present invention as demonstrated by 
the claims which follow, as one skilled in the art can, with minimal 
experimentation, extend the principles of the invention to the claimed 
scope of the invention. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 9 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acid residues 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: Protein/Peptide 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: 
(v) FRAGMENT TYPE: N-terminal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Rattus norvegious 
(B) STRAIN: Wistar 
(C) INDIVIDUAL ISOLATE: 
(D) DEVELOPMENTAL STAGE: 11 day- old/germ-line 
(E) HAPLOTYPE: 
(F) TISSUE TYPE: Proximal third of small intestine 
(G) CELL TYPE: Epithelial cells 
(H) CELL LINE: 
(I) ORGANELLE: 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: 
(B) CLONE: 
(viii) POSITION IN GENOME: 
(A) CHROMOSOME/SEGMENT: 
(B) MAP POSITION: 
(C) UNITS: 
(ix) FEATURE: 
(A) NAME/KEY: This sequence contains the first 16 amino acids 
of the mature FcRn heavy chain. 
(B) LOCATION: FcRn heavy chain (a.a. 1 to 16) from 1 to 16. 
(C) IDENTIFICATION METHOD: Purified, soluble FcRn was 
subjected to N-terminal sequence analysis. 
(D) OTHER INFORMATION: This sequence was determined to verify 
that the secreted, soluble FcRn heavy chain had the same 
amino terminus as the wild-type transmembrane FcRn heavy 
chain. 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Simister, N.E. 
Mostov, K.E. 
(B) TITLE: An Fc receptor structurally related to MHC class 
I antigens. 
(C) JOURNAL: Nature 
(D) VOLUME: 337 
(E) ISSUE: 
(F) PAGES: 184-187 
(G) DATE: 12-Jan-1989 
(H) DOCUMENT NUMBER: 
(I) FILING DATE: 
(J) PUBLICATION DATE: 
(K) RELEVANT RESIDUES IN SEQ ID NO:1: FROM 1 TO 16. 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Simister, N.E. 
Mostov, K.E. 
(B) TITLE: Cloning and Expression of the Neonatal Rat 
Intestinal Fc Receptor, a Major Histocompatability 
Complex Class I Antigen Homolog. 
(C) JOURNAL: Cold Spring Harbor Symposia on Quantitative 
Biology 
(D) VOLUME: 54 
(E) ISSUE: 
(F) PAGES: 571-580 
(G) DATE: 1989 
(H) DOCUMENT NUMBER: 
(I) FILING DATE: 
(J) PUBLICATION DATE: 
(K) RELEVANT RESIDUES IN SEQ ID NO:1: FROM 1 TO 16 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
AlaGluProArgLeuProLeuMetTyrHisLeuAlaAlaValSerAsp 
151015 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acid residues 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: Protein/Peptide 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: 
(v) FRAGMENT TYPE: N-terminal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Rattus norvegious 
(B) STRAIN: Sprague-Dawley 
(C) INDIVIDUAL ISOLATE: 
(D) DEVELOPMENTAL STAGE: 
(E) HAPLOTYPE: 
(F) TISSUE TYPE: 
(G) CELL TYPE: 
(H) CELL LINE: 
(I) ORGANELLE: 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: 
(B) CLONE: 
(viii) POSITION IN GENOME: 
(A) CHROMOSOME/SEGMENT: 
(B) MAP POSITION: 
(C) UNITS: 
(ix) FEATURE: 
(A) NAME/KEY: This sequence contains the first 16 amino acids 
of the mature protein rat beta 2-microhlobulin. 
(B) LOCATION: Rat beta 2- microglobulin (a.a. 1 to 16) from 1 
to 16. 
(C) IDENTIFICATION METHOD: Purified, soluble FcRn was 
subjected to N-terminal sequence analysis. 
(D) OTHER INFORMATION: This sequence was determined to verify 
that the secreted, soluble FcRn heterodimer consisted of 
FcRn heavy chain and rat beta 2-microhlobulin at a 
stoichiometry of 1:1. 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Sundelin, J. 
Bjorck, L. 
Logdberg, L. 
(B) TITLE: The Complete Amino Acid Sequence of Rat beta 2- 
Microglobulin. 
(C) JOURNAL: Scand. J. Immunol. 
(D) VOLUME: 27 
(E) ISSUE: 
(F) PAGES: 195-199 
(G) DATE: 1988 
(H) DOCUMENT NUMBER: 
(I) FILING DATE: 
(J) PUBLICATION DATE: 
(K) RELEVANT RESIDUES IN SEQ ID NO:2: FROM 1 TO 16 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
IleGlnLysThrProGlnIleGlnValTyrSerArgHisProProGlu 
151015 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: Protein/Peptide 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: 
(v) FRAGMENT TYPE: Internal fragment 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Rattus norvegious 
(B) STRAIN: 
(C) INDIVIDUAL ISOLATE: 
(D) DEVELOPMENTAL STAGE: 11 day- old/germ-line 
(E) HAPLOTYPE: 
(F) TISSUE TYPE: Proximal third of small intestine 
(G) CELL TYPE: Epithelial cells 
(H) CELL LINE: 
(I) ORGANELLE: 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: 
(B) CLONE: 
(viii) POSITION IN GENOME: 
(A) CHROMOSOME/SEGMENT: 
(B) MAP POSITION: 
(C) UNITS: 
(ix) FEATURE: 
(A) NAME/KEY: A 14 amino acid internal fragment of the 
wild-type FcRn heavy chain. 
(B) LOCATION: FcRn heavy chain (a.a. 265 to 278) from 1 to 14. 
(C) IDENTIFICATION METHOD: Sequence as reported by N.E. 
Simister and K.E. Mostov in Nature 337, 184-187 (1989). 
(D) OTHER INFORMATION: This wild- type sequence was referenced 
so that the reader could compare the modified FcRn heavy 
chain sequences to that of the wild-type. 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Simister, N.E. 
Mostov, K.E. 
(B) TITLE: An Fc receptor structurally related to MHC class I 
antigens. 
(C) JOURNAL: Nature 
(D) VOLUME: 337 
(E) ISSUE: 
(F) PAGES: 184-187 
(G) DATE: 12-Jan-1989 
(H) DOCUMENT NUMBER: 
(I) FILING DATE: 
(J) PUBLICATION DATE: 
(K) RELEVANT RESIDUES IN SEQ ID NO:3: FROM 1 TO 16. 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Simister, N.E. 
Mostov, K.E. 
(B) TITLE: Cloning and Expression of the Neonatal Rat 
Intestinal Fc Receptor, a Major Histocompatability 
Complex Class I Antigen Homolog. 
(C) JOURNAL: Cold Spring Harbor Symposia on Quantitative 
Biology 
(D) VOLUME: 54 
(E) ISSUE: 
(F) PAGES: 571-580 
(G) DATE: 1989 
(H) DOCUMENT NUMBER: 
(I) FILING DATE: 
(J) PUBLICATION DATE: 
(K) RELEVANT RESIDUES IN SEQ ID NO:3: FROM 1 TO 14 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
LeuThrValAspLeuAspSerProAlaArgSerSerValPro 
1510 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 amino acid residues 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: Protein/Peptide 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: 
(v) FRAGMENT TYPE: C-terminal fragment 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: FcRn from Rattus norvegicus DAF from Homo 
spaiens 
(B) STRAIN: 
(C) INDIVIDUAL ISOLATE: 
(D) DEVELOPMENTAL STAGE: FcRn from 11 day old/germ-line 
(E) HAPLOTYPE: 
(F) TISSUE TYPE: FcRn from proximal third of small intestine 
(G) CELL TYPE: FcRn from epithelial cells 
(H) CELL LINE: DAF from HeLa cells 
(I) ORGANELLE: 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: 
(B) CLONE: `p-51-DAF` cell line produces chimeric FcRn heavy 
chain with the C-terminus given in SEQ ID NO. 4. This 
C-terminus is modified intracellularly, providing 
covalent lipid attachment. 
(viii) POSITION IN GENOME: 
(A) CHROMOSOME/SEGMENT: 
(B) MAP POSITION: 
(C) UNITS: 
(ix) FEATURE: 
(A) NAME/KEY: The insertion of the DAF amino acid sequence 
after the FcRn heavy chain alpha-3 domain but before 
the transmembrane domain yields a functional, lipid- 
linked FcRn heavy chain. 
(B) LOCATION: FcRn heavy chain (a.a. 265 to 269) from 1 to 5, 
DAF (a.a. 311 to 347) from 6 to 42. 
(C) IDENTIFICATION METHOD: The cell surface expression of 
lipid- linked FcRn heavy chain was confirmed by 
immunostaining. Experiments proved that it retained its 
physiological, pH-dependent binding of immunoglobulin Fc. 
(D) OTHER INFORMATION: Intracellular modification results in 
the removal of sequence C-terminal of residue 25 and the 
anchoring of the protein in the cell membrane by the 
attachment of a phospholipid to the protein's C-terminal 
region. 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Gastinel, Louis N. 
Simister, N.E. 
Bjorkman, P.J. 
(B) TITLE: Expression and Crystallization of a Soluble and 
Functional form of a Fc Receptor Related to Class I 
Histocompatibility Molecules. 
(C) JOURNAL: Proc. Natl. Acad. Sci. U.S.A. 
(D) VOLUME: 89 
(E) ISSUE: 
(F) PAGES: 638-642 
(G) DATE: 
(H) DOCUMENT NUMBER: 
(I) FILING DATE: 
(J) PUBLICATION DATE: 
(K) RELEVANT RESIDUES IN SEQ ID NO:4: FROM 1 TO 42 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
LeuThrValAspLeuProAsnLysGlySerGlyThrThrSerGlyThr 
151015 
ThrArgLeuLeuSerGlyHisThrCysPheThrLeuThrGlyLeuLeu 
202530 
GlyThrLeuValThrMetGlyLeuLeuThr 
3540 
(2) INFORMATION FOR SEQ ID NO: 5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: Protein/Peptide 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: 
(v) FRAGMENT TYPE: C-terminal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Rattus norvegious 
(B) STRAIN: Wistar 
(C) INDIVIDUAL ISOLATE: 
(D) DEVELOPMENTAL STAGE: 11 day old/germ line 
(E) HAPLOTYPE: 
(F) TISSUE TYPE: Proximal third of small intestine 
(G) CELL TYPE: Epithelial cells 
(H) CELL LINE: 
(I) ORGANELLE: 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: 
(B) CLONE: `p51-stop` cell line produces FcRn heavy chain with 
the C- terminus given in SEQ ID NO: 5. 
(viii) POSITION IN GENOME: 
(A) CHROMOSOME/SEGMENT: 
(B) MAP POSITION: 
(C) UNITS: 
(ix) FEATURE: 
(A) NAME/KEY: The insertion of a stop-codon 3' of the codon 
for FcRn heavy chain amino acid residue 269 results in 
the synthesis of an FcRn heavy chain that has amino acid 
269 as its C- terminal residue. 
(B) LOCATION: FcRn heavy chain (a.a. 265 to 269) from 1 to 5. 
(C) IDENTIFICATION METHOD: The production of a soluble FcRn 
heavy chain implied that the new stop codon had 
effectively terminated the synthesis of heavy chain 
at amino acid 269. 
(D) OTHER INFORMATION: The truncated FcRn heavy chain 
(C-terminalresidue=a.a.269) can form water soluble 
FcRn heterodimer (with rat beta 2-microglobulin) that 
retains its physiological pH dependent binding of IgG 
Fc regions. 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Gastinel, Louis N. 
Simister, N.E. 
Bjorkman, P.J. 
(B) TITLE: Expression and Crystallization of a Soluble and 
Functional form of an Fc Receptor Related to Class I 
Histocompatibility Molecules 
(C) JOURNAL: Proc. Natl. Acad. Sci. U.S.A. 
(D) VOLUME: 89 
(E) ISSUE: 
(F) PAGES: 638-642 
(G) DATE: 
(H) DOCUMENT NUMBER: 
(I) FILING DATE: 
(J) PUBLICATION DATE: 15-Jan- 1992 
(K) RELEVANT RESIDUES IN SEQ ID NO:5: FROM 1 TO 5 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 
LeuThrValAspLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acid residues 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: Protein/Peptide 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: 
(v) FRAGMENT TYPE: C-terminal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Rattus norvegious 
(B) STRAIN: Sprague-Dawley 
(C) INDIVIDUAL ISOLATE: 
(D) DEVELOPMENTAL STAGE: 
(E) HAPLOTYPE: 
(F) TISSUE TYPE: 
(G) CELL TYPE: 
(H) CELL LINE: 
(I) ORGANELLE: 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: 
(B) CLONE: 
(viii) POSITION IN GENOME: 
(A) CHROMOSOME/SEGMENT: 
(B) MAP POSITION: 
(C) UNITS: 
(ix) FEATURE: 
(A) NAME/KEY: A 5 amino acid, C- terminal fragment of wild- 
type rat beta 2-microglobulin. 
(B) LOCATION: Rat beta 2- microglobulin (a.a. 95 to 99) from 
1 to 5. 
(C) IDENTIFICATION METHOD: Sequence is that reported by 
Sundelin, J. et al., in the Scand. J. Immunol., Vol. 27, 
pp. 195-199 (1988). 
(D) OTHER INFORMATION: This wild- type sequence was referenced 
so that the reader should compare the modified beta 
2- microglobulin sequences to that of the wild-type. 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Sundelin, J. 
Bjorck, L. 
Logdberg, L. 
(B) TITLE: The Complete Amino Acid Sequence of Rat beta 
2- Microglobulin. 
(C) JOURNAL: Scand. J. Immunol. 
(D) VOLUME: 27 
(E) ISSUE: 
(F) PAGES: 195-199 
(G) DATE: 1988 
(H) DOCUMENT NUMBER: 
(I) FILING DATE: 
(J) PUBLICATION DATE: 
(K) RELEVANT RESIDUES IN SEQ ID NO:6: FROM 1 TO 5 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 
TrpAspArgAspMet 
15 
(2) INFORMATION FOR SEQ ID NO: 7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 amino acid residues 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: Protein/Peptide 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: 
(v) FRAGMENT TYPE: C-terminal 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Beta 2-M from Rattus norvegious DAF from Homo 
sapiens 
(B) STRAIN: Beta 2-M from Sprague- Dawley 
(C) INDIVIDUAL ISOLATE: 
(D) DEVELOPMENTAL STAGE: 
(E) HAPLOTYPE: 
(F) TISSUE TYPE: 
(G) CELL TYPE: 
(H) CELL LINE: DAF from HeLa cells 
(I) ORGANELLE: 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: 
(B) CLONE: Beta 2m-DAF' cell line produces chimeric rat beta 
2- microglobulin with the C-terminus given in SEQ ID NO: 
7. This C- terminus is modified intracellularly providing 
covalent lipid attachment. 
(viii) POSITION IN GENOME: 
(A) CHROMOSOME/SEGMENT: 
(B) MAP POSITION: 
(C) UNITS: 
(ix) FEATURE: 
(A) NAME/KEY: The insertion of the DAF amino acid sequence 
after the C- terminal residue of rat beta 2-microglobulin 
results in the synthesis of a functional, lipid-linked 
beta 2- microglobulin. 
(B) LOCATION: Rat beta 2- microglobulin (a.a. 95 to 99) from 
1 to 5 DAF (a.a. 311 to 347) from 6 to 42. 
(C) IDENTIFICATION METHOD: The cell surface expression of 
lipid- linked rat beta 2-microploblin was confirmed by 
imunostaining. 
(D) OTHER INFORMATION: Intracellular modification results in 
the removal of sequence C-terminal of residue 25 and the 
anchoring of the protein in the cell membrane by 
attachment of a phospholipid to the protein's C-terminal 
region. 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Gastinel, Louis N. 
Simister, N.E. 
Bjorkman, P.J. 
(B) TITLE: Expression and Crystallization of a Soluble and 
Functional form of a Fc Receptor Related to Class I 
Histocompatibility Molecules. 
(C) JOURNAL: Proc. Natl. Acad. Sci. U.S.A. 
(D) VOLUME: 89 
(E) ISSUE: 
(F) PAGES: 638-642 
(G) DATE: 
(H) DOCUMENT NUMBER: 
(I) FILING DATE: 
(J) PUBLICATION DATE: 16-Jan- 1992 
(K) RELEVANT RESIDUES IN SEQ ID NO:7: FROM 1 TO 42 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 
TrpAspArgAspMetProAsnLysGlySerGlyThrThrSerGlyThr 
151015 
ThrArgLeuLeuSerGlyHisThrCysPheThrLeuThrGlyLeuLeu 
202530 
GlyThrLeuValThrMetGlyLeuLeuThr 
3540 
(2) INFORMATION FOR SEQ ID NO: 8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 366 amino acid residues 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: Linear 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Simister, N.E. 
Mostov, K.E. 
(B) TITLE: An Fc receptor structurally related to MHC class I 
antigens. 
(C) JOURNAL: Nature 
(D) VOLUME: 337 
(E) ISSUE: 
(F) PAGES: 184-187 
(G) DATE: 12-Jan-19891989 
(H) DOCUMENT NUMBER: 
(I) FILING DATE: 
(J) PUBLICATION DATE: 
(K) RELEVANT RESIDUES IN SEQ ID NO:8: FROM -22 TO 344 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 
MetGlyMetSerGlnProGlyValLeuLeuSerLeuLeuLeuValLeu 
22-20-15-10 
LeuProGlnThrTrpGlyAlaGluProArgLeuProLeuMetTyrHis 
5-1+1510 
LeuAlaAlaValSerAspLeuSerThrGlyLeuProSerPheTrpAla 
152025 
ThrGlyTrpLeuGlyAlaGlnGlnTyrLeuThrTyrAsnAsnLeuArg 
303540 
GlnGluAlaAspProCysGlyAlaTrpIleTrpGluAsnGlnValSer 
455055 
TrpTyrTrpGluLysGluThrThrAspLeuLysSerLysGluGlnLeu 
606570 
PheLeuGluAlaIleArgThrLeuGluAsnGlnIleAsnGlyThrPhe 
75808590 
ThrLeuGlnGlyLeuLeuGlyCysGluLeuAlaProAspAsnSerSer 
95100105 
LeuProThrAlaValPheAlaLeuAsnGlyGluGluPheMetArgPhe 
110115120 
AsnProArgThrGlyAsnTrpSerGlyGluTrpProGluThrAspIle 
125130135 
ValGlyAsnLeuTrpMetLysGlnProGluAlaAlaArgLysGluSer 
140145150 
GluPheLeuLeuThrSerCysProGluArgLeuLeuGlyHisLeuGlu 
155160165170 
ArgGlyArgGlnAsnLeuGluTrpLysGluProProSerMetArgLeu 
175180185 
LysAlaArgProGlyAsnSerGlySerSerValLeuThrCysAlaAla 
190195200 
PheSerPheTyrProProGluLeuLysPheArgPheLeuArgAsnGly 
205210215 
LeuAlaSerGlySerGlyAsnCysSerThrGlyProAsnGlyAspGly 
220225230 
SerPheHisAlaTrpSerLeuLeuGluValLysArgGlyAspGluHis 
235240245250 
HisTyrGlnCysGlnValGluHisGluGlyLeuAlaGlnProLeuThr 
255260265 
ValAspLeuAspSerProAlaArgSerSerValProValValGlyIle 
270275280 
IleLeuGlyLeuLeuLeuValValValAlaIleAlaGlyGlyValLeu 
285290295 
LeuTrpAsnArgMetArgSerGlyLeuProAlaProTrpLeuSerLeu 
300305310 
SerGlyAspAspSerGlyAspLeuLeuProGlyGlyAsnLeuProPro 
315320325330 
GluAlaGluProGlnGlyValAsnAlaPheProAlaThrSer 
335340344 
(2) INFORMATION FOR SEQ ID NO: 9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 99 amino acid residues 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: Linear 
(x) PUBLICATION INFORMATION: 
(A) AUTHORS: Sundelin, J. 
Bjorck, L. 
Logdberg, L. 
(B) TITLE: The Complete Amino Acid Sequence of Rat beta 
2- Microglobulin 
(C) JOURNAL: Scand. J. Immunol. 
(D) VOLUME: 27 
(E) ISSUE: 
(F) PAGES: 195-199 
(G) DATE: 1988 
(H) DOCUMENT NUMBER: 
(I) FILING DATE: 
(J) PUBLICATION DATE: 
(K) RELEVANT RESIDUES IN SEQ ID NO:9: FROM 1 TO 99 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 
IleGlnLysThrProGlnIleGlnValTyrSerArgHisProProGlu 
151015 
AsnGlyLysProAsnPheLeuAsnCysTyrValSerGlnPheHisPro 
202530 
ProGlnIleGluIleGluLeuLeuLysAsnGlyLysLysIleProAsn 
354045 
IleGluMetSerAspLeuSerPheSerLysAspTrpSerPheTyrIle 
505560 
LeuAlaHisThrGluPheThrProThrGluThrAspValTyrAlaCys 
65707580 
ArgValLysHisValThrLysLeuGluProLysThrValThrTrpAsp 
859095 
ArgAspMet 
99 
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