Soluble CD4 molecules modified to prolong circulating half-life

A method for extending soluble CD4 serum half-life in mammals is described. The method comprises modifying soluble CD4 glycosylation so as to inhibit clearance from serum. In a preferred embodiment, clearance by hepatocyte galactose receptors is inhibited by removal of soluble CD4 terminal sialic residues followed by oxidation of exposed galactose residues. The modified soluble CD4 molecules are demonstrated to possess extended serum half-life.

FIELD OF THE INVENTION 
This application relates to compositions for antiviral or other therapy and 
to methods for glycoprotein structural modifications which prolong the 
half-life of those proteins in the circulation by blocking specific liver 
clearance mechanisms. Specifically, this application relates to 
compositions useful in the treatment of Human Immunodeficiency Virus (HIV) 
infections. 
BACKGROUND OF THE INVENTION 
The primary immunologic abnormality in HIV-infected patients with an 
infection in the active stage is the progressive depletion and functional 
impairment of T lymphocytes expressing the CD4 cell surface glycoprotein 
(Lane et al. (1985) Ann. Rev. Immunol. 3:477). Generally, T lymphocytes 
expressing the CD4 surface glycoprotein have a helper/inducer T cell 
phenotype (Reinherz et al. (1980) Cell 19:821), but such T cells can also 
have cytotoxic/suppressor activity (Thomas et al. (1981) J. Exp. Med. 
154:459). It is believed that the loss of the helper/inducer functions in 
immunocompromised AIDS or ARC patients leads to the opportunistic 
infections and malignancies associated with AIDS. 
Molecular studies of HIV infection of T cells have shown that HIV 
specifically and selectively infects T cells expressing CD4. It was also 
observed that CD4-specific monoclonal antibodies could block HIV infection 
and syncytia formation (Dalgeish et al. (1984) Nature 312:767; McDougal et 
al. (1985) J. Immunol. 135:3151). Maddon et al. (1986) Cell 47:333 showed 
that cells normally non-permissive for HIV infection which expressed a 
stable cDNA encoding CD4 became permissive for HIV infection. These 
results showed that CD4 was required for HIV infection. McDougal et al. 
(1986) Science 231:382, demonstrated complex formation between CD4 and 
gp120, the major HIV envelope glycoprotein. 
cDNA encoding CD4 has been cloned and sequenced (Maddon et al. (1985) Cell 
42:93). Sequence analysis shows an N-terminal signal peptide sequence, 
domains which exhibit homology to certain immunoglobulin variable-region 
domains, potential glycosylation sites at about 273 and about 303 in the 
amino acid sequence, a potential trans-membrane domain from about 375 to 
about 395, and a potential cytoplasmic domain extending through the 
C-terminus of the protein. Peterson and Seed (1988) Cell 54:65 performed 
site-directed mutagenesis of the CD4 protein to determine HIV binding 
sites, and correlated this information with epitopes recognized by 
CD4-specific monoclonal antibodies. Amino acid substitutions in the region 
of amino acids 45-47 of the protein appeared to destroy both HIV binding 
and syncytium formation. 
Secreted, soluble forms of CD4 have been synthesized using truncated coding 
sequences. CD4 derivatives of about 370 amino acids have been produced; 
these are glycosylated when produced in appropriate host cells. Such 
molecules bind HIV gp120 effectively and can block HIV infection of 
susceptible cells (See, e.g., Smith et al. (1987) Science 238:1704; Fisher 
et al. (1988) Nature 331:76; and Hussey et al. (1988) Nature 331:78). 
Soluble truncated CD4 proteins as short as 113 amino acids, which are not 
glycosylated, can block HIV-mediated cell fusion (Chao et al. (1989) J. 
Biol. Chem. 264:5812). Thus it is clear that intact oligosaccharide side 
chains are not required for HIV binding or for cell fusion events. 
The use of soluble forms of CD4 has been proposed for AIDS treatment or 
prophylaxis (See e.g., EP 0 385,909, published Sep. 5, 1990). Soluble 
forms of CD4 are known to have a short half-life in circulation in 
relation to certain serum proteins. Because the in vivo plasma half-life 
of soluble CD4 has been shown to be relatively short, various strategies 
have been employed to stabilize the protein against clearance. (See, e.g., 
WO 89/03222; WO 89/02922; WO 90/01035; and WO 90/05534). Conjugates have 
been prepared in which polyethylene glycol or other hydrophilic polymers 
are attached to the CD4 either via amino acid free functional groups or 
via sugar moieties in the oligosaccharide side chains of the glycosylated 
soluble CD4 protein. A second approach to stabilizing the CD4 protein in 
circulation has been to produce fusion proteins including a soluble CD4 
portion and a portion from a protein of long circulating half-life, such 
as an immunoglobulin. Such fusions exhibited longer plasma half-lives in 
animal models than sCD4 without added domains. 
Therapeutic proteins may be removed from circulation by a number of routes. 
For some pharmacologically active proteins, there are specific receptors 
which mediate removal from circulation. Proteins which are glycosylated 
may be cleared by lectin-like receptors in the liver, which exhibit 
specificity only for the carbohydrate portion of those molecules. 
Nonspecific clearance by the kidney of proteins and peptides (particularly 
nonglycosylated proteins and peptides) below about 50 kDa has also been 
documented. It has been noted that asialo-glycoproteins are cleared more 
quickly by liver than native glycoproteins or proteins lacking 
glycosylation (Bocci (1990) Advanced Drug Delivery Reviews 4:149). The 
sialic acid residues of erythropoietin appear to contribute to its stable 
circulation (Fukuda et al. (1989) Blood 73:84). In contrast, studies of 
tissue-type plasminogen activator (tPA) showed that the oligosaccharide 
sidechains were not the primary determinants for clearance from solution, 
but rather rapid clearance was dependent on the amino acid sequence within 
one or more domains of the molecule. The presence and type of 
glycosylation made a secondary, less significant contribution to clearance 
(Larsen et al. (1989) Blood 73:1842). 
Mammalian glycoproteins often have N-acetylneuraminic acid (sialic acid) as 
the external (terminal) residue of the oligosaccharide chains which may be 
N-linked or O-linked (See, e.g., Osawa and Tsuji (1987) Ann. Rev. Biochem. 
56:21). 
Where the nature of the oligosaccharide is the primary determinant for 
clearance from circulation, generally glycoproteins with terminal sialic 
acid residues removed (asialoglycoproteins) are cleared more quickly than 
their intact counterparts. Circulating glycoproteins are exposed to 
sialidase(s) (or neuraminidase) which can remove terminal sialic acid 
residues. Typically the removal of the sialic acid exposes galactose 
residues, and these residues are recognized and bound by 
galactose-specific receptors in hepatocytes (reviewed in Ashwell and 
Harford (1982) Ann. Rev. Biochem. 51:531). Liver also contains other 
sugar-specific receptors which mediate removal of glycoproteins from 
circulation. Specificities of such receptors also include 
N-acetylglucosamine, mannose, fucose and phosphomannose. Glycoproteins 
cleared by the galactose receptors of hepatocytes undergo substantial 
degradation and then enter the bile; glycoproteins cleared by the mannose 
receptor of Kupffer cells enter the reticuloendothelial system (reviewed 
in Ashwell and Harford (1982) Ann. Rev. Biochem. 51:53). 
Studies with asialo-ceruloplasmin and derivatives showed that 
asialo-ceruloplasmin in which galactose residues were oxidized by 
treatment with galactose oxidase and horseradish peroxidase and 
asialoagalacto-ceruloplasmin exhibited extended circulating half-lives as 
compared with asialo-ceruloplasmin (Morell et al. (1968) J. Biol. Chem. 
243:155). Efficient removal by the galactose receptor appears to require 
at least two exposed galactose residues. In contrast, transferrin is a 
glycoprotein in which the sialylation state of the oligosaccharide is not 
key to rapid clearance from circulation (Morell et al. (1971) J. Biol. 
Chem. 246:1461). 
From the foregoing cited examples of glycoproteins for which sialylation is 
the key determinant of clearance from circulation and those for which 
sialylation has no bearing on clearance or for which oligosaccharides play 
a relatively insignificant role in clearance, one may conclude that the 
fate of a particular glycoprotein in circulation and its apparent 
mechanism for clearance must be determined empirically. Similarly, 
strategies for prolonging the circulation of a particular glycoprotein 
must be evaluated on a case-by-case basis. The mechanism for clearance 
must be evaluated and the strategies for slowing or avoiding clearance 
must take into account maintenance of desired biological activity or 
function, potential toxicity, potential immunogenicity and cost. 
A problem solved by the present invention is the prolongation of the 
circulating half-life of soluble CD4 derivatives, thus reducing the 
quantity of injected material and frequency of injection required for 
maintenance of therapeutically effective levels of circulating sCD4 for 
treatment or prophylaxis of HIV-infected individuals. The short in vivo 
plasma half-life of sCD4 is undesirable from the standpoint of the 
frequency and the amount of soluble CD4 protein which would be required in 
the prophylaxis or treatment of AIDS. The present invention provides means 
to prolong the circulating half-life of sCD4 with the most conservative 
but still effective change to the glycoprotein structure and with the 
substantial maintenance of gp120 binding activity. 
SUMMARY OF THE INVENTION 
It is an object of this invention to provide a means for stabilizing 
glycoproteins in circulation when the glycosylation of that protein 
provides the primary determinant for clearance. Increased half-life is 
achieved in such glycoproteins by treatments which block or inhibit 
removal of the protein by sugar-specific receptor, such as the galactose 
and mannose receptors in the liver. In particular, the prolonging of 
soluble CD4 derivatives in circulation is described. Prolonged circulating 
half-lives are desirable in therapeutic proteins because frequency and/or 
size of dose can be reduced when half-life is longer. 
It is also an object of this invention to provide a modified soluble CD4 
derivative with increased plasma half-life as compared with the unmodified 
derivative. Increased half-life of sCD4 is achieved in general by means 
which block or inhibit removal of sCD4 by galactose, mannose or other 
sugar-specific receptors. More specifically, means are provided for 
modification of terminal galactose or mannose residues of glycosylated 
sCD4 or its derivatives such that removal by sugar-specific receptors in 
the liver is inhibited or prevented. Modifications that result in 
increased half-life include, but are not limited to, exposure of galactose 
residues followed by oxidation or derivatization of the galactose such 
that binding of the modified sCD4 to galactose receptor is inhibited or 
blocked. A preferred embodiment is one in which the terminal sialic acid 
residues of the oligosaccharide side chains of the soluble CD4 or soluble 
CD4 derivative have been removed with neuraminidase treatment, and then 
the exposed galactose residues are oxidized by galactose oxidase and 
horseradish peroxidase treatment. The oxidation of the exposed galactose 
residues has the effect of preventing rapid clearance of the modified CD4 
from circulation by specific galactose receptors in the liver. It is 
understood that other structural modifications of terminal galactose 
residues, including but not limited to addition of a functional group or 
small molecule or mild oxidation treatment, which have the effect of 
blocking, inhibiting or preventing recognition of terminal galactose 
residues without destroying the HIV gp120-binding activity are 
functionally equivalent. It is also contemplated that structural 
modification of one or more sialic acid residues of oligosaccharide 
portions of a soluble CD4 molecule may be made with the result that 
terminal sialic acid residues are not removed by neuraminidase; as a 
result the persistence of the modified sCD4 in circulation is increased. 
This invention also encompasses the modification of terminal mannose 
residues so as to inhibit or prevent clearance via the mannose receptor of 
liver. 
Furthermore, a modified sCD4 may be prepared in which sialic acid (e.g., 
with neuraminidase) and galactose residues (e.g., with galactosidase) are 
removed to expose mannose residues. Those exposed mannose residues may be 
structurally altered by the addition of a functional group or small 
molecule or by mild oxidation treatment, with the result that recognition 
of this modified sCD4 by the mannose receptor and removal from circulation 
of liver is prevented or inhibited. For all modified sCD4 molecules, 
solubility in pharmacological and physiological fluids must be maintained 
and the biological activity of HIV gp120/160 binding must likewise be 
maintained. 
An object of this invention is a therapeutic composition comprising a 
concentration of a modified sCD4 effective for binding HIV gp120/gp160 and 
a pharmaceutically acceptable carrier. Preferably, the half-life in human 
circulation of said modified sCD4 is greater than about 24 hours. A 
preferred sCD4 is one which has been treated to remove sufficient sialic 
acid to expose at least two galactose residues and which has been further 
treated, e.g., with galactose oxidase and horseradish peroxidase so that 
clearance of the modified sCD4 from circulation by the galactose-specific 
receptor of liver is inhibited or prevented. 
A further object of the invention is as method for increasing the half-life 
of a glycoprotein in circulation in a mammal, where the glycoprotein is 
normally cleared from circulation by the galactose receptor of liver when 
in an asialo-glycoprotein form. The method comprises the steps of treating 
said glycoprotein to remove terminal sialic acid residues to produce an 
asialo-glycoprotein, and oxidizing any galactose residues of said 
asialo-glycoprotein to produce an oxidized asialo-glycoprotein, whereby 
removal of said oxidized asialo-glycoprotein by the galactose receptor of 
liver is inhibited. In a preferred embodiment which specifically 
exemplifies the prolonging of sCD4 in circulation, sialic acid residues 
are removed by neuraminidase treatment and the galactose residues are 
oxidized by treatment with galactose oxidase and horseradish peroxidase. 
In principle, the teachings for the structural modification of sCD4 to 
prolong circulating half-life can be applied to any pharmacologically 
active protein for which a longer circulating half-life is advantageous, 
in which intact oligosaccharide chains are not required for the desired 
biological activity, and for which the oligosaccharide carries the signal 
of primary importance for clearance from the bloodstream. 
DETAILED DESCRIPTION OF THE INVENTION 
CD4, as defined herein, is the full-length CD4 polypeptide having the 
biological activities of native human CD4. Amino acid sequence variations 
and derivatizations which allow the maintenance of the biological 
activities of the CD4 protein are included within the definition. Native, 
CD4, and recombinant CD4 which is synthesized in recombinant eukaryotic 
cells is a glycoprotein. Maddon et al. (1985), supra, notes that there are 
two sites for N-linked glycosylation, at about 275 and at about 305. EP 
372 752 gives the sugar content of a CD4 molecule, but the exact structure 
of the oligosaccharide chain(s) is not known. However, it is generally 
understood in the art that sialic acid (N-acetylneuraminic acid) residues 
will be terminal in the oligosaccharide side chain of a mature 
glycoprotein synthesized in a mammalian cell, and that galactose residues 
will be exposed when sialic acid is absent or removed (See, e.g., Osawa 
and Tsuji (1987) supra). The art knows that exact oligosaccharide 
structure of a glycoprotein may vary with respect to sugars present, the 
glycosylation enzymes present and the relative proportions of each 
according to the choice of the particular eukaryotic cell in which the 
recombinant CD4 (or soluble CD4) is synthesized. 
As defined herein soluble CD4 (sCD4) is a variant of the CD4 protein which 
is soluble in water-based pharmaceutical preparations (or pharmaceutically 
acceptable solvents or compositions which include components in addition 
to water) and in physiological fluids, including plasma, at a level which 
is sufficient to achieve a therapeutically effective concentration in 
circulation. sCD4 proteins include those in which part or all of the 
transmembrane domain of the primary structure of CD4 has been deleted, for 
example through truncation of the coding sequence; the cytoplasmic domain 
of the protein may likewise be deleted without the loss of the desired 
biological activity of HIV gp120 binding. sCD4 molecules capable of being 
glycosylated when synthesized in appropriate host cells are described in 
Smith et al. (1987), supra; Fisher et al. (1988), supra, Hussey et al. 
(1988), supra; EP Publication No. 385 909, supra; Deen et al. (1988) 
Nature 331:82-84; all of which are incorporated by reference herein. 
Unless otherwise noted and like the native full-length CD4, the sCD4 
molecule is glycosylated. In any case, the HIV gp120 binding activity of 
the native CD4 is substantially maintained in the soluble CD4 derivative 
and modified sCD4 of the present invention. Peterson and Seed (1988), 
supra, e.g., address the issue of amino acid substitutions and deletions 
in the N-terminal region of the CD4 and the effects on gp120 binding. It 
is believed that some form of glycosylation is necessary for protection of 
sCD4 from degradation in circulation and/or clearance by the kidneys. 
Thus, sCD4 of the present invention must retain some level of 
glycosylation: preferably the minimal structural modification of the 
glycosylation is made which acts to prolong circulating half-life. 
For the purposes of monitoring sCD4 molecules or derivatives in an animal 
model (e.g., in the rat, as described in the Examples), radio-iodinated 
sCD4 is prepared using, e.g., Bolton-Hunger reagent and instructions for 
use from Nuclear England Nuclear (Boston, Mass.), or according to any 
other technique well-known to the art. 
Modified sCD4 molecules of the present invention are those with structural 
alterations (modifications) of the oligosaccharide portions of the 
glycoprotein which result in prolonged circulating half-life by blocking 
or inhibiting clearance via sugar-specific receptors of the liver. 
Preferably the sCD4 glycoproteins are synthesized in a recombinant 
mammalian host. The chemical and/or enzymatic treatments to produce the 
structural modifications of the oligosaccharide should not substantially 
alter the binding reaction of sCD4 with the HIV target protein. It is 
preferred that the circulating half-life of a modified sCD4 of the present 
invention be at least about 24-48 hours in humans (or at least about 6-12 
hours as measured in a rat animal model). It is most preferable that the 
structural modification of the oligosaccharide so as to prolong 
circulating half-life is the most conservative structural change which 
will achieve this end. A variety of chemical derivatization procedures, or 
chemical and/or enzymatic procedures, as understood in the art, may be 
employed to produce the modified sCD4 molecules of the present invention. 
The modified sCD4 specifically exemplified in the present invention is 
oxidized asialo-sCD4, in which the terminal galactose residues have been 
oxidized by treatment with galactose oxidase and horseradish peroxidase. 
As for full-length CD4, there may be variations in amino acid sequence so 
long as glycosylation and gp120 binding activity are maintained. 
Asialo-sCD4 is a modification of the sCD4 in which the sialic acid residues 
are wholly or partially absent from the oligosaccharide portion of the 
molecule so that at least two terminal galactose residues are exposed. 
Sialic acid residues may be removed by sialidase or neuraminidase 
treatment; asialo-sCD4 may also be produced by synthesizing sCD4 in a host 
cell which is unable to add the terminal sialic acid residues during 
oligosaccharide synthesis. Most preferably, at least two galactose 
residues are exposed. 
Agalacto-asialo-sCD4 is a sCD4 derivative in which terminal sialic acid and 
galactose residues are absent, either as a result of enzymatic or chemical 
removal or through synthesis of the sCD4 in a host cell which is deficient 
in sialic acid and galactose addition. Where galactose residues are 
terminal in the oligosaccharide portion of a glycoprotein, those galactose 
residues may be removed with .beta.-galactosidase. 
Oxidized asialo-sCD4 is an asialo-sCD4 derivative in which the galactose 
residues have been oxidized by mild oxidation conditions, for example with 
galactose oxidase and horseradish peroxidase. Although not wishing to be 
bound by any particular mechanism, it is believed that such treatment 
oxidizes the C6 position of the galactose and prevents recognition and 
removal of the molecule from circulation by the galactose receptor of the 
liver; thus prolonging the half-life of the modified sCD4 in circulation 
so that said molecule, either injected alone or in conjunction with any 
other HIV treatment, is therapeutically effective. Mild oxidation 
conditions include any chemical or enzymatic oxidation which oxidizes the 
terminal sugar residue without substantial effect on the gp120 binding 
function of the sCD4 protein or any protective function of glycosylation 
on the protein. 
Other structural modifications of the oligosaccharide portion of a sCD4 
molecule which prolong the circulating half-life by preventing and/or 
inhibiting removal of sCD4 by the galactose or mannose receptors are 
within the scope of the invention. While the experimental results 
described in this specification are consistent with the model for the 
clearance of asialo-sCD4 from circulation by the hepatocyte galactose 
receptor, Applicants do not want to be bound by this model. It is 
understood that other mechanisms may contribute to clearance. It is likely 
that treatment of the asialo-sCD4 with galactose oxidase and horseradish 
peroxidase prevented binding of terminal sugar residues by the galactose 
receptor. Such modifications can include the structural modification of 
sialic acid of the glycoprotein so that neuraminidases cannot remove the 
terminal sialic acid residues to expose the galactose residues which could 
then mediate clearance by virtue of their recognition by the galactose 
specific receptor of liver. An asialo-sCD4 can be modified by the addition 
of functional groups to the galactose so that recognition and removal from 
circulation by the galactose receptor is prevented or inhibited. Chemical 
modification or derivatization of the C6 position of galactose is 
preferred to maximize half-life and minimize clearance without 
significantly affecting gp120 binding function and without eliciting 
negative physiological reactions; minimal and mild treatment is preferred. 
Functional groups or other structure-modifying molecules in this invention 
to be added to the oligosaccharide portion of an sCD4 exclude any 
polymeric substituents. Also contemplated are analogous structural changes 
to terminal mannose residues of asialoagalacto-sCD4 which will inhibit or 
prevent clearance from circulation via the mannose receptor of Kupffer 
cells of the liver. The binding of modified sCD4 molecules to gp120 will 
not be affected by the structural modifications to the oligosaccharide 
portion of the molecule. 
For any modified sCD4 molecule of the present invention it is most 
desirable that an immunological response will not be elicited in a human 
patient exposed to the modified sCD4. It is also required that the HIV 
gp120 binding activity is not significantly decreased to detrimentally 
affect the therapeutic function of sCD4 by the structural modification 
employed to confer prolonged circulation. It is also most desirable that 
any structural modification of a sCD4 molecule does not result in toxicity 
in a patient to which that modified sCD4 is administered. Clearly for use 
in therapeutics, the modified sCD4 should have minimal toxic, irritant or 
other side effects on administration to humans. The ability of a modified 
sCD4 molecule to bind HIV gp120 can be determined using a test such as 
that described in EP 372 752, published Jun. 13, 1990, which document is 
incorporated by reference herein. 
The circulating half-life of a protein (or glycoprotein) is the time for 
the initial blood concentration of that protein to fall to half the 
initial concentration. 
As it relates to the modified sCD4 molecules of the present invention, the 
term biological activity refers to the ability of the sCD4-related 
molecule to bind the HIV gp120 (or gp160) with substantially the same 
affinity as the unmodified sCD4 molecule. Modifications which 
substantially decrease the biological activity of sCD4 are to be avoided. 
Once the modified sCD4 has bound the HIV gp120, the bound HIV cannot 
infect a susceptible cell, thus preventing the spread of viral infection. 
Similarly, an HIV-infected cell expressing gp120 on its surface and where 
gp120 is bound to an sCD4 molecule, cannot participate in syncytium 
formation with a cell expressing CD4 on its surface. The inhibition of 
syncytium formation further contributes to the therapeutic effect of an 
sCD4-related protein in an HIV-infected individual. Bound complexes of 
modified sCD4 with HIV via gp120 or with a patient's cells via surface 
gp120 may also be targeted for removal from the circulation, for example, 
using an extracorporeal device with means for removal of sCD4-gp120 
complexes, either associated with viral particles or with cells in 
circulation. Similarly, HIV particles or HIV-infected cells may be 
targeted for destruction via bound modified sCD4 molecules. 
The modified sCD4 molecules of this invention may be purified by any means 
known to the art before formation into therapeutic compositions. 
Therapeutic compositions are formulated using a modified sCD4 with a 
prolonged circulating half-life and a physiologically acceptable carrier; 
such compositions can be sterilized by any means known to the art which 
does not significantly alter either the desired biological activity or the 
prolonged half-life in circulation. In addition, the modified sCD4 
molecules of the present invention may be used in conjunction with other 
compositions useful in the treatment or prophylaxis of AIDS in 
HIV-infected individuals. Such other compositions include, but are not 
limited to, AZT, DDC, DDI, neutralizing antibodies, immunocytotoxins, 
gp120 fragments and HIV vaccine preparations. 
For glycoproteins other than sCD4 for which a derivative with prolonged 
circulating half-life is desired, the skilled artisan can apply the 
teachings of this disclosure. In a rat model system, as described herein, 
the artisan can determine whether the galactose receptor is the primary 
means of clearance by preparing a radiolabeled, desialylated derivative, 
testing for bile excretion and also for competition by asialofetuin of 
liver-associated counts. Inhibition of clearance with co-injection of 
asialofetuin and appearance of asialoglycoprotein-associated radioactivity 
indicates clearance by the liver galactose receptor. Then, for example, 
the glycoprotein of interest can be desialylated, preferably oxidized 
under mile conditions, e.g., with galactose oxidase and horseradish 
peroxidase, and prolonged half-life can be confirmed in the animal model 
as described herein. Analogous appropriate tests and treatments will be 
readily apparent to the skilled artisan when clearance by another sugar 
receptor is the mode of clearance. For any glycoprotein in which sialic 
acid residues are normally present, treatment with sialotransferase under 
appropriate conditions to ensure complete sialylation will also prolong 
circulating half-life. 
In principle, the teachings presented herein may be applied to any sCD4 
modified to improve circulating half-life and/or HIV gp120/gp160 binding 
in such a way that glycosylation during synthesis is not prevented. 
Furthermore, mixtures of sCD4-related molecules and modified sCD4 
molecules may be combined in a therapeutic composition useful for 
prophylaxis or treatment of HIV infection and/or for alleviation of the 
detrimental effects of HIV infection. A uniform modified glycoprotein may 
be incorporated in a therapeutic composition or a mixture of modified 
glycoprotein may be formulated in such a composition, so long as the 
desired therapeutic action is achieved by those molecules and so long as 
clearance by sugar-specific receptors mediating clearance is inhibited or 
prevented by the modification or modifications made to said glycoprotein. 
This methodology is applicable to therapeutic sCD4 molecules or to other 
therapeutically useful glycoproteins which are cleared from circulation by 
sugar-specific receptors. 
It will be readily apparent to those of ordinary skill in the art that 
assays, reagents, procedures and techniques other than those specifically 
described herein, can be employed to obtain the same or equivalent results 
and achieve the goals described herein. For example, chemical means of 
oxidation or removal of sialic acid can be readily substituted for 
enzymatic means specifically described. All such alternatives are 
encompassed by the spirit and scope of this invention.

EXAMPLE 1 
Fate of Circulating Soluble CD4 
This example describes the elucidation of the clearance mechanism for 
circulating recombinant soluble CD4 (sCD4) in the rat animal model. The 
recombinant sCD4 used herein is the product of a truncated coding sequence 
of CD4. The terms [.sup.125 -I]-sCD4 and sCD4 are used interchangeably but 
only radiolabelled sCD4 was used in these experiments. The [.sup.125 
I]-sCD4 used in the experiments described herein was obtained from Biogen, 
Cambridge, Mass., and was stored at -70.degree. C. prior to use. The 
radioactive sCD4 used in these experiments had a specific activity of 
about 10 microcuries per microgram of protein. 
The rat is the model animal system used to study sCD4 clearance from 
circulation. The studies are performed using the following general scheme. 
Rats are fasted overnight and then weighed before a clearance experiment is 
begun. Then each rat is anaesthetized by intraperitoneal injection of an 
appropriate amount of an aesthetic, e.g., 5.2 mg sodium pentobarbital per 
100 g body weight. 
sCD4 is prepared for injection by preparing 0.15M sodium chloride (NS) to 
which 1 mg bovine serum album (BSA) is added. 
Then [.sup.125 I]-sCD4 is added, preferably about 100,000 cpm as measured 
in a gamma counter. It is necessary to add sCD4 to solutions already 
containing BSA and to pretreat equipment with BSA because otherwise the 
sCD4 tends to adhere to the walls of test tubes, pipet tips, etc. The use 
of BSA as a carrier protein tends to reduce the amount of 
sCD4-radioactivity adhering to tubes, etc. 
The sCD4 sample is drawn up into a BSA-coated 1 cc syringe, and a second 
syringe is prepared with 1 ml NS containing I mg/ml BSA to flush the IV 
tubing after sCD4 injection. A peristaltic pump and tubing are prepared 
with 0.15M NaCl. 
When the rat is thoroughly anaesthetized, the peritoneum is opened and the 
abdominal cavity is exposed with a 2.5 inch vertical cut. The are is 
flushed with 0.15M NaCl and covered with an NS-soaked gauze sponge which 
is kept moist throughout the experiment. An IV is secured in the tail. The 
sample is injected and the IV tubing is then flushed with NS. Timing is 
started half-way through the 1 ml NS flush. A NS flush continues at 7 ml/h 
throughout the course of the experiment. The peristaltic pump is stopped 
at the end of the experiment. For long term experiments, the sample and 
the 1 ml flush are administered under anesthesia without opening the 
peritoneal cavity. At the end of a long-term (greater than 2 hr) 
experiments, the animal is again anesthetized, and the peritoneal cavity 
is opened, the portal vessels are ligated with suture and the animal is 
exsanguinated by intracardiac puncture, simultaneously collecting blood in 
a measured volume that is placed in vials containing EDTA as 
anticoagulant. In all cases, the portal vessels were ligated after opening 
the peritoneal cavity, and this ligation signals the end of the 
experiment. 
To collect a blood sample, the heart is pierced and about 5 ml blood is 
collected. A measured volume is placed in a glass counting vial before the 
blood clots and radioactivity is determined by gamma counting. 
The liver is removed immediately after the blood sample is taken if a liver 
sample is desired. The hepatic portal vein is sutured before the liver is 
actually removed. The liver is rinsed in distilled water, blotted, weighed 
and placed in a glass counting vial. If desired, spleen, kidneys, 
intestine can be removed, rinsed, blotted and counted in a gamma counter. 
All glassware, tubing, pipets, etc. are also counted. 
For determination of [.sup.125 I]-sCD4-related counts appearing in the 
bile, the bile duct is cannulated and means for collecting bile are 
prepared in the rat before the sCD4 sample is injected. 
To look at clearance, rats were injected with sCD4 with or without infusion 
of either the glycoprotein fetuin or asialofetuin. At the noted times 
after injection of the sCD4, rats were sacrificed and the radioactivity in 
the blood samples and in livers was determined. Table 1 summarizes the 
results of this experiment. The asialofetuin infusion resulted in about a 
46% increase in blood-associated sCD4 radioactivity. 
These results suggested that there might be heterogeneity in the [.sup.125 
I]-sCD4 molecules. The relative increase in radioactivity in the 
bloodstream at 10 min after injection with sCD4 and asialofetuin as 
compared with injection of sCD4 alone or injection of sCD4 and fetuin 
suggests that a major mode of clearance is the galactose receptor in 
hepatocytes. It is proposed that sCD4 molecules with complete sialylation 
had a long half-life and that those with uncovered galactose residues have 
a short half-life in circulation. 
EXAMPLE 2 
Preparation of Control and Asialo-sCD4 
To further study the mechanism for clearance of sCD4, a desialylated 
preparation of [.sup.125 -I]-sCD4 was made for comparison with an 
untreated control preparation. 
10 microliters of [.sup.125 I]-sCD4 containing about 0.2 ug protein was 
added to 2.2 ml 0.1M sodium acetate (pH 4.5) containing 1 mg/ml BSA. 1 ml 
of this mixture was transferred to each of two 15 ml conical centrifuge 
tubes and 1 ml 0.1M sodium acetate was added to each. Then a 20 microliter 
aliquot of neuraminidase Type V (Sigma Chemical Co., St. Louis, Mo.) 
(about 0.1 units) was added to one of the tubes, and the other served as 
an untreated control. From this point on in the preparation, the tubes 
were treated in parallel. Both tubes were incubated at 37.degree. C. for 2 
hr. Each mixture was then dialyzed using tubing with an exclusion limit of 
12,000-14,000 d against 1 liter TC-PBS buffer overnight at 4.degree. C. 
TC-PBS contains 6.5 mM sodium phosphate, 3.5 mM potassium phosphate, 0.14M 
sodium chloride (pH 7.40). The dialysates were then collected, stored at 
4.degree. C., and the radioactivity in aliquots of each were determined by 
liquid scintillation counting (10 microliters of control sCD4=21325; 10 
microliters of asialo-sCD4=19490 cpm). Treatment of the sCD4 with 
neuraminidase yielded substantially desialylated sCD4 (asialo-sCD4). The 
control sample was assumed to be fully sialylated sCD4. 
In some experiments a second neuraminidase treatment followed that 
described above. After the first two hour incubation, an additional 200 
microliters neuraminidase (1 unit) was added and incubation was continued 
an additional two hours at 37.degree. C. 
To determine the radioactivity associated with relatively high molecular 
weight material, aliquots of the asialo-sCD4 and of the control sCD4 were 
precipitated with trichloroacetic acid as follows: 10 microliters of BSA 
(1 mg) was added to a borosilicate tube (5 ml size) and then a 890 
microliter aliquot of dialyzed sCD4 and TC-PBS was added. After mixing, 
100 microliters of cold 100% TCA was added, the samples were mixed and 
held on ice for 10 min. Then each tube was centrifuged for 10 min. 500 
microliter supernatant samples, pellets, and the syringe used to measure 
the sample were then counted. 
Rats R11 and R14 were injected with asialo-CD4 (twice digested with 
neuraminidase). It appears that the neuraminidase treatment to remove 
terminal sialic acid residues results in a significant relative increase 
in liver-associated radioactivity and a substantial relative decrease in 
bloodstream-associated radioactivity at 10 min post-injection. The results 
in Tables 1 and 2 suggest that circulating sCD4 is removed by the 
galactose receptor of the liver. The results also suggest some 
heterogeneity in the sCD4 preparation with respect to sialylation levels 
of the recombinant sCD4. 
To attempt to lengthen the circulating lifetime of sCD4 by preventing 
recognition and binding of asialo-sCD4 by the hepatocyte galactose 
receptor, asialo-sCD4 was treated with galactose oxidase and horseradish 
peroxidase to oxidize the carbinol residues of galactose residues to 
aldehydes. It was found necessary to incorporate protease inhibitors in 
the oxidation reaction mixtures. The oxidations were performed as follows: 
Galactose oxidase (Sigma Chemical Co., St. Louis, Mo.) was diluted to 0.5 
units/ml in 0.1M sodium phosphate (pH 7.0). Horseradish peroxidase (Sigma 
Chemical Co., St. Louis, Mo.) was diluted to 0.5 units/microliter in 0.1M 
sodium phosphate (pH 7.0). Digestions were performed in 0.15M sodium 
chloride, 45 mM sodium acetate, 20 mM sodium phosphate (pH 7.0). The 
final quantities of galactose oxidase and horseradish peroxidase were 15 
units per oxidation reaction and 20 units per oxidation reaction, 
respectively. All glassware, pipets, etc. were precoated with a 1 mg/ml 
solution of BSA. A reaction volume of 1.0 ml contained 150 microliters of 
asialo-sCD4 (about 0.5 ug), 30 microliters (0.15 units) galactose oxidase, 
20 units horseradish peroxidase, and 1 mg BSA. The following protease 
inhibitors were added to the oxidation reactions as follows: 20 
microliters 0.1M phenylmethylsulfonyl fluoride in toluene, 20 microliters 
0.1M 1,10-phenanthroline, 4 microliters 0.5M iodoacetamide in water. 
Control reactions included one without GO and HPO, and one without 
asialo-sCD4. Reactions were carried out in 15 ml conical centrifuge tubes 
precoated with BSA. After incubation for 66 hr at 25.degree. C., reaction 
mixtures were individually dialyzed to remove low molecular weight 
reaction products, toluene, etc. and to adjust the buffer environment to 
tissue culture-PBS. Immediately before injection, the control asialo-sCD4 
sample was prepared by combining the two control reaction tubes; thus the 
control asialo-sCD4 was exposed to GO and HPO for no more than about 1 
min. Analysis of the reaction products showed that there was insignificant 
proteolytic activity in the enzyme preparations if the above-noted 
protease inhibitors were included. 
Rats were injected with oxidized asialo-sCD4 and control asialo-sCD4. 
Radioactivity in liver and blood samples was determined at intervals after 
injection. The results are summarized in Table 3. At 20 min 
post-injection, there was approximately five-fold more label in the 
bloodstream for the asialo-sCD4 treated with GO and HPO as compared with 
control asialo-sCD4. The persistence of the oxidized asialo-sCD4 in 
circulation was clearly greater than that of asialo-sCD4 or sCD4 (See, 
e.g., Tables 1 and 3). 
The appearance of [.sup.125 I]-label in the bile was monitored in one rat 
injected with sCD4 and one injected with GO/HPO oxidized asialo-sCD4. Bile 
samples were collected at 15 min intervals over 2 hr post-injection and 
radioactivity was measured in each. The results are presented in Table 4. 
Over seven times as many [.sup.125 I]-sCD4-associated counts as [.sup.125 
I]-oxidized asialo-sCD4 enter the bile within the first two hours 
post-injection. The relative radioactivity associated with liver and bile 
is nearly twice as great for injection of the intact sCD4 as for the 
injection of oxidized asialo-sCD4. Thus, the oxidation of the galactose 
moieties of asialo-sCD4 appears to interfere with clearance from the 
bloodstream by hepatocytes. The results obtained in the foregoing 
experiments are consistent with clearance of asialo-sCD4 by the galactose 
receptor in hepatocytes. 
The estimated half-life of sCD4 in the circulation of rat is less than 15 
min, at least in the early phase of clearance. The estimated half-life of 
asialo-sCD4 is less than 5 min; clearance is linear. In contrast, the 
estimated half-life in circulation for oxidized asialo-sCD4 is greater 
than 6 hrs. Thus, treatment of asialo-sCD4 with galactose oxidase and 
horseradish peroxidase dramatically increases circulating half-life. 
Deglycosylated sCD4 was prepared to determine the effect of removal of the 
oligosaccharide side chains from the sCD4 proteins on circulating 
half-life and to determine the route of clearance of the sCD4 polypeptide 
from the bloodstream. 500 microliters of asialo-sCD4 was mixed with 4.2 
microliter of Endoglycosidase F in 5mM EDTA 50% glycerol (New England 
Nuclear, Boston, Mass.), 12.5 microliters of 0.2M EDTA and double 
distilled water to 1 ml. The corresponding control was identical except 
that 4.2 microliters of 5 mM EDTA in 50% glycerol was substituted for the 
endoglycosidase F. All tubes, dialysis tubing, pipet tips, etc. were 
precoated with BSA as above prior to use. Reaction and control tubes were 
incubated at 37.degree. C. for 2 hr. Then mixtures were dialyzed against 5 
mM EDTA for 5 hr and then against TC-PBS overnight at 4.degree. C. 
sCD4 was also treated to remove all oligosaccharides with a combination of 
Endoglycosidase F (New England Nuclear, Boston, Mass.) and N-glycosidase F 
(BEM, Indianapolis, Ind.). As above, all glassware and pipet tips were 
precoated with BSA. To a volume of 1 ml in 50 mM sodium phosphate (pH 
7.0), there was mixed 50 microliters of sCD4 mix and 195 microliter enzyme 
mix. The control in which oligosaccharides were not removed contained 195 
microliters 100 mM sodium phosphate, 25 mM EDTA, 50% glycerol in place of 
the enzyme mix. sCD4 mix was made by mixing 20 microliters of sCD4 with 
110 microliters of 1 mg/ml BSA. 
One rat was injected with the substantially deglycosylated sCD4 and another 
with the control sCD4 treated in parallel. Blood samples were taken at 
intervals and radioactivity was determined. The results are summarized in 
Table 5. It appears that the sCD4 polypeptide is cleared from the 
bloodstream somewhat more quickly than the corresponding intact 
glycoprotein molecule. These results suggest that clearance by the kidney, 
degradation and excretion in the urine is greater for the substantially 
deglycosylated sCD4 than for control glycosylated sCD4. 
TABLE 1 
__________________________________________________________________________ 
Distribution of [.sup.125 I]-sCD4 
in Liver and Blood Samples 
Time Input 
% Input % Input 
Rat Post-Injection 
Counts 
Counts Associated 
Counts in 
Material 
Number 
(min) (cpm) 
with Liver 
1 ml Blood 
Injected 
__________________________________________________________________________ 
R1 5 98,976 
27.2% 2.2% sCD4 
R2 5 113,076 
26.1% 2.3% 
R4 10 125,480 
12.3% 4.0% sCD4 plus 
Asialo-Fetuin 
R5 10 126,053 
19.8% 2.7% sCD4 plus 
Fetuin 
__________________________________________________________________________ 
TABLE 2 
__________________________________________________________________________ 
Distribution of [.sup.125 I]-Asialo-sCD4 
in Liver and Blood Samples 
Input 
% Input 
Time Radio- 
Radioactivity 
% Input 
Rat Post-Injection 
activity 
Associated 
Counts in 
Material 
Number 
(min) (cpm) 
with Liver 
1 ml Blood 
Injected 
__________________________________________________________________________ 
R11 10 124,954 
61.8% 0.6% Asialo-sCD4 
R14 10 129,942 
49.1% 0.6% Asialo-sCD4 
__________________________________________________________________________ 
TABLE 3 
__________________________________________________________________________ 
Distribution of [.sup.125 I]-sCD4 
in Liver and Blood for Asialo-sCD4 
with Intact and with Oxidized Galactose Residues 
Time Input 
% Input % Input 
Rat Post-Injection 
Counts 
Counts Associated 
Counts in 
Material 
Number 
(min) (cpm) 
with Liver 
1 ml Blood 
Injected 
__________________________________________________________________________ 
R26 10 141,621 
64.6% 0.64% Asialo-sCD4 
R27 20 140,393 
54.3% 0.48% 
R25 20 102,695 
23.3% 2.6% 
R28 120 144,118 
17.8% 1.9% GO/HPO-treated 
R23 360 204,864 
14.5% 1.6% Asialo-sCD4 
R29 1080 138,563 
10.5% 0.2% 
__________________________________________________________________________ 
TABLE 4 
__________________________________________________________________________ 
sCD4 Excretion Into Bile 
Time After Injection 
R3 (sCD4) R28 (GO/HPO-Asialo-sCD4) 
__________________________________________________________________________ 
15 min 1476 536 
30 min 6397 772 
45 min 8624 1094 
60 min 7611 1183 
75 min 6629 1038 
90 min 4719 928 
105 min 3716 884 
120 min 2980 760 
Total Bile: 42152 
(38% input) 
7195 (5% input) 
Total Bile & Liver: 
48963 34774 
Total Input Counts: 
111,638 144,118 
Approximate Percent of 
40-45% 24% 
Input Counts Associated 
with Bile and Liver 
__________________________________________________________________________ 
TABLE 5 
__________________________________________________________________________ 
Effect of Deglycosylation of sCD4 
(Endoglycosidase F + N-glycosidase F-treated Asialo-sCD4) 
on Distribution of [.sup.125 I]-sCD4 in Liver and Blood 
Time Total % Input 
Post- 
Input 
% Input % Input 
Count in 
Rat Injection 
Counts 
Counts Associated 
Counts in 
Urine & 
Material 
Number 
(min) 
(cpm) 
with Liver 
1 ml Blood 
Bladder 
Injected 
__________________________________________________________________________ 
R32 5 127,613 
ND* 1.3% ND Deglyco- 
sylated 
sCD4 
15 ND 0.81 ND 
30 ND 0.75 ND 
60 ND 0.53 ND 
90 ND 0.44 ND 
120 6.3% 0.23 25.4% 
R33 5 111,190 
ND 2.0% ND sCD4 
15 ND 1.13% ND 
30 13.3% 0.82% 0.1% 
__________________________________________________________________________ 
*ND = Not Determined