Note: Descriptions are shown in the official language in which they were submitted.
W095/27736 2 1 ~ 7360 F~,-l' 5- 19
Use of Agents ~hich Block T ~ - ' A~'
r~ ' ' 'Rec~ orl.. t~_ ' in the T - of
P~ Viral Infection
P '~ of the
FSeld of the Invention
The present inYention is directed to tbe use of agents which blo~k
ICAM-llreceptor as a means to iDcre~se gas exchimge m the llmgs
of a padent suffering from a Yiral infection of the respiratory trac~.
Descr~t~on of the Related Art
L Leukocyte Adhesion
l~ ukocytes must be able to attach to cellular substrates m order to
properly defend tbe host against foreign inYaders such as bacteria or Yiruses.
An excellent review of tbe defense system is proYided by Eisen, H.W., (In:
~' vb;v17~j~,3rdEd.,Halper~Row,F' ' '~' PA(1980),pp.290-295
and 381 118). Leul;ocytes attach to endotbelial cells so tbat ihey can migrate
from circulation to sites of ongorng ~ r. leukocytes
attach to antigen-presenting cells so that a normal specific immune response
can occur. Finally, leukocytes attach to appropriate target cells so that Iysis
of virally-infected or tumor cells can occur.
IL CD18 Famay
Leukocyte surface molecules involved m mediatrng such .
have been identified using hybridoma techmology. Briefly,
antibodies (nMAbs") directed against human T-cells (Davignon, D. et aL,
PrDC. Nat~. Acad. Sci. USA 78:4535-4539 (1981)) and mouse spleen cells
(Springer, T. et aL Eur. J. InununoL 9:301-306 (1979)) were identr~led which
W095/~7~36 21 87360 ~ 0'-'9
-2- ~
bound to leukocyte surfaces and ir~ubited the attachment related functions
deDcribed above (Springer, T. et al., Fed. Pr.,c. 44:266v-2663 (1985)). The
moleculeD ider~ified by these antibodies are caUed Mac-l, pl50,95 and
Lymphocyte r. Antigen-1 (LFA-1). Mac-1 iD fvund on
V~Ç~ D~ L .~i~D alld large dar 1~ . ~LD. LFA-1 iD fourld
on most 1.~ ~' ~ ,~D (Springer, T.A., et a~. Ir~noL R~v. 68:111-135
(1982)). These two molecules, plus plS0,95 (which ha~D a tissne ~' '
similar to Mac-1) play a role in cellular adhesion (l~eizer, G. et al., El~r. J.~mmunol. 15:1142-1147 (1985)). Molecules such as these three memberD of
the LFA-1 family, which are involved in the process of ceUular adhesion, are
refe~red to as "adhesion molecules. ~
The above-described leukocyte molecules were found to be ! '
sirnilar to one anvther, and to constitute members of a related family of
~ .v~l~ ' (r ~ r~ 1, F. et al., J. E~per. Med. 158:1785-1803
(1983); Kei~er, G.D. et al., Fr~r. J. I~nunoL 15:1142-1147 (1985)). This
xl~,w~. family is cvmposed of Lt~,.l having one alpha subuDit and
one beta subunit. Although the alpha subunit of each of the amtigens differs
from one member to the next, the beta subulut of each member is highly
conserved (~ l, F. ctaL, J. EJ~per. Med. 158:1785-1803 (1983)).
2v The beta subunit of the ~ I~W~lUt~ family (referred to as "CD18" family)
was fvund to have a molecular weight of 95 kd whereas the alpha subunits
were found to vary fçom 150 kd to 180 kd (Springer, T., Fed. Proc. 44:2660-
2663 (1985)). Although the alpha subunits of the membrane proteins do not
share the extensive homology s~ared by the beta subunits, close analysis of the
alpha subunits of the ~ V~IV~I ID has çevealed that there are substantial
similarities between them. Reviews of the similarities between the alpha and
beta subunits of the LFA-1 çelated x;J~v~lv~illD are provided by Sanchez-
Madrid, F. et al. (J. J~;per. Med 158:586-602 (19g3); J. Eacper. Med
158:1785-1803 (1983)).
Individuals have been identified who are unable to express normal
^Dmounts of any member of this adhesion pçotein family on their leukocyte cell
~ woss/27736 2 1 ~736û P~ slg
--3-
surfaces (Anderson, D.C., et aL, Fed. Proc. 44:2671-2677 (1985); Anderson,
D.C., et al., J. Infect. Dis. 152:668-689 (1985. The condition is known as
t'Leukocyte Adhesion Deficiency~ or ~LADt' syndrome. Leu~ocytes from
these patien~ts displayed in vitro defects similar to normal ~- ~ t l~-- - t` whose
CD18 farnily of molecules had been ~ " ' by antibodies. r.
these individuals are unable to mount a normal imtnune response due to an
inability of their cells to adhere to cellular substrates (Anderson, D.C., et al.,
Fed. Proc. 44:2671-2677 (1985); Anderson, D.C., et aL, J. Infect. Dis.
152:668-689 (1985)). LAD individuals present clinically with delayed
umbilical cord separation, recuning and ~ L ' ~, soft tissue infections, and
impaired pus formation, despite a striking blood I ~VDiD~ Studies of
LAD individuals have revealed that immune reactions are mitigated when
leukocytes are unable to adhere in a normal fashion due to the lack of
fun~ional adhesion molecules of the CD18 family.
~l1. IC4M-l
ICAM-l is a single chain ~ ,lut,~ varying in mass on different cell
types from 76-114 kD, and is a rncmber of the Ig , r ~1~ with five C-like
domains (Dustin, M.L. et al., Immlmol. Tod~y 9:213-215 (1988); Staunton,
D.E. aal., Cel~52:925-933 (1988); Sirnmons, D. etaL, Nature 331:624-627
(1988)). ICAM-1 is highly inducible with cytokines (including IFN- y, TNF,
and IL,1) in a wide range of cell types (Dustin, M.L. a al., Inununol. Tod~y
9:213-215 (1988)). Induction of ICAM-l on epithelial cells, endothelial cells,
and fibroblas~ts mediates LFA-l dependent adhesion of lr , ~, (Dustin,
M.L. et al., J. ImmunoL 137:245-254 (1986); Dustin, M.L. et al., J. CelL
BioL 107:321-331(1988); Dustin, M.L. et al., J. E)~p. Med 167:1323-1340
(1988)). Adhesion is blocked by l,lcl~ - of 1~ D with LFA-1
MAb or l,.~ic~.u...,.~t of the other cell with MAb to ICAM-l (Dustin, M.L.
et al., J. ImmunoL 137:245-254 (1986); Dustin, M.L. et al., J. Cell. Bio~.
107:321-331 (1988); Dustin, M.L. et al., J. Exp. Med 167:1323-1340
woss/27736 -7-1 87`36a 1~ 15I9
(1988. Identical rw~ults with purified ICAM-1 in ar~ficial ' or on
Petri dishw .' that LFA-1 and ICAM-1 are receptors for one
another (l\aarlin, S.D. et aL, Cell 51:813-819 (1987); Makgoba, M.W. et aL,
Natw~ 331:86-88 (1988)). For clarity, LFA-1 and ICAM-I are referred to
S herein as "receptor~ and "ligand,~ w~u~ Further ~ of
ICAM-1 are provided in U.S. Patent ~ ir9tit~nc Serial Nos. 07/045,963;
07/115,798; 07/155,943; 07/189,815 and 07/250,446, aU of which
~r are herein i~ul~ ' by reference in their enhrety.
~IV. r y ru~lnfectioA
The ability of leukocytw, especially I~L~ ~O to maintain the bealth
and viability of an animal requirw that they be capable of adhering to other
ceUs (such as endotheLal ceUs). This adherence has been fourld to reqlure
ceU-ceU contacts which involve specific receptor molecules present on the cell
surface of t_e l.J , ' , ~D. These receptorD enable a 1~ , ' ~ to adhere
15 to other 1~ w or to pn~ th~ and other .. ' cells. The cell
surface receptor molecules have been found to be highly related to one
another. Humans whose 1~ ,' J W lack thwe ceU surface receptor
molecules e~ibit defective antibody responses, chronic Dnd recurring
infections, as well as other clinical symptoms.
Acute viral respiratory iUnesses are amorlg the most common of humm
diseases, accolmting for or~half or more of all acute iUnesses. The incidence
of acute rwpiratory disease in the United Statw is from 3 to 5.6 cases per
person per year. The highest rates occur in children under 1 year of age (6.1
to 8.3 cases per year) and remain high Imtil age 6, when a ~
decrease is noted. Adults in the general population have three to four iUnesses
per person per year. Morbidity from acute respiratory illnesses accounts for
30 to 50 percent of time lost from work by adults and from 60 to 80 percent
of time lost from school by children.
.
W1~95127736 2 1 8 7360 ~ 9
--5-
It has been estimated tha1t two-thirds to three-fourths of cases of acute
respiratory iDnesses are caused by viruses. More than 200
distinct viruses from 8 different genera have been reported to cause acute
respiratory iDness, and it is lil~ely tbat additional agents wiU be described inS the future. The vast majority of these viral infections involve the upperrespiratory tract, but lower respiratory tract disease can also occur,
~, in younger age groups and in certain ~ ;, settings.
The iDnesses caused by respiratory viluses i ' "~, have been
divided into multiple distinct D,~ , such as the "common cold~,
~ croup (h~ ' ) t~acheitis, I,l, "
bronchitis, and I These general categories of illnesses have a
cert~in ~ lr.y,;- and clinical utility, e.g., croup occurs; ~ in
very young children and has a I ~ A ~ clinical caurse. In addition, some
types of respiratory iDnesses are more lilcely to be associated with certain
viruses, e.z., t~e "common cold~ with - Ylll~ D~ while others occupy
niches, such as a~llVVil~ _D in military recmits.
The syndrames most commonly associated with infection with the major
respiratory virus groups are ' in Table l. Despite these
- - -r ' -' ~ , it is clear that most respiratory viruses have the potential to cause
more than one type of respiratory illness, and frequently features of several
types of illness may be present in the same patient. Moreover, the clinical
iUnesses induced by these viruses are rarely sufficiently distinctive to enable
an etiologic diagnosis to be made on clinical grounds alone, although the
'"1 ~, setbng increases the likelihood that one group of viruses rather
than another may be involved. In general, laboratory methods must be relied
upon to establish a specific viral diagnosis.
W095/27736 ?18~3~0 r~ 9 --
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WO95/27736 2 1 8736 0 P~,l/L__ s. 1'19
--7-
S y ofthe ~ .. ~..
The present inverltion is based on the ul,~l . _ that agerhs which
block ICAM-l/receptor increase the rate of gas e~ichange in the
lungs of a mammal which is suffering from a reduction in gas exchange as a
result of a viral infection of the respiratory tract. ~ , ,,~y, these agentsdo not affect the airway ~ r ~ (e.g., ~ of asthma)
which also occurs as a result of the viral infection.
Based on these o~ v the present inverltion provides methods
for increasing the rate of oxygen absorption and CO2 - in the lungs
of a mammal suffering from a viral infection of the respiratory t~act.
~S~ , the rate at which o~ygen is absorbed into, and CO2 eliminated
from, the blood in the lungs of a mammal suffering from an infection of the
respiratory tract can be increased by providing a i' , "~, effective
amount of an agent which is capable of blocking ICAM-l/receptor
P~mples of the types of viral pathogens for which the present method
can be applied include, but are not limited to, members of the
r~u~JAuv..~, family, preferably viruses which are members of the
r- ~vu~ or r y.wvu~ genus. Specific viral pathogens include the
r~ , y Syncytial Virus and r ~ virus.
The present methods utilize agents which can be divided into two
groups based on the molecule the agent binds to (i.e., interacts with).
Group I agents are agents which bind to (interact with) ICAM-l and block the
binding of ICAM-l to a natural receptor of ICAM-l. Group II agerhs are
2~ agents which bind to a receptor of ICAM-l and block the binding of the
receptor to ICAM-l.
The agents of the preserlt inverltion include small molecules, peptides,
bullJ.' proteins and antibodies. A preferred class of agents of the
present invention are antibodies, or fragments thereof containing the antigen
.. . . . _ . _ _ _ _ _ _ _ _
w0 95127736 -2 1 8 7 ~ 6 ~ slg
--8-
binding site, which bind to ICAM-l (Group I agents) or to one or more
members of the CD18 family of l;I.~W~ (Group II agents).
Bnef D~." ~ of the Figures
Figure 1 ;' the total lurlg leukocytes recovered by whole
lurlg lavage from naive (normal) mice versus mice six days after
with control media, respiratory syncytial virus (RSV), RSV and treated with
cor~trol non-specific rat IgG (3 rnglkg, b.i.d.), or RSV and treated with the rat
anh--mouse ICAM~ anhbody YNl/1.7 (3 mg/kg, b.i.d.). Bars
represent the mean + S.E. for S animals per group. Asterisk (*) sigDifies
significant protechon by YNl/1.7 (anti-ICAM-1) compared to RSV alone as
well as RSV plus rat IgG heahment (p < 0.05 by Student's t-test).
Figure 2 ~ the lung diffusion capacity for carbon monoxide
(D~) from naive (normal) mice versus mice six days after inoculation with
cor~rol media, RSV, RSV and heated with control non-specific rat IgG (3
mg/kg, b.i.d.). Bars represent the me~m + S.E. for ~6 animals per group.
Asterisk (*) signifies significant protechon by YN1/1.7 (anh-ICAM-1)
compared to RSV alone as well as RSV plus rat IgG Ireatment (p < 0.05 by
Student's t-test).
Figure 3 ~~~~ the inbaled ' ' ' PC~ I (airway
1 -r ' V~ .. ) for mice six days after i~ c~ ion with control media, RSV,
RSV amd treated with control non-specific rat IgG (3 mg/kg, b.i.d.), or RSV
and treated with the rat anti-mouse ICAM-1 ~ 1 antibody YN1/1.7 (3
mg/kg, b.i.d.). Bars represent the mean + S.E. for 8-9 animals per group.
The RSV-induced decrease in the PC~0O (increase in airway l~ ;V~
was not prevented by anti-ICAM-1 (YN1/1.7).
WO 95127736 2 1 8 7 3 6 0 1~./1 . ~ .~19
_9_
Detailed D~s~,.r ~i. of the F~"~ F ~ '
The present invention is based on tbe ~ that agents which
block ICAM-l/receptor interaction increase tbe rate of gas exchange in the
llmgs of a mammal suffering from a reduction in gas excharlge as a result of
a viral infection of the respiratory tract. Based on these ~w.~, the
present invention provides methods for increasing the rate at which oxygen is
absorbed into and CO~ eliminated from the blood in the lungs of a mammal
suffering from a viral infection of t~e respiratory tract, prirnarily the lower
respiratory tract. S~,~;l~D~, tbe rate of oxygen absorption and CO
; " - csn be increased in a mammal suffering from an infection of the
respiratory tract by providing a i' r " 'Iy effective amount of an agent
which blocks ICAM-llreceptor
As used herein, an increase im the rate of gas exchsnge is said to occur
if the rate of exchange of gases across the lung membrane is increased. An
increase in gas exchange csn result in an increase in the rate or e~tent of
oxygen absorption and/or result in an increase in the rate of or extent of
carbon dioxide .' A skiDed ar!isan can resdily adapt known
procedures to determine the rate and exterlt of gas exchange in a particular
rnammsl in resporlse to a particular treatment.
As used herem, "a viral infection of the respiratory tract~ refers to any
viral mediated infection of cells which make up amd comprise the respiratory
t~act. Such ceDs include, but are not limited to epithelial ceDs, fibroblasts,
alveolar ~, , dendritic cells, and infiltratmg leukocytes (for a
description of the various cell types which make up the respiratory tract see
Plopper et ~1., Section I m C. , u~ Biology of ~e Normal LJ~ng, Vol. 1,
Parent, R.A., ed., CRC Press Inc., Boca Raton, FL (1992)).
The methods of the present invention are intended for use for viruses
which infect ceDs of the respiratory tract and fur~er lead to an increase or
induction of ICAM-l e~re~ r Since the present methods are directed to
' ~ a symptom common to respiratory viral infection amd are not
WO95/27736 21 87360 r~ c '519
-10-
directed at t~eating the specific viral agent, the present methods can be used
to augment the tr~atment of a wide variety of viral pathogens. Examples of
such viruses include, but are not limited to, members of the P .~AUV'
family, rnore ~ir~ viruses which are members of the r ~vi~ or
r ,~AUV 'I l.. genus. Sp~icific viruses which can be treated using the herein
disclosed metbods are the r , y Syncytial Virus and r ~ virus
(for a review of respiratory viruses see Dohn, "Common Viral r, y
Infections~ in Harrison's Principles of Internal Medicine, 11 edition,
McGraw-Hill N.Y. (1987) and Table 1).
In addition to the family of viruses, '~, described above, the
methods disclosed herein are effective in increasing the rate of gas eAchange
in the lungs for all viruses which infect cells of the respiratory tract and cause
an induction or an increase in ICAM-I expression on the surfaces of cells of
the respiratory tract (e.g.,; ~(' ' l, epithelial, r,h.,~ . alveolar
, ~ ' _ I.y , ' , h~, dendritic cells, etc.). As used herein, a virus is
said to induce or increase ICAM-l eApression when a cell produces a ~igher
level of ICAM-l as a result of the viral infection. A skilled artisan can use
known methods to assay for ICAM-l eApression in vivo or in vitro to
deter~nine if a particular virus induces ICAM-l expression (for eAample, see
Wagner et aL, Scienc~ 247:456-459 (1990)). Such procedures include, but
are not limited to, direct assays, methods which use mlcleic acid probes or
ICAM-l specific antlbodies to directly measure the level of ICAM-l
~, and indirect assays, methods which detect the presence of
cytokines known to induce ICAM-l expression. For example, interferon
garluna, ' ' 1, and tumor-necrosis factor, are cytokines which are
known to induce ICAM-l expression (Wagner et al., Science 247:456-459
(1990); Pober et aL, J. Irnm~nol. 137:1893-1896 (1986)).
As used herein, an agent is said tû "block ICAM-l/receptor
" if the agent is capable ûf reducing the rate at which ICAM-I
3û binds to a receptor. There are two targets for the agents of the present
invention. Group I agents are agents which bind to ICAM-1 and block the
wO gs/27736 2 1 8 7 3 6 û A ~.~ 519
-11-
binding of ICAM-l ho a natuIal recephor of ICAM-l. Group II agents are
agents which bind ,ho a member of the CD18 family of ~ ly~ , Group
II agents c7m be designed ho bind ,ho all members of the CD18 family of
~Iy~ h or can be designed ,ho bind ,ho a specific member of the CD18
S family (Springer T.A., Nature 346:425-434 (1990)).
Assays have been developed ho deher~nine if an ager~ c7m block ICAM-
l/receptor ;---` ~ (see Rotblem etaL, J. Inununol. 137:1270-1274
(1986); Smith et al., J. Clin. Irrvest. 82:1746-1756 (1986)) for ex7unples). In
general, these procedures compare the level of ICAM-l/CD18 im
the presence and abserlce of tbe agent which is h-sted. The forlnat of such
assays varies and c7m include the use of isolahod ICAM-l and/or CD18
prohein, cells which nah~rally express ICAM-l or CD18, or cells which have
been alh~red to express ICAM-l or CD18. A sl~lled artis7m can readily use
these methods, or a . - thereof, to isolahe agents for use im the
methods herein described.
A preferred class of agents of the present inverltion are amtibodies, or
fragments thereof containing the antigen birlding sihe, which bind ,ho ICAM-1
(Group I agents) or ,ho a member of the CD18 family of ~Iy.,~ ~ (Group
II agents). ICAM-1 and the members of the CD18 farnily of lecules are
~ molecules. Thus, a skilled artisan can routinely obtain
antibodies which bind ho ICAM-1 o} one or more rnembers of the CD18
family of molecules. Group I agerlts include amtibodies, and fragments
thereof, which bind to ICAM-l. Group II agent mclude antibodies, and
fragments thereof, which bind a member of the CD18 family of 7,1y~u,J.,
The generation of amti-ICAM-l and anti-CD18 antibodies is well
known in the art (Harlow et al."' ' ' A Laboratory Manual, Cold
Spring Harbor Press, Cold Spring Harbor, NY (1989)). In general, the
amtibody agents of the present invention may be obtained by illLlUdU~ either
a purified prohein, or cells which express the desired protem, into an
.. ~lul animal, forexampleby , ' injection, ehc. The serum
of such an animal may be removed and used as a source of polyclonal
W0 95127736 2 1 8 7 3 6 0 r~ a 1~l9
-12-
anhbodies capable of binding these molecules. ~ , a skilled artisan
can remove ~h,~ from such animals, fuse these spleen cells with a
mydoma cell li~e to form a hybridoma cell which secretes a ~
arltibody which binds ICAM-1 or a member of the CD18 family of molecules.
S The hybridoma cells, obtained in the manner described above ;nay be screened
using known methods to idenhfy desired hybridoma cells that secrete an
anhbody which binds to either ICAM-1 or to members of the CD18 family of
molecules (either the alpba or beta subunit).
Both polyclonal and ' ' arltibodies may be used in tbe metbods
of the present inveraion. Of special iraerest to the present inverltion are
anhbodies to ICAM-l or to members of the CD18 family, which are produced
in humans, or are "' '~ (i.e., non ~ m a human) by
or other technology. TT ~ araibodies may be produced,
for example by replacing an , portion of an antibody with a
wl r ' but non- _ portion (i.e., chimeric anhbodies)
(Better, M. etal., Science 240:1041-1043 (1988); Liu, A.Y. e~aL, Proc.
NatL Acad. Sci. USA 84:3439-3443 (1987)), or through the process of
,' ~' region (CDR) grafhng (Jones, P.T. et al., Nature
321:552-525 (1986); Verhoeyan et al., Science 239:1534 (1988); Beidler,
C.B. et aL, J. Immunol. 141:4053-4060 (1988)).
Another class of agent which carl be used in the present mvenhon are
soluble forms of ICAM-1 or members of the CD18 family of ~ u~ll
Because ICAM-1 binds to a CD18 molecule, soluble derivahves of ICAM-1
comprise another type of Group 11 agent Soluble derivatives of ICAM-1
which bind to the CD18 family member reduce the rate of ICA~-1/receptor
binding by competing with the CD18 found on leukocytic cells, thus
attenuating cellular adhesion.
ICAM-I is composed of 5 domains (Staunton, D.E. et al., Immunol.
Today 9:213-215 (1988~; Staunton, D.E. et a~., Cell 52:925-934 (1988);
Staunton, D.E. et al., Cell 56:849-854 (1989); Staunton, D.E. et al., Tissue
Antlgens 33:287 (1989)). Domains 1 and 2 have been found to be important
-
WO 95n7736 2 1 8 7 3 6 0 r~ 9
-13-
for the binding of ICAM-l to its receptor molecule (Staunton, D.E. et aL,
Tissrle Ar~gens 33:286 (1989); Staunton, D.E. et aL, FASEB J. 3:A446
(1989)). Fragments of ICAM-l from which the i domain hæ
been deleted, and which possess at leæt domains 1 and 2, are soluble under
S ~h, O -' conditions and can block ICAM-l/receptor (13ecker
a al., J. Imr~nol 147:4398~401 (1991)).
Soluble derivatives of CD18 family members comprise another type of
the Group I agents of tbe present invention. As used herein, a molecule is a
member of the CD18 farnily of ol.~v~.. if it cor~ains eitber an alpha
10 subunit of a member of the CD18 family of O~ ~ (i.e., a CDll
subunit), a beta subunit of a member of the CD18 farnily Of ol~
(i.e., a CD18 beta subunit), or both an alpha and a beta subunit of a member
of the CD18 family of ol~o~ Thus, as used herein, a member of the
CD18 farnily of ol~ includes molecules having only one subunit of
15 a CD18 me~nber æ well æ ~lud~ (molecules having botb an alpha and
a beta subunit of a member of the CD18 farnily). Soluble derivatives of
members of tbe CD18 farnily have been generated by deletirlg the
domair (Dana et aL, Proc. NatL Acad. Sci. USA 88:3106-
3110 (1991)). These molecules have been shown to reduce the rate of ICAM-
20 llligand binding by binding to ICAM-l.
There are r~merous procedures known in the art to æsay for ICAM-
l/receptor These can be used by a skilled artisan, without undue
~, . to identify and isolate additional Group I and Group II agerlts
for use in tbe herein disclosed methods. The agents screened in such æsays
25 can be, but are not limited to, peptides, ~I,vh,. small molecules, or
vitamm derivatives. The agents can be selected and screened at r~mdom or
rationally selected or designed using known protein modeling x , For
ramdom screening, agerLts such as peptides or ~ ' ' J.- ' are selected at
random amd are æsayed for the ability to bind to ICAM-I or a CD18 family
30 member. Alternatively, agents may be rationally selected or designed. As
used herein, an agent is said to be "rationally selected or designed" when the
, . . _ _ _ _ _ . .: .
WO95/27736 2 1 87 3 60 ~ 9
-1~
agent is chosen based on the rnolecular ~ 5, of the ICAM-l or a
CD18 farnily =ber. For example, one skilled in the art can readily adapt
currently available prccedures to generate peptides capable of binding to a
specific peptide sequence in order to generate rationally designed antipeptide
peptides (for example, see Hurby et a~., "Application of Synthetic Peptides:
Antisense Peptides~, In Synthenc Pepndes, A User's Guide, W.H. Freeman,
NY, pp. 289-307 (1992), and Kaspczalc e~al., Bi~ 28:9230-8
(1989)).
The agents of the present invention can be used in native form or can
be modified to form a chemical derivative. As used herein, a molecule is said
to be a "chemical derivative" of ather molecule when it contains additional
chemical moieties not normally a part of the molecule. Such moieties rnay
improve the molecule's solubility, absorption, biological half life, etc. The
moieties may ' ~ decrease the toxicity of the molecule, eliminate or
atter~ate any ~ ' ' ' side effect of tbe molecule, etc. Moieties capable
of mediating snch effects are disclosed in P~e~ungton's rl~, ~ '
Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980.
For example, a change inthe ' ~- ' character of the functional
derivative, such as afflnity for a given antibody, is measured by a; . ~.
type ~. Changes in ' ' activity are meæured by
the ,~ , assay. M~ -r__-- of such protein properties as redox or
thermal stability, biological half-life, h~ to
proteolytic ~." ' or the tendency to aggregate with carriers or into
multimers are æsayed by methods well known to the ordinarily skilled ar~isan.
The therapeutic effects of the agents of the present irlverltion may be
obtained by providing the agent to a patient by any suitable means (i.e.,
inbalation, i.~ v~l~, ' l~" ' l~" enterally, or
'1~). It is preferred to adrninister the agent of the present invention
so as to achieve an effectiYe t within the blood or within the
lungs. For achieving an effective . withinthe lungs, the preferred
method is to administer the agent as a nebulized solution by oral inhalation,
W0 95/27736 2 18 7 3 6 ~ r~l" ISI9
-15-
or via an oral spray or oral aerosOl. Alh,lla~ ly, intr,a-nasal or; ~ . h ,
can be employed to achieve an effective lung
To achieve an effective blood the preferred method is
to administer the agent by injection. The may be by continuous
infusion, or by single or multiple injections.
In providing a patient with~ antibodies, or fragments thereof, capable
of binding to ICAM-1 or to a member of the CD18 family, or when providing
a soluble form of ICAM-l or a member of the CD18 family, the dosage of the
~ agent will vary depending upon such factors as the patient's age,
weight, height, se~, general medical condition, previous medical history, etc.
In general, it is desirable to provide the recipient with a dosage of agent which
is in the range of from about 1 pg/~g to 10 mgll~g (body weight of patient),
although a lower or higher dosage may be - ' The ~
effective dose can be lowered by using ~ ' of the agents of the
present invention (such as, for e~ample, if arlti-ICAM-I ar~ibody is
with an aoti-LFA-l antibody).
As used herein, two or more , ' are said to be
nin ' ~ with each other when either (1) the 1' JD;ùl~tj;.,al effects of
each compound or (2) the serum of each compound can be
measured at the same time. The , of the present invention can be
, with, prior to, or following the ' of
other anti-viral or anti ~IJ' .11.~1... ~.,.~ agents.
The agents of the present invention are intended to be provided to
recipient subjects in an amount sufficient to increase the rate of lung gas
exchange and thus attenuate the morbidity ( , y distress or dyspnea) of
an infection of the respiratory tract.
The ' of the agent(s) of the inverltion may be for either
a ~ u~ yla~ ," or ~i' r '- n purpose. Whenprovided ~lulJhyla~ !y~
the agent(s) are provided in advance of any decrease in the rate of gas
exchange. The ~lu~h.~ ,~ ' of the agent(s) serves to prevent
or attenuate any subsequent reduction in gas exchange. When provided
.
woss/27736 2:1 ~7-36~ P~ 01519
-16-
~" the agent(s) are provided at (or shortly after) the onset of a
reduction in the rate of gas exchange. The IL~.a~.,t;,, of the
compound(s) serves to attenuate any ac~al reduction in gas exchange. Thus,
the agents of the present invention may thus be provided after respiratory viralS infection and either prior to the onset of a reduction in gas exchange (so as to
attenuate the anticipated severity, duration or extent of the reduction) or after
the initiation of the reduction.
The agents of the present invention are ~ to the mammal in
a l' '1y acceptable form and in a i , 'l~ effective
~ A ~ , is said to be "~ acceptable" if
its ~ can be tolerated by a recipient patient. Such an agent is said
to be -' ' in a , 'ly effective amount~ if the amount
' ' is l~. ' ,. "~, ~ ~ An agent is ~ '~
significant if its presence results in a detectable change in the physiology of
a recipient patient.
The agents of the present invention can be formulated according to
known methods to prepare l' 'l,~ useful , , whereby
these materials, or their functional derivatives, are combined in admixture
with a, 'l~ acceptable carrier vehicle. Suitable vehicles and their
r.,.. ,1--;" " inclusive of other human proteins, e.g., human serum albumin,are described, for example, P " 's rh~ Sciences (16th ed.,
Osol, A., Ed., Mack, Easton PA (1980)). In order to form a
' "~ acceptable r '- suitable foreffective
such ~ , will cor~ain an effective amount of one or more of the
agents of the present invention, together with a suitable amount of carrier
vehicle.
Additional l- methods may be employed to control the
duration of action. Control release ~ JalaLiOl~ may be achieved through the
use of polymers to complex or absorb one or more of the agents of the present
invention. The controlled delivery may be exercised by selecting a~ ul
Illa~lu .~ w (for example polyesters, polyamino acids, polyvinyl,
360
W095/27736 2 1 8 7
-17-
or protamine, sulfate) and the of ~n ' as well as the
methods of . in order to cor~rol rele3se. Another method to
control the duration of action by controlled release l ~ is to
i~Vl~ ' agents of the prewerlt invention irlto particlw of a polymeric
material snch as polywters, polyamino acids, hydrogels, polytlactic acid) or
ethylene ~-, ' co~ , instead of , ,, these
agents into polyrneric palticlw, it is possible to er~rap thwe rnaterials in
~, ' prepared, for example, by ~ ~ techniques or by
interfacial ~ yielding, for example, h~.Lu~l,l.,;h~ ' or
g~ - ,, ' and pol.~ I~_lh~ ) IIPl,.~ ulew,
~L~ , or in colloidal drug delivery systerns, for example, liposomes,
alburnin 1~ ' and , ' or in
' Snch techniques are disclosed in Remington's
rl~ ~ Sciences tl6th ed., Osol, A., Ed., Mack, Easton PA (1980)).
Having now generally described the inver~tion, the same will be more
readily understood tbrough reference to the following examplw which are
provided by way of ' and are not intended to be limiting of the
preserlt inverltion, unless specified.
h~ ,
~xample I
As mentioned above, respiratory viral infections, and in particular
infection with respiratory syncytial virus (RSV), are a major cause of
hnq-if~l in infants, the elderly and patier~s with, ~
l~ui Several Imes of evidence suggest that the morbidity of these
infections is a ~ of the immune response rather than the cytopathic
effects of the virus (Stott et al., J. Virol. 61:3855-3861 (1987); Murphy a al.,Vaccine 8:497-502(1990)). Theobjectiveofthe I~; t~ describedmthis
WO95/27736 ' 21 8~360 ~ . L s ~
--18-
example was to ~ ;,. tbe time course of the ' ~ y
response (leukocyte influx and cytokine generation) and viral replication
ir~uced by R~,V. as well as tbe lung ~ and increase in airvay
~r~ ' ;~ ;' of asthma onset and severity (Busse et al., J.
Allergy Clin. brun~nol. U:M0-775 (1988 at the peal~ of the '' y
response in mice.
Female Balb/c mice,--32 weeks of age, were inoculated by intranasal
~ ( ' ' ) - ' of 107 plaque forming units (PFIJ) of
RSV A2 strain or cor~ol (HEp-2) media. The mice were sacrificed 1, 3, 6,
15 or 28 days later and (1) lur~ lavage was perforlned to assess leukocyte
infl~x (Wegner et al., Lung 170:267-279 (1992 and cytokine generation
(EIA usrng "~ available kits), (2) lung I O were prepared
and used in plaque assays on HEp-2 cells to assess viral replication (Grah~n
etal., J. M~d. ~rol 26:153-162 (1988)), or (3)1ungs were fixed for
histologic For evaluation of pulmonary fimction, the rnice were
' with 1 ' ' ' and their tracheas camlulated with a 18 gauge
catheter. ~ , ~ system resistance (Rrs) and dynamic A~' (Crs)
were ,' ' from respiratory system impedance which was measured by
discrete fre~uency (4 to 40 Hz in 11 equal I , steps) ' ' forced
oscillations ! 1 A ~ on tidal breath n~ (WeOPner et al., Lung 170:267-279
(1992)). Diffusion capacity of the lungs for carbon nnonoxide (D,~ was
determined by the srngle breath method. Ainvay -r ~ was assessed
by ~' ., the: of nebulized and inhaled ~ ' ' to
produce a 100% increase in Rrs (PC~ This was , by
-' ~ increasing of ' ' ' (diluted with
buffered saline) in _alf 1n~rithrn;r steps (at--10 rninute intervals)
until a > 100 % increase in Rrs from baseline was obtained. The PCIOD was
then calculated by linear regression analysis of the last 2 or 3 points on the
th~:- ".. :1.-- l.n~ ' ' versus percent increase in Rrs plot.
W0951~7736 ~ ; 6~ P~ 51~ 19
_19_
Results:
Viral titers pea~ed at day 3, leulcocyte influ~ at day 6, TNF~,
GMCSF and IL,6 generation at day 1, and IFN~y at day 6 (see Table 2). On
day 6 (peak A '- ), RSV infection decreased lung diffusion capacity
S (DLCO 16.6 i 0.5 to 14.0 i Q6 ,ul/ ' TT.e p < 0.05) and increased
air~ay l--r T~II~ (decreased the ' ' PC,00: 0.?2 i 0.22 to 0.06
i 0.02 mg/ml, p < 0.05) without ,, ~ , altering Rrs (79? i 87 to 799
i 71 crnH20/l/s) or Crs (35.8 i 5.7 to 28.3 i 2.8 ul/cmH20).
Thus, RSV infection in mice induces a marked lung ~ and
~ r ,.
WO95/~7736 2 1 87 360 P~ Sl9
-20-
a ~ ~ ~ a I
O O o~
a ~ ~ ~ a
U~
,~ ~ a ~ a a a ~ -
~ j,,
L~ a ~ a a a I
æ~
O
~ + ~ ~ V
5-~ o + +~ o o ~,,
~) O O
3 ~ o
O ~
a
0. 0
WO95/27736 2 1 87360 p~" -'19
-21-
Example 2
Since RSV infection was found to stimulate the in vivo lung
generation of cytokines (INFa and IFNr) known to increase ICAM-I
expression in vitro, ~ y was employed to deternine if
' (infection) with RSV enbanced ICAM-l expression in vivo.
Female Balb/c mice, ~ 32 weeks of age, were inoculated by intranasal
~ ( ' ' ) ' of 107 plaque forming units (PFU) of
RSV A2 strain or control (HEp-2) media and then sacrificed 6 days later.
Lungs were removed, inflated with O.C.T. and then frozen in liquid nitrogen.
After being ~ l, 5-10 ~un sections were fixed in acetone, stained
with the rat anti-mouse ICAM-I ' ' antibody YNl/1.7 or rat IgG (as
a control for non-specific binding), and developed using l,luli~' ' goat
anti-rat IgG lir~ked with ~Iu~u~-conjugated ~I~c~;~ and visualized
with 3~ elh~ (AEC) (Wegner etaL, lung 170:267-279
(1992)). The sections were ' with Mayer's I .~"
lts:
As reported previously (Wegner et al., Lto2g 170:267-279 (1992);
Kang et aL, Am. J. ~e~7ir. Cell A~olecular BioL 9:350-355 (1993)), a slight
but distinct basal level of ICAM-I expression was observed on the peripheral
lung ~UCl.. L~lllal cells (mostly on type I 1 yt~ a) and alveolar
Illa.~lUI/llat,_.~ in naive (normal) mice as well as in those inoculated with
control media Infection with RSV induced a marked ~ ' of
ICAM-I expression on lung ~ cl.~L~Illa cells as well as on alveolar
.ll~lu~ t~ and infiltrating ...-- . - lr~ Ieukocytes. Linle, if any, non-
25 specific 1,.~ ' staining was observed even in the RSV infected mice.
Thus, ICAM-I expression is strikingly L~JIC,, ' ' ' on lung cells and
infiltrating leukocytes after RSV infection.
WO 95117736 ~ 9
21 ~t3~0
-22-
Exampl~ 3
T.he ' of ICAM-I tothe A ' y response, attenuated
alveolar gas exchange and increase in airway ~ w induced by RSV
infection was evaluated using the rat anti-mouse ICAM~
5antibody YNI/1.7.
Female Balb/c mice were inoculated by intranasal ~
(inhalation) ~ ' of 10~ plaque forrning units (PEiU) of R5V A2
strain or control (HEp-V media and treated twice daily (b.i.d.) beginning I
hr prior to RSV inoculation with YNI/1.7 or rat IgG at 3 mg/kg,
, l On day six after inoculation (pw~ n '- ), lung
lavage leukocyte counts ( n ' )> DLCO (lung gas exchange), and the
inhaled ' ' ' - PC,00 (airway lC~O~;~ ,) were deterluined as
described above in Example 1.
Rcs~
RSV infection induced a marked influx of ' cells tbat was
e~ rlwllly inhibited (65 - 80%) by YNl/1.7 (anti-ICAM-I) but not by
control rat IgG (Figure 1). Likewise, the RSV-induced decrease m lung gas
exchange (DL~) was ~ and completely attenuated by YNI/1.7
treatment but not by control rat IgG (FiBure 2). In contrast, the RSV-
induced increase in airway lc~ I.c.~ (decrease in ._ ' ' ' PC,00)
was not inhibited by YNI/1.7 treatment (Figure 3).
These results indiwte that the acute symptoms of dyspnea associated
with impaired alveolar gas exchange are linked to the intense r~ . y
response (leukocyte infilfr~fit-n) induced by RSV infection and can be
~ .ly attenuated by blocking ICAM-I/receptor; ~ However,
the more chronic astbma-like wheezing symptoms, which are likely due to
an RSV-induced increase in air vay Ic~ .a:~ are not the result of the
. . . n . . ., y response but rather possibly the cytopatbic effects of the virus.
WO 9Sr27736 2 1 8 7 3 6 0 ~ 9
--23-
Thus, tbe impaired llmg gas exchange tbat causes the acute morbidity
and I, ~ associated ~rith respiratory viral infections can be
, ~,1.~ attenuated by _ of ICAM-I/receptor
However, optimal tberapy which includes the prevention of tbe onset of
S as~bma would require . ' of tbis ICAM-I ~ witb the
' of an anti-viral agent (e.g. Ribavirin for RSV).
