Antibodies to ICAM-2, and fragments thereof

The present invention relates to intercellular adhesion molecules (ICAM-2) which are involved in the process through which lymphocytes recognize and migrate to sites of inflammation as well as attach to cellular substrates during inflammation. The invention is directed toward such molecules, screening assays for identifying such molecules and antibodies capable of binding such molecules. The invention also includes uses for adhesion molecules and for the antibodies that are capable of binding them.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to the intercellular adhesion molecule-2 
("ICAM-2") which is involved in the process through which populations of 
lymphocytes recognize and adhere to cellular substrates so that they may 
migrate to sites of inflammation and interact with cells during 
inflammatory reactions. The present invention additionally relates to 
ligand molecules capable of binding to ICAM-2 intercellular adhesion 
molecules, and to uses for the intercellular adhesion molecule, and the 
ligand molecules. 
2. Description of the Related Art 
Leukocytes must be able to attach to cellular substrates in order to 
properly defend the host against foreign invaders such as bacteria or 
viruses. An excellent review of the defense system is provided by Eisen, 
H. W., (In: Microbiology, 3rd Ed., Harper & Row, Philadelphia, Pa. (1980), 
pp. 290-295 and 381-418). They must be able to attach to endothelial cells 
so that they can migrate from circulation to sites of ongoing 
inflammation. Furthermore, they must attach to antigen-presenting cells so 
that a normal specific immune response can occur, and finally, they must 
attach to appropriate target tells so that lysis of virally-infected or 
tumor cells can occur. 
Recently, leukocyte surface molecules involved in mediating such 
attachments were identified using hybridoma technology. Briefly, 
monoclonal antibodies directed against human T-cells (Davignon, D. et al, 
Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)) and mouse spleen cells 
(Springer, T. et al. Eur. J. Immunol. 9:301-306 (1979)) were identified 
which bound to leukocyte surfaces and inhibited the attachment related 
functions described above (Springer, T. et al., Fed. Proc. 44:2660-2663 
(1985)). The molecules identified by those antibodies were called Mac-1 
and Lymphocyte Function-associated Antigen-1 (LFA-1). Mac-1 is a 
heterodimer found on macrophages, granulocytes and large granular 
lymphocytes. LFA-1 is a heterodimer found on most lymphocytes (Springer, 
T. A. et al. Immunol. Rev. 68:111-135 (1982)). These two molecules, plus a 
third molecule, p150,95 (which has a tissue distribution similar to Mac-1) 
play a role in cellular adhesion (Keizer, G. et al., Eur. J. Immunol. 
15:1142-1147 (1985)). 
The above-described leukocyte molecules were found to be members of a 
related family of glycoproteins (Sanchez-Madrid, F. et al., J. Exper. Med. 
158:1785-1803 (1983); Keizer, G. D. et al., Eur. J. Immunol. 5:1142-1147 
(1985)), termed the "CD-18 family" of glycoproteins. This glycoprotein 
family is composed of heterodimers having one alpha chain and one beta 
chain. Although the alpha chain of each of the antigens differed from one 
another, the beta chain was found to be highly conserved (Sanchez-Madrid, 
F. et al., J. Exper. Med. 158:1785-1803 (1983)). The beta chain of the 
glycoprotein family (sometimes referred to as "CD18") was found to have a 
molecular weight of 95 kd whereas the alpha chains were found to vary from 
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 shared by the beta subunits, close analysis of the alpha subunits 
of the glycoproteins has revealed that there are substantial similarities 
between 10 them. Reviews of the similarities between the alpha and beta 
subunits of the LFA-1 related glycoproteins are provided by 
Sanchez-Madrid, F. et al., (J. Exper. Med. 158:586-602 (1983); J. Exper. 
Med. 158:1785-1803 (1983)). 
A group of individuals has been identified who are unable to express normal 
amounts of any member of this adhesion protein family on their leukocyte 
cell surface (Anderson, D. C. et al., Fed. Proc. 44:2671-2677 (1985); 
Anderson, D. C. et al., J. Infect. Dis. 52:668-689 (1985)). Lymphocytes 
from these patients displayed in vitro defects similar to normal 
counterparts whose CD-18 family of molecules had been antagonized by 
antibodies. Furthermore, these individuals were unable to mount a normal 
immune 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)). These data 
show that immune reactions are mitigated when lymphocytes are unable to 
adhere in a normal fashion due to the lack of functional adhesion 
molecules of the CD-18 family. 
Thus, in summary, the ability of leukocytes to maintain the health and 
viability of an animal requires that they be capable of adhering to other 
cells (such as endothelial cells). This adherence has been found to 
require cell-cell contacts which involve specific receptor molecules 
present on the cell surface of the leukocytes. These receptors enable a 
leukocyte to adhere to other leukocytes or to endothelial, and other 
non-vascular cells. The cell surface receptor molecules have been found to 
be highly related to one another. Humans whose leukocytes lack these cell 
surface receptor molecules exhibit chronic and recurring infections, as 
well as other clinical symptoms including defective antibody responses. 
Since leukocyte adhesion is involved in the process through which foreign 
tissue is identified and rejected, an understanding of this process is of 
significant value in the fields of organ transplantation, tissue grafting, 
allergy and oncology. 
SUMMARY OF THE INVENTION 
The present invention relates to Intercellular Adhesion Molecule-2 (ICAM-2) 
as well as to its functional derivatives. The invention additionally 
pertains to antibodies and fragments of antibodies capable of inhibiting 
the function of ICAM-2, and to other inhibitors of ICAM-2 function. The 
invention additionally includes diagnostic and therapeutic uses for all of 
the above-described molecules. 
In detail, the invention includes the intercellular adhesion molecule 
ICAM-2, or a functional derivative thereof, substantially free of natural 
contaminants. 
The invention further pertains to ICAM-2 which contains at least one 
polypeptide selected from the group consisting of: 
(a) -S-S-F-G-Y-R-T-L-T-V-A-L-; 
(b) -D-E-K-V-F-E-V-H-V-R-P-K-; 
(c) -G-S-L-E-V-N-C-S-T-T-C-N-; 
(d) -H-Y-L-V-S-N-I-S-H-T-D-V-; 
(e) -S-M-N-S-N-V-S-V-Y-Q-P-P-; 
(f) -F-T-I-E-C-R-V-P-T-V-E-P-; 
(g) -G-N-E-T-L-H-Y-E-T-F-G-K-; 
(h) -T-A-T-F-N-S-T-A-D-R-E-D-; 
(i) -H-R-N-F-S-C-L-A-V-L-D-L-; 
(j) -M-V-I-I-V-T-V-V-S-V-L-L-; 
(k) -S-L-F-V-T-S-V-L-L-C-F-I-; and 
(l) -M-G-T-Y-G-V-R-A-A-W-R-R-. 
The invention also provides a recombinant or synthetic DNA molecule capable 
of encoding, or of expressing, ICAM-2 or a functional derivative thereof. 
The invention additionally provides an antibody, and especially a 
monoclonal antibody, capable of binding to a molecule selected from the 
group consisting of ICAM-2, and a functional derivative of ICAM-2. 
The invention also provides a hybridoma cell capable of producing the 
above-described monoclonal antibody. 
The invention includes a method for producing a desired hybridoma cell that 
produces an antibody which is capable of binding to IISAM-2, or its 
functional derivative, which comprises the steps: 
(a) immunizing an animal with an imunogen selected from the group 
consisting of: a cell expressing ICAM-2, a membrane of a cell expressing 
ICAM-2, ICAM-2, ICAM-2 bound to a carrier, a peptide fragment of ICAM-2, 
and a peptide fragment of ICAM-2 bound to a carrier, 
(b) fusing the spleen cells of the animal with a myeloma cell line, 
(c) permitting the fused spleen and myeloma cells to form antibody 
secreting hybridoma cells, and 
(d) screening the hybridoma cells for the desired hybridoma cell that is 
capable of producing an antibody capable of binding to ICAM-2. 
The invention also provides a method for treating inflammation resulting 
from a response of the specific defense system in a mammalian subject 
which comprises providing to a subject in need of such treatment an amount 
of an anti-inflammatory agent sufficient to suppress the inflammation; 
wherein the anti-inflammatory agent is selected from the group consisting 
of: an antibody capable of binding to ICAM-2; a fragment of an antibody, 
the fragment being capable of binding to ICAM-2; ICAM-2; a functional 
derivative of ICAM-2; and a non-immunoglobulin antagonist of ICAM-2 other 
than ICAM-1, or a member of the CD-18 family of molecules. 
The invention also includes a method of suppressing the metastasis of a 
hematopoietic tumor cell, the cell having a member of the CD-18 
(especially LFA-1) for migration, which method comprises providing to a 
patient in need of such treatment an amount of an agent sufficient to 
suppress the metastasis; wherein the agent is selected from the group 
consisting of: an antibody capable of binding to ICAM-2; a 
toxin-derivatized antibody capable of binding to ICAM-2; a fragment of an 
antibody, the fragment being capable of binding to ICAM-2; a 
toxin-derivatized fragment of an antibody, the fragment being capable of 
binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; a 
toxin-derivatized ICAM-2; and a toxin-derivatized functional derivative of 
ICAM-2; and a non-immunoglobulin antagonist of ICAM-2 other than ICAM-1, 
or a member of the CD-18 family of molecules. 
The invention also includes a method of suppressing the growth of an 
ICAM-2-expressing tumor cell which comprises providing to a patient in 
need of such treatment an amount of an agent sufficient to suppress the 
growth, wherein the agent is selected from the group consisting of: an 
antibody capable of binding to ICAM-2; a toxin-derivatized antibody 
capable of binding to ICAM-2; a fragment of an antibody, the fragment 
being capable of binding to ICAM-2; a toxin-derivatized fragment of an 
antibody, the fragment being capable of binding to ICAM-2; ICAM-2; a 
functional derivative of ICAM-2; a non-immunoglobulin antagonist of ICAM-2 
other than ICAM-1, or a member of the CD-18 family of molecules; a 
toxin-derivatized member of the CD-18 family of molecules; and a 
toxin-derivatized functional derivative of a member of the CD-18 family of 
molecules. 
The invention also provides a method for detecting the presence of a cell 
expressing ICAM-2 which comprises: 
(a) incubating the cell or an extract of the cell in the presence of a 
nucleic acid molecule, the nucleic acid molecule being capable of 
hybridizing to ICAM-2 mRNA; and 
(b) determining whether the nucleic acid molecule has become hybridized to 
a complementary nucleic acid molecule present in said cell or in said 
extract of said cell. 
The invention also provides a pharmaceutical composition comprising: 
(a) an anti-inflammatory agent selected from the group consisting of: an 
antibody capable of binding to ICAM-2; a fragment of an antibody, the 
fragment being capable of binding to ICAM-2; ICAM-2; a functional 
derivative of ICAM-2; and a non-immunoglobulin antagonist of ICAM-2 other 
than ICAM-1, or a member of the CD-18 family of molecules, either alone, 
or in combination with (b) an immunosuppressive agent.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
One aspect of the present invention relates to the discovery of a natural 
binding ligand to LFA-1. Molecules such as those of CD-18 family, which 
are involved in the process of cellular adhesion are referred to as 
"adhesion molecules." 
I. LFA-1 and ICAM-1 
The leukocyte adhesion molecule LFA-1 mediates a wide range of lymphocyte, 
monocyte, natural killer cell, and granulocyte interactions with other 
cells in immunity and inflammation (Springer, T. A. et al., Ann. Rev. 
Immunol. 5:223-252 (1987)). 
LFA-1 is a receptor for intercellular adhesion molecule 1 (ICAM-1), a 
surface molecule is constitutively expressed on some tissues and induced 
on others in inflammation (Marlin, S. D. et al., Cell 51:813-819 (1987); 
Dustin, M. L. et al., J. Immunol. 137:245-254 (1986); Dustin, M. L. et 
al., Immunol. Today 9:213-215 (1988); U.S. patent application Ser. No. 
07/019,440, filed Feb. 26, 1987 (abandoned) and U.S. patent application 
Ser. No. 07/250,446, filed Sep. 28, 1988, abandoned, both applications 
herein incorporated by reference). 
LFA-1 functions in both antigen-specific and antigen-independent T 
cytotoxic, T helper, natural killer, granulocyte, and monocyte 
interactions with other cell types (Springer, T. A. et al., Ann. Rev. 
Immuol. 5:223-252 (1987); Kishimoto, T. K. et al., Adv. Immunol. (1988, in 
press)). LFA-1 is a leukocyte integrin, with noncovalently associated 
.alpha. and .beta. glycoprotein subunits of 180 and 95 kD. 
ICAM-1 is a single chain glycoprotein varying in mass on different cell 
types from 76-114 kD, and is a member of the Ig superfamily with five 
C-like domains (Dustin, M. L. et al., Immunol. Today 9:213-215 (1988); 
Staunton, D. E. et al., Cell 52:925-933 (1988); Simmons, D. et al., Nature 
331:624-627 (1988)). ICAM-1 is highly inducible with cytokines including 
IFN-.gamma., TNF, and IL-1 on a wide range of cell types (Dustin, M. L. et 
al., Immunol. Today 9:213-215 (1988)). Induction of ICAM-1 on epithelial 
cells, endothelial cells, and fibroblasts mediates LFA-1 dependent 
adhesion of lymphocytes (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. Exp. Med. 167:1323-1340 (1988)). Adhesion is blocked by 
pretreatment of lymphocytes with LFA-1 MAb or pretreatment of the other 
cell with ICAM-1 MAb (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. Exp. Med, 167:1323-1340 (1988)). Identical results with 
purified ICAM-1 in artificial membranes or on Petri dishes demonstrate 
that LFA-1 and I CAM-1 are receptors for one another (Marlin, S. D. et 
al., Cell 51:813-819 (1987); Makgoba, M. W. et al., Nature 331:86-88 
(1988)). For clarity, they are referred to herein as "receptor" and 
"ligand," respectively. Further descriptions of ICAM-1 are provided in 
U.S. patent applications Ser. Nos. 07/045,963, filed May 4, 1987 
(abandoned); 07/115,798, filed Nov. 2, 1987 (abandoned); 07/155,943, filed 
Feb. 16, 1988 (abandoned); 07/189,815, filed May 3, 1988 (abandoned) or 
07/250,446, filed Sep. 28, 1988 (abandoned), all of which applications are 
herein incorporated by reference in their entirety 
II. ICAM-2 
A second LFA-1 ligand, distinct from ICAM-1, has been postulated (Rothlein, 
R. et al., J. Immunol. 137:1270-1274 (1986); Makgoba, M. W. et al., Eur. 
J. Immunol, 18:637-640 (1988); Dustin, M. L. et al., J. Cell. Biol. 
107:321-331 (1988)). The present invention concerns this second ligand, 
designated "ICAM-2"(for "Intercellular Adhesion Molecule-2"). 
ICAM-2 differs from ICAM-1 in cell distribution and in a lack of cytokine 
induction. ICAM-2 is an integral membrane protein with 2 Ig-like domains, 
whereas ICAM-1 has 5 Ig-like domains (Staunton, D. E. et al., Cell 
52:925-933 (1988); Simmons, D. et al., Nature 331:624-627 (1988)). 
Remarkably, ICAM-2 is much more closely related to the two most N-terminal 
domains of ICAM-1 (34% identity) than either ICAM-1 or ICAM-2 is to other 
members of the Ig superfamily, demonstrating a subfamily of Ig-like 
ligands which bind the same integrin family receptor. 
III. cDNA CLONING OF ICAM-2 
Any of a variety of procedures may be used to clone the ICAM-2 gene. One 
such method entails analyzing a shuttle vector library of cDNA inserts 
(derived from an ICAM-2 expressing cell) for the presence of an insert 
which contains the ICAM-2 gene. Such an analysis may be conducted by 
transfecting cells with the vector and then assaying for ICAM-2 
expression. 
ICAM-2 cDNA is preferably identified when a novel modification of the 
procedure of Aruffo and Seed (Seed, B. et al., Proc. Natl. Acad. Sci. USA 
84:3365-3369 (1987)) is employed for identifying ligands of adhesion 
molecules. In this method, a cDNA library is prepared from cells which 
express ICAM-2 (such as endothelial cells or Ramos, BBN B lymphoblastoid, 
U937 monocytic, or SKW3 lymphoblastoid, cell lines). Preferrably, the cDNA 
library is prepared from endothelial cells. This library is used to 
transfect cells which do not normally express ICAM-2 (such as COS cells). 
The transfected cells are introduced into a petri dish which has been 
previously coated with LFA-1. COS cells which have been transfected with 
either ICAM-1 or ICAM-2 encoding sequences, and which express either of 
these ligands on their cell surfaces will adhere to the LFA-1 on the 
surface of the petri dish. Non-adherant cells are washed away, and the 
adherent cells are then removed from the petri dish and cultured. The 
recombinant ICAM-1 or ICAM-2 expressing sequences in these cells is then 
removed, and sequenced to determine whether it encodes ICAM-1 or ICAM-2. 
In a preferred embodiment of the above-described method, anti-ICAM-1 
antibody is added to the petri dish in order to prevent the adherence of 
ICAM-1 expressing cells. Binding of ICAM-2 transfected COS cells to LFA-1 
is inhibited by EDTA and anti-LFA-1 monoclonal antibody ("MAb"), but is 
not inhibited by anti-ICAM-1MAb. Thus, in this embodiment, the ICAM-1 
expressing cells are unable to adhere to the petri dish through ICAM-1 and 
are therefore mostly washed away with all of the other non-adherent cells. 
As a result, only cells expressing ICAM-2 are able to adhere to the petri 
dish. 
Thus, cDNA clones are screened by expression in COS cells, and by panning 
for ligand-bearing COS cells using functionally-active, purified LFA-1 
which has been previously bound to plastic Petri dishes. After panning, 
nonadherent cells are depleted of ICAM-2.sup.+ cells, whereas adherent 
cells, released from LFA-1-coated plastic by EDTA, are almost completely 
ICAM-2.sup.+. Adherence of ICAM-1.sup.+ cells to LFA-1-coated plastic may 
be inhibited with RR1/1 anti-ICAM-1MAb. 
Thus, in accordance with this method for cloning cDNA for ICAM-2, a cDNA 
library is prepared from endothelial cells, which demonstrate both the 
ICAM-1-dependent and ICAM-1-independent components of LFA-1-dependent 
adhesion (Dustin, M. L. et al., J. Cell. Biol. 107:321-331 (1988)) using a 
suitable plasmid, such as the plasmid vector CDM8. Transfected COS cells 
are incubated in LFA-1-coated petri dishes with anti-ICAM-1 MAb present to 
reduce the probability of isolating ICAM-1 cDNA's. Adherent cells are 
eluted with EDTA and plasmids are isolated and amplified in E. coli. After 
approximately three cycles of transfection, adherence, and plasmid 
isolation and one size fractionation, plasmids may be analyzed by 
restriction endonuclease digestion. Approximately 1/3 of plasmids having 
inserts greater than 1.0 kb, when introduced into COS cells by 
transfection, yielded adherence to LFA-1. 
Alternatively, a cDNA clone of ICAM-2 can be obtained by using the genetic 
code (Watson, J. D., In: Molecular Biology of the Gene, 3rd Ed., W. A. 
Benjamin, Inc., Menlo Park, Calif. (1977 ), pp. 356-357) to determine the 
sequence of a polynucleotide capable of encoding the ICAM-2 protein. 
A clone of the ICAM-2 cDNA can also be obtained by identifying the amino 
acid sequences of peptide fragments of the ICAM-2 protein, and then using 
the genetic code to construct oligonucleotide probe molecules capable of 
encoding the ICAM-2 peptide. The probes are then used to detect (via 
hybridization) those members of a cDNA library (prepared from cDNA of 
ICAM-2 expressing cells) which encode the ICAM-2 protein. 
Techniques such as, or similar to, those described above have successfully 
enabled the cloning of genes for human aldehyde dehydrogenases (Hsu, L. C. 
et al., Proc. Natl. Acad. Sci. USA 82:3771-3775 (1985)), fibronectin 
(Suzuki, S. et al., Eur. Mol. Biol. Organ. J. 4:2519-2524 (1985)), the 
human estrogen receptor gene (Walter, P. et al., Proc. Natl. Acad. Sci. 
USA 82:7889-7893 (1985)), tissue-type plasminogen activator (Pennica, D. 
et al., Nature 301:214-221 (1983)) and human term placental alkaline 
phosphatase complementary DNA (Kam, W. et al., Proc. Natl. Acad. Sci. USA 
82:8715-8719 (1985)). 
In yet another alternative way of cloning the ICAM-2 gene, a library of 
expression vectors is prepared by cloning DNA or, more preferably cDNA, 
from a cell capable of expressing ICAM-2 into an expression vector. The 
library is then screened for members capable of expressing a protein which 
binds to anti-ICAM-2 antibody, and which has a nucleotide sequence that is 
capable of encoding polypeptides that have the same amino acid sequence as 
ICAM-2 or fragments of ICAM-2. 
The cloned ICAM-2 gene, obtained through the use of any of the methods 
described above, may be operably linked to an expression vector, and 
introduced into bacterial, or eukaryotic cells to produce ICAM-2 protein. 
Techniques for such manipulations are disclosed by Maniatis, T. et al., 
supra, and are well known in the art. 
IV. THE AGENTS OF THE PRESENT INVENTION: ICAM-2 AND ITS FUNCTIONAL 
DERIVATIVES, AGONISTS AND ANTAGONISTS 
The present invention is directed toward ICAM-2, its "functional 
derivatives," and its "agonists" and "antagonists." 
A. Functional Derivatives of ICAM-2 
A "functional derivative" of ICAM-2 is a compound which possesses a 
biological activity (either functional or structural) that is 
substantially similar to a biological activity of ICAM-2. The term 
"functional derivatives" is intended to include the "fragments," 
"variants," "analogs," or "chemical derivatives" of a molecule. 
A "fragment" of a molecule such as ICAM-2, is meant to refer to any 
polypeptide subset of the molecule. Fragments of ICAM-2 which have ICAM-2 
activity and which are soluble (i.e not membrane bound) are especially 
preferred. 
A "variant" of a molecule such as ICAM-2 is meant to refer to a molecule 
substantially similar in structure and function to either the entire 
molecule, or to a fragment thereof. 
An "analog" of a molecule such as ICAM-2 is meant to refer to a molecule 
substantially similar in function to either the entire molecule or to a 
fragment thereof. 
A molecule is said to be "substantially similar" to another molecule if 
both molecules have substantially similar structures or if both molecules 
possess a similar biological activity. Thus, provided that two molecules 
possess a similar activity, they are considered variants as that term is 
used herein even if the structure of one of the molecules is not found in 
the other, or if the sequence of amino acid residues is not identical. 
As used herein, a molecule is said to be a "chemical derivative" of another 
molecule when it contains additional chemical moieties not normally a part 
of the molecule. Such moieties may improve the molecule's solubility, 
absorption, biological half life, etc. The moieties may alternatively 
decrease the toxicity of the molecule, eliminate or attenuate any 
undesirable side effect of the molecule, etc. Moieties capable of 
mediating such effects are disclosed in Remington's Pharmaceutical 
Sciences (1980). 
"Toxin-derivatized" molecules constitute a special class of "chemical 
derivatives." A "toxin-derivatized" molecule is a molecule (such as ICAM-2 
or an antibody) which contains a toxin moiety. The binding of such a 
molecule to a cell brings the toxin moiety into close proximity with the 
cell and thereby promotes cell death. Any suitable toxin moiety may be 
employed; however, it is preferable to employ toxins such as, for example, 
the ricin toxin, the cholera toxin, the diphtheria toxin, radioisotopic 
toxins, membrane-channel-forming toxins, etc. Procedures for coupling such 
moieties to a molecule are well known in the art. 
Functional derivatives of ICAM-2 having up to about 100 residues may be 
conveniently prepared by in vitro synthesis. If desired, such fragments 
may be modified by reacting targeted amino acid residues of the purified 
or crude protein with an organic derivatizing agent that is capable of 
reacting with selected side chains or terminal residues. The resulting 
covalent derivatives may be used to identify residues important for 
biological activity. 
Cysteinyl residues most commonly are reacted with .alpha.-haloacetates (and 
corresponding amines), such as chloroacetic acid or chloroacetamide, to 
give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues 
also are derivatized by reaction with bromotrifluoroacetone, 
.alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl phosphate, 
N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl 
disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or 
chloro-7-nitrobenzo-2-oxa-1,3-diazole. 
Histidyl residues are derivatized by reaction with diethylprocarbonate at 
pH 5.5-7.0 because this agent is relatively specific for the histidyl side 
chain. Para-bromophenacyl bromide also is useful; the reaction is 
preferably performed in 0.1M sodium cacodylate at pH 6.0. 
Lysinyl and amino terminal residues are reacted with succinic or other 
carboxylic acid anhydrides. Derivatization with these agents has the 
effect of reversing the charge of the lysinyl residues. Other suitable 
reagents for derivatizing .alpha.-amino-containing residues include 
imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; 
chloroborohydride; trinitrobenzenesulfonic acid; O-methyl-issurea; 2,4 
pentanedione; and transaminase-catalyzed reaction with glyoxylate. 
Arginyl residues are modified by reaction with one or several conventional 
reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, 
and ninhydrin. Derivatization of arginine residues requires that the 
reaction be performed in alkaline conditions because of the high pK.sub.a 
of the guanidine functional group. Furthermore, these reagents may react 
with the groups of lysine as well as-the arginine epsilon-amino group. 
The specific modification of tyrosyl residues per se has been studied 
extensively, with particular interest in introducing spectral labels into 
tyrosyl residues by reaction with aromatic diazonium compounds or 
tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane 
are used to form O-acetyl tyrosyl species and 3-nitro derivatives, 
respectively. Tyrosyl residues are iodinated using .sup.125 I or .sup.131 
I to prepare labeled proteins for use in radioimmunoassay, the chloramine 
T method being suitable. 
Carboxyl side groups (aspartyl or glutamyl) are selectively modified by 
reaction with carbodiimides (R'--N--C--N--R') such as 
1-cyclohexyl-3-(2-morpholinyl-(4- ethyl) carbodiimide or 1-ethyl-3 (4 
azonia 4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and 
glutamyl residues are converted to asparaginyl and glutaminyl residues by 
reaction with ammonium ions. 
Derivatization with bifunctional agents is useful for crosslinking an 
ICAM-2 functional derivative molecule to a water-insoluble support matrix 
or surface for use in the method for cleaving an ICAM-2 functional 
derivatives fusion polypeptide to release and recover the cleaved 
polypeptide. Commonly used crosslinking agents include, e.g., 
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide 
esters, for example, esters with 4-azidosalicylic acid, homobifunctional 
imidoesters, including disuccinimidyl esters such as 
3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such 
as bis-N-maleimido-1,8-octane. Derivatizing agents such as 
methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable 
intermediates that are capable of forming crosslinks in the presence of 
light. Alternatively, reactive water-insoluble matrices such as cyanogen 
bromide-activated carbohydrates and the reactive substrates described in 
U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 
4,330,440 are employed for protein immobilization. 
Glutaminyl and asparaginyl residues are frequently deamidated to the 
corresponding glutamyl and aspartyl residues. Alternatively, these 
residues are deamidated under mildly acidic conditions. Either form of 
these residues falls within the scope of this invention. 
Other modifications include hydroxylation of proline and lysine, 
phosphorylation of hydroxyl groups of seryl or theonyl residues, 
methylation of the .alpha.-amino groups of lysine, arginine, and histidine 
side chains (T. E. Creighton, Proteins: Structure and Molecule Properties, 
W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the 
N-terminal amine, and, in some instances, amidation of the C-terminal 
carboxyl groups. 
Functional derivatives of ICAM-2 having altered amino acid sequences can 
also be prepared by mutations in the DNA. The nucleotide sequence which 
encodes the ICAM-2 gene is shown in FIG. 2. Such variants include, for 
example, deletions from, or insertions or substitutions of, residues 
within the amino acid sequence shown in FIG. 2. Any combination of 
deletion, insertion, and substitution may also be made to arrive at the 
final construct, provided that the final construct possesses the desired 
activity. Obviously, the mutations that will be made in the DNA encoding 
the variant must not place the sequence out of reading frame and 
preferably will not create complementary regions that could produce 
secondary mRNA structure (see EP Patent Application Publication No. 
75,444). 
At the genetic level, these functional derivatives ordinarily are prepared 
by site-directed mutagenesis of nucleotides in the DNA encoding the ICAM-2 
molecule, thereby producing DNA encoding the functional derivative, and 
thereafter expressing the DNA in recombinant cell culture. The functional 
derivatives typically exhibit the same qualitative biological activity as 
the naturally occurring analog. They may, however, differ substantially in 
such characteristics with respect to the normally produced ICAM-2 
molecule. 
While the site for introducing an amino acid sequence variation is 
predetermined, the mutation per se need not be predetermined. For example, 
to optimize the performance of a mutation at a given site, random 
mutagenesis may be conducted at the target codon or region and the 
expressed ICAM-2 functional derivatives screened for the optimal 
combination of desired activity. Techniques for making substitution 
mutations at predetermined sites in DNA having a known sequence are well 
known, for example, site-specific mutagenesis. 
Preparation of an ICAM-2 functional derivative molecule in accordance 
herewith is preferably achieved by site-specific mutagenesis of DNA that 
encodes an earlier prepared functional derivatives or a nonvariant version 
of the protein. Site-specific mutagenesis allows the production of ICAM-2 
functional derivatives through the use of specific oligonucleotide 
sequences that encode the DNA sequence of the desired mutation, as well as 
a sufficient number of adjacent nucleotides, to provide a primer sequence 
of sufficient size and sequence complexity to form a stable duplex on both 
sides of the deletion junction being traversed. Typically, a primer of 
about 20 to 25 nucleotides in length is preferred, with about 5 to 10 
residues on both sides of the junction of the sequence being altered. In 
general, the technique of site-specific mutagenesis is well known in the 
art, as exemplified by publications such as Adelman et al., DNA 2:183 
(1983), the disclosure of which is incorporated herein by reference. 
As will be appreciated, the site-specific mutagenesis technique typically 
employs a phage vector that exists in both a single-stranded and 
double-stranded form. Typical vectors useful in site-directed mutagenesis 
include vectors such as the M13 phage, for example, as disclosed by 
Messing et al., Third Cleveland Symposium on Macromolecules and 
Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), the 
disclosure of which is incorporated herein by reference. These phage are 
readily commercially available and their use is generally well known to 
those skilled in the art. Alternatively, plasmid vectors that contain a 
single-stranded phage origin of replication (Veira et al., Meth. Enzymol. 
153:3 (1987)) may be employed to obtain single-stranded DNA. 
In general, site-directed mutagenesis in accordance herewith is performed 
by first obtaining a single-stranded vector that includes within its 
sequence a DNA sequence that encodes the relevant protein. An 
oligonucleotide primer bearing the desired mutated sequence is prepared, 
generally synthetically, for example, by the method of Crea et al., Proc. 
Natl. Acad. Sci. (USA) 75:5765 (1978). This primer is then annealed with 
the single-stranded protein-sequence-containing vector, and subjected to 
DNA-polymerizing enzymes such as E. coli polymerase I Klenow fragment, to 
complete the synthesis of the mutation-bearing strand. Thus, a mutated 
sequence and the second strand bears the desired mutation. This 
heteroduplex vector is then used to transform appropriate cells, such as 
JM101 cells, and clones are selected that include recombinant vectors 
bearing the mutated sequence arrangement. 
After such a Clone is selected, the mutated protein region may be removed 
and placed in an appropriate vector for protein production, generally an 
expression vector of the type that may be employed for transformation of 
an appropriate host. 
Amino acid sequence deletions generally range from about 1 to 30 residues, 
more preferably 1 to 10 residues, and typically are contiguous. Deletions 
may also comprise an immunoglobulin domain, such as domains 1 or 2 of 
ICAM-2. Amino acid sequence insertions include amino and/or 
carboxyl-terminal fusions of from one residue to polypeptides of 
essentially unrestricted length, as well as intrasequence insertions of 
single or multiple amino acid residues. Intrasequence insertions (i.e., 
insertions within the complete ICAM-2 molecule sequence) may range 
generally from about 1 to 10 residues, more preferably 1 to 5. An example 
of a terminal insertion includes a fusion of a signal sequence, whether 
heterologous or homologous to the host cell, to the N-terminus of the 
molecule to facilitate the secretion of the ICAM-2 functional derivative 
from recombinant hosts. 
The third group of functional derivatives are those in which at least one 
amino acid residue in the ICAM-2 molecule, and preferably, only one, has 
been removed and a different residue inserted in its place. Such 
substitutions preferably are made in accordance with the following Table 
when it is desired to modulate finely the characteristics of the ICAM-2 
molecule. 
TABLE 1 
______________________________________ 
Original Residue Exemplary Substitutions 
______________________________________ 
Ala gly; ser 
Arg lys 
Asn gln; his 
Asp glu 
cys ser 
Gln asn 
Glu asp 
Gly ala; pro 
His asn; gln 
Ile leu; val 
Leu ile; val 
Lys arg; gln; glu 
Met leu, tyr; ile 
Phe met; leu; tyr 
Ser thr 
Thr ser 
Trp tyr 
Tyr trp; phe 
Val ile; leu 
______________________________________ 
Substantial changes in functional or immunological identity are made by 
selecting substitutions that are less conservative than those in Table 1, 
i.e., selecting residues that differ more significantly in their effect on 
maintaining (a) the structure of the polypeptide backbone in the area of 
the substitution, for example, as a sheet or helical conformation, (b) the 
charge or hydrophobicity of the molecule at the target site, or (c) the 
bulk of the side chain. The substitutions that in general are expected to 
those in which (a) glycine and/or proline is substituted by another amino 
acid or is deleted or inserted; (b) a hydrophilic residue, e.g., seryl or 
threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, 
isoleucyl, phenylalanyl, valyl, or alanyl; (c) a cysteine residue is 
substituted for (or by) any other residue; (d) a residue having an 
electropositive side chain, e.g., lysyl, arginyl, or histidyl, is 
substituted for (or by) a residue having an electronegative charge, e.g., 
glutamyl or aspartyl; or (e) a residue having a bulky side chain, e.g., 
phenylalanine, is substituted for (or by) one not having such a side 
chain, e.g., glycine. 
Most deletions and insertions, and substitutions in particular, are not 
expected to produce radical changes in the characteristics of the 
ICAM-2molecule. However, when it is difficult to predict the exact effect 
of the substitution, deletion, or insertion in advance of doing so, one 
skilled in the art will appreciate that the effect will be evaluated by 
routine screening assays. For example, a functional derivative typically 
is made by site-specific mutagenesis of the native ICAM-2 
molecule-encoding nucleic acid, expression of the variant nucleic acid in 
recombinant cell culture, and, optionally, purification from the cell 
culture, for example, by immunoaffinity adsorption on an anti-ICAM-2 
molecule antibody column (to absorb the functional derivative by binding 
it to at least one remaining immune epitope). 
Mutations designed to increase the affinity of ICAM-2 may be guided by the 
introduction of the amino acid residues which are present at homologous 
positions in ICAM-1. Similarly, such mutant ICAM-2 molecules may be 
prepared which lack N-linked CHO at homologous positions in ICAM-1. 
The activity of the cell lysate or purified ICAM-1 molecule functional 
derivative is then screened in a suitable screening assay for the desired 
characteristic. For example, a change in the immunological character of 
the functional derivative, such as affinity for a given antibody, is 
measured by a competitive type immunoassay. Changes in immunomodulation 
activity are measured by the appropriate assay. Modifications of such 
protein properties as redox or thermal stability, biological half-life, 
hydrophobicity, susceptibility to proteolytic degradation or the tendency 
to aggregate with carriers or into multimers are assayed by methods well 
known to the ordinarily skilled artisan. 
B. Agonists and Antagonists of ICAM-2 
An "agonist" of ICAM-2 is a compound which enhances or increases the 
ability of ICAM-2 to carry out any of its biological functions. An example 
of such an agonist is an agent which increases the ability of ICAM-2 to 
bind to a cellular receptor or..viral protein. 
An "antagonist" of ICAM-2 is a compound which diminishes or prevents the 
ability of ICAM-2 to carry out any of its biological functions. Examples 
of such antagonists include ICAM-1, functional derivatives of ICAM-1, 
anti-ICAM-2 antibody, anti-LFA-1 antibody, etc. 
The cellular aggregation assays described in U.S. patent applications Ser. 
Nos. 07/045,963, filed May 4, 1987 (abandoned); 07/115,798, filed Nov. 2, 
1987 (abandoned); 07/155,943, filed Feb. 16, 1988 (abandoned); 07/189,815, 
filed May 3, 1988 (abandoned) or 07/250,446, filed Sep. 28, 1988 
(abandoned), all of which applications have been herein incorporated by 
reference in their entirety, are capable of measuring LFA-1 dependent 
aggregation, and may be employed to identify agents which affect the 
extent of ICAM-2/LFA-1 aggregation. Thus, such assays may be employed to 
identify agonists and antagonists of ICAM-2. Antagonists may act by 
impairing the ability of IFA-1 or of ICAM-2 to mediate aggregation. 
Additionally, non-immunoglobulin (i.e., chemical) agents may be examined, 
using the above-described assay, to determine whether they are agonists or 
antagonists of ICAM-2/LFA-1 aggregation. 
C. Anti-ICAM-2 Antibody 
The preferred immunoglobulin antagonist of the present invention is an 
antibody to ICAM-2. Suitable antibodies can be obtained in any of a 
variety of ways. 
An antigenic molecule such as ICAM-2 are naturally expressed on the 
surfaces of lymphocytes. Thus, the introduction of such cells into an 
appropriate animal, as by intraperitoneal injection, etc., will result in 
the production of antibodies capable of binding to ICAM-2 or members of 
the CD-18 family of molecules. If desired, the serum of such an animal may 
be removed and used as a source of polyclonal antibodies capable of 
binding these molecules. 
Alternatively, anti-ICAM-2 antibodies may be produced by adaptation of the 
method of Selden, R. F. (European Patent Application Publication No. 
289,034) or Selden R. F. et al. (Science 236:714-718 (1987)). In 
accordance with such an adaptation of this method, the cells of a suitable 
animal (i.e. such as, for example, a mouse, etc.) are transfected with a 
vector capable of expressing either the intact ICAM-2 molecule, or a 
fragment of ICAM-2. The production of ICAM-2 in the transfected cells of 
the animal will elicit an immune response in the animal, and lead to the 
production of anti-ICAM-2 antibodies by the animal. 
Alternatively, anti-ICAM-2 antibodies may be made by introducing ICAM-2, or 
peptide fragments thereof, into an appropriate animal. The immunized 
animal will produce polyclonal antibody in response to such exposure. The 
use of peptide fragments of ICAM-2 permits one to obtain region specific 
antibodies which are reactive only with the epitope(s) contained in the 
peptide fragments used to immunize the animals. 
It is, however, preferable to remove splenocytes from animals (immunized in 
either of the ways described above), to fuse such spleen cells with a 
myeloma cell line and to permit such fusion cells to form a hybridoma cell 
which secretes monoclonal antibodies capable of binding ICAM-2. 
The hybridoma cells, obtained in the manner described above may be screened 
by a variety of methods to identify desired hybridoma cells that secrete 
antibody capable of binding to ICAM-2. In a preferred screening assay, 
such molecules are identified by their ability to inhibit the aggregation 
of ICAM-2-expressing, ICAM-1-non-expressing cells. Antibodies capable of 
inhibiting such aggregation are then further screened to determine whether 
they inhibit such aggregation by binding to ICAM-2, or to a member of the 
CD-18 family of molecules. Any means capable of distinguishing ICAM-2 from 
the CD-18 family of molecules may be employed in such a screen. Thus, for 
example, the antigen bound by the antibody may be analyzed as by 
immunoprecipitation and polyacrylamide gel electrophoresis. It is possible 
to distinguish between those antibodies which bind to members of the CD-18 
family of molecules from those which bind ICAM-2 by screening for the 
ability of the antibody to bind to cells which express LFA-1, but not 
ICAM-2 (or vice versa). The ability of an antibody to bind to a cell 
expressing LFA-1 but not ICAM-2may be detected by means commonly employed 
by those of ordinary skill. Such means include immunoassays (especially 
those using immunoflorescence), cellular agglutination, filter binding 
studies, antibody precipitation, etc. 
In addition to the above-described functional derivatives of ICAM-2, other 
agents which may be used in accordance of the present invention in the 
treatment of vital infection or inflammation include antibody to ICAM-2, 
anti-idiotypic antibodies to anti-ICAM-2 antibodies, and receptor 
molecules, or fragments of such molecules, which are capable of binding to 
ICAM-2. 
The antibodies to ICAM-2 (or functional derivatives of ICAM-2) which may be 
employed may be either polyclonal or monoclonal. 
The anti-idiotypic antibodies of interest to the present invention are 
capable of binding in competion with (or to the exclusion of) ICAM-2. Such 
antibodies can be obtained, for example, by raising antibody to an 
anti-ICAM-2 antibody, and then screening the antibody for the ability to 
bind a natural binding ligand of ICAM-2. 
Since molecules of the CD-18 family are able to bind to ICAM-2, 
administration of such molecules (for example as heterodimers having both 
alpha and beta subunits, or as molecules composed of only an alpha, or a 
beta subunit, or as molecules having fragments of either or both subunits) 
is able to compete with (or exclude) HRV for binding to ICAM-21 present on 
cells. 
The anti-aggregation antibodies of the present invention may be identified 
and titered in any of a variety of ways. For example, one can measure the 
ability of the antibodies to differentially bind to cells which express 
ICAM-2 (such as activated endothelial cells), and their inability to bind 
to cells which fail to express ICAM-2. Suitable assays of cellular 
aggregation are those described in U.S. patent applications Ser. Nos. 
07/045,963, filed May 4, 1987 (abandoned); 07/115,798, filed Nov. 2, 1987 
(abandoned); 07/155,943, filed Feb. 16, 1988 (abandoned); 07/189,815, 
filed May 3, 1988 (abandoned) or 07/250,446, filed Sep. 28, 1988 
(abandoned), all of which applications have been herein incorporated by 
reference in their entirety. Alternatively, the capacity of the antibodies 
to bind to ICAM-2 or to peptide fragments of ICAM-2 can be measured. As 
will be readily appreciated by those of ordinary skill, the above assays 
may be modified, or performed in a different sequential order to provide a 
variety of potential screening assays, each of which is capable of 
identifying and discriminating between antibodies capable of binding to 
ICAM-1 versus members of the CD-18 family of molecules. 
In a more preferred method, antibody can be selected for its ability to 
bind to COS cells expressing ICAM-2, but not to COS cells which do not 
express ICAM-2. 
D. Preparation of the Agents of the Present Invention 
The agents of the present invention may be obtained by natural processes 
(such as, for example, by inducing an animal, plant, fungi, bacteria, 
etc., to produce a non-immunoglobulin antagonist of ICAM-2, or by inducing 
an animal to produce polyclonal antibodies capable of binding to ICAM-2); 
by synthetic methods (such as, for example, by using the Merrifield method 
for synthesizing polypeptides to synthesize ICAM-2, functional derivatives 
of ICAM-2, or protein antagonists of ICAM-2 (either immunoglobulin or 
non-immunoglobulin)); by hybridoma technology (such as, for example, to 
produce monoclonal antibodies capable of binding to ICAM-2); or by 
recombinant technology (such as, for example, to produce the agents of the 
present invention in diverse hosts (i.e., yeast, bacteria, fungi, cultured 
mammalian cells, etc.), or from recombinant plasmids or viral vectors). 
The choice of which method to employ will depend upon factors such as 
convenience, desired yield, etc. It is not necessary to employ only one of 
the above-described methods, processes, or technologies to produce a 
particular anti-inflammatory agent; the above-described processes, 
methods, and technologies may be combined in order to obtain a particular 
agent. 
V. USES OF ICAM-2, AND ITS FUNCTIONAL DERIVATIVES, AGONISTS AND ANTAGONISTS 
A. Suppression of Inflammation 
One aspect of the present invention derives from the ability of ICAM-2 and 
its functional derivatives to interact with receptors of the CD-18 family 
of molecules, especially LFA-1 or with vital proteins (such as the 
proteins of the rhinovirus, etc.). By virtue of the ability of ICAM-2 to 
interact with members of the CD-18 family of glycoproteins, it may be used 
to suppress (i.e. to prevent, or attenuate) inflammation. 
The term "inflammation," as used herein, is meant to include both the 
reactions of the specific defense system, and the reactions of the 
non-specific defense system. 
As used herein, the term "specific defense system" is intended to refer to 
that component of the immune system that reacts to the presence of 
specific antigens. Inflammation is said to result from a response of the 
specific defense system if the inflammation is caused by, mediated by, or 
associated with a reaction of the specific defense system. Examples of 
inflammation resulting from a response of the specific defense system 
include the response to antigens such as rubella virus, autoimmune 
diseases, delayed type hypersensitivity response mediated by T-cells (as 
seen, for example in individuals who test "positive" in the Mantaux test), 
etc. Chronic inflammatory diseases and the rejection of transplanted 
tissue and organs are further examples of inflammatory reactions of the 
specific defense system. 
As used herein, a reaction of the "non-specific defense system" is intended 
to refer to a reaction mediated by leukocytes incapable of immunological 
memory. Such cells include granulocytes and macrophages. As used herein, 
inflammation is said to result from a response of the non-specific defense 
system, if the inflammation is caused by, mediated by, or associated with 
a reaction of the non-specific defense system. Examples of inflammation 
which result, at least in part, from a reaction of the non-specific 
defense system include inflammation associated with conditions such as: 
adult respiratory distress syndrome (ARDS) or multiple organ injury 
syndromes secondary to septicemia or trauma; reperfusion injury of 
myocardial or other tissues; acute glomerulonephritis; reactive arthritis; 
dermatoses with acute inflammatory components; acute purulent meningitis 
or other central nervous system inflammatory disorders; thermal injury; 
hemodialysis; leukapheresis; ulcerative coliris; Crohn's disease; 
necrotizing enterocolitis; granulocyte transfusion associated syndromes; 
and cytokine-induced toxicity. 
As discussed above, the binding of ICAM-2 molecules to the members of CD-18 
family of molecules is of central importance in cellular adhesion. Through 
the process of adhesion, lymphocytes are capable of continually monitoring 
an animal for the presence of foreign antigens. Although these processes 
are normally desirable, they are also the cause of organ transplant 
rejection, tissue graft rejection and many autoimmune diseases. Hence, any 
means capable of attenuating or inhibiting cellular adhesion would be 
highly desirable in recipients of organ transplants (especially kidney 
transplants), tissue grafts, or for autoimmune patients. 
Monoclonal antibodies to members of the CD-18 family inhibit many adhesion 
dependent functions of leukocytes including binding to endothelium 
(Haskard, D. et al., J. Immunol. 137:2901-2906 (1986)), homotypic 
adhesions (Rothlein, R. et al., J. Exp. Med. 163:1132-1149 (1986)), 
antigen and mitogen induced proliferation of lymphocytes (Davignon, D. et 
al., Proc. Natl. Acad. Sci., USA 78:4535-4539 (1981)), antibody formation 
(Fischer, A. et al., J. Immunol. 136:3198-3203 (1986)), and effector 
functions of all leukocytes such as lytic activity of cytotoxic T cells 
(Krensky, A. M. et al., J. Immunol. 132:2180-2182 (1984)), macrophages 
(Strassman, G. et al., J. Immunol. 36:4328-4333 (1986)), and all cells 
involved in antibody-dependent cellular cytotoxicity reactions (Kohl, S. 
et al., J. Immunol. 133:2972-2978 (1984)). In all of the above functions, 
the antibodies inhibit the ability of the leukocyte to adhere to the 
appropriate cellular substrate which in turn inhibits the final outcome. 
Such functions, to the extent that they involve ICAM-2/LFA-1 interactions, 
can be suppressed with anti-ICAM-2 antibody. 
Thus, monoclonal antibodies capable of binding to ICAM-2 can be employed as 
anti-inflammatory agents in a mammalian subject. Significantly, such 
agents differ from general anti-inflammatory agents in that they are 
capable of selectively inhibiting adhesion, and do not offer other side 
effects such as nephrotoxicity which are found with conventional agents. 
Since ICAM-2, particularly in soluble form is capable of acting in the same 
manner as an antibody to members of the CD-18 family, it may be used to 
suppress inflammation. Moreover, the functional derivatives and 
antagonists of ICAM-2 may also be employed to suppress inflammation. 
1. Suppressors of Delayed Type Hypersensitivity Reactions 
ICAM-2 molecules mediate, in part, adhesion events necessary to mount 
inflammatory reactions such as delayed type hypersensitivity reactions. 
Thus, antibodies (especially monoclonal antibodies) capable of binding to 
ICAM-2 molecules have therapeutic potential in the attenuation or 
elimination of such reactions. 
Alternatively, since ICAM-2 is an antagonist of the ICAM-1/LFA-1 
interaction, ICAM-2 (particularly in solublilized form), or its functional 
derivatives can be used to suppress delayed type hypersensitivity 
reactions. 
These potential therapeutic uses may be exploited in either of two manners. 
First, a composition containing a monoclonal antibody against ICAM-2 may 
be administered to a patient experiencing delayed type hypersensitivity 
reactions. For example, Such compositions might be provided to a 
individual who had been in contact with antigens such as poison ivy, 
poison oak, etc. In the second embodiment, the monoclonal antibody capable 
of binding to ICAM-2 is administered to a patient in conjunction with an 
antigen in order to prevent a subsequent inflammatory reaction. Thus, the 
additional administration of an antigen with an ICAM-2-binding monoclonal 
antibody may temporarily tolerize an individual to subsequent presentation 
of that antigen. 
2. Therapy for Chronic Inflammatory Disease 
Since LAD patients that lack LFA-1 do not mount an inflammatory response, 
it is believed that antagonism of LFA-1's natural ligand, ICAM-2, will 
also inhibit an inflammatory response. The ability of antibodies against 
ICAM-2 to inhibit inflammation provides the basis for their therapeutic 
use in the treatment of chronic inflammatory diseases and autoimmune 
diseases such as lupus erythematosus, autoimmune thyroiditis, experimental 
allergic encephalomyelitis (EAE), multiple sclerosis, some forms of 
diabetes, Reynaud's syndrome, rheumatoid arthritis, etc. Such antibodies 
may also be employed as a therapy in the treatment of psoriasis. In 
general, the monoclonal antibodies capable of binding to ICAM-2 may be 
employed in the treatment of those diseases currently treatable through 
steroid therapy. 
In accordance with the present invention, such inflammatory and immune 
rejection responses may be suppressed (i.e. either prevented or 
attenuated) by providing to a subject in need of such treatment an amount 
of an anti-inflammatory agent sufficient to suppress said inflammation. 
Suitable anti-inflammatory agents include: an antibody capable of binding 
to ICAM-2; a fragment of an antibody, which fragment is capable of binding 
to ICAM-2; ICAM-2; a functional derivative of ICAM-2; a non-immunoglobulin 
antagonist of ICAM-2 other than ICAM-1 or a non-immunoglobulin antagonist 
of ICAM-2 other than LFA-1. Especially preferred are anti-inflammatory 
agents composed of a soluble functional derivative of ICAM-2. Such 
anti-inflammatory treatment can also include the additional administration 
of an agent selected from the group consisting of: an antibody capable of 
binding to LFA-1; a functional derivative of an antibody, said functional 
derivative being capable of binding to LFA-1; and a non-immunoglobulin 
antagonist of LFA-1. 
The invention further includes the above-described methods for suppressing 
an inflammatory response of the specific defense system in which an 
immunosuppressive agent is additionally provided to the subject. Such an 
agent is preferably provided at a dose lower (i.e. a "sub-optimal" dose) 
than that at which it would normally be required. The use of a sub-optimal 
dose is possible because of the synergistic effect of the agents of the 
present invention. Examples of suitable immunosuppressive agents include 
dexamethesone, azathioprine, ICAM-1, cyclosporin A, etc. 
3. Therapy for Non-Specific Inflammation 
The present invention derives in part from the discovery that 
granulocyte-endothelial cell adherence results from the interaction of 
glycoproteins of the CD-18 family with the endothelium. Since cellular 
adhesion is required in order that leukocytes may migrate to sites of 
inflammation and/or carry out various effector functions contributing to 
inflammation, agents which inhibit cellular adhesion will attenuate or 
prevent such inflammation. Such inflammatory reactions are due to 
reactions of the "non-specific defense system" which are mediated by 
leukocytes incapable of immunological memory. Such cells include 
granulocytes and macrophages. As used herein, inflammation is said to 
result from a response of the non-specific defense system, if the 
inflammation is caused by, mediated by, or associated with a reaction of 
the non-specific defense system. Examples of inflammation which result, at 
least in part, from a reaction of the non-specific defense system include 
inflammation associated with conditions such as: adult respiratory 
distress syndrome (ARDS)or multiple organ injury syndromes secondary to 
septicemia or trauma; reperfusion injury of myocardial or other tissues; 
acute glomerulonephritis; reactive arthritis; dermatoses with acute 
inflammatory components; acute purulent meningitis or other central 
nervous system inflammatory disorders; thermal injury; hemodialysis; 
leukapheresis; ulcerative colitis; Crohn's disease; necrotizing 
enterocolitis; granulocyte transfusion associated syndromes; and 
cytokine-induced toxicity. 
The anti-inflammatory agents of the present invention are compounds capable 
of specifically antagonizing the interaction of the CD-18 complex on 
granulocytes with endothelial cells. Such antagonists comprise: ICAM-2; a 
functional derivative of ICAM-2; and a non-immunoglobulin antagonist of 
ICAM-2 other than ICAM-1, or a member of the CD-18 family of molecules. 
B. Suppressors of Organ and Tissue Rejection 
Since ICAM-2, particularly in soluble form is capable of acting in the same 
manner as an antibody to members of the CD-18 family, it may be used to 
suppress organ or tissue rejection caused by any of the cellular 
adhesion-dependent functions. Moreover, anti-ICAM-2 antibody and the 
functional derivatives and antagonists of ICAM-2 may also be employed to 
suppress such rejection. 
ICAM-2and antibodies capable of binding to ICAM-2 can be used to prevent 
organ or tissue rejection, or modify autoimmune responses without the fear 
of such side effects, in the mammalian subject. 
Importantly, the use of monoclonal antibodies capable of recognizing ICAM-2 
may permit one to perform organ transplants even between individuals 
having HLA mismatch. 
C. Adjunct to the Introduction of Antigenic Material Administered for 
Therapeutic or Diagnostic Purposes 
Immune responses to therapeutic or diagnostic agents such as, for example, 
bovine insulin, interferon, tissue-type plasminogen activator or murine 
monoclonal antibodies substantially impair the therapeutic or diagnostic 
value of such agents, and can, in fact, causes diseases such as serum 
sickness. Such a situation can be remedied through the use of the 
antibodies of the present invention. In this embodiment, such antibodies 
would be administered in combination with the therapeutic or diagnostic 
agent. The addition of the antibodies prevents the recipient from 
recognizing the agent, and therefore prevents the recipient from 
initiating an immune response against it. The absence of such an immune 
response results in the ability of the patient to receive additional 
administrations of the therapeutic or diagnostic agent. 
ICAM-2 (particularly in solubilized form) or its functional derivatives may 
be employed interchangeably with ICAM-1, or with antibodies capable of 
binding to LFA-1 in the treatment of disease. Thus, in solubilized form, 
such molecules may be employed to inhibit organ or graft rejection. 
ICAM-2, or its functional derivatives may be used in the same manner as 
anti-ICAM-2 antibodies to decrease the immunogenicity of therapeutic or 
diagnostic agents. 
D. Suppressors of Tumor Metastasis 
The agents of the present invention may also be employed to suppress the 
metastasis of a hematopoietic tumor cell, which requires a functional 
member of the CD-18 family for migration. In accordance with this 
embodiment of the present invention, a patient in need of such treatment 
is provided with an amount of an agent (such as an antibody capable of 
binding to ICAM-2; a toxin-derivatized antibody capable of binding to 
ICAM-2; a fragment of an antibody, the fragment being capable of binding 
to ICAM-2; a toxin-derivatized fragment of an antibody, the fragment being 
capable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; 
and a non-immunoglobulin antagonist of ICAM-2 other than ICAM-1) 
sufficient to suppress said metastasis. 
The invention also provides a method of suppressing the growth of an 
ICAM-2-expressing tumor cell which comprises providing to a patient in 
need of such treatment an amount of an agent sufficient to suppress said 
growth. Suitable agents include an antibody capable of binding to ICAM-2; 
a toxin-derivatized antibody capable of binding to ICAM-2; a fragment of 
an antibody, the fragment being capable of binding to ICAM-2; a 
toxin-derivatized fragment of an antibody, the fragment being capable of 
binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; a 
non-immunoglobulin antagonist of ICAM-2 other than ICAM-1; a 
toxin-derivatized member of the CD-18 family of molecules; and a 
toxin-derivatized functional derivative of a member of the CD-18 family of 
molecules. 
The invention also provides a method of suppressing the growth of an 
LFA-1-expressing tumor cell which comprises providing to a patient in need 
of such treatment an amount of a toxin sufficient to suppress said growth. 
Suitable toxins include a toxin-derivatized ICAM-2, or a toxin-derivatized 
functional derivative of ICAM-2. 
E. Suppressors of Viral Infection 
ICAM-1 has recently been shown to be subverted as a receptor by the major 
group of rhinoviruses (Greve, J. M. et al., Cell 56:839-847 (1989); 
Staunton, D. E. et al., Cell 56:849-853 (1989); Tomassini, J. E. et al., 
Proc. Natl. Acad. Sci. (U.S.A.) 86:4907-4911 (1989), which references are 
incorporated herein by reference). Rhinoviruses, members of the small, 
RNA-containing, protein-encapsidated picornavirus family, cause 40-50% of 
common colds (Rueckert, R. R., In: Fields Virology, Fields, B. N. et al. 
(eds.), Raven Press, N.Y., (1985) pp 705-728; Sperber, S. J. et al. 
Antimicr. Aqents Chemo. 32:409-419 (1988), which references are 
incorporated herein by reference). Over 100 immunologically 
non-crossreactive rhinoviruses have been defined, of which 90% bind to 
ICAM-1. 
Besides ICAM-1, the cell adhesion molecule CD4 and the complement 
receptor-CR2 have recently been found to be subverted as virus receptors 
by HIV and EBV viruses, respectively (Maddon, P. J., Cell 47:333-348 
(1986); Fingeroth, J. D., et al., Proc, Natl. Acad. Sci. USA 81:4510-4514 
(1984), which references are incorporated herein by reference). Further, a 
molecule with an Ig domain structure similar to ICAM-1 and which may 
function in cellular adhesion is a polio virus receptor (Mendelsohn, C. 
L., et al., Cell. 56:855-865 (1989)). 
ICAM-2 and its functional derivatives may act as receptors for viral 
(particularly by rhinoviruses, and particularly rhinoviruses of the minor 
serotype) attachment or infection. Thus, antibody to ICAM-2 (or fragments 
thereof), ICAM-2, or functional derivatives of ICAM-2, may be employed to 
block such attachment or infection, and to thereby suppress viral 
infection. 
F. Diagnostic and Prognostic Applications 
Monoclonal antibodies capable of binding to ICAM-2 may be employed as a 
means of imaging or visualizing the sites of ICAM-2 expression and 
inflammation in a patient. In such a use, the monoclonal antibodies are 
detectably labeled, through the use of radioisotopes, affinity labels 
(such as biotin, avidin, etc.) fluorescent labels, paramagnetic atoms, 
labeled anti-ICAM-2 antibody, etc. Procedures for accomplishing such 
labeling are well known to the art. Clinical application of antibodies in 
diagnostic imaging are reviewed by Grossman, H. B., Urol. Clin. North 
Amer. 13:465-474 (1986)), Unger, E. C. et al., Invest. Radiol. 20:693-700 
(1985)), and Khaw, B. A. et al., Science 209:295-297 (1980)). 
The presence of ICAM-2 expression may also be detected through the use of 
binding ligands, such as mRNA, cDNA, or DNA which bind to ICAM-2 gene 
sequences, or to ICAM-2 mRNA sequences, of cells which express ICAM-2. 
Techniques for performing such hybridization assays are described by 
Maniatis, T. et al., In: Molecular Clonincl, a Laboratory Manual, 
Coldspring Harbor, N.Y. (1982), and by Hayroes, B. D. et al., In: Nucleic 
Acid Hybrization, a Practical Approach, IRL Press, Washington, D.C. 
(1985), which references are herein incorporated by reference. 
The detection of loci of such detectably labeled antibodies is indicative 
of a site of ICAM-2 expression or tumor development. In one embodiment, 
this examination for expression is done by removing samples of tissue or 
blood and incubating such samples in the presence of antibodies which are 
or which can be detectably labeled. In a preferred embodiment, this 
technique is done in a non-invasive manner through the use of magnetic 
imaging, fluorography, etc. Such a diagnostic test may be employed in 
monitoring organ transplant recipients for early signs of potential tissue 
rejection. Such assays may also be conducted in efforts to determine an 
individual's predilection to rheumatoid arthritis or other chronic 
inflammatory diseases. 
For example, by radioactively labeling the antibodies or antibody 
fragments, it is possible to detect the antigen through the use of 
radioimmune assays. A good description of a radioimmune assay (RIA) may be 
found in Laboratory Techniques and Biochemistry in Molecular Biology, by 
Work, T. S., et al., North Holland Publishing Company, N.Y. (1978), with 
particular reference to the chapter entitled "An Introduction to 
Radioimmune Assay and Related Techniques" by Chard, T., incorporated by 
reference herein. Alternatively, flouresecent, enzyme, or other suitable 
labels can be employed. 
Examples of types of labels which can be used in the present invention 
include, but are not limited to, enzyme labels, radioisotopic labels, 
non-radioactive isotopic labels, fluorescent labels, toxin labels, and 
chemiluminescent labels. 
Examples of suitable enzyme labels include malate dehydrogenase, 
staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol 
dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate 
isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose 
oxidase, beta-galactosidase, ribonuclease, urease, catalase, 
glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase, 
etc. 
Examples of suitable radioisotopic labels include .sup.3 H, .sup.111 In, 
.sup.125 I, .sup.131 I, .sup.32 P, .sup.35 S, .sup.14 C, .sup.51 Cr, 
.sup.57 Co, .sup.58 Co, .sup.59 Fe, .sup.75 Se, .sup.152 Eu, .sup.90 Y, 
.sup.67 Cu, .sup.217 Ci, .sup.211 At, .sup.212 Pb, .sup.47 Sc, .sup.109 
Pd, etc. Examples of suitable non-radioactive isotopic labels include 
.sup.157 Gd, .sup.55 Mn, .sup.162 Dy, .sup.52 Tr, .sup.56 Fe, etc. 
Examples of suitable fluorescent labels include an .sup.152 Eu label, a 
fluorescein label, a rhodamine label, a phycoerythrin label, a phycocyanin 
label, an allophycocyanin label, an o-phthaldehyde label, a fluorescamine 
label, etc. 
Examples of chemiluminescent labels include a luminal label, an isoluminal 
label, an aromatic acridinium ester label, an imidazole label, an 
acridinium salt label, an oxalate ester label, a luciferin label, a 
luciferase label, an aequorin label, etc. 
VI. ADMINISTRATION OF THE COMPOSITIONS OF THE PRESENT INVENTION 
The therapeutic effects of ICAM-2 may be obtained by providing to a patient 
the entire ICAM-2 molecule, or any therapeutically active peptide 
fragments thereof. Of special interest are therapeutically active peptide 
fragments of ICAM-2 which are soluble. 
ICAM-2 and its functional derivatives may be obtained either synthetically, 
through the use of recombinant DNA technology, or by proteolysis, or by a 
combination of such methods. The therapeutic advantages of ICAM-2 may be 
augmented through the use of functional derivatives of ICAM-2 possessing 
additional amino acid residues added to enhance coupling to carrier or to 
enhance the activity of the ICAM-2. The scope of the present invention is 
further intended to include functional derivatives of ICAM-2 which lack 
certain amino acid residues, or which contain altered amino acid residues, 
so long as such derivatives posess or affect a biological or 
pharmacological activity of ICAM-2. 
Both the antibodies of the present invention and the ICAM-2 molecule 
disclosed herein are said to be "substantially free of natural 
contaminants" if preparations which contain them are substantially free of 
materials with which these products are normally and naturally found. 
The present invention extends to antibodies, and biologically active 
fragments thereof, (whether polyclonal or monoclonal) which are capable of 
binding to ICAM-2. Such antibodies may be produced either by an animal, or 
by tissue culture, or recombinant DNA means. 
In providing a patient with antibodies, or fragments thereof, capable of 
binding to ICAM-2, or when providing ICAM-2 (or a fragment, variant, or 
derivative thereof) to a recipient patient, the dosage of administered 
agent will vary depending upon such factors as the patient's age, weight, 
height, sex, general medical condition, previous medical history, etc. In 
general, it is desirable to provide the recipient with a dosage of 
antibody which is in the range of from about 1 pg/kg to 10 mg/kg (body 
weight of patient), although a lower or higher dosage may be administered. 
When providing ICAM-2 molecules or their functional derivatives to a 
patient, it is preferable to administer such molecules in a dosage which 
also ranges from about 1 pg/kg to 10 mg/kg (body weight of patient) 
although a lower or higher dosage may also be administered. As discussed 
below, the therapeutically effective dose can be lowered if the 
anti-ICAM-2 antibody is additionally administered with an anti-LFA-1 
antibody. As used herein, one compound is said to be additionally 
administered with a second compound when the administration of the two 
compounds is in such proximity of time that both compounds can be detected 
at the same time in the patient's serum. 
Both the antibody capable of binding to ICAM-2 and ICAM-2 itself may be 
administered to patients intravenously, intramuscularly, subcutaneously, 
enterally, or parenterally. When administering antibody or ICAM-2 by 
injection, the administration may be by continuous infusion, or by single 
or multiple boluses. 
The agents of the present invention are intended to be provided to 
recipient subjects in an amount sufficient to suppress inflammation. An 
amount is said to be sufficient to "suppress" inflammation if the dosage, 
route of administration, etc. of the agent are sufficient to attenuate or 
prevent inflammation. 
Anti-ICAM-2 antibody, or a fragment thereof, may be administered either 
alone or in combination with one or more additional immunosuppressive 
agents (especially to a recipient of an organ or tissue transplant). The 
administration of such compound(s) may be for either a "prophylactic" or 
"therapeutic" purpose. When provided prophylactically, the 
immunosuppressive compound(s) are provided in advance of any inflammatory 
response or symptom (for example, prior to, at, or shortly after) the time 
of an organ or tissue transplant but in advance of any symptoms of organ 
rejection). The prophylactic administration of the compound(s) serves to 
prevent or attenuate any subsequent inflammatory response (such as, for 
example, rejection of a transplanted organ or tissue, etc.). When provided 
therapeutically, the immunosuppressive compound(s) is provided at (or 
shortly after) the onset of a symptom of actual inflammation (such as, for 
example, organ or tissue rejection). The therapeutic administration of the 
compound(s) serves to attenuate any actual inflammation (such as, for 
example, the rejection of a transplanted organ or tissue). 
The anti-inflammatory agents of the present invention may, thus, be 
provided either prior to the onset of inflammation (so as to suppress an 
anticipated inflammation) or after the initiation of inflammation. 
A composition is said to be "pharmacologically acceptable" if its 
administration can be tolerated by a recipient patient. Such an agent is 
said to be administered in a "therapeutically effective amount" if the 
amount administered is physiologically significant. An agent is 
physiologically significant if its presence results in a detectable change 
in the physiology of a recipient patient. 
The antibody and ICAM-2 molecules of the present invention can be 
formulated according to known methods to prepare pharmaceutically useful 
compositions, whereby these materials, or their functional derivatives, 
are combined in admixture with a pharmaceutically acceptable carrier 
vehicle. Suitable vehicles and their formulation, inclusive of other human 
proteins, e.g., human-serum albumin, are described, for example, in 
Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton 
Pa. (1980)). In order to form a pharmaceutically acceptable composition 
suitable for effective administration, such compositions will contain an 
effective amount of anti-ICAM-2 antibody or ICAM-2 molecule, or their 
functional derivatives, together with a suitable amount of carrier 
vehicle. 
Additional pharmaceutical methods may be employed to control the duration 
of action. Control release preparations may be achieved through the use of 
polymers to complex or absorb anti-ICAM-2 antibody or ICAM-2, or their 
functional derivatives. The controlled delivery may be exercised by 
selecting appropriate macromolecules (for example polyesters, polyamino 
acids, polyvinyl, pyrrol idone, ethylenevinyl-acetate, methylcellulose, 
carboxymethylcellulose, or protamine, sulfate) and the concentration of 
macromolecules as well as the methods of incorporation in order to control 
release. Another possible method to control the duration of action by 
controlled release preparations is to incorporate anti-ICAM-2 antibody or 
ICAM-2 molecules, or their functional derivatives, into particles of a 
polymeric material such as polyesters, polyamino acids, hydrogels, 
poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, 
instead of incorporating these agents into polymeric particles, it is 
possible to entrap these materials in microcapsules prepared, for example, 
by coacervation techniques or by interfacial polymerization, for example, 
hydroxymethylcellulose or gelatine-microcapsules and 
poly(methylmethyacylate) microcapsules, respectively, or in colloidal drug 
delivery systems, for example, liposomes, albumin microspheres, 
microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such 
techniques are disclosed in Remington's Pharmaceutical Sciences (1980). 
The invention further includes a pharmaceutical composition comprising: (a) 
an anti-inflammatory agent (such as an antibody capable of binding to 
ICAM-2; a fragment of an antibody, said fragment being capable of binding 
to ICAM-2; ICAM-2; a functional derivative of ICAM-2; and a 
non-immunoglobulin antagonist of ICAM-2 other than ICAM-1, and (b) at 
least one immunosuppressive agent. Examples of suitable immunosuppressive 
agents include: dexamethesone, azathioprine and cyclosporin A. 
Having now generally described the invention, the same will be more readily 
understood through reference to the following examples which are provided 
by way of illustration, and are not intended to be limiting of the present 
invention, unless specified. 
EXAMPLE 1 
CLONING OF ICAM-2 cDNA 
In order to clone cDNA capable of encoding ICAM-2, a modification of the 
procedure of Aruffo and Seed (Seed, B. et al., Proc. Natl. Acad. Sci. USA 
84:3365-3369 (1987)) for selecting cDNAs by expression in COS cells was 
employed to pan for ligand-bearing COS cells on functionally-active, 
purified LFA-1 bound to plastic Petri dishes. 
In detail, LFA-1 was purified from SKW-3 lysate by immunoaffinity 
chromatography on TS2/4 LFA-1 MAb Sapharose and eluted at pH 11.5 in the 
presence of 2 mM MgCl.sub.2 and 1% octylglucoside. LFA-1 (10 .mu.g/200 
.mu.l/6 cm plate) was bound to bacteriological Petri dishes by diluting 
octylglucoside to 0.1% in PBS with 2 mM MgCl.sub.2 and overnight 
incubation at 4.degree. C. Plates were blocked with 1% BSA and stored in 
PBS/2mM MgCl.sub.2 /0.2% BSA/0.025% azide/50 .mu.g/ml gentamycin. 
Synthesis of a cDNA library from LPS-stimulated umbilical vein endothelial 
cells by the method of Gubler and Hoffman was performed as described by 
Staunton et al. (Staunton, D. E. et al., Cell 52:925-933 (1988)). 
Following second strand. synthesis the cDNA was ligated to Bst X1 adaptors 
(Seed, B. et al., Proc. Natl. Acad. Sci. USA 84:3365-3369 (1987)) and 
cDNA's &gt;600 bp were selected by low melting point (LMP) agarose gel 
electrophoresis. The cDNA was then ligated to CDM8 (Seed, B., Nature 
329:840-842 (1987)), introduced into E. coli host MC1061/P3 and plated to 
obtain 5.times.10.sup.5 colonies. The colonies were suspended in LB 
medium, pooled and plasmid prepared by standard alkali lysis method 
(Maniatis, T. et al., in Molecular Cloning: A Laboratory Manual, Cold 
Spring Harbor Laboratory (1982)). Ten 10 cm plates of COS cells at 50% 
confluency were transfected with 10 .mu.g/plate of the plasmid cDNA 
library using DEAE-dextran (Kingston, R. E., in Curent Protocols in 
Molecular Biology, 9.0.1-9.9.6, Greene Publishing Associates (1987)). 
ICAM-2 is trypsin-resistant on endothelial and SKW-3 cells. COS cells 
three days post transfection were suspended by treatment with 0.025% 
trypsin/1 mM EDTA/HBSS (Gibco) and panned (Seed, B. et al., Proc. Natl. 
Acad. Sci. USA 84:3365-3369 (1987)) on LFA-1 coated plates as described 
below for .sup.51 Cr-labelled COS cells. Adherent cells were released by 
addition of EDTA to 10 mM. 
Plasmid was recovered from the adherent population of COS cells in Hirt 
supernatants (Hirt, B. j., J. Mol. Biol. 26:365-369 (1967)). The E. coli 
strain MC1061/P3 was then transformed with the plasmid, colonies on plates 
were suspended in LB medium, pooled and plasmid prepared by alkali-lysis 
method. Selection of LFA-1-adherent transfected COS cells and plasmid 
recovery was repeated for two more cycles. Pooled colonies obtained after 
the third cycle were grown to saturation in 100 ml of LB medium with 18 
.mu.g/ml tetracycline and 20 .mu.g/ml ampicillin. Plasmid was prepared and 
fractionated by 1% LMP-agarose gel electrophoresis and MC1061/p3 was 
transformed separately with plasmid from nine different size fractions. 
Individual plasmids from the fraction with greatest activity in promoting 
COS cell adhesion to LFA-1 were examined for insert size by digestion with 
Xba1 and tested in the COS cell adherence assay. This yielded one plasmid 
with an ICAM-2 cDNA insert of 1.1 kb, pCDIC2.27. 
For adhesion assays, the ICAM-2 plasmid pCDIC2.27 or an ICAM-1 construct 
containing the 1.8 kb SalI, KpnI fragment (Staunton, D. E. et al., Cell 
52:925-933 (1988)) in CDM8 (2 .mu.g/10 cm plate) were introduced into COS 
cells using DEAE-Dextran. COS cells were suspended with 0.025% trypsin/1 
mM EDTA/HBSS three days post transfection and labelled with .sup.51 Cr. 
Approximately 2.times.10.sup.5 51 Cr labelled COS cells in 2 ml PBS/5% 
FCS/2 mM MgCl.sub.2 /0.025% azide (buffer) with 5 .mu.g/ml of the MAb 
indicated were incubated in LFA-1-coated 6 cm plates at 25.degree. C. for 
1 hour. Non-adherent cells were removed by gentle rocking and three washes 
with buffer. Adherent cells were eluted by the addition of EDTA to 10 mM 
and .gamma.-counted. 
The feasibility of this procedure was demonstrated using COS cells 
transfected with the previously cloned ICAM-1 cDNA (FIG. 1A). ICAM-1 was 
expressed on 25% of the transfected COS cells. After panning, nonadherent 
cells were depleted of ICAM-1.sup.+ cells, whereas adherent cells released 
from LFA-1-coated plastic by EDTA were almost completely ICAM-1.sup.+. 
Adherence of ICAM-1.sup.+ cells to LFA-1-coated plastic was inhibited with 
RR1/1 ICAM-1 MAb. LFA-1-coated on Petri dishes was stable to &gt;5 cycles of 
COS cell adherence and elution with EDTA; plates were stored with 
Mg.sup.2+ at 4.degree. C. in between use. 
To clone ICAM-2, a cDNA library in the plasmid vector CDM8 was prepared 
from endothelial cells, which demonstrate both the ICAM-1-dependent and 
ICAM-1-independent components of LFA-1-dependent adhesion (Dustin, M. L. 
et al., J. Cell. Biol. 107:321-331 (1988)). Transfected COS cells were 
incubated in LFA-1-coated petri dishes with ICAM-1 MAb present to prevent 
isolation of ICAM-1 cDNA's. Adherent cells were eluted with EDTA and 
plasmids were isolated and amplified in E. coli. Following three cycles of 
transfection, adherence, and plasmid isolation; and one size 
fractionation, 30 plasmids were analyzed by restriction endonuclease 
digestion. Of three with inserts &gt;1.0 kb, one plasmid introduced into COS 
cells by transfection yielded adherence to LFA-1. 
The isolated plasmid conferred adherence to LFA-1 on a high percentage of 
the transfected cells, similar to the percentage seen with ICAM-1 
transfection (FIG. 1B). Adherence was blocked by LFA-1 mAb, but in 
contrast to ICAM-1 transfectants, not by ICAM-1 mAb (FIG. 1B). Futhermore, 
cells transfected with this plasmid did not react with a panel of four 
ICAM-1 mAb. Thus, all functional criteria for a cDNA encoding a second 
LFA-1 ligand were fulfilled, and the ligand was designated "ICAM-2." 
EXAMPLE 2 
CHARACTERIZATION OF ICAM-2 cDNA SEQUENCE 
The ICAM-2 cDNA sequence of 1052 bp (FIG. 2) contains a 62 bp 5' and a 167 
bp 3' untranslated region. An AATACA polyadenylation signal at position 
1019, which in contrast to AATAAA, occurs in approximately 2% of 
vertebrate mRNAs (Wickens, M. et al., Science 226:1045-1051 (1984)), is 
followed at 1058 bp by a poly(A) tail. The longest open reading frame 
begins with the first ATG at position63 and ends with a TAG termination 
codon at position 885. Hydrophobicity analysis (Kyte, J. et al., J. Mol. 
Biol. 157:105-132 (1982)) and usage of amino acids around cleavage sites 
(von Heijne, G., Nucleic Acids Research 14:4683-4690 (1986)) predict a 21 
residue signal peptide (FIG. 2). 
The predicted mature sequence contains from amino acid 1 to 201 a putative 
extracellular domain followed by a 26 residue hydrophobic putative 
transmembrane domain and a 26 residue cytoplasmic domain. Four turns of 
the putatively e-helical transmembrane segment are amphipathic, with 
threonine and serine residues falling on one side, suggesting the 
possibility of self-association or association with other membrane 
proteins in the plane of the membrane. The cytoplasmic domain is unusually 
basic, and in contrast to most cytoplasmic domains which are hydrophilic, 
is of average hydrophobicity. The predicted mass of the mature polypeptide 
is 28,176 daltons which, if the six predicted N-linked glycosylation sites 
are used, would result in a ICAM-2 glycoprotein of approximately 46 Kd. 
EXAMPLE 3 
DNA AND RNA HYBRIDIZATION ANALYSES 
The isolated ICAM-2 cDNA clones were analyzed using both Northern and 
Southern hybridization. Northern blots used 6 .mu.g of poly(A).sup.+ RNA 
which was denatured and electrophoresed through a I% agarose-formaldehyde 
gel (Maniatis, T. et al., in Molecular Cloning: A Laboratory Manual, Cold 
Spring Harbor Laboratory (1982)) and electrotransferred to a nylon 
membrane (Zeta Probe, BioRad). Completion of transfer was confirmed by UV 
trans-illumination of the gel and fluorescent photography of the blot. 
The genomic DNAs were digested with five times the manufacturer's 
recommended quantity of EcoRI and HindIII endonucleases (New England 
Biolabs). Following electrophoresis through a 0.8% agarose gel, the DNAs 
were transferred to Zeta Probe. RNA and DNA blots were prehybridized and 
hybridized following standard procedures (Maniatis, T. et al., in 
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory 
(1982)) using ICAM-2 or ICAM-1 cDNAs labeled with .alpha.[.sup.32 P]d 
XTP's by random priming (Boehringer Mannheim). 
The 1.1 kb ICAM-2 cDNA hybridizes to a 1.4 kb poly(A).sup.+ mRNA and weakly 
to a 3 kb mRNA (FIG. 3A), distinct from the 3.3 kb and 2.4 kb ICAM-1 mRNA 
(FIG. 38). mRNA was examined in cells which have been characterized 
functionally for ICAM-1-dependent and second ligand-dependent binding to 
LFA-1. ICAM-1 mRNA is strongly induced in endothelial cells by LPS (FIG. 
38, lanes 2 and 3). In contrast, ICAM-2 mRNA is strongly expressed basally 
in endothelial cells and is not induced further by LPS (FIG. 3A, lanes 2 
and 3). This correlates with strong basal and non-inducible expression of 
the LFA-1-dependent, ICAM-1-independent pathway in endothelial cells and 
inducibility of the ICAM-1-dependent pathway (Dustin, M. L. et al., J. 
Cell. Biol. 107:321-331 (1988)). 
ICAM-2 mRNA is present in a wide variety of cell types including Ramos and 
BBN B lymphoblastoid, U937 monocytic, and SKW3 lymphoblastoid cell lines 
(FIG. 3A, lanes 1,4,6, and 8), as shown by moderate or long autoradiogram 
exposure. Of these, SKW3, U937, and BBN have been shown to exhibit 
LFA-1-dependent, ICAM-1-independent adhesion to LFA-1.sup.+ cells 
(Rothlein, R. et. al., J. Immunol. 137:1270-1274 (1986); Makgoba, M. W. et 
al., Eur. J. Immunol. 18:637-640 (1988)), and to LFA-1-coated plastic. The 
HeLa epithelial cell line, which exhibits only the ICAM-1-dependent 
component of LFA-1-dependent adhesion (Makgoba, M. W. et al., Eur. J. 
Immunol. 18:637-640 (1988)), shows no ICAM-2 mRNA (FIG. 3A, lane 5), even 
after prolonged autoradiogram exposure. The cell distribution of ICAM-2 is 
thus consistent with the ICAM-1-independent component of LFA-1-dependent 
adhesion. 
Southern blots of genomic DNA (FIG. 3D) hybridized with the ICAM-2 cDNA 
showed a single predominant EcoRI fragment of 8.2 kb and HindIII fragment 
of 14 kb, suggesting a single gene with most of the coding information 
present in 8 kb. 
EXAMPLE 4 
COMISON OF THE AMINO ACID SEQUENCES OF ICAM-1 AND ICAN-2 
Because of their functional similarity as LFA-1 ligands, the amino acid 
sequences of ICAM-2 and ICAM-1 were compared. ICAM-1 is a member of the Ig 
superfamily and its extracellular domain consists entirely of five C-like 
domains. The 201 amino acid extracellular domain of ICAM-2 consists of 2 
Ig C-like domains, with putative intradomain disulfide-bonded cysteines 
spaced 43 and 56 residues apart and a predicted .beta. strand structure 
(FIG. 4). Remarkably, the two Ig-like domains of ICAM-2 are 34% identical 
in amino acid sequence to the two most N-terminal Ig-like domains of 
ICAM-1 (FIG. 4), with an ALIGN score 15 s.d. above the mean, and 27% 
identical to ICAM-1 domains 3 and 4, with an ALIGN score 3 s.d. above the 
mean. 
Search of the NBRF and SWISS-PROT protein databases yielded only partial 
domain homologies with other members of the Ig superfamily, primarily with 
HLA Class II antigens. ICAM-2 shows somewhat fewer conserved residues 
characteristic of Ig domains than ICAM-1. ICAM-2 is 17% and 19% identical 
to the two N-terminal domains of the adhesion molecules NCAM (Cunningham, 
B. A. et al., Science 236:799-806 (1987)) and MAG (Salzer, J. L. et al., 
J. Cell Biol. 104:957-965 (1987)), respectively, while ICAM-1 is 19% and 
20% identical, respectively. 
Lymphocyte function associated antigen-1 (LFA-1) and intercellular adhesion 
molecule-1 (ICAM-1) were identified by selecting MAb which blocked T 
lymphocyte-mediated killing, and homotypic adhesion, respectively 
(Rothlein, R. et al., J. Immunol. 137:1270-1274 (1986); Davignon, D. et 
al., Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)). In contrast, ICAM-2 
has been defined using a functional cDNA selection procedure which 
requires no previous identification of the protein by biochemical or 
immunological techniques. 
Isolation of a cDNA for ICAM-2 confirms the postulated existence of an 
alternative LFA-1 ligand. The distribution of mRNA for ICAM-2 on a limited 
number of cells which have been characterized for ICAM-1-dependent and 
ICAM-1-independent adhesion to LFA-1 suggests that ICAM-2 could account 
for all of the observed ICAM-1-independent LFA-1-dependent adhesion. 
ICAM-2 and the two N-terminal domains of ICAM-1 are much more like one 
another than like other members of the Ig superfamily, demonstrating a 
subfamily of Ig-like molecules which bind to LFA-1. Significantly, the 
LFA-1-binding region of ICAM-1 has been mapped to domains 1 and 2 by 
domain deletion and systematic amino acid substitution. Thus, there is 
both structural and functional homology. ICAM-2 is the second example of 
an Ig-family member which binds to an integrin. Although there is little 
precedence among cell adhesion receptors, among the integrins a number of 
receptors for extracellular matrix components have been shown to recognize 
multiple ligands (Hynes, R. O., Cell 48:549-554 (1987); Ruoslahti, E. et 
al., Science 238:491-497 (1987)). 
Neither ICAM-1 or ICAM-2 contains an RGD sequence, and thus the mode of 
recognition by LFA-1 may differ from integrins which bind extracellular 
matrix components (Hynes, R. O., Cell 48:549-554 (1987); Ruoslahti, E. et 
al., Science 238:491-497 (1987)). The cellular ligands recognized by Mac-1 
and p150,95, leukocyte integrins closely related to LFA-1, may belong to 
the same Ig subfamily. ICAM-1 has recently been demonstrated to be a 
receptor for the major group of rhinoviruses which cause 50% of common 
colds. ICAM-2 may also function as a receptor for rhinoviruses or other 
piconaviruses. Thus, it may be used in a therapy to suppress (i.e. prevent 
or attenuate) infection from such viruses. 
A family of ligands for LFA-1 emphasizes the importance of this recognition 
pathway and may be a mechanism for imparting fine specificity and 
functional diversity. A number of differences between ICAM-1 and ICAM-2 
are of potential importance. ICAM-1 is inducible on most cells while 
ICAM-2 expression is not affected by cytokines on the cells thus far 
tested. The three additional domains on ICAM-1 are expected to project its 
LFA-1 binding site further from the cell surface than that of ICAM-2, 
suggesting that closer cell-cell contact would be required for 
LFA-1:ICAM-2 than LFA-1:ICAM-1 interaction. ICAM-2 transfected COS cells 
are more readily detached than ICAM-1 transfected COS cells from LFA-1 
coated plastic as the washing shear force is increased. This may be due to 
the smaller size of ICAM-2 which may make it less accessible to LFA-1 on 
the artificial substrate, or to differences in sequence which impart 
differences in affinity. 
The distinct cytoplasmic domains of ICAM-1 and ICAM-2 may impart different 
signals or may cause differing localization on the cell surface; likewise, 
signalling or interaction with the cytoskeleton by LFA-1 may differ 
depending on whether ICAM-1 or ICAM-2 is bound. 
ICAM-1 and a second LFA-1 counter-receptor, ICAM-2, thus constitute a 
subfamily of the immunoglobulin (Ig) superfamily (Staunton, D. E., et al., 
Cell 52:925-933 (1988), which reference is incorporated herein by 
reference). ICAM-1 possesses five Ig-like C domains whereas ICAM-2 
possesses two, which are most homologous to the amino terminal domains of 
ICAM-1. ICAM-1 and ICAM-2, expressed on a variety of cell types, support 
various leukocyte adhesion dependent functions incluoting induction and 
effector functions in the immune response. ICAM-1 expression is highly 
inducible by cytokines and thus the LFA-1/ICAM-1 adhesion system is able 
to guide leukocyte migration and localization during inflammation 
(Rothlein, R. J. Immunol. 137:1270-1274 (1986); Marlin, S. D. et al., Cell 
51:813-819 (1987); Kishimoto, T. K. et al., Adv. Immunol. 46:149-182 
(1989); Dustin, M. L. et al., Immunol. Today 9:213-215 (1988), all of 
which references are incorporated herein by reference). 
ICAM-1 residues which have been defined above as being important to LFA-1 
binding are conserved in other ICAMs (Staunton, D. E., et al. Nature 
339:61-64 (1989), which reference is incorporated herein by reference). 
Human ICAM-1 is 50% identical to murine ICAM-1 and 35% identical to human 
ICAM-2 (Staunton, D. E., et al. Nature 339:61-64 (1989). The residues that 
are most critical to LFA-1 binding, E34 and Q73, are conserved both in 
mouse ICAM-1 and in human ICAM-2. This is consistent with the ability of 
both mouse ICAM-1 and human ICAM-2 (Staunton, D. E., et al. Nature 
339:61-64 (1989)) to bind to human LFA-1. One D2 N-linked glycosylation 
site at N156, which influences LFA-1 binding, is also conserved in ICAM-2. 
Several residues that are important to rhinovirus-14 binding, Q58, G46, 
D71, K77 and R166, are not conserved in mouse ICAM-1 or human ICAM-2 
(Staunton, D. E. et al., Cell 56:849-853 (1989), which reference is 
incorporated herein by reference) which is consistent with the apparent 
inability of mouse cells (Colonno, R. J. et al., J. Virol. 57:7-12 (1986)) 
and ICAM-2 to bind rhinovirus-14. 
While the invention has been described in connection with specific 
embodiments thereof, it will be understood that it is capable of further 
modifications and this application is intended to cover any variations, 
uses, or adaptations of the invention following, in general, the 
principles of the invention and including such departures from the present 
disclosure as come within known or customary practice within the art to 
which the invention pertains and as may be applied to the essential 
features hereinbefore set forth as follows in the scope of the appended 
claims.