Note: Descriptions are shown in the official language in which they were submitted.
WO 94/08620 X 1 g ~ 7 3 8 PCI/US93/09777
.. - 1 -
A SUBPOPULATION OF MAC-1 (CDllB/CD18) MOLECULES
VVHICH MEDIATE NEUTROE'HIL ADHESION TO ICAM-1 AND
RINOGEN
~IELD OF THE INVENTION
The present invention relates to the field of cellular adhesion and the
use of anti-adhesion molecules as a means of merli~ting biological processes
which are facilitated by myeloid cell adhesion, migration and activation.
Specifically, the present invention discloses that specific subpopulations of
Mac-1 are activated when myeloid cells are stimulated and that upon
activation, activation specific epitopes appear on the Mac-1 molecule. Based
on the identification of activation specific epitopes, a new class of anti-Mac-1antibodies are disclosed which are capable of binding to an activated form of
Mac-1 present on stimulated myeloid cells but are substantially incapable of
binding to non-activated Mac-1 and resting myeloid cells.
DESCRIPIION OF THE RELATED ART
Under normal conditions, circulating neutrophils flow through the
vascular system making few ~tt~hments to the underlying endothelium.
Within minutes of an exposure to an infl~mm~tory stimulus, neutrophils roll
along the adjacent vessel wall, adhere firmly to the endothelium, and then
trafflc into the parenchyma (Atherton et al., J. Physiol. 222:447-474 (1972)).
WO94/08620 ~ 738 Pcr/l~S93/09777
Recent experiments have identified the mole ules on neutrophil and endothelial
cell surface that facilitate emigr~tion. ~emhers of the selectin family
(Springer et al., Na~ure 349:19~197 (1991)) m~ t~ the initial rolling and
lllbels of the intPgrin family (Hynes, Cell 69~ 25 (1992)) ~L~cllglhen
l~uLI~hil ~ r~ rl to the en(lothe-lium (L~lcnce et al., Cell 65:859-873
(1991)), and then ~lulllûLe tr~n~mi~ration to the underlying tissue (And~l~n,
et al., Blood 76:2613-2621 (1990); Smith, et al., J. Clin. ~nvest. 83:2008-
2017 (1989)).
The integrin family of heterodimeric glyco~r~t~ s me li~tP cell
adhesion and ~tt~ehm~ont to the ~oYtr~rPll~ r matrix (Hynes, Cell 48:549-554
(1987); Hynes, CeU 69:11-25 (1992)). The leukocyte integrin subfamily
includes three ...c...bera,, LFA-l (CDlla/CD18), Mac-l (CDllblCD18) and
pl50,95 (CDllc/CD18) that share a collllllol~ ,B subunit that is noncc,~lently
h~1 with unique but closely related a chains (Ki~himoto et al., Adv.
r~ .ol. 46:149-182 (1989); Sp~ ge., Naturc 346:425-433 (1990)). These
gly~lut~ ls share a co....~o-- CD18 ~B subunit (95,000 Mr) but have
individual w~ique CDl l cY s~ui~i~ (175,000, 160,000, 150,000 M,)
c~celi~.,ly, that are ~llu~:~ul~lly hor~ologous (Larson, et al., J. Cell Biol.
108:703-712 (1989)). All three ~ share two pr~ c ~ ea~ul.,s in the
eytr~R~ r region of the ll-o!e~llle, a IJuL~ , divalent cation binding region
corlci~ting of three tandem repeats of an EF-hand motif, and a 200 amino acid
inse.~d or "I" domain (~ mlt et al., J. Cell Biol. 106:2153-2158 (1988~;
Corbi, et al., J. Biol. Chem. 263:12403-12411 (1988); Corbi, et al., EMBO
J. 6:4023~028 (1987); ~ , et al., J. Irnrnunol. 147:369-371 (1991);
Larson, et al., J. Cell Biol. 108:703-712 (1989); Pytela, EMBO J. 7:1371-
1378 (1988)). The I domain is absent in all other known int~E;.iI~ a sul,ul.i~
except for the a~ and a2 ;,~u~ of the VLA subfamily of illt',~,l ;l~S (Hemler,
Annu. Rev. In~nunol. 8:365~00 (1990); Hynes, Cell 69:11-25 (1992)).
The leuko~yle i~ e several adhesive events that are crucial
for immnnlo system function. They ploIllole the adhesion that is re4uir~d for
T lymphocyte target cell Iysis (Davignon, et al., Proc. Na l. Acad. Sci. USA
78:45354539 (1981)), T Iymphocyte proliferation (Davignon, et al., Proc.
Wo 94/08620 ~ 1 ~ 4 ~ 3 8 PCr/US93/09777
Natl. Acad. Sci. USA 78:45354539 (1981)), naturdl killing (Krensky et al.,
J. ~mmunol. 131:611-616 (1983)), leukocyte adhesion to, and migration
through endothelial cells (Dustin etal., J. Cell Biol. 107:321-331 (1988);
Harlan et al., Blood 66:167-178 (1985); Haskard et al., J. Irr~nunol.
137:2901-2906 (1986); Lo et al., J. Exp. Med. 169:1779-1793 (1989); Lo
et al., J. I~tunol. 143(10J:3325-3329 (1989); Smith, et al., J. Clin. Invest.
83:2008-2017 (1989); Smith, et al., J. Clin. Invest. 82:174~1756 (1988)),
neutrophil homotypic aggl~d~ion, and neullophil çl~r!,,o~ s (~n~
et al., J. ~ nunol. 137: 15-27 (1986)).
Mac-1 is IAl,r~;,sed in an intrAr~.llular, vesicul~r compartment in
circulAtin~ nt;ull~hils and mol~ocr~s which is mobili ed to the cell surface
by i..~ ,r ~r~l;A~r~ (Todd, et al., J. Clin. Invest. 74:128~1290
(1984);Sl~linger, etal.,In: BiochenustryofMa~r~ tages (CIBAS~lllpos;u",
118), Pitman, London, pp. 102-126 (1986); Lanier, etal., Eur. J. ~rnm~ol
15:713-718 (1985); Yancey, et al., J. Immunol. 135:465-470 (1985)). This
mQbili7~ti~n ooll~la~s wi~ h,~l~d adhesi~ .css (A"dc.~n, et al., Ann.
I~ev. Med. 38:175-194 (1987)). Mac-1 cY-subunit ~nrS~e was dGt~l in
blood mOllOCy~S and PMA-in.i~cc~l myeloid cell lines, but not in most cells
of the T or B lin~es, co"~laling with Mac-1 protein surface cAI)r,_s~;oll.
Some ~lut~AiC T l~l"~ho~le clones have been found to e~press
similar qu~ntities of plS0,95 and LFA-1. Mon~clo..;~l ~n~;l~l;es to the LFA-1
and plS0,95 alpha- ,ul,u,liL~ inhibit killing by such CTL clones to similar e~tent
and are addili~ in their inhibi~o,y effects (Keizer, et al., J. Imrnunol.
138:313û 3136 (1987)). I;ul!l.f-....or~ Lil,odies to plS0,95 alpha-subulliLs
25 have been shown to inhibit ",onoc~L~ r~ to e-.-loll.P~ ." (Keiær, et
al., Eur. J. ~rnrnunol. 17:1317-1322 (1987)).
Mon~clol-AI ~ntiboAies to Mac-1 or plS0,95 inhibit "e.ll,u~hil
aggl~gàliull and adhtr,nce to endothelial cells, protein-coated su, r~ces,
bA~ ulO>~n ~ Sitl~S, and fungi (Harlan, et al., Blood 66:167-178
30 (1985); Springer, et al., In: Biochernistry of Macrophages (CIBA Symposium
118), Pitman, London, pp. 102-126 (1986); Dana, et al., J. Immunol.
Wo 94/08620 ~ Pcr/uss3/o9777--
137:3259 (1986); Bullock, etal., J. Exper. Med. 165:195-210 (1987); Mosser,
et al., J. Immunol. i35:2785-2789 (1985)).
Mac-l (CDllb/CD18) is a leukocyte adhesion gly~oprultin that has
been demo~ .t~ to bind multiple ligands in-,lutling iC3b (Beller, et al., J.
Exper. Med. 156:1000 1009 (1982)), r,b~ oge.l (Altieri, ct al., J'. Cell. BioL
107:1893-1900 (1988); Wright, et al., Proc. Nat'l. Acad. Sci. (U'.S.A.)
8~:7734-7738 (1988)),and Factor X (Altieri, et al., J. Biol. Chcm. 863:7007-
7015 (1988)) in addition to its role in cell-cell and cell-~.,l,s~ le adhesive
Pl~ l;ons. Delc,~c,ll-soluble Mac-1 and pl50,95 have been shown to bind
to iC3b-Sepharose (~icL-ko.m, et al., Biochem. J. 231:233-236 (1985)).
The ~-subunit of Mac-l is a ~ .f ~.blanc protein of 1137 residues
with a long eYtrac~ A- dom~in (1092 residues) and a 19-amino acid
cytoplasmic tail. The e-tr~- Pll~ r domain c4~ c 3 ~ulali~, divalent cation-
binding ~ en~s and 19 pbt .~I;A1 N-glyc~yl~ion sites. The a nino acid
~lu~- ~eG of Mac-l a shows that it is a .. hcr of ~e i.. ~ su~clr~llily;
Mac-1 a shows 63% identity to the a-subunit of the l~ lhP, :on
gly~lut~,;n pl50,95 and 25% to the a-~uni~. of the ~ lh)lAr matri~c
receplor platelet gly~r~)lein IIb/IIIa, the rll.lone~ilin l~)tor and the
vil~one~Lin l~p~r. The Mac-1 -subunit pu~li~ divalent cation-bindh~g
20 sites and the fl~nl-in~ regions e~hibit a high degree of identity both to theplSO,9S cY-subunit (87% idwllily at the amino acid level) and to the rest of theil,te~lh~ a-;,~ (38%). The a-s~bllnit of Mac-l, like the plSO,9S ~-
subunit, cont~inc a domain of 187 amino acids in the e~ r region
which is absent in other int~.~rinc This inse.~d or "I" domain is homologous
25 to the A ~o...~;"c of van Willebrand factor, which in turn are homologous to
regions of the C3-binding l,r~)leh~s factor B and C2. These r~n~ ,s draw
attention to this region of Mac-1 as a ~l,lial binding site for iC3b.
The functional role of Mac-l was first il~ t li by the ability of anti-
Mac-l a-subunit monoclonal ~nti~i~os (MAb) to block the rosettin~ of iC3b-
30 coated erythrocytes to macrophages and poly.-lo,L,horll~rle~r le~o~;yles (Beller,
et al., J. Exper. Med. 156:1000-1009 (1982)), demon~t~tin~ that Mac-l is
jn~iistinf~uishable from the complement l~ or type three (CR3).
WO94/08620 i~ 738 PCI/US93/09777
Subsequently, the involvement of Mac-1 in infl~ o-y processes was
evidenr~d by the inhibition of ne.ll-u~hil aggregation and adhesion to
endothelial cells by anti-Mac-1 a-subunit and anti-,B-subunit-specific MAb
(Anderson, et al., J. Immunol. 137:15-27 (1986); Dana, et al., J. Irnmunol.
137:3259-3263 (1986); Vedder, et al., J. Clin. Invest. 81:672-682 (1988)).
Recent epitope .,la~pnlg studies have suggestcd that the sites involved in iC3b-binding are distinct from those involved in ~ L,ophil ~ Ltion and
adh~l~,nce to protein-coated plastic (Al~de,so--, et al., J. r771m"~1QI. 137:15-27
(1986); Dana, et al., J. ~mmunol. 137:3259-3263 (1986), Rosen, et al., J.
Exper. Med. 166:1685-1701 (1987)). The-~,for~, Mac-l appears to be a
~ cep~or .,~lu~s at least 2 sites for its ~AhPsion-related run-~!;o~
The CA~ S:~lOn of fim~tiolLAI ae~ivily of Mac-l is ~ ~i during
leuku~;yle dirf~..c~ ;QI~ and activation. Dirf~ lion and ...~ I;orl of
myelQ.- onnc~lic cell lines ~csults in incl~d Mac-l cAyl~s~;on (Miller, et
al., J. ~.. ~.. -~1. 137:2891-2900 (1986)), whileblood ll-u~oc~dirr~ l;A~;Q~
into tiæue ll-a~ hages iS ~r~ A~ by a con~ e~able dcc-~ in ~e
~...n....~ of Mac-l on all cell sulrÇ~ces (Hogg, et al., Eur. J. ~ "ol 16:24
248 (19886)). The ~ ~s;on of Mac-1 on the surface of circnl~tin~
~e~ ù~)hils and IIIOIIG~S iS upregulated by il~n~.~.. .z~n~, ctimllli; Mac-1 is
stored in an intr~C~ r vç~;clll~r ov~ alLln~ which is rapidly mobilized tO
the cell surface by c~ .Ae~ (Todd, et al., J. Clin. Invest. 74:128~
1290 (1984)); Miller, et al., J. Clin. Invest. ~0:535-544 (1987)). Although
the ~n~ f.l~ G~ s;,ion of Mac-l can lead to il.w~sed adhe;,i~ css,
q~ it~tive çl~ gf s after cell activation may also be h.lpvl~nL in regulation
ligand bh-ling (Detmers, et al., J. Cell Biol. 105:1137-1145 (1987)). Both
the q-l~lit~tive and yuo~ cl~s may be i~ vll~ll in re~nl~tion of
leukvcyl~ billdiJ~ to post capillary c-uiQIl çlillm at i.,rlz--.--.~tol~ sites.
Thus, in s~ .y, the ability of leuku~iyt~s to m~int~in the health and
viability of an animal re~uires that they be capable of ~lhPrin~ to other cells
(such as en~otheli~l cells) and l~-v~ s (such as iC3b). This adhe.ence has
been found to require cvn~cls which involve specific .~)lo. molecules
présent on the surface of the leukocytes. Some of these cell surface receptor
WO94/08620 ~ 73~ PCl`/US93/09777--
molecules have been found to be highly related to one another. Humans
whose leukocytes lack these cell surface r~ or molecules exhibit chronic
and l~ulling infections, as well as other clinical ~,.,~IO,.,S.
I~Al,c.i.l,~ byseveralgroupshavede-"o~ Athattheadhcs,ven~ss
5 of the lc.~;yle i-l~,i, s is not col~iLuli~e, but rather is controlled by
intr~r,~ r signals ~ luc~d from other surface lCC~ . For e~ample,
LFA-l on resting T ly~ )hocyt~,s is unable to bind to its counler-.~eplor
ICAM-1 until a signal has been delivered through the antigen ,~tor (Dustin
et al., Nature 341:619-624 (1989); van Kooyk, et al., Nature 342:811-813
10 ~1989)). The signal to adhere is tr~n~i~nt, as within thirty minlltPs of
stim~ tiQn T lymphocytes lose their ability to bind ICAM-l. Similarly,
Mac-l on n~ o~hils does not bind its cellular or soluble ligands inrl~.lit~g
ICAM-l (D;au,o"d, et al., Cell6~:961-971 (1991)), Gl~lh~o~n (Altieri, et al.,
J. Cell. Biol. 107:1893-1900 (1988); Wright, et al., Proc. Natl. Acad. Sci.
USA ~5:7734-7738 (1988)), factor X (Altieri etal., J. Biol. Ch ~n. 263:7007-
7015 (1988)) or iC3b (Wright et al., J. I".,,.u..ol. 136: 1759-1764 (1986)) until
a specific signal (e.g., C1~PI~O~ ;C factor, cytokine, or l)ho,l,ol ester) has been
delivered; stim~ tion inc,~dses the avidity of Mac-1 for its ligands within
s~ ls (Andcl~on, et al., J. Irn~nunol. 137:15-27 (1986); Buyon, et al., J.
I~u~u~ol. 140.315~3160 (1988); Detm~-rs, et al.. J. EJrP. M ed. 171:1155-
1162 (1990); Wright et al., J. In~nunol. 136:1759-1764 (1986)).
While it is clPar that activation f~r31it~tPs lcuku~;y~ i~t~E;Iin i .l~l, rl;on
with ligand, ~e molecular ..~P~h~ m for the change in avidity remains poorly
nn~erstood. l~c~-~ ~ere is no inc,~se in LFA-1 t;.~,~;.sion after antigen
25 stimlll~tio~ or phorbol ester l,~~ in T cells, the avidity change of LFA-1
for ICAM-l is belie~d to occur b~aus~ of a sLlu~lul~l change in the
molecule (Dustin et al., Nature 341:619-624 (1989)). ~l~r~ ,ly, the
change in avidity could stem from a signal to cluster adhesion ~ at the
cell surface (Detmers, etal., J. Cell Biol. 105:1137-1145 (1987)). The
30 mPrh~nicm for avidity regulation of Mac-l on nc;ullophils a~Jp~lr~ analogous,but the artalysis is complicated by a greater than tenfold qtl~ live incl~se
in expression that follows stimul~tion (Berger, et al., J. Clin. Invest. 74: 1566-
WO94/08620 2~ s73~ PCl/US93/09777
1571 (1984); Detmers, et al., J. Exp. Med. 171:1155-1162 (1990); Lo et al.,
J. Exp. Med. 169: 1779-1793 (1989); Miller, et al., J. Clin. Invest. 80:535-544
(1987); Todd, et al., J. Clin. Invest. 74:1280-1290 (1984)). However, the
change in surface e~r~s~ion that occurs following stim~ tion does not parallel
S the kii.f l;es or ...~gn;l..-4. of ~hr.~ion (Buyon, et al., J. Im~nunol. 14~.315
3160 (1988); Lo et al., J. Exp. Med. 169:1779-1793 (1989)), as ion channel
antagonists ~Vedder et al., J. Clin. Invest. 81:676-682 (1988)) or ~ ...I~.Y.I...~;
co~litions (Schleirrcnh~.. , et al., J. I".".u,.ol. 142:3537-3545 (1989)) that
inhibit the ~ c increase in Mac-l e~r~,;,sion do not block Mac-l-
de~endent adhesion to enrlothp~ cells or in vitro sul~sl,~t~s. Thus. Mac-1 is
hypoth~ci7P.d to ulldel~.o additional, con&~ ;ol~l changes that f~cilitate
adhesion (Buyon, et al., J. Irnmunol. 140.3156-3160 (1988); Lo et al., J. Exp.
Med. 169:1779-1793 (1989); Philips, et al., J. Clin. Invest. 82:495-501
(1988); Vedder et al., J. Clin. Invest. 81:67~682 (1988)). Studies that show
that divalent cations (Altieri, J. ,.". .ol. 147: 1891-1898 (1991); D. ~ncl ;eld,
et al., J. Cell Biol. 116:219-226 (19~2); Dr~ncfield et al., EMBO J. 8:3759-
3765 (1989); van Kooyk, et al., J. Cell Biol. 112:345-354 (1991)) or MAbs
against Mac-l or LFA-1 (Dr~ncfiel~l, etal., J. Cell }~iol. 116:1527-1535
(1992); Hibbs et al., ,S~q~n~e 251:1611-1613 (1991); Keizer et al., J.
Irnmunol. 140:1393-1400 (1988); Robh~son, et al., J. Immunol. 148:108~1085
(1992)) "~ .en~ hrsion in the s~bs~ r~ of ~iCli~aliOn~ ~up~)ll a model in
which direct ~l.u~;lulal c~ gcs to the IAIIa~llular regions regulate le~ uc~r~
integrin avidity.
Despite the cDNA cloning and the i<le-~1;1;r~tion of an array of ligand
2S i~ l;onc7 the region(s) on the a and ,~ sul~uni~ of the leukocyte in~ .;.,.cthat r -~ liate ligand bil~ g have not been r~ h~ cd. The platelet adheJ,on
- mole~.llle crnb-~3 (CD41/CD61) is the ;~t~.g~;n farnily ,.~.. hcr that is best
cl~.a~li~d for the subunit ~iolll~il.c which CollllilJul~ to ligand ~ccog.,ilion.
Chemi(~l cross-linking e~ (D'Souza, J. Biol. Ch~em. 265:344~3446
(1990)) and functional studies (D'Souza, Na~ure 350:66-68 (1991)) suggest that
fibrinogen binds to an 11-residue peptide within the divalent cation binding
repeats on crllb~ and that R-G-D peptides bind to a site on the ,~3 subunit
Wo 94/08620 ~ ~ 4 ~7~ PCI/US93/09777--
between amino acid residues 61-203 (D'Souza, Science 242:91-93 (1988);
Smith et al., J. Biol. Chem. 263:18726-18731 (1988)). A natural point
mutation found in patients with Gl~r,7,..~nn's thromb~cll.f~-iA on residue 119
of the ,B3 subunit abrogates ligand recognition and disturbs binding of divalentcations (Loftus, et al., Science 249:915-918 (1990)). However, ~1.~ Ll,er the
results can be genePli7PA to the leukocyte il~ ls is not clear. ~lthough
Mac-1 was reported to bind to R-G-D-like ~.Je~-r~s in some of its ligands
(Ross, etal., J. Leukoc. Biol. 51:109-117 (1992); Wright, etal., J. Erp.
Med. 169:175-183 (1989); Wright, et al., Proc. Natl. Acad. Sci. USA
84:1965-1968 (1987); Wnght, et al., Proc. Natl. Acad. Sci. USA 85:7734-
7738 (1988)), recent studies have demol.c~ ed that it does not utilize R-G-D-
like ~.~uel-rr,s to bind iC3b and fibrinogen (Altieri, et al., J. Biol. Chem.
265:12119-12122 (1990); Altieri, et al., J. Cell. Biol. 107:1893-1900 (1988);
Taniguchi-Sidle et al., J. Biol. Chem. 267:635-643 (1992)). Similarly, the
binding sites on ICAM-1 for LFA-1 (St~llnton, et al., Cell 61:243-254 (1990))
and Mac-1 (D;h~ni~l> et al., CeU 65:961-971 (1991)) do not contain R-G-D
res~ les Thus far, no data has been lc~d on the 1~1i7~tior~ of ligand
binding sites for any of the in~ c that contain I do.l.aills. ~
domains are ~llu~;lu~ally homologous to ligand binding modlllPs in von
Willebrand factor and complement factor B (Larson, et al., J. Cen Biol.
108:703-712 (1989); Pytela, EMBO J. 7:1371-1378 (1988)), it has been
speculated that this region is involved in the l~og~ ;Qn of ligands by i~
(Hynes, CeU 69:11-25 (1992); Larson, et al., J. Cell Biol. 108:703-712
(1989); Pytela, EMBO J. 7:1371-1378 (1988)).
Mac-1 is one of tne most i-lt~ of the i"t~ c that contain an I
domain as it IJ-u--~ u~u..ly binds several soluble and cell surface lig~ndc.
Rer~ e previous studies had demo..~ ~ that distinct Mac-1-dcp.,ndw
adhesive functions could be blocked by sli~.z.~l.; groups of MAbs to Mac-1
a (Ande.son, et al., J. Immunol. 137: 15-27 (1986); Dana, et al., J. In~nunol.
137:3259-3263 (1986); Diamond, et al., ~Dirr~ Lial Effects on Leukocyte
Functions of CDlla, CDllb, and CD18 Monoclonal Antibodies," in
Leukocyte Typing IV, Knapp et al., eds., Oxford University, London, 570-574
Wo 94/08620 2 1 ~ ~ 7 3 8 PCr/US93/09777
(1989); Wright, et al., Proc. N~l. Acad. Sci. USA 80:5699-5703 (1983);
Wright, et al., Proc. Natl. Acad. Sci. USA 85:7734-7738 (1988)), we
hypothPci7~d that se~ dte ~LIuclul~l domains of the m~le 1~1e might confer
fim~tion~l crecificity~ ~ of the high degree of s~ c~undl identity,
5 ~ntigenic dirf~ ce. and a~c,nL functional rlictim~.tinn bel~.~n Mac-1 and
plS0,95, we ~lesignp~ r~ cal sets of Mac-l/plS0,95 cl.;.... ;~c to map the
ligand binding sites. In this report, four function~lly distinct ligands are
eY~minPA We find that the I domain on Mac-l is critical for the ,ccognilion
of all of these ligands.
SUMMARY OF l~lE INVENTION
The present invention is based on the novel obse.valion that when
resting myeloid cells are stim~ ted, not all Mac-1 molecules which are present
15 on ~e cell surface be~ e &Clivat~d. Only select sub-popnl~tionc of Mac-l
û'~ es b~4~ a~ àt~d and gain the ability to bind ligand. The present
invention further t~ oces that upon activation, activation :,~c-;fic el)iLo~s
appear on the Mac-1 molecule. These activation ~;fic epit~pe can be used
to dirf~ "lLiale &;li~ràted Mac-1 ~lecllles and ~timnl~tp~ myeloid cells from
20 non~ d~d Mac-1 mnleclll~P.s and resting myeloid cells.
Based on these activation spP~ific c,pi~cs, the present ill~ ion
discloses the ge l~r~liQrl of a novel clæs of ",~ onql ~nti~i~s~ Mabs,
which bind to a~ilivdt~d Mac-1 molecllles present on stim~ t~d myeloid cells.
These ~ içs were also found to ~s~css the novel ability to bind to
25 purified Mac-l ,,~b~ tes, as well as ~ l solubilized cell lysates.
A ~ ~ber of this class of alllibodics, hc.~inal~r CBRMl/S, is
l~scribeA. CBRM1/5 binds to a subset (1~30%) of Mac-1 m~le~lles present
on stimlll~t~d n~ u~)hils (those that are acLi~aLed and are capable of binding
ligand). CBRM1/S was further found to be capable of blcc~ the binding of
30 a.;livaL~d l.e,~ .hils to the Mac-1 1i~nr1c, GblinGge-l and ICAM-1.
Wo 94/08620 ,~ 7 ~ ~ PCr/US93/09777 ~1
- 10 -
Another member of this class of antibodies, hereinafter CBRMl/19,
was additionally isolated. This antibody, though binding to an activation
specific epitope, I~J~ S a dirrt;~ d ~.1 ~.l1,.1;. ,.- ~en ~at bound by CBRMl/5.The present invention further provides mf~thr~c of isolating other
5 ...~...be.~ of this class of ~fntihodies Specifif ~lly~ anlibo~lies which react with
the same ~ubpopulation of stim~ ftf~ myeloid cells or the same subpopulation
of a~LivaL~d Mac-1 can be identified by their ability to bind the same
subpopulation of cells, or the same ~,opulation of a~livat~d Mac-1
molecules as does CBRM1/5 or CBRM 1/19.
The present invention further provides meth~l~ of sele~Li~ely inhibiting
the binding of stim.ll~t~A myeloid cells to a ligand of Mac-l. Spec-ific~ily~
stim~ t~l myeloid cells can be blocked from binding to Mac-1 ligands by
supplying to the myeloid cells an antibody, antibody r.~ or antibody
d~ raLivt; which is (1) capahle of binding to acLiv~t~d Mac-1 present on
15 stimlll~t~d myeloid cells, (2) is ,.II,sl;...l;~lly in~able of billdi.lg to non-
a~ at~d Ma~1 and resting myeloid cells, and (3) is c~ablc of inhi~iLing the
ligand/Mac~ t ".rl;nn which is being targeted. For e~cample, CBRM1/5
when supplied to a myeloid cell, blocks ICAM-l and rll,li"c~en binding.
The present invendon further provides ..clho~ic of treadng various
20 p~tholog~ ;ol~c which are ...~Ai~l~l by myeloid cell ~(lhP~ion,
mi~r~tinn, and ligand il~t~ ;on. S~ r~lly~ .n,...~ OIy l~ se
which is ...~I;~IP,d by the non-s~ir~c derellse system can be treated by
~h..;l~ h.;n~ an err~ mol~nt of an andbody based agent to a padent in
need of such ~ l The ~ntibo.~iy based agent used in ~e present metllod
25 is an anlil~ly, an ~ntil~ody r~ e~, or all ~ntil~ocly de.ivali~., which is
ç~p~hle of ~inding tO ~livdt~d Mac-l present o~ stim~ tp~l myeloid cells but
is s~lbslz~rl~ y incapable of binding to non-a~ilivatcd Mac-1 and resdng
myeloid cells.
The present invendon further provides mP,th~c of lor~li7ing the
30 ~resence of stim~ tPA myeloid cells within a subject. Speeifie~lly, the
presence and location of cells expressing an ~livated form of Mac-l can be
determined within a subject by ~ministering to the subject a detPct~hly labeled
WO 94/08620 ;~ 1 ~ 4 ~ 3 ~ PCr/US93/09777
antibody, or antibody fragment. which is capable of binding to activated Mac-
1 present on stim~ t~l myeloid cells but is subst~nti~lly inr~p~hle of binding
to non-activated Mac-1 and resting myeloid cells. The location of the labeled
antibodies or antibody r~A~",rnl~; can then be detP~I using known ...-,ll,~lc
The present invention further provides meth~lc of id~ irying agents
capable of activating Mac-l present on resting myeloid cells. Spec;r.c~lly,
resting myeloid cells, or deactivated Mac-l molec~les are conLac~d with an
agent which is to be tested. The cells are then CO!~ d with an antibody
which is capable of binding to a~;livaLed Mac-l present on stim~ te~ myeloid
10 cells but is s~,bs~l;Ally ic~ kle of billding to non-~liva~ed Mac-l and
resting myeloid cells. The cells, or pllrified Mac-1 are then P~;....;l-r~l to
....ine v~helller the antibody binds to the cells.
The present i~ ,nlion further provides meth~$ of ide~ ing agents
capable of d~c~i~aling ~ilivat~d Mac-1 which is present on stimlll~t~l myeloid
15 cells. Spe~ific~lly~ A~d myeloid cells, or l,u-iGed aclivdt~d Mac-1
~ 'e~nles are C4~ ~I with an agent which is to be tested. The cells are ~en
co..l~.~l with an ~..l;lx)~.ly which is capable of bil~ding to a~;liva~d Mac-l
present on stimlllzt~ myeloid cells but is ~-~hsl~r.l;zlly i.~pzhle of l,;~,ding to
non-acli~at~d Mac-1 and resting myeloid cells. The cells, or purified Mac-1
20 molecules are then ~ ~ to det~ ~ whelher the cells bind the ~..I;lwl~.
The present in~ ioll further provides ...~ yls of s~lecL~,~ly killing
cells t,Ayl~S~;ng a~ dt~d Mâc-l. .S~ lly, cells which have ac~i~at~ Mac-
1 present on their cell surface can be s~ lcclh,~ ly killed by c0,~ such a
cell with a toxin de.iv.~ ntibo~ly which ~---~.iscs a toxin moiety and an
25 antibody, or ~ r. ,.r ~ which is capable of l~hl~ing to aclivat~d Mac-1
surface but is ;,..l,s~ lly inr~p~hle of binding to non-aclivd~d Mac-1.
The present il.~,.-lion further provides m~tho~.c of selecli~,ly .~...oving
cells eA~ ,~;ng d~ dtl~d Mac-l from a solution or fluid such as blood.
Spe~ific~lly, stimlll~t~ myeloid cells which contain aclivd~d Mac-1 on their
30 cell surface can be selecli-~,ly removed from fluid by pas~ing the fluid over an
immobilized antibody, or antibody fr~m~nt which is capable of binding to
activated Mac-1 but is subst~nti~lly in~p~ble of binding to non-aclivdted Mac-
7 3 ~ ~
WO 94/08620 PCr/US93/09777
- 12 -
1. Such a method can be used in standard leukophoresis type procedures to
remove stim~ t~d myeloid cells or myeloid tumor cells which express
activated Mac-l from a p~tientc blood without effecting non-stim~ t~d
myeloid cells.
BRIEF DESCRIPTION OF l'~ IGURES
FIGURE 1. A. T... ~-)fluo~ nr4 flow iy~~ ,Lly of peripheral
blood neutrophils that were nn~tim~ ted or treated with PMA (100 ng/ml) for
t~n ~ ~s at 37C. N~ILl~hils were stained with either a I~Cgati~e control
(X63), a MAb to Mac-1 a subunit (LM2/1), or CBRM1/5. B. Immlln~
iL~Lion of Mac-1 from Triton X-100 d~ gel~ lysates of '2sI-~
peripheral blood ne.~llo~hils. Tl~ ulJr~ i~L~s with the following MAbs
were ;,~I,j~t.,d to l~Juc;~g SDS-5% PAGE and ~tor~ ogla~hy. Lane 1,
CBRIC2/2 (ICAM-2); I~ne2, LM2/1 (Mac-1 ~); Lane 3, CBRM1/5
(Mac-1 ~Y). M~ r weight st~ddr~s are il~ t~d to dle left.
FIGURE 2. .~tur~tion binling of CBRM1/5 IgG and Fab. A,
unL.~t~d lleullu~hils; B, .~ ophils that were stim--l~t~ with PMA (100
ng/ml) for ten .n;n~t~ s at 37C. I`~ l.aphils were il~c~ib~ 1 with l25I-I~be-ll~
CBRM1/5 either in the ~hs~ (open circles) or ~ Ce of excess unlabelled
CBRM1/5 (open ~ gleS) for 4 hours on ice. Specific binding (closed
circles) was ll~ uc~ by s~~ !;n~ the non-~ec-ific. from the total binding.
Panel B, Inset A Se~tr~ d plot of the spe-rifir, bindil~g from the curve in
Panel B. The r~leul~t~ Kd is 15 nM. The ~ t~ (lOlO pM) is
equivalent to 60,800 sites per ~ l-o~hil. C, Co...~ of l25I-CBRMl/S
IgG by CBRM1/5 Fab r~...F .I~. ralafollllaldehyde fi~ced l}~ lul)hils that
had been stim~ t~ with PMA (100 ng/ml) for ten ~ Jt~,s at 37C were
i~cu~ rll with a 5~h~ ¢ co~ e~ ;on of l2sI-CBRM1/5 IgG (1 nM) and
30 increasing col-r~ ;ons of lunl~hell~i CBRM1/5 Fab ~la~,llle~ for 60
minl~teS at 37C. Bars inriir~te the standard errûr of the mean for triplicates.Panel C, Inset. SDS 6%-PAGE of IgG and Fab fragments of CBRMl/5.
wo 94/08620 ~ 1 4 ~ 7 3 ~ PCr/USs3/09777
- 13 -
Samples (3-5 ~LgS) of CBRMl/5 IgG (Lane 1,4), CBRM1/5 Fab'2 (Lane 2,5),
or CBRM1/5 Fab (Lane 3,5) were subjected to SDS-8% PAGE under non-
reducin~ (50 mM iod~ le, Lanes 1-3) or red~lcin~ (5 %
e.~loetl.~nol, I~nes 4-6) con-litions and silver st~inin~. Mole~ r
S weight s~nd~ds under non-,~uc;.~ and lc~ ;n~ con~litirJnc are in~;rA~ed.
FIGURE 3. KinP.tir,s of e~r,i,;,ioll of Mac-1 el)ik)l)cs. I~Te.lllophils
that were ~liva~d at 37C for the i~ l times with no stimuli (open
triangles), fMLP (1~7M, solid squa,~s), IL-~ (25 ng/ml, solid circles), or
10 PMA (lOO.ng/ml, open circles) were im...ll, o~ ~ with A, CBRMl/5 or B,
LM2/1 and subjected to immunonuor~se~-re flow ~l~",~ . Data are
e,L~r~,ssed as relative linear fluo,~ -nr~ units and ,~,esc~" the mean of three
cA~c,i",e,nls. The bars i.~ tP, the ~ rd error of the mean.
FIGURE 4. T.he effect of ~ , on Mac-1 ~,A~.~,s~ion on A,
pe~il he.al blood lllolloe~tLs and B, ll~ ullvl>hils. Cells were i~ A at 4C,
25C, and 37C in the ~b~ ~r~ or ~sence of stimuli (fMLP, 1~" PMA, 100
ng/ml) for ten .. ;.. ,~ s and then i.. i-o~; ineA with either a negali~, control
(X63), a MAb to Mac-l a subunit (LM2/1), or CBRMl/S. Repr.,~.,~tive
20 his~gl~l,s show the log lluGl.,s~f-~ in~.,~ r versus ell nw"bcl.
FIGURE S. .Sç~ ;c l~pl~ n and flow ~k~lll' hiC analyses of
Mac-l/pl50,95 chimeric mn~Cll4S A, MAbs used for the flow C~ lllf h
were X63 (non-bindi,~g control), LM2/1 (anti-Mac-1 a), OKMl (anti-Mac-1
25 a), BLY6 (anti-plS0,95 a) and CBRMl/S. R~,~,se-~ hi~t~gl~ulls show
the log llu~ ,s~ r~ hlt,~ versus cell .~ .ber. B, Chhllc,ic ms)'erllles
were consL.uct. d by the ligation of ,'~ c from either plS0,95 or Mac-l
cDNA, and t;Ayr~,j~d stably in CHO cells. Those parts of dle chh.l~.~ from
Mac-l are repl~sel~t~ by open bars, those from plS0,95 are r~..,sell~d by
30 shaded bars. The region from the EcoRV to the BglII rectriction site
constitutes the I domain.
Wo 94/08620 ~ 7 3 8 PCI~/US93/09777--
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FIGURE 6. Divalent eations affe t expression of Mac-1 MAb
epitopes. A, Neutrophils were in- ~IhAt54i at 4C (resting) and 37C (fMLP,
10'M) for ten ~ .S in HBSS that was supple........... ~ i by the inf~ t~l
divalent cation or rh~l~tin~ agent. Cells were i.... ~ o l~ A with either a
ncgati~e control (X63), a MAb t~o Mac~ subunit (LM2/1), or CBRM1/5.
Al~ subsequent washes were ~Irollllcd in the ~,.~nce of the inAi~t~l
divalent cation. Repl~n~ti~e l~;~t~.. c show the log lluor~ sc~ t~ llSiLy
versus eell "u",~r. B, Purified Mac-l was ~dsc~l~cd to plastic and non-
specific sites were bl~xked after ~ e,.l;~l washes with HSA and Tween 20
10 in the p,~ se~ of the il.fl;c~ divalent ation (see Materials and Methods).
Solid-phase Mac-1 was il.c~ ed with MAbs (CBRIC1/11, anti-ICAM-l;
LM2/1, anti-Mac-1 x; CBRMl/S, anti-Ma~ x) and developed by an EUSA
with hor~ h EJe~OAi~ couple goat anti-mouse IgG. This l. ~r~ ;ve
eA~,;..~f..-l was ~IÇG~ ed in triplicate and the bars i~.l;r~le the s~ldar~l error
15 of the mean.
FIGURE 7. SDS-5% PAGE analysis of i.. ~.~i~i~t~ s from
neutrophil Iysates. Io lin~tPA ~ l~hils were Iysed in 1% Triton-X-100 and
~liquot~ (60 ~1, 1:4 ~ JtiQrl) were il..;vl~te~ with dh~clly coupled Sepharose
(Lane 1, LM2/1; Lane 4, CBRMl/5: Lanes 7 and 10, Mouse IgG) for 2h at
4C. The beads were p~ d, ~e pre-cleared ~U~Il~ S were l~ .cd
and l~ ,i~t~ with ~lition~l di~lly coupled S~haluse (Lane 2, LM2/1;
Lane 5, CBRMl/5; I~nes 8 and 11, Mouse IgG) for 2h at 4C. After
pelletin~ the s~ were again collected but the LM2/1 ~,ecl~cd
Iysate was pl~ i~t~ with CBRMl/5 (Lane 3), the CBRMl/5 ~r~lealcd
Iysate was pl~i~i~d wi~ LM2/1 (L~ne 6), and the mouse IgG ~)l~cl~d
Iysates were L"~cle~d wi~ LM2/1 (Lane 9) or CBRM1/5 (Lane 12).
Material eluted from the beads was run under l~l.~c;~ and
subjected to ~lltor~-liog.~l l,y. Molecular weight s~ndar~s are shown at the
left.
WO 94/08620 2 1 4 4 7 ~ 8 PCr/US93/09777
FIGURE 8. The effect of CBRMl/5 on neullophil adhesion to its
lig~ntic. A, Neutrophils were allowed ~o bind to purified sICAM-1 in 6 cm
Petri dishes for four ...i....~s at room ~"~r~l~u~t; in the presence of fMLP
(10-'M) after a ten minute preinrub~tion with the following MAbs: TS1/22
S (anti-LFA-1 a, 11250 dilution of ascites), LPM19c (anti-Mac-1 ~, 1/250
dilution of ascites), CBRM1/5 (anti-Mac-1 ~Y, 25 ~g/ml IgG and Fab'2; 50
~g/ml Fab), CBRM1/23 (anti-Mac-1 ~, 25 ~g/ml). B, The effect of
CBRM1/5 on ICAM-1+L cell adhesion to purified Mac-1. ICAM-1+L cells
were allowed to bind to purified Mac-l in 6 cm Petri dishes for 60 ...i.~.,t. s at
37C after a 25 minute ~ rub~l;on with the following MAbs: TS1/22 (anti-
LFA-1 a), LPM19c (anti-Mac-1 ~), CBRM1/5 (anti-Mac-1 a), and
CBRM1/23 (anti-Mac-1 a). Data is ~ ,r~,~ as the ~clcenl cell binding
relative to a media control. Da~oinLs are the &~ age of five lllic.ùseopic
fields and the bars in-lic~le the st~nd~ud error o~ the mean. C, Dose
depc~-~lenl jnhihjti~n of n~ulluphil binding to fb,ino{~n by CBRM1/5.
Neutrophils were allowPd to bind to pllrifiP~ r~ ~e~ in 6 cm Petri dishes
for four ~ tcs at room L.ll~alul~, in the p~nce of fMLP (1~'M) after
a ten minute ~ ."l;o.- il,.;,~asil-g co~ ..l .,.t;ons of CBRM115 IgG or Fab.
The bin~ was de rr l ...i l~r.d by light micr~seo~ (40X) after the ul~bound cells
20 were l..ll~,..,d by serial washes witn a Pasteur pipette. Data is C~ SSed as
dle ~.~;e,.,L inhibition of cell bindil~g 1~,laLi~e to a media control. n~ oi"~iare the ~_r~e of five llliCl~SCOpiC fields and the bars i le ~e ~ d~l
error of dle mean. The graphs are l~ _ P~ ~at were
.~.pe~lrA several times.
FIGURE 9. Sçh~ l;r, ~ I;or~ of the le.lkc~L~ ~lin ~Y chain
and Mac-1/plS0,95 ch;~ c. ResL-iclion sites (e, EcoRV; b, BglII; a, AflII)
that f~rilit~tPcl r~i~r~cal e .çh~l~ges are in(iir~tf~A With arrows or in the name
of each cl-i...el,~
FIGURE 10. SDS-PAGE of Mac-1, plS0,95 or Mac-l/plS0,95
chimer~c imm--n~ l~ipitated from l251-COS cell de~r~cnl Iysates. COS cells
Wo 94/08620 ~ Pcr/uss3/o9777--
- 16 -
were cotransfected with the ~B subunit and (A) Mac-l, (B) pl50,95, (C) M-e-
X-b-M, or (D) X-e-M-b-X cDNA, surface l~he.lle~ with '~5I, and
imml-noprecipitated with the X63 (Lane 1, negative control), TS 1/18 (Lane 2,
anti-CD18), LM2/1 (Lane 3, anti-CDllb), Mn41 (Lane 4, anti-CDllb),
S VIM 12 (Lane 5, anti-CD 1 lb), OKM 1 (Lane 6, anti-CD1 lb), OKM 10O,d (Lane7, anti-CDllb), SHCL3 (Lane 8, anti-CDllc), BLY6 (Lane 9, anti-CDllc),
L29 (Lane 10, anti-CDllc) as described in the M~t~ri~l~ and MethoAc.
Material was boiled in SDS sample buffer with 5% ~ l.e.~aploeth~nol,
electrophoresed on a 7 % polyacrylamide gel, and autoradiographed. Molecular
weights of the protein standards are intlir~t~d in the center.
FIGURE 11. Flow c~ leL~ profiles of CHO cells ~AI"c;s~ing Mac-l,
plS0,95 and Mac-1/plS0,95 chi...~",~c. CHO cell ~ r~c!~nl~ (in-lic~t~ on
right) were im~ osl;~in~ with either a ncgaLi~-, control (X63), a MAb to the
lS I domain (LM2/l) or C-tf ...il-~l (OKMl) region of the Mac-l a subunit, a
MAb to the C ~ ;n~l region of the pl50,95 a subunit (CBRplS0/4G1), or
a MAb to the CD18 ~B subunit (TS1/18). Cells were subjected to
of lu~ s~nl flow ~lle~ Each histogram shows the negati~e control
(X63, light line) and the MAb intlir~ted at the top of the column (dark line).
FIGURE 12. SDS-PAGE of Mac-l, pl50,95 or Mac-l/pl50,95
cllill.F.~ i..,r...~..oprwi~ ed from '25I-CHO cell d~,~e,I~ Iysates. CHO cells
t~ncr~.".; (A) Mac-l, (B) X~-M, (C) M-e-X, (D) X-a-M, (E) X-b-M, (E~
X-e-M-b-X, (G) M-b-X-a-M, (H) plS0,95, (I) M-b-X, (" X-b-M-a-X, (K)
M-a-X, and (L) M~-X-b-M were surface l~hell~A with l25I, and
op~ at~d with a I~egdi~ e control MAb (X63, Lane 1), a MAb to the
I domain of Mac-l (LM2/1, Lanes A2, B2, F2, I2 and K2, a MAb to the C-
terminal region of Mac-l (OKMl, Lanes D2, E2, G2 and L2), a MAb to the
C-terminal region of pl50,95 (CBRpl50/4Gl, I~nes C2, H2, J2), and a MAb
to the CD18 ~ subunit (TSl/18, Lane 3) as described in the Materials and
Methods. Material was boiled in SDS sample buffer with 5 % ~B-
mercaptoeth~nol, electrophoresed on a 5 % polyacrylamide gel, and
WO 94/08620 ~ ~ 4 4 7 ~ 8 PCr/US93/09777
- 17 -
autoradiographed. Molecular weights of protein standards are inflic~ted to the
left.
PIGURE 13. Summary of MAb r~aclivily as de~l,llined by
S i~n~ nuo~ flow ~k~ elly with CHO cells l-; n~r~ l witn wild type
or chim~oric Mac-l and plS0,95 mole ~les (+++) ;l-fiir~tf-s that 100% of
cells stained tne MAb witn a pattern similar t~ that shown in Figure 3. (+)
intiie~teS that 100% of cells stained positively with the MAb but with a
si~nir~Anlly lower nuol~ y. (+/ ) in~iic~tes that tne MAb stained
10 a ~b~o~)ulation of cells of cells ~,Gs;~ ly. (-) in~iir~tes that the MAb s~ -hlg
was not si~ ;fc~ y dir~ from the nfgalive control (X63).
~IGURE 14. S~ y of the functiQn~l effects of MAbs on Mac-l
l;on with lig~nf~i~, The assays for the i~t~ cl;Qn with the four ligands
15 are de.sel ;I,e~l in the Mell~yic~ The inhihition data with OKM 10O~d was o~incd
from iC3b ~ to t.~ r~ l COS cells instead of ~,ulr~hils. The data
iS e~ ,SSed as the pel~llL inhil~iLion by each MAb and is the average of at
least three i~ep~ e"l e~ t~n i~rd error of the means are in~ii~t~d
after the + sign. ND i~ t~! S that the value was not de~.lllined.
PlGURE 15. Agg~ (t~ profiles of neullol.hils ~ ,d with
PMA. Ne.lllul)hils were ~l~h.l,.Jb~t~d with the in~;r~t. cl MAbs for 25 .~in~lt~s
at room ~ ~ralul~" eq~ilibr~t~ at 37C, and a~ d~ed with PMA (100
ng/ml, t = O min). Individual l~lC,SCnl~l;Ve ~ c.in~,~ are shown for
25 e l-c- ;~ n~ ~at were p~-rull-led at least three times. I/Io l~ ~nl~ the
relative light ~.;.,.~...i~-:,.l~
FIGURE 16. CHO cell lri.n~ ;.nl binding to ICAM-1 (A) and iC3b-E
- (B). Panel A. Mac-1, pl50,95, Mac-1/pl50,95 chi.~ c, and ICAM-1
transfected CHO cells were ~et~rhecl, l.,~ ed (8 ~c 105 cells) in 1 ml,
stimulated with PMA (100 ng/ml), and bound to immllno~ffinity purified
ICAM-l adsorbed to plastic for 90 minlltes at room temperature. Unbound
WO94/08620 ~ 3~ PC~/US93/09777--
- 18 -
cells were removed by five washes with a transfer pipette. Bound cells were
r.~ t~i by visually scoring the number of cells in five micloscopic fields
(40x m~gnifie~tion). Bac~uund binding to plates lacking ICAM-1 was
de~l",ined for each L~ar,sr~c~nt and sul,L~ d. The data is the average of
S three e~ e"~ and is norm~li7PA to the bin lillg of wild type Mac-1. Bars
intlir~te the standard error of the means. Panel B. T~ cr~t~A CHO cells
were det~rh~A replated on tissue culture treated 6 well plates (4 x 105
cells/ml, 0.5 ml/well), and adhered for greater than 3 hours at 37C at 10%
CO2. Erythrocytes (iC3b-E, 50 ~1 of 2 x 108 cells/ml) were added and
10 ineub~l~ with ll~nsr~clan~ for60 ..~il...t~-sat37C. Non-adherenterythrocyteswere ,~lllo~ed after eight washes with a transfer pipette and ~sel~s (> 10
elylhlo~s/CHO cell, ~ 100 cells e~ ~) were scored by light
ll~icluscu~y at 100X m~r ;r~rs~l;on. The data is the average of three
e,.~el,ll,e.~ e the s~ndar~l error of the means.
DESCRIPIION OF ~1~; PREEl~RRED E~MBODIMEN'!S
The present invention is based on the novel o~selv~lion that when
resting myeloid cells are stim~ t~A not all of the Mac-1 ~ ~lo~cnles present on
20 the cell surface bc~..ç &_livd~d and capable of ~hldi.lg ligand. Only select
sub-pop~ l;ol~ of Mac-1 mo'ecnles as well as ~ubl~ûpulaLiOI of myeloid cells
b~lllc aclivat~ and capable of binding to ligands of Mac-1. Upon activation,
a~;livdlioll ~l~C;I';C ~ilO~S appear on the Mac-1 m~'~c~ . which di~l~,nliaLG
the a~livdled Mac-1 ~ les and stimlll~t~ myeloid cells from the non-
25 ~;li~dt~i Mac-1 mol~cllles and resting myeloid cells.
Based on this ~1;G~1OS~e~ ~e present invention .l;~loy,s the ~,ene~
of a novel class of ...nl~lon~l antibodies, MAbs, which bind to acLiv~t~,d
Mac-1 ~le,~lPs present on stimlll~t.oA myeloid cells but are s~l,s~ lly
h~cdpablc of bindh~g to non-~livaled Mac-1 .llole~,ules on stimlll~t~A and
30 resting myeloid cells. These al~Libodies were also found to possess the novelability to bind to purified Mac-l ~ubsL,~Les, as well as deler~enL sûlubilized
cell lysates.
~ WO 94/08620 2 1 ~ 4 7 3 ~ PCr/US93/09777
19
Anti-Mac-1 MAbs are generated using routine procedures known in the
art (Harlow et al., An~ibodies: A Labora~ory Manual, Cold Spring Harbor
Press (1988)) using either purified Mac-1 or cells e~cpressing activated Mac-1
a_ an immlmogen. Once anti-Mac-1 antibodies have been geller~ted antibodies
5 which selecli~,ely bind to acliv~.~d Mac-1 can be identifie~ in seve~ f~chionc~
First, ~ntiboAies which selecli~ely bind stim~ ted myeloid cells can be
identified by screening the antibody for the ability to bind to stim~ t~
myeloid cells such as ".,~n,phils, and the inability to bind to resting llly.lD~d
cells.
Alternatively, antibodies which bind stim~ t~l myeloid cells can be
identified by scr~nillg the antibody for the ability to bind to purified ac~ivatcd
Mac-1, and the inability to bind to purified d~cliv~ed Mac-l.
As used herein, a mony~lonal ~ntibo~ly is said to "selectively bind~ to
~li~at~ Mac-l or stim~ t~~ eloid cells when the ~nti~ody binds to
stim~ ted Ill~.lQI~ cells or acli~at~d Mac-l but is s~h~ y ~ p~hle of
~illdi-lg to non-a~ ~d Mac-l or resting l"~ cells. An A~liboJ~ is said
to be "subst~nti~lly i.~r~r~ble" of binding to resting myeloid cells if the level
of binding to the resting myeloid cells is less than 10% of the level of bindingto stim~ t~d cells.
As used herein, a myeloid cell is said to be stim~ t~d when the cell is
capable of bi~lding to a ligand of Mac-l or is capable of ~l;c~ t;,~g in a
Mac-l d~en~ biological fui~c1;0.~ Thelefo.~, a stim-~l~ted myeloid cell is
a cell which is capable of binding to ICAM-l, iC3b, rll.linogcn. and/or is
capable of l,~l;~;p~ g in Mac-l flr.~ndel-l r~i~rl;onc such as h~.l.,otyl.ic
ag~l.,~l;on Tl~ which stimulate myeloid cells inclu(le, but are not
limited to agents such as fMLP and PMA.
As used herein, a myeloid cell is said to be resting when the cell is
h,~ydble of billdillg to a ligand of Mac-1 or is in~ ble of pa~ l;ng in
a Mac-l ~lepen-lent biological fuQclio~ fol~" a resting myeloid cell is a
cell which is j~ .Ahle of binding to ICAM-l, iC3b, rlblh~ogen, and/or is
incapable of Mac~ pentlent hollloLyl~ic agg~,gdtiOn.
Wo 94/08620 Pcr/uss3/o9777 ~
73~
- 20 -
In detail, differential screening is performed using two populations of
myeloid cells, or purified Mac-l molec~lles. Myeloid cells, such as
neutrophils, and Mac-l molecules (acli~ated and de-~liv~ted forms) are
i~ol~t~A using known methor1c. When using ~ irled cells, the purified cells are
S se~ .t~ A into two pop~ tion~ The two populations are p~ u~.At~ under
conditions which keeps Mac-1 on these cells in a resting conrc~ aLion, for
example at about 37 C for about five mimltes in HHMC. One of the
population of cells is inr.ub~t~d under co~tlitions which a~ a~e Mac-1, for
example, in a media cont~ining 100ng/ml PMA, while the other population is
10 m~int~in~d in the resting m-oAillm
The ~ntiboAies which are to be sc~ d are then ir.r.ubAt~A with a
sample of the two poplll~tionc of cells, or a sample of p~l;ried a~iLivated and
de-a.~livaLed Mac-1, and antibodies sele~-te~l which selccli~ly bind to the
aClivat~d population of myeloid cells, or purified scLi~dt~d Mac-1 mol~.~lles
15 Any me~od for scl~ning ~ntibody binding to a cell or molecule can be used.
These include~ but are not limited to, flow C~rL~IlleLl ~, ELISA, and RIA. W.henlarge nu",l~.s of anlibo.l;cs are sc.~.-ed, it may be ~.~fe.~ble to fi~ced ~e
myeloid cells prior to i.~cvl,~l;on with the antibody, for c~-~"ple by in~-ub ~ g
the cell in 2 % p~arol.llsldehyde.
~ AI;~1Y~ the above l)r~dul~,s can be pe.[~.. ned with two
populations of cells e"~ g ~on~bi~anL Mac-1. Any cell capable of
expressing Mac-1 can be used. These inr~ e, but are not limited to yeast,
bacL~lia such as E. coli, ...~ ti~n cells and insect cells.
Using such a ~l~lur~, two ~nti~ies de,~ ted as CBRMl/5, and
25 CBRM1/19 were gene.àt~ and ide~-lil;~d.
Using CBRM1/5 or CBRM1/19 as a marker of a distinct ~ opulation
of stim~ t~od myeloid cells and a~;Li~aLed Mac-1 molecules, other ~F.~tr!e,~ Of
this class of antibodies can be identifieA.
Antibodies which bind to the same activâtion c~-ific epitope, or to a
30 sterically o~"lal,ping activation specific epitope on the acLi~ated Mac-1
molecule as that bound by CBRM1/5 or that bound by CBRM1/19 can be
identified using a two antibody binding assay or a competitive binding assay.
WO 94/08620 ~ ~ 4 d 7 3 8 Pcr/usg3/09777
Such antibodies may recognize the same sub-population of Mac-l molecules
as that bound by CBRM1/5 or CBRMl/19 or may bind to a different
- subpopulation of a~;livaLed Mac-l mo~ es.
As used herein, "activation specific epitope" refers to an epitope which
5 is present on a~ a~d Mac-l mClDcllles but is not present on non-a~ at~d
Mac-1 mole ~llles Such ~;L~f S are often l~fe.l~d to as a nC~piLo~C.
As used herein, two ~il~s are said to be the "same" or "st~oric~lly
o-v~lla~ping" if ~nti~lies to ~e two C~iL(~S C4~ h- with and exclude each
other's ability to bind to the ~ntig~o.n,
Antibodies which bind to ~irf~ or non-sterically o~,.la~ g
activation spe~-ifir, e~;~opcs can be iclentified by their ability to non-
col..l~liL;vely bind to the same su~poplll~tion of stimulated myeloid cells or
the ability to non cc~ ly bind to the same sub-population of acli~,d~d
Mac-l as that bound by CBRMl/5 or that bound by CBRMl/19.
Any of the known ~ ~ for testing v~ er two ~ es bind to
the same population of cells or ~e same ~ lle can be ...-Yl;l;ed by one
skilled in the art to idenliry ~nti~ies which bind to the same popnl~tio~ of
cells or mole ~lrs in either a co~ or non~4~ f~chion, as ~at
bound by CBRMl/S or ~at bound by CBRMl/l9. Such assays include, but
20 are not limited to, flow c~,~,"~ r and ELISA analysis.
The present invention further ~-c,.ides the ~ ;es CBRMl/5 and
CBRMl/19 in s ~l,a~r.l;~lly ~ irlcd form. As used herein, an antibody is said
to be "svbs~r~l;Ally pilrifie~" if it purified to the level nF~eC~r~/ in order to be
err~ ,ly :I.n;l-.c~,~d to a subject or to be of use for a desired pu,~o~. In
25 some cases the ~ ;boJy will be svl,r~ ;Ally free from all cellular co",poncnls
while in other ~ nces the ~-.l;bo~ly will only be free from other
immlmoglobulin like proteins.
The present invention fur~er provides a hybridoma capable of
pro~ucing the CBRMl/5 antibody. The hybridoma pl~ Cing this antibody has
been depoa;tl,d at the ATCC and d~Psi~n~tP~ .
Wo 94/08620 2 1 4 4 7 3 8 pCr/US93/09777 ~
- 22 -
The present invention further provides a hyblidoma capable of
producing the CBRMl/l9 antibody. The hybridoma producing this antibody
has been deposited at the ATCC and design~ted
The present invention further includcs fr~mentc of the antibodies of
5 the present invention which m~int~in their ability to bind to stim~ t~ myeloidcells or to acLivaLcd Mac-l. Such rl~glllf'nt include, but are not limited to, the
Fv, the Fab, and the Fab2 r,~ . One skilled in the art can readily
genfli.led antibody r".~ f..lc which ...~ b;lldi.lg ability using routine
mf thoclologY~
The present invention further il~cludes de.ivdLivcs of the ~ntibotiies of
the present invention (antibody de~ivaLi~c). As used herein, an "antibody
deli~,alive" wlll;~ c an allLibody of the present invention, or a r. ~ Ul of said
anLil)ody, as well as an additional moiety which is not normally a part of the
antibody. Such ...oie~ips may il--~ ._ the ~l;hof3ies~ solubility, abs~-~Lion,
15 biclcgir~l half life, etc, or may ~Ir~ ly decl~se the toxicity of the
ibody, el;lll;~ or ~ .u~ any ~ ble side effect of the ~nt;bo)y,
etc. Moieties capable of m~Ai~tin~ such effects are ~ os~A in Renungton's
Phann~r~ Sc ences (1980).
"Toxin-dc,ivati~d" antibodies co--~lilu~ a special class of antibody
20 delivali~ s. A "toxin-dc.ivali~ed" a,.lil~ly is an allli~ly, or an~ibody
fr~m~.nt which Gonl-inc an ~-lAition~l toxin moiety. The l~indillg of such an
agent to a cell brings the toxin moiety into close IJloAilllily- to the cell andthereby ~)lUlllOtl~S cell death. Any suitable ~oxin moiety may be employed;
ho. ~ r, it is ~ fcl~ble to employ toxins such ac, for example, the ricin
25 toxin, the dirhtheri~ toxin, li..lioicl?lo~ic toxins, ~e ~hl ~ne ch~nnel-forming
toxins, etc. Pluc~lul~ s for ~,~nf ~ such toxin de.i~dii~-,s are well known
in the art.
nnetrct~hly labeled" antibodies C4~ 1t; a,lulller special class of the
antibody delivaLi.~es of the present invention. An antibody is said to be
30 dete~t~hly labeled if the antibody, or r.,.~ .l thereof, is ~tt~rh~ to a
molecule which is capable of identifir~tion, vi~u~li7~tiQn, or loc~li7~tion using
known methods. The detecf~hle labels of the present invention include, but are
~ ~14~13~
WO 94/08620 PCr/US93/09777
not limited to, radioisotopes labels, affinity labels (such as biotin, avidin, etc.)
fluorescent labels, p~r~m~netic atoms, etc. P~OCedUIGS for accomplishing
- such labeling are well known to the art.
The present invention further includes l~...n~ d forms of the
5 antibodies of the present invention. Hum~ni7PA forms of the antibodies of the
present invention may be prod~lc~d, for example by replacing an imm-lm)genic
portion of an antibody with a co~ -.l;n~, but non-i.. l.. ogellic portion
(i.e. chi-l-eric ~ntil~ies) (Cabilly, et al., European Patent Application
125,023; Better, et al., Science240 1041-1043 (1988); Liu, et al., Proc. Natl.
Acad. Sci. USA 84:3439-3443 (1987); Liu, et al., J. Immunol. 139:3521-3526
(1987); Wood, et al., Nature 314:446 449 (1985)); all of which reÇG,."lces are
incollJo.~lGd herein by rel;,.Gnce).
~lle~ ly~ suitable "I~ A" antibodies can be produced by
CDR or CEA ~ rl;~ ,l,s~ l;Qn (Jones, et al., Naturc 321:552-525 (1986);
Ve,l-o~an etal., S~ e 23*1534 (1988); Beidler, et al., J. I~ruu~l.
141.40534060 (1988); all of which l~f~ ,nces arG i~O~ '~ herein by
rGçGr~nce).
The ~lc,scnL invention further provides ~ s of s~ ,ly illhibiling
the binding of stim~ t~l myeloid cells to a ligand of Mac-1.
As used herein, a ligand of Mac-1 is defined as a non-antibody
m~kcllle which is capable of bi.~ g to Mac-1. T .i~n-lc of Mac-1 inrll~de, but
are not limited to, iC3/b, ICAM-1, and fil~ Ogf n
In detail, stim~ t~ myeloid cells can be b'nrl~P~l from binding to a
Mac-1 ligand by supplying to ~e myeloid cells an ~ntibo~ly~ antibody
fr~gmto.nt or ~nt~ y de.i~a~ which is capable of biiiding to a~ Ld Mac-
1 present on ~ t~ myeloid cells and is s~s~ lly i... ~ hle of bintlin~
to non-a~tivat~d Mac-1. For e%ample, CBRM1/5, when supplied to a
stimnl~t~ myeloid cell, will bind ~o the a~ilivaled Mac-1 clsc~lec present on
the cell and block the binding of the cell to ICAM-1 and rlbl in~ ~l However,
30 activation ~e~ific anti-Mac-1 antibodies which inhibit other Mac-111igand
inter~r,tio~c, such as Mac-1/iC3b binding, can be isolated.
Wo 94/08620 PCr/US93/09777 ~
2~ 4~7~8 24-
The adhesion, migration, and biological activity of circul~ting myeloid
cells such as neutrophils and monocytes results from interactions involving
Mac-1 and it's lig~n-l.c Ce~ r adhesion has been found to be required for
neutrophil and mono~ migration to sites of infl~.."...l;on and for various
5 neutrophil and ~..onoe~ err~lvr functions contributing to i,.n~ ;nl-
Non-activation ~;peeifie anti-Mac-1 antibodies which inhibit such
cellular adhesion, have been de."oncl.~ to be erf~ at ~ ellLing an
infl~mm~tory response when ~tlminictered to a human subject. In ~ itiQIl,
anti-Mac-l antibodies have been demonstrated to inhibit phagocylv~is of
10 foreign material by lllono~yt~,s. Further, antibodies which bind to Mac-1 have
been demol~ .led to be err~li~c at m~ ting other I~lVI~V~rle and neutrophil
activities such as ch~omn~in~cic~ and c-l~P-~o~liC both in vitro æ well as in
vivo.
nc~..~ of their spe~3fieity and sele~Livi~y, the ~r~l;l~;cs~ the antibody
15 r. i1g~ ;, and the ~ , d~iva~ s of the present invention agents provide
an illl~rv.~.lle~ll over ~ ivusly ~ cloc-~ anti-Mac-1 ~ ies for use æ an
anti-i..n~ agent, an anti-m~l~ri~l agent, an anti-HIV agent, and an
anti-asthma agent, for e~ample, see Fisher et al., Lancet 2: 1058 (1986), Perez
etal., BoneMarrow Transp. 4:379 (1989), Pdt~lu,~o etal., Scan J. ~ ol
30.129-164 (1989), Lindbom et al., Clin. Immun. 1,.,. uo Patholo. 57:105-
119 (1990), R~ ,r, Amer. J. Pathol. 136:3-11 (1990), Falanga et al. Eur.
J. Irnmunol. 21:2259-2263 (1991), Dreyer et al. Circulaion 84:400411
(1991), Simpson et al., Circulation 81:22~237 (1990) and Wegner et al.,
Chest 101:345-395 (19g2), all of which are herein incGl~la~ by r~ cc.
25 As ~ iousl~ ~eo~ 't~ , anti-Mac-1 ~ ;es are err~~ in treating
inll~,n",~liQn caused by a ,~lioll of ~e non-specific ~efensP, system.
The term "il,ll~.,..,~l;on " as used herein, is meant to include only
reactions of the non-~ecific defense system. A "reaction of the non-~e~ir~c
defense system~ is a ,e~ol~ mPAi~t~ by myeloid cells incapable of
30 immllnological Ille~llol~r. Such cells include ll~ r~hils and macrophages. Asused herein, infl~mm~tion is said to result from a response of the non-specific
defense system, if the infl~mm~tion is caused by, me~ t~rl by, or ~c~cj~ted
~ 4~3~
WO 94/08620 PCr/US93/09777
with a reaction of the non-specific defense system. Examples of infl~mm~tion
which result, at least in part, from a reaction of the non-specific defense
system include inn~ ;on ~soci~led with conditions such as: adult
respiratory distress ~yl~drul~le (ARDS) or multiple organ injury syndromes
S secondary to se~ lia or tr~l~m~ rusion injury of ll-yocaFdial or other
tissues; acute glomerulol~ephlilis; Ic~;li~e arthritis; de~ oscs with acute
infl~.. ~lo~y CO~ OI.P~IL ~ acute purulent .~p l~ing;l;c or other central llelvvus
system itln~.,.".~to.y disor~er~: thermal injury; h~mo~ lysis; leukapheresis;
ulccr~Live colitis; Crohn's disease; necrotizing ellLer~colitis; granulocy-te trans-
10 fusion ;~ tPA ~yll~;llollle~5; and cytokine-in~uc~d IOA;C;IY.
The ac~iv~lioll s~ 'ic and-Mac-l antibodies of the present invention,
r.~.g...P .I~ of said a,~Lilx)dies, and dc.i~,~.ti~,~,s thereof, are ~ ,d to a
patient as an anti-il.llAI.-..\zl-,ly agent to treat the above recited con(litiQn as
well as others oQn~lition~ which I~.rA;~ by the non-s~ ;c ~er~
15 system. Such agents differ from general anti-i~n~ c.ly agents in that they
are capable of sc,l~~ ly ~lii~g stim~ tP~ myelûid cell pop~ tions and
inhibit Mac-l/sperific ligand int~ ioll. Such s~ y will only affect
c biological function of the myeloid cell and will not cause side effects
such as nephr~Aicily and myeloid cell depletion which are found with
20 CUII~ ;On~I and less "~ ;c ant~ n~ Al~,lr agents. Further, such agents
differ from other previously known anti-Mac-1 alllib~l~r agents in that they do
not affect non-acliv~t~d myeloid cell populations. Thererolc, the ~ntibo~lies7
antibody fra~m~ntc, and ~n~ibody dc~i~alhr~,s of the present invention provide
an hllL~Iuvelllerll over all ~e pr~iuusly ~4g..;7~d usage of anti-Mac-l
25 antibodies such as the ~ .l of il~n~ zl;r~n resultin~ from a reaction of
the non-specific dcfc l-~ system.
The ability of activation s~ific anti-Mac-1 ~ntibo~lies to inhibit t~e
interactions llr~c~y for an i~.n~.. ~OI~ reaction provides the basis for their
therapeutic use in the l~ ..r ~l of chlùl~ic i~.n~...ll.~c."~ ses and
30 aulo;.. ~ e ~ es such as lupus eryfkf ..~ -s, aul~ u~s thyroiditis,
experimental allergic encephalomyelitis (EAE), multiple sclerosis, some forms
of diabetes Reynaud's syndrome, rhf;l....~tc-id arthritis, etc. Such antibodies
WO 94/08620 2 1 4 4 7 3 8 - PCr~USs3/09777--
- 26 -
may also be employed as a Iller~>y in the tre~tm~ont of psoriasis. In general,
the monoclonal antibodies capable of binding to activated Mac-1 may be
employed in the ~ ."F.~ of those ~ ce5 cu~ ly treatable through steroid
ILe~ and in which previous know non-activation specific anti-Mac-l
S antibodies have been suggest~l.
Of special interest to the present invention are antibodies to a.;~ivated
Mac-1 which ~ e the same subpopulation of s~im~ t~ myeloid cells or
activated Mæ-1 as that bound by the antibody CBRM 1/5. Such antibodies can
be used to block myeloid/ICAM-1 and myeloid/fibrinogen binding and hence
can be used in vivo as a means of treating an i~.n~.. ~tOIr response of the
non-specific defense system without ~fr~l;ng the reactions of the specific
defense system and without the ~t~ l side effects of depleting non-acliv,.led
n~,~lr~hils.
The present invention further provides mPth~c of deterlnining the
15 loc~tir~n and l;C~ .,. of G~ ing &C~ d Mac-1 cells within a subject.
The ~ .,inicl.;.l;oll of det~l~hly labeled activation ~ irie anti-Ma~l
~ntiholies to a patient provides a means for i...~g;ng or vicu~li7ing ~e
location, migr~tion~ and ~glegalion of cells expressing aclivaltd Mac-1 such
as stim~ ted myeloid cells and myeloid tumor cells. Clini~l application of
20 antibodiesin~ ,l;e i..-~jngarG~,i..~.~l byGr~s~ n, Urol. Clin.North
Amer. 13.465~74 (1986)), Unger, ct al., Invest. Radiol. 20:693-700 (1985)),
and Khaw, et al., Science 209:295-297 (1980)).
In such a use, the antibodies of the present invention, or r.
thereof, are ~ l...;,-;~,~d to a patient in ~le~ hly labeled form. As desr,ribe~25 earlier with regards to antibody de.ivdli~.,s, anlibodies can be ~ .~Ahly
labeled II.rou~ll ~e use of radioisolu~s, affinity labels (such as biotin, avidin,
e~.) nuol.,scenl labels, p~ gn~l;e atoms, etc using pr~lul.,s known to
the art for use in ~e present in~e.lLion.
The present invention further provides mtoth~lc of selc~ ely killing
30 e~ es,;ng ac~ivaled Mæ-l cells. In detail, cells e~ s~ing ac~iv~ted Mac-1
can be selccliv~ly killed by co~ l;n~ them with a "toxin derivatized
antibodyr which is capable of binding to activated Mac-1 but is subst~nti~lly
Wo 94/08620 `~ 1 4 4 7 3 8 Pcr/uss3/o9777
- 27 -
incapable of binding to non-acLivd~ed Mac-l. As descnbed above, the antibody
derivatives of the present invention include antibodies which are conjugated
to toxic molecules such as the ricin A chain. The present procedure provides
a means of both treating Mac-1 depel-.lel~l biological pl~cess by sele~ ,ely
5 killing the myeloid cells which "~ ;AIe the pr<~ces, as well as a means of
selectively killing tumor cells CA~ illg a~;livd~tid Mac-l.
In providing a patient with antibodies, antibody r,~ or antibody
de.ivalivt;s capable of binding to ~livated Mac-l, the dosage of the agent
which is to be ~flm;,~ d will vary ~epen(ling upon such factors as whether
10 the antibody agent is being ~Amini~tered as a ll;A~no~l;e agent or a Il~Cla~)euliC
agent. Factors such as the patient's age, weight, height, æ~c, general meAir~l
con-litiQn~ iouS mPAir~l history, etc will also affect the dosage
~,~ministe~ed.
T~hniq~les of dosage det~rmin~ti~n are well known in the art for
15 di~noctic ~nl;l~l~r agents as well as ~l-e~ c z~ ~ly agents. In ~Pner~l~
it is desi~ble to ~1.J.;de the ~ enl with a dosage of ~ntibody which is in
the range of from about 1 pg/kg to 10 mg/kg (body weight of patient). As
below ho~ r, the lt~f . ~ y e~r~~ , dose can be lc,.._l~cl if
the anti-Mac-l antibody agent is ~A~lition~lly Z~ in;rt --ul with an anti-ICAM-
1 antibody, or its equivalent.
As used herein, one co~ l is said to be ~ l;l;O!~AIIY ~ ich~d
with a second ~ oui~-i when the r-lmini~l.,.l;oll of ~e two co."poullds is in
such ~u~ y of time that both cc,.ll~ullds can be ~ at the sarne time
in the patient's serum.
The acli~,~ioll ~;I;e anti-Mac-l anlil>odics of the present invendon,
or r.~ ..l thereof, wl~ er for uæ as a l;~..osl;c or lilrl~ul;e agent, may
be ~ c~ ,~d to ~ti~nt.~ O~ y, il-l-~ y, i~l-~ll~--~;--l~rly,
s~lb~ ~ucly, ente~lly, or l,~enlerally. When r~l,,,inic~ g such
mole-llles by injection. the ~ inicl~l;on may be by co~ .o~c infusion, or
30 by single or multiple boluses.
The anti-i~n~ toly agents of the present invention are intenlle l to
be provided to recipient subjects in an amount sufficient to ~u~llle~s the
WO 94/08620 ~ 1 ~ 4 ~ ~ ~ Pcr/US93/09777--
- 28 -
binding of activated Mac-l to the Mac-l ligand which mtoAi~t~s the biological
function which is to be inhibited. For example, antibodies used to treat
infl~mm tti~n are ~-lmini~tered to a patient in s ~rli~ie!~l cO~ f ~ ;on so as to
inhibit ICAM-1/Mac-l binding.
S An ~mo~nt is said to be sllffirient to ~u~r~,ss" Mac-1/ligand
int~ ction if the dosage, route of ~tlminictr~ttion~ etc. of the agent are
s~-ffiriertt to ~t~..r...,.~e or ~>le~,n~ Mac-l/ligand intPr~rtiQn Such ~ J~l;oll or
pre~,elllion can be assayed by P~mining whether the biologir~l runclio
m~ t~d by the targeted interaction is oc~iul.ing in viw, for example by
10 eY~mining neutrophil infiltr~tion into a site of infl~tmm~tion, or by c~ ,laling
in vitro blocking studies with predicted in uvo erG~.
The activation s~ec;Gc anti-Mac-l ~llibodies of the present invention
may be ~z.1,1,ini~ ,d either alone or in co",~il~lion with one or more
?~d~1itinnz~ y."s~;~e agents (esl~eri~lly to a ~ ,ienl Of an organ or
1~ tissue ~.,.r.~lJl~n~). The ~-I...;t~ .,.l;on of such CQ~ (s) may be for ci~er
a ~ o~hyl~lic" or ~ll.r.~.JI;e~ ~u,l~o~. When l,lc..ided plu~y4c1;e~1ly,
~e co- .l-o~ (s) are provided in advance of any h~nz~ "~y ~ ,onse or
...plol.- The prophylactic ~k~ini~ on of the co~ n~1(s) serves to
~r~,cnl or ~ tr, any s.~ ,enl il~ll,.. n~tol~
When provided tk~".~ A11y, the colll~ul~d(s) is provided at (or
shortlyafter)theonsetofaS~lllpLC~ ofaclualinll~.. ~1;0n. The~ ~.J~;C
~l.... in;~l.i.liQnofthec~ >ound(s)servesto~llr.. ~ eanyactuali,.n;.. ~
The anti-i--ll~----- AIOly agents of ~e present invention may, thus, be ~,~.idedeither prior to the onset of i~n~ ;on (so as to ;~u~llr~ an z..l;- ;l.~t.,d
i~.n,.. ; lio~) or after the initi~tir n of inllz.. ..~lion
A u~ osilioll is said to be "~hz........... n~cQl~ir~11y acce~L~ble" if its
~tlminictr~tioll can be tr)ler~t~l by a re~il,ie.lL patient. Such an cc"nl)osiLion is
said to be ~ c~P,ed in a "tke i~ lir~lly err~;~ivG a",-,unl" if the ~--ounL
~r1minict~ored is physiolo~fr~l1y ignifir~nt An agent is physiologically
30 cignifir~nt if its ~,l~,~.lc~ results in a dctecl; hle change in the physiology of
a recipient patient.
W0 94/08620 ~ 7 3 8 PCI/US93/09777
- 29 -
The antibody based agents of the present invention can be form~ t~
according to known metho~ls to prepare pha~ ee~lir~lly useful co~ o,ilions,
whereby these materials, or their functional deli~alives, are combined in
~mil~tme with a ph~ x.Jl;c~lly acceplable carrier vehicle. Suitable
S ~ehicles and their formnl~tiQn, incll,si~ of other human pr~teins, e.g., humanserum ~ lmin, are described, for example, in Renungton's Pharm~r~er~tr~ rl
Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)). In order to
form a ph3~ ..l;c~lly acce~)t~ble f~...pûs;!;on suitable for efÇ~ ad-
ministration, such compositions will contain an errec~ arnount of anti-Mac-1
10 antibody.
Additional IJh~ xl~l;c~l methfplc may be employed to control the
duration of action. Control release p~a~ions may be acl~ ,d lhnougl. the
use of polymers to complex or absorb the ~ l;c agents of the invention.
The ontrolled del;~_,y may be e~ ~d by s.~ lF~ a~
15 ,l.ac.ù...olecules (for example pûl~_st~r." pol~ o acids, polyvi..~
pyrrolidone, ell-yle~ yl~ x ~le, methyl~llnlQse, c~l~Ay-l-e~ lose, or
,r~!~...inP, sulfate) and the co~ -n~ l;on of ~l~ac~ ies as well as the
.nell~oAc of ineo,l,o,~llion in order to control release. Another possible method
to ontrol the duration of ætion by controlled release ~,,p~ o .C is to
20 incc,.~ola~e anti-Mac-1 ~ntihQAip~s into particles of a poly."e,ic material such
as ~ol~ st~r~, polyal"ino æids, llydlu~cls, poly(lætic æid) or clllylene vinyl
acetate copolyllle,s. Al~.l-a~ ly, instead of i-lco,~l~ling these agents into
polymeric l,~licles, it is possible to entrap these materials in micr~ ..les
yl~,p~d, for example, by coa~. ~a~ion t~hniques or by il-~lÇ~
25 polyn-e ;,~t;on ror~A~.l~le,l.y-i~Ayll,~Illylr~ seor~ e-lllic,~ les
and poly(",~ yl~ell~æyl~le) ,l,ic,~ .les ~,;,~cli~.,ly, or in colloidal drug
delivery Sy~.llS, for example, lil,os<,n.es, albumin ~licl~*,heres,
l..ic.ue...~lQions, n~n{~rlicles~ and nAI~ s~les or in llac,~ ...~lQi~nS. Such
techniques are ~liQ~losed in R~"../~gt~n's Phann~ uti~71 Sciences (1980).
The present invention further provides metho~l~ of identifying agents
capable of activating Mac-1 present on resting myeloid cells. Agents can be
assayed for their ability to stimul~te resting myeloid cells or activate Mac-l by
Wo 94/08620 ~ ~ ~L 4 7 3 ~ PCl/US93/09777--
- 30 -
ex~mining treated cells, or purified Mac-l molecules which have been treated
with the agent for the appearance of activation specific epitopes.
In detail, isolated myeloid cells such as neutrophils or isolated de-
aclivaled Mac-l molecules are first conlacLed with the agent which is to be
5 tested. The treated cells or Mac-1 ~le ~11es are then con~cled with an
antibody which is capable of bindillg to ~liv~led Mac-1 but is ~bs~ 11y
incapable of binding to non-~;Livaled Mac-1 and resting myeloid cells. The
cells or Mac-l molec-1lP,s are then c ;~ 1 to determine whether the cells or
molecules binds to the activation specific antibody. If the cells or Mac-l
lO molecules are able to bind such an antibody, the agent which was placed in
contact with the cells Mac-1 or molecules are said to be a "stim~ ting agent."
The present invendon further provides methoflc of idc.~ ing agentc
capable of deacli~àling Mac-1. In detail, s~im~ tP~ myeloid cells such as
PMA treated ,l~ uphils, or ico1~t~~ at~d Mac-1 mohp~ es~ are fir_t
15 co..l~r~d with the agent which is to be tested. The treated cells or Mac-l
mo1co~u1P~s are then c~ ~l with an ~ which is capable of bhlding to
ae~iva~ Mac-1 but is ,t~l"~ 11y in.-~p~hle of binding to non-a~liva~d Mac-
1 or to resting myeloid cells. The cells or Mac-l "o!oc~1es are then P~mine~
to deteLlllill~ v~rhelller ~e cells or Mac-l molecules binds to the antibody. If20 the cells or molecules a.~ no longer able to bind to the antibody, the agent
which was first placed in contact wi~ the cells or Mac-1 mole~llles are said
to be a "dea~livaling agent."
The present in~lllion further provides metho~l~ of ide~ hlg agents
capable of blocking the activation of Mac-1 present on resting myeloid cells.
25 In detail, resting ".~eloi~ cells, such as n~ )hils, or ico!~t~A de-~cli~atedMac-l molecules, are first co~ d with ~e agent which is to be tested. The
con~;led cells or Mac-l molecules are then treated with an agent capable of
stimu1~ting myeloid cells, or activating Mac-1 molecu1es, for e~ample PMA.
These cells or Mac-l m~l~cnles are then in~ub~1ed with an antibody which is
30 capable of binding to a -~ivated Mac-l but is ~.bs~ 11y incapble of binding
to non-activated Mac-1 or to resting myeloid cells. The cells or Mac-1
molecules are then eY~mined to determine whether the cells or Mac-1
wo 94/08620 '~ 8 PCr/US93/09777
molecules binds to the antibody. If the cells or Mac-l molecules are not
capable of binding to the antibody, the agent which was first placed in contact
with the cells or Mac-1 molecules is said to be an "activation blocking agentn.
The present invention further provides mPth~ of sele~;lively removing
5 cells from fluids or tissues which contain a~;li.,aLcd Mac-1 on their cell surface.
In detail, stim~ ted myeloid cells which contain ~cLivaLcd Mac-l can be
sel~lively removed from a fluid or tissue, such as a patient's blood, by
passing the fluid over an immobilized antibody which is capable of binding to
at;liv~lted Mac-l present on stimlll~t~d m~yeloid cells but is subst~nti~lly
10 incapable of binding to non-acLivated Mac-1 or resting myeloid cells. For
example, stimnl~tçd myeloid cells or tumor cells cA~r~,~hlg activated Mac-1
can be ~ o~ed from a patient's blood by subjecting the patient to
leuk~pho,~sis in which the lc~lk~hGl~ sis con' ~ c the patient's blood with an
immobilized, a~Li~alio., sper3fic anti-Mac-1 ~nt~ y.
The present ill~.. lLioll is further based on the novel obsc.~dLion that a
majority of the ~ ir,s which block Mac-1lligand ;~ ;OnC bind to
regions within the I dom~in of ~e Mac-1 molocllle (see e~cample 2). Further,
it was found ~at antibodies could be ~ el~l~ which block ~ subsets
of Mac-1/ligand i,.~Lion, for example CBRM1/1 completely blocked Mac-
20 1/ICAM-1 il~.t.,.~ ;on but had no effect on Mac-1/n~ r~hil bin~ling Based
on this Ob~.valioll, the present invention provides -.- l1.~5 of idenlirying
agents which are capable of blocking specific Mac-1/ligand il~ cl;~ n
In detail, the ~ 1ies desclibed in Figures 13 and 14, or an
equivalent set of anlil,odies, can be used to map the peptide l~ .,;dU~S within the
25 Mac-1 mole~llle which bind to ~ific Mac-1 lig~n~. A similar a,)pl~ach to
epitope mapping is ~çc~ l in co-~.-~ g U.S. Ser. No. 07/618,286, filed
No~e",ber 28, 1990, herein il~col~la~ed by ~ r.,nce.
The present hl~enlion ~ closes that the sites of il-t ,~cl;on between
Mac-1 and the various Mac-1 ligands (iC3b, ICAM-l, and fibrin~en) are
30 located within the I domain of Mac-1 and are ~ tinrt and mostly do not
overlap. Based on this disclosure short peptide fragments of the Mac-l
WO 94/08620 ~ 4 47 3 ~ PCr/US93/09777--
- 32 -
sequence can be generated which are subst~n~i~lly capable of blocking spe~cific
subsets of Mac-1/ligand interaction.
As used herein Yspecific subsets of Mac-1/ligand inter~rtion" refers to
an agent's ability to block one or more Mac-l/ligand i~ler~rl;ons. For
S example, a peptide may block a specific subset of Mac-1/ligand i~ ions
by blocking Mac-1 binding to a single ligand such as ICAM-l.
Among the p~rell~,d short peptide r.,.~,...r .l of the present invention
are peptides which bind to the Mac-l contact site on ~e Mac-l ligand, which
comprises amino acid se~.P-~-r4s derived from the amino acid sequen~e of
10 Mac-l, as well as peptide which bind to the ligand binding site present on
Mac-1 which comprise amino acid se~lue .r~s derived from the Mac-l ligand.
The monoclon~l antibody blorl~ing data ~l~nted herein can be
combined with routine ".~ ,r,;c studies in order to further map MAb
~ilopes to distinct regions within the I ~orn~in of Mac-l. In ~ ition arnino
15 acid ~lbs~ l;on and dornain deletion mllt~onesic of Mac-1 can pc~&,Lll.cd,
for ~".alllplc by oligo.~ r!~ c dil~:~d mllt~enr~;c as dcs~-l;he~ by Klln~
(Proc. Natl Acad. Sci. USA, 82:488-492, (1985)), as mo~1ified by ~l~on et
al. (Nature, 329:842-846 (1987)), in order to ~,er~ e Mac-1 deletion and
~ul~s~ l;Qn ....,l;...l~i which lack the ability to bind to a ~ec-ific Mac-1 ligand
20 or antibody. By using such l,ro~lu,cs, short peptide ~.,~ ~s which serve
as the ligand contact site on Mac-1 can be ~eh- --;nr~.
The present invention fur~er provides chimeric molecules comprising
a fusion be~ween Mac-l and plS0,95, see ~nplc 2. Such chimeric
molecules are useful in mapping epitopic do.,~h.s and ligand contact regions
25 within the Mac-1 and plS0,95 molecules. Further, c~ lle ic mol~p~ es can be
gf..n~ P,d which m~int~in the ability to ~csocia~o with the ~B-subunit (CDl l) of
the protein dimer and ~a~ aLP in a select set of a-subunit ligand binding.
For example, a chimeric Mac-1/plS0,95 a subunit can be ~,enf.~tP~I which
inc the ability to form a dimer with CD18 as well as bind ICAM-l and
30 fibrinogen, but is incapable of n~ L~u~hil a~ gation or Mac-1 d~,t,c..denL
homotypic aggregation. Methods of gc,~e,aling such chimeras, as well as
WO 94/08620 i~ 1 4 4 7 3 8 PCI/US93/09777
suggestions of their use are described in co pending U.S. Ser. No.
07/618,286, filed November 28, 1990, herein illco~ ed by c;f~,.,.lce.
Having now genP,r~lly described the invention, the same will be more
readily ulldc~ ood through ~fe.~ ce to the following e~tamples which are
5 provided by way of ill,.~ l;on, and are not intPn led to be limiting of the
present invention, unless specified.
EXAMPLE 1
IDENTIFICATION OF A NOVEL CLASS OF ANTI-MAC-l
ANTIBODIES
Materials and Methods
MAbs
~e following murine MAbs against human ~nti~m were used: TSl/æ
(anti-CDlla, IgG1, ascites)(S~nr-h~7-Madrid et al., Proc. Natl. Acad. Sci.
U5~45 79:7489-7493 (1982)); LM2/1 (anti-CDllb, IgGl, protein A-purified)
(Miller et al., J. Inup~7Ql- 137:2891~2900 (1986)); CBRMl/23 (anti-CDl lb,
IgG2a, protein-A p~rifip~d); OKMl (anti-CDllb, IgG2b, protein-A purified
(Wright et al., Proc. Natl. Acad. Sci. USA 8~:5699-5703 (1983), LPM19c
(anti-CDllb, IgG2a, a gift of Dr. K. Pulford, Oxford, UK (Uc;~l~ i et
al., In Leukocyte Typing IV: W~ute Cell Differen~ianon Antigens, Kr~pp editor,
Oxford Uni~ y Press, Oxford, pages543-551 (1989)); BL~6(anti-CDllc,
igG1, ascites (Uc;ccl~c,w~i et al., In Le~yle Iyping IV: W~uite Cell
Differenti~lion Ant~gens, Knapp editor, Oxford Uni~ ;Ly Press, O~cford,
pages 543-551 (1989)); ~;6.5 (anti-CD54, IgG2a, gen~luus gift of Dr. R.
Rothlein, Boehlinger-lngelh~im~ Ridgefield, CT) (Smith etal., J. Clin. Invest.
82:1746-1756 (1988); CBRIC1/11 anti-CD54, IgGl, protein-A purified); and
My4 (anti-CD14, IgG2b, purified) (Griffin et al., J. Clin. Invest. 69:932-941
(1981)) (Coulter T.n...~.nf~logy~ h FL). X63 (nonbinding antibody, IgG1)
and 3G8 (anti-CD16, IgG1) (Fleit et al., In Leucocyte Typing IV: W~ute Cell
Wo 94/08620 PCr/US93/09777
3~
- 34 -
Dif~erentianon Antigens, Knapp et al. editors, Oxford University Press,
Oxford, pages 579-581 (1989)) were used as culture s~
Protein Purification
S Mac-1 was purified from leukocyte lysates by im~ nc~ rr~ y
chru~l,d~al,hy after d.,~.~,en~ solubilization as decr,rihe(l (Diamond et al.,
Cell 65:961-971 (1991)). Soluble ICAM-1 (sICAM-1, genelo~s gift of Dr. S.
Marlin, Boehringer-IngelhPim ph~ eeu~ efield7 CT) was purified
from s-.l.el..,.li.nts of ICAM-1 tr~n~fect~1 CHO cells by immllno~ffinity
c~llolllalogldl,}ly as described (Marlin et al., Nature 344:7~72 (1990)).
Tissue Culture. Tld~lsr~lion. and Cell ~ aratio~
N~ phils were ;~Q1~t~ from the whole blood of healthy vvl--
~by ~e~t~n ~r~ .on at room ~ u.~, with Ficoll ~die-nt
15 cf~ ;r~ ;on and ll~ onic lysis at 4'C as ~lescribe~ (l~.~li~h et al., J.
I~,u,.u,.ol. Methods 5:249 (1974), Miller et al., J. Clin. Invest. 80.535-544
(1987)). Prior to l25I-MAb bi~dil-g experiments, n~ ophils (2 x 10~ cells/ml)
were stored at 4 C at in HBSS, 10 mM HEPES pH 7.3, 1 mM MgCl2, 1 mM
CaCI2 (HHMC) in polyL~Iop~lene tubes (Falcoln 2097, Becton Di~Lin.con,
20 Lincoln Park, NJ). For time course and t~ studies the l~ ocyle rich
~ e.l-alall~ was used after ~e~t~n s~ l;Qn Cell ~ y~ onc were
placed on ice i... ~ lP.ly and washed four times to l~ , platelets in HBSS,
10 mM HEPES pH 7.3, and l~ CIh. n~l~ at 5 x 106 cellslml in HHMC.
Neutrophils were ide~lirled during flow c~ lllet~ by 1~lW~ 1 and 90 scatter,
25 and conri.l~ed by i"""nl~n~st~ g with a MAb to CD16. Mo..o..~ r cells
were isolated from ~e i~ r~ of ~e Picoll y.,~lie.l~l and washed five times
in RPM1 1640, 5 mM EDTA, 2.5% FCS (heat-h~ a~ed, low endoio..,n,
Hyclone, Utah), and l~i,u~ d~d in L15, 2.5% PCS, 1 mg/ml human y-
globulin (ICN ~..... "nBiologicals, Costa Mesa, CA) at 5 ~c 10 cells/ml at
30 4C. Mononuclear cells were i~ -o~in~d according to the ~r~tocol for
neutrophils (see below). The monocyte subpopulation was determined by the
7 3 8
WO 94/08620 PCr/US93/09777
flow cytometric forward and 90 scatter pattern and confirmed by
immllnost~ining with the a MAb to the monocyte-specific CD14 antigen.
The gel u .~liQn and selection of CHO cells eA~ ,ssing wild type and
chimeric forms of Mac-1 and plS0,95 will be described. They were
S m~int~inPA in ~Y-MEM, 10% dialyzed FCS, 16 ~M thymidine, 0.05 ~LM
methotl~Aate, 2 mM gll~t~minP, and 50 ~g/ml ~el~-...icin
Generation and Labellin~ of CBRMl/5 IgG and Fab r;~n~...e~tc
The g~l-e. ~l;on of MAbs against Mac-1 are described in Example 2.
10 In brief, hybridomas were pl~ar~,d from BALB/c mice that were iml~ni,~
with imml-no~ffinity purified Mac-1. Hybridomas (500) were screened as
follows. Purified ne.~ hils were isol~tP~ z.i l~d into two groups, and
preil-r.,l,At~l at 37C for five minlltes in HHMC. One popul~tion was treated
with phorbol esters (PMA, 100 ng/ml) for ten ~";"'JIt'~S while the other
~ inr~ ~d. Cell ;,.~ olls were placed on ice for five l~.inu~es
;l.~;~.;-A~ with hybridoma ~ Z~ , and ~ l by flow Cy~ l.,hy. In
some cases, nellL.o~hils were fiAed with 2% ~aidfolll,aldehyde prior to
b-liQn with MAbs. CBRM1/5 was identifiecl for its ability to bind PMA
stim~ t~l but not resting n~,.lLIophils. It was cloned twice by limiting dilution,
and purified from culture su~.l~ll by protein-A S~har~se affinity
chl~,lllal~la~hy after NH~SO~ i~Lion (Harlow et al."4ntrbo~i~s A
Laboratory M~ rrn/ ~Cold Spring II~l. Cold Spring Harbor Labolatolr
(1988)). CBRM1/5 was determined to be of the IgG1 ~ c~ (~mmunoPure
Monoclonal Antibody Iso~y~illg Kit, Pierce, Roc~ld, IL)
Fab r. ~",~ n of CBRM115 was ~.r~ ed by pepsin ~ estion
followed by cysteine reduction (P~l.~ll, P., In Irnmuno~ogical Methods in
Biomedical Sciences, Weir et al. editors, Blacl~ ll, O~cford (1983)). Fab'
were sep~r~t~ by 1.5 cm x 75 cm S-200 Seph~cryl (I~l~lllaciâ, Piscataway,
NJ) size exclusion chrolllatography. Fab' eluted from the column as a single
homogeneous peak with an apparent Mr of 40,000. Purity of Fab'2 and Fab'
fr~gm~ntc was conrlrllled by reducing (5% ,B-lllel~,utoeth~nol) and non-
reducing (50 mM iodo~c~t~mide) SDS-8% PAGE. Proteins were visualized by
wo 94/08620 ~ PCr/US93/09777--
- 36 -
silver st~inin~ (Morrissey, J.H. Anal Biochem. 117:307-310 (1981)).
Unlabelled (Bio-Rad, Richmond, CA) or '~C-labelled (GIBCO BRL,
Gaithersburg, MD) protein standards included myosin (M, 200,000), ,B-
g~l~r~tos~ (Mr 116~000)~ phosphorylase B (M, 97,000), serum albumin (M,
66,000)~ ovalbumin (Mr 43,000), carbonic anhydrase (M, 31,000), trypsin
inhibitor (Mr 21,500), and lysoLyllle (Mr 14,000).
MAbs (100 ~Lg) were labelled with Na'2sI (1 mCi) using 1,3,4,~
tetrachlor~3~ diphenylglycouril (Pierce C~hPmi~ Co, Rocl~rold, IL)
(Fraker et al., Bioichem. Biophys. Res. Commun 8~.849-857 (1978)). The
specific activity of the ioflin~ted MAbs was determined (protein ~ e.y
averaged 83 ~gs, specific activity was ~7 x 106 CPMl~Lg).
I-MAb Binding Assays
Purified neutrophils (2 x 10' cells/ml in HHMC) were either kept on
ice or warmed to 37C for five ,,.il,~,t~r,s and stim~ t~A (PMA, 100 nglml;
fMLP, 1~7M) for ten "~ tes. A~;liv~t~ n.~ .hils were i...~ ;ly placed
on ice and S00,000 cells were ~liquott~l into U-bottom plates (96 well non-
tissue culture treated Illie~li~l plates, Linbr~Titertek, Flow Labo-ato.ies,
~'c~ ~n, VA) c~nl~;..;n~ h-~l~as;"g co.~ I;ons of io lin~teA MAb (0.109
nM - 112 nM) in the ~hsenr~ or ~I~,~nce of 20-100 fold e~ccess cold MAb.
The binding media cont~inp~ L15, 2.S% PCS, 1 mg/ml human y-globulin.
Neutrophils were il~ h~lr~i with iodinated MAbs for 4h on ice, washed five
times wi~ L15, 2.5% FCS by 21 gauge needle aspiration, lysed with L15,
2.5æ FCS, 0.2~ NaOH, and cou~led for y~mission. Specific bindin~ was
delc~ ~ by ~ acling the CPM in the ~I~,~nc~ of e~ccess cold MAb.
S-'At~h~.~ plots were gene.aled (SCAI~ L G. Ann N. Y. Acad. Sci. 51:66~
672 (1949)) and site densi~içs were d~ ter...i,.cd using the data point at whichthere was no ~llrlitiQn~l increase in s~il;c binding of l~helle~l MAb.
WO94/08620 ~ 73~ PCI/US93/09777
- 37 -
Sufface Labellin~ Immunoprecipitation. and Gel Electrophoresis
Purified neutrophils (2 X 107 cells) were washed twice in PBS, 1 mM
MgCI2, 0.5 mM CaC12 and r~su;,~cnded in 2 mls; fMLP (10-'M) was added
to some aliquots of neu~lophils. The cells were iodil-At~A, Iysed and precleared. 5 as described (D;alllond et al., Cell 65:961-971 (1991)).
T~ oprecipi~lions were ~Irullned one of two ways: (i) indirectly
by il-. .,b~;n~ 250 ~1 of MAb (neat s ~ nl or 20 ~glml purified IgG) with
1 ~1 of 1 mg/ml purified rabbit anti-mouse IgG (Zymed, San Fr~nci~co CA)
for 4 hours at 4C. Protein-A-Sepharose (20 ~1 of a 1:1 slurry) was added for
10 overnight in~lb~tion Eppendorf tubes were ce~ u~ged, s~pc~ ntc were
aspirated, 50 ~1 of iorlin~tloA I~ Ll~hil Iysate was added and the ~ ul~ was
il.r..h~tlA for 2 hours at 4C while ch~kir~ vigol~usly, (ii) di~;lly by adding
60 ~LI of a 1:4 diluted Iysate to 60 ~LI of dil~lly coupled MAb-Sepharose (2-3
mg pulir~ed MAb per ml CNBr~ ivd~d Sepharose CL 4B) and i~ ub~ for
15 2 h at 4C while ch~kin~ vigo.~usly. The ..~I.illg and elution of the
i.. u.~ s has been described (Di~llol~d ct al., C~ll 65:961-971
(1991)). Samples were loaded, and subjected to SDS-7% PAGE (T ~emmli,
U.K. Nature 227:68~685 (1970)) in the pl~,sY,nCe of ~ lle~loe~ ..ol, and
~ olA.1iographed with in~ll~irying screens (Laskey et al., FEBS Letters
82:314-316 (1977)). P~cl~hihlg eA~lilllcnls were ~,rulllled with directly
coupled Se~har~se as follows: after the initial ;.. ~l-.>pl~;~i~tion with
control (mouse IgG) or sperific Sepl~l~se (LM2/1, CBRMl/5), the beads
were pelltot~oA, the s~ .-Al~..l (60 ~1) was ~ r.-.~ to a second aliquot of
the same MAb-S~h~, and il~o~ t~A as clc~-il~A~ above. S~lbsequently,
25 these beads were pe~ t~A the s.l~.l~l~ll~ (60 ~ ;,r~"~ to an aliquot of
either LM2/1 or CBRM1/5-Sepharose, and i~ ub~A After each round of
i.. ,m~l~i~itation~ the beads were washed and the bound protein eluted as
desclibed (Diarnond et al., CeU 65:961-971 (1991)). Sarnples were loaded,
and sul)jecled to SDS-5 % PAGE (~ ~Pmmli, U.K. Nature 227:680-685 (1970))
30 in the l.lesellce of ~-lllel,_aploe~ ol, and autoradiography (Laskey et al.,
FEBS Let~ers 82:314-316 (1977)).
Wo 94/08620 Pcr/uss3/o9777 ~
~4~7~ `
- 38 -
ELISA with Purified Mac-l
Tmmlmo~ffinity purified human Mac-1 was diluted 1/20 in 25 mM Tris-
HCI, 150 mM NaCI, 2 mM MgCI2and adsorbed to individual wells of a non-
tissue culture treated microtiter plate (Linbro-Titertek, Flow Laboratories,
5 McLean, VA) for 1 h at 37 C. Plates were washed five times and blocked for
one hour at 37C in HHMC supple...e,..l~A with 1% human serum albumin.
Plates were then washed twice with HBSS, 10 mM HEPES pH 7.3, 10 mM
EDTA, 0.05% Tween 20 and twice with HBSS, 10 mM HEPES pH 7.3,
0.05% Tween 20. MAb solution (100 ~1 of 20 ~glml of purified MAbs) in
HBSS, 10 mM HEPES pH 7.3, 0.05% Tween 20 supple.. c.~ with either 1
mM MgCI2, 1 mM CaC12, 1 mM MnCI2 or 5 mM EDTA (HHTcation) was
added to the wells and allowed to inc~ le for 1 h at 37C. The plates
s~bs~quc- ~liy were iced for five ...i~..lf s and wæhed four times with
HHTcadon at 4C. Enzyme linked second antibody (50 ~1 of 1/400 dilution of
15 hG~ Ai~h pero~ e goat anti-mouse IgG, Zymed Labordt4lies, San
n~iG~, CA) was added in HHTcation, jl..`~ t d for 1 h at 4C, and the
plates were washed five times with HHTcation at 4C. Sub~kdte (2,2-azino-
di(3-ethylbe~ ,oline) sulfonic acid, Zymed T ~holi~lolies~ San Fr~nr.icr,o,
CA) was added in 0.1 M citrate with 0.05% H2O2, and the results qu~ntit~tY
at 414 nm on an ELISA plate reader (Titertek l~fulti~n MCC340).
Flow Cylu~ -Y Studies
The time course and ~e~ "~u.c; studies were pclrol.lled as follows:
P~.irled neutrophils or leukocyte rich s.~ll-a~lls after dP~tr~n s~li...P ~ l;on(5.0 ~c 106 cells/ml in HHMC) were ~ r,d in ep~"do.r (1.5 ml) tubes
at 0, 25, or 37C for five ~ tes before stimu~ was added (1L,8, 25 ng/ml
(72 amino acid form), PeproTech, Rocky Hill, NJ; fMLP, 1~7M; PMA, 100
ng/ml). After a time point was re~ch~A, ~-lples were placed on ice
immP~ t~ly and aliquots were added to individual wells of a U-bottom
microtiter plate on ice cont~inin~ MAb. In some cases, after a time point was
reached cells were fixed imm~li~tely with paraformaldehyde (2 %) and
in~ub~t~ for 30 mimltes on ice. Tmmlmofluor~srence and flow cytometry was
wo 94/o862n 2 1 4 4 7 3 8 PCI/US93/09777
- 39 -
performed as described (Diamond etal., J. Cell. Biol. 111:3129-3139 (1990)).
The protocol for cor,~ ion of logarithmic fluolGs~l-r~ into linear
fluorc~cnce has been deselibcd (Schmid et al., C~C,..~ry 9:533-538 (1988)).
5 Adhesion Assays
Purified fib,illogen (2.0 mg/ml, in PBS, Sigma C~hPmir~l Co., St.
Louis, MO) or purified sICAM-1 (200 ~g/ml in PBS) were spotted (25 ~l)
onto 6 cm bac~.ial Petri dishes (Fisher 1007) for 90 minl~tes at room
Ltlll~cldlulc. Plates were blocked with the delcr~,e.,~ Tween 20 as described
(Diamond et al., Cell 65:961-971 (1991)). Neull~hils (4 x 106 cells in 1 ml)
were Ics~y~l-tled in HBSS, 10 mM HEPES pH 7.3, 1 mM MgCl2 and
ple;~ ub~l~A with MAbs for 10 ...;..~t~,s at room tt ~ ~.f'l~.,. S~ ~u~ ly,
cells were added to the dishes in the ~l~,~nce or ~hsenr4 fMLP (10-' M, final
volume of 3 ml), and allowed to adhere for four ...i..~t~s. Non-adherent cells
lS were n,~llu~d by twelve washes with a Pasteur pipette after gentle ~irling
with same buffer supple ~ with 0.5% BSA. ~in~ was ~ ;.tr~ by
scoring the nu~r of adlle~ cells of at least four dirr.,.c"l~ fields using an
ocular grid at lOOX ,..~.~;r~l;Qn The ~f~.ll inhibilion by MAb was
delcl...h~cd upon col.-~;soll with the media control.
The bindil~g of ICAM-1+ L cells is a .. ~ir.r~;on of ~ iously
de~libcd l)lolocols (D;~...on~l et al., J. Cell Biol. 111:3129-3139 (1990)).
Briefly, l,ulir,cd Mac-1 was diluted and adsoll ed (30 ~l~ to 6 cm Petri dishes.After a 90 minute ;~e~ ;o~ at room ~.n~lalulc~ non-~l~irc binding sites
were blocked with 0.5% heat-treated BSA. ICAM-1+ L cells, after removal
25 from tissue culture plates with ~ypsin-EDTA (GIBCO, Long Island, NY),
were washed twice and l~ ~n-l~d in PBS, 2 mM MgCI2, 0.5% heat-treated
BSA (0.5-1.0 x 106 cells/ml). Thir~y ~n;...~s prior to the bhldil.g assay, the
Petri dishes or cells were ~ .;..l.AI~ at room ~IlllKlalul-, with MAb (1/200
dilution of ascites or 20 ~g/ml of purified MAb) in PBS, 2 mM MgCI2, 0.5 %
30 heat-treated BSA (2 mls). Cells (1 ml) were added to Petri dishes, and
incubated for 60 mim-tes at 37C. The Petri dishes then were swirled and non-
adherent cells were removed by tipping the plate at an angle, removing the
Wo 94/08620 PCr/US93/09777 ~
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binding buffer with a Pasteur pipette, and adding fresh media (3 ml) to the
edge of the dish. This procedure was repeated five times. Subsequently, the
number of adherent cells was determined by light micloscopy at 100X using
an ocular grid. The ~.~,'~ge binding was norm~li7~d to a media control.
RESULTS
MAb to a nevepitope on Mac-1
If a confo"l,ational change in Mac-l was involved in the regulation of
its adhesivt;ness, specific epitopes should be expressed on activated but not
10 resting cells. To gene.dte MAbs that lecognize these activation~ependent
epitopes, i~ u~ffinity purified Mac-1 that was judged functional by its
capa~iiLy to bind several ligands (Diamond et al. In Leukocyte Typing IV,
Knapp et al. editors, O~cford Uni~ y, London pages 57v-574 (1989);
Diamond et al., J. Cell Biol. 111:3129-3139 (1990)) was used to i..""II";~
15 BALB/c mice. Hybridomas were ~;~ ed for their dirr~ .. .,lial ability to bind to resting and phorbol estera_~iv~t~cl n~ul~vphils. One MAb, CBRM1/5,
showed little binding to resting ll~uLIophils but bound to neutrophils that werestim~ t~A with phorbol esters (Figure la). CBRM1/5 bound to aclivat~d cells
at a si~nific~ntly lower level cs~ ,ar~d to previously ch~,;rle~iLed MAbs to
20 Mac-1. Tm,,,~ ~;ipi~lion from det~ .~e.ll Iysates of surface I~heIIed
n~lLIvl)hils (Figure lb) or tlansr~ct~d COS cells (data not shown) cvllrl"l,cd
that CBRM1/5 l~cogniLed Mac-1.
The lower level of t;~r.s,ion of the CBRM1/5 epitope on PMA-
stimlll~t~d lle~ hils was not due to i~ ;cie~l alllvunls of MAb, as studies
25 by flow ~v~ tly s I~,est~ cl that saturation was l~ cd at 20 l~g/ml (data notshown). This was conrl~ d by ~inding e~.i".ellls with '25I-CBRM1/s and
l25I-LM2/l. CBRM1/5 bound spe~ifi-~IIy to a~;livdt~d (fMLP or PMA)
neutrophils but not to resting lleulr~hils (Figure 2a and 2b, note dirÇelt;nce
in scale, and Table 1). Despite the lower absolute nulllber~ of binding sites,
30 the binding of CBRM1/5 was saturable, and of collll,a,able affinity (Kd =15
nM) to LM2/1 (Kd =2 nM). Neutrophils stim~ ted with fMLP or PMA had
a total Mac-l site density of 139,000 and 197,000 sites/cell (deterrnined with
WO 94/08620 ~ 1 ~ 4 7 3 ~ PCI'/US93/09777
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LM2/1) but a CBRM1/5 neoepitope site density of 13,000 to 57,000 sites/cell,
respectively (Table 1). Bivalent binding of CBRM1/5 to Mac-1 was not strictly
required, because monovalent Fab' fr~gm~onts of CBRM1/5 bound cpe~ific~lly
to activated neutrophils and displaced (K~ of ~ab' = 200 nM) bivalent
5 CBRM1/5 IgG (Figure 2c, and data not shown).
Kinetics and Te~ ~,alu,c; Studies
IL-8, fMLP, or PMA prompted a time ~.pen~e~t increase in
e~r~ssion of the CBRM 1/5 epitope that pl~t~ued after 10 min~lt~s (Figure 3).
10 In all cases, CBRMl/5 reacted with a subset of the LM2/1-reactive Mac-1
molec~lles on the cell surface.
MAb binding studies were carried out at ~lirrer~ n~l~lul.,s to
detel"~ine whether the CBRM1/5 ~iL~pC eA~ .;on co~lela~ed with an
increase in surface tA~l~,s~.ion or the ~ilivation of Mac-1 (Figure 4). Elevating
15 the Ir~ )f"~ f.~m 4 to 37C in.il~s the surface eAI,r~,ssioll of Mac-1
but does not l ~e-c~ ;ly ~r~n,ut~ Mac-1~,pe~ ent ~her:o,. (Buyon et al., J.
Irnmunol. 140.315~3160 (1988); Lo et al., J. Ex~. Mea'. 169:1779-1793
(1989), and SchlP rr~ .b~... et al., J. Immunol. 142:3537-3545 (1989)). This
lç~ er~ e~-~r-.l increase is primed by pr~p~..ti.~e procedures. We
20 found that raising the t~ from 4C to 37C ci~ ly in~l~sed the
overall ~mo mt of surface Mac-1 wl,~,~as eA~ ~ion of the CBRMl/5 epitope
rçm~in~d virtually absent on both ,nor,~;~s (Figure 4a) and neutrophils
(Figure 4b). In cûllllaâl, in both IllOl~O~;y~S and lle~ u~hils~ fMLP triggered
;,aion of the CBRM1/S epitope. These ~ e.i~ s~ -st that the
25 expression of CBRMl/5 is s-C.~ci:~t~d with the activation of Mac-1.
Epitope Mapping of CBRM1/5 on Transfected Cells
We eY~min~A the expression of CBRM1/5 on CHO cells that were
- transfected with wild type and chimeric Mac-1 molec--les (Figure 5). A subset
30 of Mac-1 molecules on transfected CHO cells constitutively expressed the
CBRM 1/5 epitope. This agrees with our previous observations that the
activation MAb NKI-L16 reacted con~Li~Lively with a subset of LFA-1
WO 94/08620 Pcr/US93/09777 ~
73~
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molecules on transfected COS cells (Larson et al., Cell Reg. 1:359-367
(1990)Keizer et al. . ~. Irnrnunol. 140.1393-1400 (1988)). CHO cell
transfectants that cA~leis wild type Mac-l are functional as they rosetted with
iC3b coated elylhru~y~es, bound Leishmania l.r~ as~igotes, and adhered to
5 purified ICAM-l (data not shown). CBRMl/S lor~li7~ specifir~lly to the I
domain of the Mac-l ~ chain as it, like LM2/1, bound to CHO cells that
eAI,.essed the wild type Mac-1 and chimera X-e-M-~X which corl~ins the I
domain of Mac-l on a pl50,95 frame. In COll~ , CBRMl/S did not bind to
wild type pl50,95 or the reciprocal chimera, M-e-X-b-X which contains a
pl50,95 I domain on a Mac-l backbone (Figure 5).
Effect of Divalent Cations on CBRMl/5 l:;A~ ,s~ion
For int~E lins to bind to their lig~ntls, divalent cations are required.
~tlhpsio~ is ~holichPA ~.~,iÇol,llly by the ~ ,sel~ of EDTA and altered
15 yuA~ ,ly by the type of divalent cation that is present (Gailit et al., J.
Biol. Chem. 263:12927-12932 (1988); Diamond et al., In Leuko~te Typing
IV, Knapp et al. editors, London, Oxford Uni~,L~;Iy, pp. 57~574 (1989);
Altieri, D.C. J. Immunol. 147:1891-1898 (1991) and D.;.r.c~ld et al., J. Cell.
Biol. 116:219-226 (1992)). We tested the CBRM1/5 and LM211 cpil~es for
20 their sen~ilivily to dirr~ t divalent cations. For bi"ding to cell surface
(Figure 6a) or purified Mac-l (~;igure 6b), the pl~sel~ of divalent cations was
required for CBRM1/5 epitope ~Ap~i,;,ion but not for LM2/1. EDTA (S mM)
ab.v~at~ the cA~ ,s ~iOl~ of CBRM1/5 but only slightly l~dUCed eA~ ssion of
LM2/1. Neutrophils Stim~ ted with fMLP eA~ the neoepitope better in
25 the ~ of Ca2+ ~an in Mg2+ alone but in other st~ es~ CBRM1/5 was
~ressed better on l~.lllol)hils stimnl~tpA with PMA in the ~ ce of Mg2+
alone (data not shown). Mn2+, which is reported to e-nh~n-,e Mac-1 adhesive
function in vitro (Altieri, D.C. J. Immunol. 147: 1891-1898 (1991)),
~--g.. ~l~ the eAI,.c ";on of CBRMl/S on purified Mac-l and l)ro.l,oled the
30 e~r~i,sion of the epitope on resting ~ ophils. Unlike the Mac-l on
neutrophils, Mac-1 that was purified by immnno~ffinity chromatography
e~r~ssed equivalent levels of the CBRM1/5 and LM2/1 epitopes in the
WO 94/08620 2 1 ~ ~ 7 3 8 PCl/US93/09777
- 43 -
presence of divalent cations. In contrast, Mac-l that was purified by iC3b-
Sepharose chromatography (Hermanowski-Vosatka etal., Cell 68:341-352
(1992)) e~ ,ssed the LM2/1 epitope but lacked the CBRMl/5 epitope, and
was unable to sustain ligand binding (MS Diamond, KPM van Kessel, SD
S Wright, and TA Sl"ing_r, unpublished obselvalions).
Sllucluldl subsets of Mac-l
To d_Le-l.linc whether CBRMl/5 r~ i7~d a subset of Mac-l
mole~ulPs, sequenti~l i,.,l..l..~ol~r~ ion experiments were l.e,~,l,ed
10 (Figure 7). After preclearing a l.e~ ~hil Iysate twice with
CBRM1/5-Sepharose so that negli~ihle ~mollnts of CBRMl/5 ~acli~e Mac-l
rem~inPA LM2/1-Sepharose i~ opr~cipilal~^cd a si~nifi~nt 4u~ of
Mac-1. However, if the Iysate was l)L~,cl~d with LM2/1-S~h~usc first,
CBRMl/S-S~llalu~c was unable to i~ opl~i~Jlla~ any additional Mac-1.
15 These e~ .f,.~l~ s..~g~,s~ that CBRM1/5 l~cogni~s a subset of the LM2/1
l~,aC~i~,_ Mac-1 "-olecl-lçs in the cell Iysate.
Functional effects of CBRM1/5
Bivalent IgG, Fab2, or ~ûnovalent Fab fr~m~nt~ of CBRM1/5
20 inhibited greater than 80% of the Mac-1~epeul~-.l b;.ldillg of fMLP-
stim~ t~l "~ ~hils to ICAM-1 (Figure 9a). CBRM1/5 was cc,...~ ble in
efr~li~.~n_ss, to LPM19c, a MAb to Mac-1 that binds a similar nu~,l)cr of
sites/cell as LM2/1 and completely blocks Mac-l-ICAM-1 il~f "~ ;on
(Figure &, (D;~ d, et al., J. Cell Biol. 111:3129-3139 (1990)), and data
not shown). As a control, a third MAb to Mac-1, CBRM1123, had no
$ipni~ hibil~ly effect on ~ul~ hil ~lhçsion to ICAM-1. When
CBRM1/~ was added to a blocking MAb to LF~-1 ~TS1/222), similar to the
case with LPM19c (Diamond, et al., Cell 65:961-971 (1991)), the coml>inalion
of MAbs virtually abolished læull~hil binding to ICAM-1. The ability of
CBRM1/5 to inhibit Mac-1 dependent n~,u~ hil int~ra~lion with ICAM-l was
confil..led in binding assays with purified Mac-1 (Figure 8b). Prein~llh~tion
of purified Mac-l with CBRMl/5 completely abolished ICAM-1+ Ladhesion.
-
WO 94/08620 ~ 3 8 PCr/US93/09777--
When we tested the effect of CBRM 1/5 on fMLP stim~ t~d neutrophiladhesion to purified fibrinogen, nearly complete abolition of binding was also
observed. S~tl-~tin~ co..~ lions of CBRMl/S IgG, Fab'2, or Pab blocked
adhesion to fibrinogen ~p~r~im~tely 90%, and half-m~sim~l blocking was
S observed at an IgG co~ tion of a~p~im~tP,ly 1 ~g/ml (Pigure 8c, and
data not shown). Again, other MAbs to Mac-1 (CBRMl/23) used at similar
con~ l;ons showed little inhibitory effect (data not shown). Thus, the 10%
subset of Mac-l molecules present on neull~hils after fMLP stim--l~tiQn
which are identified by CBRM1/5 m.oAi~tes at least 90% of the adhesive
10 activity of Mac-1.
DIS~USSION
Di~closeA herein is the ch~ t~ ;7~l;on of a novel MAb, CBRM1/5,
that binds to a ;~u~ ulation of Mac-1 mole~-les on aeliv~t~d, but not resting
15 peripher~l blood ~ opl~ils and ll-onoc~s. This MAb r~cogl~.~s an
activation~e~ r..l n c~;lol)c and acts as a .~l~r for the activation state
of Mac-1. Our results demQ~ lr ~at the subset which eAl,r~sses the
CBRM115 ~eocpilo~c c4~ s the stlu~;lul~lly active Mac-1 molecules that
sustain ~tlh~sion to at least two of its ch~ i~d li~ntlc, ICAM-1 and
20 fibrinogen. Evidence for this includes the following: (i) CBRM1/5 does not
react by i.. m~ ol~ wi~ Mac-1 on the surface of resting lleull~hils
or ",onocy~s. However, stim~ tiQn with the ~l~e.~o~ c~l~ fMLP, the
chemo~ll.,.cl;~,e ~lu~ine IL,8, or phorbol esters rapidly ~),o--~o~s e~L),. s~ion
of the epitope on a subset of Mac-1 inolec~ .s. The time course of eA~ s,ion
2~ of the neGe~ ~ and adhesive a.-livil~ of Mac-1 (Ande.~n, etal., J.
~mmunol. 137:15-27 (1986); Detmers, etal., J. E~xp. Med. 171:1155-1162
(1990)) are parallel as both occur within two ...i...~s. (ii) The .~ ;on of
site de-ncities by ~tllr~tion binding experiments with '2S~ he~ MAbs to
Mac-1 demor.~ s that resting neuL~hils express app,~ ly 12,000
30 LM2/1 binding sites/cell but no specific CBPM1/5 sites/cell. Tre~tme.nt with
fMLP and PMA inc,~s the overall site density of Mac-1 (as reported by
LM2/1) greater than tenfold as has been described (Berger, et al., J. Clin.
WO 94/08620 ~ 7 3 ~ Pcr/US93/09777
- 45 -
Invest. 74:1566-1571 (1984); Lo et al., J. Exp. Med. 169:1779-1793 (1989);
Miller, et al., J. Clin. Invest. 80 535-544 (1987); Todd, et al., J. Clin. Invest.
74: 1280-1290 (1984)), and i~ ces e~cpression of the CBRM1/5 neoepil~y~ on
10-30% of these molecules (13,000 57,000 sites/cell after stim~ tion)~
S (iii) Sequenti~l i.. ,.. ~o~r~~ ion conr~ s that CBRM1/5 recognizes a
structural subset of Mac-1 molecules on l~eull~hils. Lysates that are
precleared of the CBRM1/5 epitope contain residual Mac-1 ~nole~lles that are
immllnop~ iLa~d by LM2/1 but Iysates that are ~ clear.,d of Mac-1
cont~ining the LM2/1 epitope do not contain residual qll~ntities of CBRM1/5
10 precipitable material. (iv) F~-nctio~l studies reveal that despite binding to less
than 15 % of Mac-1 molecules on n~ L,ophils stim~ tpcl with fMLP,
CBRM1/5 (IgG or Fab fr~gmP,ntc) inhibits almost completely (>85%) the
Mac-1~e~,en~e~ hp~s;on to rll,~ Ggell and ICAM-1. Ad-litio~ ~.hllC.Il~
col.r--..P~ the bloc~-in~ aci~ of CBRM1/5, as it ~ ,gat~s ~lhPQion of
15 ICAM-I+ L cells to l,ulirled Mac-1.
While several MAbs have been cl~r~t~i~d that block Mac-1-
dependent adhesion to ICAM-1 and fibl;..o~f-n (Altieri, ct al., J. Biol. Ch~
265:12119-12122 (1990); Diamond, et al., J. Cell Biol. 111:3129-3139
(1990); Smith, etal., J. Clin. Invest. 83:2008-2017 (1989); Wright, etal.,
Proc. Natl. Acad. Sci. USA ~S:7734-7738 (1988)), CBRM1/5 is unique as it
g~ .S the subset of Mac-1 molerlllP~s that mP~ t~ps ligand jl,t~ ;ol-
CB~ maps to the I domain on Mac-l which a~ to contain the major
.~ cog~ilion site for ICAM-1 and fib. ;.~ (desc.ribe~ below). Not all MAbs,
however, which bind to subsets of Mac-1 mo!~ s IGCO~ .~ filnr.tiOn~l
epitopes. We have ~ ~ two addidonal MAbs (and-CD18, CBRM1/19;
and-CD1 lb, CBRM1/28) thatbind to a~Liv~led but not resdng neul.ol)hils that
do not block function (MS Diamond and TA Springer, unp~-blichYi data). The
finding of ~ A11Y distinct subsets of Mac-1 molecules may explain why
neutrophils do not show signifir~nt changcs in the redistribudon of surface
Mac-1 after activation with fMLP (Detmers, et al., J. Cell Biol. 105:1137-
1145 (1987); Smith et al., J. Clin. Invest. 65:804-812 (1980)).
Wo 94/08620 ~ , PCr/USs3/09777
- 46 -
Although activation of neutrophils and monocytes with chemotactic
factors, cytokines, nucleotides, or phorbol esters increases the avidity of
Mac-1 for several of its ligands (Altieri, et al., J. Cell. Biol. 107:1893-1900
(1988); Altieri et al., J. Biol. Chem. 263:7007-7015 (1988); Diamond, et al.,
J. CellBiol. 111:3129-3139(1990); Philips, et al., J. Clin. Invest. 82:495-501
(1988); Smith, et al., J. Clin. lnvest. 83:2008-2017 (1989); Wright et al. J.
Immunol. 136:1759-1764 (1986); Wright, et al., Proc. Natl. Acad. Sci. USA
85:7734-7738 (1988)), the molecular basis for this change has reln~ined
unclear. The 4u~r.~ tive in~ ase in Mac-l expression on neutrophils does
not correlate dh~lly with adhesive functions (Buyon, et al., J. Immunol.
140:3156-3160 (1988); Lo et al., J. E~cp. Med. 169: 1779-1793 (1989); Vedder
et al., J. Clin. Invest. 81:67~682 (1988)) but until now, no direct evidence
has been obtained for a structural change. Studies at 4C, 25C, and 37C
show that an in.;.~se in overall Mac-1 e"l,r~,~sion does not induce the
CBRM1/5 epitope on ~ .o~hils or lllonocy~s. Similarly, an h~c.~ in
Mac-1 surface ~ylession is not sufficient to plulllole Mac-1~eye~den~
adhesion to ICAM-1, rl~ ~f U, or other ligands (Buyon, et al., J. ~mrnunol.
140:3156-3160 (1988), Vedder et al., J. Clin. Invest. 81:67~682 (1988)),
and MS Diamond, unpublished observations). Adhesion to fibrinogen and
ICAM-1, and ~,~yr~,ssion of the CBRM1/5 epitope both require cell
stim~ tiQn. We s~ggest that activation y-u.-.~l~ a subset of Mac-1 m~leculF s
to acquire the functionally lelc~rcul~ CBRM1/5 epitope, and to become
co~peh~ slluclul~lly to bind to ICAM-1 and fibrinogen.
Several other MAbs against activation~ie~nllenl neoepitopes on
in~lill Sl~ye;~r~nily ~ ,llbGlS have been described recently. MAbs against the
platelet integrin gpIIb-IIIa have been reported to monitor SlluC;lu~ c-h~n~s
that are in~ ce~ either by cellular activation or ligand binding (Du et al., Cell
65:409 (1991)). Indeed, the initial evidence for a Slluelulal change in
integrins came from the development of the PAC-1 MAb that l~;coglli~es
gpIIb-IIIa on ADP, epinephrine, or thrombin-treated but not resting platelets
(O'Toole, et al., CeU Reg. 1:883-893 (1990); Shattil, et Rl., J. Biol. Chem.
260:11107-11114 (1985)). PAC-1 binds to a subset (20-50%) of gpllb-IIIa
W094/08620 ~ 7~ PCr/US93/09777
- 47 -
molecules after stimulation and inhibits fibrinogen-m~i~tecl platelet
aggregation (Shattil, et al., J. Biol. Chem. 260: 11107-11114 (1985)).
Furthermore, CHO cells that express gpIIb-IlIa are unable to bind soluble
fibrinogen or the PAC-1 MAb until a sllu~;lul~l change has occurred (O'Toole,
S etal., Cell Reg. 1:883-893 (1990)). Additional evidence for sllucluldl
changes has come from the ch~ri~t~ dlion of a class of MAbs that r~og~
ligand-in~ bill.ling sites (LIBS) on gplIb-IIIa (Frelinger et al., J. Biol.
C~em. 265:In press (1990)). The gcnc~ation of MAbs to multiple
conformations of gpIIb-IIIa ~ug~c,l~ that cell activation and ligand binding
10 may effect distinct ,L u~;luldl changes that f~'ilit~tP adhesion and ,,Ai~t~function (Frelinger et al., J. Biol. Chem. 266:1710~17111 (1991)). Rer~
PAC-1 and CBRM1/5 share similar bil-dil~g ~ ~.lies, we suggest that the
le-lkocyLt; h.t~~ s, like the platelet i~ , require similar SL~uCLu
changes to ~vr~lllote ~ihesio~
Several ~ilivaLio~ ~ndell~ ~;lo~s on l~lwcy~ int.o.grinc also have
been desc,il)cd. The MAb 7E3, t~hat was raised originally against gpIIb-IIIa
(Coller, J. Clin. Inuest. 76:101-108 (1985)), binds to all Mac-1 mol~llles on
ADP-stim~ ted but not resting mol~o~yLs or "~Ol~y~ cell lines (Altiere et
al., J. Imrnunol. 14I:2656-2660 (1988)). Unlike PAC-1 or CBRM1/5, it has
no reported functional effects. The NKI-L16 MAb l~cogl-i>~s the lc~yle
intcgli,l LFA-l on resting T Iylll~h~s only weakly (I~rson et al., Cell
Reg. 1:359-367 (1990); van Kooyk, et al., J. Cell Biol. 112:345-354 (1991)),
but binds to T Iylll~ho~ s ~at have been a.;livat~ with phorbol esters, IL-2,
or MAbs that cr~)sslink CD3. NKI-L16 ;. .J.,ces ho,-..,ly~,ic ~Ahesion of B cell25 lines and T cell clones and l~ ,S partially bil dh-g to purified ICAM-1 of
COS cells that have been ~ r~ with a wild type LFA-1 chain and a
defective CD18 ~B subunit (Hibbs et al., Science 251:1611-1613 (1991)). The
NKI-L16 epitope, ho..e~er, is not s~lrl~;e..l for cell binding since cloned
T cells express high levels con~ u~ ely but do not aggl~egale spontaneously
(van Kooyk, et al., J. Cell Biol. 112:345-354 (1991)). The MAb KIM 127
recognizes the common CD18 ,B subunit of the leukocyte integrins and
promotes LFA-1 and Mac-l dependent adhesive events (Robinson, et al., J.
WO 94/08620 - PCr/US93/09777
- 48 -
Immunol. 148:1080-1085 (1992)). Binding of this MAb may trigger a
confol",ational change that mimics activation, and inclt;ases avidity for ligand.
In comparison with these activation-de~ndelll MAbs against leukocyte
inLe~ s, CBRMl/5 ap~ unique as it binds to a subset of molecules on
stim~ t~A cells, blocks rather than pr~not~s adhesion, and has an epitope
whose pr~,sence correlates with, but does not induce, the highly avid form of
Mac-1 on "eul,~phils and mollocy~s.
Recent studies have begun to address the SLI~CLul~l events that effect
the increase in avidity. One model is that stim~ tion of cells promotes or
alters the divalent cation binding by an integrin, and this event triggers ~e
high avidity conl~.~l,a~ion (Altieri, J. Immunol. 147: 1891-1898 (1991);
Dr~n~fiel~l et al., J. CeUBiol. 116:219-226 (1992); Dr~ncfield et al., EMBO
J. 8:3759-3765 (1989)). ~ntr.~,-;n a chains contain a region of e,.~,lsive
homology to the divalent cation binding sites on Ca2+ regulatory ~ t~
(Argraves, etal., J. Biol. Chcm. 261:12922-12924 (1986); Corbi, etal.,
EMBO J. 6:4023 ~1028 (1987); Poncz, et al., J. Biol. Chem. 262:847~8482
(1987); Suzuki, et al., J. Biol. Chem. 262:1408~14085 (1987)), and
numerous studies demoll~ e a le(luil~.llel~t for divalent cations in integrin
function (Altieri, J. Immunol. 147:1891-1898 (1991); Bajt, etal., J. Biol.
Chem. 267:3789-3794 (1992); Dr~ncfiel~, et al., J. Cell Biol. 116:219-226
(1992); Elices et al., J. Cen Biol. 112:169-181 (1991); Gailit et al., J. Biol.
Chem. 263: 12927-12932 (1988); Hynes, R.O., Cell 48:549-554 (1987);
Kirchhofer etal., J. Biol. Chem. 266:4471-4477 (1991); Loftus, etal.,
Science 249:915-918 (1990); Martz, H~nan Immunol. 18:3-37 (1987);
Rothlein et al., J. E~p. Med. 163:1132-1149 (1986), Somlel~ ,, etal.,
Nature 336:487~89 (1988)). Several activation-dependent neoepilopes on
leukocyte integrins have been shown to require the presence of divalent
cations. Binding of ~he MAb 24 (Dransfield, et al., J. Cell Biol. 116:219-226
(1992); Dransfield et al., EMBO J. 8:3759-3765 (1989)), which recognizes a
common deterrninant on the a chain of the three leukocyte integrins, requires
either Mg2+ or Mn2+, but is abolished in the presence of Ca2+. In contrast,
expression of the NKI-L16 epitope requires Ca2+ as it is abolished in the
W0 94/08620 C~ 7 ~ ~ PCr/US93/09777
- 49 -
presence of EGTA or EDTA (van Kooyk, et al., J. CeU Biol. 112:345-354
(1991)). CBRM 1/5 is the first example of an activation-sl-ecific MAb against
an integrin a subunit whose expression requires divalent cations but is known
to bind to a region outside the divalent cation binding repeats. Mg2+ or Ca2+
S is sufficient and EDTA abolishes t-he e~l,r~s ,ion of the CBRM 1/5 epitope; onneutrophils, Mn2t h~cl~ses the CBRM1/5 epitope eAyl~;.sion in the absence
of stim~ tion, and thus may impoæ SIlU~;lU~ .on.c that mimic cell
activation (Altieri, J. Immunol. 147:1891-1898 (1991); Dr~ncfiel~l et al., J.
Cell Biol. 116:219-226 (1992)). Although these studies s~lgge~l that divalent
10 cations modulate integrin function, it remains unclear if they are per nissive
for ligand binding or whether divalent cations are involved actively in the
regulation of ~lhesion.
While leukocyte tr~ffl~lring is a ~lolec~ e l~,S~OllSe of the host defense
system against foreign ~ll.o~el)c or tissue repair, under some ci~ ,es
15 le~k~s can ~"r~l;ale cig,.if';r~ d~l~s of v~s~;ular and tissue injury.
Neutrophils have been implicated in the l~tl~el~F~;c of a l.. ~r of clinical
disorders inclu~ling adult r~ ilalc~l~r distress sylldlulnc~ iG~ reperfusion
injury following IllyOc~iâl infarction, v~c~lliti~, and shock (for review see
Carlos et al., I~ununol. Rev. 114:1-24 (1990)). Already, sevelal groups have
20 de.l.o~ .l~ that the ;~.n~..,...~c,l~ injury r..fAi7.lr~d by leul~ ~s can be
?llen..~t~d by antibodies against the l~.lk~yl~ e~l~i, etal.,
Surgery 108:20~212 (1990); Rosen, et al., J. Ejcp. Med. 169:535-548 (1989);
Simpson, et al., J. Clin. Invest. 81:624-629 (1988); Vedder, et al., J. Clin.
Invest. 81:939-944 (1988)). MAbs lil;e CBRM115, which I~cQg~ cli~led,
25 and not resting le.,koc~yles, are useful clinic~lly to target, Illonilor or deplete
the highly adhesive cells that effect iI~lls.n~ loIy injury.
WO 94/08620 ~ PCr/US93/09777
- 50 -
T~BLE 1
Site Densit~y of Mac-l on Neutrophils
S ~ i251-LM2tl ~ - ; '25I~BRMl/~
Sit 1~1~ .Site J eil ;~
Un~tim~ tf'd 11,300 i 1600 No sites detected
fMLP 138.700 i 21000 13,200 i 1400
PPMA 196.900 i 800 57,200 i 1300
The site densides were ~ from thc amount of MAb bound at ~
Data (i standard error of the mean) are the average of two ;~ c
p~.r~ in i "' - on a single donor.
EXAMPLE 2
15 IDENTIFICATION OF TEIE LIGAND BINDING DOMAINS OF MAC-1
Materials and Methods
MAbs
The following murine MAbs agcunst human antigens were used from
ascites: TS1122 (anti-CDlla, IgGlj (S~n~hP7-Madrid, et al., Proc. Natl.
Acad. Sci. USA 79:7489-7493 (1982)), LPM19c (anti-CDllb, IgG2a, a gift
of Dr. K. Pulford, Oxford, UK) (Uciechowski and Schmidt, "Cluster Report:
CDll," in Leucocyte Typing IV: W~ute CeU Differennation Antigens, Knapp
et al., eds., Oxford Uni~e~ y Press, Oxford. 543-551 (1989)), TMG-65 (anti-
CDllb, IgGl, a gift of Dr. I. Ando, Sæged, Hungary) (Uciechowski and
.Cchmi(it "Cluster Report: CDll," in Leucocyte Typing IV: White Cell
Di.~erentiation Antigens, Knapp et al., eds., Oxford U~ ,r~ity Press, Oxford.
543-551 (1989)), Mn41 (anti-CDllb, IgGl, a gift of Dr. J. Buyon, New
York) (Eddy, etal., Clin. Immunol. Imrnunopathol. 31:371-389 (1984)),
OKM10 (anti-CDllb, IgG2a, a gift of Dr. P. Rao, Ortho Pharm~r~ltic~
Raritan, Nn (Wright, etal., Proc. Natl. Acad. Sci. USA 80:5699-5703
(1983)), VIM12 (anti-CDl lb, IgGl) (Bernstein and Self, "Joint Report of the
Myeloid Section of the Second International Workshop on Human Leukocyte
Wo 94/08620 i~ 7 3 8 PCT/US93/09777
- 51 -
Differentiation Antigens," in Hurnan Myeloid and Hematopoietic Cells,
P~inherz et al., eds., Springer-Verlag, New York. 1-25 (1986)), 14B6E.2
(anti-CDl lb, IgM) and SA4.C5 (anti-CDl lb, IgGl), gifts of Dr. L. ~hm~n
A~el~ide, Al-ctr~ (Uci~how~i and SchmiAt, "Cluster Report: CDll," in
5 Leucocyte ~yping IV: W7u'te Cell Dif~erenna~ion Antigens, Knapp et al., eds.,
Oxford University Press, O~ford. 543-551 (1989)), CBRplS0/4Gl (anti-
CD11c, IgG2a) (Stacker et al., J. Immunol. 146:648-655 (1991)), 1~29 (anti-
CDllc, IgGl) (Ucie~how~l~i et al., "Cluster Report: CDll," in Leucocyte
Typing IV: W~u'te CeU DiJ/~ ic~ion Antigens, Knapp et al., eds., Oxford
10 Unive~ y Press, Oxford. 543-551 (1989)), BLY6 (anti-CDl lc, IgGl)
(Uciechow~ki et al., ~CIuster Report: CDl 1," in Le~-o~le Typing IV: W~u'te
Cell Differentiation Antigens, Knapp et al., eds., O~ford Uni~r~lly Press,
Oxford. 543-551 (1989)), TSl/18 (anti-CD18, IgGl) (S~n~h~z-Madrid et al.,
Proc. Natl. Acad. Sci. USA 79:7489-7493 (1982)). The following MAbs were
15 used æ pul;rled IgG: R15.7 (anti-CD18, a gift of Dr. R. ~ thlPin
Roch.;i~ger-Tng~lhP-im, Ri~fi~p~ n~ et al., J. Clin. InYest.
85:1497-1506 (1990)), OKMl (anti-CDllb, IgG2a) (VVright, etal., Proc.
Na~l. Acad. Sa. USA 80:5699-5703 (1983)), OKM9 (anti-CDl lb, lgGl, a gift
of Dr. P. Rao, Ortho, Pl.~ ls, Raritan, NJ) (Wright, et al., Proc.
20 Natl. Acad. Sci. U5A 80:5699-5703 (1983)), LM2tl (anti-CDllb, IgGl)
(Miller, etal., J. L,.".~.ol. 137:2891-2900 (1986)), SHCL3 (anti-CDllc,
IgG2b) (scliwalling~ et aL, Blood 65:974-983 (1985~). M1187 (anti-rolss,llan
~ntig~n IgM) (Springer, et Ql., Eur. J. Irnmunol. 8:539-551 (1978)) was used
as a culture ,-~ after II~ PnI at 60DC for lh. X63 (non-binding
25 MAb, IgGl) was used æ culture ~up~ L.
Generation of MAbs against human Mac-l (CBRMl series) was based
on a previously published ~r~JIocol (Stacker et al., J. Irrununol. 146:648-655
(1991)). In brief, female BALB/c mice were i..,.,,,.l-i,~ with purified Mac-l
- (2~g/im.. "l-;,,.tion)(D;~nond,et al.,J.CellBiol. 111:3129-3139(1990))on
30 day 31 (illlla~liloneally) and day 3 (inl,~c.il~neally and illLIa~ sly) priorto fusion with the non-secreting murine myeloma P3X63Ag8.653 (ATCC CRL
1580). Mac-l was prepared for the first immunization by combining 10 ~g of
WO 94/08620 ~ 8 PCI/US93/09777--
purified Mac-1 with trehalose dimycolate from Mycobacterium phlei,
monophosphoryl lipid A from Salmonella minnesota R595, PBS, 0.2 % Tween
80, and sq--~lene as described in the m~ r~c~ r~s illsll uc~ions (RIBI
T.. u~ f,.. ir~l Research, Hamilton MT). The second i.. n.~ ;on was
5 ~c.rul...ed in PBS. Mice were test bled on day 8 prior to fusion and the titerof the ~nti~ by ELISA against purified Mac-l was 1/5000. The ~r.,l(Jcol for
fusion and hybridoma m~ n~nce has been described (Sl,lingcr, et al., Eur.
J. Immunol. 9:301-306 (1979)).
Indirect i~ nl~ofluol~,~erl~. flow cytometry was used to identify
10 hybridomas that produced MAbs which bound to peripheral blood neutrophils
(Diamond, etal., J. CellBiol. 111:3129-3139(1990)). Ofthe500hybridoma
au~e~ e ;....i.l~l a~ .ly 20% c~nt~in~l MAbs that bound to
l~hils. These were tested ~ ly for their ability- to bind to
perirhP.Pl blocd lr~ ho~;~s, and Mac-l or pl50,95 l~ r~t~ COS or CHO
15 cells to ~ .e wLell,er the MAb ~cog";~d the a or ,B chain of Mac-l. 24
MAbs were s.ll~loned and detel",i"ed to be .~ ;c for the Mac-l ~Y subunit.
MAbs were isoly~ed using an ~ lU~Ul~ MAb IsolyL,ing Kit (Pierce,
Roc~r~,ld IL).
20 Generation of Mac-l/pl50.95 Chimeras
Por pl~lu~:lion of chi"lclic Mac-l and pl50,95 a chains,
oligonuclootide directed mutagenesis (E~unkel, Proc. Natl. Acad. Sci. USA
82:488-492 (1985); ~Icl~on et al., Nature 329:842-846 (1987)) was used to
introduce ,~ ;lion sites into homologous regions of the cDNA willlou
25 altering the coding ~enl4 of aM (Mac-1) (Corbi, et al., J. Biol. Chern.
263: 12403-12411 (1988)) and ax (p150,95) (Corbi, et al., EMBO J. 6:4023-
4028 (1987)) in pCDM8. The nucleotide scql~enr~ ~ulllbe~ing was according
to the published cDNA clones (Corbi, et al., J. Biol. Chem. 263:12403-12411
(1988); Corbi, et al., EMBO J. 6:4023-4028 (1987)). Changes were
30 introduced at the following nucleotide se~uell~s to facilitate r~ r~,cal
e~eh~nges: EcoRV (aM bp 520-525, GACATT is changed to GATATC; cyX bp
515-520, GACATT is changed to GATATC), Bglll (cyM bp 1064-1069,
W094/08620 ~ 7~ PCI/US93/09777
AGATCT is native; ~x bp 1059-1064, AGATCT is native), and AflII (~M bp
1908-1913, GCTCAG is changed to C~AAG; ax bp 190~1905, GCI'CAG
is changed to Cl'rAAG). These sites were scl~cle~1 to bracket the I domain
and divalent cation binding regions based on amino acid s~Ucl~r~ homology
5 I)aLkllls among the leukocyte intP.~rin family (Larson, et al.. J. Cell Biol.
108:703-712 (1989)). t~e~omie cloning of plS0,95 (Corbi, et al., J. Biol.
Chem. 26~:2782-2788 (1990)) j~lentifi~d the exon-intron boundaries that
(listing~ h these regions and conr~l"~ed that EcoRV and BglII bracket the I
domain: the EcoRV site is located 23 mlcleotides into the first e~on (exon 7)
10 of the I domain and the BglII site is 15 nucleotides before the end of the last
exon (exon 10) of the I ~ n The BglII and AflII ~i,Lri~;lion sites define a
region that c4r.l7,i..c e~cons 11 to 13 and the three divalent cation binding
repeats (exons 14 to 16): for simplicity this will be l~,fcl~ to as the divalentcation binding region: the Bgl II site is 10 r~ kol;~es before the end of e~con
15 10 and the AflII site is l).cci~ly at the end of the last e~con (e~on 16J of the
divalent cation bil~dill~ repeats (Corbi, et al., J. Biol. Chem. 2~i5:2782-2788
(1990)). Mac-1 a ~ul,uni~ that cont~in~ silent I~ Al;Ouc were intact
~nti~ni-~ily and fim~ti~n~lly as they bound iC3b-E and applop.iale a chain
srecific MAbs after ~ ~r~i~ion into COS cells (data not shown).
For all ctl;-l.. "~, M l~ 3CJ1kj a region of the Mac-1 a svb-mit, X
r~l~,~n~ a region of ~e plS0,95 cr subunit, and e, b, and a refer to the
l~ s~ ion sites EcoRV, BglII, and AflII. M-e-X and X-e-M were COn~lluCt~,d
by lig~tin,~ the ~coRV/Not I r~ of pCDM8-aM (4.7 kb) or pCDM8-~YX
(4.1 kb) wi~ ~e NotI/EcoRV f.i.~...e.l.~s (3.5 or 4.7 kb) of pCDM8-aXor
25 pCDM8-~M. M-b-X and X-b-M, or M-a-X and X-a-M were cor~l.uc~d
similarly using the BglIllNotI or the AflII/NotI r.,.~,..P ~ of dle co~ ,or ~
pl~mi~i~, M-~X-a-M and X-b-M-a-X were con,l-u.:led by li~tin~ ~e 0.8 kb
BglII/AflII of ~M or ax wi~ the AflII/BglII r.~y...f~ of pCDM8-crX (6.7 kb)
or pCDM8-~M (7.2 kb). M~-X-b-M was Collsll ucted by li~tin~ a SalIlEcoRV
(4.5 kb) r.~ ." of pCDM8-~M with a BglII/SalI r~ig~.~P.~t (3.0 kb) of
pCDM8-cYM and a EcoRV/BglII (0.5 kb) fr~gm~.nt of aX. X-e-M-b-X was
constructed by lig~ting a EcoRV/Bglll fragment (0.5 kb) of cyM with the
Wo94/08620 ~ 7~8 Pcr/US93/09777--
- 54 -
BglII/EcoRV fragment (7.0 kb~ of pCDM8-~X. The con:,L.ucLion of each
chimera was confirmed by restriction digest analysis, hybridization with
applul"ia~e segmPntc of cDNA, and by seql-enrin~ through the junctions
(~ani~tiS, et al., Molecular Cloning: A Laboratory Manual, Cold Spring
S Harbor Labol~dlol~, Cold Spring Harbor, NY (1982)).
Tissue Culture. T,il,.s~lion. and Cell P~ ar~Lion
Neutrophils were isolated from whole blood of healthy volunt~ers by
dextran sP~ Fn~ on~ Fjcoll gradient centrifugation, and hy~lonic Iysis as
described (F.ngli~h et al., J. Imml~nol. Meth.ods 5:249 (1974); Miller, et al.,
J. Clin. Invest. 80:535-544 (1987)). Prior to ~A~c.;...P- ll;.l;on, cells were
stored at room ~ .e in HBSS, 10 mM H~.~ S pH 7.3, 1 mM MgCI2
in polyl,lo~ylene tubes (Falcon 2097, Becton I:~icLllsoll, T.inr41n Park, NJ).
COS cells were grown on 15-cm tissue culnlre treated plates in RPM~
1640, 10% FCS aRH Rjosr;en~s~ ~ ~.nP~, KS), 2 mM ~hJI;~r.. ;.~r-~ and 50
~Lg/ml gc~ ;.., and then co ,.;~,.cr~c~ wi~ CsCl purified t~!M, ~yX or
çhirl.e~ ;c aM~Xand the common ,B (Ki.chimoto, et al., Cell 48:681-690. (1987))
cDNA in pCDM8 by the DEAE-De~tran method as desc,ibed (Staunton,
et al., Cell 61:243-254 ~1990)). Surface e~pression was monitored three days
after l ~.cr~l;Qn by flow ~ellr as ~s~,il~cd (Di~mon-l, et al., J. Cell
Biol. 111:3129-3139 (1990)).
CHO DG 44 cells which have a deletion of the DHFR gene were
obtained from Dr. L.Chasin (Columbia Uni~ ily, New York NY) and
m~int~in~ as deseli~ (Qu, etal., J. Exp. Med. 167:1195-1210 (1988)).
Cells were col ~r~r~ by e~ opolal~ at 380 V and 960 ~F (Chan ~t al.,
Mol. Biol. Cell 3:157-166 (1992)) with 25 ~g of wild type or clli.nc.ic a in
pCDM8, 25 ~g of wild type ~ in pCDM8, and 5 ~g of pDCHIP vector that
co~ l~;n. a CHO DHFR mini~n~, Cells were initially s~ t~cl in ~-MEM,
10 % dialyzed FCS aRH Biosr,ienr~s, T erlloY~ KS) that lacked hypo~ inç
but was supple .~e- ~l~ with 16 f~M thymidine, 2 mM glu~;t.. i.,~, and 50 ~g/ml
gent~mir,in After ten days, selection was augme.-~ed by the addition of
metho~ ate (0.02 ~LM). After two weeks of growth in media cont~ining
WO94/08620 ~1 4~738 PCI/US93/09777
metholleAate, cells were det~rhçd with HBSS, S mM EDTA and in~ub~t~
with MAbs against either Mac-1 or plS0,95 (~epen-ling on the chimera) at 4C
for 30 minlltes. Cells were washed thrice in a-MEM, 2.5 9 i dialyzed FCS, and
panned for five minutes at room ~.,~Iu.e on 10 cm non-tissue culture
S treated plates (Lab-Tek 4026, Nunc, Inc., N~.v-ille, IL) that were coated
with 50 ~g/ml rabbit anti-mouse IgG (Zymed Laboratories, San rldnciseo,
CA) and blocked with 10% FCS. Non-adherent cells were removed by eight
changes of media, and adherent cells were ,c~.~,r~d after tll~ Prlt with
trypsin-EDTA. Cells were cultured and the p~nnin~ I,r~,cedu,ci was repeated
10 several days later. High eA~res;,ing cells were gelle.~Lted by h~w~sing the
con~ lion of methotreAate (0.02-0.20 ~M) in culture. As controls, CHO
cells expressing a domain deleted form of ICAM-1 (Domain 3 deleted, F185)
(St~nnt n, D.E., etal., Cen 56:849-853 (1989)) were ge~ ~ by
c4~ r~cl;on of F185 cDNA in pCDM8 with pDCHIP followed by seleclion
15 and i...l.n.n~nnil,~ as described. For all !.~.,c~ lc, ~silive surface
eA~l.,s;,ion was ~-o~,ilurcd by in~n~ ol~n~ flow c~ ehy.
The ICAM-1+ L cell stables were ,,.~i..l~i,~ on 15 cm tissue culture
plates in DME, 10% heat il~livdt~d FCS (Low endot~sin serum (defined),
Hyclone Labo~ olies, Logan, UT), 2 mM gl~l;....inP., and 50 ~g/ml
gent~micin as previously le"~ ib~l (D;~m- lul, et al., J. CeU Biol. 111:3129-
3139 (1990)). Prior tO ~flh~$;~n assays, cells were ~ ,ed from tissue cultune
plates with trypsin-EDTA, and washed twice in PBS, 2 mM MgCI2, 0.5 %
heat-treated BSA (D;~nol~d, et al., ~. Cell Biol. 111:3129-3139 (1990)).
25 Surface Labelling. T,n~ ol~l~ipi~lion. and Gel Electrophoresis
T~ r~-l~l COS or CHO cells (1-2 X 106 cells) were ~et~h~ with
HBSS, S mM EDTA, washed four times with PBS, and l~,s.ls~nded in 2 mls.
The cells were io lin~t~d, Iysed, and ~leclear~d as deselibed (Diamond, et al.,
Cell 65:961-971 (1991)). Tm~n~moplceipil~lions were l,~rolmed as follows:
Tmmnn.o complexes were formed by i-~-ul~ling MAb (250 ~l volume of neat
supen~ , 1/100 dilution of ascites, 25 ~g/ml purified MAb) with purified
rabbit anti-mouse antibody (5 ~l of 1 mg/ml, Zymed Laboratories, San
wo g4/08620 ~ 4 7 ~ ~ Pcr/US93/09777 ~
- 56 -
Francisco, CA) for 4 hours at 4C. Protein-A Sepharose (Pharmacia KLB,
Piscataway, NJ) (25 ~l of a 1:1 slurry) was added for overnight incubation.
Eppendorf tubes were centrifuged, supellldldn~s were aspirated, and i~lin~t~d
Iysate (50 f~l) was added and iu(~ul,A~ed for 2 hours at 4C while sh~king
5 vigolc,usly. The WdSllillg and elution of the i~....~....o~ ild~s have been
cle.~. ;Ixd (Diamond, et al., CeU 65:961-971 (1991)). Samples were loaded,
and subjected to SDS-PAGE (T ~.rnmli, Nature 227:680-685 (1970)) in the
presence of 5 % ~-lllel~toeth~nol, and autoradiogr~rh~d (Laskey et al. FEBS
Letters 82:314-316 (1977)).
Purified rl- ~ch~s
Mac-l was pulified from leukocyte Iysates after d~ ,en~ solubilization
by.LM2/1-Se~har~se i~ n...~ffi~ yclllulllalogl~hy asdes.,l;be~l (Di~mon~,
etal., J. Cell Biol. 111:3129-3139 (1990)). ICAM-1 was ~ulirled from
15 dc~.~ll~ Iysates of the ery~rcl ~ l~k~omie cell line K562 by RRl/l-Sephar~se
i.. u~Affinity clllul.. Ato~ .hy as deselil~cd (Marlin et al., Cell 51:813-819
(1987)). Fibrinogen was o~h~ed from Sigma Chlo.mi~l CGI~.P;~ (St. Louis,
MO)
iC3b-E ~ )aldlion and Rimlin~ Assay
IgM and iC3b coated elylllluc~ ,s (IgM-E, iC3b-E) were ~l~p~ed as
described p~iuu~ly (RothlPin etal., J. Irnmunol. .135:2668-2672 (1985)) with
the following moAifir~tiQns~ In some eA~ llelll~ sheep e. ~ll-roc~s
(Colorado Serum Company, Denver, CO) were washed once in PBS, and
l~he.lled for 2 h at 37C with a 0.22 ~m filtered solution of ~l l C (0.8 mg/ml
in PBS after being di~s~)lv~d in 30 ~I DMSO) E~ylhrucyt s were washed
thrice in HBSS, 10 mM Hl~S pH 7.3, 2 mM MgCI2 l~s ~ ~l (6 ~ 108
cells in 10 mls) and i~.cul~tucl with a 1/128 dilution of heat inaclivated anti-For~ .an IgM MAb (M1/87) for 60 ~ ,s at room lC~ Clalulc; while
ch~king gently. IgM coated el)~llll~yl~s (IgM-E) were washed thrice,
res--cpen-led (1 ml) in HBSS, 10 mM HEPES, 1 mM MgCI2. 1 mM CaC12
supplemented with C5 deficient human serum (100 ~1) (Sigma Chemical Co,
Wo ~4/08620 ~ 7 3 ~ Pcr/US93/09777
- 57 -
St. Louis, MO) and incub~tçd for 60 minutes at 37C while rotating. iC3b
coated erythrocytes (iC3b-E) and IgM-E were washed thrice and resuspended
(4 x 107 cellslml) in HBSS, 1 mM MgCI2, lmM CaC12, 10 mM HEPES pH
7.3.
Neutrophils and transfected CHO cells were assayed for binding to
iC3b-E. Peripheral blood n~ l~hils (1.25 ~ 105 cells/well in 50 ~1) were
seeded onto 96-well tissue culture treated plates (Corning Glass Works,
Corning, NY) in HBSS, 10 mM Hl~S pH 7.3, 1 mM MgCI2, lmM CaC12
for 20 min~ltes at 37C in a 5% CO2 inrub~thl~ non-adherent cells were
removed after two washes with the same buffer. Adherent ne.lll~hils were
plr;~ b,.l~ with MAb (~A~iition of 50 ~1 of diluted ascites or purified MAb
in PBS, 1 mM MgCI2, 0.5% BSA) for 20 "~;".Jt~5; each c~ndilion was
,~,e.rolllled in quintuplicate. S~ J - ~lly~ iC3~E or IgM-E (2 ~t 106 in 50 ~
and PMA (65 ng/ml final c~-.c4.~ ;0n) were added; the e.~lllr~c~s were
ce~ rug~ (300 RPM, 5 .. i.. ~s, 4C) onto ~e ne~ )hils and ~e plates
were in~ t~l in a 37C water bath for 15 ,~ ~s. Non-adhc.elll
ely~ ~y~s were le.llo._d by llirL ;n~ the plates si~c times after addition of
PBS, 1 mM MgCI2, 0.5% BSA (175 ~l/well). Bound cells were .~u~..l;li.~ in
the 96 well plates using a Pandex nuO~ c4n~ alion analyær (Ba~cter
20 ~lthc~re Corp., M~ndelein, IL). The ~.c~"~ge of inhibition was
nonn~liæd as follows:
X Inhibition - 100 X (X iC3b-E bindinD Yithout ~b - X 1C3b-E bindin~ l~ith ~Ab)
(X ~C3b-E binding l.lithout H~b)
T.i...`l;~l~ CHO cells (2 x 105 cells/ml in 250 ~1) were seeded onto ~well
tissue culture treated plates in L15, 10 mM HEPES pH 7.3, 5% heat-
indeliv~Led FCS and adhered for greater than three hours at 37C.
Suhsequently, iC3b-E or IgM-E (2 x 108/ml, 50 ~1) and PMA (100 ng/ml final
30 co~ .,.l;on) were added and cells were bound for 60 ...i~.--t~,s at 37C. Non-
adherent elylhrwyles were removed after si~ to eight washes and r~Jscl~s
(> 10 erythrocyteslCHO cell, > 100 cells P~min~) were scored by light
microscopy at 200X magnification.
Wo94/08620 ~ 7~`~ Pcr/uss3/o9777--
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Neutrophil Homotvpic Adhesion Assays
Previously, two methods have been utilized to q-~ntit~tP the effect of
MAbs on neutrophil aggregation: micfoscopic determination of the number of
free neutrophils (those not found in cell conjugates) (Diamond, e~ al.,
5 "Di~l~nlial Effects on Leul¢~cyle Functions of CDlla, CDllb, and CD18
Monoclon~l Antibodies," in Lt~hyle Typing IV, Knapp et al., eds., Oxford
University, London, 570-574 (1989); Patarroyo, et al., Scand. J. I)rununol.
22: 171-182 (1985)) or aggrego--lclly, which measul~;s the relative incl~ase in
light tr~n~mic~ion that occurs when cells go from a single cell dispersion to
cell clusters (Anderson, et al., J. Immunol. 137: 15-27 (1986); Buyon, et al.,
J.Immunol. 140.3156-3160(1988);Hammerschmi~t et al.,Blood~5:898-902
(1980)). While both mpthods are reproducible, the former is labor-il~ler,si~."
and the latter only indirectly ",easu,~s the ~ her and size of cell agg~gat~s.
We devised an alterr ate mPth- d which uses a Coulter COull~l~ it is ;~nlo~ r~,
15 r~lodueible and scores ~ rh~n~ lly the total number of cell particles in a
fixed volume. Since the Coulter counler treats cell agg"~ga~s, regardless of
size, as single particles, small numbers of two- or three-cell agg~gat~s
tlr~m~ti~lly will affect the particle count. Thus, this assay is sensili~ in
distinguishing MAbs which block weakly from those which block sl~o"gly.
20 Neutrophils (2 x lo? cells/ml) were ~sua~nded in HBSS, 10 mM HEPES, 1
mM MgCI2, 1 mM CaC12, 0.5% HSA (HHMCH). Aliquots (0.325 ml) were
pr~ir..-ub~t.,d with MAb (1/200 dilution of ascites, 20 ~g/ml purified MAb) for
25 minntes at room ~ t;. These cells (0.3 ml) were then added to a
pre-wet polypropylene eppendorf tube (1.5 ml) that cont~ined 0.1 ml of
25 HHMCH. PMA was added (100 ng/ml final COl~ tion) and the tubes were
rocked on their sides gently for 15 minlltes at 37C on a Bellco Rocker
Platform (Bellco, Vinel~nd, NJ). Subsequently, an aliquot (0.2 ml) was
~n~re~ d to a conical tube (3 ml, Sarstedt, Gel,~ ) that contained HBSS,
10 mM HEPES, 2% paraformaldehyde (0.1 ml). The number of particles
30 (single cells or cell aggregates) in a fixed volume was determined with a
Model S-Plus Coulter counter (Coulter, Hialeah, FL). The average of two
readings was used as an individual datapoint. Each condition was repeated at
7~8
WO 94/08620 PCr/US93rO9777
59
least three times with different donors. The percent aggregation was
determined accor.ling to the following equation:
X A,.~ tion = 100 X C1 - ( # of D~rtic~es in ~ s~nPle ~timul~ted ~ith PtU)
~# of pr~rticles in ulstin~lnted sanple)}
The percent inhibition was determined accol iing, to the following equation:
0 % inhibition ~ 100 X ~X ~ ~. Lion P~U s~nple - % a . ~ tion t~Ab ~le I P~)
tX ~ssre~ation PM.~ ~a~le)
Neutrophil aggregometry was performed as described
(Hanlllle.~llmidt et al., Blood 5~:898-902 (1980)). Briefly, purified
neutrophils were washed twice and r.~ leA. (10' cells/ml) in HHMCH
(wiLlloul phenol red). Aliquotc of cells (0.5 ml) were p~ ,Jb~t~ ll with MAbs
for at least 25 .~in~lt~s at room IG~ UIe. Neutrophils (0.4 ml) were stirred
(700 RPM) at 37C in a sili~4~ pA cuvette in a SL~d~d platelet aggl~,go"~eLcr
(Model DP-247F, Siencio, Morrison, CO). A b~ce-!inP- was recorded for three
minl~teS at which time PMA (100 ng/ml) was added and the change in light
tr~ncmiccion was Illo,li~r.,d on a strip chart lecor~er.
Adhesion of Neutrophils to Purified Fibrinogen
Purified rlblinogen was l~s~n~leA. in PBS (2.0 mglml) and spotted
(25 ~1) onto 6 cm bacL~"ial Petri dishes (Falcon 1007, Becton DieLinson,
Licoln Park, NJ) for 90 ...;...,t~,s at room ~ cl~Lu,G. Plates were blocked
with the deter~nl Tween 20 as described (Diamond, et al., Cell ~5:961-~71
(1991)). Neutrophils (4 x 106 cells in 1 ml) were ~ ~n-lPA in HBSS, 10
mM HEPES pH 7.3, 1 mM MgCI2 and ~ ub~le~A~ with MAbs for 25
minU~eS at room t~ ...pe.~ G. Sll~seque-ntly~ cells were added to the dishes in
the ~ ce or ~hse~ e of fMLP (10-7 M, final volume of 3 ml), and allowed
to adhere for three mimltes and forty-five seconds. Non-adherent cells were
removed by twelve washes with a Pasteur pipette after gentle swirling with the
35 same buffer supplelllenLed with 0.5 % BSA. Binding was ql-~ntit~t~i by scoring
the number of adheren~ cells in at least four different fields using an ocular
W0 94/08620 æ ~ 7 3 ~3 Pcr/US93/09777
- 60 -
grid at 100X magnification. The percent inhibition by MAb was determined
upon comparison with the media control.
Binding of ICAM-l+ L Cells to Purified Mac-1
The binding of ~CAM-1+ L cells to Mac-l is a mo~lific;tion of
previously described ylo~ocol (Di~mon-l, et al., J. Cell Biol. 111:3129-3139
(1990)). Briefly, purified Mac-1 was llihlted~ adsoll,ed (30 ~Ll) to 6 cm Petri
dishes and blocked with 0.5 % heat-treated BSA as describe~ (Di~mon~i, et al.,
J. Cell Biol. 111:3129-3139 (1990)). Thirty mimltps prior to the binding assay,
the plates were ~ b~t~ at room ~.llpc.alule with MAb (1/200 dilution
of ascites or 20 ~g/ml of purified MAb) in PBS, 2 mM MgCI2, 0.5% heat-
treated BSA (PMBSA, 2 mls). ICAM-1~ L cells were removed from tissue
culture plates with trypsin-EDTA (GIBCO, Long Island, NY), washed twice
and l~ r~ ed in PMBSA (0.5-1.0 ~ 106 cells/ml). Some ~ uotc were
15 preir.cul~t.~ for 25 it"s at room ~ ,"~c with control or anti-ICAM-1
MAbs. The l~ dillg assay was pc.rull.,ccl as de~,l;l~ccl (~irlrlem~ e~ al., J.
Irnmunol. 144:2295-2303 (1990)). The ~.~cnl inhibition by MAb was
ieLr~ ..PA upon co...l)~r;~ll wi~ the media control.
20 Binding of CHO Cell Chilllel~s to P~lirled ICAM-1
The billdil-~, of Ir~ r~t~ CHO cells to ICAM-l is a n~lifir~tion of
a previously described p~ ~locol for COS cell ll~lsr~l~ (Di~monri et al.,
J. CellBiol. 111:3129-3139(1990)). Briefly, purified ICAM-l wasdilutedand
adsorbed (25 Jul) to 6 cm Petri dishes. After a 90 minute i...-~.b~l;on, non-
2~ s~ecirc bh~ g sites were blocked with 0.5% heat-treated BSA. CHO cell
~ n~fect~ , after removal from tissue culture plates with HBSS, 10 mM
HEPES pH 7.3, 5 mM EDTA, were washed twice and ~ u*)l.lded (8 ~c 105
cells/ml) in HBSS, 10 mM HEPES pH 7.3, 1 mM MgCl2, 0.5% heat treated
BSA, and bound to ICAM-l coated Petri dishes for 90 minutes at room
30 ~ llpel~lul~. Non-adherent cells were l~.llov~;d after five washes with a
Pasteur pipette and the number of adherent cells was determined by light
mic,oscopy at 100X using an ocular grid.
WO 94/08620 ~ 1 4 ~ 7 ~ 8 PCrlUS93109777
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RESULTS
Generation and Expression of Mac-l/plSO.9S Chimeras
Naturally Occ;~ c;,L,iclion sites and sites illlrudu~d by silent
S mutations were used to divide the Mac-1 and plSO,9S a s~, "iLS into four
s~ c~ lly distinct regions (Figure 9). COS cells ~l~nC;el~lly Lldnsrectc;l with
the chimeric a ~ bullil~ and the common CD18 ~B subunit were subjected to
jm ~ of lllolescenc~ flow cytometry and ~ ..o~rci~ ioll with a panel of
previously cha~ i~d MAbs to the a sul,u"î~ of Mac-l and pl50,95. Most
10 of the chimeras were e~ ,ssed on the cell surface and were stained with a
unique pattern of MAbs to Mac-l and plSO,9S (Table 2). The data obtained
by imm~-nofl~lol~ r~ was co..l~ ~l by i~ pr~,~ lion and showed
that most of the rl~irl~ ;c cr s~ c~~ stably with the ~B subunit
(Figure 10, and data not shown). Unrolluna~ly, not all of the c~ Pr; c were
15 e~ scd in COS cells: two (X-b-M and X-b-M-a-X) were S,~ llF,;.;,f.d in a
form that did not ~Ccoriqte with the ,B subunit and were r.,~ined in the cell
~data not shown), and a third ~X-e-M) which showed IllOdcla~e cell surface
expression had an a~nol-"al pattern of il--....J~r~cipi~ion, as MAbs to the
ar subunit p~ ed only the ~ chain, and MAbs to the ,~ subunit
precipitated neither the a nor ,~ chains (data not shown).
Since we were unable to ~l,r~ . a subset of cllimePC in COS cells, we
auGnlp~ed to express the molecules in stable cell lines to map definili~_ly the
sllucLu~l epitopes of MAbs and ~.r~"", fimrtion~l analyses. Chimeric a and
wild type ,B s~uniL~. were co~An.~rected with the pDCHIP plasmid into CHO
DG 44 cells (Qu, etal., J. Exp. Med. 167:1195-1210 (1988)) that lack
fimctic!n~l dih~d~Çolate ~-Ic1;r~e. After drug selection, ~sili~rrc cells were
identifi~, IGcc~e~Gd by iln,....,-o~,...ni.,g (Aruffo etal., Proc. Natl. Acad. Sci.
USA 84:8573-8577 (1987)), cultured, and l~p~ d until CHO cell lines that
e,~lr_ ,sed high levels of individual chimeric molecules were obtained. Flow
30 cytometric analyses del"on~ ~ that all of the chimeric molecules were well
expressed on the surface of CHO cells (Figure 11) and for each chimera levels
of the ~ and ~ chains were equivalent (Figure 11). In one case (M-b-X-a-M),
WO 94/08620 2 1 4 4 7 3 ~ Pcr/US93/09777
- 62 -
the level of ,~ subunit on the surface e~c~ l the level of ~ subunit. Despite
expressing all of molecules on the cell surface, i~ u~oprecipitation studies
revealed an i",l.r~pcr ~c~ci~tion be~ ~n the a and ,~ subu~ in three of the
chimeras. ~.. ~-opl~,cillitations of surface l~hell~ X-e-M, X-b-M, and X-b-
S M-a-X Cl~illlf,l~ C with the ~B subunit MAb (TS1/18) were either weak or absent,
(Figure 12, Groups B,E, and J; Lane 3). These three chi."elas appalt;rlly lost
the TSl/18 epitope on the ~ subunit after de~l~,cl l solubilization. r~ iously
we have shown that TSl/18 immnn~pleci~i~t~s LFA-l, Mac-l, and plS0,95
from ~ complexes but not from the lln~coci~rA ~ subunit (Springer, et al.,
J. Exp. Med. 160:1901-1918 (1984)). Despite the change in antigenic
~,aclivily, the ~ subunit was still ~ccoci~sf d with the a subunit to some degree
in these ~hi....,".c as it was copl~cipilaled by the a subunit spe~ific MAbs
(Figure 12, Groups B,E, and J; Lane 2). The we~kness of the ~ ;on
~cl~ ~n s~unils also wæ s ~c,Jh4 by the change in the ~ichio~"ctl ~ of the
1~ ~Icci~i~ co~ T.... nop~ iol~ of X~-M, X-~M, and X-b-M-a-X
with MAbs to the a subunit differed from the other Cl C æ there wæ a
~ig,~.r.~ l" greater ~lualllily of cr subunit l~laLi~,~, to the ,~ subunit.
Epitope Mappin~ of MAbs to Mac-l
ne~ ~ an illlpl~cr ~csoci~l;oll of ~ and ~ subuni~. in a subset of
ch;- Cl~ could affect r .".~ "~l assays and our SubSC4uenl analyses, we used
MAbs to ~ f .,~te a sL-uclul~-func~ion map of the ligand binding dol"ains on
Mac-1. MAb epilo~s were loc~li7PA on the Mac-l a subunit by their ability
to bind to a particular subset of che,~ s and the results coJI~la~d with the
ability of the MAb to block distinct adhesive functions of the native Mac-1
mol~ . To s-,~lelllclll the ten MAbs that had been ~le~iously cll~r~ctc~
24 new MAbs against the ~ chain of Mac-1 were ge~ 1r,d (Figure 13) by
im...~ ion with purified Mac-1 and scl~~ g by im ---- ~onuo~ell~ flow
cytometry on neuLl~Jphils~ Iymphocytes, and l,~r,srecLed cells.
To map the slluclul~l epitopes of the 34 MAbs against the Mac-l
subunit, the CHO cells e~ ssing wild type and chimeric Mac-1 and plS0,95
were tested for their differential ability to bind to MAbs (Figure 13). Of the
WO 94/08620 ~ I ~ 4 7 ~ ~ PC~/llS93/09777
- 63 -
34 MAbs ~ P~, 19 1oc~li7l~d to the I domain, 11 to the C-~l,l,hlal region,
and one (CBRMI/20) to the region cont~inin~ the divalent cation binding
repeats. Two MAbs (CBRMI/32 and OKMlOo,d locali_ed to a discontinuous
epitope that ~ uhed the presence of both the N-Le~ al and divalent cation
S binding regions. The OKM10",~" that was ~ cd from Ortho
p~ ic~ls is distinct from the OKMlOo~d. The new OKMI0 does not
block iC3b-E b.h~ding to Mac-1 as was de,s,.ibe~l above and as we have
verified for OKMlOo,~" and the two MAbs do not 1~1i7~ to the same region
of Mac-1 (Figure 13)).We were unable to loc~li7~ the sllùelu~l epitope of two
MAbs (OKM10",~, and CBRM1128). Three MAbs against the plS0,95 cY
subunit also were m~pe~i with the CHO cell chime~: CBRplS0/4Gl mapped
to the C t~....;.L~l region, Bl,Y6 loc~li7~ to the I domain, and SHCL3 mapped
to sites both in the N ~ l and divalent cation l)indillg regions.
Inhibitory Activit,v of MAbs
iC3b-E Binding to Mac-l
l~e abili~ to inhibit distinct Mac-1~epe-n~lent adhesive ~u,.clions vas
investi~tf~l with the ~ f~ panel of MAbs. To test which MAbs block
iC3b bit~ -g to Mac-l, a ~ e assay was developed in which FITC-
lf,d iC3b e.y~ ~s were ~ t ~ with adherent n~ ophils in dle
presence of MAbs to Mac-l (Figure 14). MAbs to ~e I domain inhibited
iC3b-E binding to l~f .ll,~hils wi~ a mean of 72.6% ~ 24.9, wl.e.~as MAbs
to the C t~ al blocked with a mean of 33.6% ~ 16.6. Eleven of ninf t~n
MAbs that ",a~l,ed to the I ~nm~in blocked ~llul~gly (>85%), wher~s no
MAb that ~ i7~1 to the C ~,.--;n~l region showed greater than 60%
inhibition. The one MAb (CBRMl/20) which ."a~lJed dil~liy to ~e divalent
cation binding region had little inhibitory effect. The two MAbs (OKMlOo~
and CBRMI/32) that mapped to sites in both the N t~l",inal and divalent
cation regions blocked 81% and 65 % of dle binding (Figure 14). These results
agree with previous reports that used a limited number of these MAbs to
inhibit iC3b-Mac-1 interactions (Anderson, etal., J. In~nunol. 137:15-27
WO 94/08620 ~ 7 3 ~ PCI~US93/09777--
- 64 -
(1986); Diamond, et al., "Differential Effects on Leukocyte Functions of
CDlla, CDllb, and CD18 Monoclonal Antibodies," inLeuk~yte TypinglV,
Knapp et al., eds., Oxford University, London, 57~574 (1989); Mosser,
et al., J. Cell Biol. 116:511-520 (1992)).
Neutrophil Homotypic Adhesion
Several groups have rltomo~ ed that ~CIivat~ neullol)hils bind
homotypically, and that this agglt;~,à~ion response is blocked by MAbs to the
~ or ,B chains of Mac-l (Anderson, etal., J. Imm~nol. 137:15-27 (1986);
Buyon, et al., J. Irnmunol. 140.315~3160 (1988); Pa~ uyO, et al., Scand.
J. Immu~wl. 22:171-182 (1985)). The ligand on neutrophils for Mac-l that
m~Ai~t~s hol.lo~yl,ic agg-~,~,alion l.,,l~lls .~ t~ li~d. To de~.lllil,c the
regions of Mac-l that were illl~l~nL for neu~l~hil h~l--o~yl~ic adhesion, we
tested the panel of MAbs for their ability to inhibit this i~ cl;on in a newly
d~elolxd Coulter coull~cr assay (see Meth~ls). This assay which counts the
number of p~r~irles is more ~I. ;n,~e.~l than the cl~CQ;~l a~gl~ol~try æsay;
MAbs that inhibit in this assay by 20-30% such as CBRMl/2 and Mn41
(Figure 14) show 56 and 67% inhibition in the light s~, lh~ g aggl~gollle~ly
assay (Figure 15). MAbs to the I ~ m~in blocked the ag~l~ation assays with
a mean of 25.9% i 16.1, ~llel~ds MAbs to the C ~llli"al region blocked
with a mean of 1.8% :t 1.1. Two MAbs (LPM19c and CBRMl/29) to the I
domain blocked lleu hil holllo~y~:c ~Ah~Qio~ s~,ongly by 81% and 51%.
The CBRM1/32 MAb to the N ~llllil~al and divalent cation binding regions
blocked 21 %, and the CBRMl/20 MAb to the divalent cation binding region
inhibited 7%.
Neutrophil Adhesion to Purified Fibrinogen
When ~euL,~hils are stim~ t~ with f~ILP (1~7M) they bind to
fibrinogen in an almost exclllsi~,ly Mac-l~e~ e~l lllanl~er. MAbs to the I
domain of Mac-l inhibited binding to fibrinogen sllùngly (Pigure 14) with an
average 77.6% + 13.2, whereas MAbs to the C-terminal region inhibited
binding weakly with an average of 5.4% + 22.1. One MAb (CBRMl/32)
WO 94/08620 ~ 1 4 4 7 3 8 PCr/US93/09777
- 65 -
which mapped outside the I domain to sites in both the N-terminal and divalent
cation regions blocked binding to fibrinogen (85 %). None of the MAbs against
plS0,95 that were e~;....;~.Pd (Figure 14, and data not shown) inhibited bindingto fib,;llogen of neuLl~hils that were stim~ tPA with fMLP.
ICAM-l+ L Cell Adhesion to Purified Mac-1
Ne,uLl~hils that are stiml~l~t~p~l with fMLP use Mac-1 and LFA-1
cooperatively to bind to ICAM-1 (Diamond et al., CeU 65:961-971 (1991);
Smith, et al., J. Clin. Invest. 83:2008-2017 (1989)). To identify blocking
10 MAbs more easily, we switched to an assay that e~...ined Mac-l interaction
with ICAM-1 exclusi~,~,ly: ICAM-1~ L cell ~Ahp~cion to purified Mac-1
(Di~m~n(l et al., J. Cell B~ol. 111:3129-3139 (1990)). MAbs to the I domain
inhibited bindin~ with an a~cla~c of 82.4% + 26.1, ~I,el~as MAbs to the C-
te. ,~ l region i~hib;~ by an a.~lage of 24.1 % + 26.8 (Figure 14). 12 of
15 the 19 I ~lom~in MAbs i~ib;t~d by greater than 90%, wl,e~as the sLIongc~,~
inhibition by a MAb to the C A,.l~ al region was 52%. One MAb which
ed to the sites in both the N t,....il~l and divalent cation regions inhibited
binding s~.o.lgly (CBRM1/32, 97% reduction). The one MAb (CBRM1/20)
that bound di.~clly to the divalent cation bindi"g region did not block ICAM-1
20 binding to Mac-1.
Functional Studies With Mac-l/nlS0.95 Chihlle.as
To P~minP wllelllcr the I domain of Mac-1 was e~ ;Al for ligand
reco~nition, we tested the CHO cell e~ f~c for their ability to bind ICAM-1
25 and iC3b-E. ne~ ~ control ~ roc~d or ~ rcct~ CHO cells bound
cor.sl;~ ,ly to fibrinogen, this ligand was not tested.
The l-i n~l~ CHO cells cont~inP~ a functional form of Mac-1 as
~tim~ tion with phorbol esters p.~ ed Mac-1 ~ r~ s to bind to
purified ICAM-1 (Figure 16A). In CQnll~aSL to previous studies with L.~lll ,r~ted
COS cells (Diamond, et al., J. Cell Biol. 111:3129-3139 (1990)), little b;nding
to ICAM-1 was observed in the absence of stimulation (data not shown). Two
chimeras (M-a-X, M-b-X) which contained the I domain of Mac-l bound to
WO 94/08620 ~ 1 4 ~ 7 3 8 PCI`/US93/09777--
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ICAM-1, but more weakly than ~nticip~tPd. UnPYre~tP~ly~ the CHO cells that
expressed wild type plS0,95 also bound to ICAM-1. Despite the low level of
binding, it was reproducible, inhibited by MAbs to pl50,95, and not obse.ved
with control CHO cells tran:,r~ with ICAM-1 (Figure 16A, and data not
5 shown). Although we are uncer~ain of the physiologic relevance of this
pl50,95-ICAM-1 inter~cti~)n~ plS0,95 acts as a counkr~ or for an
Imr,h~ "i~d adhesion rnol~P~Ille on stim~ tP~ endc~ f-li~l cells (Stacker et
al., J. Immunol. 146:648-655 (lg91)c), and L cell ~ sr~c~l~ that e~press
ICAM-1 bind weakly to purified pl50,95 (Stacker and Springer, unpublished
10 data). C`himPr~c in which the I domains of Mac-l and pl50,95 were
eY~h~nged (M-e-X-b-M, X-e-M-b-X) showed no .~igl ific~nt dirL,.tince in
adhesion to ICAM-1. Although both wild type Mac-1 and plS0,95
l.~rcr~nl~ adhered to ICAM-1, three chi...e-, c (X-e-M, X-b-M, X-~M-a-
X), did not bind to ICAM-1; these three rl~i...~.,.c, ho..e~,r, were eA~..,ssed
15 improperly on COS and CHO cells (see above). Thus, despite cA~-~,s;,ion of
all of the ch;~c~c at l~la~ ly high levels on CHO cells, no readily
intel~ able pattern of bil-Ai.~g to ICAM-1 was o~
We tested the CHO cell ~ r~ r.~-j for their abiliy to bind iC3b-E
coated elylhro;y~s. Almost all of the CHO cells CA~ ,ssi-lg wild type Mac-1
20 r~sel~d with iC3b-E (Figure 16B). ~ith~~ the bhlding occullt;d in the
Aksen~4 of stimul~tiQn, ~e a~elage .. ~...~,r of iC3b coa~ icles per cell
inc,~d after phorbol ester l.~~ .l (data not shown, and D;~.l.ond,
Mosær, and Springer, unpublished data). The pattern of binding of iC3b-E to
the Mac-1/pl50,95 c~ f,~ again did not loc~li7~ the binding region
25 definitively (Pigure 16B). The three c~ pli~c (X-e-M, X-b-M, X-b-M-a-X)
that were not LA~re~d p..,pe.ly in COS and CHO cells and did not bind to
ICAM-1 in CHO cells, failed to rosette with iC3b-E. Four chimeras that
cont~inPA the I domain of Mac-l (M-b-X, M-a-X, X~-M-b-X, and M-b-X-a-
M) roætted with iC3b-E, but at sig~-i I ;~r.l Iy lower levels than wild type Mac-
30 1 despite similar expression levels. Une~l,ecledly, one chimera (M~-X-b-M)
which contained the I domain of plS0,95 bound iC3b conci~t~ontly. In
WO 94/08620 ~ 1 4 4 7 3 ~ PCr/US93/09777
- 67 -
independent studies with transfected COS cells, we observed that the same
subset of chimeras rosetted with iC3b-E (data not shown).
DISCUSSION
In this report, using chi...ç--c of Mac-1 and plS0,95, we loc~li7Pd the
epilo~es for 34 MAbs to Mac-1 and c~rrelated this with their ability to block
distinct adhesive functions of the native Mac-l molecule. We find that the 200
amino acid I domain on the cY subunit of Mac-l is a major site of ligand
10 l~og.lilion for iC3b, ICAM-1, fiblh~ogell, and the u,-dern~ co~ t~r-l~p~or
for ne~ ~hil aggltg~lion. One MAb (CBRM1/32) that tn~.ped outside the
I domain to an epitope shared by the N ~.ll~h~al and divalent cation bin.lillg
regions also blocked billdil~g to iC3b, ICAM-l and fi~ f -- Thus, ~e N-
and p~ s divalent cation binding regions may c~ .;b~le to15 re~o~nition of several of the lig~ntls~ MAb c~i~pcs were used to i~li7.e ~e
ligand l~o~n;~;on sites be~ ,et~lion of the results from ru~ ;o~
assays with the Mac-l/pl50,95 çl~i..,.,,..c was not str~ lrolwa~d~
The I domain ~ ~nl in the ~cogl.ilion of all four ligands
by Mac-l be~A.~ in each of the e~cperim~nt~l ~y~t~.ns tested, several di-Ç~,r~ t20 MAbs that mapped to the I dom~in blocked Mac-l-de~ d~ binding Sh~ gly
(> 80%). Furthç ..~ " inall cases, the individual MAb thatab~g~
most completely ...~ to the I do..lain. Two-thirds of the MAbs that
mapped to the I tlom~in blocked Mac-l i~ . Iion with iC3b, fibrino~çn, and
ICAM-l by greater than 70%, and all of the MAbs that blocked n~ lu~hil
25 homotypic adhesion strongly, I-~ cd to the I domain.
While the majority of MAbs to the I domain were sLlullgly hlhibilu-y
in at least one of the assays, not all of the MAbs were able to block several
ligand i~ ons. For exarnple, only five MAbs (LPM19c, TMG-65, Mn41,
- CBRMl/29, CBRMl/34) blocked all Mac-l-ligand interactions ~l~ungly. Many
30 of the MAbs that blocked binding to fibrinogen, ICAM-l and iC3b, only
partially inhibited neutrophil homotypic adhesion. There were, however,
several exceptions. Two MAbs (OKM9 and CBRMl/27) blocked binding to
WO 94/08620 ~ 1 4 4 7 3 8 PCI/US93/09777--
- 68 -
fibrinogen binding strongly but inhibited interactions with the other three
ligands only weakly, whereas three MAbs (CBRM 1/1, CBRM 1/2, CBRM 1/32)
that blocked adhesion to fibrinogen and ICAM-1 inhibited binding to iC3b
only partially. The dirr~ ces in palLG...5 of inhibition among MAbs that map
S to the I domain suggest the ~.~,3ence of several ;~ ~Li~e and functional
subdomains. For each individual ligand, some or all of these subdomains may
contribute to the archi~ of the binding site. Thus, we spec~ te that the
ligand lec4~ ;0n sites within the I domain are not identir~l, but are
o~erlapping.
Despite sCI`~nillg for new MAbs to Mac-1 only by their ability to bind
to peripheral blood ~.c.lL-~hils, the majority of new MAbs -.a~>pcd to the I
domain. This was ~ ZIll;~ .At.,d bc~,,sc the ~ niL~ of human and murine
Mac-1 are 74% idPntir~l at the amino acid level (~rn~ lt~ et al., J. Cell Biol.
106:2153-2158 (1988); Corbi, et al., J. Biol. Chem. 263: 12403-12411 (1988);
Pytela, EMBO J. 7:1371-1378 (1988)), with the divalentcation region sl-vwi.~g
the ~ dt,sl ide,-lily (84%) and the C ~ ..in~l region ~e least ide"li~y (74%)
among ~ ,.r~ r dQ.--~ . It is not clear why the I domain is
imm~m~lGi~.i..~l.l it is more homologous (81% identity) and smaller in size
(200 co...~Gd to 500 amino acids) ~han the C t~ inal region. Not
20 surprisingly, the strong identity bc l~,.~n I dolz;c of murine and human Mac-1 conse-~,s sites of ligand lC~o~ ;oll as both species of Mac-1 bind to human
and murine iC3b coated particles (Beller, et al., J. E~p. Med. 156: 1000 1009
(1982)) and to COS cells ll~ r~t~ with murine ICAM-1 (Diamond, et al.
unpublished).
While the homology beh. ~n human Mac-1 and p150,95 is ~ r~
lower than murine and human Mac-1, the residues ~at confer ligand
specificity appear to be cor~.~ed. Human Mac-1 and pl50,95 have a 63%
overall protein se~luence identity with a 55% identity bet~.~n co.l.,s~ohding
I domains (Corbi, etal., J. Biol. ~hem. 263:12403-12411 (1988); Corbi,
et al., EMBO J. 6:4023-4028 (1987)). None of the MAbs studied cross react
between the a ~ubuniL~ of Mac-1 and pl50,95. However, increasing
experimental evidence suggests that this degree of consel vc-tion is sufficient to
Wo 94/08620 PCr/US93/09777
~6~9~4~3~
conserve sites of ligand recognition as plS0,95 binds to a similar repertoire
of ligands as Mac-1 including fibrinogen (Loike, et al., Proc. Natl. Acad. Sci.
USA 88:1044-1048 (1991); Postigo, etal., J. Exp. Med 174:1313-1322
(1991)) and iC3b (~ lern et al., Biochem. J. 231:233-236 (1985); Myones,
S etal., J. Clin. Invest. 82:640-651 (1988); Ross, etal., J. Le~coc. Biol.
51: 109-117 (1992)). LPA-1 and Mac-l, which are 36% ide .~ l at the amino
acid level share ICAM-1 as a ligand, ~lthol~gh the binding a~ distinct as
LFA-1 binds to the first Ig-like domain (St~--nton, et al., Cell 61:243-254
(1990)) and Mac-l binds to the third Ig-like domain on ICAM-l (Diamond,
et al., Cell 65:961-971 (1991)). More refined mutagenesis studies are required
to dete~ ~ whether Mac-1, pl50,95, and LFA-1 utilize related IGcog~ ion
sites to bind their shared lig~nfls.
MAbs that l,layped to the 493 amino acid C-t~-rmin~l region showed
little ca~acily to block any of the ligand i~ ;ol.c with Mac-1. The a~ ~e
15 inhibition ranged from a low of 1.8% for l,elr~hil holllu~y~ic ~rlhesion to ahigh of 33.6% for bil~ding to iC3b. Thus, we su~,gci,l that the C ~lllii~l
region of Mac-1 does not contain ~ ilion sites that are critical for a direct
interaction with any of the four ligands eY~min~.
ne~nsG we g~'ne'~led only one MAb that maL,l~ed to the 362 amino
acid region that col.~il.Fd the 294 amino acid divalent cation binding repeats
and the 68 amino acids h~ r~,ly N-te~ l to them, little co.lclu~
sl~lt;...Fu~ can be made about the role of this part of the molecule in ligand
recognition. The a~al~,n~ lack of i.~.. ~el~cily may be attributed, in part,
to the 84% amino acid col~l~ation I~L..~.~ the col~ Jonding regions of
25 human and murine Mac-1. The one MAb that ~ li7rd strictly to this region,
C B R M 1120, did not block adhesion in any of ~e æsays by greater than 30%.
However, we cannot ~Acludc the e~ n~e of l~cog~ ion sites that were not
blocked by C B R M 1/20. TntleeA, eA~ (D'Souza, J. Biol. Chem.
265:3440-3446 (1990); D'Souza, Nature 350:66 68 (1991)) suggest that the
30 related platelet integrin ~,Jb ,B3 uses a site within the divalent cation binding
repeats to bind to fibrinogen. Future studies with additional MAbs to this
region are required to clarify whether the strong sequence conservation among
Wo94/08620 ~ Pcr/uss3/o9777--
- 70 -
divalent cation binding modules m~int~inc receptor conro.",aLion or sl-ct~inc
a direct interaction with ligand.
Although we did not s.le~es,rully map any of the MAbs to the N-
terminal region exclusively, two MAbs to Mac-l (OKMlOo~d and CBRM1/32)
S and one MAb to plS0,95 (SHCL3) bound to discontinnous ~ilopcs that
required the presence of both the N-terrninal and divalent cation binding
regions of Mac-l or plS0,95. SHCL3 difÇel~d from CBRMl/32 and OKMlO
as it bound to chime~c that cont~inP~i only the N-terrninus of pl50,95;
however, it bound more strongly when both the N-terrninus and divalent cation
binding region of pl50,95 were present. Importantly, these MAbs were the
only ones that mapped outside the I domain and blocked ligand intç~ction
strongly. CBRMl/32 blocked (70-95%) binding to iC3b, fibrinogen, and
ICAM-l, and OKM10O~d blocked binding to iC3b (Figure 14 and ~n.lerso
et al., J. Irnrnunol. 137:15-27 (1986); Wright, et al., Proc. Natl. Acad. Sci.
USA 80:5699-5703 (1983)), and inhibits binding to fibrinogen (Wright, et al.,
Proc. Natl. Acad. Sa. USA 85:7734-7738 (1988)). SHCL3 blocks binding of
pl50,95 to stimul~tp~d en-lQthPli~l cells (Stacker et al., J. In~nunol. 146:648-655 (1991)) and iC3b (Bilsland, Di~monfl~ Mosser, and Springer, unpublished
observations), but not to fibrinogen (Postigo, et al., J. EJCP. Med. 174:1313-
1322 (1991)). The lllap~ g of three MAbs to ~ c4nlin~ c regions of the ~
chain provides ;.. ~-olo~ir~l evidence for z~ ;ons be~ the N-
terminus and the divalent cation binding regions, which are ~ P,d by the
200 arnino acids of the I domain. Previous electron mic,usco~y studies on the
related integrins a!nb-,B3 and a5-,1B, have shown that the ~Y and ,~ subu-li~ form
25 a globular head at the N-~,...i~l edge of the m~le~l-le that is distal to the membBne sp,.nnin~ region (Carrell, J. Biol. Ch~m. 260.1743-1749 (1985);
Hynes, Cell 69~ 25 (19~2); Nermut, et al., EMBO J. 7.40g3~099 (1988)).
The ~ tions from dispaste regions of Mac-1 and pl50,95 suggei~ that
the I domain does not intercalate into the globular head to form a string of
30 physically autonomous modules. The structure of the leukocyte integrins thus
differs from the linear array of domains that is proposed for members of the
immllnoglobulin and selectin f~milies of adhesion molecules (Bevilacqua,
WO 94/08620 i~ l 4 4 7 ~ 8 PCl`/US93/09777
et al., Science 243: 1160-1165 (1989), Driscoll, et al., N~ure 353:762-765
(1991); K~nc~c, et al., J. Cell. Biol. 114:351-358 ~1991); Williams et al.,
Annu. Rev. Im~nunol. 6:381 105 (1988))
Although the e~.~eli-l-enL~ with MAbs in~ te that the I domain is a
5 primary ligand ~cog,.;lion site, no discernible pattern was obs~.ved when theMac-1/pl50,95 chimpr~c were tested in functional assays. Several factors may
have contributed to the failure of these eJ.~.i---enls to map directly the ligan d
binding sites on Mac-1 for iC3b and ICAM-1. As suggesLed above, others
have recellLly den~or.cl ~P~l that Mac-1 and plS0,95 a~pear~ to bind to some
10 of the same lig~ntls. Attempts to gcneratc Mac-1/LFA-1 chinle.~s which have
less overlap in ligand l~elLoire were . ~cv~c~s~rul as these molecules after
transfection into COS cells were synthP~ci7ed in ~r~ul~r form, but were not
e~ ,~d on the surface. ~lthol-gh others have le~l~d complement l~e~t~r
activity for pl50,95 (~irl~lPm et al., Biocfi~m. J. 231:233-236 (1985);
Myones, et al., J. Clin. Invest. 82:640 651 (1988); Ross, et al., J. Le~c.
Biol. 51: 109-117 (1992)), in our experiments, iC3b coated e~ h~;~s did not
bind to ~ r~ed CHO cells e,.~ .ing pl50,95 (Figure 16B, and data not
shown). Possibly, in the Llal~sr~t~A cells, the I domain of plS0,95 may
contain the ~Llul;lulal in~llllc.Lion to bind iC3b but lack proper inl~racLions
with other parts of the ~ or ~B s.lbu.ii~. In favor of this, a human~hirl~PIl
plS0,95 hybrid mol~P~llle that was C~ iS~l of a human ~ subunit and
chir~n ,l~2 subunit and e,.~ scd in COS cells rosetted sllungl~ with iC3~E
although the human ~ that was paired with the human ~B2 subunit did not
(Bilsland and S~ringer, unpublished obselv~lions). Our e,~c ;.nr-l l~ also
suggest that at least in vitro, plS0,95 binds weakly to ICAM-1 (Figure 16A).
The I dom~in of pl50,95 in the contPYt of a wild type pl50,95 frarne bound
to ICAM-1 poorly, but in the context of a Mac-1 frame (M-e-X-b-M) where
secondary sites may be present, the binding to ICAM-1 was enh~nr~d.
The analysis of the functional assays with the chimeras was complicated
also by the il,lplu~cr ~ r~i"ion or association with the ~B subunit by three of
the chimeric cY subunits (X~-M, X-b-M, X-b-M-a-X) in COS and CHO cells.
Immunoprecipitations of surface labelled CHO cell transfectants expressing
WO94/08620 i~ 38 PCI`/US93/09777--
- 72 -
these three chimeras with the ~B subunit MAb (TSl/18) were either weak or
absent although the ~ subunit was still associated with the ~ subunit as it wac
coprecipitated by the ~Y subunit specific MAbs. In light of this, it is not
surprising that X-e-M, X-b-M, and X-b-M-a-X did not bind to iC3b or ICAM-
5 1 when ~ r~,~d in CHO or COS cells. Two other rh;~ ,.c (M-a-X and M-
b-X) that con~il ed the I domain of Mac-l and bound to ICAM-1 and iC3b
poorly, show evidence of subtle '.IIUCIUI~I anomalies based on changes in
~nti~nic 1~ ViIY. M-a-X and M-b-X show a reduction or loss of binding to
two MAbs that map to the I domain, CBRM1/2 (Figure 13) and CBRMl/5
10 (Diamond and Springer, submitted). It is IIO~WO1 Ihy that CBRM 1/5 recognizesa subpopulation of functionally active Mac-l molecules on neuL,~hils and
monocyl~s (Diamond and Springer, submitted), and is ~Ayr~ssed on other
chimer~c (X-e-M-b-X, M-b-X-a-M) that contain the I domain of Mac-1 and
bind ligand (data not shown). Thus, despite the high levels of ~,A~ss;on in
15 CHO cells, M-a-X and M-b-X may not be in a con~,...alion that ~ c
ligand binding; ~e molecular p~cl~in~ of the I domain may be dislul,Led
sl-fficiently by ~e i,..p,o~r context of surrounding domains so that it is unable
to ~ e ligand. On l~ ~;yLes, to recognize lip~nr~, Mac-1 and LFA-1
must convert from a low avidity to high avidity state (Buyon, et al., J.
Irnmunol. 14~.3156-3160 ~1988); Dustin et al. Nature 341:619-624 (1989);
Lo, etal., J. E~tp. Med. 169:1779-1793 (1989); van Kooyk, etal., Nature
342:811-813 (1~89)). It is ~r,.pLing to spe~ t~ that integrinc require a
physical "o~elung or closing" of the molecule that enables ligand binding and
that the j~uL~osilion of homologous, but non-native domains in some of the
~himer~c yr~ this change from OCCullillg. Mol~ov~;r, the ~B subunit could
m~dlll~t~o, the sLIu~;lul~l change in the ~ subunit. This could e~cplain why
deletion or svb..~ ion of residues in the cytoplasmic domain of the ~B subunit
abolishes LFA-1 binding to ICAM-l (Hibbs, et al., J. Exp. Med. 174:1227-
1238 (1991); Hibbs, et al., Science 251: 1611-1613 (1991)), why X-e-M, X-b-
30 M, and X-b-M-a-X do not bind to iC3b and ICAM-1, or why a f hi~l~en but
not human ~2 subunit allows plS0,95 to rosette with iC3b coated particles
WO 94/08620 X ~ 8 PCr/US93/09777
- 73 -
In summ~ry, the studies presented here demonstrate that the I domain
on Mac-1 is a major l~co,~"i~ion site for both its cellular and soluble lig~n~l~This is the first study to provide a definitive role of the I domain on integrincY s~bunils. Previously, bc~..se of its homology to the ligand l~ ding regions
S in von Willebrand factor, cartilage matrix protein, and complement ~ins,
the evolutionarily inserted I domain has been spP~ tPcl to be i-..~.~,~ in
ligand l~cog--ilion (Larson, et al., J. Cell Biol. 108:703-712 (1989); Pytela,
EMBO J. 7:1371-1378 (1988)).
W094/08620 ~14~3~ PCI`/US93/119777--
- 74 -
~pplicant'soragent'sfilc ¦Ir ir~n~
l referencenumber 1ù11.070PC01 ¦ : ~ 3 ~ 0 9 7 7 7
INDICATIONS REI~'I'ING TO A DEPOSITI~ T~ ol~G ~NISM
(PCT Rule 13l~is)
A. Ibe i~ " - made be10w relate to thc mb,~uu.~ ;s~ referred to in the dc~.,.;~,liu
on page 9 , line 26
B. IDENTIFICATION 0~ DEPOSIT Further deposits are identified on an additional sheet O
Name of J.! oS;~
American Type Culture Collection
Address of d~,~,ùs;t... ~ . (includin~ pos~ol codc and coun~ry)
12301 Parklawn Drive
Rockville, Maryland 20852
United States of America
Date of deposit Accession Number
C. ADDITIONAL INDICATIONS (leav~ blank if nol applicahle) This f'~ is continued on an additional sheet O
Hybridoma CBRM 1/5
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if t/u~ indications arc notfor all dcstgnatcd S~atcs)
E. SEPARATE FURNISHINC;: OF INDICATIONS (leavc blank if not appticablc)
The- ' Iistcdbelowwillbesubmittedtotbel ..~.ti,a~.~.lBureaulater(spcci*thc~cncralnaturcof~' ' cg., ~cccsston
Number of Dcposit~)
For receiving Of fice use only For I - I Bureau use only
¦~ This sbeet was received with the - ' a,~ li - O This sheet was received by the I - t I Bureau on:
~ -' ' officer ~ / Authorized officer
'.V-~P~V
.1`!.~0~D;`~"~,'.C'.
Form PCI~/RO/134 (July 1992)
~WO 94/08620 ~ 3 8 PCl/~S93/09777
I Applicant'soragcnt'srilc ¦Inlcrnational?F~rli~lio.~ l
¦ rcfercnccnumbcr 1011.070PC01 ~ ~ 9 ~ ~ O 9 7 7 7
INDICATIONS I~EI~TINC, '10 A D~POSITED MICROORGANISM
(PCT Rulc 13l~is)
A. The ' - made bclow relate lo the ~ .;sl" referred to in lhe dc;.. ,.i~Jtiu
on page 10 , line
~. IDENTIFICATION OF DEPOSIT l:urther deposits are idenlified on an ~d~ nnql sbeet O
Name Or deFnasil..~ y inslilulion
American Type Culture Collection
Addressofdcposilaryi -i~ ~ (including postalcodcandcounlr~)
12301 Parklawn Drive
Rockville, Maryland 20852
United States of America
Date ot dcposil Accession Numbcr
C. ADDITIONAL INDSCATIONS (Icavcblonkif not applicablc) This ;~fnrm~ is conlinued on an d~ I sheel O
Hybridoma CBRM 1/19
D. DESIGNATEDSTATESFORWHICHINDICATIONSAREMADE (if~hc;ndicationsarcn~foralldcsigna~cdStatcs)
E. SEPARATE FURNISHING OF INDICATIONS (Icavc blank if not opplicablc)
The ~ listcdbelowwill besubmitted lo the I ' -' I 8UrC,dU bter(spcci*thcgcncralnaturcofl' ' '' ' cg7 ~cccssion
Numbcrof Dcposit~)
For receiving Of ~ice use only For I - .. dl;u.. dl Bureau use only
Cl This sheet was received wilh the international 7F F ~ This sheet was received by thc I ~tiu.. dl Bureau on:
Authorizcd officer ~1 Authorizcd officer
RA Y.~I~EI A~
~mFFr~ ~ 3
Forrn PCr/R01134 ~July 1992)
