Note: Descriptions are shown in the official language in which they were submitted.
W~' 93/02192 PCI/US92/05903
I''J~ 32i3
DESCRIPllON
MODIFIED PF4 COMPOSITIONS AND METHODS OF USE
Cross-Reference to a Related Applicatiop
ll~is application is a continuation-in-pan of co-pending application Serial No.
07/316,333, fi1ed JulS 6,1989.
s
Backg~ound of the Invention
Angiogenesis, the development of new capilla~y blood vessels, is an important process
in the d~veloping ~etus and growing human. Howev~r, in 11ealthy adults, angiogenesis occurs
signific~ntly only during woul~d hea1ing and in the menstrual cyc1e.
It is now wide1y rccognized that much of the angiogenic activity occurring in adults
is pathological in nature. For example, pro1iferation of vascular endothelial cel1s and
~ fonnation of new capil1aries is esserltial for growth of solid tumors beqond a few cubic
; ~ millimeters ~n vo1ume (Folhnan et al. [19831 Ciba Found. Symp. I00:132-149~. We now
understand that deve14ping tumors secrete growth factors which stimulate neighboring
~dothelial cells to divide and migrate toward the tumor.
In additi~n to growth o~ solid tumors, other conditions involving angiogenic
dysfunctions ~nclude diab~tic ~eti~opathy, retrolen~a1 fib~op1asia, neo~ascular glaucoma,
psoriasis, angioflbromas, immune and norl-immune infla~unatioll ~including rheumatoid
anh~itis3, caplUa~y proliferlldo~ tl~ ~therosclerotie plaques, hemangiomas, and Ka~s,i's
20~ ~ Sa~rna have also se~nd~ reoQgfli~d as diseases poss~si~g sharac~etistics of
dss~laUd endofnolla1 cell divis~o~ and cap~11ary gro~th. These conditio~ alongwith growth
of solid tumors ~Ire co~ Yel~ referred tO as asngiogenic diseases~ (FoJkman, J., and M.
K1agsbr~ Science235:442-447).
In adâition to ang3O~or~ic ds~, there are other condi~ions where endothelial cell
proliferatiotl is pathological or, at lesst, ullwanted. For example, endometriosis is
' ~ characte~i2ed by the~ abnorms1 proli~eration a~d positioning of oertain endo~helial cells which
norn~1~J line the inner wall of the uterus. Colltrol of ~e angiogenic process could help to
preveM or alleviate endometriosis. ~Iso, prevention of endothe1ial cell grou~th in the uterus
cou1d be ~ means of birth contwl.
~ Endothelia1 ce11 growth is associated ~ith wou~d healirlg. This growth is ~ndesirable
dunng exte~ded surgical proceedings and where excessive scar formation may occur.
Therefore, a means of cQntrolling endothelial cell proliferation wos1d help pre~ent or reduce
unwan~ed scar fo~a~tion.
:
WO 93/02192 PCI /US92/0590~
7~113'~06 2
The mechanism of angiogenesis and endothelia~ cell proliferation has not been
completely characterized. It has been established that mast cells accumulate at a tumor site
before new capillary growth occurs; however, mast cells alone cannot initiate angiogenesis.
Heparin, a mast cel1 product, has been shown tO significantly stimula~e the capilla~y
S endothelial cell migration which is necessary for angiogenesis (Folkman, J. 11984]
Angiogenesis: Initiation and Modulation. ln Cancer Invasion and Metastasis- BioloQic and
Therapeutic Aspects. G.L. Nico1son and L. Milas, eds. Raven Press, New Yor1c, pp. 201-208).
Several substances are known to have the capability of inhibiting endothelial cell
growth in vitro. One of the most extensively studied inhibitors of endothelial cell growth is
protamine, which is a protein found only in sperm. Protamine has been shown to inhibit
tumor angioge~esis and subsequent tumor growth (~ylor, S. and J. Folkman [1982] Nature
297:307-312). Protamine's anti-angiogenesis activity has been attributed to its well-~nown
capacity to bind heparin (~ylor and Folkman 11982], ~). Clinical expenments withprotarnine have not been pursued because of the toxicity associated with protamine injection.
Protamine, which is usually isolated from salmon sperm, is known to be antigenic in humans,
and anaphylactic reactions to ~his protein have been observed wi~h secondaly exposures.
At least two other compounds have b en studied in regard to their heparin-binding
activity: platelet factor 4 (PF4) and major basic protein. Major basic protein has
demonstrated heparin-binding activity but is of little pra~ical utili~ because of its high
toxi~ity.
Platelet ~c~or 4 is awell -kDow~ proteinwhich has been completely~equenGed (Deuel,
T.F., P.S. Keim, M. Pa~mer, and R.L. Heînrikson ~ oc. Natl. Acad. Sci. USA
74(6):225~22583. It is a 7~residue secretable platelet protein with a molecular weight of
appm~mately 7.8~Kd. Although there is evidençe of hepann billdi~g acti~rity and some
indi~tions of anti-angiogenesis acti~i~r (F;ollcmaII 11984], ~, PF4 has never been shown
to have cli~i~l utili~. ~
A compound whic~ has been described as "o~costati~l ~ and which appears to ~ the~e9 or si~iiiar to, native PF4, has been implicated as affecting hhe ~owth of tumors tlJ.S.
Patent Nos. 4,645,828 and 49737,5~, both issued to l~Nardzik et aL~. However, the e~ects
reported in these patents pertain to slowly growing human ca~cer cells in immunodeficient
mice. The results of 1hese experiments cannot be reliably extrapoLated to predict the effect on
apidly glOWblg tumors which are native to the host a~l. Furthennore, the e~periments
reported in these patents in no way pred~ct or disclose ~ny angiostatic properties.
Various peptides from PF4 have been purified and theil properties studied. None has
been shown tO have any roae in the inhibition of angiogenesi~. It is known that the ~13
peptide of PF4 is chemotactic for neutrophils and monocytes (Deuel, l:F., R.M. Senior, D.
Chang, G.L Griffin, RL Heinrikson, and E.T. Kaiser f1981~ Proc. Natl. Acad. Sei. USA
~) 93/02192 ~ 1 ~ 3 2 o ~j Pcr/usg~/0sgo3
78:45854587; Osterman, D.G., G.L Griffin, R.M. Senior, E.l: Kaiser, and T.H. Deuel [1982]
Biochem. a~d Biophys. Res. Comm. 107(1):130-135). lt is significant to note that the
irlfiltration of monocytes would be expected to stimulate the proliferation and migration of
local endothelial cells by the secretion of angiogenic factors. Thus, peptides of PF4 could be
S expected to stimulate, rather than inhibit, angiogenesis.
Tbere is a significant and very long standing need to locate an e~ective and non-toxic
inhibitor of angiogenesis and endothelial cell proliferation. Angiogenesis plays a major role
in ~he initiation and progression of widespread catastrophic illnesses, including cancer. An
effective, non-toxic agent which can be administered locally and/or systemically to treat these
illnesses would be highly advantageous and has long eluded identification.
B ef SummarY of the Invention
The subject invention relates to compositions obtained through chemical modifications
of PF4 or recombinant PF~ (rPF4). For example, P~4 can be modified through its free amino
groups with fluorescein-isothiocyanate and retain tbe capability of inbibi~ing allgiogenic activity
and endothelial cell proliferation. Similar modifications ean be made with PF4 analogs,
mutants, or fragments.
A fu~ther aspect of the subject invention is the targeting of the biological activity of
PF4 ~o s~ic loca~ion3 where that activi~ is needed. T~is can be done by con~ugating PF4
(or an appropriate f~agment, analog, or muta~t) to a mo~oclonal or polyclonal an~ibody,
carrier protein, cell receptor molecule, or other binding protein sequence.
A further aspect of the subject in~ention is extending the half-life of biologically active
PF4 (or appropdate ~agments, analogs, or mutaIIts) ~y conjugating said PF~ to a poly~per
oDmpound. The pol~!mer may be, for e~ample, a polyamino acid such as polyglutamate, or a
polysa~adde such ~s polyetbylene gbcol (PEG).
In addition to treating angiog~nic disorders ~d inhibiting endothelial cell
pr~lifera~ion, ~odified PF4 can also be used to target to~ to spe~fic cell populations.
Various other modifications of PF4 and rela~ed compounds are descnbed here. These
modificatioDs can be made ~ order to enhance biological activity or othe~e increase -lhe
tility of the PF~ compound.
Brief Description of the Drawings
Figure 1 shows the i~hibition of angiogenesis resulting ~om treatment with ~PF4 and
various related peptides.
Figure 2 compares the amino acid sequence of rPF4 with rPF4-24:1.
Figllre 3 depicts the c~-helical configura~ions of rPF4 and rPF4-241.
.
WO 93/U21g~ ~ ~ 1'3 ,1 0 ~ PCl'/US92/0590~-.
Figure 4 c~mpares the inhibition of angiogenesis resulting from treatment with rPF4
and rPF4-241.
Figure ~ shows inhibition of human endothelial cell proliferation by rPF4 and rPF4-
241.
S Figure li compares the inhibition of human umbilical ve~ endothelial cell proli~eration
resulting from treatment with rPF4 or rPF4 241.
Figu~e 7 shows the ability of rPF4 to inhibit tumor growth.
Figure 8 depicts the possible chemical structure of the ~termi~al end of FrPF4.
Figure 9 shous inhibition of human endothelial cell proliferation by FrPF4.
Figllre l0 shows inhibition of human endothelial cell proliferation by FrPF4-241.
Figllre 11 shows inhibition OI tumor growth by FrPF4.
Detailed Description of the lnven~ion
The subjec;t invention concerns the discovery that PF4, rPF4, and fragments and
analogs of these compounds can be chemically modified to create new compounds with highly
desirable charactenstics. For example, chemical modification of rPF4 and its fragments has
resuited in the identification of compounds wh~ch show surprising ability to inhibit angiogenic
actnity as well as the capabili~ to inhibit endothelial cell proliferation. One specific chemical
modiScation which resulted in altered biological properties i~volved modification of the ~ee
ami~o ~roups of rPF4 with fluoresce~n-isothio~yanate (E~ITC). The result~ag adduct, FrP~4,
lacl~; heparin bindi~g activity because of modi~cation of l~sine residues ~nthin the heparin
bL~ding do~ but, s~ mgly, retains the ability to inhibit angioge~esis as well as suppress
HUV13C proli~eraaon in vitro.
~ic activi~ i~ also found in PF4 ~agments and mutants which have been
~odified v~th the bulkJ~ and ~ydrophobic nuoresceLn moie~. In addition to their biologic~l
a~, t~e ~ PF4 sequences are useful for visual detection of PF4 molecules.
hermore, the a~ to modii~r PF4 and its fragments wit~ large moieties without loss of
the relevant bioI~gical activity pTovides a basis for conjugating PF4, its ~agments, mutants, or
derh1atiYes with to~ins, mo~oclonal antibodies, polyclonal antibodies~ fluo~ophores, cell
receptor molecules, non-prole~naceous i~iological e~ector molecules, chelators, carrier proteins,
polysaccharides; polyamino acids, and o~her large entities. The conjugation may occur through,
for e2~ample; modiScation of functio~al g20ups on the protein, such as free am~no, carbo~yl,
slllfhydryl, or guanadinium (argi~ine) groups of PF4 or a variant of PF4. ~e modi~cation of
these moieties necessaly to effect the desired conjugation can be ~ried out by chemica1
3~ proeedures well know~ to those skilled in this ar~.
: ~ ~
One of the uses of the compounds described here is in the treatment of angiogenic
diseases. As used in t~is application, the tenn "angiogenic disease" refers tO growth of solid
:
,
~ 93/021g2 ~ 1 1 3 2 0 6 PCI/US92/~)5903
S
tumors, and other conditions involving angiogenic dysfunctions in&luding diabetic retinopathy,
retrolental fibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune and non-
immune inflamma~ion (including rheumatoid arthritis), capillary prolifera~ion within
atherosclerotic plaques, hemangiomas, and Kaposi's Sarcoma. The subject invention also
S oollcerns the use of rPF4 and PF4 fragments, analogs, and mutants for treatment of diseases
of dysregulated endot~elial cell proliferation.
As used in this application, the term "analog" refers to compounds which are
substantially the same as another compound but which may have been modified by, for
example, adding additional amino acids or side groups. "Mutants" and nvaliantsr as referred
to in this application refer to amino acid sequences which are substantially Ihe same as another
sequence but w~ich have amino acid substitutions at certain locations in the amino acid
sequence. "Fra~nents" refer to portions of a longer an~ino acid se~uence.
The subje~ invention embra~es the specific arnino acid sequences and other
compositions which are speci~cally exempli~ied. The subject inventioll further embraces
analogs and mutants of ~ese sequenees, as well as fragments of the sequences, and analogs and
mutants of the ~agments. These analogs, mutants, and fragments are embraced within the
subject invention so long as the analog, ~agment, or mutant retains substantially the same
relevant bio~ogieal activity as the originally exemplified compound. ~:or example, it is well
within the skill of a person trained in this art to make conservative amino acid substitutions.
These sub~titutions are discussed Ln more detail below. To the extent that these substitutioDs
do not substantially alter the relevant biological activity, then the resulting compounds f~ll
in the scope of the subject invention. The tenn "rele~ant biological activityY refers to the
activi~ of interest for a particular application of a compound. For example, several u~,es of
` PF4 are dis~ below. l'hese uses include inhibi~ion of angiogenesis and endotheLal cell
2S prolifelation. When PF4 is bein8 used in these w~ys the~ ranalogs" would refer to compounds
whe~e PF4 has ~een modi~ied (by a conservat~e amino acid substitution, for example) without
s~bstan~y altering ~he compound's ability lo inhibit angiogenesis or endotheLial cell
proliferation. Consen~ative amino acid substitutions a~e only one G~ample of the type of
m~difications which are wi~hin ~e scope of the subjec~ maner of this ~vention.
The subject invention ansesi from ~he unexpected discovely that chemieally modified
rPF4 inhibits in vivo capiL1aly formation iand e~ ~yo~ic neovassularization. It ~as also been
delermined that full length recombinant P~-- inhibits growsh factor~ependent human
endothe1ial cell prollferation in viiro. Significantly, it has also been de~ermined that the
angiogenesis-inhibiting activity of PF4 is retained by synthetic peptides corresponding to
3~ sequences of PF4 as small as 10 amino acids in length. In particu1sr, a synthe~ic peptide of
13 amino acids corresponding to the carboxy terminus of P~4 (C-13) has displayed potent
-
WO 93/02192 ~ PCI /US92/0590~
angiostatic activity. A peptide of 41 amino acids corresponding to the carbo~y terminus of PF4
(C41) has also shown angiostatic activity.
The activity of ~he ~13 peptide is especially surprising in light of its inabilitY tO affect
the anticoagulant activity of heparin. The use of the C-13 peptide o~ers several advantages
over whole rPF4 such as reduced dosage (weight basis), reduced likelihood of antigenicity, and
greater likelihood of effectiveness in novel dosage forms.
The C-13 peptide of P~4 also retains the ability to pre~/ent Con-A induced
immunosuppression in mice, an actiYity which is unaffe ted by heparin and probably
independent of the ability of the peptide to inhibit angiogenesis.
It ~s well understood that angiogenesis is required for solid tumors to grow beyond a
~ew cubic rnillimeters. Thus for the treatment of solid tumors, use of rPF4, or modiScations
thereof, to cause tumor rejection by inhibiting angiogenesis presents a novel and highly
ad~antageous means of therapy. The ~act that the C-13 peptide inhi~its angiogenesis without
affecting the anticoagulant activity of heparin demonstrates that this small pep~ide would also
haYe the benefit of not interfering with concurrent anticoagulant ~herapy~ ~ditionally, small
peptides are gene~ally less antigenic than larger proteins, and, thus, the PF4 ~ragments can be
used a~vantageoosly for oral and transdermal administration. These types of delivery are
particlllarly useful in the treatment of gastrointestinal capillary proliferation (e.g., Kaposi's
Sar~oma) and skin lesions, respectively. Intralesional, as well as sy5temic, administration of
PW ~agme~ts are~also appropriate for treatment of these conditions. ~pical or aerosol
sdminislration of PF4 ~agments is appropriate for skin or pulmona~y lesions, Iespectiveb~ (e.g.,
si's sarcoma and lung cancer).
An analog of PF4 which exhibits enhanced abili~ to in~it angiogenesis has ~en
:~ ~ synt~izut. ~ a~nalog, k;nown as rPF4-241, was created by cassette mutagenesis sf a
Z5 ~thetic PF4 gene wb~Ly ~our Iysine residues of the carbooy termînus of PF4 were conv~rted
to two ~ Glu couple~s in order to eliminate heparin binding acti~i~ while retaining the a-
helical secondaly structure. If rPF4241 (or FrP~4-241) i~ administered intralesio~ally, it can
be applied such that the ~dosage is between about 1,~ esion and about 4 mg~esion. For
systemic administratio~, the dssage of rP~241 ~or FrPF~241) ca~ be bet~veen 0.5 mg/kg of
body weight and about 100 mgll~g of body weigh~ Similar and higher dosages can be used for
the ad~tration of ~Dative sequence rPF4 (or FrPF4) as we}l as peptide ~gments. F~r
; example, dosages of rPF4 (or FrPF4) and fragmea~s thereof may be ~wice that of rPF~241 (or
- E;rPF4-241) or higher.
The ~ompounds of the subject invention can be ~mbined ~th a suitable
pharmaceutical earrier. For e~mple, FrPF4 or FrPF~241 ean be formulated in physiologically
acceptable carAers, such as phosphate buf~ered sa1ine, distilled wa~er, excipients, or the like,
or may be administered neat.
.
::~
W~93/02192 ;~ 1 ~ 3 2 û fj PCr/VS92/05903
Materials and Methods
Chicken Chorioallantoic Membrane (CAM! Assav. Fertile eggs were incubated in a
stationary position for 3 days at 37C and 70-80% r~lative hun~idi~r. During this time, the
embryo rose to ~he ~lpper sufface of the egg contents. At the beginning of the 4th day, the
S eggs were cracked without inversion and carefully deposited int~ stenle plastic petri dishes
such that the emb~yo remained on the upper surface. The shel1-free eggs were incuba~ed for
an additional 72 hours at 37C, under an atmosphere containing 2.5-3.5% CO2 after which
the growing embryos developed a recognizable CAM. Discs, made by mixing test samples with
1% (wh) methylcellulose were dried and placed on the CAM between major vei~s andappro~nately O.S cm from the emblyo. Following another 48 hour incubation at 37C (2.5-
3.5% CO2), the samples were scored for their ability to inhibit angio~enesis. I~ibition
appears as an avascular zone surrounding the implant and can often include elbows formed
by veirls avoiding the disc and a red~ced number of capillaries in the region of the implant.
Endothelial Cell Proliferation AssaY~ Human umbilical vein endothelial cells were
cultured in Medium 199 (Gibco) containing 10% (v/v) fetal bovine serum (FBS), lS0 mcg/ml
endothelial cell growth supplement (E(::GS) and 5 units/ml heparin at 37C and 4-5~0 CO2.
Every 34 days, the cultures were ~arvested by trypsin treatment, diluted, replated, and grown
o conflua~ Prior to the start of sn expenment, the cells were centrifuged and resuspended
~; in heparin-J~ee media and incubated Y~n~h the test su~stance for 3 days under standard culture
conditio~s. At the end of the incubation period, the cells were harvested and counted.
Statistical signifi~nce ~etween means was determined by a standard Student t-tes~ for unpaired
data.
~ *~4 Prod_ion. Recomb~t PF4 was producgd i~ E coli as an N-termi2Al f~ion
plotein; conulining ~ methionine immediately preceding the PF4 sequence. l~e insoluble
sioI~ p~o~tein was eleaved with ~yanogen bromide trea~ent and puriiied by hep~ agarose
~init~ onLttography.~ The isolated protein was buffer excha~ged into 20 mM sodium
aoetate, pH 4.Q and either ~ozen or ~rophilized for storage.
Prod~ on of Peptides. Peptides ~ere prepaTed by standard solid phase s)mthesis
procedures, cleaved ~om the solid support a~d deblocked, and purified by reve~se phase
' 30 HPLC ~
Following are~ examples which illustrate pro~edures, including ~he best mode, for
practicing the inYe~tion. These alunples should not be sonstrued as limiting. All peroentages
are by weight and all solvent mnYture proportions are by volllme unless o~helwise noted.
~: ~
WO 93/02192 21 1~ 2 0 ~ PCl`/US92/0~90~ ~
ExamPle 1
Chicken eggs, prepared as descnbed above, were treated with discs containing several
concentrations of recombinant PF4 or peptides derived fiom the sequence of PF4. rPF4 and
C-terminal peptides as smaU as 13 amino acids inhibited angiogenes~s on the CAM (Figure 1).
S In each case, the inhibition was dose-dependent and the response approximately equiva1ent
(molar basis) for the inhibitors containing the ~tenninal region of PF4. An N-terminal
peptide of PF4 (N-29) did not inhibit angiogenesis even at the highest concentration tested,
suggesting that all of the anti-angiogenic activity of PF4 is probably associated with the G
terminal portion of the molecule. Since the C-terminus of PF4 is rich in Iysine, polylysine was
tested in this assay system and found not to cause inhibition at 6.5 mnol dosages.
ample 2
The Iysine rich region of PF4 (residues 61 66) is also the domain associated with the
binding of heparin by PF4. Hepam~ u known to play a role in modulating angiogenesis, which
1~ can also be affected by protamine, another well characterized heparin-binding protein. To
assess the ability of PF4-based synthetic peptides to bind heparin, we assayed the activity of
coagulation cascade en~ymes which are inhibited by heparin. Protamine and platelet factor
4 ar~e able to prwent the beparin inhibition of thrombin and Factor Xa at appro~nately
equimohr conoen~trations. The 41 amino acid Gtermi~al peptide of PF4 (C41) prevented
hepa~in inhibition ~less effectively, but the C-13 peptide was unable to prevent the inhibition
of thro~mbin even at concentrations~ ten tirnes that of an effective level of rPF4. Tnis
unexpected finding su8~es~s that the C~13 peptide inhibits angiogenesis by some method other
than hep~uin bindL~g.
El~ample 3 - ~ ~
Endothelial cell division and growth is tightly controlled and strictly dependent on the
presence of growth factors We eYaluated the a~ility of rPF4 and related peptides to inhibit
growth factor-stimulated human endothelial cell p oliferation vitro. rPF4 sigllificantly
inhibited endothelial cell growt~ in a dose4ependent ~shion at a concentradon as low as 10
30; mcg/ml. Inhibition was compbte at 25 mcg/ml in the heparin~eficient medium employed
here.
:::::: : :
~ ~,
Example4
lb assess the imponance of the hepann binding activlty of PF4 in the inhibition of
end helial cell proliferation, cells were incubated in media containing or lacking 5 unitslml
heparin. The presence~of héparin stimulated proliferation of these cells during the three day
93/02-g2 ?.. 1 1 3 2 0 6 PCI/US92/05903
incubation of this experiment. rPF4 significantly inhibited both control (lOO~o) and heparin
stimulated (45%3 endothelia~ cell growth (l~ble 1).
Table 1. Attenuation of rPF4 inhibition of endothelial cell growth by heparin.
rPF4 %
Addition -- 50mcgtml Inhibitiona
144 ~ 2.5 b60 + 0.6 ~ 100
S utml heparin 18.9 + 1.2 b14,0 + 0.4 45
8Based on seeding of 8 x 104 cellstwell
bSigni~cantly different from appropriate control (p~ 0.005)
ample S - Construction of rPF~241
Cassette mutagenesis of a synthetic PF4 gene was used to convert the four lysine2û ; r~sidues at the carboxy terminus of PF4 to two Oln-Glu couplets (see Figure 2). T~is
construction apparently retains the ~-helical secondary struc~ure (Figure 3) for this region of
the molecule wieh the concurre~t loss of heparin binding activi~.
The gene~ for rPF~241 was expressed as a fusion protein in E. coli with the same N-
, ~
terminal amino acid sequenoes as with the paren~ rPF4 molecule. The protein was cleaved
2 5~ om the E coli fusion peptide by GNBr and formic acid and purified to near homogeneity by
DEAE-sepharose~chromatography. The protein was reactive with polyclonal antibodies to PF4
and was delermined to~ possess the appropria~e modifications by an~ino acid aa~ysis.
Significantly, ~he~ purified mutant protein lacked hepalin-binding activity in the ~actor Xa
: iIthibiliOII assay.
The substitutio~s described here~can be made with the peptide fragments as well as
with the full length PF4 molecule. For e sample, C-13-241 has the follo~ing sequence:
Pro-Leu-~r-Gln-Glu-Ile-Ile-GI3l-Glu-Leu-Leu-Glu-Ser
~: : : : :
Example 6 - Inhibition of an~io~enesis bv rpF4-241
35 ~ Pusified rPF4-24i~was dried in methylcellulose discs and tested for its ability to inhibit
capilla2y gr~w~h in the ChiCkeII chorioallantoic membrane (CAM) ~y. Ev n at the }owest
concentrations tes2ed (1.25 nmoUdisc) rPF~241 exteDsively in~bited angioge~esis in the CAM
system (Figure 4). This i~ibition was even more effective than that caused by equal
concen~rations of native rPF4 as suggested by larger avascular zones on the membrane~ The
inhibitoly eff~a of rPF4-241 was not reverséd;by heparin.
~ :
: :: :
WO 93/02192 2 i ~. 3 ~ ~) 6 P~IJS92/059~
ample 7 - Inhibition of human endothelial cell Proliferation bv rPF4-241
At concentrations where native rPF4 complelely inhibits endothelial cell proliferation,
mutant rPF4-241 was at least as effective in inhibiting cell growth (Figure 5). Further tests
suggest that rPF4-241 was inhibitory at concentrations as low as 0.5 mcg/mL, a level at which
native rP~:4 has little or no effect.
Ln a test of inhibition of human umbilical vein endothelial cell proliferation by native
rPF4 and mutant rPF4-241, the rPF4 241 was shown to be much more effective than the native
rPF4 at inhibiting the prol~feration of these cells. The results of this test are shown in Figure
6.
These resul~s are remarkable in that previous theories of PF4 inhibition of
angiogenesis assumed that the PF4 effects were due to heparin binding. We have designed a
protein, retaining most of the structural features of native PW but lacking detectable heparin
binding acthrity, which is apparently more active than nztive PF4 in inhibiting angiogenesis in
vivo and endothelial cell proLiferation in vitro. Additionally, the mutant we have designed
would not be expected to interfere with heparin anticoagulant therapy.
ample 8 - Inhibition of In V~vo l~mor Growth
Normal C57BL/6J ~emale mice (~8 weeks old) were inoculated subcutaneously with
5 x 105 log phase cells of a B1~F10 melanoma tumor line. This protoeol led to progressive
~umor grow~b ~esulting i~ large ~300 r~m3) necrotic tumors after appro~matel~ 10 days,
foUowed by death of untreated animals usuaLy within three weeks of tumor inoculation.
In an experime~t to test the efficacy of rPF4 i~ preventing 1n vivo tumor growth and
; ~ ~ angiogenesis; tumor bearing animals were divided into two groups. One group was inj~ed
unth 50 f~g rPF~ (natNe sequence) in 100 f~l uf ~0 mM sodium phosphate, pH 6.5, 50 mM
s~dium chloride directly into ~he nascent tumor, dai~, ~ginning one day af~er tumor
inoculation. A control group was treated identically with carrier buf~er laclcing rPF4. I~mor
volu~ ~ measwed a~ regular intervals with digital calipers by la~oratory personnel
unin~rmed of the~ spe~c treatment received by each subject al~imal.
seven days of tumor ~noculation, co~trol animals posssssed ob~ious three
dimensional tumols, while rPF4-treated animals were essentially tumor-free ~Figure 7).
Continued treatment with rPF4 completely suppressed t~mor growth lmder these conditions
where control animal tumors became necrotic and large as seen previously with untreated
mice. The same effect was observed when rPF~241 was used as the inhibitoly agent.
This finding supports the proposition that rPF4, as an inhibitor of angiogenesis, will
possess clinical usefulness in the management of malignant melanoma and other cancers.
Progressive growlh of tumors requires new blood ~7essel forrnation which, if inhibited, may not
Z1132Q~
~1V~ 93/02192 P~/US92/05903
11 .
only restrict tumor growth, but stimulate regression of e~sting vessels, as well as enhance other
responses to malignant invasion.
The ~nding that rPF4 inhibition of in vivo tumor grow~h was apparent within three
days of the initial inoculation (of rPF4) indicates that rPF4 acts to modulate tumor growth by
S local mechanisms rather than by immunomodulation which would require a longer time course.
Additionally, rP~4 did not directly inhibit tumor cell growth in vitro. It appears, therefore,
that rPF4 was modulating the hosl's angiogenic response to the growing tumor.
It has been shown that proteins of identified structure and function may be
constructed by changing the amino acid sequence if such changes do not alter the protein
secondary struclure (Kaiser, E.T., and FJ. Kezdy l19~4l Science 223:249-235). Thus, the
subject invention includes mutants of the amino acid sequences depicted herein which do not
alter the protein secondary structure, or if the structure is altered, the biological activity is
retained.
We have conducte~ extensive research to determi~e what mutations can be made to
the carbo~y terminus of the PF4 sequence and still retain biological activity. l~ble 2 provides
a listing of examples of several mutant sequences and their biological activity.
bie 2. Mutant sequences and their biological acti~
CA~
Desi~nation Sequence Activitv
60 7~
rPF4-211 [PF4 AA 1-57] - Pro Leu 13rr Lys Lys Ile Ile Lys Lys Leu Leu Glu Ser pos.
rPF4 231 lPF4 AA 1-571 - Pro Leu l~r neg.
rPF4 241 lPF4 AA 1-57l - Pro Leu ~r Gln Glu Ile Ile Gln Glu Leu Leu Glu Ser pos.
` rPF4 302 lPF4 AA 1-571- Pro Leu ~r Gln Gln lle Ile Gln Gl~ Leu L~u Glu Ser neg~
rPF4 303 ~PF4 AA 1-571- Pro ~u ~r Lys Lys Gln Glu Lys Lys Gln Glu Glu Ser pos.
rPF4 3a7 lPF4 AA 1-57l--Pro Leu ~r Gln Ile Glu lle G~ u Glu Leu Glu Ser pos.
rPF4 308 lPF4 ~1-571 - Pro Leu l~rr Asn Asp lle lle As~ Asp Leu Leu ~;lu Ser pos.
3Q rPF4 315 ~PF4 AA 1-571--Pro Leu Tyr Gly Glu Ile Ile Gly Glu Leu Leu Glu Ser pos.
Construction of the various mu~ants was accomplished via cassene mutage~lesis OI a
synthetic gene as described in Example S above. This process is well known to any person of
ordinary skill i~ the an. The results of the research demons~rate that a high pércentage of the
mutants retained angiostatic activi~ in the CAM assay. Although not every mutant retains this
activi~, from the teachings herein it is well within the skill of a person trained in this art to
make desired mutations and dete~ine whether such activity has been retained.
In particular it should be understood that conservative substitutions of amino acids
may be made. For example, amino acids may be placed in the following classes: basic,
hydrophobic, acidic; polar, and amide. Substitutions whereby an amino acid of one class is
replaced with another amino acid of the same type fall within the scope sf the subject
WO 93/OZ192 P~/IJS92/059
21~,32l3& 12
inventioll so long as the substitution does not materially alter the bio10gical activity of the
compound. l~ble 3 provides a listing of examples of amino acids belonging to each class.
3/02~92 2~ 6 PCI`/US92/05903
ble 3.
Class of Amino Acid Example of Amino Acids
Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, T}p
Polar Gly, Ser, Thr, C~ys, Tyr, Asn, Gln
Negatively charged Asp, S31u
Positively Charged Lys, Arg, His
.... ....... .... . . . .. , " ,, ,,, .,,,, . ,.. , __.
In some instances, non-conservative substitutions can also be n~de. For example,lysi~e ma~ be substituted ~or with any of the following amino acids: Glu, Gln, Asp, Asn, Met,
Ala, Leu, and Ile. The critical factor is that these substitutions must not significantly detract
from the biological activi~ of the rPF4 or the rP~4 ~agment.
The following s~quence helps provide some additional guidance to a person skilled in
this art in maldng various substi~utions. It should be noted that this sequenc~ is meant to be
illustrative a~d not; exhaustive, and that there may be other substitutions which eliminate
heparin binding and re~ain angiostatic activity and, thus, are within the scope of the subject
:: :
vention.
A- Pro Leu l~r a10 a9 a8 a7 a6 a5 a4 a3 Glu Ser - COOH
wherein A represents all or part of the polypeptide sequen~e consisting of residues 1 through
57 of PF4; A may be or may not be present;
qvherein a10 is Ly$, Gly, Glu, GID, Asp, Asn, Met, Ala, Leu, or Ile; ~-~
:
ag is L~s, Glu? ~, Asp, ~sn, I~Set, Ala, Le~, or Ile;
a8 is C;lu, &14 Met, Ala, Leu, ne, Val, Pro, Phe, l~p, or l~r,
a7 u ~;lu, Me~, Ala, Leiu, Ile, ~l, Pro, Phe, l~p, o~
a6 is Lys, Gly, Glu, G1n, Asp, Asn, Met, ~la, Leu, or Ile~
; ~ ~ a5 is Lys, Glu, Gln, Asp, Asn, Met, Ala, Leu, or Ile;
~t4 iS Lys, Glu, Met, Ala, l~u, Ile, ~al, Pro, Phe, l~p, or l~r, a~d
,~ a3 is Gl~, Met, Ala, I~u, Ile, Val, Pro, Phe, ~p, or 13rr. 3 5~ wherein, most prefe~ably
a8 is lle, Glu, or Gln;
a~ is lle or Glu;;
a6 is Leiu, GL~, or Glu; and
~ ~ aS is L~u or Glu.
:: :
WO 93/021~2 ~ 3 2 0 6 PCI`/VS92~0~90~.
14
The amino terminus of the proteins of the subject invention can also be modified in
a variety of ways while retaining the biological activity fundamental tO the subject invention.
Most notably, the len~th of the amino terminus can be modified while retaining angiostatic or
endothelial cell inhibitory activity. Our reference here to the "amino terminus" refers to amino
S acids 160 from the arnino terminal of PF4 or its variants. As we have shown herein, up tO
57 of these amino acids can be removed with a retention of angiostatic activity. The remaining
peptide is the biologically active C-13 peptide. We have also shown that ~he C~I peptide has
biological activity. Thus, various active fragments of PF4 can be readily produced and used
according to the subject invention. It would also be readily apparent tO the skilled artisan that
other modifications of the amino terminus can be made. For example, additional peptides or
pro~eins can be added to that terrninus, and various standard chemical derivatives can be made.
Such modifications are within the scope of the subject invention so long as biological activity
is reta1ne~.
It should be emphasized that the critical feature of the subject invention is the
pr~vision of polypeptides and polypeptide conjugates which have angiostatic aetivity or
antiproliferativ~ activi~ but do not bind heparin. As used herein, "angiostatic activi~ refers
to a level of angiostatic activity which is characteristic of PF4. This level of angiostatic activity
is, for example, at~ least about 75% (and preferably, greater than about 90%) of the angiostatic
activi~r exhibited by PF4 as me:asured, for example, in the CAM assay. Methods for
determining angiostatic acti~ity are described herein and are well known and readily performed
by those skilled in this art. As used herein, the term ~tiproliferative activity" refers to a level
of antiproli~erative activitywhich is characteristic of PF4. ~his level of antiproliferative activity
iS Dt least, for c~ample, about 75% (and preferably, gre~ter than about 90%) o~ the
antiproliferative activi~ ~hibited ~y PF4 as measured, for example, by the H~JVEC assay.
Methods for dctermi~nc antiproliferative ac~ivity are described herein and are well l~nown and
; readil3r per~ormed by those skilled in this art. As used herein, ~he term "lack of heparin
binding aclivi~y refers to a relative lack of abili~ to bind heparin u~der no~mal physiological
conditioDs compared to PF4. This lack of heparin binding activi~ is, for cxample, less than
about 25% of PF4's heparin bindi~g actiYity and, preferabJy, less than about 10% of PF4's
~, 30 heparin binding activiy. The ability to bind heparin can be readily determined by various
assay~ as described herein a~d as is known by those skilled L~ this ar~
Example 9 - Modification of PF4 and rPF~-241 with Fluorescein-Isothiocvanate LE~ITC~
Purified rPF4 or rPF~241 ~5 mg in 50 mM Na2CO~ pH 9.3, 25 mM NaCI) was
treated w~th 5 mg of nuorescein isothiocyanate in a volume of 5 ml tQ modii~ the ~ee an~ino
groups. After incubation for 3 hours at room temperature in the darlc, ~e labeled protein
.
Y~'')93/02I92 ~1~320~ PCl/US92/05903
(FrPF4 or FrPF~241) was separated ~om unbound FITC by gel filtration and dialyzed into
50 mM acetic acid. A possible structure of the C-terminus of FrPF4 is shown in Figure 8.
ample 10 - Inhibition of Angio~enesis bY Fluorescein-Isothiocvanate-Con~u~ated rPF4
FrPF4 was tested for activity in the CAM assay as described above. Although FrPF4
lacked heparin binding activity, it retained full activity as an inhibitor of angiogenesis on the
CAM. The results of these assays are shown in Table 5.
ble 5. Activity of FrPF4 in the CAM assay.
Amount per Inhibithn f~%)
disc f~ ,e) rPF4 FrPF4
0 o Q
~ 22 17
33 33
. _
~ E~cample 11--Inhibition of Endothelial Cell Proliferation by FrPP4 and FrPF~241
FrPF4 and F~PF4-241 were tested separately to detern~ine their abili~r to inhibit
;~ endo~hel;.al cell p olifelation. H UVE cells w~re tested for their sensitivity to FrPF4 as
desaibed above e~cept t~at [3H]-thymidine was added to t~e cultures 24 hours after the
addition of FrPF4. l~e cultures were then incubated an sdditional 6 hours Cells were
hane~ted, washed, and radioactive thymidine incorporation into DMA was measured.,;,
As shown~ Figore 9, ~1~conjugated rPF4 was very effective, even at low dosages,
in inhibitil~g DNA~s~mthesis in human l~mbilicalvein endotheLial cells 8nd therefore i~hibhing
Ge11 prolifehtion. ~ Similar~ results were oblsi~l usin~ FrPF~241. In this GIse, the in}libition
of ~HWE cell~ prolif~tio~ w~th in~si~ concentrations of rPE;4^241 was tested using the
30~ EndothelialCell~Proliferation~Assayasd~iheda~ove. Theresultsofe~perimentsusing
FrPP4-241 are shown iD Figure 10.
: ~
- ~ ,
Exampk;12--Inhi~ition of ln Yivo~l~mor Growth bv FlTC-rPF4
B^16 Melanoma tumors were grown in C~7BL6/J mice as described pre~ously.
eatment was begun 24 hours following implantation of tumor cells (Day 1) and consisted
of ~ g/day of FrPF4 in 100~l of sodium acetate buf~er, pH 4Ø Cor~trol rnice were injected
with 25 ~g/d~y of FlTC labeled cytochrome^C in the same buffer. A statistically significant
suppression of tumor growth by FrPF4 was observed by Day 11 (E~gure 11).
-
W0 93/02192 2 1 1 3 2 0 ~; P~/US92/0590~-~
16
ample 13 - Delivelv of PF4 Activity to Specific Sites
For treatment of certain conditions, it is sometimes advantageous tO direct biological
activi~y to a specific location. For ~xample, in order to inhibit solid tumor growth, it may be
desirable to send PF4, or an analog with angiostatic properties, directly to the tumor site. l~is
S can be accomplished by coupling the PF4 (or analog) to an appropriale antibody, preferably
a monoclonal antibody. The monoclonal antibody, which can be produced using techniques
that are well-known in the art, will selective1y seek out the target site. As the antibody moves
to the desired location, it brings with it the PF4. Thus, the PF4 activity can be concentrated
at a specific location.
General means of conjugating antibodies to polypeptides such as PF4 are well known
to those skilled in the art and are discussed, for example, in U.S. Patent Nos. 4,671,95
(Rodwe11 et al.) and 4,?92,447 (Uhr et a1.). The PF4 may also be targeted ~o specific locations
via analogous conjugation with binding proteins (e.g., thrombospondin or fibrob1ast growth
factor), cell receptor mo1ecules (e.g., CD4, lymphocyte function associated antigen-1 [LFA-1],
and von ~111ebrand Pactor 1YWF~) or the complementary ligands, and non-proteinaceous
biological effector mo1ecules (e.g., ICAM-I, tumor associated-antigeDs, and prostag1andins).
For example, the monoclonal antibody, or other moiety, can be associated with PF4
at one or both pairs of 1ysine r~sidues 10cated near the carbo~cy terminus of PF4. By
associa~ing the monoclonal antibodies at these residues, the angiostatic acthity is retained
while hepann bi~ding is eliminated. Also, other amino acid residues may be substituted for
the Iysine residues be~re conjugation with appropriate moieties at these and other positioDs.
; ~ Therefore, the compou~ds descnbed here can be represented as foUows:
~ :~
F G H
: ::~:25 1 1 1 1
A Pr~ Leu ffl ~B C Ile Ile D E Leu Leu Glu Ser COOH
where:
~a) A represents all or pan of the polypeptide sequence consisting of residues 1through 5~ of PF4; A may or may not be present on said hybrid polypeptide;
(b) B, C, D, and E can be any amino acid haYing a functional group suitable for
covalent attachineltt; and
~c) F, G, ~I, a~d I are selected ~om the group consisting of monoclonal
antibodies, polyclonal antibodies, fluorescein-isolhiocyanate, fluorophores,
to~ns, ~ceU receptor molecules, non-proteinaceous biological effector
3 5 molecules, polyamino acids, polysa~harides, and chelatoss; at least one of the
moieties designated F, G, ~I, and I rnust be present on said hybrid
polypepude.
.
wn 93/o~1s2 ~ 3 2 0 PCI /US92/05~03
17
In the above representation of the compou~ds described here, the venical lines
represent chemical bonding interactions as do the spaces between the amino acids on the
horizontal line. The e~astence of specifically illustrated moieties associated at B, C, D, and E
does not exclude the possibility OI conjugation occurring at other r~sidues.
Example 14 - Conju,~ation of Carrier Proteins to PF4
It may be desirable to increase the circulating ha1f-life of PF4 tO improve its
e~ectiveness as a systemically active angiostatic complex for tumor and angiogenic disease
therapy. For example, PF4 can be crosslinked to a large carrier protein, e.g., human serum
albumin ~HSA) or immunoglobulin, by disuccinimidyl suberate (DSS) through free primaly
amino glOUpS (i.e., lysine E-amino groups or N-terminal a-amino groups; see Montesano et
al. [1982] Biochem. Biophys. Res. Comm. 109:7-13).
Purified rPF~ and HSA (10 mg and 100 mg, sespectively) were incubated with 25 mMDSS for 4 hours at room temperature. The reaction was terrninated by the addition of l~is
buffer, pH 8.0 to a final concentration of 100 mM. The resulting composition was a
heterogenous mixture of crosslinked molecules which lacked heparin binding activi~, but
retai~ed the ability to inhibit HUVEC proliferation. A control sample in which HSA was
to cytochrome-C did not i~hibit HU VEC growth.
Examp!e 15 - ~nju~ation of PEG to PF4
An alter~ative means of e~ctending the ~ life of PF4 can be achieved by covalentliDkage of PE~ ~to one or more units of a polymer such as a polyamino acid or a
polysaccharide. The polyami~o acid may be, for example, polyglutamate, whil~ t~epolysaccharide may be, for example~ polyethylene glycol ~PEG).
PEG ~s a water-s~luble, ~on-immunogenic, linear po3y~er which is available in many
~e~4e~ned molecular weight ~anges ~ O,OOO dalto~s). The dramatic ~ncre~se in molecular
veight~of PEG~ ugates~slgnifiGmtly loduces glomeru1ar Sltering of the protei~ by the
Icidne~. Also, PEG polymers may s~elically ~inder attack by proteolytic enzsnnes and
immunoglobulL~ molecules, aga~ adding to the serum life of the protein.
~ A~ivated estérs of PEG aro readily coupled to Iysine ami~o groups on a pr~tein. We
have successfully`employed pH controlled aa~l~tion (Katre, N.V., M.J. Knauf, WJ. I,aird [1987]
;; Proc. Nstl. A~ad.~ Sci. USA 84:1487-1491) to selectively modi~y the N-termi~al amine OI
peptides in tbe presenoe of up`to four unproteeted lysines. Using ~he teachiIlgs descnbed
herein, synthesis of rPF4-PEG conjugales with currently available reagents is straightforward.
: ~ ;:
:: "
~ .
WO93/02t~2 ~ 3~06 P~/US92/05
18
PEG ~an be covalen~ly attached tO rPF4 by free amino groups using a modificationof the procedure out1ined by Katre et al. (supra). Ln brief, PEG-glutaryl-N-hydrol~ysuccinimide
(MW-50009 Polysciences, Inc.) is added at a 100-fold molar excess to ~PF4 in 0.1 M sodium
borate, pH 9Ø The reaction is allowed tO proceed at 37C for 48 hours. Experiments us~g
S this prot~ol haYe yielded rPF4 with varying degrees of PEG addition. The extent of reaction
can be conveniently monitored by SDS-PAGE. Products of the reaction are clearly visible as
a series of bands, each differing by 5000 molecular weight and representing species with one
or more PEG mo~lifications.
Four lysine residues located near the C-terminus of PF4 have ~een shown to be
essential for binding of PF4 tet~amers to heparin. Since these residues are li~keiy sites of PEG
modificat~on based on surface accessibility, a subpopulation of rPF4-PEG molecules is
sy~thesized with reduced affinity towards heparin. One possible mechal~sm for the rapid
clearance ~ine~ics observed for PF4 is binding to heparin suL~ate present on the surface OI
endotheLial cells. Thus, molecules with reduced heparin binding which retain their anti-
angiogenic properties are highly advantageous for systernic effica~y. Coupling reaction
products can be fractionated by affinity chromatography on heparin agarose. Major products
eluting at different Sall co~centrations can be analyzed by SDS-PAGE to determine the range
of PEG addition in each pool. If desired, each salt fraction can be further separated by other
chromatographic ~ iques (gel ~ltration or hydrophobic interaction chromalography~ to
separate molecules with a defi~ed PEG:rPF4 ratio.
s desired to retain heparin binding for the rPF4 conjugate, the conjuga~ion
process can be carried out in the presence of heparin. The prese~ce of heparin prevents
modification of the hepann binding site of PF4 and results in a conjugate which will still~bind
heparin.
am~le 16 - Delive~l~ Molecules to PF4 Specific làr~ets
It is sometimes advanlageous to use PF4 as the targeting molecule for di~ecting the
activity of a to~ac agen~ tO a particular cell type. For ~mple, PF~ can be chemically or
genetically crossli~ked to the t~ licin A or the diphtheria t~n.
~A fusion protein comprised of PF4 and ricin A can be produce~ recomb~nantly in a
proka[yotic or eukar,Yotic host. The resulting purified tOxiII will have the high specificity for
endothelial cells or cells in close pro~mi~ to endothelial cel1s, e.g., tumor cells. Alternatively,
PF4 and ric~n can be linked with cross linkers. DSS can cross link PP4 and liCi~ A while
retaining both PF4 and ricin A activities.
3~ PF4 can also be covalently linked vnth a cross linker to photoactivatable molecules,
for example, hematoporphyrin de~ivative (HPD~. ~ter soluble carbodiimides (e.g., EDC) are
most useful in linking the acid side chains of HPD tO the amino groups of PF4. The reSUItillg
. 3 2 ~ ~
wn 93/02192 PCl /VS92~05903
19
conjugate will concentrate a~ ~ites rich in endothelial cells (such as solid tumors) and can be
activated by relatively non-toxic laser or phototherapy focused directly on the tumor site.
A~ivated HPD is known to generate active oxygen species which non-specifïcally kill nearby
cells.
s
ample 17 - Modific on of the steine Residues of rPF4
During preparation, the disulfides of rPF4 are reduced by dithiothreitol (DTI') to free
sulfhydryls, but the heparin binding activity is retained. Ib assess the biological activity of PF4
requires rernoval of the Dl~ which, by allowing the disulfide bridges to reform, obscures
whether or not they are essential for these ac~ivities.
T~e su1fhydryls of rPF4were s~ically and irreverslbly modified by prereduction with
Dl~ followed by treatment with fluorescein maleimide (~M). The ~educed and puri~ed rPF4
(5 mg in sodium carbonate bu~er, pH 8.5 lSCB]) wa~ treated vnth 10 mg of F~ for 3 hours
at room temperature. Resid~ I was removed by gel ~Itration in SCB and then dialyzed
1~ against the same buffer. The FM-rPF4 partially retained hepann binding activity. When
tested in the CAM and endothelial cell proliferation assays, FM-rPF4 exhibited inhibitory
activity indicatmg that neither free sulfnyd~yls nor correct disulfide bonds are required for the
angiostatic activity of PEi4.
This FM-m~dified rPF4 may possess some utility as an alter~ative endoth~Lial oell
:
labeling or inhibiti~g colnpouDd but, most importantly, it indicates that the 6~ysteine residues
of PF4 are also appropr at0 targets for conjugating or cross lin~g PF4 to other molecules f~r
diagnostic~or therapeutic applications.
It should be underst~d that the ~xamples and embodimen~ described herein are for: ~ :
illwtrative p~s o~ and that various mod~cations or changes in ligh~ thereof will be
; ~ suggested to persons s~ilbd i~ the art and ~re to ~ included vnt~in the spirit and putview of
this appl.;calion and t~e scop~ of the appended claims.
~::: : :
~ , .
~ .
::: ::: : ::
; '
. ~
