CA2182494A1 - Antibodies which bind the g-csf receptor extracellular domain and methods of treatment - Google Patents

Abstract

The present invention relates generally to cytokine interactive molecules, such as antibodies and other immune reactive molecules, agonists and antagonists. The present invention also provides methods for assaying for the presence of cytokines or receptor associated proteins such as kinases and their function.

Description

~824 WO 95/21864 ~ ~ P~ 5 1696 ANTIBODIES WHICH BIND THE G-CSF RECEPTOR EXTRACELLULAR DOMAIN AND
METHODS OF TREATMENT
E~ELD OF THE INVENTION
5 The present invention relates generally to cytokine interactive molecules, such as antibodies and other immune reactive molecules, agonists and ~ The present invention also provides methods for assaying for the presence of cytokines or receptor associated proteins such as kinases and their function.
Throughout this ~ , unless the context requires otherwise, tbe word "comprise", or variations such as "comprises" or " , ,,", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elemems or integers.
BACI~GROUND OF THE INVEN~ON
(~ ' ~colony- - factor(G-CSF)stimulatestbel,.. ,l;F ~;.. and ;.. of neutrophil precursors via interaction witb a specific cell surface receptor, the G-CSF receptor (G-CSF-R).
Altbough the G-CSF-R has been cloned (1) and is ru~Liu~lly active in several different cell types (2), little is known about the of signal 20 1~,..._-1`.. 1;-,.- The G-CSF-R is believed to consist of a single chain tbat is activated through ligand induced l ~ ;. ,. . (3) as has been shown for the c.~ il..u~vk,.ill and growth hormone receptors (EPO-R, GH-R) (4). The G-CSF-R does not contain an intrinsic protein kinase domain (1) although tyrosine kinase activity seems to be required for i of the G-CSF signal (5). JAK kinases (6,7) are receptor-25 associated proteins which are rapidly PhOD~LV~ after receptor activation. Inparticular, Tyk2 is 1' .' ~- ' following interferon ~-receptor aE~ R) activation (8) and JAK2 following the binding of EI'O (9), GH (10) and ' 3 (IL,3) (11) to their respective receptors.
In work leading np to the present invention, the inventors v~ i,, ' early 30 signal events resulting from the association of G-CSF with its receptor and the role of JAK1 and JAK2.

i `2 1~4~ ~

- 2 --In accord~mce wilh the present inverltion, antibodies were prepared to the , ~r~ r domain of G-CSF-R. It has now been ~ ~yl;DLI2;;y discovered that G-CSF interaction with G CSF-R is required for tyrosine yLvDyLulyla~iuu of JAK
kinases. The antibodies of the present invention now provide for a method of 5 inhibiting G-CSF bindir~g to its receptor and, by ~ r~ , pLùjylluly' by JAK kinases. The present invertion A ~ ' , therefore, a method for treating G-CSF related disease conditions or JAK1 and JAK2 yhuDyllulylaLiull associated disease conditions which result from G-CSF mteraction with its receptor.
10 SUMMARY OF THE ~ TION
One aspect of the present invention is directed to a c~ ;.... comprising arltibodies or pa~ts, fragments or derivatives thereof to G-CSF-R l~YhP~ r domam.
Another aspect of the present invention relates to amtibodies to the deflned above.
Yet ~mother aspect of the present invention ,' a method for inhibiting, reducing or otherwise decreasing tyrosine yll~DyLulylaLiuu of JAK1 or JAK2 in a mammal, said method ~ to said mammal a binding effective ar~ûunt of an arltibody or a part, fragment or derivative thereof interactive with G-CSF-R eY~r.'ll ' domain.
Still yet another aspect of the present invention ~ ,' a method for inhibiting, reducimg or otherwise decreasing G-CSF interaction with G-CSF-R in amammal, said method ~ to said rnammal, a binding effective amoumt of an antibody or part, fragment or deriYatiYe thereof interactiYe with G-CSF-R ~Y~ r domain.
In still yet another aspect of the present invention, there is provided agorlists and: ~ to G-CS]F-R.
BRIEF DESCRIPIIO~ OF THE EIGURES
Figure 1 is a ~ h I~r showing tyrosine l' , ylaLiu 30 of JAK kinases in respol~se to GCSF (AML~193 cells).
AML-193 cells ~were incubated with rhG-CSF (100 ng/ml) for the times indicated (minutes) and Iysed. Tyrosine y~J~yLvly' ' proteins wee ?~
WO 95/21864 PCT/US9!j/01696

- 3 -' with Al~ IU;~IIIC antibody 4G10 (aPY), Upstate r ' ' ' O~Y Inc. (usr,. The r '~ proteins were separated by sodium dodecyl sulfate-pvl~ y' ' gel ~' , ' (SDS-PAGE) 6% w/v gel and analysed by Western blot using either JAK1 antiserum (M7) (oJAK1, JAK2 S antiserum (UBI) (aJAK2) or TYK2 antiserum, Santa Cruz Inc. (aTYK2). The mobilities of the pre-stained molecule weight markers are shown on the left.
Figure 2 is a ~ IC~I~ showing tyrosine, ' ,' y' of JAK1 and JAK2 in response to G-CSF (CHO-6All cells).
CHO-6A11 cells were incubated with rhaCSF for the times indicated 10 (minutes0 and Iysed. JAK1 and JAK2 proteins were , , ' using purified JAKl antiserum (M7) (left panel) and JAK2 antiserum (UBI) (right panel).
I~c were separated using SDS/6% w/v PAGE and subjected to Western blot analysis using i~ ~ antibody 4G10 (aPY) (upper panel).
The .l was stripped of antisera and re-probed using M7 and JAK2 15 antiserum (lower panel) ~' ~ equal sa~nple loading in each lane. The mobilities of the pre-stained molecular weight markers are shown on the left.
Flgure 3 is a r 1. ~ showing JAKl and JAK2 autokinase activity in CHO-6All cells.
CHO-6All cells were incubated with rhG-CSF for the times indicated 20 ~minutes) and Iysed. JAKl and JAK2 proteins were , , ' using purified JAKl antiserum (M7) (left panel) and JAK2 antiserum (UBI) (right panel).
were washed and suspended in an equal volume of kinase buffer (20) containing [y-32P]ATP (0.25~Ci/ml) for 30 minutes at room i, T , ~, were analysed by SDS/6% w/v PAGE. The gels were treated 25 with 1 mol/L KOH 55C for two hours to remove 1' ,' and ive bands were visualised with IMAGEQUANT software on a rl`~.~l-l.l~.T.. ~,. system (Molecule Dynamics).
Figure 4 is a, ' O ,' I~l c~ showing association of JAKl and JAK2 with the G-CSF receptor.
AML,193 cells were incubated with rhG-CSF (lOvng/ml) (+) or medium (-) for 10 minutes. Cells were Iysed and G-CSF-R proteins , , ' using the ' ' antibody LMM741. Following SDS/6 % w/v PAGE analysis, proteins ;

4 2~ .`O.CS6 were cl~L~ ly transferred to nitrocellulose and ~ ' ' with either JAK2 antiserum (M7) (left panel) for JAK2 antiserum (UBI) (right par~el).
Figure 5 is a l' ,~,.~L~ ..C~ " showing that different cytokines result m tyrosine phJ~ u~ iull of specific JAK kinases in AML-193 cells.
AML-193 cells were incubated with either G-CSF, GM-CSF or IL-6 at 100 ng/ml or medium (control) for 10 minutes and I , ' with t~lu~ amtibody 4G10 (~py). T r 1 ' ' proteins were separated by SDS/6% wlv PAGE and analysed by Western blot with either JAK1 antisrum (M7) (IYJAKl), JAK2 ar~tiserum (UBI) (~JAK2) or TYK2 antiserum (Santa 10 Crus) (CYm2). The rlobilities of the pre-stained moelcular weight markers are shown on the left.
m~,TA~ n DESCR~rION OF l~ ) EMBODIMENTS
The present inventiorl relates generally to antibodies to G-CSF-R Preferably, 15 the antibodies are direo~ted to the P~ AI domain of G-CSF-R and more ~cu~ul~ a ,c~ form of this molecule. The present mver~tion is also directed to parts, fragments or derivatives of such amtibodies which still interact with the G-CSF-R. The antibodies of the present invention are generally in isolated or purifled form meaning that a, , comprises at least 25%, more preferably at least 35 %, even more preferably at least 45-50%, still more preferably at least 60-70% and even still morc preferably at least 80-95% of the antibodies as determined by weight, ~ii~i~y or other converlient means. Alternatively, the arltibodies may be present in the form of isolated culture . t, tissue extract, serum or whole blood or ascites fluid.
Preferably, the G-CSF-R is of " origin such as from a h =, livestock aDimal (e.g. cow, horse, sheep, goat or dor~key), laboratory test animal (e.g.
mouse, rat or rabbit), companion animal (e.g. dog or cat) or captive wild animal (e.g.
dingo, fox, wild boar or k~mgaroo). The most preferred receptors are of human and laboratory test animal origin (e.g. murine species).
Where the antibodies a~re polyclonal antibodies. they may be generated in any convenient host including a human, livestoclc animal, companion animal or captive wild animal as ~ above. Where the antibodies are ~ ' antibodies, wo 95/21864 2 1 8 2 ~ 9 4 ~ . 1696 they may be prepared in any convenient hybridoma such as of murine (i.e. mouse or rat) origin.
The receptor used to generate tbe antibodies may be the whole receptor such as in purified, partially purified or isolated form including in the form of isolated

5 membrane ~ The receptor may also be produced by procedures or syntbetic procedures or a ~ ' thereof. In a ~
preferred b~ ' t, a fragment of the receptor is uæd which, in an even more preferred: b~ " t, is fused to a suitable carrier or marker molecule such as FLAG proteinoraL~aline l' ~1' (AP). ~ S-transferase (GST) may 10 also be used.
According to tbis preferred ~ there is provided an antibody or part, fragment or derivative thereof interactive with a non-full length G-CSF-R fused to a carrier mokcule. The non-full lengtb portion of the receptor acts as a hapten and is or forms part of the; " ' domain. Preferably, the carrier molecule is FLAG
15 or AP. ~ , the carrier molecules is GST.
The resulting fusion molecule may then be used to generate polyclonal or ' antibodies which may undergo ' ' procedures to provide a ~ , of ' "~" for example, ~ domain-reactive receptor antibodies.
The terms "antibody" or "antibodies" are used herein in their broadest sense and include parts, fragments, derivatives, I..- -lne" ~ or analogues thereof, peptide or non-peptide equivalents thereof and fusion molecules between two or more antibodies or between an antibody and another molecule. The antibodies or other v~ molecules may also be in ' or synthetic form.
~ ,, the present invention , ' mutants and derivatives of the antibodies which interact with G-CSF-R; "' domains. Mutants and derivatives of such antibodies include amino acid ' , deletions and/or additions. r~ i~.. ~ amino acids may be replaced by other atnino acids having like properties, such as h.~L~IJLvbi~ y, L~ J, cl~l~ ., vil~, bulicy side 30 chAins, interactive and/or functional groups and so on. GI~ L;V,I variants and hybrid antibodies are also . ,' ' by the present invention.

~4~ 4 WO 95~21864 . ; 2 PCT/US95/01696 ,.

- 6 -Amino acid ~ are typically of single residues; insertions usually will be of tbe order of about 1-10 amino acid residues; and deletions will range from about 1-20 residues. Dcletions or insertions preferably are made in adjacent pairs, i.e: a deletion of 2 residues or insertion of 2 residues.
The amino acid variants refenred to above may readily be made using peptide synthetic techniques vell known in the art, such as solid phase peptide synthesis and the like, or by l~ ' DNA , ' Techniques for making ~
mutations at ~ sites in DNA having known sequence are well known, for example through M13 ~ The , of DNA sequences to produce 10 variant proteins which n~anifest as ~ l, insertional or deletional variants are well known in the art.
Other examples of ~c ' or synthetic mutants and derivatives of the antibodies of tbe presenl invention include single or multiple ' ddetions and/or additions to any molecule associated with the ligand such as ~
15 lipids and/or proteins o~r ~ iJ~. Naturally occurring or altered ~Iy~,u;,y' forms of the subject antibodies a~re ~ , ' ' by the present invention.
Amino acid alterations to the subject p~ly~li 1~ , ' ' herein include insertions such as amino acid and/or carbo~ryl tenninal fusions as well as in~a-sequence insertions of single or multiple amino acids. Generally, insertions within 20 the amino acid sequenc~ will be smaller ~an amino or carboxyl tenminal fusions, of the order of about 1 to 4 residues. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a l~r~ J site in the protein. Deletional variants are ~ l by the removal of one or more amino acids from the sequence. S ' ' variants are those in which at least one 25 residue in the sequence ~las been removed and a different residue inserted in its place.
Such ' may be made in _ ' with Table 1.
. .

W0 95/21864 ~ .. .. 6~;

- 7 -S Original Residue Exemplary S.. h-l;~,l;
Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro; Val His Asn; Ghn lle Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val lle; Leu The terms n ~ and "~livali~w" also extend to any functional chemical , v of the ântibodies ~ by their increased stability and/or 30 efficacy in vivo or in vitro. The terms "analoguer and ~d~,livdtivw" further extend to any amino acid derivative of the antibodies as described above.
Anh~ody analogues ,' ' herein include, but are not limited to, ...1;1~.-:;....~ to side chains, iu.,UI~ '' of =tural amino acids and/or d~liv ~ the molecules and the use of cLu~ali~la and other methods which 2~
WO 95121864 ~ PCT/US9S/01696

- 8 -impose . ~ ' constraints on the antibodies. Examples of side chain ".,.. I.r~ , ' ' by the present invention include . - -~ of amin~
groups such as by reductive aL~ylation by reaction with an aldehyde followed by reduction with NaBH4; A.. 1 - ;1... with ~_lly'~ ' ~ acylation with acetic 5 anhydride; ~alballuylaLiullOf amino groups withcyanate; Lli~,lu~.,~ylaLiunof amino groups with 2, 4, 6-i ub~,~ sulphonic acid CI NBS); acylation of amino groups witb succinic anhydride and Lhal~Lu~llLI~I;~ anhydride; and ,uyli~uAylaliull of Iysine with pyridoxal-5'-phosphate followed by reduction with NaBH~.
The guamdine group of arginine residues may be modified by the for~nation 10 ûf h~,~,u~ products with reagents such as 2,3- ' IY~A~I and glyoAal.
Thc carboxyl group may be modified by ~,albù~h~PIl~ activation via O-~lii~u. ca formation followed by subsequent ~ ;v;~iDaLiull, for example, to a amide.
Sulphydryl groups may be modified by methods such as calbuAy~, tlylaLiu with iodoacetic acid or ~ '7, performic acid oAidation to cysteic acid;
formation of a mi-Aed ' 'i ' with other tbiol ~ , ' reaction with maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- ' ' U..._ll " ', 4- ' ' , .' y:s~~L~ acid, 20 ~1~1., y chloride, 2- - u~."u.i 1 ~ ., ,' and otber ~bull,u~l~,Liu,l with cyanate at aL~aline pH.
Tryptophan residues r~ay be modified by, for eAample, oAidation with N-b~ or alkylation of the indole ring with 2-hydroAy-5~ ub~yl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered 25 by nitration with i , ' to form a 3 ~IUDiUC derivative.
l~f~ ;r~ of the imidazole ring of a histidine residue may be ~
by alkylation with iodoaoedc acid der:ivatives or N~b~ u~y- with Examples of i I ,, unnatural an~ino acids and derivatives during 30 protein synthesis include, but are not lilnited to, use of nnrl~..r;n~ 4-amino butyric acid, 4-amino-3-hydroxy-5-~.1J.~ . acid, 6 ' acid, t-wo g~/21864 ~ ~ 8 ~ ~ 9 4 ~ 1696 g L ~Igly~,~, nonaline, ~ ly.,~, ornithine, sarcosine, 4-amino-3-hydroxy-6-~,ILy'', acid, 2-thienyl alanine and/or D-isomers of amino acids.
Crosslinkers can be used, for example, to stabilise 3D ., r.- ;.~--- using homo~ ' such as the ' ~ ' imido esters having (CH2)n 5 ~pacer groups with n = 1 to n = 6, ~ hyde, N ~Lui~ ' esters and hetero-' r '- ~ reagent which usually contain an amino-reactive moiety such as N hJLu~ ' and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or ~,~uL~vLh lid~, (COOH). In addition, peptides could be ~ ~ '~ cnnc~inPd by, for example, ilI~.UllJUlati~ of C~, and N"-10 11.., h~lallPillu acids, ~ ' of double bonds between C,, and C~, atoms of aminoacids and the formation of cyclic peptides or analogues by _h ' ' ~, covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
The present inverltion, therefore, extends to amino acid and/or chemical 15 analogues of the subject antibodies having the identifying ~ of being interactive with the eY~rar~ll ' domain of G-CSF-R.
Accordingly, reference herein to an antibody includes the naturally occurring molecule, ' t, syrlthetic and analogue fornns thereof and to any mutants, derivatives and human and non-human ~ . thereof including amino acid and 20 ~ ly~,u~y' variants.
The ar~ibodies of the present invention may be used to develop a new range of IL~ ...~ and diagnostic agents. For example, the antibodies or fragmerlts or derivatives thereof may act as 1 l and be useful, for example, in the treatment of G-CSF related disease conditions which result from G-CSF interaction with its25 receptor, including JAK family ~Lu~Luly' related disorders (e.g. some cancersand tumours). They rnay also be used for screening for agonists useful, for example, where G-CSF interaction (or JAK1 or JAK2 ~Lu~h~ly~ ) is to be promoted.
Normal, abnormal or mutant receptor structure or receptor expression may also be,'t ' tbrough -vily studies.
According to this latter ~ there is ~ ,' ' a method of detecting a GCSF-R on a cell in a biological sample, said method ~
cor~acting said sample with an antibody capable of binding to the; ~" ' domain W0 9512186~ 2 ~.8 2 4~ .cgc of said G-CSF-R ' " ' to a solid support for a ti~ne and under conditions sufficierlt for an antibody G-CSR-R complex to form and then detecting the preserlce of said complex.
In one preferred method, the complex is detected by contacting the complex 5 with a second antibody agaiDst tbe first mentioned antibody with tbe second antibody being labelled with a reporter molecule. Alternatively, the first antibody itself is labeiUed with a reporter molecule.
The first and second antibodies may be polyclonal or ' ' and botb are obtamable by of a suitable ani~nal with the mteractive molecule and 10 either type is utilisable i~ the assay. The methods of obtaining botb types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory anilnal with an effective amount of ~ molecule, or antigenic parts tbereof, coUecting ser3m from the animal, and isolating specific antl~odies by any of the known; ~ ."1 15 techniques. Altbough antibodies produced by this method are utLisable in vL~aUy any tyype of assay, they are generally less favoured because of the poter~ial L.~lu~_~;.y of the product.
The use of ",...,.,~ antibodies in the above J is L~ ul~uly preferred because of ~he abLity to produce them Ln large quantities and the 20 I ~ ~ of the prodluct. The ~ I of hybridoma cell lines for )L
ar~tibody production derived by fusing an Lmmortal cell line and IY~ JhJ~Y~j~
sensitized against the i..,..--- .,,. .:~ I , can be done by techniques which are well known to those wiho are skilUed in the art. (See, for example Douillard andHoffinan, Basic Facts about Hyl~idu~s, in f. I - ' of T ~c~ Vol II, ed.
25 by Schwartz, 1981; Kohler and Milstein, Na~ure 256: 495-499, 1975; European Jou~ of T ~z 6: 511-519, 1976).
The presence of a G-CSF-R may be ~ " ' Ln a number of ways such as by Western blo~ting and ELISA procedures. A wide range of techniques are avaLable as can be seen by reference to US Patent Nos. 4,016,043,3û 4, 424,279 and 4,018,653. These, of course, Lnclude both sLngle-site and two-site or "sandwich~ assays of the non~ ., types, as weU as in the traditional ; ~f82~
WO95/21864 P~.1. . 1696 Cuuu~ binding assays. These assays also include direct binding of a labelled antibody to a target.
Sandwich assays are among the most useful and commonly used assays and are L _ '- ' ~.~/ useful in the present invention. A rumber of variationS of the5 sandwich assay technique exist, and all are intended to be ~ by the present invention. Briefly, in a typical forward assay, an antibody is brought into corltact with a biological sample ~ ~ cells potentially carrying G-CSF-R. After a suitable period of - for a period of time sufficient to allow for~nation of an antibody G-CSF-R complex, a second antibody specific to the complexed antibody, 10 labelled with a reporter molecule capable of producing a detectable signal, is then a~ded and incubated allowing sufficient time for the formation of a tertiary complex.
Any unreacted material is washed away, and the presence of the second antibody bound to the first antibody is; ' by UbD~ ' of a signal produced by the reporter molecule. The results may either be qualitative, by simple Ub..~ UU of lS tbc visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include using a first arltibody (against G-CSF-R) labelled with a reporter molecule. In addition, the antibodies or cells carrying G-CSF-R may be ' onto a solid support.
Suitable solid supports include glass or a polymer, the most commonly used 20 polymers being cellulose, p~lyd~1y' ', nylon, ,UUlyDiy1c1~, pol,vvinyl chloride or pUlyy1u~l~,llc. The solid supports may be in the form of tubes, beads, discs of UUL.II~' ', or any other surface suitable for conducting an y. The bindir~ processes are well-~nown in the art and generally consist of cross-linking covalently bmding or physically adsorbing molecules to the polymer.
~Reporter molecule", as used in the present, is meant a molecule which, by its chemical nature, provides an '~icdly ;,~ .l signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or ~ ~. The most commonly used reporter molecules in this type of assay are either enzymes, flUUll ~ ~ or " " ' containing lecules (i.e. .
30 and ' ' molecules.
In the case of an enzyrne y, an enzyme is conjugated to the ~, molecule or an antibody thereto generally by means of W0 95121864 P~ 696 g ' ' ' ~ or periodate. As will be readily recoglused, however, a wide variety of different, ~ _ techr~iques exist, which are readily available to the slcilledartisan. Commonly use~ erc~ymes include l~ peroxidase, glucose oxidase, bet~ and aDcaline l' , , amongst others. The substrates to be 5 used with the 4pecific er~nes are generally chosen for the production, upon hydrolysis by the Cu~ enzyme, of a detectable colour change. Examples of suitable enzymes include allcaline l- .' and p~ co It is also possible to employ 11 ...,~,. substrates which yield a fluorescent product rather than the ~1.1, ,, substrates noted above. Generally, the enzyme-labelled antibody is lû added to the ~., molecule-reccptor complex, allowed to bind, and then tbe excess reagent i~ washed away. Alternatively, an enzyme labelled molccule is used. A solution corltairling the a~l)lu~ substrate is then ad~ed to the terti~ry complex. The substrate will react with the en2~ne linked to the dlll.ibOdy/- ' '-~. molecule, givmg a qualihtive visual signal, wbich 15 may be further ~ l, usually ~ ...r~ ly, to give an indication of the amount of hapten which was preserlt in the sarnple. rReporter molecule" alsoextends to use of cell ~_ or inhibition of __' sucb as red blood cells on latex beads, and the like.
Al~ernately, fluorescent , . such as fluorescein and ' , may 20 be chemically coupled to antibodies without altering their binding capacity. When activated by -- with light of a particular Wd~ l, tbe n,....~. 1..~, ..
Iabelled antibody adso:rbs the light energy, mducing a state to excihbility in the molecule, followed by emission of the light at a 1 ,.. ~. .;-l;. colour visuallydetechble with a light Ilil,~V~._VIJli:. T r~ and EIA techniques are botb 25 very well established in the art and are yolL~,~ddll~ useful for the present method.
However, other reporter molecules, such as ' ., ~ - or ~: ' molecules, may also be employed.
lBy detechng G-~CSFR by the yll ' aberrant receptors may be discerned tbus providing a usefuT screening procedure for potential disease condihons.
The present invenhon also provides a l~h - ~ '1 _ , '- Cul~
an effechve amount of anhbodies capable of binding or otherwise associating witb the r~r~ r dor~ldin of G-CSF-R and one or more ~ y accephble ,, W095/21864 ~182g4 1~ . .C~6 carriers and/or diluents. The active ingredients of a p~ C~
c, , ~ the antibodies are - .' ' to exhibit excellent therapeutic activity, for example, in the treatment of aCSF disease conditions such as some cancers, in an amount which depends on the particular case. For example, from about 0.5 ~Lg 5 to about 20 mg per kilogram of body weight per day of antibody may be ~ ' ' Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be r ' ' ' daily or the dose may be ~u~ reduced as indicated by the exigencies of the i' situation.
The active compound may be ' ' in a convenient mar,ner such as by the oral, ~ILav~ (where water soluble), ~ , - ir¢ranasal, or ~r. y routes or i~nplanting (eg using slow release molecules).
Depending on the route of ' the active ingredients which comprise the ~ molecules may be required to be coated in a material to protect 15 said ingredierlts from the action of enzymes, acids and other natural conditions which may inactivate said irgre-liPntc In order to administer the molecules by other than parenteral: ' they will be coated by, or ' ' with, a material to prevent it~ i~liv_ For example, the v~ molecules may be ' ~ in an adjuvant, co--' ' 20 witb enzyme irlhibitos or in liposomes. Adjuvant is used in its brvadest sense and includes any immune stimulating compound such as interferon. Adjuvants ~ hPrPin ~ ~ r~C~rrinnlc~ non-ionic surfactants such as p~lyv~ ~
oleyl ether and n ~Aad~,yl p~ ether. Enzyme irlhibitos include pancreatic trypsin irlhibitor, d~.Jylu~y'r' I, ' . ' (DEP) and tras,vlol. Liposomes include25 water-in-oil-in-water emulsions as well as cu.... ' liposomes.
The active , ' may also be l "y or hdlalJ. ' ~.y. Dispesions can also be prepared in glycerol, liquid ~,ul~.,lhjl~,~
glycols, and miAtures thereof and in oils. Under ordinary conditions of storage and use, these ~ corltain a ~ ", v~ , to prevent the growth of 30 ~ .,lU~ ~
The l' forms suitable for injectable use include sterile aqueous solutions (where water soluble) or .l~ and sterile p~wdes for tbe W09i5/21864 ~Z4~4 r~"~ 696 _ 14 -l~ r~ Or sterile injectable solutions or dispersion. In all cases the form must be sterile i~nd must be fluid to the exterlt that easy a.~lhl~i~iliLy exists.
It must be stable under the conditions of ,.. -.r~ ~I..G i~nd storage and must be preserved against tbe 1 action of lUil,lU"'~ '' --- ` such as bacteria and S fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid pol~d~l~.c glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be i, for example, by tbe use of a coating such as lecitbin, by tbe o~ tbe required particle size in tbe case of dispersion and 10 by tbe use of .,. . r,.. ~ The yl~ lLiulla of the action of ' _ can be brought about by various ~ ;,.l and antifungal agents, for example, parabens, c' ' ' l, phenol, sorbic acid, tbimerosal, and the like. In many cases, it will be preferable to inciludc isotonic agents, for example, sugars or sodium chloride.
Prolonged absorption of tbe injectable _ , can be brought about by the use5 in the , af agents delaying absorption, for example, aluminium and gelatin.
Sterile injectable solutions are prepared by , ~ the active I ---r in the required amount in the ~y~l, solvent with various of the other ingredients ' above, as required, followed by filtered ~ ' Generally, 20 dispersions are prepared by ;~ the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those ' above. In the case of sterile powders for the of sterile injectable solutions, the preferred methods of I , are vacuum drying and the freeize-drying technique which yield a powder of the active 25 ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
When the antibodies iare suitably protected as described above, the active, compound may be orally ~ ' i, for exaunple, with an inert diluent or with an ' '- edible ci~rrier, or k may be enclose~ in hard or solt shell gelatin cap~ule, 30 or it may be c~ l into tablets, or it may be i r ' ~ directly with the food of the diet. For oral therapeutic the active compound may be i~Cul~ ' with excipier~ts and used in the form of ingestible tablets, buccal tablets, ~ w09s/21864 2~2~ 1696 troches, capsules, elixirs, ! ~, ' , syrups, wafers, and the like. Such C~ and l"'Y~ ;""` should contain at leasit 1% by weight of active compound. The percerltage of the , and l y ,' ;~ - ~ may, of course, be varied and may . .. -~y be between about 5 to about 80% of the weight of the S unit. The amount of active compound m such Ih '1' -- ;- ~11y useful ~ r "' in such that a suitable dosage will be obtained. Preferred , or I
accordrng to the preserlt inverltion are prepared so that an oral dosage umt form contarns between about lO ~g and 2000 mg of active compound.
10 The tablets, troches, pills, capsules and the like may also contam the followrng: A
br~der such as gum i ~ ~, acacia, corn starch or gelatin; excipients such as dicalciumphosphate; a .~ ;.¢ agent such as corn starch, potato starch, alginic acid and tne like; a lubricant such as ~ stearate; and a sweetening agent such a sucrose, lactose or saccharin ~nay be added or a f~avouring agent such as15 I~,Y~ t, oil of Willt~ ,...4 or cherry flavouring. When the dosage urlit form is a capsule, it may corltain, in addition to rnaterials of the above type, a liquid carrier.
Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active 20 compound, sucrose as a sweetening agent, methyl and ylul~yly~a~ as yl~ D~ a dye and flavouring such as cherry- or orange flavour. Of course, any material used in preparing any dosage unit form should be 1 'ly pure and non-toxic m the amourlts employed. In addition, the active compound may be , ' imto sustamed-release ~ ~y ~ and iri ' As used herem "~ y acceptable carrier and/or diluent" includes any amd all solvents, dispersion media, coatings, ~- -;1 r ;_1 and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for l)h " .,1;. ~1 active substances is well known in the art. Except rnsofar as any . ~, ' media or agerlt is , ' '- with th~e active ingredient, use 30 thereof rn the Ih ~ . , is . ~ ,,' J active u~gredierlts cam also be , ' iDto the wo 95121864 ~X 4~3 P~ 696 -- l6 -It is especially ~1~ ~ to formulate parerlteral c~ .. in dosage unit form for ease of: and uniformitv of dosage. Dosage unit forrn as used herein refers to physically discrete units suited as ur~tarY dosages for the subjects to be treated; each unit containing a I " ' quantity of 5 active material calculated to produce the desired therapeutic effect in association with the required l3. ~ carrier. The ~ . for the novel dosage unit forms of the invention are dictated by and directly depeDdent on (a) the unique ;, of the active rnaterial and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of C~ r _ such an active 10 rnaterial for the treatlnent of disease in living subjects haYing a diseased condition in wbich bodily health is irnpaired as herein disclosed in detail.
The principal active ingredient is ~.. ~1.., , l ;J for convenient and effectivein effecti~le amounts with a suitable rl -- .- - -- ; ~11J acceptable carrier in dosage unit form æ 1 r ~ disclosed. A unit dosage form can, for example, 15 corltain the principal active compound in amounts ranging from 0.5 llg to about 2000 mg. Expressed in ~IU~J~ ' , the active compound is generally present in from about 0.5 ~g to about 2000 mg/ml of carrier. In the cæe of . containing active i ligref~ ntC~ the dosages are ~' by reference to the usual dose and manner of ~ .,..;.... of the said ~ ntc In a most pref~rred e l - t, the antibodies used in a r.1.~.. ----.. l;. 1are antibodies or mutants or derivatives thereof. Most preferably, the antibodies are .~ .'1 antibodies.
The preserlt invention is further described by reference to the following non-limiting example.
EXAMPLE .
To generate a model system for analysing signali i by the human G-CSR-R, CHO-Kl ceDis were transfected with the human aCSF-R (CHO-6Al 1). The ceDi line CHO-6All was generated by i ~ with the pEE6.HCMV.GS expression 30 vector (12) carrying a full length cDNA encoding the G-CSF-R. Theæ cells expressed a~ 60,~00 receptors per cell (as ~- by binding of Il2~G-CSF). ~ alld polyclonali antibodies to the G-CSF-R were produced to ~ W0 95~21864 ~ ~ 8 ~ 4 ~ ~ r~ c~ -enable detection of receptor-associaoed proteins. Polyclonal rabbit antiserum, designated R55, was generated by with a pGEX/G-CSF-R bacterial fusion protein, , _ the 1 ~ ; receptor domain. A BamHI fragment of the receptor cDNA encoding amino acids 17-345 was ioserted into the BamHl site S of PGEX-l (13). The ' ' antibody to the G-CSF-R, LMM174 was raised against CHO-6All cells.
- The ' I antibody LMM741 and rabbit serum R55 detected the receptor in cell Iysates from CHO-6Al 1 and the human monocytic leukaemic cell line AML,193 (14) (Figure lA). Two protein of apparent molecular mass 130 and 110 10 kDa were detected in CHO-6All Iysates (Figure lA, lane D) and were not present in, r ' ~ CHO-Kl cell Iysates (Figure lA, lane C) or when rabbit serum was used (Figure lA, lanes A and B). Three proteins of apparent molecule mass 145, 135 and 115 kDa were detected in AML,193 cells (Figure lA, lane E).
The different sized proteins observed are likely to be due to differently ~ .va,y- ' ' 15 forms of the receptor (2). AML~193 cells ~ in response to G-CSF whereas CHO-6All cells did not (Figure lB).
S ' of CHO-6Al 1 cells with G-CSF induced a tyrosine l~hva~lluly' band of 130 kDa which was ~ , ' by antiserum to JAKl (M7) (Figure 2A). M7 was generated by with a pGEX/JAKl bacterial fusion protein 20 spamling the first kinase-like domain of JAKl (amino acids 576-825) (8). Whencompared with JAK2 antiserum, M7 detects a protein which migrates slower on SDS-PAGE than JAK2, suggesting tbat it is specific for JAKl, despite the close homology of domain 1 between JAKl and JAK2 (15).
rLva~JLul~' of JAKl was also observed by ~ , witb M7 25 and '' ~ with anti-~ a~ (Figure 2B). When this blot was stripped and re-probed with M7, the result (Figure 2B, lower panel), showed thatthere was equal sample loading in all lanes. Tyrosine ~J~Lv~yl~ of JAKl was observed after 2 minutes of G-CSF ' was maximal between 10 and 20 minutes and still evident, although ' _ after 60 minutes.
Tyrosine ~Lo~},Lu-.y of JAKl in response to GCSF was also obsened in the AM~193 cells (Figure 2C). Increased levels of l - , ~ I~ have proved to be an excellerlt index of the ~..JI~, of particular protein tyrosine kinases in wo ssnls64 ~ O.,. 1696 signal ~ - pathways (15). The h~ lv. of JAK1 in ;~ events triggered by G-CSF was thus strongly indicated by these data.
An in vitro kinaso assay (11) was used to test whether the increased tyrosine yl~-~.iuU of JAK1 correlated with an increase in irltrinsic kinase activitv.
S When incubated with [ y-3~P]ATP, JAK1 showed an increased capacity to ~ ~1 y' inresponsetoG-CSF ' (Figure3A). Ahighermolecular weight band of unknown ider~ity was also r' A- y' ' ~ The kinase activitv appeared to be ma~imal. at 30 minutes. r ~1~ acid analysis from kinase assay samples of 0 and 30 minutes after G-CSF ! ' comSrmed that JAK1 was lû I ' . ' y' ' on tyrosine residues (Figure 3B). Some increase in I ' I ' yLIllu on serine residues was also observed. This is in cor~rast to the ui,.~. V~iUIl that JAK2 , 32p into tyrosine and threonine residues (9). Thus, the elevated kinase activity of JAKI upon activation of CHO-6A11 cells by G-CSF, ' the likely ill.ulv.~,... of this molecule in G-CSF-mediated signal i To determine wllether JAK1 is physically associated with the G-CSF-R, a receptor )~ , was analysed by blotting with M7. JAK1 co-~ ' ' with the G-CSF-R both before and after G-CSF treat[nent of CHO-6A11 cells (Figur~ 4, lanes A and B). This band was not observed when G-CSF-R , , were blotted with M7 pre-ilnlnune serum. The amount 20 of JAKl co ~ r 1~ ~ ' remained constant for at least 20 minutes (not shown) and appeared to be a relatively small percerltage of total ~ ~blDIIIil~ JAK1 (Figure 4, lanes C and D). Receptor , c, also shown in vi~ro kinase activity to a 130 kDa protein.
'` ' of CHO~All cells with G-CSF also resulted in ~LuDl~Lu~y'a 25 of the G-CSF-R on tyrcsine witbin ~ minutcs of G-CSF ' (Figure 5). The IJLoDlJLoly ~d receptor migrated at a higher apparent rnolecular mass (150 and 135 kDa) that the two r~ain bands detected by LMM741 a~d R55. Differerltial migration f 1' ~- y' ' pro ein has been observed with other proteins, for e~ample, STAT91 (16) and middle sr~ed tumour antigen (17). r~ . y~u~ of the murine 3û G-CSF-R in a transfected h- -~ cell line (32D) has also been detected recently by Pan et ~1. (18).

~2~9~
~W095121864 r~ .C1696 The G-CSF-R shows i ' ' homology with gpl30 (46.3 % amino acid similarity) (2), signal ~ L component of the IL,6, oncostatin M, LIF, CNTF
and Il-ll receptors. Of interest, two of the three highly conserYed regions of homology between gp-130 and the G-CSF-R have been shown to be necessary for 5 signal l . . ~ .. . most not~dbly a 99 amino acid region of the ~ L~r ~ domain (2). Consistent with the notion that this homology indicates similar function, it has been recently obserYed JAK1 association with gp-130 following IL-6 and LIF
.. . ..
The datd presented here are strongly suggestiYe of am important role for JAK1 10 in G-CSF receptor signal l,.-- ~ l;.~-- pathways. The co-l , of G-CSF and JAK1 ~'- a close association between these molecules. r...~ ...,,c, Jhulyldliull of both within two minutes of G-CSF binding makes it likely that JAK1 is the kinase lc~ for G-CSF-R, ' , ' .y' Recent reports indicate that JAK1 is ~ll~, ' - with at least one other JAK family kinase in signalling 15 pathways for IFNcY and IFN~ receptors.
Although the CHO-6A11 system is an artificial one, it is clear that AML-193 cells, which proliferate im response to G-CSF, share the same JAK1 response (Figure 2C). This suggests that these ~.._l ~ - can also be applied to signal i from the native G-CSF-R.
2û Those skilled in the art will appreciate thdt the inverltion described herein is susceptible to variations and ' ~ other than those specifically described.
It is to be understood that the invention includes all such variations and The invention also includes all of the steps, features, A dnd ~ , ' referred to or indicated in this -~ . ., h.~ or collectively, and any and 25 all ' of any two or re of said steps or features.
.

WO95121864 2~8~ ~9 4 ~ 1696 REFERENCES:
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87: 8702 (1990); Larsen et al., J. FY~!. Med. 172: 1559 (1990); R. Fukunaga, E.
Ishizaka-lkeda, Y. Seto, S. Nagata, Cell 61: 341(1990).
5 2. R. Fukunaga, E. Ishizaka-Ikeda, C. Pan, Y. Seto, S. Nagata, EMBO 10: 2855 (1991).
3. R. Fuhmaga, E. Ishizaka-lkeda, S. Nagata, J. Biol. Chem. 265: 14008 (1990); E. Ishizaka-Ikeda, R. Fukunaga, W.I. Wood, D.V. Goeddel, S. Nagata, Proc. Natl. Acad. Sci. USA 90: 123 (1993).
4. S. Watawichetal., Pr~c. Natl. Acad. Sci. USA 89: 2140 (1992); G. Fuh etal., Science 256: 1677 (1992).
5. R.J. Isfort and J.~. Ihle, Growth Factors 2: 213 (1990).
6. A.F. Wilks et al., Mol. Cell. Biol. 11: 2057 (1991).
7. A.G. Harpur et al., Oncoge~e 7: 1347 (1992).
8. L. Velazquez, M. Fellous, G.R. Stark, S. Pellegrilu, ~11 70: 313 (1992).

9. B.A. Witthuhn et al., Cell 74: æ7 (1993).

10. L.S. Argetsirlger et al., Cell 74: 237 (1993).

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Claims (29)

CLAIMS:

1. A composition comprising antibodies or parts, fragments or derivatives thereof to G-CSF receptor extracellular domain.

2. The composition of claim 1 wherein the G-CSF receptor extracellular domain is in recombinant form.

3. The composition of claim 1 or claim 2 wherein the antibodies are polyclonal antibodies.

4. The composition of claim 1 or claim 2 wherein the antibodies are monoclonal antibodies.

5. The composition of claim 1 or claim 2 wherein the antibodies are labelled with a reporter molecule capable of providing an identifiable signal.

6. An antibody to the composition of antibodies defined in claim 1.

7. An antibody according to claim 6 labelled with a reporter molecule capable of providing an identifiable signal.

8. A method for inhibiting or decreasing tyrosine phosphorylation of a JAK kinase in a mammal comprising administering to said mammal a binding effective amount of an antibody or a part, fragment or derivative thereof interactive with G-CSF receptor extracellular domain.

9. The method of claim 8 wherein the antibody is specific for a recombinant form of the G-CSF receptor extracellular domain.

10. The method of claim 8 or claim 9 wherein the antibody is a polyclonal antibody.

11. The method of claim 8 or claim 9 wherein the antibody is a monoclonal antibody.

12. The method of claim 8 wherein the mammal is a human, livestock animal, companion animal or laboratory test animal.

13. The method of claim 12 wherein the mammal is a human or murine species.

14. A method for inhibiting or decreasing G-CSF interaction with G-CSF
receptor in a mammal comprising administering to said mammal a binding effectiveamount of an antibody or part, fragment or derivative thereof interactive with G-CSF
receptor extracellular domain.

15. The method of claim 14 wherein the antibody is directed against a recombinant form of G-CSF receptor extracellular domain.

16. The method of claim 14 or claim 15 wherein the antibody is a polyclonal antibody.

17. The method of claim 14 or claim 15 wherein the antibody is a monoclonal antibody.

18. The method of claim 14 wherein the mammal is a human, livestock animal, companion animal or laboratory test animal.

19. The method of claim 19 wherein the mammal is a human or murine species.

20. An antagonist to G-CSF receptor.

21. The antagonist of claim 20 wherein said antagonist is an antibody or part, fragment or derivative thereof directed against G-CSF receptor extracellular domain.

22. The antagonist of claim 21 wherein the antibody is directed against G-CSF receptor extracellular domain in recombinant form.

23. The antagonist of claim 21 or claim 22 wherein said antagonist is a polyclonal antibody.

24. The antagonist of claim 21 or claim 22 wherein said antagonist is a monoclonal antibody.

25. A method of treating a condition resulting from G-CSF interaction with its receptor comprising administering a pharmaceutically effective amount of antibody specific for G-CSF receptor extracellular domain.

26. The method of claim 25 wherein the antibody is directed against a recombinant form of G-CSF receptor extracellular domain.

27. The method of claim 25 or claim 26 wherein the antibody is a polyclonal antibody.

28. The method of claim 25 or claim 26 wherein the antibody is a monoclonal antibody.

29. The method of claim 25 wherein the mammal is a human, livestock animal, companion animal or laboratory test animal.