CA2397169A1

CA2397169A1 - Targeted ligands

Info

Publication number: CA2397169A1
Application number: CA 2397169
Authority: CA
Inventors: William Herman
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-08-13
Filing date: 2002-08-13
Publication date: 2004-02-13

Abstract

The invention contemplates a composition containing a multispecific ligand containing at least a first ligand binding moiety and a second ligand binding moiety. The first ligand binding moiety specifically binds with a pre-selected first affinity to at least a first ligand. The first ligand has a first biodistribution.
The second ligand binding moiety specifically binds with a pre-selected affinity to at least a second ligand.
The second ligand has a second biodistribution. The affinity of first and second ligand binding moieties are selected to bias the biodistribution of the multispecific ligand in favour of a selected location of one or both of the ligands.

Description

Tarsreted Lit~_andg Field of The invention The present imeMion relates to muItispecific ligands, for example a heterofunctivnal ligand comprising at least first and stcond binding moieties which have cooperating functional affuuties including a multispecific ligand, for CxBntpIe, a bispecitic antibody, having at (east a first portion which binds to a 'lymphatic vessel associated' antigen/receptor and a second portion having at least one immune-affecfi'rtrg functionality including, without limitation, tunttiozls rtlated lo Sntii;en presentation, immune signaling, suppression or enhancement of itmnune tolerance or immurlt stimulation, or binding to a target molecule, for example a cell surface antigen, receptor etc.
Backaroulod bf the lnVetltlo Immunotherapy has gained wide acceptance as a promising measure to address several disease states including autohnmune disease, iransplant rejection, infectious disease and cancer. Despite rapid and exciting progfess in appronehes to ueatment, the disease burden attributable to such illnesses has not significantly abated. The Complex nature of the normal and pathologic immunologic processes associated with such diseases, coupled with logistical problems in evaluating and implementing methods for invnunotherapy in human subjects, wntinue to be same ofthe obstacles to sueGGSSfi~l advances in treatment.
Successful approaches to imrnudadterapy are predicated on the ability of the immunvtherapeutic molecule to be delivered in a therapeutic, sub toxic dose at the desired therapeutic frequency. In the process of selection of a suitable therapeutic molecule, it is recognized that sub-toxic doses may be insufficient for the desired therapeutic effect, especially where the antibody binds incidentally to cell populations other than the tarmet population. In the case of an lnjcctable preparation and especially au intravenous made of delivery, in contrast to readily self-,administered modes of delivery, the optimal dosing frequency for therapeutic purposes could it~OSe an undesirable burden on the patient and care-giver, assuming that such optimal frequency is to begin with deemed convenient for clinical uials.
Numerous research effods are wrderway to identify and test ligands including antbodies, biologic effector iigands (e.g. eytokines, ehemokines, growth factors colony stimulating factors) receptor agonisls orantagonists etc. which wUl bind to or otherwise itrtetact with or trigger responses in or towards target entities, including patho$enie organisms, tissue speaiffe celJs,dlseesed cells, Immune cells etc. A recent example is a renewed interest to find molecules and methods of trtggering an interaction witli C1~45 (see for example Nature (2001) Vol. 409 p. 349-354). Evaluatini the biological effect of interactions with such target ligands is ofTett obfiiscated and retarded by the biodistribution of such ligands on cells other than the target population which results in undesired and/or confttsing plelouoplc effects.
The present invention fBellitltes SCiWltitIC a55os5tnCnt, dCYelOptnGnt, role evaluatipn, therapeutic evaluation, and delivery, particularly targeted delivery of molecules that exert biologic funcilons and particularly immune relatedftmctions. In particular, the targeting agents and methods which are the subject of the invention herein fhcilitate scientific evaluation of the biological effects of a more targeted biodistnbution ofsuch targeting agelrts, by limiting undesired or confusing side efFects_ 1n preferred aspects the invention contemplates compositions of matter and methods of delivery, in some tests using hgands that but far the targeting methods herein defined would be ineffective or have a broader effect than is desirable; or similarly, but for the severity of the disease or i'he absence of other thenpeutle alternatives for which such ligands are useful, dtey would otherwl5e be inappt~opriate far therapeutic u;e. The present invention aeaomroodazes evaluation of the biological role and/or vffccts of such li~ands for therapeutic or other scientific purposes using such targeting strategics. In particular, the present invention provides a vehicle to preferentially target, on a sub-population of. cells far which there is a cell-associated marker, a receptor or receptor li;and which is present on a more heterogeneous population of ce113.

SummBry 0I Tile )(IA~entieo The invention contemplates a composition containing a mult3specific ligand containing at least a first ligand binding moiety and a second ligand binding moiety. The first ligand binding moiety specifically binds with a preselected first afTmity to at least a first ligand. The first ligand has a first biodistribution.
The second ligand binding moiety specifically binds with a pre-stleettd afflttity to at least a second ligand.
The second ligand has a second biodistribution. The affinity of first and second ligand binding moieties are selected to bias the biod9s~bution of the multispecific ligand in favour of a selected location of one or both of the ligands.
The invention contemplates a composition conLalnirig a mtlltlspecific ligand.
The multispeciflc Ligand contains at least a first ligand binding moiety and a second ligend bindltlg moiety- The first ligand binding moiety specifically binds to a first ligand having a first biodistribution.
Tht second llgand bindin~ moiety specifically binds to a second ligand having a second biodistribution. The second biodistribution is different frbm that of the first biodistribution, and the aflinity of the first and second ligand binding moieties to their respective ligands are different and selected to bias the biodistribution of the multispecific ligand towards the first or second biodistribution.
The invention contemplates further, a composition containing a multispecific ligand. The multispecifle ligand contains a first li;;and binding moiety end a second ligand binding moiety. The first ligand binding moiety specifically hinds with a pre-selected first affinity to a first ligand. The first ligand has a first biod;stribution_ The second ligand binding moiety speci$eally binds with a pre-selected affinity to a second ligand. The second ligand has a second biodistribution. In this ambvdiment of the rnultiapeeitic ligand, the affinity of first and second ligand binding moieties are selected to bias the biodistribution of the multispeciftc ligand.
The invention farther contemplates a composition containing a multispecific ligand. The multispeeifie ligand speci$cally binds to a target llgand. The target llgand is specific to a selected sub-population of a heterogeneous cell population. This embodiment of the multispecif c ligand contains a Crst ligand binding moiety and a second ligand binding moiety. The first ligand binding moiety specifically hinds to a poll sub-population associated ligand. The second ligand binding moiety binds to the target li$and. Tn this embodiment, tl,e first ligand binding moiety has an affinity for the sub-population associated ligand higher than the affinity of the second li~nd binding moiety for the target Iigand.
The invention further contemplates a composition containing a bispacitic ligand containing a first ligand and a second 1'igand. The first ligand binds to a first target ligand end the second ligaod binds to a second target ligand. In this embodiment of the bispecific ligaad, the affinity of the first ligand is selected to enable binding to the first target ligand independently of the ability of the second ligand to bind to the second target ligand. Further, the affinity of the second ligand is selected to substantially reduce the probability of its binding to the second target ligand without the first ligand binding first or substantially contemporaneously to the fir$t target ligend_ The invention further Contemplates a composition containing a bi8p0C1J:1C
BGtlbOdy C4ntalilln$ d flYSt antibody compone~,t and a second antibody component. 'fhe first antibody component binds to a first torso ligand and the second antibody component binds to a second target ligand. In this embodiment, the affinity or avidity or both the affinity and avidity of the first antibody component are selected to enable binding to the first target ligand independently of the abflity of the second antibody component to bind to the second target ligand. The avidity or affinity or both the affinity $nd $vidity ofthe second ligand are selected to substantially reduce the probability of its binding to the second target ligand without the first ligand binding first or substantially contemporaneously to the first target liaand.
The invention further contemplates a multispecific ligand cbnt$iiliilg a fu'st moiety and a second moieiy.
The first moiety binds to a first target ligand. The second moiety binds to a second target ligand. The affinity or avidity or both the affinity end avidity of the first moiety are selected to enable the first moiety to bind to the first target ligand independently of the ability of the second moiety to bind to the second target ligand. The avidity or affinity or both the affinity and avidity of the second moiety am selected to substantially reduce the probability of its binding to the second ttu'get ligand without the first moiety, first or substantially contemporaneously, binding to the first target ligand.

The Invention further contemplator a muriispecifc ligand containing a first moiety and a second moiety.
The first moiety binds to s first target ligand. The second moiety binds to a second target Ligand. The affuury or avidity or both the ai~inity and avidity of the first moiety ate selected to enable the first moiety to bind to the first target ligand independently of the ability of the second moiety to bind to the second target ligand. The avidIry or affinity or both the aiflnity end avidity of the second moiety are selected to substratially reduce the probability of either moiety binding for a sufficient duration or series of durations tn its respective target ligand to accomplish a therapeutic function without the other moiety, first or substantially contemporaneously, binding to its respective target ligand.
The invention further contemplator a composition containing a multispeciftc ligand containing a first moiety and a second moiety. The first moiety binds to a first target llaand.
The second moiety binds to a second target ligand. The affinity or avidity or both the affinity and avidity of the first moiety are selected tp enable the first moiety to bind to the first target Iigand independently of the ability of the secppd moiety to bind to the second target liøattd. The avidity or afFtnlty or both tire affinity and avidity of the second moiety are selected to enable the stCOrid moitty to bind to the second rarity in preFc-ren~e to the fir3t moiety binding to the first entity when both fast and second moieties are substantially contemporaneously bound to the respective first and second e>~ities.
The invention contemplates a composition containing a multispecific ligand containing a first moiety, a second moiety and a third ligand binding moiety. The first moiety hinds to a first target ligand and the second moiety binds to a second target ligand, Ia this embodiment, the affinity or avidity or bpth the aih'mity and avidity of the fast moiety are selected to enable the first moiety to bind to the first target ligand in prCftr6oCe to the second moiety binding to the second entity when both first and second moieties arc substantially contemporaueously hound to the respective first and second entities, and the avidity or affinity or both the affinity and avidity of the second moiety are selected to enable the third target ligand to bind to the second entity in pref~errace to the second moiety binding tp the aecpnd entity when both the third target ligand and the second moiety are substantially contemporaneously bound to the second entity.
The invention further contemplates a composition containing an antibody which specifically binds to an epitope on a tigand. The lisattd recosnimd by the antibody exerts a biologic effect by binding to a tttrget site on a target ligand. The epitope bound by the aan'body is proximal to the binding site of the ligand far the target ligand, an that binding of the anttbody reduces hut does not prevent the affinity of the ligand for its target Ligand.
The invention fiutlter conbompIatcs a Composition containing a multispecific ligand containing a first ligand bIndlng m8iety and a second moiety. The first ligaltd binding moiety specifically binds to a lymphatic endothelial cell associated marker. The second moiety contains an independent thernpeutie fttnetion-The invention further contemplates a composition containing an immunocytokine containing an anti-idioryple antibody cotatpoudent and a cytokine eompvnerd. The anti-idiotypic antibody component recognizes the paratope of an antibody wlttch binds to a lymphatic vessel associated ligand.
The iJtvention further contemplates a composition containing a bispeci;fic ant,~Qdy containing as antl-idiotypic tmtibody component and an anti CI)3 antibody or art anti-CD2$
antibody component. The anti-idiotypic antibody recognizes the paratope of an antibody which binds specifically to a lymphatic vessel associated ligand.
The Invention additionally contemplates pliysiologically aceeptabla compositions ofthe compositions encompassed by the invention.
The lttvention likewise contemplates methods of use of the compositions encompe.ssed by the itavendan.
A conaposition comprising a muttispecifc ligand comprising at loser a fixer ligand bhtding moiety which specifically binds to a first ligand having a first biodistrlbutian and a second ligand binding moiety which specifically binds to a second Iigand having a second biodistnbution different from that of rite first ligand, and wherein the affinity of the first and second ligand binding moieties are different and selected to bias the biodlstribution of the multispecific ligand.

ed Description of Preferred As exempl3tied above, the dual "affinity" based targeting strategy of the invention, may be understood in one aspect, up terms of a strategic allocation of the respective affinity properties of the rnuItispecific ligand to at least ollC "targeting" function and at least one "effector" Function.
Accordingly, with respect to some embodiments of the invcntioa, the term'~ultifunctianal" ligand is used interchangeably.
Thus according to one preferred embodiment, at least one of the ligand binding moieties is a "targeting"
arm in the sense that it at least preferentially recognizes a marker that is associated with one or more specific target entities eg. cell populations, and the other llgattd binding moiety is an "efl°ecto~" arm which binds with relatively Ices affinity or functional affinity to a target ligand which has a more diverse biodisttibution. in this case, the biodistnbution ofthe multispecific li8and is biased in favour ofthe locations) of both ligands rolative tb the locstion(s) of the target Iigand so as to limit the big distribution to non-target entities.
Such binding or recognition is understood throughout to be specific, in contrast to non-specific binding.
The term "effectar" is used to refer to the ability to effect a biolo;ical consequence through binding, fbr example effecting a signal transductivn event by activating a receptbr, yr blocking the tbrget ligand from associating with a complementary tigand, for example blocking a receptor from associating with a complementary ligand (op. its natural ligand) and thereby, for example, preventing a signal trausductlon, or far exatngle is the case of a decoy receptor greventing the 6ialogical consequence (eg protective effect) associated with the iuoction of such receptor, yr btocidng a ligand from associating with a complomeatauy ligend eg. receptor on another entity eg. a cancer cell, infectious agent or immune ceU.
A biased biadlstributian is preferably accomplished by the nutltispeclflc llgand contEmporaneously recognizing both ligands on the same entity eg cell, and may be accomplished by such contemporanous recognition occurring on adjacemt entities or by increasln4 the propanslry of the multlspecflc ligand to locaDe In proximlry to a target entity in virtue of the relatively high at>6nity targeting arm. The targetipg arxn may i~elfbe an e$ector .
In another embodiment, the biological consequence accomplished by the etfector arm is at least minimally retatyetin~ for example wherein the lesser sflinity or l5tnedonal affinity ofthe i5rst binding moiety is selected to permit the multispecific ligand to prefer~tjp]ly bind to an adjacent entity, for example, a circulating entity which circulates in praxitnlty to a lymphatic endothelial cell tb which the multifunctional ligand is bound with lesser affinity. Again, the relatively high affinity first binding moiety may itself be an effeclor.
to another embodiment the biological cousequeace accompUshcd by the ofFoct~
arm is minimally cooperative targeting, for example whero the biodish~butions of at least one of the first and second ligands extends to a diverse population of cells ocher than target cell population and where binding is only possible or consequential if both llgands are available for contempornncous binding, in this case due is the afiltllties ofthe first and second Ligaad binding being individually Iusuffieient for effective targeting (eg. insufficient for other than ephemeral binding). 1n the context of this embodiment of the invention, the "cooperative targeting" is not simply ameliorated by the effector arm, it is predicated and reliant on this arm. One or both lJgand binding moieties may exert additional effector properties.
)t will also be appreciated that Spy multispecitic ligand of the invention or any cvntponant thereof may be fused or cotljugated to a separate effector as exemplified Ixlow, including toxins, eytokines, adhesion molecules etc.
The ligand binding moiety is preferably an antibody or a sequencs Qr sequences of amino acids etc.which arc the natural Iigand far the target ligand, for example where the ligand 1s a cytolcinc or lymphalcitte receptor, such as ILr2 receptor, the ligand binding moiety may comprise a sequence of amino acids which is IIr2. The ligand binding moiety rnay also be a mutated or a newly developed form of the natural ligand (eg_ developed through combinatorial Ubraries) or a natural or synthetic chemical ligand (developed through combinatorial chemistry).

In one aspect, the invention contemplates a composition containing a multispecifIc ligaud containing at least a first ligand binding moiety and a second ligsnd binding moiety, tire first ligand binding moiety specifically binding with a preselected first affinity to at least a first Jlgand, having a first blodistribution and the second Llgand binding moiety specifically bind;ng with a pre-selected affinity to at leest a second ligand with a second biodistrtbutlon and wherein the alfWry of first and second ligand binding moieties are selected to bias the biological site of biologic nativity o~the rapltispecific ligand; and wherein the first ligand binding moiety preferably binds with htgtt af~'tnity (preferably nanatnolar afldnity or greater) to a specific cell associated marker (e.g. a CD marker a marker assoCieted with diseBSCd CClls, a a merkCr associated cells in a particular physiological state (e.g. activated T cells, B cells) arc. (such markers may be associated with a particular class of cell or a subclass thereof (if applicable) or particular subpopulation within the subclass (if applicable), however classified, sucli as epithelial cells, endothelial cells, hnmuue cells (lytt~phOCytGS, tPem4ry cells, efFector cells) mtmocytes,Teelh (CD4+, CD8+, CD45RQ+), hepatocytes, stow cells, etc.(expand) and wherein said second ligand bittdinm bindin; moiety binds with relatively low, yr medium sf$nity (preferably 0.1 mictomal8r or Ices) to a receptor (cg. chcmakinc, growth factor, cytolcine) involved in cell signaling or a decoy receptor, a cell surface receptor ligand cg. the ligand for such receptor which effe~s a si~tal or inhibits a si~aI [cg. CTLA4), a ligand involved in cell adhesion, a receptor or channel (ion channel) for a molecule involved in toll rC)~ttlation or homeostasis ate, The invention cpntemplates that the differemco in afFnity will in mast cases be an essential clement itr biasing the location of action of the mult4spec18c ligand to yield an acceptable or desired safety profile and that the high affinity ofthe first Iigand binding moiety for the cell associated marker will be optimised for this purpose insofar as the safety profile of the multispeci$c lipand dictates maxlm7zing Its afl5niry characteristics. The invention also recognizes that choosing the relatively lower nffnity o~the second ligand binding moiety rnay assist in this regard up to a point whsre its e~'ectivenass to bind to the second ligarid is sigoi~CSntly Camprlmised. id this n:gsrd, the invention also contemplates that factors othier than the choice of affinityof the first and second ligand binding moieties (and of course the avidity effect resulting from having two ligands on the target cell and only one on the non-Carpet cell) may be taken into consideration or optimized to balance the safety and effectiveness profiles of the muttispeci$c liiand, especially if such cyuefitl balance is requjred-Examples of such fhctors, one or more of which can be employed in various combinations, are described hereafter.
1 ) the selection of call asso~ated marker, in terms of its call surface density relative to cell density of the second ligand. The number of first and second ligand can readily be assessed by radiolabelling studies or approximated by flow cytometrio methods relative to a standard.The selection of the cell surface marker in this respect will depend on the function of the relatively low affinity binding meiety (whether it causes a signal transducrion (directly or indirectly cg. through bindirg to a receptor (where Less emph,ttsls on relative cell density may be watraaroedxagonist antibodies are well known in the art and include those described in US 6,342,220, US b,331,302, US 5,635,177, US 6,099,841 see also Cancer Res 2001 Mar 1;61(5).1846-8 and can be made according to routine screening techniques, especially using a~ttibodies capable of cross-linking receptor components (see references below) including antibodies in which the YH and YL are capable of binding individually to different receptor components), binding to a decoy receptor, binding to an inhibitory receptor arc.) or prevents a sfgnal transductlon (d;rectly or ;nd;reedy, e.g. binding to a receptor, binding to receptor ligand) and in the final analysis how many binding events per cell are required to cause or prevent the sought-after biological effect. This can be assayed in vitro through well known assay methods established in the art for measuring responses to external stimuli such as cytoklncs, chtmok3des, growth faetars, colony stimulating fetters using various immunostaining techniques including, flow cytometry (cg, to measure apoptosis (e.g. annexin V binding assay) signal transduction (e.g. using phosphospeciftc antibodies that detect phosphorylation of serine, tyrosine, threonlne), differential gene expression etc.) depending on the type of effect that is being measured (see for example ;3iosource Method Booklets at httu:l/www.bso~urce.tom/ccmtent/techCorncrContentlmethodpbFs/indcx.acp; see also ~44Z
Amersham Biosciextce catalogues, and those of other well known sttppliars etc.) or via animal studies. Far example, some growth factors, lymphokines or ntoleculeslions required for homeostasis are in more delicate balance and can more easily disrupted. 1L-2 depletion will cause apoptosis of activated T cells, which can be measured. For example, it may also be necessary or desirable for the cell specific marker to approximate (preferably the the number of cell specific markers on the target cell population Is no less in number than 50°!° more preferably no less than 90°i6 in number relative to the second ligand - as stated above, which will depend nn what degree of causation or prevention ofthe signalinB/interactiptt will cause the desired biologic effect) or preferably out-number (by greater than 50°!0, preferably by greater that 100%
(greater than two fold), preferably by greater that 200°!e (greater than 3 fold), by greater than 300% (greater than 4 fold)) the tergtC ligand for the relatively low atZznity binding moiety especially, for example, if the goal is to block interaction of a receptor with a high atl:lNry ligand that exerts a biological effect in low concentration (as, a cytokLte).
2) Furthermore, in the latter case the nffmity of the fast binding moiety will preferably be selected to approximate (preferably no less than one order of magnitude, more preferably no less than S fold less, more preferably no less three fold less, more preferably uo Less two fold less, more preferably no less one fold (100%) loss), and will preferably equal or exceed the affinity of the natural ligand.
3) Furthermore, the concentration (in virtue of tire choice of administered dose) of the multispecific ligand in the target cell microenvironment may also be selected to exceed that of the nattu-al ligand (MTD
petvnitiug).
4) the choler of carstruct will maximize the static blacldttg of tht target (igG ar F(ab' )' vs diabody).
Furthermore, in soma mamtnalian systems (as. mice) the hinge raglan is naturally logger and this effect can be mimicked for human antibodies through a hinge extension on the N-terrnittal side of the hinge region using well known neutral linkers (gly4ser) or a repeat of all or a portion pf tl,c natural hinge sequence. This cxtensioa will also permit a ~atcr span between first and second llgands to be bridged.
5) the choice of construct will include nn Fc portion or partial Fc portion (cg. Ctl2 or minibody-CH3) or weighted Fc eg. by pe~vlatioa (site specific pegylation is well known in the art) or 1gG subtype naturally having additional Fc domains (e.g. an 1gE) (which Fc if it includes the Cl-I3 is preferably mutated tp preclude Jts binding andlor increase its half life as is >mpvyp 1n the art sec USP 6,121, 022) so as to maximize the shear otFcets on tire muItispeclflc ligand which will be most consequential in the case of univalent binding in order to minitttize the duration of such binding (maximun shear force is also prefetTed where there is an excess in the total number of bioavailable targets of the second llgand binding moiety relative to the total number of bioavailable to pets of first llgend bi"riro tneiety(greater numbet of CClls andlor greater number of targets per cell andlor increa9rd biottv~lablity of such targets cg. on nam,sl cells relative to cancer cells).
6) Optionally, the muItispecific ligand will include a 3'~ binding moiety which binds to bnd neutralises the natural Llgsnd for the rtetptor sought to be blocked. Such formats are well known in the art (see for example particularly Schaonjans R et al_ A new model for intermediate molecular weight reeombinam bispecific and trLspecific antibodies by efficient heterodimeri~ian of single chain variable domains through fusion to a Fab-cltain. Biomol Eng. 2041 Jun; l7(6):193-202.
Schoanjans R at al. Fat chains as an efilcient htteradimerir~lion scfffold Cot the production of recombinant bispecific and trispeeific antibody derivatives..T lmmunol_ 2000 Dec 15;165(12):7050-7. 5choonjans R, et al. -DfFtcient heterodlmerixatipn 4f recombinant ti- and trispccifie antibodies. H;Qaeparation. 2000;9(3):179-83.
see also French RR.
Production of bispecIfic and trispecliic f(ab)2 and F(ab)3 antibody derivatives. Methods Mol Biol.
1998;80:131-34; US Fabent Application No. 20020004587; Kortt AA, Dimtrie and trimetie antibodies:
high avidity scFvs for cancer targeting. Biamol Eng. 2001 Oct 15;18(3):93-108).
7) Optian311y, two multispacific liganda each binding to different cell specific markers and each having a second ligan~d binding moiety which binds to the same second ligand cg. a receptor, optionally to a different polypcptide/component of the receptor, may be employed to achieve the desired biologic effect. One or both may also be vispecific as discussed above.Accordittg to eaother embodiment the muItispecifc ligand binds is used to protect a first target cell population in virtue pf its high affinity first ligand binding moiety from the effects of a therapeutic entity which desirably binds to s second target cell population via eht second ligand but also undesirably binds to the first target cell population.
Thereforo the second ligand binding moiety can be used to selectively block the binding of the therapeutic entity e_g. an interleukin, interferon, itnmunatoxin, ate. to the ttrst target cell population In virtue of the relatively low affmiry second ligand binding moiety. In rl,is case, the muttifpecifc ligand may also comprise a third ligand braiding moiety which binds to the therapeude entity, particularly where the mtLltiSp~iftC ll~dnd is f>fSt adminlstt.,red t7rst, optionally an anti-idiotypic binding Inoitry component where the thetapt:utic comprises an antibody coznptmentSome sample targets are listed immediatety below, while others are Usted later.
Greater targeting using a high affinity first ligand binding moiety which binds to a cell associated or specific marker tray be imparted to a variety of existing atmbodies with suitably diminished al5lnlty including ihoae marketed or in clinical trials or listed below which era the subject of the patent and scientific literature, inctudiog those listed in PhartnaBusiness June 2002 LIo.5l:
~atepory FunctionalCcu tocaliaing Mode Of nationCotoments ligand li8attd for bi8b for low af<salty nrta affinity arm loratiatl aril sad Growl'h IL2. CD4 T ctll9 drawth fatlor11r2 rcquircrJ
faClor blockade tQr naYVC (;I,7Q
for a 0r CDf blotktde (aolubk) spcei6cally and memory CD4 for Cl>4 T call raapanses:.
T cell subset; i.e, Binding ofC.D4 sekccivc or CD8 would also Lnlnunosuppressionblxk internation with antigen aria talk.

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ccus InhibitoryI'c canonsFc npstlon RI Enhancx ossucintionRit of scone receptor li.ll of of allergic ois~~ce.

aeiivalitm(Mast cells)(Mast cells) tmttvaring lldvantagcs over end inhibitorybispesitic Pc fusio receptors. reagents because of more spaclfic ci Iargatino . See Ghu D et al.Nat Med 2(102 May;B(5):518-7.1) lnhtbitayCfI.A~ CD8 t3lock CDBOI86Fnhana specifically receptor Interbttion GTL mediated blockads with C'fLA~i anti-tumor ras;pansee witltotn global coil activatlatt_ wnti-CTL,A4 ubs nu thaeapautia trials ut onhancc rtlmor imtnuni Adhesion VCAM-1 CD31 (antlulhelialHloek YL.A.4.dtpenUentnautc Rx of MS
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blockade (activatedsdaadn (aWvarcdtell and monncytcis is trial as endothelium) sdhcsion Rx for MS.
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towarels effects another (soluble) endothcliat not clear if therapeutic: odls during IH?JLl R Or IL-2 IFNO is bat i.e. immunotoxins them for tumorstar at.

frowct IFN-DR CD3l Block 1FN-0 bath ligands from torticity On same cWl.
towards Cd1 another (cndothdial(endmhelial endothelial safecti~o block therapcudc:cell) cell) ins during aflFNl7 et3bcta;
n..2 not i.e. immunotoxins theca for clear if~NU R
tumors or IFIYrJ v bon tar InhibitoryTGF-O CD4 6! CD8 Block immunOS~pprCSSivcTOFCJ mode of rcccptcx immunosupprassiut blockade c$cccs of not dour tumors: anhanca anti-tumor immunity Cell typeCu3 Cri45Rp Trtvalcnt Anlitumot Rx;
spxific ab so CD3 saldeLivaly eau be anhartca activation (mCnOry/pffccwrstoss-linked memory T cells;
T Calls) reduce nonspecitic activation oYitrclvent T rolls C9tcgory FunctionalCell loaalixiapMode of ocdou Possible therapeutic Ilgaed liaand for ucee biRb (or low n~tnity arm affinity as~o lotstiou o(Il nd Growth 0.L5 CI78 T coils Growth tactvr Fa Rx of aqograft factor biOGha~ fps rcjsction: T
can blockade specifually subset apeciFtc for CD8 T suppression Will cdl limit subset; l.c. infectious complications selective or uninlendztl immunoqu sessioninhibition nfCd4 ' rc loco 'C
cells Cetl octiwetionCb$U/$d CD$3 (dendritiai3lonk ineeraetionAt time of siio~ng tells) with Cb2g; to-induce blockade (dMdrlHe itnmunosttppressiontolerance. Targeting mtigcn to deadritic tens re entin will enhance etTicienc ocllv /

Adhesion V AM-t CD31 (endothelialBlock VL,A-0-dGp~udmlAunts itx o M
meleeu calls) or T a~up. Anlr-VLA-4 ~e E-blockade (activatodselectin (acr'rvatedcell and monocyreis in trial as endcttltelium)adhe9ion ao Rx for MS.

endothelial VCAM-1 on endadtelial cell) cells.

Cdl death CD95L (FasIigand)CD25 (activatedBlock activationIruhancc anti-lamas ligand T cells) induced cell immunity blockade T edl death of'f cells Protect>fiorrt1FN-aR CD31 Call selectivecloak IHnt-n toxicieytotvsrds blockoftFNo another (endothelial(endothelial endothelial calls therapetrtia:call) coil) durieg IL2 rharapy i.e. tM~NOnAtOxins for tumors without impairins other useful IFN-n efrecls InhibitoryFa ~nrnma Fe epsilon Enhenoc associationRx of atttte or receptor Rlf RI a( allergic disease.

aCtIVatiOn(MtISI (Mast CtIIS) ~cliveting AdYanta Cells) and inhihittxygcs over bispcciTic Fc fusion rccepttus. reagents because of morc spocittc cell targeting . Sae Zhu D at uLNm Maul 2002 M '8 5 :51821 The invention also contemplates that FAS can be selectively blocked on various different types oFt:ells such as pancreas beta cells using markers such as GAD65,1A.2,1A2-H, ICA-12.
Type 1 Diaboxs is characterized by the destruction o~insulin producing $eta Cells In rite pancreas. one method in which Beta sells tire destroyed is thought to ba through apoptosia mediated by CD95 receptors on Bcta cells. CD-95 seems to be upregulated in Rata cells of those with Type 1 diabetes (see Ann N
Y Acad ScI 2002 Apr 958 297-304; J Clin Immunol 2001 Jan;21(1):15-&). Sitnilarly, using Tg, TPO
ligands as cell associated markets C1793,TRAILR1, TRAILR2 can be blocked oa thyroid cells. Hashimoto's ThyroidJtis (HT) is characrtet;zed by ille desauctson of thyroid horatoue producing ells acct therefore hypothyroidism. It has been observed that soma oPthis cell desttuctian is due to apoptosis. The CD95 receptor wluch is responsible for apoptosis is up regulated in thyroids a>~Cted by 1-IT_ Blocking the CD95 receptor by the RLAA may reduce the amount of apoptosis_ The IiAA can target either Tg or TPO
which are unique to thyroid tissue. ' There are also 2 other receptors suspected to lx involved with apoptosis in thyroid cells:
T~AI'Ll~t and TRAlLR2 (sec Nat Rcv Immunol 2002 Mar;2(3):195-204) Fan can also be selectively activated on distinct subsets of disease mediating immune cells associated with sutoimmune and inflatxunatory disorders such as activated T cells, regulatory T cells, CD4+ cells, CD8~i~
cells era Antibodies that era directed to the ligands identified herein can be routinely generated by phage display and similar display systtms, for example, bacterial or cellular display, ribosome display or by immunizing mamtnttls, including transgenic mice, acct by screening lymphocytes having such potential. $oth the light and heavy chains can be vatic=d screened for their ability to bind to one or multiple components of a rrceptor sifnult$neousty.
The Invention contemplates that each of the first ligand binding moieties herein identified generally (whether by class, subclass, catololtue reference etc.) that are cell associated or specific can bt: combined in any permutations with each of the ligande through which a bioJogioal effect i$
excrtad by the relatively low affinity binding moiety, to assess the in vitro in vivo biologics! effects of biasing the distribution of the multispeciFc ligand to the target population identified by the first ligands as accomplished thzouglt the direct instrumentality of a relatively low affinity binding arm herein described, whether the effect is directly or indirectly througli bindiag a ligand or one of its biological correlative Iigulds.

Antibodies to such liganda are known in the art and listed patents, scientific literature and in various commercial catsloguts and on-line databases well known tv those skilled in the art.
Definitions The term "associated" in relation to markers tbat are dominantly distributed on one or more particular antit1ca is used to inane excIusivcly expressed, primarily expressed, or over-expressed to advantage from a targeting 9t'andpOini.
The term "receptor ligand" means a target ligaa,ud which is 8 ligand for a receptor, for example, a receptor on a cell or infectious agent or a receptor which circulates independently of another entity.
The term afl'mity is contrasted to fltttctioual afHuity which may result firom avidity.
The term cpitope though technically understood to be specific for a i;iven antibody, is uxd in a preferred embodiments to refer to antigenic determinants that are situated proximally to one another so that two aacibodles will be considered to bind to the same epitopa if one competively inhibits the binding of the other through any probative competitive inhibition experiment Irnown to those skilled In the art.
fhe ltlVCntlan Contemplates that two antibodies with the same epitope specificity may have substantially the sautc amino acid composition is with possible exception of one or more additions, deletions or substitutions includitte conservative amino acid substitutions which do not substantially affect the specificity and amino acid composition of the paratope The terra appmxlinately in the context of orders of magnitude variations in afFmiry refers a variability that is up tv a half an order yr magnitude.
Without limiting the scope of the claims it is generally understood that biodistribution of a multisp~ifie ligand in contrast to that of a ligand will be predicated on the bioavailability of Its target ligand.
The tame "overlap" and rotated terntS connot8 that notwithstanding the dilTerence in distributions of the first and second ligands the f,rst and second ligands are bioavailable for recognition on the same entity.
This term and related terms, exemplftled below, are intended t4 exclude a situation where both ligands arc preferenttally expressed on substantially the same entity, for exempla two different tumor associated antigens associatod differentially with a differentiatod population of cells within a tumor , most particularly in the case where they are individually suitable targets for delivery of a toxic payload- Thu stha terms "ditfarent" in regard to biodistributions and "heterogeueaus" and "diverse" in reference to populations of entities are similarly understood to exclude such a common distribution, in the appreciation that the iwentipn prip7a~y represents an improved strategy fpr taTg~pg two different ligands, is which one ligand has a broader d3arribution than the other or bout have distributions that may overlap but are different from that of the tataet population. It will also be appreciated that the invention has particular application to a situaiiort in which at Icast one of the non-target populations is one on which one of said first and second ligaeds Is substntttIally represented (in contrast to one on which h simply en3oys limited expression).
The term ''receptor ligand" means a target ligaad which Is a ligand for a receptor, for example, a receptor on a cell or infectious agent or a receptor which circulates indepCndently of another entity.
The term "antigen binding fragment" refers tv a polypept;de or a plurality vFassvciated polypeptides Comprising one or morn portions of an antibody including at least one VH or v1. or a functional firagmcnt thereof.
A moiety that exerts a biologic function is understood to be a "biologic effector" in the sense that its intended interaction with an entity in the lymphatic system or elsewhere in the organism has a biolosical consequence.
The tam neutralizing in regard to an an immune function is used broadly to refer to gory interposition, interference or impediment which affects the function of the mrget entity Tht terms modulating, mediating, neutralizing fuxtion etc. arc not intended to be mutully exclusive end are each used broadly, for cxatnple, without lhxlfting the generality of the scope accorder herein or by those skilled In the art the team modulating preferably refers to effecting a change, and the term mediating preferably connotes an indirect affect achieved through the iasttumentality of auother entity, for example a cell, cytoltine, chemokine etc__ The term ~prtferentially binds" recognizes that a given Ugand binding moiety might have sotnc non-defeating cross-raactivities.
The term biologic effeetor ligands is used to refer to any ligand for which there is a complementtuy target ligand on a target entity, alai wherein binding of the biologic effector ligand to the target ligand exerts a biologic efFect. For example the target ligand is typically a receptor and the biologic eFfector ligand may be any complementary ligand such as a cytokine, chemokilte, hormone, colony stimulating factor, growth factor, receptor inhibitor, agonistor antagonist, which binds to the receptor with resulting biologic etfect.
The term "pre-selected" in reference to the affinity of ligand binding moiety refers to any selection or choice of diLferential or cooperative affinities relative to a second ligand binding moiety which is generated as a result of a mental or physical process or both, preferably through a process of prediction or post-facto validation of the effects of the choice of the first and sxond a>~nities andlor more preferably through an empirical evaluation of different choices for at least one of the first and second affinities, and preferably boCh_ The term multi means at least two and the term ligand is used broadly to refer to any tntiry ar pan thereof which can participate in an intBrmolecular interaction that cao result in specific binding of suitable affinity for the interaction in question.
'rhe term entity Includes without limitation any molecule including without limitation, antibodies, camptex or assoclo~ou of molecules, drugs, drug carriers (eg.Yssiclea eg. liposomes, tuutoperticles,ctc.) or any cell a8 wCll a8 any infrscti0u8 agv~l: bY paYaSitC (including, without limitation, spores, viTUSea, baceria, funp,i ) as well as any other immune or therapeutic target.
The term "low affinity" means an al~nity of approximately (this term is defined herein) 10 '~ molar to micrometer afHafty, preferably (subject to safety considerations), approximately, 10-' molar affinity, more preferably (subject to sslely ConSideYetions), approximately micrvmolar affinity, the teen '5nedium affinity" means approximately 10-'to tlanomolar affinity, preferably approximately 10'° molar affinity, more preferably approximately nanomolar affinity, and the term "high si~nity"
means approximately 10''0 nffinity or greater. Thus is one embodiment rhc invention contemplates that the muItispecific ligand comprises a "t$rgtt-ligand" biedittg moiety which binds with low or medium afiznity to a target ligand present on a diverse population of cells (preferably this moiety is an effectvr moiety ie. one which exerts a biological effect attributable to Its bindlag eg. blocking or activating a receptor or blocking a cell membrane channel) and a ~'taraeting", liaand binding moiety, which binds with medium or high afftalty to a Iigsnd associated with a sub-population of those cells so as to bias the biodish-ibution of the multifunctional ligand in favor of said sub-population. Preferably the mulcispecific ligand is adapted to be bound contemporaneously to the same call.1n another embodiment the first and second ligands binding moieties each bind to ligands present on diverse overlapping populations of entities eg. cells ( Ie. neither ligand being preferentially associated with a target cell population) and are adapted to be bound cantemporancously and to both bind individually with low affinity, so as to bias the distribution of the multispecific Iigand to the population of cells bring both ligands.
As discussed elsewhere the teen approximately, in reference to "order of magnitude" increments in a$'mity, refers to up to a half order of magnitude in alT'ulity.
According to another embodiment, the invention is direetcd to an antibody termed a °coybody". A
"coyhody" is an antibody in which the on-rate contribution to affinity of the antibody is proportionally less than the Off rate Contribution relative to a reference antibody of the same specificity and a greater afFmity of up to several orders of magnitude, preferably a reference antibody of approximately one to thhee orders of magnitude greater af~iniry, preferably a reference antibody of medium affinity or preferably high 1o at~ity. AS dlStussGd above the reference antibody preferably comprises cooperating light and heavy variable regiotu in which at least at least one ofthe C~Ra of at least one pfthe chains, preferably at least the CDR3, preferably that of the heavy chain, is exclusively or primarily responsible for the binding affinity of the eoybody preferably in conjunction with the contribution of at least one of the CDRs of the other chain, such that alterations in the length and or amino acid compositions of one or more other non-contributlng CDRs can be leveraged to diminish the oo-rate, for e~mple due to ste~ric andlor eleck~ostatic hindrance. Tn pne embodiment the on-rate is reduced by a factor of 2 to 100x.
Tn one embodiment the coybody binds to a ligand which is over-expressed on a ta~et population of entities (eg. cells) relative to n non-target population of entities such that the biodistnbution of the cdybedy t0 the non-tttrgta gdpul8tion (and target population) is diminished in a given increment of time following adtninisira4ion. 'this targeting strategy is w~derstandably adapted to situations where the resulting delay in biodistribution is preferable for diminished toxicity attributable to reduced non-target entity binding in a given unit of time especially where the effectiveness threshold ht that same amount of time is not significantly if et all compromised or is prefceable due to a sustained release effect (for example using a larger antibody format that is not headily Cleared) A5 di9CU59ed below, advantages accrue particularly when this anfbody is coupled to a higher afJ~aity antibody (in the form of a multifuaetional limand) which binds to a different ligand assoclaroed with the target population. The invention contemplates that cvybvdies have multiple independent applications, including tempering the effects through antibody mediated neutralisation of an over-production or sensivity to biologic efFector ligends (eg. cytokines eg. TNF,,~", chemokines eg. lL-16 (crohns disease) etc. which acs over-produced aadlor mediate or aggravate eg, a chronic medical co"dit;on (which for example is not an acute phase) by binding to such ligands, over a prolonged periods, preferably using larger ontlbody formats which are cwt readily cleared, especially where such tempering lies side effects which are hotter spread over time and/or where effectiveness is not a limiting factor andlor where a second therapeutic with different non-cumulative side-effects sliares the therapeutic burden and/or where a the same antibody with a higher on-rate is used in combination The term "antibody" is used broadly, unless the context dictates otherwise, to refer without limitation, to a whole antibody of any class or biologic origin, or chimerIc combinations of antibody regions or domains (eg.1?Its gad Gl7lts) of different origins or species eg, hutnaniud, atri combination of one or more an~'body fragments or recombinant reconstntcdong (scFvs) of antibodies including dimers, diabodies, triabodies, a myriad ofknown bispecific, trispecific, tetraspecific antibody formats or monovalent, dival~t, trivalent, tetravalent~or outer multivalent antibody formats (see for example review In Kriangkum !, et al-Bispeciiic and bifirnctional sit~le chain recombinant anttbodics.131omo1 Eng 2001 Sep;18(z):31-X10 and others herein directly or otherwise refCrenced) or any Fragment, portion, or reconstruction of one or more portions of an antibody (scFv) or any truncated forth a ligend binding entity, such antibody typically comprising at least a VFI or VL portion or both or a functional portion of same (eg microbodies), includinb single domain antibodies, F(ab')Z_ Fab, Fab', Facb, 1c, etc. The term antibody also includes fusions of such an anh~ody gp defined and other funCtiotta) moieties (eg. tpxinS, ~okines, chempk1t1e5, 5tieptavidi7t, adhesion molecules).
According to one aspect, the invention is directed to a multispoci8c ligand with at least two different binding specil9cities far different target ligartds on the same target entity eg_ a Bell and which is preferably adapted to bind contemporaneously to (se. there ere no geometric or other constraints which preclude both moietiss from fut,ctionally interacting with their respective target ligands at the seine time)the diff~ront target ligands, said multispecific ligaud comprising a fust target binding moiety which preferentially(some cross-reactiviry(s) does not preclude the utility of the invention) racogtizes a fu~st target lia~and and a SeCOnd target biI1d1I1$ LltOiety which preferentially recpljnizes a gecpnd target ligand, and wherein the ability of the second target binding moiety to bind to the second target ligand is diminished relative the ability of the first target binding moiety to bind to the first target ligand, the first target binding moiety having an ability to bind to the first target ligand which is at least sufficient for the first target moiety to bind to the first target ligand indepeDdently pf the second target binding moiety binding to the second target ligand and an off rate (with respect to the fn~st target ligrsnd) which at least suf~,ciently cxCCedS the on-rate of the second target binding moiety for the second target tigand to at Irsst provide oppottuniry for the second target moiety to bind the second target ligand when the first target binding moiety is bound to first target ligand, the second target binding moiety having a relatively diminished ability to bind andlor stay bound to the second target ligand independently of the binding of the first target binding moiety to the frost target ligand (such that a phvality of the rnultispecitlc llgand will bind to a population of cells bearing both target Iigands ln,prefarerfce re a population of cells bearing only the second target ligand (se. at least in part due to the first target binding moiety assisting (sc. providing oppommlry) the second target binding moiety to 's 1 bind to the Second target ligand and preferably out of propordon to what could be stotistically attributed to tkte presence of two targets ligands on tile target cell cg, the binding of the first target binding moiety providing necessary assistance for the second target moiety to bind Is rclatIvely increased (ic. relative to the situation where both of arc of comparable affinity) Tt will be appreciated that relative number of bioavailable second target ligands relative to the number of the bioavailable first target ligands will influence the scleetion of affinities of the first and second target binding moieties. For example, from the standpoint of safety, the afi-tuity of the fast target binding moiety for the ftrsc target llgand may well be sui~cient if initially approximatingnanomolar affinity and the affinity pf the second target binding moiety for the second target ligand will be selected tp limit the tfutnber of effective binding events on the population of cells bearing only the second target moiety; an afffniry which is inversely proportional to the nurnba3r of biuavailable second target ligands on the population of cells bearing only the second target ligand re. the non-target population (relative to the number of first target Ggands on the target population of cells). For example, this may be assessed by determining the amount of labelled multispecific ligand on the target and non-Target populations of cells in vivo (ar in vitro where the number of bloavailable firs, t and second target ligands can be roughly estimated). This selected afl3niry, Crom a effecdvcncss point of view, will then be assessed as to whether it is sufficient for the second ligand binding moiety to bind to the second to bet ligand on the target population of tolls, with the bentfit of the fast ligand binding rnoicYy bound br having been bound to first target ligand.
For example, whore the binding of the second target binding moiety may be assessed through an in vitro assay (eg. an assay in which the blocking or activating of a receptor is measurable eQ, through inhibition of binding of the natural ligand for a target receptor or ehrough some measurable parataeter associated with effective binding for example the rtlease of cytokines or otter biologic ePfcctor ligand. The effect of binding tuay be also be assessed by comparing the effects over time relative to a higher affusity second binding moiety which is not associated with a 6n,t ligand binding moiety.1t will be appreciated that a more ubiquitous second target llgand may require selecting a higher initial affinity et: the first target binding moiety for the ~r~ tprg~
ligand eg. picotnolar affinity, and selecting an afllnity of the second target llgand which may for example be of micromolar affinity plushninus approximately one order of ma(~uitude. It will also be appreciated tlwt the deleterious effects of non-target cell binding will vary as will the degree to which the first target ligand is uniquely found on the target population of cells. In the final analysts a suitable difference in a~niry between the two binding affinities may well 6c et leapt, approximately, one, two, three, four, ffvC, six, seven or eight orders of magnitude. In this connection the term approximately refers to +/- up to a half order of magnitude (~Sx). As discussed below, the invention contemplates that variants of a dual affinity multispecific ligand may be assessed in a high throughput screen or series of soch screens with a view to selecting a variant that has one or mare predefined properties, alluded to above such as a) the ability to mediate a biologic attest on a target population relative to a negative control; b) the ability to mediate an improvtd or diminished biologic effect on $ target population relative to a positive control. This ability may also be assessed in a competition experiment of any probative type well-known to those skilled in the art; c) the inability or diminished ability to mediate a biologic effect on a non-target population relative to negative and positive controls. Such dirrtinisbed ability may be also assessed in a competition experiment of stay probative type well known to those skilled in the tuft d) the abilikty to target a target population through binding relative to controls and in a competition; e) the inability or diminished ability to target a non-target population relative to controls and in such competition experiement.
In one embodiment, said first target binding moiety r~ognizas an entity-associated ligand ag. a target cell-assaciated* target ligand, for example a ligand which is exclusively expressed, primarily expressed or over-expressed to advantage on the target cell population and said second target binding moiety recogulzes a non-target cell-associated target ligand which is present on target cells and non-target cells, for example a receptor, including a decoy receptor eg. for TItATI.,. The multispeci8c li,gaud is thereby adapted to block or activate the receptor primarily on the target population of cells.1n this connection, the invention is also directed to methods of evaluating or implementing the effects of this enhanced selcctivily for the receptor on the target cell population and can be employed to diminish the adverse consequences and evaluate the beneFtts associated with using a ligand balding moiety that would otherwise undesirably bind to receptors on non~target cells.
The invention contemplates that a variety of different strategies that can be used alone, or in any variety of compatible pennutations to differentiate bttwecn target cells andlor between target and non-target cells.
The choice of strategies, may depend at least in part on the circumstances, including the nature of the fluid environment in question, including the rapidity and pressure of flow and the direccion(s) ofth;s pressure, the method of delivery, the medical condition for which the molecule is being evaluated, whether the target is moving or stationary, or both, the location or various locations of the target, the targeting venue or venues that islare most ei~ective and the importartcc of the size of the molecule for reaching the target as well as bioavailablility, and the importance of creating immunocotguaatas and immunofirsions with other molecules (insofar as this affects the size and distribution of weight in the molecule). The invention contemplates that employing more than one than one type of construct may be desirable and the invention Is therefort directed to the various combinations and permutation of constructs according to the invention, in combin$tion with tech other and other thefapeutic trtolecules or modalities. one of constructs contemplated by the invention, is a multispecific antibody, far example a blspecific antibody having a configuration which allows for binding to two antigens on the same cell, for example a traditional four chain ftnmunoolobulin configuration having a hinge region (inchrding F(ab')z minibodies etc.), a diabody configuration (depending on the relative positions of the target ligends) and others herein referenced and known to those skilled in the alt. 1t will also be appreciated that the mode of action of the multlfuoctional ligaad tray be cof7tmbuted to by fusing or conjugating the multifunctional ligartd to another ftrnctional moiety, for exatriplG, as dCSCribed in t~ liternttrre referenced below_ These supplementary strategies are set forth below:
Additional Strategies For Modifying Targeting Capabilities Accordit>~ to one embodiment, the intrinsic afftttity of the 5rst target binding moiety fiyr the first target is Beater than the intrinsic affinity of the second target binding moiety for the second target. The term "intrinsic" affinity connotes a measure of the affuuty of a given target binding moiety for its target ligand which 1s Independent of the affinity of the at least one other target binding moiety ftir its target ligand end as used heroin could theoretically be evaluated in the context of the muhispecific lignnd as a whole, if the ether target binding moiety had an Irrelevant specificity and therefore could eat bind to its target tigaad.
The invention contemplates that at least approximately one, two, three, four, five, six, seven or eight orders of magnitude differences in "intrinsic affinity" may be required to accomplish the targeting objectives of the invention_ According to another embodiment, the relative on-rate' of tile first target binding moiety is greater than the relative on-rate of the second target binding moiety. The farm rolativc on rate is used to coonota an e~esrtlve difference In oa-rate tliat may be instr3nsic to the individual target binding ligand or may attributable t4 its configuration Or relationship vie-i<-vie other parts of the molecule.
Whero the intrinsic on-rarer of the fnat target binding moiety is greater than the invinsic on-rate of the second target binding moiety, the invention contemplates that tfie off rate contribution to the affinity of the second target binding moiety may be proportionally greater than the off rate contribution to the affinity of the first target binding merely. The invention contemplates that the binding of the second target ligand binding moiety to its target ligand may be more eiTxtive if its lower affinity is attnbutable in part due its reduced on-rate. The invention contemplates methods for reducing the atllnity a target binding moiety by reducing its on rate for example by mutating or adding amino acid residues in regions of the VH or Vl. that don't directly cvntnbute to the off-rate (of a relatively high affinity binder for the target, for example, as determined by modeling and stwctiu~al analysis, for example, by evaluating x-ray crystal structure and evaluating NMR data of the herding, or by rnutagenesis, preferably by introducing a diversity of changes in a high-throughput manner (eg_ phage display, ribsome display,microarray or other expression library) including substitutions, addhions and deletions within various regions of the VH or VL and determinin4 their effect. For example, the invention contemplates that the second target binding moiety is generated using a library characterized by members in which one of the regions of VH or VL, including particularly the CDRI and CDlt2, for example the CDRI of the VH or CDR2 of the VL., is shortened and/or mutated in a manner to reduce the prpbability of its having any direct contribution to the affuxity of the selxted molecule (through molecular interaction), for example mutated to introduce amino acids that are least important for intermolecular interactions, for example by minimizing the occurrence of amino acids that an:
impotTSnt for electrostatic interactions and optionally also hydrogBn binding, gencratirtg a bhlder whose affinity will be postulated to be independent ofthe contribution ofthe modified CDR, andthcn optionally evaluating the success of this latter step through further mutagenesis (this step is most revealing if the CDR
is shortened but not mntatad or mutated to introduce amino acids important for intermolecular interactions) ' The actual on-rate if the on-rate was to be measured indapendetttly of the on-fate of the other binding moiety and then using the library to incrementally lengthen the region and/or introduce aminq acids important for intermolecular interaction at a distance (cg. electrostatic intcrasctions arid optionally also hydrogen binding) to introduce minimal steric hindrance or intermolecular repulsion. The invention also contemplates that introducing amino acids that have the greatest potential for hydrogen bonding may introduce an aqueous cushion into the interface region with the target ligand to diminish the on-rate contribution to affinity. The invcriliori also contemplates modifying the amino acid composition of an existing binder by introducing or one yr amino acid8 or lriu~ttions into a fCamewprk region at a location which is proximal to the binding region or a region which borders the itttttface of approach to the binding region or any interface between the target binding moiety and the target lig9nd. Tilt iriverition contemplates that the an-rate and pff rate can be routinely measured using various technologies (cg. Biacvre) known to those skilled in the art, including various techniques of measuring these rates in real-time, for example those that measure the deflection pattern of an incident farm oFradiatlon (cg. Biosite). In one embodiment of the method the antibodies each have unique preferably cleavable peptide tags that are generated for example through a random or partially random insertion of nucleotides into the DNA encoding the antibody and that serve to link them to their DNA cg a phase (as per techniques known to those skilled artisans or published in the art) and the antibodies ere evaluated independently of a phase (cg. they may even be cleavable from the phase) or other expression system linkage which allows a more accurate measure of their true on rates and oft=rates. The invention also contemplates that FR.1 could be lengthened in a relatively high afI'trtity second target binding moiety to reduce its on rata. The cleanable peptide could be a unique identifying CDR.
In another aspect the invention contemplates that the multispecific li8and may comprise an Fe portion and a hinge portion and that one or both of a) the length, amino acid composition br"' molecular weight (or various combinations of these interrelated factors) of the Fa6 or Fc portion;
and b) the amino acid composition (including length) of the hinge portion (cg. any polypeptide segment that provides means for linking two typically heavy chains, cg. throu6tt one or morn disulfide bonds, teucine zipper fns j en, optionally a flexible hinge typical of an IgGl or having one to several more disulfide bond3 cg. Tg(33) arc selected to reduce the circumstaniial(shear rate, presence of degrading enrymes) affinity of the second ligand binding moiety whore the first tigand binding moiety is unbound relative to the circtunstantial affinity of the second ligand binding moiety where the first ligand binding moiety is bound. The term circumstantial affinity broadly contemplates that the length and molecular weight of the Fc and the flexibility of the hinge region will individually and colJcctively contribute to the affinity of the molecule in proportion the shear rate ofthe fluid environment to a degree depending on whether the target is stationary or moving, once the lnttl#ispecifie ligand ie bound. If bound via the second target binding meicty, any increase in the molecular weight especially a distribution of the molecular weight towards the Fc or first ligand binding moiety will serve as a lever in a moving fluid environment, to favor disengagement from binding especially since the off rate of this binding arm is relatively tow to begin with. This same lever effect will impinge on the binding of the first ligand binding moiety but to a lesser functional degree due to its higher affinity. To sri extent deprading on the context in which binding occurs, the invention also contemplates that the high affutity ligaitd binding moiety will draw the multispecifzc ligand from the circulation into a desired target tissue and that the low affinity binding arm will then have greater appartttnity to bind even if it does net bind simultaneously with the high affinity binding arm. Where the hinge region is extra flt7tible or has Several regions of flexibility (far exa,nple where the heavy chains are linked through several d;sulfide bonds with regions of Plexibile linker therebetween) the disenqagittg effect on the individual and paired binding of both the first and second ligand binding moieties wih be Ic:3s Similarly, using a truncated Fc portion (Chl3 dek;ted, F(ab'): or minibody format) will assist the first ligand blndinm moiety to remain bound or foster binding of the second liaand moiety and will assist the second ligarid binding moiety to remain bound. This construct may be preferred from an effectiveness standpoint (getting bath ligand binding moieties bound), where the affinity of the second ligaud binding moiety is low to begin with.Gn the other hand, decreasing the flexibility of the hinge region by alteration to its length andlar amino acid composition and increasing the molecular weight distribution towards ehe "Free" and of the Fc will affect ail binding scenarios to a greater extent. The latter strategy may be less desirable where tha Fob of the first ligand bindine moiety is lengthened (cg. has $ lOripCr hitlge region at the N-tetTtlinu5 Of the disulphide bond linlaug the heavy chain,, than the low a,ffin.iry binding arm) to increase its propensity for individual binding. For example, in a conventional four cliain or heavy chain antibody (two heavy chains but no light chains) the hinge region could be lengthened or shortened on the amino terminus side of the disulfide bond linking the heavy chants to an extent that does interfere with the simultaneous binding to both the first and second Larget bindles moieties- The invention also contemplates that the target cell environment, naturally or through intervention, is a fluid environment (low shear rare) or enzyme environment which will favor a greater impact on disengagement of the second ligand binding moiety, in the case of an enzyme, one which will cleave off an Fc into wlvch a cleavage site has been introduced so that disengagement due to the lever effect wilt primarily ixnplltge on binding of the seGOad ligand moiety to the non-target cell population (cg. low shear rate or proseace of MMP typE
enzymes in a targeted solid tumor environment).
The invention also contemplates that second ligand binding moiety may be selected in an environment in which there is a selac>ive presence (moderate fluid flow cg. using live cells or tissue, candidate ligtmd binding molecules or pairs of the target ligeads on latex beads, where the substrate to whiCll they aTC hound is on an incline or otlierwise snbj~ to fluid flow (optioaally with rigid or b'lgh mol. weight l~c), for simuhaneous binditsg so that the aiHtlIty of tile second llgand binding moiety is seh;cted on the basis of its ability to augment the binding atY'uuCy of 8 fr9t Iigbnd binding moiety of preselected affinity for the fast target lignnd (after or optionally before its affinity maturation, depending on the shear force and affinity in question) and thereby augntant the affinity of the multispecific binding ligattd as a whole, while the first ligand binding moiety is bound, In dtis way, the strength oftha binding affinity ofthe second Iigand may be predicated on the fast ligand moiety being bound. The foregoing strategy may have accentuated or at least equal application where the first ligand bindinb moiety has a longrx 1.-"ab or for example where both the 'First and second ligand binding moiety are devoid of a light chain ie, where having the correct binding interface for the second target binding moiety might be more acute. The invention contemplates that the individual affinity of second Ilgand binding moiety selected !n the above manner would be tested to ensure that its individual aflir>ity was ant sufHcieat for substantial indepcndsat targeting.
The invention also contemplates that engineering a suitable affinity antibody for solid tumor targetitta in which the on-rate contribution to af5nity is reduced (accordlrtg to the strategy suggested above) may assist a dose of such antibody In achieving better tumor penetration An antibody having a reduced oa rate could be fused to a toxic such as a truncated version of PE or conjugated to a rsdionuelide, etc. the reduced on, rate contnbution ensuring that the antibody will be less ltlcely to bind at sites proximal to the point of entry to relieve congestion in that area and better ensure its diffusion throughout a tumor. The invention contemplates thnt the strategies decribed above wlll better permit the affinity to be more suitably apportioned between the on-rate and the off rate. The inventton contemplated that a higher on-rata lower off rate Ab could be delievcrcd in alternating days or other cycles oftreatTnent. Thus the Invention is directed to an antibody conjugated or fused to a functional moiety, wherein the on-rate conttibutlon to the affinity oCthe antibody is anywhere between 3x and two order ofmagnitudes less than typical molecules having suitable properties for tumor penetration through difftisiarl, far example molecules having anywhere (any increments) between 10-t and 10-t° molar a~lnitIas (tg. 5x 10-', 3x10'0 preferably increments between 0'' to 10't° (molecules whero the ou rate is twtmally approx. I0-3) molar af6nitias, more preferably itxremants between 5x10$ and 5xI0-~
1t will be appreciated that the foreQoina strategies could to employed for designing a muItispecifie ligand which will primarily target cells which have both the first and second target ligand (cg. where the ligands together are present primarily on the target cell population) even where neither target ligand Is individually found primarily on the target X11 poputatioo. by employing a multispeci>ic ligaad in which neither target ligand Is of BuFficient a~fl5rtity iri the eiretu»stanees tb effectively (with effect) bind or remain bound without the other target ligend lxing available for simultFUteous binding_ As suggested above, it will be appreciated that a relatively higher affinity ligand could initially be employed on one of the IIgaed binding arms to select a second lignnd binding arm which improves the binding properties of the t,aultispeai5c ligatld under a suitable biologically relevant shear stress and which is selected or later modifted so that it is individually insufficient for targeting its target on non-target cells in the circumstances in which it will he employed, and that the high affinity ligand binding arm can subsequently be reduced to moderate affinity with similar lack of individual effect. In one embodiment, this construct can be employed to evaluate the effect of blocking two t~ecepmrs on the same cell, for example chemokine receptors cg.
CCR7 and CXClt4 on a breast cancer cell. In one embodiment, the off rate of one or optionally both ligand binding moities is sufficient in the circuntstanoes to permit the moiety to remain bound for a sufficient duration for the oilier moiety to bind ie_ it exceeds its effcetiva or intrinsic on-rate. In one embodiment, both arms of such multispacific llgand, bind to their respective ligands with low affinity. In one embodiment, one such arm is a "cpybody'".
In connection with the foregoing and ensuing strategies It will also be appreciated that the hinge region may be lengthened on the N-terminal side of the most N-terminus linker between the heavy chains so a2 to perntit greater flexibility in the binding of dil3erent antigens at dilyercnt possible proximities to one another.
The invention also contemplates that the two heavy chains of an IøG (with or without light chatns andlor CH1/CL domains), minibedy/ F(ab',)z (with Or without light ChaidB and/or CH1/CL doalaios), may be linked (whether they have a full size or fully truncated Fc or elongated hinge regions) tluough a flexible peptide linker (such as used for making scFvs i.e. multiples of gly4ser) in order to ensttrc correct pairing of the heavy chains by expressing the linked heavy chains in E. Colt, for example, as inclusion bodies, which are refolded in refolding solution according to well estflbllshed techniques in the art. In a construct employing light chains, the light chsias may be linked through a disulphide hand linking according to wall known methods of making disulphide stabilized Fvs (ds>r~rs) and the same light chain may be employed for both the high and low ai~nity arms.
With respect to each ofthe preceding aspects ofthe invention, the invention also directed to a multispeciiic ligand comprising a first Llgand moiety which recogni~s a lust target ligand~that is ova expressed on a disease associated entity for example a diseased or disease-causing or mediating cell or infectious agent and a second ligand binding moiety that recognizes a forgot llgand aed wherein the first target ligand is ~baracterizc:d in that it does dot lend Itself to facililatiag ar pannitting inttmaliz8tion of the second ligand binding moiety.
The invention also contemplates that a target Iigand can be distributed in various concetmations for testing purposes on cell slzxd latex beads, columnar paclang inateriaLa or flat substrates having a high density dispersion of both target ligands.
The invention is also directed to combination therapies with the foregoing multispecifle ligands including, without limitation, immunotoxins, drugs, therapies wilt other multispecific Iigands herein described and particularly for cancer therapies dirceted at interfering with the integrity of tumor cell vasculature.
Delivering ~iologlc Ltlcector Ligands To A 1 argot Etttlty W ith respect to each of the preceding aspects of the invetttian, the invention also contemplates that the second Ligand blndino moiety may be constituted in whole or In part by a llgend which binds to a biologic etFector ligand (such as a rytoka~e, colony stimulating for, chemokine, growth factor etc. ar related exrracellularly expressed regulatory molecules that control their expression ouch as inhibitors, agonists, antagonists of same, which may have corresponding biological receptors), the ligand optionally having a higher aFfmity for the biologic effector ligand than the affinity of that biologic effector liaand for Its receptor, and wherein the ligand, combined with the bioiogie effeetor ligand (ie_ bound thereto), lies a relarively dhninlshed ability to bind and/or stay bound to the receptor (the second target ligand) independently of the binding of the first target binding moiety to the first target ligand eg. a lower affinity of approximately one, two, three, four, five, six, seven or eight orders of magziitude. The invention contemplates that the foregoing construct can be used to deliver the biologic effector ligand more selectively to the target cell population recapiized by the first ligand binding moiety. The second ligand binding moiety may be an a>ttibvdy portion of a multispecific ligand of the invention and the invention contemplates that a library of second ligand binding moieties, recognizing mulliple different epitopcs on the biologic efFactor ligand, can be screened far tlieir ability to bind to the biologic effector ligaud, while it i5 bond in gitu to its receptor, For example, using a miCroarrary oFsuch anri'bodies, and the affinities of the binders cen b~ evaluated.1'he invention also contemplates that suitable antibodies could be generated by "panning" (with an expression library, eg. phage display, ribosome display, or other similar display systems including yeast, bacterial, viral, cell based or eel l-free display systems) or otherwise screening (eg. using antibody microarrays) against the biologic et»etor ligand while bound to its recc.~ptor and seretming for their ability to bind to the biologic effecwr ligand independently of its receptor. Again, the af)itiittes of the antibody coupled to the-biologic effector Iigand far the target receptor could b~ evalueted_ More generally, the invention contemplates that an array of antibodies which recognize all different epitapes on a given biologic effector ligand could be generated and tested for their ability roc accommodate binding of a biologic effector Ligand to a first but not a second in a related Family of receptors. This could be accomplished by screening the array for one or more members that bind to the biologic effector ligand 1 ti ($EL) wltile bound to it9 receptor, and testing the identified members for their ability to bind to the seeped receptor, preferably by loading the biologic effector Ligand onto an array of those members pre-bound with BEL and dttecting those 8EL bound members For those which do and do not bind to the seed receptor.
Therefore the invention is also directed to an antibody which accommodates binding of the 8EL to one receptor but hinders the binding to at least one second receptor, preferably by steric, charge or other inter-molecular hindrance, attributable to the proximity of the antibody epltope on the EEL to the HEL's receptor binding site and optionally also the annino acid composition of the antibody at that interface.
ThC invtnti0n contCtnplBtCS that flUld flout Can be Sinfulattd in $
puriFtCatlon or immunoaffinity column packed with one or more known packing materials to simulate flow over a ligattd coated substrate.
The invention also contemplates an apparatus and method for testing ligand binding in a circulating fluid environment in which the multispeci6c ligands of the invention can be tested and wherein a trontiinous flow of Llgands, Including target ligands, ligands of the invention andlor ligand bearing entities (cg, cells or synthetic cg. Iatex spheres which can be adjusted to a cell size) to which one or types of ligands have been affixedly associated accordingly to known methods) can be generated. 'fhe fluid contact interface of the apparatus has a ganet~ally circular shape and is convex or otherwise capable of containing the fluid pad thereby preferably permits fluid to ~Iow around the surfhce continuously. For example, this surface may be enclosed with a bagel-shaped cylinder which is optionally open at a location opposite the fluid contact surface for introducing and/or removing its contents, or it may completely enclosed with the exception of an access port, from which airy air may optionally be displaced or evacuated.
The invention contemplates that the apparatus (at least the fluid contact vessel) can be rQtat4d or oscillated (og. it1 an elliptical, oval or similar shape well known to those skilled in floe tA<ts of fluid ZYleChanics and related engineering arts) in a Variety of different planes or with rocking-like naoda~n in multiple planes yr subject to peristaltic pressure (ie. where flexible tubing is used) to generate a continuous, optionally turbulence fi-ea fluid flow over the fluid contact surface at selected rates simulating the various shear rates of arterial, venous, intro-lymphatic flow (including different diameters of such vessels) or Interstitial flow. The invention also contemplates that the fluid contact surface may be provided with a 1) substrate for lhtlclng llgands of the invention or target ligands or IIgand bearing entities to permit fluid flow across the substrax in a plane substantially parallel or conforming to the axis of flow.
In another asptet tht invtttlion is direct~d to methods of making a muItiapecifie antibody in which:
a) the light chains are the same for both the VL domains . For example, the light chains (assuming the construct laa'two light chains) are generaud for a First target binding moiety cg. in one aspect of d1c invCntion, the relatively high affinity binder, optionally from a light chain gerntline sequence, and this light chain is then coupled with a diversity ofheavy chains to select a pair of chains which bind to the second target ligand, thereby constituting the seeped libarid binding moiety, which may be a relatively low aif'mity b~der. An alternate or concorriitent strategy to generate rl lower af3bnity second ligattd binding moiety Would simply be to substitute the light chain of fhb first ligand binding mnicty for that of the second ligaad binding moiety and to test the affinity. In die case of a multispeciFe which target l3ELs to particular target cells, where for example, two high affinity binders are preferred, the heavy chain and light chain binding to the BEL. can be truncated correspaadingly at the ~H1ICL region so that the VHNL
interfaces and cysteints pairing these heavy and light t:bains are similarly spaced but spaced differently from the other VI-1/VL chains. By lutldng the heavy chains as explained above, all chains will pair correctly. It will be appreciated that the foregoing production strategies could be applied ep the production of heavy chain aotibQdies (two chains structures without associated light chains), wherein the heavy chains are from human or other spccits and that production iu this case could be adapted to E. Coli. 1t will also be appreciated that deletion of a substantial part of the CH 1 and GL domains can be measured to provide a space for the BEL to sit in line with the other Fab which can be Iengthenod in the linker or CH 1 domain, as shown in Figure C.
The invention contemplates that evaluation of a diversity afthe first target binding moiety cats be accomplished with the 13EL place to bust accommodate selcctioa in the context of the entire structure its a whole.
b) With respect to othCr methods to makC bispCciftc and bispCCific fusivns see Antibody Fusion Proteins Wiley-Liss 1999 (infra) cg. particularly p 131 at seq., and Chapter 7 and the discussion, Methodologies improving the correct pairing of heavy chains are well-known in the art.

Such a construct could also be employed in conjunction with other flmctional moieties fused or conjugated thereto, for example toxins, cyWkines, ettzymes, prodrugs, radionuclides etc.
In one prefetTed embodiment, t)re invention is directed to a mulrispecific ligand* witb at least two different binding spec'tftcitiea for different target liganda* on the same target cell*
and adapted to bind contemporaneously to the different igrgct ligands, said multispecific ligand comprising a first target binding moiety which preferentiaily* recognizes a first target Llgand and a second target binding moiety which preferentially recognizes a second target ligand, and wherein said first target binding moiety recognizes a target otll-~lSaociatGd'' target llgand and said Second targOt binding moiety rttOgnirts 8 non-cell-associated target ligand which is present on target cells and non-target cells; and wherein the ability of the second target binding moiety to bind to the second target is diminished relative the ability of the first target binding moiety to bind to the first target ligend, the first target binding moiety having an ability to bind to the first target ligand which is at least sufficient for the first target moiety to bind to the Ever target Iigand independently of the second target binding moiety binding to the second target ligand and en ofF rate which at least sufficiently exceeds the on-rate of the second target binding moiEKy for the second target ligand to provide oppot~tunity for the second target moiety to bind the second target ligand when the first target binding moiety is baued to first target ligand, the second target binding motery having a relatively diminished ability to bind or stay bound tv the second target ligand independently of the binding of the first target binding moiety to the first target ligend, such that the multifunctional ligand will bind to the target population of cells In preference to the non-target population of cells. As suggested above, the strategy embodied in this preferred embodiment can also be employed is conaectfon with any one or any combination of compatible strategies referred to above, to dinninish in degt~ee the requirement of using a low affinity second ligand binding moiety.
In another aspect the invention is directed to hettrofunetional ligand comprising a first moiety which binds to a first target ligand and a second moiety which binds to a second target ligand, and wherein the affinity or avidity or both the affinity and avidity of said first moiety are selected to enable thefirst moiety to bind to the fuse target ligand independsntty of the ability of said second moiety to bind to rhc second target ligand and wherein the relative avidity or affinity or both rite affinity and avidity of said second moiety era selected or adjusted to substantially reduce the probability of the second moiety binding to the second target ligand without the first moiety, first or substantially contemporaneously, binding to the first targ~
ligand. For example, in one embodiment the first moiety is divalent and the second moiety i$ moaovtyent, In one embodiment the affinity of the first moiety for its target llgand Is for example up to several orders of magnitude greater that llx aF>mi.Cy of the stooad moiety for its target ligand. as discussed below. In a prxtisrred embodiment both moieties are capable of binding to different target ligattds on the same cell, for example as hereinafter speciFed, although in the case of tumor call targeting, particularly with respect to cells that are growing adjacent to another the invention contemplates that ehc first moiety may bind tp one cell and the second moiety may bind to a neighbouring cell. Accordingly, in ~e cast of rcecptorg requiring cross-linking for biological activity 'the invention contemplates that such same cell interactions and adjacent cell interactions are optionally accomplished when Ihc second moiety is bivalent. In one embodiment, at least one of said first and second moities comprise one or more antibody components. In enpther embodiment, s$id first moiety binds to et least one cell-surfaoe llgand which differentiates bctwaen cells of the same population or sub-population, for example, at least one Iigand which diffentiares which between populatious or sub-populations of inunuae cells (eg. see GV~ 01/21641, US
6156878), for example, activated vs. non-activated, di3eage-a;a0ciated or non-diaeas~associated (over-expressing or uniquely expressing certain receptors or other ligands [for example cytokine or growth factor receptors, pattiet<1ar intmunoglobulin like molev.-ules or MHC peptide complexes] or other differentiating markers hereinafber exemplified or apparent to those skilled in the art), and said second moiety, in virtue of its binding to the second target figand, directly or indirectly exerts a biologic effect og. a therapeutic effect, for example an immune modulating effect- Tn a further preferred txrtbodiment said second moiety has a broader target cell population than said first moiety Eg. see Whey H. et al. Expression of CC
Chemokine Receptor-7 and Lymph N4de Metastasis..., J. Natl. Cancer Inst. 93;1638-1643; Maore MA
Bioassays 2001 Aug;23(8):674.
6. (The invention contemplates that by targeting CCR7 receptor stltClivtly an tumor arils, fOt txatnple using a relatively high affuuty binding moiety for a tumor associated antigen and a relatively low affrnity moiety which binds to and blocks CCR7 receptor, eg. when combined in therapy with a chemotherapeutic agent ar an Ltatntutotlxtut for the same tumor, metastasis can be inhibited).
For example, in one embodiment said first moiety binds to a tumor associated antigen on a tumor cell and said second moiety binds to a receptor which is found on the tumor till but also on a braadtr population of tolls. In another embodiment said first moiety binds to an anrigen associated with particular population of leukocytes and paid gecpnd moiety binds tp a receptor which ;5 found on that population of cells but also on a broader population of cells (eg. apoptosis mediating receptors Journal ol"Immunology 1998 160.3-6, Nat Mad 2001 Aug; 7(8)954-960, WO 01185782; ICAM-R WO 00129020; see also WO O1 J85768, WO
01185905; WO
O1J83755, WO 01/83560, WO 01129020: Vitals et al. Prpc. Nat. Aced. 5ci. 2001 May 8; 98(10):5754-5769;
CCRZ see also USP 6312689; USP b,294,655 Anti-inicrlcukin-1 receptor antagonist antibodies and uses thereof-, USP 6,262,239; USP 6,268,477) . Iv another embodiment the second moiety does not necessarily bind with lower affinity to its target however it may bind to a first lisand which in turn binds to a second ligand on a target cell (eg. a receptor on the target cell eg. a cytokine, ebemokine or growth factor receptor), for example the receptor being on the same cell to which ho first moiety binds, and Lt binds 111 a manner in w6iCh it partially interferes with tht binding of the first ligand to the second ligand and thereby directs or retargets that first ligand to the second ligattd in a manner which accomplishes the intended interaction of the the first with the second ligand (eg a signal transduotion or blocking interaction ic. the second moiety causes the ee. cytoklne to bind to its recepmr without engendering the biological effects attributable to receptor binding eg. signal transduction, which may be assessed by assaying for effects of eg. signal transductioa according to well established techniques In the art) but less competitively relative to the first moiety so that the fast moiety exerts a targeting function ie. where the first ligand bound by the second moiety binds to a broader than desired population of cells. The binding of the second moiety tnay also be compatible with the first ligand binding to one cell surface ligand but not another eg. gee Wp Op/ø494( the conxats of which are hereby incotpotaxd by reference. The abillry to identlf~
ligand residues of importance to binding or residues other these, the alteration of which might interfere with binding is well established in the art The invention contemplates varylno, by high throughput techniques eg. phage display, residues oFan antibody that are not involved in first ligend binding 1Q create variants wliich eaa ba tested for partial interference with first ligand binding t0 tile second llpitnd eg. rGGeptor binding.
Exareples of receptors for blotking or activation by the targeting methods described herein include tyrosine kinase type recptors, serine kinase type receptors, heterotrimerio G protein coupled receptors, receptors bound to tyrosine kinase, TNF family receptors, notch family receptors, gttaaylate cyclase types, tyrosine phosphatase types, adhesion receptors etc. (for example receptprs sea those discussed in Caucer: l5rinciple5 and Practice of 4acvlogy 6'" Ed. De Vita et al. Eds Lippincott 2001, lnclnding particularly Chapter 3, 7 and 1 S, The AutOimmune Dise;tses, Academic Frass Third Edition, RoselMaekay ISBN:
0125969236, Immunology 6'" Edition, Masby 2001 Roitt et al. Eds; Molecular Mimicry, Microbes 8c Autoimmuniry by Madelcino W. Cunningham (E.ditpr), Rpbrrt S. Fujinatnl (EdltOr) December 2000, among other references horeininbelow idantifted). Further mention may also be made of interleukin end interferon type receptors, HGF receptor (see for example USP 6,214,344), CD45, CXC family receptors including CXCR1 and CXCR2 receptors including IL-8 receptor, ECFI~s, receptors for molecules with fnnetions in apoptosis or homeostasis, receptors such as FGF which sensitize tumor cells 1v chemotherapeutic agents, ate. It is known for example to modify receptor ligartds in a way which does not interfere with a signaling function (the residues important for signaling may be known or can he readly ascertained eg. see Retargeting interleukin 19 for radiointmuuodetection and radfoimmunotherapy of human high-grade gliomas, pebiaski W, l7tompson JP.CIin Cancer Res 1999 Ocr;S(10 5uppl)-3143s-31475) but reduces the affinlry of the ligand for this receptor (sec also Wp d 1119$61 ). Alternatively, the second molery may be an antibody which is alanistic or antagonistic and used to block, activate. neutralize etc the receptor. With respect to EGFR family,TNF family and other receptor tar~,eting antibodies which are capable of causing apoptosis directly or indirectly, see US 5,876,158, WO 00120576, W096108513, WO 01144808 (P75AlRM1 ), WQ
00/29020 (1CAM-R), WO 99112973, CA 2234913 etc. The invention also contemplates that the second moiety may also be targeted to a specific portion of a receptor which differentiates it from other receptors of its class and more generally contemplates that the second moiety may contribute 2a the targeting ability of the multifunctional ligand.
lri another aspect, the invention also contemplates that the first moiety binds to a target cell and said aeeond moiety binds to a ligattd, for example a natural ligartd, (eg_ a cytokine or chemokine circulating at normal levels or at higher levels attributable to a disease or treatment of a disease with another therapeutic molecule) and retargets that !baud (for example, the ligand may be retargened from circuletian) to a targeted cell. For example the invention comtemplates that TL~2 tray be retargeted to 1: AR Culls or CTLS
via a high a~niry L,eu-19 binding first moiety. for example, antibodies including fragments thereof which bind to cytokines or ether natural ligands for retargeting purposes (eg.
single domain antibodies) can be made by phage display against the cytokine or ligand while bound in situ to its receptor. The invention also contemplates that the affinity for Cha cytokinu may be adjusted to regulate the degree of targeting and that scnan samples may be evaluated to assess the degtt;e of bound cytokine and the relative degt~e ofbound altd unbo~d Cyboi~11t0. Arnvng other methods, for exgmple, the invention contemplates that a radioltlbClled multifunctional ligand may be used assess the amount of label associated with tho muhifwtctional ligand when bound to the cytokina, by capturing tl,a 'complex' with an antabody that recognizes both antigenic determinants on both the cytokinu ttttd an adjacent portiout of the ligattd binding thereto ie. forming g composite epitope), such ag may be generated by phage display and assessing the amount of label relative to the amount of captured complex.The invention also contemplates administration of supplemental amounts of nrttural llgand to compensate for the degree In which the llgand is retargeted Insofar as such retargeting rnigttk impact negatively on immune or other phygiQlogical processes.
In another aspect tba invention contCmplates that patients treated with anh~odies to a particular biologic effector ligand eg. a natural ligand eg. a cytoldtte, for example TNFa, may preferably be treated with a muhi~ctiottal ligand having a first moiety which binds to at least one cell type and a second moiety which binds to a natural lilgand suslt as et cytokino for retargeting that cytokiae to that cell type, as in a preventative method for treating a disease, eg. cancer. !a this respect: the invention contemplates that the antibody is capable of binding to the cytokine but once bound the cytokinc, the cytokine is incapable and/or only weakly capable of binding to 'rts receptor and/or that the multifunctional ligand also comprises a higher at33oity receptor blocking moiety to minimize retargeting of the primary disease site. in one embodiment, the first moiety binds with relatively higher flutctional affinity (lo. avidity, af$nlty, andlor Telatively advantageous binding capacity in virtue ofmultiple ligand binding arms, each l7inding to different llgands on the target cell) to enstu~ binding to the retarget cell.1n another embodiment the bound cytokine is capable of binding to the cytokine receptor at the retarget site but incapable of binding to tIu reeepror at the disease site owing to differences in the receptors at the two sites. The nvention also tOnterriplBteS using antibodies which interfere but do not prccladc binding of the b;alagic eft'ectar to provide a less toxic effect.
Far example, patients with GYohn's disease that are treated with anti-TNFa (see for example, Expert Opin Pharmacother 2000 May; l (4):6I 5-22 and references cited therein) may be treated according to the invention with a bisp~eiti~ antibody having, In addition to an anti-TNFa binding moiety, which reduces the aft-,pity of the bound T1VF for the receptor, but also an antibody moiety which binds to tumor antigun which is expressed on many different tumor types or optionally a trispecific antibody which additionally binds to a second multi-carcinomic antigen, preferably one which broadens the range of targeting against prevalent cancers. W itb respect to ttunor antigens rnentlon may be made of EGFR, EPCAM, Mt,ICTNs, TL~G-~2, CEA, H11 among ether known multicarcinomic antigens (see also Cancer:
Principles and Practice of Oncology be Ed. De Vlta et 81. Eds LippiriCOtt 2001 Chapters 18 and 20.5).1n another embodiment, the second moiety differentially retargets a cytokine to one receptor in prefrrence to another, for example, to a ThtF receptor over-expressed on tumor cells in preference to a TNF receptor associated with Crohns disease. l;a a related but also indeporadent aspect, the invention contemplates a method of screening for an antibody which prefererrtialiy hinds to a ligand when bound to a first receptor relative to another second receptor by screening for antibodies (eg. by phage display, ribosome display, eto.) which bind to the ligand eg. a eytokine, when bound in situ to the first receptor, and selecting among them those that bind to the ligand eg. cytakine but da oat bind (substractive screening) or bind with lesser affuyiry to the cytakine when bound to the socond receptor, as well as to antt'bodies end multifunctional ligands created by this method (see also USP 6,046,048 and WO 99112973 and references ched therein with respect to TNh family pf receptpr9). Variations in the extracellular domains Qf such receptors are known and can be ascertained by methods known to those skilled in the art.
Further with respect to multifunctional ligands having a higher affinity targeting moiety relative to the second ie. effector moiety, the second moiety may be for example an antibody or other tigand which interferes with the binding of the regular ligand for this receptor. For example, the invention contemplates a first ligand binding moiety which recogtizes activated T-cells and a second ligand binding moiety which blocks the 1L-16 receptor for testing the efliect on Crohns diseass (or altcrnativCly activ$tCS an IL-16 receptor on those cells eg. by using a high affinity JL-l6 bound second moiety which becomes relatively low affinity IL-16 receptor ligand when bound to the antibody, again to test the effect on Crohn's disease (see C3ut 2001 Dec. 49(6) 795-803) Far example, in one embodiment, the invention contemplates that the second moiety blocks a receptor that ere found on cells other than the target cell, the blockage of which loads to the apvptvsie ofor destruction afthe cell eg. CD95 (ea. see Jung G, et at.,Target cell-restricted triggering of the CI795 (APO-llFas) death receptor with bispecific antibody fragments Cancer Res 2001 Mar 1;61(5):1846-8). With respect to blocking insulin like growth feeler receptor, insulin receptor ate. see 'I'he IGF system in thyroid cancer: new concepts, Vella V., Mol Pathol 2Qpl Jun;54(3):121-4; Insulin receptor isoform A, a newly recognized, high-affinity insulin-like $rOWth factor 11 receptor in fetal and cancer cells. Mol Cell dial 1999 Ivi0.y;19(5):327$-$$; F,~cprtssian of the insulin-like g'owth factors and their receptors in adenocaeciaoma of the colon. Freier S Gut 1999 May;44(5):7D4-8; Pattdini G., insulin and insulin-like growth factor-1 (ICP-I) rtetptor vverexprrssion in breast cancers leads to insulin/IGF-I
hybrid receptor overexpression: evidence for a second mechanism of IGF-I
signalingClin Cancer Ices 1999 JuI;S(7):1935-44. With respect to targeting bite-1 integrins see eg. Masumoto A, et al Rola of betel integrins in adhesion and invasion of hepatocellular carcinoma cells.
Hepatology. 1999 lan;29(1):68-74.
Aran S, et al. Betel itttegrins play an essential role 1n adhesion end invasion ofpancreatic carcinoma cells.
Pancreas. 2000 Mer~O(2):129-37. Xic Y, X;e H. Characterizadan of a novel monoclonal antibody raised against human hepatocellular carcinoma. Hybridoma. 1998 Oet;17(5):437-44. Pang H, et al production and Cltaraettriiation of anti-human hepatotCllular carcinoma monoclonal antibodies. Hua Xi Yi Ke Da Xue Xue Boo. 1990 Sep;21(3):259-b2; Whittard JD, Akiyama SK. Activation of betel integrins induces ceil-cell adhesion. Exp Cell Res. 2001 Peb 1;263(1):65-76 Nejjari M, et al.
atpha6beta) iutegtin expression in hepatocarcinoma calls: regulation and role in cell adhesloa and migration. lnt J Canecr_ 1999 Nov 12;83(4).518-25; Yao M et a1 Expressiaa of the iategria alphas subunlt and its mediated cell adhe3ian in hepatocellular carcinoma. J Cancer Rts Clin Oncvl. 1997; I23(8):435-40.
The invention also contemplates a method of optimizing the cooperative atflnities of respective binding ligands of a multifunctional IIgand described herein and the length of a linker therebetween for the above end apphcatiotts described below by pha~o or ribosome display ate. in which the multifunctional ligand is a single polypeptide chain, for example, two single chain Fvs or single domain antibodies linked in sequence, or a diabvdy (see USF 5,837,242), by varying the DNA sequenec corresponding to amino acids that represent linker endlor for example CDR regions that are postulated to impact on affinity according to methods and strategies that well known in the art far afFiniry mnturatipn.
These same strategies can be employEd for engineering lower affinity molecules. Accordingly, mart generally the invention is directed to a phage display or similar library (cg. a ribosome display library or a microarray) in which the population of variants is a multispecific ligand, including a multispecific ligand according to the Invention herein defined.
In another embodiment blockage of a receptor does not necessarily lead to cell death but may lead only to decreased or inct~sod release of curtain cymkines ate, fbr example as mediated vin the 1L$ receptor. In another embodiment the second moiety may achieve the desired therapeutic effect by constituting the normal ligand for that receptor or a functional substitute. The tnultispecitic ligand may also be fused or eonjugattd to a toxic moiety yr other efFaGbor. In another or further preferred emboditnent, said first moiety comprises two binding ligands (eg. one or both of which may be an antibody) which respectively bind to two different target lt~ands each of which contributes to its total binding capacity and neither of which arc sufficient to efl5ciently target the the cell, for exeirple a ligand which binds to a specific MHC peptide complex and a second reduced affinity ligaod which binds to a ligand on an APC. Th;s approach also obviates the need to create high affutity ligand for a particular MI~IC petide complex, although this can been accomplished. In another or further preferred embodiment the target cell is an immune cell and the second moiety binds to a molecule involved in cellular adhesion, a cytokine receptor, a ligand which stimulates the activity of said immune cell, a ligand which inhibits the nativity of said immune cell, a ligand which causes one or mare cytakines to be released, a ligand which prevent one or more cytokines tram being released, a ligattd which causes or faeilitatts apoptosis of said immune cell or a ligand which permits internalization of said multispecific ligand. In enotlter prefetrcd embodiment the heteroftinctional ligand is fltsed or conjugated to a therapeutic agent or a moiety that binds to a therapeutuc agent (exempliized below) or a ligaud which effects binding to another immune Bell, for example a T cell. tn another preferred embodiment, the multispecific Ligand is a bispecific antibody, a trispeefic antibody or a tetraspecific antibody. In a further preferred embodiment the first moiety binds to but is incapable of modulating the achVity of said immune cell and said second moiety modulates the activity of said immune cell Independently of said first moiety. in another pteferted embodiment the multispecific l;gand futttter comprises a moiety that binds to at least one ligand located on the intraluminal surface of a lymphatic vessel, preferably a Lymphatic vessel associated ligand, as hereinafter dot-used. In other aspects thu invention is directed to a pharmaceutical composition comprising such a multispecific ligand and a pharmaceutically acceptable carrier, a method ofusing the heterofunctlonal ligand in the preparation of a pharmaceutical composition for treating a disease, and to a method of treating a subject by administering same in a therapeutically effective amount.

The fnveatlon is also directed to a multispecifIe ligand which comprises a first ligeryd binding: moiety which net>rralizxs a li$and eg. a natural lig,and such as a cytokinc, chemokine, colony stimulating actor ~ growth factor and a second ligaad binding moIcry which binds to a cell marker associated with a cell through which the natural ligand exerts a deleterious a~'ect. Preferably the amity for the first ligand binding moiety for the natural ligand will be greater than that of the second ligand binding moiety for the cell associated marker. Optionally the construct will be selected so that the binding interfaces are pofnted in opposite directions and the lever effect is maximized, for example a bispecific construct where the heavy chains are joined directly or through an inflexible linker and are optionally linked to their respective (optionally common respective light chains) through a disulphide linker via framework regidttes as is well knpwn In the an. 4pdOnally, the molecular weight pfthe first llgand blndiztg moiety is substantially less or morn (by at least 10%, preferably at least 20°~, more preferably at toast 25°~) and preferably lcaa than that of the second ligand binding moiety as may be effected or maxImtztd through mutating from higher to lower (on the first ligattd binding nloltty) and lower to higher next. wt residues (on the second ligand bindhtg moiety), the native residues which era not exposed ( to avoid immunogenicity) and not essential for proper folding and function of the VHIVL (as may be determined from the degree of conservation of sucli residues among immunaglobullns of the species, through % &enutsncy tables available through the Kabat database and well knows published determinations in this re*ard (see for example yV0 02/40545).
Examples include tteutcalizing lLrz via a marksr on aetivattd T cells, blocking IL-15 via CD8+ T cells, blocking TNFalpha on mast cells; binding thrombin via activated endothelial etlls ate.
The term hetrrofunational is used broadly to refor to a Iigand: 1 ) comprising at IGa~ two fitactional moieties that have di~rent functions or different capacities to pcxform the same funct;on and 2) which is typically and preferably heterospeciC~c (having two binding specificities).
Unless the oodictates otherwise the term avidity when used in a comparative, qtlantiflable or controllable sense is used to refer the valency of the binding entity or moiety. The term functional afftruty is used a composite term referring to a quantitative and aontollable (though not necessarily quantifiable, especially when its consists ofboth avidity and affinity components) propensity to speciRc binding atvlbutabla to one or both of avidity and affinity efFecu, In another aspect, the invention contemplates that cells, particularly immune cells, that are expected to be prc3ent at or proximal to a disease site (eg. at the site where an immune Cell crosses the vascular endothelial cell wads), in virtue of the disease or a therapeutic modality which is employed in relation to the disease or a concurrent disease, including cells that directly mediate the disease, may be targetted in virtue of a marker associated with such cells, eg_mark~s associated with ~jvat~ ~nune culls or disease mediating immrme cells eg. LEU-19, a marker associated with activated or kilIcrT-cells, etc for example with an antibody, which is linked to a moiety that is capable of exerting a therapeutic effect in relation to the disease, for example, an immunoliposome or an antibody linked to another tlterepeutIc delivery system (~Qr example example strcptavidin or biotin fused, coated or conjugated entities or other payload carrying entities (see for example US patents 5439686, 6007845, 5879712, 5456917, 6165502, 5079005, 5888500, 5861159, 5193970, 6190692, 6,077, 499, WO 00169413, WO 01/07084, arc.). For example, an immunoliposome may carry one of or a combination of eytoltines, chemokines, toxins or other therapeutic molecules suitable for heating the disease directly or indirectly, foe example by attaacting or preventing the attraction, activating, energizing or otherwise modulatinc the activity of immune cells for therapeutic or related purposes. Thus according 1o anothc,~r aspect, the invention is directed to a multifunctional l3gand characterised in that it exerts an independent biologic ftutction said multifunctional ligand comprising a ligand which binds to a nou~Iseased disease associated cell and: a) a Therapeutic entity; b) a ligand which binds to a ~erapevtic entity; or c) a ligand which binds to a disease mediating entity cg. a biologic effcctor moltcul which is reltased by the disease mediating eeeity or the dise$sed tall eg. a oytokine or other 13EL
which mediates or a~ravates a disease process. Preferably said multifunctional ligand comprises at least two of a), b) ar c) and prefetttbly all three.
The terns "independent" re Fers to a function which is primarily exdted in relation to an entity other than the entity That is targeted (save foe possible entity associated side cfftcts).
The invention contemplates that targeting a cell which localizes to a disease site will better localize the independent effect of the targeting ligand to that locale. For example, an antibody which hinds to and neutralizes a eytokind or other BEL
associated with Crohn's disease eg. TNT alpha at the disease locale if targeted to an activated CD4+ T-cell using a marker which idenri8cs activated T-cells.

In another aspect the invention is directed to a heterofunctional ligand comprising a first moiety which spec9ficatly binds to at least a first rrtrgct ligand on a first entity and a sscond notary which specifically bIads to at least a second target ligaad on a second entity, and whtrtitt tilt af6nicy or avidity yr both the affinity and avidity of said first moiety are selected to enable the fast moiety to bind to the at least one first target ligand independently of the ability of said second moiety to bind to the at least one second target Iigand and wherein the avidity or affinity or both the affinity and avidity of said second moiety are selected to enable the second moiety to bind to the second entity In preference to the first moiety binding to the first clarity when bob first and aecand moieties ere subsrar><ially cotrte~mporaneously bound to the respective first and second entities. In one tmbadiment the first moiety comprises at least ono ligand preferably at least one antibody which binds to a liter cell, for example an intrahuninal lymphatic endothelial cell and the second moiety comprises a ligand, preferably at Least one antibody which binds to a different cell, for example a disease associated cell (hereinafter exemplified and meaning, unless the context implies otherwise, diseased sells or disease causing, mediating (la. having a role which is known to be intermediary or indirectly facilitating cg. antigen presenting calls) or mitigating cells (sells, typically Immune cells, which directly or Indirectly counteract the diseased or disease causing or mediating cells). In ocher aspects the invention is directed to a phatvtaceutical composition comprising such a heterofunctional ligand and a phamtaceutically acceptable carrier, a mothod of using tho heterofunctional ligand itt the preparation of a pharmaceutical composition for treating a disease, and to a method of treating a subject by admitdsterlng same in a therapeutically affective amount.
In another aspect the invention is dirttttd to at multispecific ligand comprising a first moiety which specifically binds to at least one first target ligand on n ttrst entity (cg.
s lymphatic endothelial cell, a diseased coil or a cell proximal to a site of disease) and a second moiety which sp~Iffcally binds to a second target ligand or site on a second entity, end wherein the second entity binds tp a third target ligand, and wherein the first and third target ligamds may be on the saint or different entities tg. the saint or different cells, and whtrtin prtftrably tht affinity or avidity or both the affinity attd avidity of said first moiety are selected to enable the first moiety to bind to the first target ligand independently of the ability of said second moiety to bind to the second target ligand and independently pf the ability of the second moiety to bind to the t~rd target 1i>itutd (the f rat moiety optionally comprising more than one ligand (which may He the same ligand or a diflbrertt lig:tnd) one or more of which are necessary for binding and optionally each of which is sufficient far speciFtc binding) to corresponding first target ligands) and preferably wherein 1) the avidity or afir"rtrtiry er both the affinity acrd avidity of said >3rst moiety islets stltcttd to enable it to bind to the at least l3rst target ligand in preference to the second moiety bindittr to the third target ligand when both said first acrd second moieties and the second entity are substantially contemporaneously bound to their respective targes ligettds cg, to effect a Iranfar or 2) wherein the avidity or afHalty or both the affinity and avidity of said second moiety for the second entity are selected to enable the first moiety to bind to the first entity in preference to the second moiety binding to the second entity aadlor 3) wbtrtin the avidity or affinity ac both the a>~nity and avidity of said second moiety for the second entity are selected to Cnablt the 3acond moiety to bind to the third target hgand itt preference to the second moiety binding the second entity when both first and second moieties are substantially eoaatemporaneously bound to the respective first and second entities and the second moiety is substantially contemporaneously bound to the third target ligand), or 4) wherein both 1) and 2) above are both operative conditions. In one embodiment, the first entity is a diseased or disease causing, mediating or mitigating till, for example an immune cell (the first moioty preferably binding to a particular population or sub-population of the first target entity eg_ the immune cell, for example activated T cells), the first moiety optionally comprising two or more Iigands which may be the same or different and which bind to two Qr more respective first target cell surface ligartds (though hat neetssarily to arty particular tffeet (arid in oat embbdiment to no effect at all) ty Irer rhon to better bind to and thereby target the cell, preferably in competition with the second entity, which in a preferred embodiment tat Gets a broader population of cells), and the socond entity cg, a biologic cffectore ligand is an eattlty that binds to a third target ligand, the third target ligand preferably btIng expressed on the surface of a cell for example the same immune cell, for example a natural cell surface ligand, to Which bittdittg yields a desired effect, for example a therapeutic advantage, the second moiety being, for example, the natural tigand for the cell surface tigand or functional mimitope or antagonist or agonist tbereoi; For exatuple a cytokine, the third target ligand in this case being a cytokine reCCptor on the immune cell. The invention is also directed to a method of "tat vebed delivery' of a therapeutic entity to a cell in need of such therapy by administering said hcterofunctional ligand. In this respect numerous therapeutic entities will be apparent to those skilled in the art, only same of which are mentioned herein by referring to the therapeutic entity itsttf or by referring to the third target linand foe which such entity is known and available or rondily made by routine skill in the art. Optionally the heterofunctional llgand (and simJlarly in the case of other multispeci$c ligands ofthe invention described above which are adapted to deliver a BEL) is delivefOd with the second entity, preferably in the same composition (preferably bound).
In the case wbcrc the second entity is a nettu~tttl ligand circttlatlng in the path of delivery ofme heterofrmctional hgand, some proportion (0-100°/a) of the hetarofunctional ligand may he delivered without supplied second entity, particularly if the treamtent or the disease generates art abundance of the natural ligand. In another embodiment the ptrst moiety hinds to a target ligand on a stationary cell (for example a vascular endothelial cell or a lymphatic endothelial cell), preferably a tissue or cell type "associated" ligend (more abtutdttntly rxpressed uniquely expressed on target cells relative to non-target cells), and the third target ligand and the secpnd mpiety ere cell-surace target and ltgand therefor as stated above, for example rite second moiety binds to a cytoisiue and rite third target ligand is a cytokine receptor, for example oa ad imm,ute eth. In one embodiment the first moiety binds to at least one target ligand which differentiates between populations or sub-populations of itmnune cells and the second entityin vhtue of its binding to the third target ligand, directly or indirectly exerts a therapeutic effect, for example by modulating the activity of said immune cell. In another or fiuther preferred embodiment the first moiety is incapable of modulating the activity of said imnmne cell and said second entlry modulates the activlry of said lmmtttte cell independently of said Crst moiety, Tn ar~thGr or ftirther preferred embodiment the second entity binds to a molecule Involved In cellular adhesion, a cytokine receptor, a ligand which atimulatES the activity of said immune ~Il, a ligaod which inhibits the activity of said immune cell (eg, via anerey or toIe~tance mechanisms), a ligand which causes one or more cytokines to be released, a ligand which prevent one or more cytokines from being released, a ligand which causes or facilitates apoptosis of Bald immune celll, a tigand which permits internalization of said heterofuactlonal ligand. In another preferred embodiment the het~xofw~ctional ligand is fused or co>;jugated to a therapeutic agent or a moiety (eg. biotin, avidin) that binds to a therapcutuc agent (exemplified below) or a ligand which effects binding to another immune cell, for example a T
cell. In another preferred embodiment, the heterofimctional llgand is a bispecifie antibody, a trispecfic antibody pr a teo-aspccific antibody. In another preferred embodiment the heteroflmctional ligand fiuther comprises a moiety that binds to at least one ligand located pa the intraluminal surface of a lymphatic vessel, preferahly a lymphatic vessel associated ligand, as hereinafter de~rted_ In othrr aspects the invention is directed to a pharr~,ar~uttoal composition comprising such aforemcntione;d hetcrofunetiotlal ligand and a pharmaceutically acceptable carrier, a method of using the heterofunctional ligand in the preparation of a pharmaceutical composition for treating a disease, and to a method of treating a subject by administering same in a therapeutically eff'ectlve amount. As suggested below, the fpregping strategy could be used in combination with other targeting strategies herein mentioned or known in the art.
The invention contemplates malting antibodies to second entities, for example, while bound to their natural receptor, by phage or n~bosome display, by methods as hereinafter disclosed.
In anotlier aspect the Invention is directed to a heteroiitttctIonal ligand comprising at least a (first moiety which spe~fically binds to a first tnrl;et li~ad an a cell and a second molery which spcel~cally binds to at least a see0nd target li~and on the same cell, and wherein the amity or avidity or both the affinity and avidity of said first moiety and the affinity or avidity or both the afHalty and avJdlty of the second moiety are selected to substantially reduce the probability of the either moiety singly binding to its respective ligand for a suf~tcient duration or series of dttrations to accomplish the function of said heterofunctionai ligand unless both first and second moieties ate substantially contemporaneously bound to the eell_ In a preferred embodiment the first moicaty binds to at least one target ligand which di$'erentiates between populations or sub-populations of immune oelJs and the second moiety in virtue of its binding to the secpnd target ligand, directly nr indirectly exerts a t~rapeutic effect, far example by mpdulating the aC.tlviry of said itmnune cell. la another or further prefbrred embodiment the first moiety is incapable of modulating the activity of said immune cell and said second moiety modulates the activity of said inunune cell independenrly of said fast moiety, In another or further preferred embodimmnt the second moiety binds to a BEL, for example a molecule involved in cellular adhesion, a cytokine receptor, a ligand which stimulates the activity Of Said It4,Tmme cell, a ligand which inhibits the aptivity pf gaid immune CCII (CQ, via anCTgy 4r tolerance mechanisms), a ligand which caugC~ pne Or mpTC cytokines to ba mleased, a tigand which prevet~
one or more cytvkines from bring released, a ligand which causes or facilitates apopcosis of said immune cellt, a ligand which permits internaliTation of said heterofitnctional ligand. In another preferred embodiment the heterofiutctional ligand is fused or conjugated to a therapeutic anent or a moiety (eg.
bl4tln, aYidln) that binds t4 a tllCfdpeutuc agent (exCmpli~ed belpw) pr a ligand which effects binding la another immune cell, for example a T cell. In another preferred embodiment, the heterofunctional ligand is a bispeci$c antibody, a tr~ispe~c antibody or a tetraspecific antibody, in another preferred embodiment the ?4 heterofunctiorial ligand further comprises a moiety that b;nds to at least one hgand located on the intraluminal surface of a lymphatic vessel, preferably a lymphatic vessel associated hgand, as h~e;"after defined. In ether aspects the invention is directed to a pharmaceutical composition comprising such a heterofunctional ligand and a pharmaceutically acceptable can-ier, a method of using the heterofunctional ligand in the preparation of a pharmaceutical Composition For heating a disease, and to a method of treating a subject by administering same in a therapeutically effective amount.
1n other aspects the Invention is directed to a method of in oleo modeling or testing using one or more foregoing targeting strategies by admitilstCring a hcttrofunctional /
multifuriCtional ligand as htreinbelow disclosed as well as a method of infra-lymphatic drug delivery employing such ligand and such strategies including adaptations thereof for such purposes, as hereinafter described. In related aspects the invention is directed to a test ligand iii the form of such a heterofunetional /
multifunctional ligand and compositions Thereof.
In one aspect, the invention is dsre~ed to a heterofunctional ligaitd, comprisinm a first moiety which specifically binds to at least one ligand located on the intraluntirial swfaco of a lymphatic vessel and a second moiety which specifically binds to a disease associated cell and the use of such heterofunctional ligand in treating or preparing a pharmaceutical composition for treating disease associated cells, including diseased cells or disease causing, mediating (1e. having a role vvhien is known to be intermediary or indirectly facilitating eg. antigen presenting cells) or mitigating cells (cells, typically immune cells, which directly or indirectly counteract the diseased or disease causing or mediating cells), within a lymphatic vessel. Preferably, the ligand located on the intialuminal surface of a lymphatic vessel is a lymphatic vessel associated ligand.
1n another aspect, the invention is directed to a pharmaceutical composition comprising a phannaceutically acceptable carrier and a heterofunctional iigand comprising a $rst moiety which specifically binds to a ligand located on the intralumiiuil surface of a lymphatic vessel and a second moiety which specifically binds to said d;sea~ associated cell and the use of such ligand in ireatirig treating disease associated cells, including distased cells or disease causing or mediating cells, within a lymphatic vessel. Preferably, the ligsnd located on the intralununal stuface of a lymphatic vessel is a lymphatic vessel associated ligand.
In another aspect, the inveutton is directed to a method of treating disease associated cells, including diseased tolls or disease causing or mediating coils, within a lymphatic vessel comprising administering to a subject a heterofunctional ligand comprising a first moiety which specifically binds to a ligand located on the intralum;naI surface of a lymphatic vessel and a second moiety which specifically binds to said disease associated cell.
It is to be understood that disease causing cells as used herein includes diseased cells and pathogens, including micro-organisms and viruses.
In another aspect, the invention is directed tp a heterofunctional llgand, comprising a first moiety which specsfically binds to at lea.4t one iigwd located on the intraluminal surface of a lymphatic vessel and a sCCOnd moiety which specifically hinds to a therapeutic entity for example a eytotoxin or cytotoxin-liiiked-entity or a non-toxic entity which is present in toxic amounts arid to a method of reducing the toxic effect of such entity in a subject by admielstering said hcterofunctional ligand to said subject.
In another embodiment the invention is directed to a method of tlierapeutic evaluation and/or targeting /
intervention in which such hetexofiinctional ligand is administered substantially contemporaneously with a eytotuxic subs~p~ for example a cytotoxic substance useful for treatment of cancer. The term substantially contemporaneously is used in this connection to mean in a time frame that permits Loth to exert their respective effects, preferably one or both exerting their respective effect optimally, or one exerting its effect dominantly. Tt will be appreciated that this might entail that one such entity is advanced in its delivery over the other. Optionally, one or both of these cooperating entities are delivered proximally to their respective target cells, for example by cannulating pne yr more blood vessels as proximally as possible to the sites) of a turn4~' andlor acn,al or anticipated sites) of metastases (as discerned by using one or more tumor and vascular imaging agents, for example, one or a combination two or more agents selected from a vascular opaQuing agtnt, a radi0nuC11dC conj ugalCd anti-engi0genic antibody, and a i-adionuclide conjugated anti-vascular endothelial cell marker antibody, which canuulation may occur for example in the course of initial surgical intervention with respect lo the primary tumor site) and/or at the same time cannulating one oT more lymphatic vessels (which may optionally be located which the help of a radionuelide eov;jugatCd anti-lymphatic vessel marker ttnb'bpdy) leading to or frpm such tumor sites or metastases. The invention contcrnplates that small sections of vascular prostheses, well latown to those skilled the art (eg. Decree types) may be grafted into those locations to permit a prolonged and secure attachment of such prosthesis to an intro-vascular cannuta for secure delivery to such vascular or lymphatic locations fbr repeated and/or prolonged administration, optiomlly while the patient is mobile, optionally using one or more portable Infusion devices, including micropmnps designed for such purpose (see For example 1 NeuroSci Methods 1997 Mar;72(1):35-8, US 5180365:Implantable infusion device. See also cancer: Principles and Practice of 4neology (infra). Numerau$ embodimsrtts and improvements in vascular prosthesis and in such portable infilsion devices and nticopttmps ors described in the relevant scietrt;fic and patent literature lmown to those skilled in the art. The invention also contemplates delivering any multifunctional ligand herein disclosed in the above matmer.
It is to be understood that targeting strategies employing the cooperative action of ligands wrth different ai~nitigs far their targets exemplified above, may preferably have afTrnities which differ, depending on the application and their avidity, by a factor of 20% up to a number of orders of magtutude which may one, two, three, four, five, six and even seven or eight order ofmagnitudc, in order to achieve substantial advantage, as herefter detailed in connection with one such strategy.
In mother aspect the invention is directed to a heterospeciflc llgand comprising a first moiety which 5peci$cally bind9 to at least a first disease assecintcd Iigmd located on a diseased or disease causing, mediating or mitigating cell for example a cancer cell or an la?muno toll, as well as on non- diseased or disease causing, mediating or mitigating cells (non-target cells) and at least a second moiety which specifically binds to a second different disease associated ligand an the same cell and whet-oin each ligand is expressed on a substantially (see definition below) different, n-overlapping, subset of non-target cells, so that functional binding to a nott-target tissue is substantially (see definition below) precluded. In another embodiment the functional affinities of the respective ligands may be selected in accordance with the strategies suggested above, to further facilitate targeting. In another embodiment, both different Iigands are inquired far internalization. In outer related embodiments, the lieterofunctional ligaud comprises at least two different pairs of binding moietie8 (vg, a trispecigc yr tetraspecific antibody which depending on its construction will permit 2, 3 or 4 such different pairs eg. a tetraspecific single domain type antibody (ie.
consisting prhnatziy of the heavy or light chain variable region or a functional fra~nant themof) (see discussion below regarding its construction) allowing the greatest variation in such geametr;es and preferably simultaneous binding of mare than one pair), wherein 1) at least three such ligands are expressed on a substantially (see definition bClow) different, preferably non-overlapping, subset of non-target cells, so as to further limit binding to non-tau~get cells andlor 2) wherein at least two difTerent pairs of ligands target a substantially different subset of cells wlthirt the same target population eg. dJfferent eel Is within the $amc tumor (eg. pmliferating vs. non-proliferating cell-the respective amounts ofthe different types ofcelis will dictate the perecentage of the dose that will be targeted to ono population or another). In other aspects the invention is directed to a pharmaceutical composition comprising such a heterospeci6c ligand and a pharmaceutically acceptable carrier, a method of using the heterospecific ligand in the preparation of a pharmaceutical campositiora far treating a disease, and to a method oftreat;ng a subject by administering same in $ Cherapeutic$tly etifcctive amount, It Will be apprteiated fh3t the foregoing ~nerat strategy can be accomplished with two or mom different antibodies have differing and preferably non-overlapping aottnal is, non-targeted cell distnbutions, preferably administered in the same composition and preferably cross-linked by biotin-avidin like complementary pairs to facilitate cross-linleing for internalization or targeting of therapeutic agents. In a preferred eroboditnent each such independent antibody carries a different complimentary aspect of a toxic payload eg. a difFerant liposome (or other payload carrying entity for example a micro or none particle or spbcxe or albumin) which complement each other in virtue of their respective contents (eg,. one carries the prodrug and the other the necessary converting enzyme).
Tn another aspect, the invention is directed to a multifunctional ("muld~
rrieariing at least tWo) ligand having, at least, a first portion which binds to a lymphatic vessel associated ligand add a second portion comprising an immune function-exerting moiety.
The term lymphatic vessel is used to fhcilItate broader reference to ligands (eg. antigens l receptors) present on cells bordering the infra-ittminal pathway through the lytnphtttic system including preFerably the lymphatic vessels and optionally also parts of the lymph nodes, and refers in the case of the lymphatic vessels, primarily (from a Functional standpoint) to the infra-Inmittal cell surfaces (not necessarily to tht exclusion of non-laminar surfaces) on the infra-luminal endothelial cells (not necessarily to Lhe exclusion of non-lumdnal lymphatic endothelial cells) of those vessels.
The term 'associated' with raferencc to lymphatic vessels, Is used to mean diff'ercntially expressed on the surface of endothelial cells of those vessels for targeting purposes, such as to accomplish an abject afthe invention, but unless otbe~wise expressly indicated in a particular instance, it is used !imitatively, to reference ligands that are predominantly, if not exlusively, found on the aforementioned endothelial cell 5t>tfact (as wall aS in lymph nodes), such that the first portion of the multifunctional ligand is for all intents and purposes functionally targeted to the intro-luminaI surface of the lymphatic system. For instance, it is appreciated that tht ligand ill i]uestion may be targetted to a limitOd extant elsewhere cg. in the case of preferred LYVE-1 ligand discu55td below, to parts of the spleen (which also provides a venue fpa~ immune cell u,teractions).
The invention is not concerned with imparting effects to or simply blocking a receptor on the intraluminal lymphatic endothelial cell. In this context, the multifunctional ligand of the invention is intended to exclude only, unless otherwiso specifically stated in the claims, only those embodiments disclosed in WO 98106839 or other references describing ligands, antagonists or antibodies which bind to a lymphatic vessel associated ligand or receptor (see examples of such references below), insofar as such embodiments comprise lymphatic vessel associated ligands as hereinabove !imitatively defined, and to this limited extent only, the term therapeutic function exerting moiety or immune function exerting moiety preferably txcludts: 1) an antibody Fc. receptor, insofar as such limitation excludes from the ecopt of the multifunctional ligand (per se) aspects of the invention, substantially intact naked antibodies whioh simply bind to a lymphatic vessel associated ligands, as well as preferably excluding 2) cytotoxins or dings, insofar as this excludes from~the scope of the mutifimctional ligands of the invention an antibody pr fragment thereof which is fused or coniuJ~ated att. exclusively to a cytotoxic molecule (including an atom) or drug (re. an antibody linktd to a eylotoxin or drug only, which is not per se an or is not integrated with un indESpendent blolugie or immune function oxerting component) so as to accomplish a function in relation to cells or other entities (including other multifunctional ligat,ds) within the lymphatic system other than the cell pr liga>Zd to which the multifunctional ligand is anchored.
Similarly, the invention is wa concerned with multlft,uctional liøadds which are adapted to be internt,lized into a lymphatic endothelial cell and the invention i3 apBCifteelly eOriCerned with targeting a lymphatic vessel associated marker which does not promote internalization and/or in which the first portion has an affinity (high or medium) which limits this effect (re. to a side effect) In the; same vein, tho term immune function is broad in intent (as discussed below, and includes particularly any fimction, including binding, capable of being exerted by an ligand preferably an antibody (cg.
multifunctional ligands which are bispecific antibodies) however it 1s to be understood that the invention and particularly dm immuna function exerting moiety does not have a5 an obJect (despite possibly incidental effects) evaluating or exerting a diseaso respousive or immune function vie-~-vie ligands ! cells lining the infra-lamina! surface of the lymphatic system lc. insofar as such ligands have a role in disease (other than simple binding exclusively for anchoring purposes which is attributable not the immune funHon exerting roaiety but to the first portion) but rarher, as evident in preferred aspects of the invention, prCferably an irrdependenl blOldgiC of immune function which is not prtdie$tcd on blockin8 the lymphatic endothelial receptor or treating cells bearing the receptor re. exerted vie-~-vie targets other than the lymphatic endothelium target, for example 1) in the case of stationary diseased cells or disease causing cells or molecules, targets at the site of the disease (which may optionally be effected, for example, in case of immunization or other immune cell stimulation, inhjbptioo att. in the lymphatic system); and 2) in the case of non-stationary dise83dd or disease eauBing cells or molecules, at the site Of those cells I molecules including, preferably, within the lymphatic system, for example by binding to or signaling those cells in the lymphatic system.
1n one embodiment, the first portion of the multifunctional ligand is an antibody.
Tn another embodiment, the immune function exerting moiety binds to a target ligand and thereby directly or indirectly accomplishes its effect (in whole or part). For example, the target ligand may be a cytoldne, for example in order to target immune cells to the lymphatic system to assist in, diseased, disease causing or other target cell ablation or phagocytic type activity (cg- by the cytoldne in tuns bidding to a ligand, for example on an immune eel! having phagocytic activity) yr exerting a ehemotaetie effect within the lymphatic system, or to mop up cytokines, for example, when released in toxic amounts due, for example due to effects of a disease or particular irnmunotherapy (such as anti-CD3 therapy; see for example USP
6193969, )Guttlnler ~7- et al., Immmol L.ett 2001 Jan 1; '15(2):153-158) (with respect to removing disease associated antibodies from circulation see for example a bispeci8c dsDNAx monoclonal antibody construct for clearance of and-dsDNA 1gG in systemic lupus erythematosus. J Irrtmunol Methods. 2001 Fob 1; 248(1-2):12$-138). [see also, for example, US 5,95$,5 LO with respect to antibody-CTLA~I fusion proteins for use in binding to various target ligands).
3. In another embodiment, said immune function exerting moiety comprises an antibody and optionally both the first portion and the immune function exerting moiety are antibodies (with respect to bispecifio antibodies, and a reeeut review of some of the technologies referred to or applicable to various asptcts of the invention (sec particularly, Journal of Immunological Methods February 2001 Vvl. 248(1-2) page 1-200) In another embodiment, said immune function exerting moiety binds to an immune cell, a diseased host Bell or a disease causing cell or entity (eg see US6193968). The term disease is used broadly to refer to any undesirable condition. The term diseased host cell includes but is not limited t8 a Cancerous (in the broadest sense of that term) cell and a virally infected cell (these exampltS 8Ye glVen inasmuch as the invention in a preferred embodiment involves t-argcting such cells for destruction) and the term disease causing toll includes but is not limited to a virus or other infcetious agent and as well as inunune cell which is directly or indirectly involved in mediating or causing a undesired, deleterious or pathologic Consequence, includia,g but not limited to autoimmune disorders, transplant rejection, and other immune system linked diseases. The term disease causing entity is used to refer, without limitation, to any molecule, atom, peptide, ligand, complex, chemical, component, epitope ate. that is directly or indirectly involved or associated in mediating or Causing a disease or disease causing tvent including an antibody. Such binding to the entity may be effected through the instrumentality of one or more (same or different) multifunctional ligands and through bindinb to any ligaud or set of ligands, including receptors, multi-component epitopes ate, including far example, tumor "arrociated" (ie. differentially expressed to advantage for targeting purposes) epitopes which may or may not or may only be partially present on tumor associated arnigens, or commonly, for example antigens I epitopes I ligands l receptors ate.
which arc over-expressed in association with cancer cells; or for example, antigens I epitopes / ligands / receptors ate. involved in imnmne sip aling, stimulatory, co-stimulatory, inhibitory, adhesion or other interactions, including without limitation, cytokine receptors, ligands associated with immune cell adhesion (sec for example US 5,747,035), ligands to which binding results in stimulation, activation, apoptosis, W Orgy or oostimulati8n, or ligands which differentiate between different populations or subpopulations or immuue cells, including sub-populations of B cells and T cells, activated versus non-activated iympocytes, diseased or disease-causing cells versus non-diseased ! disease causing lymphocytes and specific immune ctll clones for oxatuple those having specific Ig type and MHC-peptide type ligands 1 acrd correlative limands. Examples of such li~ands include CCliS, CTLA-4, LFA-1, LFA-3. ICAMs op. ICAM-l, SLAM-1, CD2, CD3, CD4 (eg see US 6,136,310), CDS, CD6, 01518, CD22, CD40, CD44; CD80, CD86, CD134 and CD154, to name only a few (see also US6087475: FF4A receptor, US6135941, WO o1I13945 Such ligand may also selectively be targeted using any dual affinity strategy according to the invention,.
5. The invention is also directed to a multifunctional ligand and a method which comprises using the multifunctional li~~and to assess the toxicity of directly or indirectly tarmeting, for ~ca~nple, primarily within the Lymphatic vessel system (see discussion below), tolls having will known markers that are associated with immune cells, for example, those exclusively associated with activated immune cells, in-so-far as such targeting has a role in prolonging or cbunteracting the a~vattd state, destroying the cell (eg. where the multifunctional ligand is a immunotoxin) causing the cell to be destroyed (cg. through apaptosis (eg. see WO 01119861, fns - fast, U.5 6,046,048) or assisting another molecule or Cell for example a T-cell or other lulling or immune modulating cell re do the modulation or killing (markers such as CD23, CD25, CD26, CD28, CD30, CD38, CD49a, CD69, CD70, are just some ofthc markers associated with acti~ed immune cells) ate. (for a complete listing of marker associated with activated immune cells see for example Roitt 1 et al. Immunology, sixth edition, Mosby referenced below end Fatcyclopedia of Immunology (J99$), Abbas et at. Cellular and Molecular Immunology 2000, Harcourt & Brace, the contents of which are incorporated by reference herein). Antibodies for many such ligands are known or could be re$diiy made by eg. phage display (see references herein including J Irnmunol Methods 1999 Dec 10;231 ( 1-2)_65-81), and natural ligands for such markers or functional analogues thereof are in some cases known or could be made by recomblnatrt DNA technologies referenced herein (set also Cellular & Molecular lmrnunology a'~ Edition, Abbas Ak et a1_ WS Sounders and Company ?600, Antibody Fusion Proteins, Steven M Chamow , Av1 Ashkenazi Eds. ISBN 0471 May 1999 Wiley; I~ontel7uann. R., et a1-{><d5-) Antibody Engineering, 5pringer 2001. 1S13N 3-540~1354-S; Anybody Engineering, Carl A.'E3orrcbaeck Oxford University Press, 1995;
Antibody L~rtgineering:A Practical Approach David 1. Chiswtll, Hermit R.
Hoogenboom, John MeCafferty Oxford University Fress,1996; Antibody Engineering Protocols, Sudbit Paul (1993) Humans Press; Antibody Expression & Engineerinø (1998) hienry Y.
Wang, Tadayuki Imanaka, American Chemical Society). The term modulation is used broadly to rifer to any cban;e, directly or indirectly, in an immune function ar effect, as broadly understood. Many such forms of modulatiatt art will known in the art (some ai'e exemplified herein), and therefore these need not be specifically recited (far a review of such effects see for example Itoitt I et al. Immunology, sixth Edition, Mosby 2001;
Encyclopedia of lmmttnology ; (1998) Morgan Kaufittann Publishers, LS>3N:o1222b765b)-6_ In one aspect the invention contemplates that the multifunctional ligand exerts its function substantially (1e. upon gaining entry into lymphatic system and when bound to the lymphatic endothelial cells, which is dependant on the mode of administration) within the lymphatic system, on a~lla anchor molecules circulating through the lymphatic system, for example with respect to some embodiments, for greatest effect, to avoid an undesired degree of immunosuppression (for example, embodiments where immune cells are targeted for ablation andlor apoptosis). Preferably, such effect, is exerted at least in part, and preferably substantially to the exclusion ofregions within lymphatic system that house at the time of administration non~irculatinQ cells (eg. thymus, bone marrow, and various parts of the secondary lymphoid tissues) or/and with respect to some embodiments (excluding for example those related to immunization or mopping up toxins or antibodies) preferably, non-activated cells. This specificity of targeting can be accomplished in part to the natural distribution of the lymphatic endothelium associated marker of choice, the mode of administration and various targeting strategies herein described.
For example, the invention eonremplates modes of delivery that to varying degrear ensure a greater degree of lymphatic system targeting, for example administration directly within the lymphatics, adminstration is tissues that drain ro the lymphatics or parts thereof, intravenous delivery, es are well known to those skilled in the art, preferably in each individual case at strate~;iC sites of administration that are most pertinent or selective For the disease in question, to the extent that selectivity is desirsd. The invention contemplates a variety of different size multifunctional ligsads (MRU, single domain, acFv, Fab, minibodies, ;F(ab),_, F(ab')Z_ substantially whole antibodies etc. and la~own or obvious multimers thereoF
referenced herein and in the referenced literature) that are most suitable (cg_ for small enough or, for example, hawing longest half life In circulation) for particlar modes of administration to the extent that this IS a llmltat101r (eg- size, where drainage into the lymphatic system is Sought to be increased or optimized).
In a preferred embodiment the invention contemplates that the immune function oxerting moiety of the multifunctional ligand comprises (eg, by way of recombinant fusipn, conjugation etc.), or binds to (such antibodies are known or may be made by pbage, ribas4me or other such 'display' methods), so as to present the functional part of an adhesion molecule (molecule involved in cellular adhesion), for example an endothelial adhesion molecule such as a selectins, LCAMs (eg. ICAM-1,1CAM-2) V-CAM, MAdCAM-I or functional analogues or portions thereof (see for exatnpie USpb143298, 5512660, 5861151, 5489533, 5,538,7?5, 6037454, 5565550, Circulation 2001 Feb ?7; 103($)'- l I 28.1134, and speciFc exarnples/references recited below) in order to control cell traffic including facilitating cell anchoring within the lymphatic system, including for example to f~cilitatt interaction with another'°arm" (functional moiety) of the multifunctional ligand or a second ctcamtltifunctional ligand pr an immune cell (or a sell-sized letex sphere as described herein -for this pupose the adhesion molecule may be on the surface of anotber, preferably multifunctional-liaaud-anchored latex sphere or on a similarly anchored cell) as well as combination therapies, for example, with therapeutic entities that enhance or inhibit leuconyte adhesion, or multifunctional ligands or antibodies that bind to one of their cowespondirtg ligands on immune cells (eg, incergrins) or other ligands eg. C1~A4, w facilitate control andlor some selectivity of cell entry into the lymphatic system, for example, for reactivity witli the multifunetianal Ligands of the invention. The invcntian also contemplates that such adhesion molecules may be the subject of targeting with dual affinity ligands of the invention and that such ligands may include a moiety which binds to a lymphatic endothelial cell.
The invention also contemplates that ono or snore multiflmctional ligands in which the immune function exerkina moiety comprises an entIbody type molecule targeted to a particular cell surface ligand may be able to mimic eff~et of such adhesion moleculCS, erg discussed belpw (any such discussipn of au antibody mimicking this function is unless otherwise stand not intended to lltnit the broader canetpt of utilizing any class of molecule that would facilitate anchoring or contmlling, erg. slowing the passage of cells through the lymphatic vessels). It is to be understood that there may be limitations in the number of cells that cart be targeted for ablation in the lymphatic system by slowioo the passage of cells, particularly for the purpose herein specif e:d of allowing them the requisite period of residence within the lymphatic system for immune tell targttlag or inttractiad err pro longed interaction with multifunctional ligands of the invention for binding purposes while bound to the lymphatic system endothelium, for example, certain end stage lymphomaslleukemlas. In this particular context it ?s to be understood that; 1) the invention ratty have greatest applltation when the multlfunctlanal llgand is administered sv as to primarily target cells within the circulatory system, or as an adjunct therapy, or for remission or near remission conditions, or when combined with hyaluronic acid therapy_ For example, the invcnfipn contemplates that an ei~'ective amount of hyaIuronic acid Is pre-administered to tissuES
draining to the lymphatic system so as to initially occupy binding sites vn LYVB-I ptirnarfly in the smallest lymphatic vessels and thereby minitniu excessive binding within the narrowest vessels.
1 o. In a prefErred embodimem said first portion binds to LYVTrI or podoplantin described below.
1 I . In a preferred embodiment, said first portion is fused, conjugated or otherwise linked directly or indirectly to an immunizing moiety, for example an antigen, epitope, mimotope or peptide era. presenting~incorporat:ing encity/scaffold that generates by 'itself or with the help of one or more cytokines, CaBtIItlultltory molecules andlor adjuvants ere. an immune response to a desired asttigvnle dtttrmLtant (this term is used broadly to correspond at least in scope to the overlapping groupings: antigen, epitope, mimotope or peptide), for example an anti-ldiotypic anh-hody, an antibody component which is capable of binding to a T cell activating ~tity for example a cell (eg. an A'PC see Inrlmrnanol 2000 Joe; 12(1)_~57-d6 or other cctl having eg, immune modulating activity eg. see USP 6,004,811 ) which is for example genetically engineered to express relevant ligands far activating (or with respect tp funetipns not necessarily related to immunizing, anelglzlng, tolerizing err othcrwist modulating the activity of), an immune cell for example a B cell or T-cell, for example an MHC-peptide and B7 co-stimulatory nwlecules for activation of T-cells ( see for tacample Proc Nat!
Acad Sci US A
2001 Jan 2; 98(1):241-?46 see also Tharo EL et a1.1 ofltmnunological Methods Vol. 249(1-2)(20D1) pi l I-119 with respect to latex spheres that can be used for this purpose), pr for example a C'Ix.A,-4 scafFatd, a peptide fused to an Fc domain (sec WO
01115203) a >;kS!'-peptida complex/cOnjugetC, an MHC prCtein or peptide complex ere. Anh~body-cvmplsx fusions and antibody-B7 cast~nulatary tusian moltculcs are latown and the invention contemplates that fusion molecules with anti-lymphatic marker antibodies could be made and used together for immunization purposes. It is also contemplated that the absence of costimulatory molecules for ptrsetttation in a co-stimulatory fashion with an MHC peptide complex will cause a toletizing effect. Accordingly the invention is also directed to a multifunctional hgand comprising as immune tunetion txetting moiety which comprises an MHC, preferably complexed or otherwise linked to a peptide. Peptide linking may for examplt be effected independently, naturally or for example through causing release of peptides lion an MHC peptide or HSP peptide eamplex by injecting a weak acidic solution into tumor eg. just prior to excision. Suitable such solutions which may for example be Combined with a cytokine , eg.lT..-12 andlor adjuvant are known in the art.
12. In a preferred embodiment said immune function exerting moiety comprises an anti-idiotypic antibody, for example an anh'body that a) mimics, for exaldple, a colt surface expressed tumor associated epitope, a virus or other infectious agent assaciate'd surface epitope, a toxin, ad immune stimulatory, Cost3mulatory, inhibitory, or otherwise interactive ligand; or b) serves to hind to the idiotype (ie. paratopo) bearing antibody to which it binds as an anti-idiotype, for example an autoimmtme antibody, ete_ or an armbody bearing a toxic moiety for removing such antibody from passage into the circulation.
13. In a preferred embodiment, the invention contemplates that the terse multifunctional ligand is used f4r dtvClopmerlt, therapeutic evaluation or combination therapy in cotljunction with a second different multifunctional ligand of the invention, to achieve a cooperative effect (for example, in the same composition or substantially contemporaneously administered (ie. to reach the same or an interrelated destination in a cooperative time frame) or in necessary or dtsirtd stqutnet/lnttrval, ate.). An example of such cooperative effect is an interaction (not necessarily simultaneously) with two difFor~ent immune cell surihcs Iigands (for example via an anh-body binding interaction), or to deliver different payloads eg. toxins, to a diseased cell see (USp 4,077,499). The invention also contemplates a method oftffecking substantially ooardinnttd inttrnctiwu of differing temporal and spatial complexities, ranging from a somewhat proximal and contemporaneous delivery (eg. in the same composition) of a first multifimctional ligand having, for example, a cancer call binding second portion, and a second multifunctional ligand having, for example, a cytokiane bidding Ab, eg.
to rennet am toxic effects associated with toxic levels of cytoktnt release, a cytokine component (for example to harness the t~tet of such component as a means to attract one or more immune calls to kill a diseased toll or to harness the intubitory effect of such component (eg. using ottc or more cytoIiittes employed by Cancer cells to evade immune cell targeting) eg. on unde$ired immune cell elimination or immune tail elimination oftlte multifunctional llgand, or a T-cell binding cornpo~t (eg. anti-CD3) to harness the effects ofsuch component on cancer cell kiting optionally vnth a concomitant object of assessing possible counterproductive immune cell elimination (eg. as would be enabled by using a radiplabeIled multiftlnGtlonal ligand and determining the disposi~ivn of the label over timE) of the multifunctional ligand_ 14. Also contemplated are methods to implement more spatially and/or temporally setfsidve interactions. l~or example, when admitdstered in empirically determined suitable proportions and in empirically determined Sufficient total amounts for, at least, partial ancvor local lymphatic-vessel-associated-ligand saturation or partial saturation to achieve proximal binding of a first to second multifunctional ligavd (having regard to the route of administration cg. local saturation can be more readily accomplished by administration into the Inmen of the lymphatic vessel). Two difparent such rnultifitnctlo»ttl lig$nds may be used.
for example, to deliver two different immune flldCtiOn txCrting moieties in substantial proximity to ono another for eontemporanevus interaction with two different ligands on an immune cell f ie. when it approaches the luminal wall of a lymphatic vessel).
Per example, this approach may be used to implement one or more effects including increased avidity to the cell for prolonged cell anchoring, which may positively impact oa desired (in some embodiments) transfer of the multifunctional ligand from the lymphatic vessel wall to the target cell eg. for achieving an inhibitory e~bct via lIgand binding (eg. assessed via duration of muItifixnetional ligand binding eg. quantitative or radtoimage approximated label elimination)(N.B. this effect may be assessed with multiple apples Of the stone multifunctional Ligand), delivery of a cooperative payload eg. different entities which contribute to the same or a different mechanism of cell killing, counterparts in a two component interaction (biotin-avidin), which preferably yields evidence (preferably quantifiable evidence) of the lnteractIon, for example an enzyme-substrate inroeractiort to quantitatively assay rite amount of an enzyme converted substrate (eg_ using a conjugated prpdrug and pro-drug ConveCSioa akin to ADEPT and assessing the extent of prodrug conversion og by labeled anti-drug sptciGt antibbdy). For example, the invention Contemplates the use of $ rtspettivtly linked catalytic antibody component (see for example US5658753:Catalytic antibody components) and labeled subs~te or lZN~e and labeled RNA ete.for this pWp45C- Another example, discussed in more dctatl below is the use of one multifunctional tigand for targeting (sclECtiviry) purposes and another for implementing directly or indirectly a desired therapeutic effect, both ligands optionally being rCquired to to give rise to a substantial probability of binding (the invention also contemplates that this strategy could be used with a single ntultifi~nctional ligand having two intro-iumlnally directed bindiur moieties).
15. ~'hs invention contemplates that such interactive entities may be conjugated fused or otherwise lirikcd to a respectiVb fiYSt and ascend multifunctional ligand for achieving a cooperative interaction between adjacently bound such ligands.
16. The invention contemplates that adjacently interacting multifunctional ligands yielding detectable evidence of the interaction, could be use in a method to assess eg.
a) Iuminal ligand saturation for doslrtg, b} multiple simultaneous binding interactions, and c) perhaps most spatially sernitive, development of a process to achieve cross-linked binding with multiple eg.
immune cell ligands e». a costlmulatory inunune effect (is, the effect of different simultaneous interactiOna eg. on stimulation, inhibition etc.af eg, an immune call for example combining a first multifunctional ligaud capable of selectively binding to, cot~ugated to or fused to a 87component (sea J Immunother ?001 tan-Feb; 24( 1 ):37-36;
Jlmntturol 2001 Feb 15; 166(4):2505-2513; Challtta PM et al. J. Imrntmol. 160:3419-3426) and a second muItifi~nctianal ligand capable of selectively binding to, cot>,f ugated t4 or fused to an MHC
molecule delivered initially with or without peptide. For example, the invention contemplates using various amountslproportions of multifunctional ligauds having antibody compo~m fused or conjugated to or capable of binding selectively to, for example an MHC
cIaSS 1 or 11 peptide complex slid YGCOmbinant H7-I-Fe artd/ar H7~2-Fc respectively (see >:ur J
lmmunol2001 Jan; 31(1):32-38; EurJlmmtano12001 Fcb; 31(2)_440-449) (for tumor reactive peptides see For example Jlmtnunother 2001 fan-Feb; 24(1).1-9). In this ia~r connectioa f crass-linking type interactioa), andlarfor permanence of bindutg or aria of attaching otlurc cooperative entities (for example biotin coated or cotlju(~ated radionuclides, liposomes or other payload carrying entities (eg. see for example U5 patents 5439686, 6007845, 3879712, 3436917, 6163502, 3079003, 5888300, 3861159, 6193970, 8190692, WO
00/89413, WO 01/07084) the invention contemplates biptinyla~ng the two multifi~mCtional ligands atad linking the two biotinylated cooperative mulfitactional liga>ids with evidin, atttpGwid111 (or athtr tdadit'itd farms thereof eg. deglycosysylated avidin or using other complementary linking Components- see eg. U9 Fatant (USF) 6,077, 499).
17. 'fhe invention also contemplates enhancing the cross-linking ofthe multifunctional li,»nds of the iaveation through complementary components such as biotin and avidin.
18. Preferably, with respect to, for example, increasing selectivity of targeting certain cells (eg.
to induce immuee tolerance), the invention also contempl><tes that a first multifunctional ligand is used to bled to a marker sptcifie to $ particular kind of cell (eg, activated immune cells) and a second multifunetiona! ligand (which may not be specific for activated immune cells) Is used to modulate the activity of the immune cell (for example inactivate it pr reduce its disease causing capability directly or indirectly by binding to it ). For example, where the marker is used to determine the selectivity of the targeting but cannot be used for modulatiua its activity, it is contemplated that the functional affinity of one or both the first portion and second portions of one or both of the cooperating multifunetlonal ligands can be selected to at least partially control the selective modulating affect of the pair, for example both interactions would be required for the second multifunctional ligand to have an optimal opFroriunity to bind. For example, flee L'fmctiana! affinity far the target cell is relalivCly weak for the purpose of attaching to the eg. immune tell for a suf~ciartt duration (eg. So as t0 yield the ttl:CCt of becoming attached to the immune cell in preference to the lymphatic vessel), compared with that of the first multifunctional ligand (is tits one that accomplishes the selective recognition through binding) to reduce the likelihood that the second moiety will bind in the absence of binding of the first moiety (notably a similar type of coardineted interaction ic. two bind'sng interactions, is naturally used far sell adhesion). (1VH, this typo of coordination has application is. both specificities air optimally required for binding, to a single multifunctional ligand, having a divalent immune function exerting moiety eg a triabody or tetrab4dy or for cross-linking and other types of coordinated interactions). in a pt~eet~rred embodiment, if transfer of binding of the first multifinctiQnal ligand to the immune cell is not desired its functional affinity of the first portion to the lymph vessel caa be greatrr than that of its second portion, while the reverse could be true for the ascend multifunctional ligand. It will also be appreciated that antibodies which cross-link for example an integrin and a marker of inunune cell activation could be used to limit the number of activated immune cells that migrate through the lymphatic system. For example bispecific d Abs, diabodies, etc. m which the fimctional atlfutity of each specific binding portion individually does net srongly Favour binding, could be used to selectively target spaci~c sub-populations of immune cells or even specifically activated lmmtllte cells (for example antibodies that recognize particular antigen l peptide specific T call ar S cells).
19. Accordingly, more generally speaking, the invention is directed a bispecific ligand, preferably a bispecific a~ibody, having a first portion whicli binds to a ligand which differentiates betw~n members of the same immune cell population (eg a particular type of T
cell) and a second portion which binds to a second llgand on the same cull, which binding axe,-t5 directly or indirectly a desired effect, wherein the functional amity of said first and second portions are selected so as to substantially increase amount of immune cells in which both such portions are bound to their respective ligat,ds relptive tp those which a single such portion is bound to s single llgand and preferably wherein the amount of immune cells to which the bispecific lig;tud is not bound is substantially greater than the number of inunune cells that are not bound when compared to using a bispacific ligand having the same specificity and for example a 10~ to 107 (preferably 10~ to 106, preferably 10'to 106, preferably 10'to 10') increase in affinity oFune or borh portions. This intention alas contemplates that binding w the ligand which di~rentiatos between members of the same population (a particular type of T cell) dbC$ not have a negative consequence other than to cause the molecule to be ineffectual unless both of its portions are bound end that its binding is itself sufficient fnr binding andlor stronger relative to the second portion by two fold to 5 orders afmagrlitude ,preferably 1 to 3 orders oFmagnitude. The term substantially greater imparts medical signiftc~ttco and may prtftrably be 15% - 10000% greater. The foregoing examples ere not meant to be litnitative.
20. In a preferred embodiment, the invention more broadly speakjog contranplates a two ligand interaction (using one or more multifunctional ii$ands) wherein for example both are required or increase the likelihood of interaction and wherein the interaction of at least one conrrlbtues to specificity, though not necessarily to modulation, thus permitting a broader selection of modulators including those that but for the selectivity enhancing effect of the cooperating ligoutd and the Iymphadc system venue, would be toxic in the desired theraqrautic dose.
Examplc3 afmarkers that could assist in selectivity include those are unique to, for example, activated B cells or T cells or those having particular specifieites in vhtua of unique Ig type receptors. Examples ofligands on, for exempla Immune cells, through which modutationlinhibitionlstimulation etc. (including, for example apoptosis), for example by antibody binding or supply of a natural interactive ligand, era wcl! known.
Some examples are provided htt~t:in. Combinations and permutations of markers and ligands for selectivity and exerting an immune effect such as modulation/inhtbitionlstimulation referred to herein or in the literature incorporated herein by reference or well known in the art are contemplated to he within the scope of the inveation_ 21. It will be appreciated that a combination of factors, such as dose, using additional molecules that increase or decrease migration or adhesion optionally in a tissue targeted manner, rqute pf administration (eg within tissue that best drain to lymphatic vesssels or a portion thercofj, use of cytokines, etc. and immune modulating drub, as well combination therapies with known entities, can b~ employed in various combinations for strategies of harnessing the unique properties of the multifunctional ligand of the invention, to achieve a seleetiviry enhanch o and/or modulatory/inhibitorylstimulatory etc or otherwise cooperating effects with respect to the desired target popalatiou of cells. Unless their fimction arc self evidently conflicting die invention eanttxdp18IC8 a1117CYtntIIaLL4ri5 OFThe In111ItftlnCii011a1 ligands disclosed herein or in the literature incorporated by reference hererin as well as tha3C evidmnt to pCrsonB skilled in the art whose mention is onutai.

32. In a preferred embodiment, the immune functipn exerting moiety binds with greater functional afCrniry to its target IIgand rhea said first portion binds to ItR
target ligaad. For example said immune function exerting moiety may bind with greater avidity (preferably at least 2 times greater (divalent vs. monovalent) and lesser or greater affinity (eg. within a rangy of I x 10~ to 1x106 fold) or with the same avidity and greater affinity (eg_ up to1x106 fold)_ In applicable aspects, the invention contemplates that this increased functional affinity can be employed to effect transfer of a lymphatic vessel bound multifitncrlonal ligand (eg. a bispeciflc antibody) to a cell passing through lymphatic system. The invention al,5a contemplates a method catnprising radiolabelling the tnutifimcfional 11ltand to assess, for e~cample, the degree to which Immune coils at a disease site have passed through the lymphatic system. Certain 38pCCts afthe invention, diSCUSSed herein, rClatC to a multifunctional ligand based system vftargeting a particular immune cell ligand fur stimulation, inhibition etc. predominantly within select portions of tho lymphatic system that contain migrating cells (although some general targeting ten controllably occur before the multifunctional ligand binds to the lymphatic system or when the multifunctional lisand xcleasas Crmn the lymphatic system without having fpund its target within the lymphatic system) will have at least a partially selective effect on targeting disease causinglmediating immune cells (ag, activated with a specificity that causes the disease) as opposed to non-disease causinglmediating Cells, in the case where such ligand is also expressed on such other immune cells eg, of the same type eg. T cells. This permits targeting of immune cells primarily within the partiona of the lymphatic system that contain migrating culls particularly disease causinglmediating cells while minintizin~; immune system dysfunction.
This effect con be even more sela~,-tively accomplished, for example, by delivering the multifunctional ligand directly into the lymphatic system and within a time frame which is shorter that the normal duration ofbinding ofthe multifunctional Ligand determining the degree to which the multifunctional ligand is touted to such diseased related tolls at the disease site and similarly the degree to which it is bound to the cells twrelated to the same disease e8.
via radlolabel. As discussed more fltlly below, the Invention also contemplates amultifimctional li~and based system of assessing the effects of certain typos of immune stimulation og. how stimulating enhanced migration or adhesion, wil! differentially affect disease activa#ed cell migration through the lymphatic system to enhance such disease cell targeting within the lymphatic system_ For example, For tumor cell targeting and stimulation of disease-activated immune CC11S the 111VCdtlan COntemplate8 eValtlatlrig cytakine (eg. TNFa) linked anti-angiosenic marker antibodies, optionally, Preferably in camtbination with anti-tumor vaccination strategies, to direct disease activated immune cells to tumor site and the lymphatic system for further immune stimulation. Dosed on a "bait and trap" type approach, Iigands such as OX40L
aad 01744 may also be assessed for this purpose.
33_ In this connection and more generally the invention also contemphtbes using a bi-speeific antibody, for example having a lymphatic endothelial binding first portion and for example a cytakine binding second portion, wherein the cytokine binding portion has a lower functional afI'rnity for the cytokine (for exe.mple 1 x 10 -6 to 0.9 fold) compered with that of its natural receptor vu an immune cell. It is contemplated that a muhIfitnctional ligand of the invention could be used optionally in conjunction with a multifynctional liQand which displays a functional adhesion molecule (a selcctin, ICAM, etc.) to assess the optimal parameters far transfer of the cytoldne, for example, as is lmown to occur by monitoring the effects of cytokine release attributable to such cytokine transfer. 1t will be appreciated that this information or approach could be used to optiroi~e the binding parameters for other ligands as well (eg. anti CI53) end could be employed not only in lymphatic system but cu locally deliver lahibitory or stitnnIatory cytokines or other ligands to certain tissue targets, for exutnple new blood vessels forming within tumurs or other tissue specific markers.
24_ The foregoing strategies could be used as pare of a primary, adjunct or low disease burden therapy.
25. In a preferred srnbadimcnt, the sccand portion comprises a ligand which is capable of binding to an immune call for example B cells, T calls etc, preferably in one embodiment t0 assist in cell klllhig or iturnune modulation of a target cell (re NK cells see for example US

5770387xsec also US6071517_Bispceibe heteroanhbodies with dual affactor fllndioos;
Sispecific antibody-mediated destruction of Hodgkin's lymphoma cells.
Jlmntunol Method 2001 Feb 1; 248(1-2): I 13-123; Bispecitlc antibody-targeted phagocytosis ofHER-2lneu expressing humor cells by myeloid cells activated is vivo_J lmmunoi Math. 2QQ1 Peb 1;
248(1-Z):167-182 as well as ~Im~a! Methods 2001 Feb 1; 248(1-2):103-111).
2G. With respect to avidity, afFinity and other elements of design including sin, blood clearance, additional functionality etc.dre multifunctional lig,and may be, for acample, a bispeeifie antibody having a mvnavalent first portion and a monovalent second portion, a bispeciRc antibody having a divalent fast portion and a divalent second portion, a trivalent trispeei~c antibody having a monavalent first porrion and a Second portipn comprising a monovalent immune function exerting moiety which binds, for example, to a target ligand on a target diseastd, disease causing or immune cell, and for example, a monovalent portion which binds to an immune cell which esSiBLS in killing or modulation for exempla anti-CD3 or a~tti-CD28 antibody component, a betravalant trispecific antibody having a monovalent first portion and a second portion comprising a divalent immune fzmetion exerting moiety which binds, for example, to a target ligand on a target diseased, disease causing or immune cell, and for exempla, a tnonovalent anti-CD3 or arni-CD28 antibody coraponent (it is contemplated that this orieritatiod might advarttageotrsly position the anti-CD3 component for interaction with a T-cell almost exclusively when the first portion is not bound to the Iuminal wall of a lymphatic vessel), a trivalent bispeciflc antibody having a monovalcnt first portion and a second portion cotilprising a divalent immune fimcdon exerrJng molery, for example, one which binds, her examplC, to a target ligand on a target diseased, disease causing or immune cell. The antibody subunit may be for example, a Fab, a substantially intact antibody, a single domain antibody (see also Hui3on SE. Dis Markers 2000;16(1,2):37 Single domain human immuneglobulin fold-based biomoleculea; Antigen 9peCifiClty and high a!$111ry binding provided by one single loop of a camel slnglo-domain setibody. J Hiol Chem.
2D0I Jut 13,276(28):26285-90. Optimal Design Features of Camelized Human Single-domain Antibody Llhraries_ J Biol Chcm. ZODI JuI 6;276(27}:24774-24780; )Zecogoitlon of antigens by singl~-domain antibody fragments: the superfluous luxury afpaired domairis.Trends $iochem Sci. 2001 Apr;26(4):234-5; Llama heavy-elisin V rtgions consist of at least four distinct subfamilies revealing novel sequence features. Mol ltnmunol. 2DDD
Aug;37( 10):579-90} a minibody an scFv or a minimal reco~ition unit (MRU eg see US6174891:Minimum rccagnit;en unit of a PEM mucin tvndem repeat specific monacloiial antibody).
27, In apreferred embodiment, the multifunctional ligand binds to an immune cell which is associat«1 with an autuimmunc reaction, fbr example a CCRS-expressing cell.
(see also Apoptosis genes and autoimmunity. Curr Opin lmmunol_ 2000 Dcc; 12(6):719-24, for application herein) 28. In a preferred embodiment, the second portion comprises a eytokine component.
29. In a preferred embodiment, the second portion comprises a eytotoxie component 30. In a preferred ambadiment, the second portion of the multifunctional ligand comprises an internalizing antihody and a cytotoxic component.
31. In a preferred embodiment, the second portion consists of as antibody which binds to 1 cells, for example, an anti-CD3 antibody or an anti-CD28 antibody.
32. Tn a preferred embodiment, the second portion consi~ of a cytplcine catnponent.
33. 1n a preferred Crribodintent, the 3eCdnd portion comprises an antibody which binds to a target diseased, disease causing or immune cell end further comprises one or more eomponenta selected from the group eonsistlag of a cytokine component, a cytotoxlc component and an anti-CD3/CD28 component.
34. Tn another aspect the invention is dixected to a Gon~pasit;an comprising a multifunctional ligand and a pharmaceutically acceptable excipient.

35. In mother aspect the invention Is directed to a composition comprising a plurality of different muttifirnctional ligands.
36. In another aspect the invention is directed to methods and compositions for developing and evaluating the therapeutic value of stimulators, mediators, inhibitors ctc_ of immune cell signaling (cg. stimulatory, inhibitory, cootimulatory), adhesion, rnigration,etc. including the effects of llgandlt~eceptor blooding and supply of specific cooperative ligands, using we multifunctional li~dS of Sht invention.
37. In a preferred aspect, the multifunctional ligands of the invention may be used to assess the effects of such compositions on the sub-populaton of cells that migrates into lymphatic vessels. In partiouler, the invention is directed to assessing the expectation that some disease causing, mediating or afherwise disease native immune calls have an enhanced abilirylopportuniry (andior can ba enhanced in their abilirylopportuttiry to make their way into the lymphatic systenn) so that targeting of relevant lilyands on that sub-population of cells within the lymphatic systxxn will cause at least a partial selective tatgetinb effect, profeJably with positive effect on the dosing capability and choice of ligands ie. in terns of litnitinp morn universal andlor deleterious consequences. The uvention is also directed to a method of reducing the toxic side effects of a phnrmaeeutical cpmpoaitiott comptisitlg a multiftulctional Llgand In which the immune function exerting moiety is targeted to a ligand that is not found exclusively on disease causing, mediating or otherwise disease aotive immuorse cells, by administering said composition in a mariner in which it enter more directly Inm the lumen of a lymphotic vessel. (It contemplated that immunization within the lymphatic system Can also be enhanced in virtue of suc>t selective targeting.) In particular, the invention is directed to a multifunctional ligand, a pharmaceutically acceptable composition therof and method of using same for assessing enhanced migration or enhancing migration of disease-active immune cells, sold multlfltnctlonal Ligand comprising an immune function effecting moiety which has an immune effect on an immune cell surface lignnd it. effects including signaling (ag.
stimulatory, inhibitory, costimulatory, antagonistic, agonistic), including for adhesion and migration effects,etc. This may be accomplised practically, for example through ligandlraceptor blockhts eg. via antibody, or by arm'bady fvsions/cpujugates etc. that supply the natural ligand or a functional fragment yr chemicallbiologicai mimotope thereof. 1n a preferred embodiment the irtvertion is directed to a multifunctional ligand in which the immune function exerting moiety is an antibody that binds to a ligand selected, for example from the group consisting of CTLA,-4,1L-2 receptor, CCRS, CD44, CD134, CD3, CD28, CD2.
38. In another aspect the invention is directed to a composition comprising a plurality of different multifunctional ligands which exert a potentially cooperative immune effect with respect to an immune cell, for example binding to two or more different ligands on an immune ceU, wherein said ligands are selected, for example from tl,e group consisting of CTLA-A, IL-2 receptor, CCltS, CD44, CD 134, from any of the ligands herein mentioned or referenced or preferably CD3, CD2R, CD2.
39. The iitvtn2lon 18 also dirGCted to a method Of inhibiting metastasis during the course of surgical removal of a tumor comprising administering to a patient prior to surgical treaiment of the tumor site, a pharmacetical composition comprising a multSCunetianal ligand in which the itttmune function effecting moiety binds to a tumor associated epitope on a cancer cell.
40. In another aspect the invention is directed to an immunocytokine having as anti-idiotypie antibody component which recognizes the paratope of an antibody which buds to a lymphatic vessel associated ligand and a cytakine component fused therewith or conjugated thereto. For example the vytoltine component comprises IL-~ or a functional Fragment thereof and/or IL, 12 or a functional fragment thereo>i In addition to their individual use in fitslon proteins for tumor Cell killing, combinations of 11-2 and IL-12 have bean used successfully for this purpose. It is contemplated thar such cytokine fusion could be used to target T-cells or phagocytic cells to a multifunctional ligand that has bound to its disease causing or diseased cell target, preferably having left the lymphatic vessel endothelitun in preference for binding its target. In this corutecdon it is coattmplated that tht 8tuctionsl aff7alry of the anti-idiorypic Ab for the first portion would be less than that of the first portion to the lymphatic endothelium, so as to minimize competition between the two. It is also contemplated that the delivery ofthe immunocytokine occur substarrtially contemporaneously but separately and after that of tile multi~nctional liga~,d, optionally by a different route of administration-41. Similarly the invention t:ontcmplatts for the same purport, a biSpecific antibody having an anti-idiotypic antibody component which recognizes the pafatope of an antibody which binds specifically to a lymphatic vessel associated ligaad (preferably with lower affinity than that of the Ab for itg target) and for example an immune cell binding portion eg_ an anti-CD3 and'body or an antI~D28 antibody component.
43. Thus the invention is directed to a method of targeting a diseased or disease causing cell for destruction by the immune system compristitg adrrr,inistering stparately but subat~ntially contemporaaeousIy to a subject hosting the diseased or disease causing cell, preferably in sequence with en interpoacd interval and/or by different routes of administration, first a multiiitactional ligatui In which the immune function eirecting moiety binds tp a diseased or disease causing can surface associated epitope, gad an immunocydokItu ar bispecific antlbady as decribed in tht irilmtdiately prECcding two paragraphs.
43_ Ia a preforrcd embodiment the Lnveation contemplates modification of rite mufti-fw,ctional ligand to substitute acre or tnorc amino ncida which reduce without functional impact on the molecule the number of imttumogenic MHC II class peptide sequences within the molecule.
This can be accomplished through procedures available to those skilled in the aft, for example through the l7eImmuuisstion services ofBiovation hltttittd (sec also U5 5821123 and rolated Xoma pattnts).
44. Inasmuch as the invention is predicated on Intraluminal lymphatic systetn targeting suCh~
lymph associaton may be alternatively implemented, in suttable cirCUmstetlCts by the mtthOd of delivering the muhiRuietional ligand, for example into the lumen of s lymphatic system vassal or (where the multifunctional ligand is not of en unsuitable sine (see for example Ikomi, F_ et al. LymphoIogy 32 (1999) 90-122, within a portion of body that drains to the lymphatic system (ie a portion of the lymphatic system), for eventual migration to the lymphatic system. Particularly, with respect to trnbodiments ofthe invention in wliich the immune function exerting moiety is targeted with greater functional affinity to a therapeutic target {ie_ not the lympluttic system target), such lymphatic system oriented modes of delivery coupled with preferred targeting to the thcrnpeutic target may combine, absent saturattd binding to the therapeutic target, to bitter aeaamplish functional lymphatic targeting.
Accordingly, in a broader aspect the invention is directed a lymphatic system targeted multifunctional ligand in which the second portion is as described herein and In which the speci$city of the first portion exclusively for a Lymphatic system is inessential. Tn this conaect;on, the invention contemplates targeting markers on lymphatic vtssels that art also present, for example on blood vessel endothelial cells (ca. YEGF2). (with respect to lymph specific markers see also Birner P. et al. Clin Cancer Res 2001 den; 7(1).93-7 "Selective immunohistochemical staining ofblood and lymphatic vessels reveals Independent prognostic influence of blood and lymphatic vessel invasion in early-stage cervical cancer" and publuhcd references to the markers therein mernioned.) 45_ In the case of purely sustained mlease aspects of the inventipn where the fugt portion is temporarily anchoring a second portion for eventual release back iota the circulation, the use ofterm immune function affecting moiety with reference to the role vfthe second portion does not adequately accommodate the breadth of the invention since any form of disease palliating active moiety or entity which exerts its effect elsewhere than at the lympharic endothelial cell may gain advantage from this form of delayed delivery (depot effect) or anchoring.

46_ Furthermore, in another preftrrtd asptet, tht second pordon is capable of binding directly or indirectly (eg. binding tb an Entity which in turn binds to a target Gdtity) to a target Entity, for example a therapeutic entity (for example to mop up excess such entity that does not immediately reach its target (eg, an entity that is toxic elsewhere in the body), a toxic entity including an entity which is not per se toxic but the presence of which is undesirable at a particular time or In particular amount or concentration (eg. a cytokiue, for example when released as a result of anti-CD3 therapy), to redireca an an entlry to a target, for example a therapeutic entity, for exrunple through the iastrumeatnlity of en antibody portion that is directed to that target (eg. a multifunctional ligand in which the second portion comprises as anti-tumor antibody portion that is conjugated to straptavidin, to retarget biotin conjugated radionuolide back to the tumor (see Martin J. et al. (199' Cancer Chemother, Fharmacol.
40: I $9-X41 ), to temporarily anchor liposomes or other carriers of entities (eg. drugs) having an direct or indirect beneficial effect elsewhere.
In a preferred embodiment, the invention provides a multifunctional ligand having, at least, a first portion which binds to a Iymphatie vessel associated antigen/receptor (and thereby exerts, not necessarily to the exclusion of other e$~ts) at least an anchoring function, and a second portion having at least one independent immune function. The form "immune functipn" ig broad in intent in~h~ditfg but R4t limited to direct or indirect and primacy or corollary effects related to siarlple targeting, tolerance, immunimtion, stimulation, inhibition, modulation or various other immune related effects (other than simply forming part ofthe entity which blocks the lymphatic eatdothelial associated ligand)_'fhe term independent is used to exclude only an etYect specifically targeted towards the Ilgand (blocking) or cell bearing the ligand to which the first portion of the multifunctional ligand is bound, which is not contem lp ated as an object of the invention. The invention contemplates rather that the immune function is exerted, for example, vise-vis immune cells or molecules or against cancer or infected cells to affeot an immune function that relates to assessment, diagnosis, therapeutic modeling, or treatment of various disease states such as autpimmru~e disease, transplant rejection, cancer and infectious disease. In a preferred embodiment, the invention contemplates that the independent immune function is exerted through a physical ligand-Ilgand interaotlon_ In a preferred embodiment the murlNfimetional ligand has an ability to bind in the manner of an antibody in virtue of at least one of the first or second portions, and preferably at least the first portion. The lymphptic system du~etted first portion may in some embodiments (LYVE-1) be hyaIuronlc acid or aealoguts lhercoF
that have the appropriate binding capacity, In a further preferred embodiment the second portion binds to a target ligand on a cell or molecule (eg_ a cytokine or autoimmune antibody) which passes through the lymphatic system. In a more prefbrred embodiment the multifunctional ligand Is a bispecifc antib4dy. The term antibody is used tQ refer to nny antigen binding ftagtneot of an antibody that substdtltially has the binding capability of err antibody Including anti-idiotypic antibodies, and therefore the term bispecific antibody is used (unless the context implies a more specific usage) in a functional sense to refer to at least two different speci8citles (including trispecife antibodies etc.) and Includes well known entities which err diabodies, triabodies, tetrabadies, minibodies, scFv dims, etc" iced entities in which One Or both binding moieties atx scTv or side domain type antibody fragtnelus or dlmtrs etc of such Fragments (with respEct to single domain antibodies see for example Camel single-domain antibodies as modular building Entire in J
Biol Chem. 200D Oct 25, & Mulligan-Kehoc U_S_ patents )-The term "anchoring fttuction" is used broadly to refer to physical attachment for a period which renders the BECOnd pvrtian ofthE mull-funttioeal ligand capable of exerting its inunune function. For example where; the function of the second portion is to interact with a cell passing through the lymphatic vessels, for at least a period which permits sufficient interaction for the desired effect.
The term ligand is used very broadly herein to refer to any moiety, preferably in some cases, a specifically interacting moiety including binding moieties (eg antibodies. receptors etc.) and bound moieties (eg antigens, epitopes ete) andlincluding otherwise interacting moieties (eg.
chemotactic interactions or interactions that require multiple points of Interface ee. cross-linking or mufti-component epitopes). In other wot'ds, the teen ligand is used broadly to refer to any entity or part thereof which can be subject to an Lturmolecular interaction that can result in birtding_ -I~,e term moiety is used broadly and nonaimitatlvely to refer primarily to a functional part of an entity, and may rifer to even the whole of the entity depending on the context in light of the broadest concept ofthe invention. .

Optionally, dependltta an the mode of delivery and the relative functional affinity of the respective first and second portions, the multl-fhnctional li$ands of the present invention, may txett their imtt»rte funetion primarily in lymphatic system and also signiFcantly before and optionally after entry into the lymphatic system, In a preferred embodiment the multifitactional ligand is capable of simulating a depot effect by binding for a prolonged period to the infra-laminar lymphatic endothelium for later t~elease over time back into the circulatipn_ The choler (avidity effeet resulting from multiple binding "arms") and affinity pf the binding molecule as well as vafious, preferably cootxollable factors impacting on any "undulatinø"
movements of the lymphatievessels (eg. water consumption)or competitive bindla$ is contemplated to impact the binding time.
With respect to the depot and delivery aspects of the invention discussed herein" ii is contemplated the second portion of the muIii-ftmctional ligand of the invention may have at least primary medicinal effects that are not immune function related as broadly understood.
It is to be understood that a use of a slash (n means the broader of "or" or "and/ot~' unless to the context dictates otherwise.
Some immune interaotions require, prefer or are capable of being enhanead via coordinated ligand interactions, for example for optimal immune stitnttlatlon, for example, specific eostimulatpry ligttnd interactions eg. CD801CD8G interactions with CD28, or for example, interactions aimed at tvlerizanQ or otherwise inhibiting or reducing immune effects or preventing such inhibition (for example using anti-CTLA-4/CD152 see related U.S. patents, for example 6,051,227, 5,844,095) (see also Hedge JW et al_ Emst Schering Rae Found Workshop 2000 (30): 23-~2 and hamtutole$ieal Reviews Vol 1'12 Dec 1999, Entire Issue).
The invention cot>templates modeling, evaluating and/or effecting these interactions for therapeutic intervention within the lymphatic system through the substantially comentporancous use of different multifunctional ligands of the invcnt3on. Furthermore, control of the relative proportion of each of the dIiferent ltgands permits different spatial interspersion of three ligands on the intraluminal sufaco of the lymphatic system (primarily) so as to provide controlled variability of spatial conirtgurations appropriate for optimizing the coordinate interaction with multiple lipands on another entity, fior example Lnmurte cells or cancer cells. This strategy also perrnlt9 controls on avidity that extend beyond the choice of valency for a given single multifllnctional Iigand for conttroIlittg the nature and duration of the coordinate interactions including the duration of temporary anchoring, for example to allow cancer cells to be killed by immune cells, as well delivery of, fix example, cytokines (through cytokine antibody fusions), superatttigens etc. to the site of interaction. Such coordinate tateracttons may be Substantially contemporaneous or sequential, for exvtnple the effect of a first intetxcdon with a first multlfltnctional ligand slowing the progression of a cell or infectious agent though the lymphatic system for eventual rection with another first muhifunetional ligand (ie of the same type) or reaction with a second type of multiflmctional ligand. The invention also contemplates as a strategy, alone or in combinnHon with other strategies: 1) delivery of a multifunctional ligand of the invention to a particular site of action For the purpose afexerting, for example a local effect, with the result of causing the multifunctional ligand (whether err not it has exerted its effect, provided that or to the extent that it remains funetioattl in at least one aspect) to subsequently be targeted to the lymphatic system far exertieg a second effect (be it the same or a different disease counteracting effect) includitta simply elimination, or rettu~t back to the circulation (la, where the ligaud is selected (eg. bastd an sloe, immunogenicity ate.) to be preferably minimally eliminated (at least not maximally eliminated) by the body in the course of its circulation, having regard to competing design considerations) for example, in the case of multifunctional l3gattd which is an anti-tumor ligand that has some residual binding to normal tissues, to set up, in e~ct, a site of competitive bindinS that advantageously impacts ( ie.reduces) undesired binding more than desired target binding; 2) delivery of a multifunctional tigand of the invention or an entity that binds to a multifunctional lil;and of the invention to a particular site of aotion eg, local disease mediating immune cells, for ttte propose of simple binding with the expectation that a delayed immune or other effect will be exerted within the lymphatic system. Accordingly, the invention is also directed to a composition comprising at least one and optionally a plurality of di$erent muhi.functional ligands of the ievention. The invention is also directed to such a composition when combined with a pharmaceutically acceptable carrier for example those that may be suitable for one or more of the various well known and heretofore used routes of administration including intravenous, intradermal etc which (for present purposes) arc preferably not incompatible with delivering a multiiitrtctional ligand of the invention to the lymphatic gystem_ The invention is also directed to therapeutic compositions comprising a multifunctional ligand of the invention and to methods of treatment using such compositions. The invention is also directed to method of _ 1 ) evaluating the therapeutic efFtct of a particular therapeutic entity against a particular target with reduced effect on undesired targets; 2) facilitating elimination a therapeutic entity; - by administ~ng the therapeutic entity as part of or in circumstances which permit interaction with, a multifunctional ligand of the invention.
'Ilte utvention also conttmplates cattnulating particular portions of the lymphatic system to localize the delivery of a multifunctional ligazid (set lJnited States Patent 4,911,690 ) , for example 1) to accommodate or further accommodate the treatrnent of conditions in which the immune affecting molecule has an undesirable systentic or localiGed side-e$ect i~ delivered otherwist; 2) for the localized delivery, as required, of larger molecules, complexes {eg. for temporarily anchoring MHC-ptptidt complexes) or otherwise associated (at least temporarily) entities (re. associated other than through complex formation) Cte. aed/or 3) for tile lo~lized delivery of additional compositional aletnents eg. adjuvants, cytokines (sec Immunological Reviews 2440 Vo1 177 p. 5-246; Nature Immunology Feb 2001 Vol 2 No. 3 page 89), or for affecting only subsets of populations of cells or ~lecules that pass through the lymphatic system or a desu-ed portion of the lymphatic system yr are found with greater concentration within the lymphatic system. The invention also contemplates methods of selective, enhanced or localized, targeting/ delivery by administering multifunctional ligands of the invention as well as methods (including methods directly yr indirectly employing the multifunctional ligands of the invention) of enhanclngl inducing entry of cells or maleculte, particularly immune cells (re. cells having an immune system function as broadly understood) or subsets thereof, to the lymphatic systettt or a portion of the lymphatic system, for example for the purpusc of direct or indirect interaction with the multifunctional ligands ofthe invention (in order to be acted on directly or indirectly, by multifunctional ligands of the invtntion) or for recruiting cells that will for example kill or modulate the activity of ether cells, for example hill cancer cells ar infected cells that will have, are having or have had direct or indirect interaction with the multifunctional ligands of the invention, as discussed further below, for example in the cars of cancer, by t~geting immunocytokines to the discast affected tissue eg. using cytpkines eg. TNFn fused to antibody that binds specifically to tumor cell markers or markers for angiogtntsis. Similarly tissue targetted as opposed to disease targeted immtmocytokines could be used selectively recnuc immune cells within that tissue for examplC a distastd tissue to enter the tymphatie system for such purposes including for example interaction with a multifunctional ligand ofthe invention.
It is also contemplated that a single multifunctional ligand can have multiple requlsitt interactive funetioaalities for example to stimulate, attract, anergirx (or otherwise inactivate) sub-populations of B-cells of T cells via the use, Far example, of trivalent or tetravalent antibodies and antibody conjugates/fusions thereof having multiple ligand interactive capabilities (see also for example technoloSies being developed for selection of sucetssful binders by phagt or ribosome display (see for example w0 01/00866; fJdv Protein Chem 2000; 55:367-403). A particular application of this technology for application to this invention are antibodies which retarget T-cells to tumor cells (see for example Man~ke O. et al. int. J. Cancer $2, 700-708 (/ 999); Br J Cancer 2000 Jau; 82(2):472-9; J Control ReleQSe 2000 rob 14; 64(1-3}:229-39 as will as related references, cited therein or citing these publications.
The present invention accomwodates such twehnology through multispecific anh'bodies or alternatively obviates the nerd for combining a T-cell receptor type molecule with the primary immune f nciion effecting moiety (eg. a canetr cell binding moiety) by using a separate multiftmctional ligand which combines, for example, a first portion and a second portion comprising a'f-cell interacting moiety (eg. anti-CD3). This is accomplished by administering in the same composition or substantially contemporaneously an amount of LhC second ntultiftinctional ligand that provides, as may empirically predicted by assessing the dispersion ofthe marker on the endothelial cell, a strong grob~itity (eg_ .001-100%, optionally 1-100%, optionally S-I 00°!0, optionally 10-100%, etc) that the T cell will be tftrgetcd in the vicinity of a given lymphatic endothelial Cell that happens be proximal to the Cell sought be targeted eg the cBnCtr Ctll. It i5 self evident that a 5050 proportion of the first and second multifunctional ligand will yield a strong chance that a second rnultifiutctional lig~d will be immediately adjacent on a particular given side (assuming far the sake of argument that there are sides when in reality the dispersion of the lymphatic cudothelial marker is governing). It is also contemplated that adjacent multifunctional ligands may be linked for example through linkage effective pairs of ligands (avidin-biotin), the sttond portions having an antibody Component which binds to a common iigand (eg on a liposome (see US 6197333 and refs, therein cited) or other pharmaetutically acceptable microlnano particle/sphere ofpreferably selectable size for optimal spacer or endothelial cell protective purposes) and that such entities could optionally also be employed to house and deliver a payload to a given target vicinity.
In one aspect the rttulti-Puttetlonal ligands of the present invention provide for a method and preferably a means for evaluating and/or inducing immune tolerance (with respect to S cells see strategies discussed in Imrounological RevicvVS 2000 'foI. 176 pp- 5-247).
It is believed that immune tolerance is enhanced or pralanged through prolonged lsttategic exposure to tolerance inducing and/or enhancing molecules far example prolonged antigen exposure (see Waup Y et al.
Eur. J. hnmunol. 2000; 30(18):2236-2234; Encyclopedia of Immun4logy ; (1998) Morgan Kauhronnrt Publishers,1SBN:Ot 32267656; Hoyne G)r et al. Immanolop~ 2000 Jul; 100(3):2$1-$; C.~ner CG et al. J
Invnuttol. 2000 Apr. 15; 1 b4(8): 3996-4002; Grossman Z. et al. Semin lmmunal 2000 Jun; 12(3): t 97-203; discussion 257-344 Textbook of ihc Autoimmune Diseases by l.ahita R. et al. ISBN: 47 $1715059 Lippincott Williams & Wilkins; M'ulti-Systemic Auto-hnmune Diseases : An Integrated Approach Dermatological & Internal Aspects ISBN: 0444$ l $960 Elscvier Science ;
Arthritis and Allied Conditions - A Textbook of Rheumatology, Thirteenth aYtd Fourteenth Editions, William J.
Koopman, MD 14'~:1SBN:
0-7817-22A0-3, November 2000; Principles of Drug Development in Transplantation de Autoimmunity Landts Bioscience, ISBN:0412100614; Cancer & Atrtoimmunity by Gershwin M, of aL.,ISBN:
0444503315 Elsevier 5cienct ; JAutottnmtrn 2000 Jtm; 14(4).278-82; The multi-functional ligands of the present invention, depending on their mode of administration (dirtct application by cannulating a lymphatic vessel or conventionally cg. intradermally or intravenously), can be advantageously employed to provide prolongedlstrategic exposure to tolerance enhancing molecules (for example by employing a multivalent cg. bi-specific Ab fragmwc or diabody which 1~ a first portion which >ainds to a lymph associated antigen and second portion which optionally comprises anti-idiotypic Ab portion mlmicldng the desired Ag or the antigen itself or a suitable portion thereof fused or conjugated to the first portion) on the infra-luminal surFace of tl~se lymphatic vessels, optionally, in addition to its conventional effects, when administered intradermally or intravenously, etc.. It is anticipated that the multi-fttnctional ligands of the present invention would be useful to assess and/or effect tolerance induction (sec Hassadonn GP et al. Proc Natl Acid Sci USA 1998 Mar 31; 95(7):3821-6; USP 6,106,834; UST' 6,099,$3$;
US6010942: Antibody heteroconju8ates and bispucirie antibodies for use in regulation of lymphocyte activity; as well as additional examples cited below with reference to examples of suitable anti-idiotypic antibodies).
It is also contemplated that a rmlltlspocific contract as described is W099/37791 could he used with respect to various aspects aspects of the invention..
Additional Ao~hcations of 'Varl4ns Aspects of the 1 v ' l it is contemplated that tire presort invention could be used to straugicelly mediate, CD45 (or varianr$/ather PTPs) related "cell signaling", far exempla through signaling molecules (cg, inhibitors) using multifunctional llgands of the iztvention including but not limited to bispeci8c antibodies, antibody fusions/conjugates cg. where the immune aFfCCting atlLlbody p41'tlon 4r 4tlter moiety is conjugated, fused etc. to an antibody or fragment that binds to an entity associatied marker t8.
LYVE-1 (1999) Journal of Cell Biology Vol 144 No 4 p. 789-801) (set for example USP 5,914,111 Sievers EL, Cancer Chcmather lyharlnaCO120D4 4b Suppl s18-22 W09946268, Neel BG Curr Opin Inmunol 1997 Jan 9(3) 405s120;
Front Biosci 1998 Nov 1 3:D-1060-94, 5li~a MK et al. J. Mol. Med 2000 78(2) 74-80 Goodnow CC Ciba Found Symp 1997 204: 190-202; Mustelin T. et ah Fr4ut Biosci. 1998 Nov 1; 3:
01060-96; Gaya A, Letxl: Lymphoma, 1999 Oct 35 (3-4): 237-43; Sievers EL, Curr Opic~ Oncol. 2400 Jaa 12(1): 30-S;
Thomas ML, et al. hnmul. Today 1999 Sep 20(9); 406-411; Appelbaum FR, 5etnin.
Htruatol. 1999 Oct;
36 (4 suppl. 6): 2-8; Ulyanava T; lmmul. Res 1997 Feb; lb(1}. 101-13; re PP32 for example USP
5,846,822 and Brody JR, ~t al. ) Biol Chem. 1999 Ju1 14; 274(29):20053-5 regarding the functional moiety of PP32 which is necessary for interaction with Cn45, and for example USP
5,9$1,251 with respect to methpds ef identifying such molecules}.
In preferred embodiments the invention is directed to muhifimctional ligands that comprise immune function exerting moieties having functionalities of molecules currently In clinical trials or proposed for clinical trials (see for example Glennie M1 et al. Aug 2000, Immunology Today 408 Vol 21($); see also Journal of lmmunological Methods 237 (2000) 131-145; Mo1 Immunol 2000 Jun;
37(9) 515-526; Anna Rev Med 2001; 52:125-145; Antm Rev Ivied 2001 52:63-78; Q 1 Nucl Med 2000 yep;
44(3) 268-83) including those that have an anti-CD2 Cunctionality (see USP 5,795,572) anti-CD4 functionality (see for ' example U5P 6,136,310 Flerzyk D, J infect Immun 2000 Feb; 69(2): 1432-1043) anti-CD3 functionality (for example WO D0J41474; WO 98139363; USP 6,113,901; Transplantation 2000 Dec 27 70 (12) 1707-12); Anti-CD44 functionality see for example Weiss L, 1t ad., Pros Nat A~Cad Sci 1,15A 200D; Jan 4 97(1) 285-z90; Sugiyama K, Immunol invest (1999) Mer-May 28(2-3) 185-200;
Srocke 9. et al. Pros Nat Acad Scl USA 1999 Jun 8 96(12) 6896: Mickecz K et ai. Nat Med 11995 Jun;
1(6); 558-63;
Ahrens T et al., I Invest Dermal. 2001 Jan! 16(1) 93-101); with respect to control of migrafion ofT-cell lymphocytes see Nohara C, 1t al. J Immunol. 2001 Feb 1; 166(3) 2108-2115), anti-CD20 functionality (see Crit Rev Oncol IIematol 2001 Jan 37(1):13-25) etc. anti-CD22 functionality see for example Newton DL, et al- Blood 2001 Jatt15; 97 (2): 528-535, USP 5,1$4,$92; Anti.CD40ICTLA~
see for example .l lmmuno12000 Oet 1; 165(7):3612-9; Microsurgery 2000; zc ($); 448-452; USP
5874082; USP
6056959; USP 5,801,227: USP 6004552; USP 5677165; USP 6087329; USP 5961974;
USP
6051228; White CA,et al. Annu Itev Med. 2001; 52: 63-78 (see also reviews and speti~lic applicatlotts referred to in Ditzel 1t al., lmmunal Res. 2000; 21(2-3):185-93; USP
6,010,902, USP 5876950; USP
5876718; USP 5,601,819, USP 59$1251, USP 5$$5579 and 5885796; Cancer lmmunollmmunother 2000 Jun; 49(3).173-80; OmarK, I Nturoirnttxunol2001 Feb 1, 113(1) 129-141;
Bellido M, Eur 1.
Haematol 2041 Feb. 66(3) 100-106; l3roeren et al. 1 Immurlol (2000) DeclS
165(i 2) 69D8-14;
AlexandroffAB et al Mol immunol 200D lone 37(9) 515-526; Werkerle T J lmmunol.
2001 Feb 15 166(4) 2311-2316; Howard LM J:hmnunol 20D1 Feb; 116(3) 1547-53 anti-C1~154;
JPharmacokinat Biopharm 1999 Aug; 27(4}.397.-420, J Clin Oncol 2000 Apr; I 8(8).1622-36, Leukemia 2000 Mar;
14(3):474-S, Clin CallcBr Rea 20Q0 Feb; 8(2):372-80, Leukemia 2000 Jan;
14(1):129-35, JNucl Med 1999 Nov; 40(11):1935-46, Blood 1999 Nov 15; 94(10):3340-8, Blood 1999 Aug 15;
94(4).1237..47, L'ancor-Res 1999 May 1; 59(9):2096-101, Vaccine 1999 Apr 9; 17(15-16):l$37~5, Blood 1998 Dec 1; 92(11):4066-71, JRheumatol 1998 Nov; 25(1 I);2D65-76, Clin Pharmacol Ther 1998 Stp; 64(3):339-46, Muh,$cler 199fi JnI; 1(6):339-42, Cancer Immunol Immunother 1997 Jul; 44(5):265-72, Transplant Proc 1996 Dcc;
28(6):3210-1, Arthriti5 Rheum. 1996 Jul; 39(7):1102-8, Immunology 1996 May;
88(1):13-9 and USP
5,876,718)-The invention contemplates assessmtnt and therapeutic benefit of lymphatic localization in the case of antibodies and multispeciFe Ligands which are toxic to non-target cell populations which express the targeted ligand to a limited extent or in the case of toxic cross-reactivity of the second portion cg. antibody far its desired target with etn uadesi;ed targee (see cg. Lancet 1999 Nov 13;
334(9191 ):1691-5). It is contemplated that the toxic effect of a given eff'ector moiety of a multifunctional ligand of the invention could be alternated using net additional binding arm far 8 lymphatic marker, Antibody 5trutture and Function Antibody structure and fraction ha,~c bee extensively desCxibed h1 the literatue. Far example see Antibody Engineering 2°° ad. Carl A.K. Borrcbaeck, dxford University Frees 1995 p 3~4.
Production of Bispecifit Antibodies A variety of different constructs have been developed for the production of bispecific antibodies including conventional four chain antibodies (including truncated version thereof such minibodicy (see USP
5,837,$21 ), F(ab')z (see Antibody Fusion Proteins, Steven M Chautow , Avi Ashkenazi Eds. ISBN
04711835$X May 1999 Wiley p.136-144; or using C1I3-truncated heavy chains), diabodies (see USP
5,837,242 Multivalent and multispecific binding proteins, their manufacture and use) constructs in which of one or two diabody molecules are heterodimerized by crtating a fusion protein with the CL and Cl-11 itntnunoglobulin constant domains (see Wt7 02102781).
In recent years, a variety of chemical and recombinant methods have been developed for the production of bispeeifte andlor multivalent antibody fragments. For review, see: Knangkum J, et al. Bispecific and bifunctional single chain recombinant antibodies. Biomol Eng 2001 Sep;l8(2}:31-40, Holliger P, and Winter, G., Curr_ Opin. ,Blotechnol. 4, 446-499 (1993);_CaCter, P. BI al., J, Hematothcrapy 4, 463-47D
(1995); PluGkdlan, A. and Pack, P., lmmunoteehnologyr 3, 83-105 (1997).
~ispeciflCity andlor bivalency has been accomplished by fusing ~o seFv molecules via flexible linkers, lcucinc zipper motifs, C,ICL-.
heterodimerization, and by association of scFv molecules to form bivalent tnonospecific diabodies and related structures. Muitivalency has been achieved by the addition of multimeriration sequences at the carboxy or aminb terminus of the scFv or Fab fragmenu, by using for example, p53, streptavidiu and helix-tura-helix motifs. Pvr example, by dimerization via the helix-turo-helix motif ofan scFv fusion protein of the form (seFv 1 )-hinge-helix-turn-helix-(5cFv2), a tetravalent bispecific is produced havirte two scFv binding sites for each of two target antigens.
Production of IgG type bispecific antibodies, which resemble IgG antibodies in that they posses a more or less Complete TgG constant domain structure, has been achieved by chemical cross-linlciug Of tWO different 18G molecules or by co'expresslon of two antibodies from the same cell. Both methods result in production pf signllYCant anl4llnt5 of undesired and non-fttnctional species due to mispairing among the component heavy and light chains. Methods have been employed to reduce or eliminate mispairing_ One strategy developed to overcome unwanted pairings between two different Sets of TgG heavy abd light chains co-expressed in transfected cells in modification of the Ci,3 domains of two heavy Chains to reduce homodimerizati4n between like antibody heavy chains. Merchant, A. M., et of., ( I 998) Nat. BiotechnUlogy 16, 677-681, In that method, light chain mispairing was eliminated by requiring the use of identical light chains for each binding site of those bi5peciftc antibodies.
To produce bispecific antibodies, Kostelny et al (J. Immunology 148:1547 (1992)) Rtsed rob $agments of antibodies to the lancing zipper portions of fos and jun proteins in the absence of a single chain construct for the antigen combining region. Those methods are well described in the lher~tture and summarized with t~eferenoes in Antibody Fusion Proteins, Steven M Chamow , Avi Ashkenazi EdS.
ISHN 0471183585 May 1999 Wiley; Kontermann, R., et al.(Eds_) particularly at pages 139-145. Pack and PIuckthun, fused a single chain antibody to amphipatlua helices from a four helix bundle or from lancing zipper proteins-l3ispecific antibodies that are in a cnnvenbonal IgG-like and Fab-likt format have been developed by Zhu as tetravalem or bivalent molecules, respectively with each of the chains serving tb anchor a binding moiety (sec WO 01/90192 and Fio re 1 therein), preferably consisting ofa scFv. In the bispeci6c TgG-like construct, each $ide of the molecule comprises a CH I domain and a CL domain and oath CH and CL
domain is linktd through its N-t~minua to a scFv of different specificity. The invention herein contemplates that this construct can readily be adapted to have each each half of the molecule associated with a polypeptide eg. a scFv of the same specificity so that each half of the molecule is mooospecitlc (or to have each half of the molecule associated with different pairings of scFvs) so that each half of the molecule i5 eff'ectiveIy monospe~fia- The invention herein contemplates that a bivalent relatively low affinity second ligand binding moiety is used to activate receptors that redttire crass-linldag for activity.
The invention also contemplates that numerous permutations in which the functional affinity of the first ligand binding moiety whether monospeeiflc or bispecific can be accentuated relative the functional affinity of the second ligand binding moiety including employing a first ligand high affinity xcFvs for a single aatinstances in which the second ligand binding moiety is effectively monovalent (has one, or one useful binding moiety). The iavefttion also contemplates that this construct can have a ttvncated Fc portion and various known methods in the fll't fOr imprOVIng the pall'Ing efFlIGiency of the heavy chains- The invention also contemplates that the CH1 and CL domains of the second ligand binding moiety can be truncated as in camelid ant-bodies for efficient delivery e8. of biologic effector ligands.
Methods of Generating Antibodies 'that Bind To Sslected Target Ligands A variety of technologies for generating antibodies with desired specificity have been extensively developed and beGOtne well known to and routinely practiced by those skilled in the art including phage display (see review in Basic Methods in Antibody Production ~t Ch~cteriaion G.C. Howard et al. ads.
CRC Press ?001 p. 105) and other display systems (ribosome display, display on the surface of various cells), immunising mice, including particularly nliee having human T8. genes, and antibody microarrF~y teehnalogies. These methods have also been extended to making antibodies with dual specificites such as diabodies (USP 5,837.42 Multivalent and multispecific binding proteins, their manufacture and use) and are the subject of extensive scientific and patent literature. For example, see US patents of Whiter at al.
6,291,650; 6,291,161; 6,291,158; 6,017,732; 6;?25,447; 6,172,197; 6,140,471, b,410,$84 5,969,14$, 5,871,907, 5,858,657; 5,733,743, 5,723,287and those ofDyax, Morphosy5, arid Cambridge Antibody Technology .

Affinity Maturatipn Methods of codon based mtztagenesis have been extensively developed for ett~neerlng the antibody binding silo. Far example, the use of such methods in a filamentous phage display system is described in Antibody Engineering 2°° ad. CeYI A.K. BorrtbaCCk. Oxfbrd University PreSS 1995 p 1 l7-128 see also pp.53-84 with respect to techniques of phage display of antibodies (see also Kanrermarrr~ R; Diibel. 5..
(Eds.): l2llOJ)Antibody Enginexrine 15BN: 3-540-X11354-5, Methods of Generating Single Domain Ligands The ability of a single variable fragment of an antibody to bind with speclflcity and stiltable selected a$tnities in the nanomolar+ range has bcsen extensively demonstrated using camelid and human VH
fraEments. Methods of generating VHs with the desired specificity have been extensively described (see USP 6,248,516 Single domain ligends, receptors comprising said ligands methods for their production, and use of said ligands and receptors). (see also literature referenced herein on this subject).
Methods of Making Antiuodks In E. Coil The expression of recombinant antibodies, including diabodies in E. Coli has become routine_ General precepts, and methods are discussed in Antibody Engineering 2"a ad. Carl A.K.
Borrebaeck, Oxford Unlversiry press 1995 p229-266 see also Aatibady Therapeutics WJ Harris et al.
ads. CItC Press 1997 p.
221; see also review in Biotechnology, Volume 5A, Recombinant Proteins, Monoclonal Antibodies, and Therapeutic Genes A. Mountain, U. Noy, Dietmar Scbomburg ISBN: 3-527-283 L3-3, January 1999, Antibody PraducHon_ ES!iential Techn9que.~ Pete.~r3. Delves 1$FJN; p~4~1-9701Q~? Wiley7une 19)7 and Antibody Therapeutics Production, Clinical Trials, and Strategic lssubs, By Rathin C. Des, Ph.D., M.B.A. & K_ Jphn Morrow, Jr., Ph.D., D&MD Publications October 2001 Chapter 3.
Eu~a ryotlc & Other Expression & Production Systems Approaches for the eukaryotlc exprassloa of antibodies and antibody ittsIon proteins and the preparation of vectors far use in such methods are well lmawn and extensively described in the literature. General precepts, and methods are discussed is Antibody Engineering 2"' ad. Carl A.K.
Hotrebaeck, Oxford University Press 1993 p~67-293 (see also Antibody Therapeutics WJ Hams et al.
eds_ CRC Press 1997 p_ 183-220; see also review in Biotechnology, Volume 5A, Rccombinaat proteins, Monoclonal Atttitrodies, and Therapeutic Gems A. Mountain, U. Nay, Dletmar Scyomburg 1SBN: 3-527-28315-3, Wiley, January 1999 and Antibody Production: Essential Techniques Peter J.
Delves ISBN: 0-4?1-97010-7 Wiley June 1997 and Antibody Therapeutics Production, Clinical Trials, and Strategic Issues, Hy Rathin C. Des, Ph.D., M.H.A. & I~. Joha Morrow, Jr., Ph.D., D&MD
Publications October 2001 Chapter 3.
With respect to a review of immunotoxins sea also Antibody Therapeutics WJ
Harris et al. ads. CRC Press 1997 p 33 With respect to Methods for producing recombinant vectors see also 5,962,255 Methods for producing recombinant vectors FdrmuLati0tl, purif"tCation atld analytic ntethpds involving antibodies are well knoen to those skilled Irt the art and have been extensively reviewed. With respect to fo>znulation, purification and analytic methods see for example, reviews in Antibody Therapeutics Production, Clinical Txlals, arid $tCategiG Issues, Hy ltathin C. Des, Ph.D., M.B.A. & K. John Morrow, Jr., Ph.D., n&MD Publications t7ctober 2oal, Ghaptcr d.

With respect to methods of generating antibodies against self anibodlzs see USP 5,885,793 Produerion of anti-self antibpdie5 from antibody segment repertoin-__s and displayed pr phage Antibody Conjugates Methods ofchemical manipulstiou ofantibodies for attachment of ligands (eg.biotin), radionuclides etc. are well lmown in the art and have been extensively reviewed (for example see review in Basic Methods in Antibody Production & Characterizvion (3.C. Howard et al. eds_ CRC Press 2001, p. 199; with respect to therapeutic principles see for example, Antibody Therapeutics WJ Hauls et al.
eds. CRC Press 1997 p 53-$$).
The applications ofbispeciftc antibodies, including methods ofmaking and usingtltam have beat extensively reviewed (ee for example van Spriel A13, van Ojik HH, van De Winlvel JG. lntmunotherapeutic perspective far bispecihc antibadies_ Inununol Today_ 2000 Aug; ? 1 (8):391-7;
Weiner LM. Bispecific antibpdies in cancer therapy. Career J Scl Am. 2000 May; 6 5upp13:S265-71_ Barber 1, et al. Pretargeting with the afFlniry etlbancement system for radioIntmunotherapy. Cancer Siother Radiopharm. 1999 Jun;
14(3):1 S3-66. de Wolf FA, Brett GM. Ligand~binding proteins: their potential for application in systems for controlled delivery and uptake of iigands. Ph$tmacoi Rev. 2000 Jun; 52(2):207-36.: Wang H, Liu Y, Wei L, Guo Y_ Bi-specific antibodies in cancer therapyAdv Exp Med Biol. 2044;
4b3:369-$0; Staerz UD, Lee DS, Qi Y. litduetion of specific immune toltrance with hybrid antibodies.
Immunol Today. 2000 Apr;
21(4):172-6: 1999 Dec; 43(4):336-~13. Elsasser D, Stadiek H, van do Winkvl JG, Valerius T. GM-CSF as adjuvant for immtmotherapy with bispecific antibodies. Eur J Cancer. 1999 Aug;
33 Suppl 3:525-8.
Molema G, lCroesen 13J, Helfl~Ich W, Meijer 17K, de Leij LF_ The use of bispecific antibodies in tumor cell and tumor va5C111atUre direCltd immunotherapy. J Control Release. 2000 Fcb l4; 64(1-3):229~39. Bodey B, Bodey B, Siegel SE, Kaiser HE. Genetically engineered monoclonal antibodies for direct anti-neoplastia treaitnent and cancer cell apeciftc delivery of cht>motherapeutic agents. Cure Phann Des_ 2000 Feb;
6(3):361-76. Kudo T, Suzukl M, Katayose Y, Shinoda M, Sakurai N, Kodatna H, lchlyama M, Tekentura 5, Yoshida H, Saeki 1'l, Saijyo S, Takabashi J, Tominaga T, Matsuno S. Specific taTgetlag trnmunotherapy of cantle with bispeci~fic antibodies. Tohoku J >rxp Med. 1999 Aug; 188(4):275-88. Koelemq R., et al.
Bispeeific antibodies in cancer therapy, from the laboratory to the clinic. J hnnmnother. 1999 Nov;
22(6):514-24. Segal DM, Weiner GJ, Weiner LM gispecific antibodies in cancer therapy Cure Qpia Immunol. 1999 Oct; l i(5):558-62. $udsan PJ_ Recombinant antibody constructs in cancer therapy. Cure Opin Immunol. 1999 Oct;
11(5):548-57. Berth RF' et al, $oron neutron capture therapy of brain tumors:
an emerging therapeutic modality. Neurosurgery. 1999 Mar; 44(3):433-50; Fleckenstein Q, Qsmers R, Puchta J. Monoclonal antibodies in solid tumours: approaches to ti~erapy with emphasis on gynaecological cancer, Med Oncol.
1998 Doc; 15(4):212-21. Guyre CA, Fanger MW. Macrophage-targeted killing and vaccines. Res lmmunol. 1998 yep-Oct; 149(7-8):655-GO Cao Y, Suresh MR l3ispecific antibodies as novel biocoujugates. Bloconjuo Chem. 1998 Nov-Dec; 9(6):635-44. Fareh RA, et al, The development of monoclonal antibodies for the therapy of cancer. Celt Rev Eukaryot Gene Expr.
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18(4C):2905-17. Rouard H, et al, Fc receptors as targets fbr immuaotbtrapy.Int Rtv Immtazol. 1997; 16(1-2):147-8S. Fan Z et al.
Therapeutic application of anti-growth father receptor antibodies; Glur Opin Oncol. 1998 Jan; 10(1):67-73: da Gast GC, et al,Clinical perspectives ofbispecific antibodies is cancer.
Cancer Immunol lnvnunother. 1997 Nov-Dec; 45(3-4):121-3. Carter P, Merchant AM. Engineering antibpdies for Imaging and therapy.Curr Opin Biotechnol. 1997 Aug; $(4):449-54. Pluckthun A, ct al, New protein engineering approaches to multivalent and bispecific antibody fragments_ Immunotechnology.
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et al; l~unor vascular endothelium: barrier or target in tumor directed dntg delivery and immunotherapy.
Pharm Res. 1997 Jan; 14(1):2-10. Hodey H, et al, Human trotter detection and immunotherapy with conjugated and non-conjugated monoclonal antibodies. Anticancer Rcs. 1996 Mar-Apr, 16(2):b61-74 Harmtann F et al, Treatment of Hodgkin's disease with bispecific antibodies.
Ann Oncol. I99b; 7 Suppl 4:143 ~b, wels W, et al, Tnterventian in receptor tyrosine kina$e-mediated pathways: recombinant antibody fusion proteins targc;tcd to ErbB2. Cure Top Microbiol Lt~unol. 1996; 213 ( 15t 3):113-28.: lCalremo TCJ.
Radioimmunothetapy of solid cancers: Acta Oncol. 1996; 35(3):343-55. Verhoeyen ME, et al, Antibody fragments for controlled delivery of therapeutic agents. Biochem Soc Trans.
1995 Nov; 23(4): I OG7-73.

Heagen IA. Performance of CD3xCD19 bispecific monoclonal antibodies in B cell malignancy. Leuk [.ymphoma. 1995 Nov; 19(5-6):381-93.
In mother aspect the invention is dimmed to presenting antigen within the lymphatic system (eg. in the form of an anti-idiotype antibody) such aS to facilitate a desired immune response eg. vaccination type responses). Optionally, adjuvants can be conventionally employed to assist initial immune stimulation eg.
intradermally when appropriately delivered. Activating eytokines for cxatnple a5 Specified above, can also be employed to enhance the immune response. Examples of antibodies having an anti-idlotyplC Coiulterpalt or for which an anti-idiotypie counterpart could made by well lrnown techniques in the art (and that art capable of exerting the desired anti-idiotypic effect) arc numerous and numerous such antiidiotypic antibodies have application to immunization as well as applications relating to tolerance (see for example U5 patents: 6,146,627 Method for reducing T cell-mediated cytotoxlciry in HIV
using anti-idiotypic antibody; 6,063,479 Anti-idlOtypic mpnpclonal antibodies and compositions Including the anti-idiotypic monoclonal antibodies; 6,460,449 Surrogate tolerogerlesis for the development of tolerance to xenografts; 6,042,827Anti-idiotypic antibody induction of anti-tumor response;
6,047,815 Anti-idiotype vaccination against diseases resulting from pathogenic responses by specific T
cell populatibna; 5,981,502 Methods and compositions for inducing apoptosis in tumor cells; 5,766,588 Tumor immunotherapy using anti-idiotypic antibodies; 5,728,812 Anti-idlorypic antibody composition for inhibiting acute complement-mediated cytotoxicity.
According to another aspeu~t of the invention the multi-functional ligand comprises a first portion which binds to a lymph associated antigen and a second portion which hinds to a trtmor cell infected cell or infectious agent. This embodiment of the invention can be used for example, to assess and affect the ability of the tumor-binding portion to more advantageously inhibit metastasis.
Optionally, for example, the portion which binds to a lymph associated antigen has a lower affiniiy andlor avidity so that the tumor cell binding portion preferentially binds to the tumor cell and is therefore more likely to accompany its passage through the lymphatic system. This strategy also has application to bi-specific antibodies of tlZe invention in which the second portion is for example targeted to an immune cell.
Optionally, multiple such muti-ilrnctional ligands may permit sufficient tumor call anchoring to permit the tumor cell to be killed within tile lymphatic SysbCIn via a toxic payload carried by the ixsuttjftmctional ligand or through the recruitment of immune cells which accomplish this end (cg using the same or a different multLflutetional ligand fttsed or conjugated to a suitable cytokine (eg IL-2, TL-12). The prolonged presence of these cells could be advantageously used to assess methods of ittamunizfrtion directly against the rumor cell using, for example, cytoldnes including cytolcines fused or conjugated in whole or functional part to a lymph targeted Ab on the Same, or a different multifunctional ligend d~livcred in a suitable dose (with respect to genoratian of anti-tumor antibodies and other antibody fragments for application herein as wolf as Important related technologies see also WO 00150008; WO OlIOI 137; WO 97137791; WO 99137791; WO
97/10003;
Hoogenboom et al. Nat. Biotechnology ! S(2) Feb 1997 p l25-12G; Fell H. et al.
Journal Of lmmunolgy Vol 146(7) Apr 1991 p244d-2452; Anderson D. et al l3ioconjugate Chemistry 14(1) Jan 1993 ptD-18;
USP 6, 172,197; USP 6,171,782; Immunological investigations 2000 29(2) entire issue). bptionally tht tumor binding portion internalizes and/or delivers a toxic payload, for example a radionuclide, or other toxin, or a eytokine to the tumor cell (with respec# to selection of tumor interttalizin g human antibodies see for example Pool M et al. J Mol Biol. 2444 Sep 1; 301(5):1149-61, see also Kohl )v~ et al, J Mol. Biol.
l3iotechniques (2000) Vol 28(1) p 16~ In this way the multi-functional ligands of the invention, for example, when provided in a sufficient dose to both target the tumor and line a ponion of the lymphatic system to which the target tumor is lileely to drain, acts as a cancer treatment as well as a sentry system for assessing I augmenting (for example as an adjunct therapy) the ability ofthe tumor binding portion withlwithaut payload to inhibit metastasis. There are nurncmus exomples of functional eytolcine and toxin fusions used for example in cancer therapy that may have application to the invention herein (for examples and reviews see references herein cited as well as WO 99137791; W099 WOOO/Ofi6D5 ; WO
99/52562Wb 99/37791 MULT1PURPOSF ANTI190LfY; Proceeding of the IBC's 11'"
Annual Intemadonal Coz~f~ence on Antibody Engineering State of the Art Science, Technology and Applications, December 3-6, 2000; Ampli$cation of T cell-mediated immune responses by antibody-cytokine fusion proteins. ImmunoI Invest. 2000 May; 29(2):117-20; Cancer Res.1999 flay 1;
59(9):2159-66.;
Phannacokinetics and stability of the ch14.18-interleukin-2 fusion protein in mice. Cancer I177munoI
Tmmunother. 1999 Aug; 48(5):219-29. Phase I study of single, escalating doses of a superantigen-antibody fusion protein (PNU-214565) in patients with advanced colorectal or pancreatic carcinoma. J lntmunother.
2000 Jan; 33(1):146-53. Targeted toxin therapy for malignant astrocytoma.Neurosurgery. 2000 Mar;

46(3);544-51 ; Targeting cytokines to tumors to induce active antitumor immune responses by recombinant fvsian proteins. Hum Antibodies. 1999; 9(1):23-36; Lode HN, et e1.
Tumor-targeted IL-?
amplifies T cell-mediated immune response induced by gene therapy with single-chain TL-12. Pros Nat1 Aced Sci U S A. 19991u1 ~0; 96(15):8591-6; Cancer Vaccines and Irnntunotherapy 2000 (textbook) ;
Immunotherapy With intravenous lmnumoglobulins P. Imbach ( 1991) Academic Press; Molecular Approaches tp Tumpr Tmmunothetapy (1997) GVorld Scie171tiiaG Publishitlg Comlpany, IncpYpordted;
Vaccines & Itttmunptherapy S. l_ Cryz ( 1991 ) McGraw-FIill Ryersan, Limited Llrith respect to internalizing antibodies see eg Biological Effects of Anti-ErbB2 Single Chain Antibodies Selected for lnternalioing Function; Biachem Biophys Res Commun. 2001 tan 12;
280(1);274-279 and references cited therein, Immunoconjugates of geldanamycin and anti-HER2 monoclonal antibodies:
arltiprpliferative activity on human breast carcinoma cell lines J Natl Cancer Tnst_ 2040 Qct 4; 9?(19):1573.
8I; Foulon Cr, et al., Radioiodinafian via Iy-atraiuo acid peptide enhances cellular retention and tumor xcnagraft targeting of an internalizing anti-epidermal growth factor receptor variant III roanocional antibody. Cancer Res. 200D Aug 15; 60(16):4453-60. Poul MA, Becerril B, Nielsen UB, Morisson P, Marks Selection of tumor-specific ittterualizing human antibodies from pbege libraries J Mol Biol. 2000 $ep l ; 301 (5). I 149-61 _Vrouenraets MB, et al_,Targeting of a hydrophilic photosensitizer by use of internalizing monoclonal antibodies: A new possibility for use in photodynamic therapy. 1nt J Cancer.
2040 4ct l; 88(1):108-14.
In yet another aspect, the invention contemplates that the passage of tumor cells can ba inhibited within the tumor vasculature using a bispecIflc llgand, optionally a bispecific antibody, which targets on the one liand a well known vascular endothelial marker and one the other hand binds to a lig$nd on the surface of the tumor. Other aspects of the invention related to tumor cell targeting are understood to described in reference to this aspect of the invention as well. It is also contemplated that markers which are present on both the lymphatic endothelium and the tumor vaseulature can be simultaneously targeted with bispecif,,c ligands of the ir<ventipn tp inhibit tumor meta3tasis andlor immunize a subject against tumor tolls.
It is contemplated that the multifunctional ligands ofthe invention when used to inhibit metastasis, for example, in the manner descn-bcd shove, could be advantageously employed in combination with other well known therapies Foe txample cytoxic dings, other tumor targeted antibpdicg and CpnjugatG/iitslons therewith used or currently being evaluate for immunotherapIes, angiogenesis targeted drugs cat. (re angiogeuesis see for example Angiogenesis in cancer and other diseases.
Nature. 2000 Sep 14;
407(G 801 ).249-57).
Similarly, a bi-specific antibody of the invention could ba used to bind to andgens/ligands on lymphocytes which are known or become known to inhibit or enhance immune function or mediate a disease eg. CD45.
With respect to target receptprs related to the inventipas defined herein see also USP 6,277,962.
As discussed above, as used herein the term "lynaph associated antigen" refers to antigens that arc expressed significantly on lymphatic endothelial cells but not significantly expressed, if at all, on other tissues. Examples of such antigen include LYVE-1 a CD44 receptor analpgue which binds to 1~IA (February 22, 1999, Banerji et_ a1_, Jotunrtl of Cell Biology Vol. 144, fi4, p789-HD1) and which is expressed primarily on lymphatic endothelial cells. LYVE-1 specific antisera have been shown to inhibit binding of IiA. The invention contemplates research and treatments using multi-functional ligands ofthe invention with respect to non-human mammals, including preferably agricultural animals, canine species, primates and mice bavin~ similar receptors/antigens_ For example, a marine counterpart to LYVE-1 (published in Prevo R
et al. 2001 Feb 20, J. Bial. Chem.; Manuscript M01100430D) can be omployod to implcmtnt the various methods and embodiments described herein in a mouse model, for example to assess the extent of inhibition of metastasis effected by a multiftmctional Iigand (optionally comprising for example to a toxin, GytOkine T Celt reteptOr etc) which has a fiat ppmpn which binds tp LYVE-t and a sGCpnd ppttipn which binds ta, far example to G1-101, a breast tumor which is known to metastisize to the lung {see USP
6037520 and 5, 693, 533 see also US patents 5,643, 551, 5491284, 5569812, 5917124 and 6 I 07540 and references cited in these patents, particularly with respect to other metastatic models and methods of evaluating anticancer drugs in mice). LYVE-I counterparts in other mammals can be idtmtifled in the manner described by Prevo R et al. (see also Strobe M. et al. Trtduckion oftumor lymphangiogenesis by YEGF~ promotes breast cancer metastasis Nat- Med. Fob; 7(2) 192.8.) Qther models of metastasis in animals are well known in the art (see for example CItirgwin JM, Cuise TA.Molecular mechanisms of tumor-bone interactions In osteolydc metastases.
Crit Rev Eukaryot Gene Expr. 2000;1 Q(2):159-78. 3: Kobaek-Larsen M, et all~eview of colorectal cancer and its metastases In rodent models: compatati~e aspects with those in humans. Comp Mad. 2000 Ftb;50(i): 1 s-26. 5: Magnano M, et aLA physical-bastd triodel for tht slmulSdOG of ncoplastic grbwth and mxtastasiis. J Surg Oncol. 2000 Jun;74(2):122-9. 6: HotT:man RM. Orthotapic metastatic mouse models for anticancer drug discovery and evaluation:a bridge to the clinic. Tnvest New Drugs. 1999;17(4):343-59.
Russo J, Russo IIi.The pathway of neoplastic transformation of human breast epithelial oells.Radiat Res.
2001 Jan;155(1 Pt 2):151-154. Dutty M!, McCarthy K.Matrix metalloproteinases In cancer: prognostic markers and targets for therapy(review).Int J Oacol_ 1998 Jun;12(6):1343-8.
22: Batterjec A, Quirks P.Experimeiztal models of colorectal cancer. Dis Colon Rectum. 1998 A~pr,41 (4):49p-505.
Wu TT et al.Establishing human praatate canctr cell xenograits in boat:
intlaction of astcoblastic traction by prostate-specific antigra~-producing tumors is atlrytnic and SCID/bg mice using LNCaP and lineage-derived metastatic sublines. Tat J Canoer_ 1998 Sap 11;77(6):887-94.61: Molpus KL, et alCharacterization of a xcnogmlFt model of human overlap carcinoma which produces intraperhoneal carcinomatosis and metsstasca in mica. hrt J Cattctr. 1996 Nov 27;68(5):58$-95.65: Pages !C, Sardat B, Bautlsta D, Costa J, Benhattar J. Detection of rare citeutating human colon tumor cells in a nude mouse xenograft model.
Cancer Lett. 1996 Aug 23;106(1):139-04.66. Sakakibera T, ~K al.Doxorubiein eneapeulated in stcrically stabilized Iiposomes Is superior to free drug or drug containing conventional liposomes at suppressing growth and metastases of hut» lunl: tumor xeno~afts. Cancer Res. 1996 Aug 13;36(1:3743-6.
With respect to modifying an antibody to inctbaso its affinity see also Crystal structure of Fab198, an efRcient protector of the at:etylcholine receptor against myasthenogenic antibodies_ Eur 1 Biochem. 2001 lu1;268(13):3b85-3693.
rot example, in one embodiment the inveution contemplates a bispecific antibody comprising an antigen binding component specific for a tumor cell associated antigen and a relatively low amity anti-II~6 recoptor antibody component. Wlrh rospect to the anti-armor role of LL-6 see WeI LH et al. lnterleukin-6 in cervical cancer: the relationship with vascular endothelial growth factor.
Gynecol Oncol. 2001 Ju1;82(1):49-56.
The inveatIon contemplates that TCRs and modified TCRs (see for example, Wa 01148145) may be used as ligands. in place of anfbody fragments far binding to target ligends such as paptide/A~C ligands_ .
Techniques for generating antibodies, and methods, for example of subtractive screening useful to identify other lymphatic vessel associated antibodies, including those optionally having smaller scFv, Fab and dAb (single domain antibody or functional fragment thereof] component (more easily passagiag to lymphatic vassals from tissues particularly when constructed in tht form of bispeciflc antibodies eg. diabodies ate.) by phagc or ribosome display era well known in the art (see for exatnpIe lloogenbom 1Ht et al. lmmunol.
Today (Aug. 2000) Vol 8 p 371; Schaffttzel C. et al. J ltmnunol. Methods (Des.
10, 1999) 2310-2) p.
119; Roberts RW et al. Curt Opin Chem Biol. 1999 Jun; 3(3):268,73; Winter C.
et al. Anau Rev Immuool 1994 12:433-55; Kaatennann 1~ et al. Nat Blotechnol. 1997 Jul;
15(7):629-31; Phage Display of Peptides and Proteins, A Laboratory Manual Kay HK et al. Eds 1996 Academic Press;
Tmmunology Mechads Manual Le&ovits, I ad. 1997 Academic Press;Hoogenboom et al.
lmmunotechaolagy 4 ( 1998) 1 ~20;
"With respect to making sinEle domain anh-bodies see for exatnpIe USP
5,824,520, USP 5622836, USP
5,702,892, USf 5,959,087, Unique single-domain antigen binding fragments derivod fi~om naturally oCCUrrin)i Camel heavy-chain antibodlas.J Mol Recognlt. 1999 Mar-Apr; 120):13 J -40. An antibody singlo-domain pltage display library Of a native heavy chain variable regia~n:
isolation of ftmecional sIn$le-domain VH maltcults with a unique interface. 1 Mo1 Biot. 1999 Jul l6; 290(3):685-98 and references cited in khese references.
Methods far making antibody fusion proteins and bi-specific antibodies including diabodies arc. and Fusion prattins thereof ore well e9mblished is the art (for reviews and particular applications see for example Adams QP et al. Journal oPltnmunological Methods 231 (1999) 249-260; tJSY
6,121,421, 6,027,723 and 6,025,165; EP 0654085; Hudson P. Exp. Opin. lnvCSt. DnlgS (2000) 9(6): 1231-124; Anybody Fusion Proteins Steven M Chamow , Avi Ashkenazi Eds. ISBN 047118358X May 1999 Wiley;
Antibody Engineering, Carl A. Botrebaeck oxford University Press, 1995; Antibody Engineering:A Practical Approach David J. Chiswell, Hennie I~ Hoogenbovm, John McCatJerty OxfordUniversity Press,1996;
Antibody Engineering Protocols, Sudhlr Paul (1995) Humaea Press; Antibody Expression 8c Engineering (1998) Henry'Y. Wang, Tadayuki Tmanaka, American ptGp7jqaI Society; ~hu 2:.
>3iptechnplogy (NY) 1996 Feb.; I4(2): 192-6; Nielsen UB of al. Cancer Res. 2000 Nov 15;
60(22):6434-40; Lawrence LJ.
fit al Febs Lett. 1998 Apr. 3; 425(3) 479-84; Hollinger et al., Dancer ln,munvl Immunother 1997 Nov-Decc 45 (3-4) 1?8-30; Immunotargeting of tumors: stale of the art and prospects in 2000 Bull Cancer.
2000 Nov; 87(11):777-91; Hellfrich Wet al Int J. cancer 1998 Apr 13 76(2): 332-9; Wu AM, Q J Nuc Med. ?004 Sep.; 4-4(3):268.83 KrebsH. Et al. J Interferon cytokine Res 19985ep 18(9): 783-91; Takemura 91, et al. Protein l;tag. 2000 Aug.; 13(8)' 583-$; Cochlqviu; B et at. 1 lmrnunol. 2000 Jul 15;
165(2):888-95; Atwc111 JL et al. Prouin Eng. 1999 Jul; 12(7) : 597-604;
kiprivauov SM et al. J. Mol Biol. 1999 Oct 15, 293 (1): 41-56; Alt M. ct a1 FEES LeH. Jul 2 454 ~1-2) 90-4. Hudson PJ et al. J
llnmunol Methods 1999 Dec 10; 231 ( l-2):177-89 Ardnt MA et al. Blood 1999 Oct 15 94(8): 2562-8;
Lu D. et al. J Immunol. Methods 1994 Nov. 19; 230(1-2):159-171; Saotos AD et al, Clin Cancer Res 1999 pct 5 (10 supply: 31 185-31335 Kontermann RE et al. Nat Biotcchnol_ 1997 Jul; 15(7):629-31;
Doles et al. Protein eng. (2000) Aug 13 (8): 563-74; Adams CyP et al. Nucl.
Med. Diol (2000) May 27 (4); 339-46; Williams LE et al. Med phys 2004 may 27(3) 988-94; Fitzgerald K.
Protein Ettg 1997 oct 10(10): 1221-5 and the various rBfireoC05 eitBd thErEin) as are various methods for identifying internalizing antibodies and creating toxin, radionuclide and cytokine fusions / conjugates (see ao Y et al Bioconj. Chcm 1998 Nov-Dec; 9(6): 635-X14) for ft~lly exploiting various aspects of the invention herein defined (see for example Becerril H et al. l3iochem l3ivphys lees Gamm 1999 Feb 16; 233(2):386-93 sec also additional references below.
Triabodies and other known multivalent antibodies etc. (sec for example lliades P et al. FIrBS ~.Gtt. I 997 June l6; 409(3):43741) etc. could advantageously be employed to provide additional functionaIitica, as well a5 variation in avidity etc. for the purposes of variously exploiting the invention herein.
Methods of expressing and identifying new molecules like LYVE-1 are also well known in the art (see WO
98/06839) Technologies for rendering the multifunctional ligattds of the invention less immtmogenie (cg such as employed by Biovation) are preferably applied to the multifunctional ligands of the invention.
For recent progess in the treatment of lupus nephritis see Zinunertnan R. Annu Rev. Med. 2001; 52:63-78.
With respect to targeting Fas-L see US6068841:AntibodIes to Fas-L for treaunent oFhepatitis, The invention also contemplates using chemoltines and variously targeted antibodies and fragments thereof fused or conjugated to chemokines or other molecules with for example, lymphocyte or other immune cell attractant properties (see for example Sun J. et al. Lympho,logy 32 (1999) 166-170; and Gerard C. et al.
Nature Tm»unalopy (2041, Feb.) 2(2)- p 148; Immiurological Re~~iews 1999 Vol 170 p 5-197) to attract imrtlune CcllS into targCt ti$SUes for CVerltLtal penetration into the lymphatic vessels for activation, signalling, binding to, inhibition, etc.. For example, for cancer treatment antibodies that bind to augiogenesis markers fused to such type such molecules eg. TNF-a can be advantageously employed optionally in conjunction with various vaccination strategies (including the use of the muti-functional ligands of the present invention) to attract immune cells including, optionally, vaccination-activated tumor targeting lymphocytes to the tumor site. In an indirectly related aspect (having independent applications as Well as for Comblnatiotf therapy with a multifuctional ligan~, the invention is also directed to an antibody that targets an angiogenesis marker fustdlconjttgated to a cytakirie or antibody (ie a bispeci6c antibody) which binds to a oytolcinc, which cytokine augments adhesion of immune cells to blood vessels and method of using same (by administration to a subject), alone, in combhtation with multifunctional ligands ofthe invention or with other vaccination strategies to increase immune cell targeting to a solid tumor. In the case of a bispeciftc antibody it i5 COt7ltemplated that the cytpkine binding portion has a relatively low functional affinity to the cytokine so as to compete unfavourably for its binding to its natural receptor.
With reference to modulating bindiltg of leucocytes to endothelial adhesion molecules see for example U5 Patent No. 6,123,915 and the references thercm creed.

It is well lanown to those in the art to make bispecific antibodies which are adapted to bard two different ligands on the same cell, for example so called mrtigen-forks as dixlosed in USP 5,705,614 (see also Shi T
et al. Murine bispecisc anybody 1A10 directed to bwnan transferrin receptor and a 42-kDa ntmor-associated glycoprotein also Clin lntmunol Immunopathol 1996 Feb;78(2):188-95;
Amorpsp AR et e1., Binding charaettriscics and antitumor properties of 1A10 blspecific antibody recognlzlng gp4D and human transferrin receptor Cancer RCS 199fi Jan 1;56(1):113-20; Ring DB et al., AntigCn forlc~: bispecific reagents that inlu~bit cell growth by binding selected pairs of tumor antigens, Cancer Immnnol Tmmunother 1994 Ju1;39(i):41-8; Lu D et al., Cotnplete inhibition of vascular endothelial growth factor (VEG~ activities with a bifunetional diabody directed against both VEGF kinase receptors, Eras-like tyrosine kinase receptor and kinase Insert domain~ontaining receptor. Cancer Res 2001 Oct 1;61 (19):7002-8; Schmiedl A, Hrcitling F, Dube1 S. Expression of a bispecific dsPv-ds'Ev' antibody fragment in Escharichia cola. Protein Eng 2000 Oct;13(i0):725-34 see also Park 55, et al., Generntion and characterization of a novel tetravalent bL5pec1flC alltibOdy that biDdS to hepatitis H virus surface antigens Mol Immune! 2000 Dec;37(18):1123-30;
ICriangkum I et al., Bispeciflc end bifunctiooal single Chain recombinant antibodies $iomol Eng 2001 Sep; 18(2):3 I-40; USPs 4,474,893, 5,989,830; WO 00/29431).
With respect to antibodies to nutoantigcos, ADEPT, use of anti-~otHx9n antibodies, DeImmunitation, antibody-cyookine fusions, ribosome display, xenamouse technology; cutting edge phage display techniques, construction of hwnan antibody fragment based phage display libraries, saldctibn of internalizing antibodies by phage-display, cancer targeting, antibodies, antibody urays, plantibodios, desip Of mutant 1GSF domains of CD2, CD38 and TCR; oligopepyde cg. paratope mimetics, diabodies, m3nlbodies, trlabodies> tetrabodies and related siie/k'ntetics issues. caspase activatable pro-drugs, dellvety of Bismuth 213 vie scFv and diabodies,atlti-angioaeaeais marker strategies, immunocnzype therapy of cancer (cg. with ltnases) pancarcinonuc antigens llkt CEA (TAG")-'72; and related teclutologies see the papers and references in Proceedings of 1BC's 11e' Annual International Conference on Antibody Engineering, State of the Art, Science, Technology and Applications Dec 3-6 300D La Jolla, CA.
With respect to bioloEy of the lymphatic system having practical appHcatian herein see acami, F.
lynpholosy (1999) 32:90-102; Shield JW. Lyrnphology 1999 32: 118-123 and Lymphology 33 (2000) 144-147, as wall as the references cited therein_ The invention also contemplates control of such migration by inhibition ofreceptors that mCdiate Such migration (see for example Sun J. et a1_ T~ymphology 32 (1999) 166-170) for controlled application of the multifitnccional ligaads afthe invencion.
With respect to recern developments with respect to target llgands and/or hnmunotherapy having applicayon herein see also WO 01112224, WO 01/14550, WO 01/11059, WO 01//0205, WO 01/04679, W0029445 WO 01114885, WO114564, WO 01114558, WO 01/14224, WO 01113945, WO
01112840, WO
01112781, Wo 01/12674, WO 01/12470, WO 01112224, WO 01/12646; WO 01112223, WO
01112218, WO 01112217, WO 01112216, WO 01112154, WO 01114357, WO 01 /11059, WO 01110912, WO 01/11040, WO 011108$8, WO 01/10460, WO 01110205, WO 01/09611, WO 01/09328, WO 01/09186, WO 41/09192, WO 01/08635, WO 0110?481, WO 01107082, WO 01107084, WO 01/07081, WO O1/D7484, WO 0//07466, Triggering Fc alpha-receptor 1 (CD89) reerults neutrophils as effector cells for CD30-directed antibody therapy. J Immune!. 2000 Nov 15; 165(10):954-61. CD47 cttEag~nent inhibits cytoklne production cud maturation of human dendritic cells. J Immune!. 2000 Feb 15; 164(4):2193-9.
'fhe invention also cortt~nplates that a multiftmctional ligand that recognizes an immune cell as a target in virtue of a particular cell marker and will be able to deliver a toxic payload to the cell, for exempla, in virtue of its second portion comprising ouch toxic component fbsed or Con;lu8atad thereto- The invention Also contemplates attracting or supplying other immune cells or molecules to kill, or othCrwise inmate the target immune cell (cg. lymphocytes co. by TH cell modulation or CD4 cell modulation or using antibodies Including anti-idiotypic antibodies. The invention therefore contemplates that treatment of such immune cells caa~ be accomplished by a combination of different mechanisms or drugs depending on the disease sa as to reduce imrnunosuppressiott due to immune cell ablation where this is the dominant consideration. Such interactions may trqaire late;-action with one or more Iigands an the surface of the targeted immune cell, as Pacititated via anchoring Interactions of varying a~nity/avidity/duratiQn. The invention also contemplates using mulyflmctional ligands comprising or bound to salectins end lCAMs etc.
to facilitate such targeting, far example co-a~instering same in a proportion which is for example 0.01%

to 25% of the targeting muitifunetional ligand. The relative amounts of the selectinIICAM ate. (including antibody mimics) being multifunctional ligand as compared with the tsrgetin;
multifunctional iigand can be determined empirically by varying the proportions and assessing any objective indicator of svccas,~fu1 targeting in a disease related or purely experimental context. For example successful targeting (cg. antibody binding to eg. C03, CD28, CD2) usln; multifunctional ligands of the invention could be monitored by evaluating levels of cytokines nonnalIy atAtibutable to such binding (see for example CD8 T cell activation after intravenous administration of CD3 x CD I 9 bispccifiC 8ntlbOdy in patients with non-liodakin lymphoma. Cancer Immtmol lmmunother. 1995 Jun; 40(6}.390-6. Definition of a lamina propria T cell responsive state- >~nh~ced cytokiue responsiveness of T cells stimulated through the CD2 pathway. 7 Immunol. 1995 Jan 15; 154(2):664-75.
With respect to multifunctional ligauds that are used to directly or ind'rreecly exert an immunization fitnction, other examples of disease associated peptides that can be presented as immunogens or inhibitor/modulators of immune activity or disease progression in one of the fashions Suggested above include, examples as well as technologies referenced in, for example, Knuth A, Cancer Chemother Phatmacol (2000); 46 suppl: 546-31; Engelhard VH, Cancer J Sci Am 2000 May; 6 Suppl 3: 5272-80;
PietCrsz GA tt al, Cell Mol Life Sci. 2000 Feb; 57(2): 290-310; Alaarta I et al, Hum Immunol. 2000 Jan;
61(1}: 65-73; Tumour vaccines: a new itnmunvtherapeutic approach in oncology.Ann Hematol. 2000 Dec;
79( 12).651-9; Human tumor-rejection antigens and peptides from Bones to clinical research Nippon Geka (',akkai Zasshi. 2000 Sep; 101(9):612-7. Pinilla-lbarz J, et al CML vaccines as a paradigm of the specific irnmunotherapy of cancer. Blood teen- 2000 Jun; 14(2).111-zo).
In order to present an MHC-peptide complex in proximity to a B7 co-stimulatory molecule, the invention contempi~eS using, in addition to varying amounts (varying from a 30/30 proportion) of adjacent multifurlCLional Iig821ds (which may be a dAb, diabody ate.) preferably cross-linked by an avidin component, -- as a different strategy-- cross-linking with avidin or the like $djaeent arms afa single diabody, triabody or tetrabody ate. which binds to yr has been fused or conjugated individually to respective H7 and ME1C peptide components (with respect to recombinant B7 and MHC molecules and fusion proteins thereof including antibody tltsions end related technologies see references above and EP
99197477 WO 99/42597, W4 97 28191, US 6, 197, 302, US6015884 US6140113, US
6,045,796, US
5580756, EP0935607, WO 9806749 W09803552, EP 1054984, US 5$69270, Construction and characterization of bispecific costimulatory molecules containing a minimized CD86 (B7-2) domain $nd single-chain antibody fragments for tumor targeting; method is useful for cancer therapy Rohrbach 1= et al., CIin.Cancer Rcs.; (2000) 6, 11, 4314-22 WO 00/008057 17 Feb 2000; WO 9921372 6 May 1999; WO
99I3095 18 Mar 1949; WO 9742329 13 Nov 1997; WO 9720048 5 Jun 1997; W4 9640915 19 Dec 1996; WO 001023087; EF 610046 10 Aug 1994, USP 6056952 as well as references therein cited).
In a related aspect, the invention similarly contemplates usinE or more antibodies (optionally biotinylated and cross-linked by an avidin component) th8k bind to the same or different Cpitapes an 8 tumor including, where two such anfibodies are used different proportions of MHC and B7 linked (re fitsed, conjugated or capable ofbinding to) antibodies as well as different proportions ofdifferent epitope-specific antibodies to Qptimize the di$tributien ePsueh cross-linked B7 and MHC peptide complexes for 1'-cell recognition. In this way any strongly immunapenic peptide may be used in conjunction with Suitable vaccination strategies to create a universal cancer antigen. Using a tumor urtrel$tcd peptide is advantageous to avoid any tolerization effects resulting from T-cell binding to the MHC-peptide alone and does not preclude immune system recognition of a different epitope or other therapies. In a preferred embodiment, a single multifunctional Iigand or pair of multifunctional ligands optionally biotinyIated and cross-linked by an avidin (or variants), is used to bind to both the lumen of the lymphatic system and to a tumor cell. (using for example a trispeeifie antibody with monovalent linkage to both the cancer cell and lymphatic endothelial cell and a third antibody component having respective fusions to ono of MHC-peptide and B7 art heavy and light chain, or a trispecifie or tetraspeci~fic tctrabody having an antibody component devoted to each or the 137 and MHC Linkages) 'This permits a single molecule to be used for both the immunization within the lymphatic system and the tumor targeted antigen display. Howev~.r, It wilt be appreciated that presentation of MI-IC-peptide complex on a tumor does not necessarily require costimulatory B7 presentation to induce a cytotoxic T cell response which is specific for the peptide and that multiple such presentations, preferably in a Gross-linhable fashion may be preferable.
Accordingly, strategies herein for castinlulatory presCntatlon of MI-iC-peptide and H7 may be differently applied to a lymphatic endothelial cell surface for immuni7~ltian purposes gad alld a tumor cell surface (primarily for recognition proposes), for example by using avidin facilitated cross-linking of in the fottner but not the latter.(tnmor) context or using different sets of molecules in each case or using modularly reconstructing the tumor cell suface with a bispefic antibody that binds to a separately administered MHC and/or B7 component Subject to the latter proviso, in 'preferred cmbodnnents, the invention contemplates using as senate counterparts 1) separate trispecific Abs, tacit Including for example, one antibody component which binds to the each oFthe respective B7 and MHC dtoleculcs which are preferably together, separately administered. Such multiftmctional ligands are preferably biotinylated for cross-linking - both batwttn adjacent trisptcific Abs and adjacent T-cell stimulaiory/co-stimulatory arms;
or 2) separate bispecific pairs of Abs each respectively having 1 ) either a B7 and lymphatic vessel oi' B7 end tumor binding portion or 2) a MHC peptide cbnlplcx and a lymphatic vessel or MHC complex and tumor binding, portions which again are prcFereably cross-iirtlted by an avidin,sueptavidin or a variant (lc. using biotInylated antibodys) This latter embodiment permits smaller size anfbody molecules to be used for better tumor targeting.
Antibody components which recognise thn non-T cell izttetactive portion ofthe B7 or MHC molecule eau be readily generated by phase display, for ex;tmple in the cast of a known peptide specific antibody to an MIdC peptide complex (see Chames et al. Proo Nail Acad Sci USA 97, 7969 and Cbamcs et. al. "Affinity Maturation of TCR-Likt MAC-peptide specltIc antibody- peptide specificity is possible over a wide aB'inity range" Proceedings of IHC Couftrcnce on Antibody Engineeering Dec.
2000) cg. by first causing binding of the "peptide specific" antibody and thtn doing the phase display cg, using an array of multiple (cg. repeats ofthe same anf6ody) such peptide specific MHC antibodies, applying the; MHC peptide Complex to afF~t binding and then performing the phatae or ribommt di9play.
AltCmatively a TCR (ed cell bound) or analoguelmimotope could be used for the oriemation. Similarly antibodies could be generrted which in etrect do not compete with CD28 or a mimotope thereof to create suitable anti-H7 rypc antibodies. Anti-87 antibodies see known in the art. The invention also c~templates that the MHC-peptide binding fimction may be supplied using a linked superaatigen [US 6197299, WO
9601650 25 Jan 1996;
froe.Natl.Acad_Sci.U.S.A.; (1994) 91, 19, 8945-49) in both the tumor and lymphatic system binding sites.
Optionally, tht tumor antigen ar one or both of the antigens arc a pan-carcinomic antigen like TAG-72, CEA, Hl 1 (WO 9'1144461). ThB invention also contemplates using one or more phase display libraries to optimize the development of MHCIB7 costimulatory bispocific anCibodICS, by using Cell sized latex spheres coated with an antigen cg. CEA in various surface dispersions (or a cell) and using a array of preferably biotinylatad antibodies which recagnit;e the antigen and have a "oppositely located"portion fused, conjugated or capable of binding to one yr both of Ml-IC and B7, the library optionally also presenting also variations and combinations of lengths (truncations) of one or more constant regions or for example the CDR2 generated by phase display, depending on the choice of antibody, and with microarray technology, using a signalling means be detect T-cell rccogaition and evaluating cytotaxicity with for example a Cr51 release assay. {With respect to protein chip or mieroarray technology see WO 00/63701 references, for example in the Proceedings of IBC's conference on Pratsln Mierparray Technology March 19-21 Santiago California The inventiau also contemplates use of recently published antibodies iu the context of the invention (see WO O I I198G1, WO 0//19990, WO 01119860, WO 01//9987, WO 01/19990, WO
99/58678, WO 00/59943, WO 01!18014, WO 01/18016, WO 01/18204, WO 01118042, WO 0111$421, WO 01118014, WO 01!18046, WO 01/141 ø6, WO 01113731, WO 01/15728, WO 01/16183, WO 01/16170, Wb 01/15732.
The invention is also directed to a method of evaluating dosing, llgaud saturation, avidity effects of simultaneous ligand binding an prolonged anchoring and ass,xiated benefits (cg-to delay a cancer cell for targeted killing or facilitate transfer of the multifunctional ligand to the tgrgetcd Cell), Cooperative intEractions, cross-linking interactions (See flmmtusol 2001 Mar 1; 166(5)3256-3265; Nippon Rinsho.
1999 Dec; 57 Suppl:428-32; Harefiish, 2040 Jun 15; 13$(12)' 1046-50. Leuk Lymphoma. 1998 Mar; 29(1-2)-. I-LS ) and costlmulatory interactions by administering to a test subject two ditfierent muhitluictional ligands of the invcudon with cooperating second portions.
With respect to the displ8y of iliactional peptides oa an antibody type scaffold see Nuttal SD; et al."
Proteins (1999) 36: 217-227; ate also Sktrra A_, !. Me!. Recognition 3000 Jttly-Aug 13(4): l67-187. The invention also cantcmplates bispecifte multifunctional ligands in which the intmtmo function exerting mokty ex~ its fiuiction through binding to an iinmunogeuic component or carrier for such component as discussed above, for example an Fc domain fused to a peptide, a heat shock protein (see for example Wang XY, Iinmunol Invest 2000 May 29(2): 131-7 and referc'~tees cited therein as well as USb 1G$793;
US6071956 ; US0598170fi; US95948646 Methods for preparation of vaccines against cancer comprising heat shod: protein-peptide complexes; US05830464 Compositions and methods for the treatment and growth inhibition of cancer using heat sh~klstress protein-peptide compirxes in combination with adoptive immunotherapy as well as patents, scientific articles and patent applications referenced in these patents; with respect to MHC peptide Complexes( see for example W O 99/64597, W 0 98/03552, Wp 9$/06749 oral references cited therein).
As described above, the invencion also contemplates that the lower af>:'tniry ligatrd binding arm of the aforementioned multi$utctlonal ligand (Ie, having a high affinity targeting 9rr11 and a lower affinity effector arm) is constituted by a high e>~n$y iigand, for example an high affinity antibody or functional fragment thereof, which binds to a target blologice7 etfeecor (eg. a cytokine, ehmmokine, growth factor, hormone yr other biologcal response modiftar or divg) With high affinity, in a manner which permits the effector to continue to bind to its desired target receptor white bound tv the erse'body (ie. the antibody binds to a portion of the offector which is not critically involvtd In the ofPector binding to its receptor) provided that when bound t0 the efFectot the antibody or fragment thereof has, when combitsed with the effector, a suitably lower affinity for the receptor than the ligand binding arnt which fltnctions as the high affinity binder has for its target cell marker. In one embodiment the binding moiety which binds to the biological effector binds to it with higher amity than the affuliry that the effector has for the effector receptor. The invention also contemplates that this binding amt can bind to biological e+Fector in a manner which permits It to bind to one receptor but not a related receptor to wluch the efl'ecter would otherwise bind (see examples btlow). The invention also contemplates that antibody arrays ere used to screen for antibodies which era cepabte of binding to ouch biological effestors, while bind in situ to their receptors. The invention also contemplates that such binders, when bound to tht bioleglcttl effcctor, can be used to test their ability to bind to related receptors, such as those within the same family eg, within the same family of TNF like receptors. With respect t4 ani,body microarrays see for example Cahill DJ_Protcin and antibody strays and their medical applications.J Lmmunol Methods, 2001 Apr;2S0(1 2)-81-91. MacBeath G.
Proteomlcs comes to the surface.hlat Biotechnol. 2001 Sep;19(9):8~ 8-9.
Clewley Jf.ltecombin>trtt protein arrays.Comtnun I)is Public I~ealth. 2000 Dec;3(4):311-2; Hoh L.f, Enever C, de WiIdt RM, Ttmtlinson IM.
The use ofreeombinattt antibodies in preteomics.Curr Opln HiotecImol. 2000 Oct; t 1 (5):445-9. Walter G, a aL.Protein arrays for gene axpressIoa and molecular interaction sct~ting.Curr Opln Microbiol. ?000 Jun;3(3):298-302. de Wildt RM, Mundy CR, Gorlck BD, Tomlinson IM.Antibody arrays for hIgh-throughput screening of antibody-antigen interactions.Nat Hiotachnol. 2000 9ep;1$(9):989-94.Holt LJ, et al.By-passing selection: direct sct~eening for antibody-antigen interactions using protein arrays. Nucleic Acids iZCS. 2400 Au$ 1;28(l5):E72 and the references cited therein- Tho term receptor as used herein for Beater ce>t$inty inelude5 decoy rCxptars.1~xamplas of decoy receptors InchxdeTRAIL decoy receptors (APp-3L), CD44 decoy h7ce receptors (hyatoranan), inttrleukin receptor like protein (IL- I7) ( see J Biol Chew 2001 Nov 12), CD95-Fe decoy receptor, TRAMP, IL,-1 RII receptor, osteoprotegerin (OPG), ii., l3ItAlpha2.
""'Affutity Maturation Techniques for affinity maturation ruing high throughput sct~eerting techniques to evaluate mutants am well known in the art. Femtomolar amities have been achievtd and it is quite Common to obtain nanomolar to picomolar affinities as a result of an affinity maturation process. Fvr example it well knows to use techniques of parsimonious mutagenesis to engineer amino acid change at selected "hotspots". With respect to a~n(ty maturation see for exareple Cola G, Iludson PJ, Irving RAProtein afFinity maturation in vivo using E, colt mutator cells. J itnmunol Methods. 2041 May 1;251(12):187-93.
Manivel V, Sahoo NC, Salunke DM, Rae KV. Maturation of en antibody response Is governed by rnodulatians in flexibility of the antigen-combining site. Immtutity. 2000 Nov; 13(5):611-20. Boder ET, Midelfort ICS, Wittrup K15.
Directed evolution of antibody fragments with monovalent femtomolar antigen-binding aff'uury.Proc Natl Aced Sci U S A. 2000 Sap 24;97(20):10701-5. Holler PD, Hohnan PO, Shasta 8V, O'Herrin S, Wittrup KD, Kranz DM.In vitro evolution of a T cell rsceptor with high affinity for peptideIMHC. Proc Nail Acad Sci U S A. 2000 May 9;97(10):5387-92. Dmtgherty P5, Chen G, IverSOn DL, Georgiou G.Quanticative analysis of the affect of the mutation frequency on the affinity maritratlon of single chain Fv antibodies.
Proc Nail Acad 6ci U S A. 2000 Feb 29;97(5):2029-34. Van.-Arttwerp JJ, Wittrup KD. Fine affinity discrimination by ytast surface display and flow cytometry.Biotecltnol Prog.2000 Jan-feb;l6(1):31-7.
Adams GP, 5chicr R.Generating improved sLtgie-Chain Fv molecules for tumor targeting.1 Tmmunol Methods. 1999 Dec 10;23 t(1-2):349-60. Daugherty PS, Chcn G, OLsen MJ, Iverson BL, GeorgIou G.
Antibody affinity maturation using bacterial surface display. Protein Eng.
1998 Sop;11 (9): 825-32. Wong YW, Kussit PH, Parhami-Sin B, Margolies MN. Modulation of antibody affinity by an engineered amino acid substitution. J Iillmttnol. 1995 Apr 1;154(7)'3351-8. Belint 1RF, Larrick lW.AntiBody engineering by parsimonious tnu~genesis_ Gene. 993 Dec 27;137(1):109-18.
Schillbach 3F, Near Rl, Bruccalcri RE, Haber E. Jeili-cy PD, Novotny l,5heriff s, Margolies MN.
Modulation of antibody affinity by a non-contact residue. Protein Sci. 1993 FCb;2(2):206-l4.Chames P, Gary D.
Enbineering of an anti-steroid arrt~bndy-. amino acid substitutions cttangeanttbody fine 9pecificlry from cortisol to estradiol.Clin Churn Lab Med. 199$ Jun;35(6):333-9. Kussie PH, Parhami-Seren B, Wysocki LJ, Margolies MICA. single engineered amino acid Substi~on changes auribody fine specificity. J
ltnlnunol. 1994 Jan 1;153(1}:14b-52, as well as references cited thercin_ With respect to generation ofhigh affinity antibodies and affinity maturation of antibodies see also Hanes J.
Nat. Biotechnol. 2000 Dec; 18(12): 1287-92; tC&rences in Hudson PJ Exp. Opin.
invest. Drugs (2000) 9(6) 1231-1242; Toran 1L et al Evr. J. I'mmunol. 20D1 Jan; 31(1) 12$-137.
NielSOn ~ et al. Cancer Reg 2000 Nov IS; 60 (22) 6434-40 Adams Gp, Journal of lmmunologiCal MCLhods (1999) 249-2fy0;
Chowdhury PS et al (June 1999} Nature Biotechnology Vol 17 p. 568With respCCt to strategies and recent technologies which have appiieatlon to the invention see references in Hudson PJ Exp. Opin. Tnvest Drugs (2000) 9(6) 1231-1242 and in particuIarref~ences relating to strategies to achieve multivalency and multispecificity; recruitment of viruses, ADEPT, photoactivation of cytotoxic radionuelides; surface receptor cross-linking; (see also Ear. J. lmmunol 2000 30(10) 3006), use of anti-J3 antibodies;
itamunocytokines (see also Lode HN )mmul. Res. 2000, 21 (2-3) 279-88; Gillies SD Cancer Research 59 2159-2166 May 1999; 1~ode HN et al Drugs of Today 2000 36(5) 3221-33G).
With respect to practicial size limitations sad pharmacokinetIcs of various typos of antibodies and fragments see Coleber D. et al. G.1. Nucl. Mad ( 1999) 43: 132-139; Wu AM Ct al ti.J. Nucl. Mad 2000 Sep; ~14(3): 268:83; Williams LE et al Mcd Phys 200D May 27(5): 988-941 Ikonu F lymphology 32 (1999) 90.102.
With respect to the construction of diabodies sec also Takemura S1 Ct al.
Protein Eng. 2000 Aug;13($) 583-8; Biomol. Eng. 2001 Sept;18(3):31 ~0.
With respect to anti-cancer antibodies see also 6,1$0,357.
With respect to technologies to produce multivalent and/or tnultispecific antibodies see also USP G, 172, 197; WO 92/41047; WO 93/11161; WQ 94107921; Wp 94113804; Helfrich W. et aI_ Journal of hnmunological Methods 237 (2000) 131-145. ProcCeding9 of I 1'~ 1HC Conforence on Antibody Engineering; WO 01185795 Monoclonal antibodies may be routinely produced as taught by Harlow, E. and D.
Lane, ( 1988) ANT1SODLES: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.ll.
Humanized antibodies may be routinCly produGCd a8 taught, for Cxemplt, by U.$.
Pat. No. 5,585,0$9 and U.S. Pat. No. 5,530,101. Techniques for engineerhtg antibodies are well known and described in Winter and Millstein (1991) Nature 349:293, and Lan-ich and Fry (1991) Hum. Antibod, and Hybridomas 2:17.
One having ordinary skDl in the art may use well known techniques and starting materials andlor commCrcially available expression vectors and systems that are readily available and known in the art. See e.9., Sambrook et al., Molecular Claming s Loboraaory Manual, Second Ed. Gold Spring Harbor Press (1989).
Examples of radionuclides usefrtl as toxins 1n radiation therapy are wolf known. Some examples are referred to below. Auger emitters may be preferred for internalizing antibodies. As suggtsted $bovc, the term antibody is used interchangeably with antibody fragment and antigen binding fragment and includes a whole antibody; antibody >iagnent a portion of an antibody Such as a scFV
F(ab~) 2 Flab) 2. Fab~, Feb, dAb, microbodies (W000 29004) or the like or multivalent such fragments, including those itemized or referenced herein. l~egardle~ss of structure, an antibody fra~nent can be made to bind with the same antigen that is recognized by the intact antibody. More partictdarly, in addition to fragments formed by enzymaic digestion of an intact Ab the term antibody or "antibody fragment" unless otherwise stated also includes any synthetic Or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex includingles applicable, cysteine noose peptides and minimal recognition units consisting of the amino acid residues that mimic tilt hypCi'vat'iable re~lon. Although fully human antibodies, for example, antrbodies generated via human-human hybridoma5 or through phase display using human antibody based libraries, are preferred, the invention does not preclude other strategies to avoid a HAMA, type responso.

A cl7i~o antibody is a recombinant protein that Contains the variable domains and complemcotary determining regions dived from, for example, a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody.
With respect to stability engineering of seFW fragments for enhanced mulfunetional ligands comprising scFvs see J Mnf Biol 2001 Feb ?; 305(5):9$9-1010.
humanized antibodies are recombinant proteins in which marine LDR's of a monoclonal antibody have been transferred from heavy and light variable chains of the marine immunoglobullri lrlto a human variable domain.
The term therapeutic age>rt as used her~zt, ss a moteeule or atom which is conjugated ate. to an antibody moiety to pruduee combination intluding a cort~ugate which is useful for therapy. Examples of therapeutic agents include drugs, toxins, immunomodulators, ehclators, baron compounds, photoactive agents or dyes, and radioisotopes.
The term "a naked antibody" tray be used to refer specifically to an entire antibody, as opposed to an antibody fragment, which is not conjugated with a therapeutic agent. Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombinant antibodies, such as chimeric and humarti2,ed antibodies.
The tet'm imrounoconjugate may be used to refer a coqjugate of an antibody component with a therapeutic agent.
As used herein, the term antibody fusion protein refers to a recombinant molecule that comprises an antibody component and a second functional component for example a therapeutic agent. Examples of therapeutic agents suitable for such fusion proteins include immunomodulators {"antibody-imtnunomadulator fusion protein") and toxins ("antibody-totcin fusion protein").
Production of Antigen - 5peciftc Monoclonal Antibodies, Rodent monoclonal antibodies to antigen can be obtained by methods known to those skilled in the art. See generally, for example, Kohler and Milstein, Nature 256.495 (1975), and Coligan et al. (ads.), Current Protocols in Immunology, Vol. l, pages 2.5.1-2.6.7 (John wiley 8c Sons 1991) ["Coligan"]. $riefly, monoclonal antibodies can he obtained >ay injecting mice with a composition comprising the antigen in a question (Ag), verifying fhG presencC of anCibcdy production by removing a serum sample, removing the spleen to obtain 8-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hylxidomas, cloning the hybridomas, selecting positive clones which produce anti-Ag antibodies, cultt>riug the clones that produce antibodies to the antigen, and isolating the antibodies Pram the hyhridoma cultures. Transgenic mice having for example engineered immune systems to create human antibodies such those used by Medarex and Abgenix are also contemplated for use herein to Create suitably targeted antibodies.
Monoclonal antibodies can be isolated tutd purred from hybridoma Cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A
5epharose, size-exclusion chromatography, and ion-exchange chromatography.
See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see l3aines et al., "lyurificatlon of ImmunoglobuIin G
(1gG) ," in Methods in Molecular Hiology, Yol. 10, pages 79-104 (The Humane Press, lna. 1992).
With respect to relevant molecular biology techniques See also, for example, At>SUbel et al., (ads.), CfJRItENT PR4TOCO>r5 TN MOLECLiLAR BIOLOGY, pages 8.2.8 to 8.2.13 (1990) ["Ausubel"]. Also, see Wosnick et al., Gene 60:115 (19$7); and Ausubel et al. {ads.), Short Protocols in Molecular Piology, 3rd Edition, pages 8--8 to 8-9 (John Wiley & Sons. Tnc_ 1995)_ Established techniques wing the polymerase chain reaction provide the ability to synthesize genes as large as 1.8 kilobases in length. Adang et al., Plant Molec. $iol. 21:1131 (1993) Bembot et al., PCR Methods and Applications 2:266 (1993);
Dillon et al., "Use of the Palymerase Chain Reaction for the Rapid Construction of Synthetic Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ad.), pages 263-26$, (Humane Press, Inc. 1993).
Techniques far codStntcting chimetic antibodies are well-known to those of stall in the art. As an example Leung et al., ELybridoma 13:469 (1994).
1o yet another embodiment, an antibody of the prcsopt Invention is a "h~ni2ed"
monoclonal antibody.
That is, mouse complementarity determining regions are transferred from heavy and light variable chains of the muuse inununoglobulin into a human variable domain, followed by the replacement of some human residues in the framework regions oftheir marine counterparts. Humanized monoclonal antabodies in accordance with this invention are sulLable for use in therapeutic methods.
General techniques for cloning mutZne immunoglobulin vaiiable don~ins art described, for example, by the publication of Oriandi et al., Proe_ Nat'1 Acad. Sci. USA 86: 3833 (1989). Techniques for producing humanized monoclonal antibodies are desCCibed, for ~~ple, by Jones et a1_, Nature 321:522 (1986), Riechmann et al., Nature 332:323 (1988), Verhoeyen et al-, Science 239:1534 (1988), Carter et al., Proe. Nat't Acad. Sci. USA 89:4285 (1992), 5andhu, Crit. Rev. Hivtech_ 12:437 (1992), and Sinner et al., J.
Immun. 150:2844 (1993). The publication of Leung et al., Mol. Itnmunol. 32:1413 (1995), describes the construction of humanized LL2 antibody.
In a preferred embodiment of the invention the multiftmctionaI ligand has a unique portion which differentiates it from outer antibodies and preferably other co-administered different multifunctional ligands, which unique portion, allows ~e multifimctional ligand to be efficiently segregated on an immunoaffmity column. In the case of differentiating a siltqle multifitnctional ligand en anti-idiorype (assuming the 1-trst portion consists of an antibody) or other antibody uniquely recognizing the first portion could be employed. Modifyinn a portion ofthe first portion, for example in the case where it is antibody component and creating a antibody thereto, for example by phage display, is a matter ofroutine skill in the arts of antibody engineering and phase dispiszy.
In another embodiment, an antibody of the present invention is a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific hmnat, antibodies in response t0 antigenic Challenge, In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenie mice een synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybtzdomas. Methods for obtaining human aetibodies from transgenic mice are described by Green et al., Nature Genet. 7:13 {1994), Lonberg et al., Nature 358:856 (1994}, and Taylor et al., Int. Immun. 6:579 (1994).
Examples of Production of Antibody Fragments Antibody fragments can be prepared, for example, by proteolytic hydrolysis of an antibody or by expression in E. cell of the DNA coding for the fragment.
Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. Eor example, antibody fraatnents can be produced by enzymatic cleavage aCantibodies with pepsin to provide a 5 S fragment denoted F(af)i. This fragment can be further cleaved using a thiol reducing anent, and optionally a blocking group for the sulEaydryl groups resulting from cleavage of disulfide linkages, to produce 3.5 S Faf manovalent fragments. Alternarively, as enrymatic cleavage using pepsin produces two monovalent Fab fragments and an Fe fragment directly.
These methods are described, for example, by Goldenberg, U.S-'Pat. Non. 4,036,945 and 4,331,647 end references contained therein.
Also, see Nisonoff et al., Arch Biochem. $iophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959).
Edelinan et al., in Methods in Enrymology Vol 1, page 422 (Academic Pmss 1967), and CoIigan at pages 2.8.1-?.8.14 and 2.10.-2.10.4-Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enrymativ, chtmitsl 4r gertet;c techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
For example, Fv fragments comprise an association of Vu and VLChains. This association can be noncovalent, as described in mbar et al., free. NafL Acad. Sci. U5A 69:2659 (1972). Alternatively, the variable chein5 Can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, for example, ~andhu, supra.

Preferably, dte k'v fragments comprise VH and VL chains which ara connected by a peptide linker. These single-chain antlgeo binding proteins (seFv) are prepared by constructing a structural gene Comprising DNA sequences encoding the VH and VL domains which are connected by on ollgonucleotide. The struen~ral gene is inserted into en expression Vector which is subsequently introduced into a host cell, such as B. coli_ The recombinant host cells synthesize a singly polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sCFvs ere descl'ibed, for example, by Whitlow et al., Methods.
A Companion to Methods in Enzymology 2:97 (1991). Also see Bird et al., Science 242:423 (1988), I,adner et al., U.S. Pat. No. 4,946,778, Pach et al., $io/Technology 11:1271 (1993), and Sandhu, supra.
Another form of as antibody &~agmeut is a peptide coding for a single complementarity-det~xminirtg region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest_ Such genes are prepa~od, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-produeing cells. See, for example, Larrick et al., Methods: A Companion to Methods in Enzyrnology 2:106 (1991); Cvurtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antbvdics: Production, Eughleering and Clinical Application, Bitter et al. (ads.), pages 166-179 (Cambridge University Press 1995); and Ward tt al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies:
Principles and Applieatioos, Birch et al., (ads.), pages 137-1$5 (Whey-Liss, Inc- 1995)_ **~repararion of lmmunoconjugates The present invention contemplates immwtoeonJugates to assess and e~'ect treatment of various disease conditions. Such lmmunoconjugatcs crut be prepared by indirectly conjugating a thorapeutie agortt to an antibody component. For example, general techniques are described in Shih et al., int. J. Cancer 41:832-839 (198$); Ship et al., int. J- C~~Er 46.1101-1106 (1990); and Shih et al., U.S. Pat. No. 5,057,313. The general method involves rf;actirtg an antibody component havin~ an o7;idiz~d carbohydrate pertion with a carrier polymer that has at least one free amine function and that is loaded with a plurality of drug, toxic, ehelator, boron addends, or other therapeutic agent. Thin reaction results iu an initial Scliiff base (imine) lin]tage, which can be stabilized by reduction to a secondary amine to form the final conj ugate.
The cattier polymer is preferably an aminodextran or polypeptide of at least SO amino acid residues, although other substantially equivalent polymer carriers can also be used.
Preferably, the final immunoconjugate is soluble in an aqueous solution, such as mammalian serum, For ease of administration and effective targeting far use in therapy. Thus, sotubilizing functions on tJte carrier polymer will enhance the serum solubility of the final immunoconjugate. In particular, an amhiodextran will be prefetxed.
The process for preparing an immunoconjugate with an aminodextran carrier typically begins with a dextran polymer, advantageously a dextran of avet'age molecular weight of about I0,o0o.100,000_ The dexiran is reacted with an oxidizing agent to effcGt a contrOllcd oxidation of a portion of its carbohydrate rings to Denerate aldehyde groups. The oxidation is conveniently effected with ~,Iycolytic chemical reagents such as NaTO₄, according to conventional procedures.
The oxidized dextran is then reacted with a polynmine, preferably a diarnine, and more prefer~rly, a mone-or polyhydroxy diatnine. Suitable amines include ethylene diamine, propyltde dlatnine, or other like polymerhylene diamines, diethylene triamine or like polyamines, 1,3-diamino-2-hydroxypropane, yr other like hydroxylated diamincs or polyamines, and the Like. An excess ofthe amine relative to the aldehyde groups of the dextran is used to insure substantially complete conversion of the aldehyde functions to Schiff base groups.
A reducing agent, such as Nal3H,, NaBH~ CN or the like, is used to effect reductive stabilization oC the rtsuhant ~chiff base intertnediatc. The resultant adduct can be purified by passage through a conventional sizing column to remove cross-linked dextrans.
Other conventional methods of derivatizing a dextran to introduce amine furtetions can also be used, e.g,.
reaction with cyanogen bromide, followed by reaction with a diamine_ The aminodextran is then reacted with a derivative of the particular drug, toxin, chelator, immunomodulator, boron addend, or other therapeutic agent to be loaded, in an activated form, preferably, a carboxyl-a~vated derivative, prepared by conventional means, e.g., using dicyclvhexylcarbodiintide (DCC) or a water soluble variant thereof, to form an intermediate adduct.
Alternatively, polypeptide toxins such as pokeweed antiviral protein or riein A-chain, and the like, can be coupled to aminodtxtratl by glutaraldehyde condensation or by reaction of activated carboxyl groups on the protein with amines on the aminodtxtrtm.
Chelators for radiomutals or magnetic resonance enhancers are well-lmown is tfte art. Typical are derivatives of ethylenediamittetetraacetic acid (EDTA) and diethylenetriamineptntaaattie acrd {DTfA).
These ehelators typically have groups on the side chain by which the chelator can be attached to a carrier.
Such groups include, e.g., benzylisothiocyanate, by which the DTPA or EDTA can be coupled to the amine group of a carrier. Alternatively, carboxyl groups or amine groups on a chelator can be coupled to a cm ier by activation or prior dedvatization stud then coupling, all by well-lmown means.
Botron addends, such as carboranes, can be attached to antibody components by conventional methods. For example, carboranes can be prepared with carboxyl functions on pendant side chains, as is will h~nown in the ate Attachment of such carboranes to a cancer, e.g., aminodexttan, can be achieved by activation of the carbonyl groups of the carboranes and condensation with amines on the carrier to produce an intermediate conjumate. Such intermediate conjugates are then attached to antibody components to produce therapeutically useful inuuunoconjugates, as described below.
A poIypeptide carrier can be used instead pf aminodextran, but the polypeptide carrier must have at least 50 amino acid residues in the chain, preferably 100-5000 amino acid residues. At least soma of the amino acids should be lysine residues or glutamate or aspartate residues. The pendant amines ef lysine residues and pendant carboxylares of glutamine and aspartate are convenient for attaching a drug, toxin, immunamodulator, chelator, boron addend or other therapeutic agent. Exfunples of suitable polypeptide carriers include polylysine, palyglutamic acid, polyaspartic acid, copolymers thereo>y and mixed polymers of these amino acids and others, e.m., serines, to confer desirable solubility properties on the resultant loaded carrier and immunoconjugate_ Conjugation of the intermediate conjugate with the antibody component Is affected by oxidizing the carbohydrate portion of the antibody component and reacting the resulting aldehyde (and ketone) carbonyls with amine groups remaining on the carrier after loading with a drug, toxin, chelator, inununomodulator, boron addend, 0T other therapeutic agent. Altemativcly, an intermediate conjugate can be attached tp an oxidised antibody componr:nt via amine groups that have been introduced in the IntermEdiate: conjugate after loading with the tlterepeutic agent. Oxidation is conveniently effected either chemically, e.g., wirh NaIO, or other glycolytic reagent, or enzymatically, e.g., with neuraminidase and galactose oxidase. In the case of an aminodextran carrier, not all of the amines of the atninodextran are typically used for loading a therapeutic agent. The remaining amines of aminodexitan condense with the oxidized antibody component to form Schiff base adducts, which are then reductively stabilized, normally with $ bvrohydride reducing agent.
Analogous procedures are used to produce other itrununoconjugatcs according to the invention. Loaded poIypeptide carriers preferably have free lysine residues remaining for condensation with the oxidized carbohydrate portion of an antibody component. Carboxyls on the polypeptide carrier can, if necessary, be converted to amines by, e.g., activation with DCC and reaction with an excess of a diamine.
The final immunoconjumate is purified using conventional techniques, such >ts sizing chromatography on Sephncryl S-300.
Alternatively, immunoconjug$tes cart be prepared by directly conjugating an antibody component with a therapeutic agent_ The general procedure is analogous to the indirect method of conjugation except that a therapeutic agent is directly attached to an oxidized antibody component.
Fpr application to linking MHC 1111 peptide/H7 molecules to a latex which has previously conjugated to biotin, for avidin assisted linking to a multi~fiutctional ligand, it will be appreciated that biotin can be conjugated to a paYC of a latex sphere which is then linked to MFIC peptide and B7 molecules by placing the spheres in a confluent layer or ire the spheres in a microwells such that only part of the sphere is exposed for conjugation and then coating the spheres onto avidin coated plates for the )~7 and MHC linka4e.

It will be appreciated that other therapetitie agents can be substituted for the chelatars describtd herein.
Thane of skill in the art will be able to devise conjugation schemes without undue experimentation.
As a further illustration, a therapeutic ageat Can be attached at the hinge region of a reduced antibody component via disulfide bond formation. For example, the tetanus toxoid peptides can be constructed with a single cysteine residue that is used to attach the peptide tb $n antibody component. As an alternative, such peptides can be attached to the antibody component using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pytzdyldithio)proprionatt (SPDP). Yu et al., Int. J. Cancer 56:244 (1994). General techniques for such conjugation are well-known in the apt. See, for example, Wong, CHEMISTRY OF PROTEIN
CONJUGATION ANp CROSS-LINKING (CRC Press 1991); Upeslacis et al., "Modification of Antibodies by Ghemlcal Methods," in MONOCLONAL ANT1B417IFS: PRJhTCIPLE5 AND
APPLICATIONS, Birch et al. (ode.), pages 187~23D (Whey-Lies, Inc. 1995);
Priet, "Production and Characterization of Synthetic Peptido-l7erived Antibodies," iii MONOCLONAL
ANT180D1E5:
PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, hitter et al. (eds.), pages (Cambridge University Press 1995).
As described above, carbohydrate moieties in the Fc region of an antibody can be used to conjugate a therapeutic agent. However, the Fc region is absent if an antt~bvdy fragment is used as the antibody component of the immunoconjugate. Nevertheless, it is possible to introduce a carboliydrate moiety into the light chain variable region of an antibody or antibody fragment. See, for example, Leung et al., J, lmmunol.
154;5919 (1995); Haasen et al., U.S- Pat_ Nq_ 5,443,953 (1995). The engineered carbohydrate moiety is then used to attach a therapeutic agent.
In addition, these of skill in the art will recognize numerous possible variations of the conjugation methods.
for example, the carbohydrate moiety can be used to attach polyethylene; lycal in order to extend the half life of an intact antibody, or antigen-binding fragment thereof,. in blood, lymph, or other extracoliular fluids. Moreover, it is possible to construct a "divah:nt immunoconjugate" by attaching therapeutic agents to a carbohydrate moiety and to a free sulfhydryl group- Such a free sulfliydryl group may be located in the hinge region of the antibody component.
Methods for determining the binding specificity of an antibody are well-known to those of skill in the art.
General methods are provided, for example, by Mole, "Epitope Mopping," in MOLECULAR HIOLOGY, VOLUME 10: IMMTJNOCHEMICAL pItOTOCOLS, Manson (ed.), pages I OS-1 16 (The Humans Press, Ine. 1992). More specifically, competitive blocking assays for example to determine CD23 cpitope specificity nre described by 5tcin is al., Cancer Inununol_ Immunother. 37:293 (1993), and by Tedder et al., U.S. Pat. No. 3,484,892 (I996).
Tn another aspect the invention is directed to a bispecific ligand, preferably a bispecifc antibody, comprising at least a first ligand, preferably en antibody component, which binds specifically to a first cell surface associated ligand end at least a second llgand, preferably a second antibody component which binds specifically to a second cell surface associated ligand on the same cell, and wherein the functional affinity ofat least one and preferably bath ofsaid antibody components is selected so as to substantially limit functioned binding unless both of said first raid second antibody components arc substantially contemporaneously bound to said cell- It is known to provide bifunctional ligands wherein fimctional binding, for example, to accomplish signal transduction, is predicated on both ligands being bound Qr cross-linking.1-lowtver this effect is not contemplated to be predicated on differentially controlling the functional affinity of the respective ligands. According to a broad aspect of this invention (in which inclusion of a ligand which binds to a lymphatic vessel associated marker is optional), the invention excludes known such bispecific tigands which inherently have a suitable differential functional affinity.
5~ch bispecific llgaed are mentioned herein. Hy controlling the affinity of at least one of said llgands, for example where the functional affinity of one said ligands is substantially less than that of the other ligand the inventioe contemplates that a substantially greater percentage of the administered dose of the bispecific Ligand will affect cells in which only both Iigands are present, and/or that a reduced percentage of the dose administered will functionally bind to the cells in virtue only Qf the reduced functional affinity ligand. The invention also contemplates that functional affinity of one ligand is groatly increased to establish the functional affinity differential and that the functional affinity of both ligands is reduced relative to that of a standftrd, for example relative to that of a comparable ligands in hand or known in the art or identified by phage display, ribsome display or other comparable techniques using a single such ligand. The invention also contemplates that a micraarray (or library) afbispecific ligands in which for example, the bispecific tigand is "tethered" (la. immobilized) directly or indirectly in virtue of one or mare amino acid residues which ar'e positioned within the molecule to preferably minimally interfere with any binding, and in which the signal (cg its intensity) associated with a single Ligand binding interaction can be differentiated from a two Or marC ligand iatCractioTlS, FOr sample cell surfape [finding (alternatively the ligands Qr cell may be immobilized) and that ribsome and phage display could be adapted to bispeciflc single domain antibodies constituiiog a single chain (see references herein) by elongating the end of the chain from which the molecule is tethered. The invention contemplates that the affinity of one such )ignnd may be fixed and that the variability in members of the library lies in the permutations of certain key residues to which binding is attributable which can readily be identified by persons skilled in the art.
The invention also contemplates esscssiitg single ligand binding capability of successfbl bl-ligaed binders for example by blocking the other (non-assessed at that rime) ligand (eg_ with correlative ligand or n mirnotope thereof) and for example deterntinlng limited or non-existent such binding to as well as using inclined ligand testinS swfaces for washing over the wrrclative ligend, for example of defined surface area, including preferably defined lengths and widths and concentrations l distributions I amounts of the bound ligand, where the decree of incline is selected to roughly simulate the micro-environment of the comparable in vivo target, be ii a stationary cell with a roughly defined average shear force of bathing fluids cg, within a tumor or in the lymphatic system, or a mobile toll within a vein, artery, or lymphatic vessel, including those of different sixes. Tire invention is also dSr-ected to a mthod of generating a target ligand or improving the target specificity of any ligend by using a population of variants of that ligand within a micro-envnvnment simulated micrearray system in which the at least one of the follwlng factors Is simulated: concentration or amount or distribution of cairelative Ligand, shear ford and shape using length and width peramaters to simulates intraluminal diameter and Ieugth.The invention also contemplates in the case of a raultifunctional ligand or in the case of a bispecific or multispeofic ligand (as herein described) that the effrnity of its component binding llgands may be selected for venous or arterial tageting or to accommodate lymphatic system targeting or targeting within or through tissues or combinations of trio aforementioacd cg, median, average or or weighted compromi3cS to improve desired targeting. In a preferred embodiment the fi1'st ligand is selected on the basis of its ability to at least partially discriminam between a target population of cells (co. a ligattd that is ''associated" with a target population of cells) and a non-target population of cells (in one embodiment it is selected so as to have no otlier effect other than binding for targeting purports) end trio sCCOnd ligead is seltcted for its ability to modulate the activity of the targeted cell, optionally in virtue of binding alone cg. without delivering a payload (the term modulate referring broadly to a~ desired effect on the cell or its functionality) 1n this case the functional afllnlry for the llgand which is targeted for modulating the activity of the cell is selected sv as to reduce the likefhood of binding unless binding has first or caatemporeneausly occurred to the first ligand targeted for selectivity (cg, the second ligand would have monovalent as opposed to divalent binding to the ligand required for selectivity andlor from 0.20 to 10~ 'fold reduction in affinity (for example as measured by $Iacore) relative to the binding affinity for the first ligand. This reduction in affinity is preferably greater than a 104%
reduction in a~lnlry (multiply by 0.1), preferably greater than a 2409° reduction in affinity, preferably greater than a 300% reduction in affinity, preferably greater than a A00°r6 reduction in afhniry, preferably greater than a 500% reduction in affinity, preferably heater than a 600% reduction in aflIniry, preferably greater than a 700% reduction in affinity, preltrably greater than a 800% reduction in affinity, preferably greater than a 900% reduction in affutiry, preferably greater than a 1000% reduction in affinity, preferafly greater than a 2000% reduction in affinity, preferably greater than a 3000% reduction in affinity, preferably greater than a 4000% reduction in affinity, prefbrably greater than a 5000% reduction in a>fittiry, preferably greater than a 6000°r6 reduction in affinity, preferably greater than a 7000% reduction in affinity, preferably greater than a 8040% reduction in ai~nity, preferably greater than a 9004% reduction in af37Nry, preferably greater than a 10000°~ reduction in affinity, preferably greater than a 20000% reduction in amity, preferably greater than a 30000%
reduction in affinity, preferably greater than a 40000% reduction lo af5nity, preferably Beater than a 50000% reduction in affinity, preferably greater than a 60000% reduction in affinity, preferably greater than a 70000% reduction in af>fuuty, preferably greater than a 80000%
reduction 3n affinity, preferably greater thaw a 90000% reduction in ai~nity, preferably greater than a 100000%
reduckion in affinity, preferably greater than a 500000% reduction in affinity, preferably greater than a 100D0009~° reduction in affinity, preferably heater than a 10000000°/0 reduction in affutity, preferably greater than a 20000000%
reduction in affipity, preferably a greater than 3000000% reduction in efGniry, prelc.~rably a greater then 40,044,000% YCductiOn II1 af'.f7nity, prefera>aly a greater titan 50000000°i6 reduction in affinity, preferably a greater than 600000009. reduction in afftrtity, preferably a gxcater than 70000000% reduction in affinity, preferably a greater than 80000000% reduction in affinity, preferably a gre$tvr than 9000000D% reduction in affinity preferably a greater than IQ0,000,000% reduction in afFrrtiry, preferably a reduction in affinity of between one and two orders of tttagnihrde, preferably a reduction in affinity of between two and three orders of magnitude, preferably a reduction in affinity of between three and four orders of magnitude, preferably a reduction in affinity of between four and Hve orders of magnitude, preferably a reduction in affinity Of between flee slid six orders Of magnitude, preferably a reduction in off-miry of between six and seven orders of magnitude preferably a rtductlon in affinity of between seven and etgttt orders of magnitude, preferably a reduction in affnity of between eight and nine ord~.-rs of magnitude, prtftrably a t~eduction in affinity of between nine and ten orders of magnitude.
It wiD be appreoia~d that a suitable reduction in affinity, If any, will depend on the valency of the rt5pti;tivG flt5t and seCOnd Ilgands and the Selected affinity ofthe first ligand, which for example may have been augmented. Z'he invwtion also contemplates a tri5pecific (and triavalent) ligand In which two ligands differently define its specificity to reduce the likelihood of au undesired effect attributable to the function exerting moiety binding alone. In terms of the physical constitution of a ligand having a trispecific binding capability, the invention also contemplates linking three monovalent dabs, MRUs or the like or mixed combinations thereof or two bivalent dabs, MRUs or the Like or mixed combinations thereof (see WO
99/42077, (JS 6174691. WO4029004, Camel single-dpmain antibodies as modular building units in J Biol Chem. 2000 Oct 25, & Mulligstt-Kehoe U.S. patents including t1S 5702892, US
5824520; se also US
6040136 ) (in the latter case optionally one or both may be bispecific and linked by well known methods in the art (see WO 99142077, Celltech's TFM, leucine zippers, U5 5,910,573, US5892D20, F.P 06540858, see also EP 0318554B). The term functional binding is used to refer to binding which yields the desired effect, for example a therapeutic effect on a target cell population attributable to the binding to one or both ligands. Using the previous example, one ligand, eg. the first ligand, may be used to target activated invnune cells, and the second ligand may be different and may upon being bound to, for example result in inactivation, aueray, apoptosis or reduced capacity for endothelial adhesion of the immune cell. In this case, the invention contemplates that the fimetlonal affinity of the antibody component which hinds to the second ligand is selected such that binding is unlikely to occur without binding to the specificity dictating ligand, for Example the ratio of targeted relative non-targeted cells affected by the dose administered is approximately 1.10 to 1, preferably approximately 1.15 to 1, more preferably approximately 1.20 to 1, more preferably approximately 1.2$ to i, more preferably approximately 1.30 to 1, more preferably approximately 1.35 to 1, more preferably approximately 1,40 to l, more preferably apptmcimately 1.45 to 1, morn preferably approximately 1.50 to 1, more preferably approximately 1.55 to 1, more preferably approximately 1.6D to 1, more preferably approximately 1.60 to 1, more preferably approximately 1,65 to 1, more preferably approximately 1.70 to 1, morn preferably approximately t.75 to 1, more preferably approximately 1.80 to 1, more preferably approximately 1.85 to 1, more preferably approximately 1.90 to l, more prelcrably approximately 1.95 to 1, more preferably approximately 2 to 1, more preferably greater than 2 to 1, more preferably approximately greater than 3 to 1, more preferably approximately greater than 4 to 1, more preferably greater than 5 to 1, more preferably greater than 6 to 1, more preferably greater than 7 to 1, more preferably grcattr than 8 to 1 , more prcfi>rably greater than 9 to 1 , more preferably greater than 10 to 1 , more preferably greater than 20 to 1 , more preferably greater than 3D to 1 , more preferably greater than 40 to 1 , more preferably greater than 50 to 1 , more preferably greater than 60 to 1, more preferably greater than 70 to I, more preferably greater than BO to 1, more preferably greater than 90 to 1, mere preferably greater than 100 tv 1, more preferably greater than 500 to 1 , more preferably greater than 1000 to l, morn preferably greater than 10,D00 to 1, more preferably greater than 100,D00 fo 1, more preferably greater than 500,000 to 1 more preferably greater than 1,000,000 to 1.
It will be appreciated by pEr5ott5 Skilled in the art that the foregoing aspects of the invention apply to a variety of different combinations of immune function or other therapeutic function exerting Iigands and specificity dictatin8 li;auds including those involved in immune si~aIing, stimulatory, co-stimulatory, inhibitory, adhesion or other interactions, including without limitation, cytokine receptors, ligands associated with immune cell adhesion, ligands to which binding results in stimulation, activation, apoptosis, energy or costimulation, or ligands which differentiate between different populations or subpopulations or immune cells (see eg. US 6135941, WO 00163251, WO 00/61132, US 6120767), including sub-populations of 13 cells and 'f cells (see for example US 6147524) activated versus non~activated lympocytes, diseased or disease-causing cells versus non-diseased I disease causing lymphocytes (see for exampleWO
O1I13945A1, US 5132980, ) and specific immune cell clones for txamplt those having specific Ig type and MHC-peptide type ligands I and correlative ligands. Examples of such li~,ands include CCRS, CTLA-4, LFA-1, LFA-3. lCAMs e4. 1CAM-1, CD2, CD3, CD4 (eg see US 6,13b,310), CD18, CD22, CD40, CD44;
CD80, CT786, CD134 and CDt54, to name only a few (see also US6087475: PF4A
receptor) (see also Glennie MY et al. Clinical Trial of Amibody'fherapy. lmmunolegy Today Aug 2000, Vol_ 21 (no. 8) p.406).

The invention also contemplates that the therapeutic function or itmnune function effecting ligand is also a specificity imparting ligand, which in the case of for example, an antigen presenting cell may be an atttr~pdy which recognizes and binds to a specific MHC peptide complex, as is established in the an (see perti>s~tt Chames tt al. references herein, see also WO 97102342 , Direct selection of a human antibody fragment directed against the tumor I-etll cpitope HI.A-A1-MAGE-A1 from a uonimWuriizBd phago-Fab library. !?roc Nat! Acad Sci U S A. 2000 7u15; 97(14):7969-74). In this ease it wilt be appreciated that the APC iargeting ligand assist the particular MHC peptide binding antibody to bind to its tarmet.
See also WD 97!07819 which is hereby disclaimed with respect to all relevant aspects of the invention herein insofar as inherently disclosed therela. See also U5 5,770,403 with respect to antibodies which bind tp cyt~kine5-In vnc embodiment, rhc respective antibody companent9 of the muItispecific llgand recognize a substantially different subset of non-targetted tissues so that functional binding to a non-targetted tissue is substantially precluded. It will be appreciated that this strategy can be accomplished with two different antibodies have differing and preferably non-overlapping normal ie. non-targeted tissue distributions. In a preferred embodiment the target cell is a cancer cell and the respective fast and second cell surface associated llgands are expressed on different subsets of normal cells, which are non-overlapping subsets, so as to minimize deletetlous normal cell targeting and distibute the undesired effects or normal cell targeting (eg. with a toxin), to diFFtrcnt eth popul3tion5. For cx8rnple in the case of tumor cell targeting one or both ligands may be expressed exclusively on a single tumor type (eg. a human sarcoma or carcinoma, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosareama, Iymphangiosarcoma, lymphangioendotheliosareoma, synovioma, mesothelioma, Ewit~'s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, paltcreatlc cancer, brtast eatteer, 6vari~n cBnCtr, prostBtC C3nCtr, squamous ell! tartinbma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenoearcinomas, cystadenocarciztoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoroa, embryonal carcinoma, Wilins' tumor, cervical canetr, testicular tumor, lung eareiaoma, small ee:ll lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendrogliorna, mettittgioma, melanoma, neuroblastoma, retinoblastoma;
leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (mycloblastic, promyelocytic, myelvmvnvcytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytie (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia ogre, lymphoma (Hodgkin's disease and rton-FIod&kizt's disease), multiple myeloma, Waldenstrorn's macroglobulinernia. and heavy chain diseflse) or a particular category of tumor types (eg. adenocarcinomas, tumors oFneuro~ctvdermaI origin, or on multiple difterent tumor types or categories of tumor. One or both components (they may be the same or different) may be a dAb, a scFv, an Fab, a minibody moiety or a substantially Intact antibody, lbr example both may be scFvs and the resulting product may be a diabvdy, triabody, or tetrabody. For example ie a preferred embodiment the bispecific antibody comprises two dAb components comprising linked via a linker (see above) and having at least at least part of a constant region for fusion for example to a toxin (eg_ at least a partial hinge region, and preferably also at least a partial Cfl2 domain (optionally also at !cast a partial CH3 domain). In another embodiment, a trispecific anu'~ody pr tetraSpGcif~C at7tibvdy with at least two different and preferably 3 or 4 subsets (preferably at least one ar mort of such subsets being non-overlapping subsets) ofnon~targcted tth re$ctivities may be employed in the form of a trispecific or tetraspecific autibody respectively whereby up to three or four different pairs of ligands era targeted, so as further miniznizx normal cell targeting and also preferably target a heterogenous population of cells within the same tumor. Ligands with distributions on normal tj59PeS are well known, some being referenced htrein, for example CEA, CD-20, P53, epidermal growth factor, ineludieg Irnown multicarcinomic and pancarcinomic ligands (eg. see USS, 171,665, US 4349528.
The ~ funCti0nal binding ig used to mean binding for the purpose vFaceomplishing the object of the binding, for example binding for a suffcitat duration to inhibit or tnhartet a particular effcet, such as cell killinb, for example in the case where one both antibody components are selected for their ability to internalize, binding for a sufficient duration to permit internalization, for example to deliver a toxic payload. As discussed above, the teen substantially in reference to therapeutic advantage is used to refer to a degree which provides a significant benefit from a therapeutic standpoint.

Examples of tumor associated antigens (eg. WO O 1121828) and targets and related antibodies ere referenced throughout the disclosure and the foregoing aspect of the invention is for grOater certainty directed to bispecific antthodie5 (including trispecific and tetraspecific antibodies, optionally including a component which also binds to a lymphatic vessel associated ligand), which target each of the combinations and permuatipns pftt,e target cell (diseased, diyeage cau3ing or immune) associated antigens, ligands, epitopes or receptors well known to those skilled in the art, herein directly or lridlrectly referenced or referenced in the materials herein incorporated by reference (ie, permutations and combinations of pairs or where a tri-or tetra- specific antibody is used possibly permutations of (3 or 4) groups of pairs including for example pairs in which one member is used for targeting and the second is used for modulation puposes such mpdulatio~ including without limitation, simple binding eg_ to deliver a payload, apoptosis inducing (eg. anti-fas), modified vascular adhesion propet'ties (eg, anti-CD44), modified cytokine binding (anti-CCRS) etc.(re: relevant Iigands/markers see also U5P 6,010,902 and me refeteriees cited thtreiri, 5amtezas Immunologic Diseases, Fifth and Sixth Edition, Lippincott, Frank Austen, MD
Michael M. Frank, MD
John F. Atkinson, MD Harvey I. Cantor, MI) (6'''-ISHN: 0-7817~2120-2);
Fundamental Virology, Third and Fourth Edition, Lippincott David M. lCnipe, PhD Peter M. Howley, MD Diane E. GrifF'm, MD, PhD
Robert A. Lamb, FhD, ScD Malcolm A. Martin, MD Bernard Roimtan, ScD Stephen E.
5traus, MD (4~'-ISHN: 0.7817-1833-3); Arthritis and Allied Conditions - A Textbook oflZheumatology, Thirteen26 and Fourteenth Editions, William J. Koopman, MD 14~':ISEN: 0-?817-2240-3, November 2000; Cantor -Principles and Practice of Oncology, Fi'Rh end Sixth Editions, Lippincott, Vincent T. D~Vita, Jr., MD
Samuel E~lellman, MD Steven A. Rosenberg, MD, FhD ISBN: 0-7817-2229-2; Dubois' Lupus Erythematosus, Fifth Edition, Daniel J- Wallace, MD ISBN: 0-683-08663-0, December 1996; Cytokine Therapeutics in Infectious Diseases, Steven M. Holland, MD: PhD, Lippincott, 15HN: 0-7$1'7-1 G25-X, U$
6054561 ), in each of their permuatations of size/valency (ie. dabs, scP'v, diabodies etc heroin referenced) as applied to each of the applicable disease conditions herein referenced or otherwise lrnown to those skived in the art.
With rtspect to recombinant techniques for producing Fv fragments see also WO
88101649, WO 88106630, WO 88/07085, WD 88/07086, and WO 88109344.
With respect to preparing Ligands for speciSc MHC peptide complexes sec also WO 01/22083; Direct selection of a human antibody fragment directed against the tumor T-cell epitope HLA-A I-MAGE-A 1 from a nonimmunized phage-Fob library. Proc Natl Acad Sci U 5 A. 2000 Jul 3;97(14.):7969-74.
with respect to bispecific antigen binding constructs that are Suitable for for binding to more illari one antigen on tile same cell see also Scltmiedl A et al. Protein Eng 2000 pct 13(10):725-34.
Preferred irtununoconjugates include radiolabeled antibody components and conjugates of an anti-Lyve-1 anribody component and an antbody component which comprises an immunotnodulator.
A radiolabeled imrnunoconjugato may comprise 3n .alpha.-emitting radioisotope, a .B-emitting radioisotope, a gamma emitting radioisotopE, an Auger electron emitter, a neutron capturing agent that emits alpha-particles or a radioisotope that decays by electron capture, Suitable radioisotopes include lie Atl, 32 p, 125 l, ~3~ 1, f70 fir, X86 Vie, 188 Vie, 67 ~, z11 At, and the Like.
As discussed above, a radioisotope can be attached to att antibody component directly or indirectly, via a chelatang agent. Far example, 6' Cu, considered one ofthe more promising radioisotopes for radioimdlurlother$py dut to 1tS 61.5 hOlu' hall life arid abundant supply of beta particles and gamma rays, can be conjugated to an antibody component using the chelating agent, p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA) . Chase, "Medical Applications of Radioisotopes," in Remington's Pharmaceutical Sciences, 18th )edition, Gennaro et al. (ads.) pages 624-652 (Mack Publishing Co. 1990) (see also 19'" edition of Reminton's). Alternatively, ~° Y, which emits an energetic beta particle, can be coupled to an antibody component using diethylenetrismine~entaacetic acid (DTPA). Moreover, a method for the direct radiolabeting of the antibody component with " I is described by Stein et al., Antibody hnmunoconj. Jtadiopharm. 4: 703 (1991) (see also ilSP 6, 080, 384).
Alternatively, boron addends such as carboranes can be attached to antibody components, as discussed aUove.
In addition, thcrapeude immunoconjugates coo comprise an immunomodulator rnoiety suitable for application for the purposes herein. Broadly speaking, the tern, "immunamodulator" includes cytvkines, stern sell growth factors, lymphotoxins, such as tumor nr~crosis factor (TNF~, and hematopoietic factors, such as inteileukins (e.g., interleukin-1 (TLrl), TL-2, iT~-~, ILb, 1L-10 and IL-12), colony stimulating factors (e.g., granulocyee.colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), iaterfemns (e.g., interferonsalDl,a, -beta and gamma.), the stem cell growth factor designated "51 factor," erythropolttin and thrombopolttin. Examples of suitable immunomodulator moieties include TL-2, IL-6, IL-10, IL12, interferon-gauuna., TNF-alpha., end tht like.
A related form of therapeutic protein is a fusion protein comprising an antibody moiety and an itnmunornodulator moiety.
Methods Of naakitlg antibody-immtutomodulator fusion proteins are known to those of skill in the art as discussed herein. Far example, antibody fusion proteins comprising era interlcukin-2 moiety are described by Bvleti et al., Ann. Onool. 6:945 (1995), Nicoltt tt al., Cancer Gene Thtr.
2:161 (1995), Backer Lt &L, Proc. Nafl Acad. Sci. USA 93:7826 (1996), Hank et al., Clin. Cancer Res.
2:1951 (1996), and Hu et al., Cancer Res. 56:4998 (1996). In addition, Yang et al., Hum. Antibodies Hybridomas 6:129 (1995), describe a fusion protein that includes an F(ab''ya frngrrlent and a tumor necrosis factor alpha moiety.
Such inununoconjugates and antibody-ittuuunomoduIator fusion proteins provide a means to deliver an immunomodulator to a target cell and are particularly useful against tumor cells. Tht cytotoxic effects of irrunuuomvdulatvrs are wall known to those of skill in the art. See, for example, Kle et al., "Lymphokines and Monakines," in Biotechnolopr and Pharmacy, Pessuto et al. (eds.), pages 53-70 (Chapman & Hall 1993) as well as other roferences herein cited. As an illustration, interferons can inhibit toll proliferation by inducing increased expression of class I histocompatibility antigens on the surface ofvar;ous cells and thus, enhance the rate of destruction of cells by cytqtoxic 'T lymphp~eg.
Furthermore, tumor lttCrOSis faCtots, such as TNF'-alpha., art believed to produce cycatoxic effects by inducing DNA
fiagmentation.
Moreover, therapeutically useful immunoconjugates can be prepared in which an antibody component is conjugated to a toxin or a chemot6erapeutic drug. Illustrative of toxins which are suitably employed in the preparation of such conjugates are ricin, abrin, ribonuclease, DNase I, Staphylococcal enttrotoxin-A, pokeweed antiviraI protein, gelonin, diphthetin toxin, Fseudomooas exotoxin, and Pseudomonas endotoxin.
See,rcferences herein as well as for example, Pastan et al., Cell 47:641 (1986), and Goidenberg, CA-A
Cancer Journal for Cliniciaias 44-43 (1994). Other suitable toxins are lalown to rhost of skill in the art.
With to i-espeet to bispecifie antibody constructs which are capable of binding simultaneously to two ligands on the same cell see also W096I32841. Various such constructs am known in the art.An altematlvc approach to introducing the combination of therapeutic antibody and toxin is provided by antibody~toxin fusion proteins. An antibody-toxin fusion protein is a fusion protein that comprises an antibody moiety and a toxin moiety. Methods for making antibody-toxin fusion proteins are known to those of skill in the art (see references cited herein); antibody-Pseudomonas cxotoxin A fusion prpteins have been described by Chaudhary et al., Nature 339=394 (1989), l3rinkmann et al., Proc. Naf1 Acad.
See. USA 88:8616 (1991), $atr8 et al., Pr4c. Nsfl ACad. Sci. (JSA $9:5867 (1992), Freedman et al., J.
Tmmunvl. 150:3054 (1993), WeIs et al., Int. J. Can. 60:137 (1995), Fon,inaya et al., J. Biol_ Chcm.
271:10560 (1996), iCuan et al., Biochemistry 35:2872 (1996), and Schmidt et al., Int. J. Can. 65:538 (1996).
Antibody-toxin fusion proteins containing a diphtheria toxin moiety have been described by Kreitman et al., Leukemia 7:553 (1993), Nicholls et al., J. $iol. Chem. 268:5302 (1993), Thompson et al., J.
Biol. Cbtm. 270:28037 (I995}, and Vahera et al., Blood 88:2342 (1996). Deonarain et al., Tumor Targeting 1:177 (1995), have described an antibody-toxin fusion protein having an RlVase moiety, while Linardou et al., Cell Biophys. 24-25:243 (1994), produced an antibody-toxin fusion protein comprising a DNasc I
cpmponent. Gelpnin vyas used as the toxin moiety in the antibody-toxin fusion protein of Wang et al., Abstracts of the 209th ACS l~Iacional Meeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005. As a further example, Dohisten et al., Proc.
Naf1 Acad_ 5ci. USA 91'8945 (1994), reported a~, antibody-toxin fusion protein comprising Staphylococcal enterotoxin-A. Numerous other examples have been reported in the literature.
Useful cancer chemothcr~aptulic drugs for the preparation of immunot:onjugates inCludt nitrogen mustards, alkyl sulfonates, nitrosotueas, triazenes, folic acid analogs, pyrimidiue analogs, putine analogs, antibiotics.
epipodophyllotoxins, platinum coordination complexes, hormones, and the like_ Suitable chemotherapeutic agents arc described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co. I 995), and in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 7th Ed.
(MacMillan Publishing Co.

1985). Other suitable chemotherapeutic agents, such as experimental drugs, are known to those of skill in d,e art.
In addition, thorapcutically useful immunoconjugates can be obtained by conjugahug photoactive age"ts or dyes to an antibody composite. Fluorescent and other ehromogens, ar dycs, such as parphyrins sensitlvc to visible light, have been used to detect and to treat lesions by directing tho suitable light to the lesion. In therapy, this has been termed photoradiation, phototherapy, or photodynamic therapy (Jori et al. (ads.), Photodynamic Therapy of Tumors and Other Diseases (Libreria Progetto 1985);
van den Bergh, Chem.
Britain 22:434 (1986)). Moreover, monoclonal antibodies have been coupled with photoactivated dyes for achieving phototherapy. Mew et al., J. lmmunol. 130:1473 (1983); idem., Cancer Rts. 45:43$0 (19$5);
Oseroff et al., Proe. Nat/. Acad. Sci. USA 83:8744 (1986); idem., Photochem.
Photobiol. 46:83 (1987);
FIasan et al., Prog. Clin. Biol_ Res. 288.471 (1989); Tetsuta et al., Lasers 5urg. Med. 9:422 (1989);
Pelegrin et al., Cancer 672539 ( I 991 ). However, these earlier studies did not include use of endoscopic therapy applications, especially with the use of antibody fragments or subfragments. Thus, the present invention contemplates the therapeutic use of Immunoconjugates comprising photoactive agents or dyes.
With rcspect to a multifunctional ligaud having a first portion that binds to both lymphatic endothelial cells and tumor vasculatx~re, the invention contemplates using phage display or ribosome display to generate an antibody that hinds co vefgr-3 as well as one or both of of vegfr-2 or vegfr-1, having regard to the sequences of those respective receptors (see USPs 5,776,?55, 5877020, 5952199, 6107046, 6130071, 6221839, 6235713, 6245530; see also WO 00121560, WO 95133772, WO 97/05250, WO
9$/33h17).
Freferably the antibody does not internalize, particularly in the case where the multifunctional ligand is fused or conjugated to a toxic moiety. The invention also contemplates, for example, fitsing the binding domain of VEC,F-C Qr VEGF.D to antitumpr antibody, The invention also contemplates that the risk of retargeting cancer cells to non-tumor sites of sngiag~.rnesis, can be minimized by employing one or more of the following strategies pre- and/or co-treatment with inhibitors of angiogenesis, providing the multifunctional ligand with an effector fitnction, such as a toxic moiety, cytokitte or antibody component which retargets immune cells capable of kilIiog such canetr eelLs. The invention also contemplates using in combination or alone a multifunctional ligand liaving a second portion Hurt comprises an anti-VEGF
antibody portion which binds to one or mare of the VEGF family of ligands in order to inhibit lymplurnglogenesis and/or angiogenesis. (see also for example, Wp 00137025, WO
98133917, USP
6130071, WO 41/12669). With respect to angiogenesis and particularly lymphsngiogenesis sec also: /:
Shibuya M. Structure and function ot' VEGF/VEGF-receptor system involved in angiogenesis. Cell 5truct Funct. 2001 Feb;26(1):25-35: Yonemura Y, et al.Lymphangiogenesis and the vascular endothelial growth factor receptor (VEGFR)-Sin gastric cancer Eur l Cancer. 2001 May;37(7):918-23.: Iljin K, et aIVECtFR3 gene structure, regulatory region, and sequence polymorphisms FA$EH I. 2001 Apr;lS(6):102$-36.: Tang RF, et al4verexpressian of lymphangiogenic growth factor VEGF-C in human pancreatic cancer. Pancreas.
200I Apr;22(3):285-9?: Kadambi A, Carreira CM, Yun CO, Fadera TF, Dolmans DE, Carmeliet P, Fukumur,rD, Jain RK.Vascular endothelial growth factor (VEGF)-C differentially affects tumorvascular function and leukocyte recnritmenr role of VE4',F-receptor 2 and hostVEGF-A.Cancer Res. 2001 Mar 15;61(6):2404-8. Karpanc.~n T, Ct 21Va5CUlar endothelial ~owth factor C
promotes tumor lymphangiogenesis and intr-ttlymphatic tumor growth. Cancer Res. 2001 Mar 1;61(5):1786-90: Baldwin ME, et al The Specificity of Receptor binding by Vascular EndoJ~ehal Qrnwth Factor-D Is Different in Mouse and Man. J Biol Chem. 2001 Jun 1;276(22):19166-19171: Niki T, et al J
Pathal. 2001 Apr;193(4):450-7: Veikkola T, et al Signalling via vascular endothelial growth factor rerxptor-3 is sufficient forlymphangiogenesis in transgenic mice. EMHO J. 2001 Mar 15;20(6):1223-31 Achen MG, et a1 Localization efvascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis. J pathol. 2001 Feb;193(2):147-54 Stttcker SA, et aIVEGF-D
promotes tho metastatic spread of tumor cells via the lyurpliatics. Nat Med. 2001 Feb;7(2):186-91 Plate K.1~rom angiogenesis to lytnphangiogenesis. Nat Med. 2001 Feb;7(2):151-2. . Joukov V, et al; A novel vascular endothelial growth factor, VEGF-C, is a ligattd for the Flt4 (VEGFR-3) cad KDR (VEGFR-2) receptor tyrosine kinases.EM$Q .1. t996 Apr 1;15(7):1751. Lee J, et al, PTOc htatl acad $ci U$A.
799 Mar 5;93(5):19$8-Multimodal therapies are also contemplated within the present invention, including particularly for cancer, therapies which can be determined to be useful complementary therapies for the anti-metastatic embodiments of this invention such as anti-angiogenic Ab conjugates In another form ofmultitnodal therapy, subjects receive the multtfuncttOnal ligands of tfto present inventionanct standard cancer chemotbera y. For example, "CVB" (1 _5 g!m_sup.2 cyclophosphemfde, 20U-404 mglm= etoposide, and 150-20D mglm~carmustine) is a regimen used tn treat non-Hodgkin's lymphoma.
Path et al., Eur. J. Haetttatol. s 1:18 (1993). Other suitable cotnbinatiqn chemotherapeutic regimens are well-known to those of skill in tbc> art. Sao, for example, Freedman of al., ''Non-Flod~lan's Lylx~pl7omas," in Cancer Medicine, Volume 2, 3rd Edition, Holland et al. (ads.), pages 2028-20b8 (Txa & Febiger 1993). As an illustration, first generation chemotherapeutic regimens for treatment of intermediate-grade non-Hodgkin's lymphoma include C-MOPP (cyclophasphamide, vincriatinc, procarbazine and prednisone) and CfI01' (cyclophospltaznide, doxorubleln, vincrlstlne, and predttlsone). A
useful second generation chemuthcrapeutic regitnelt is m-~ACOD (methotrexabe, bleomycin, doxombiein, cyelophospbamide, vincristina, dexamethasone and lcucovOrln), while a suit~7ble third $eneiatlo>a regimen is MACOp-H
(methotrexare, doxorubicin, cyclophosphamide, vineristine, p~radnlsone, bleomycin and leucovorin).
Additional useful drugs include phenyl butyrate and brostatin-I.
In general, the dosage of administered multifltuctlonal ligands, hnmunoconjugates, and fusion proteins will vbry depending ttpob such factors as the patient's age, weight, height, sax, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of antibody component, lmmunoconjugate or fusion protein which is generally at least in the range of from about I
p$Ikg t4 10 mg/kg (am0urit of agent/body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate, partlculst'ly to take advantage of the depot effect of the inveetlon.
Adnvniswation of the invention including, imm~moconjugates or fusion proteins to a patient can be intravenous, intraarccrial, intraperitoneal, lntramuscular, subcutaneous, Intrapleural, intrathecai, by perfu5i01t thr0ugkl a regional catheter, or by direct intrnle;tonal injectipn.
When administering therapeutic proteins by itljection, the adminisu~ation may be by continuous lnfitsion or by single or multiple boluses.
Those of skill in the art are aware that intravenous injection provides a useful mode of administration due to the thoroughness ofthe circulation In rapidly distributing etnibodies_ intravenous administration, however, is subject to limitation by a Vascular barrier compri5iag endothelial cells oftlie vasculature and the subondothelial matrix. Still, the vascular barrier is a more notable problem for the uptake of therapeutic antibodies by solid tumors. Lymphomas have relatively high blood flow rates, contributing to effective antibody delivery. Intralytnphatrr~ routes of adrnirttst~on, such as subcutaneous or inttamuscular injection, or by catherization of lymphatic vessels, also provide a useful means of treaties lymphomas.
With regard to "low doses" of ~3~ I-labeled immunoconjugates, the Invention includes a dosage is in the rare of t 5 to ~0 mCi, 20 to 30 mCi. In co~rast, a prcfem;d dosage ofs°
Y-labeled immunoconjugatcs is lit the range from 10 to 30 mCi, while the more preferable range is I O to 20 mCi.
Immunoconjugates having a boron addend-loaded cattier for thermal neutron activation therapy will nermally be effected in similar ways. However, it will be advantageous to wait until non-targeted itnmunocoztjugate clears before neutron irradiation is performed, Clearance can be accelerated using an 8ncibudy that binds to the iritmuilocOtlJugare. See U.S. PBt. N4. 4,624,846 for s description of this general principle.
The immunoconjugates, and fusion proteins of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, wliereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier. A composition is said to be a "pharmaceutically acceptable carrier" if its adrninistratlon can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a phat'maceutically acceptable carrier. Other suitable carriers are well-lcnown to those in the art. See, for example, IZEMINGTON'S PHARMACEUITCAL SCIENCES, 19th Ed. ( 1995).
For purposes of therapy, antibody components (or imrnunoconjugates/fusion proteins) and a phat'ntaceutiCally acceptable carrier are administered to a patient in a therapeutically effective amount A
combination of apt antibody componey opfionally witlt atr immunoconjugate/fusion protein, and a pharmaceutically acceptable carrier is said to be administered In a "dterapeutically effective amount" ifthe amount adminishered is physiologically sigtificant. An agent is physiv)vgically significant if its preseriCe results in a detectable change in the physiology of a recipient patient. In one aspect, an agent is physiologically significant if its presence results in the inhibition of the growth of target tumor cells.
as Yet another therapeutic method included in the invention is a method of treating cancer by administering to an animal suffering from caacer a pharmaceutically effective arnount of one or more multifunctional ligands Capable of binding to cancer cells, wherein the compound Is associated with a substance capable of damaging canctr CC11S.
Pharmaceutical compositions herein described ar alluded to ioeludt multifiutctional ligands of the invention or therapeutics used in combination therapy which may be administered by a variety of routes o'f adminstratlon.
By administxatian of an "effective amount" is intended an amount of the compound that Is sufficient to enlranct or inhibit a reSpotise, is some embodirnetats partlCUlafly an immune response or cellular response to a multifunctional ligaud. one of ordinary sllll will appreciate that effective amounts of a naultifbnct;onal ligand can be determined empirically and may be employed in port 'Form Or, when SUCK fOxmS exist, i11 pharmaceutically acceptable salt, ester or prodrua form. The multifunctional ligand may be administered in compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage of the compounds and COmpOSltlOns of the present invention will be decided by the attending physician within the scope of sound medical,judgemtnt. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the type and degree of the cellular response t0 be achieved; activity of the specific multifunctional ligand employed; the specife composition employed; the age, body weight, general health, sex and diet ofthc patient; the time of administration, mute of administration, and race of excretion of the agonist or antagonist; the dur$tion oC the trcatrnc~xt; drugs used in Combination or eoineidenml with the specific agonist or antagonist; and like factors well known in the medical arts.
On administration parenterally, for example by i.v. drip or infusion, dosages optionally at least on tht ordtr of from 0.01 to 5 mg/kglday, optionally 0.05 to 1.0 mglkglday and more preferably 0.1 to 1.0 mglkgldtty can be used. Suitable daily dosages for patients are thus on the order of from 2.3 to 500 mg p.o., optlonatly ~ tp 25D rng p.o" optionally 5 to 100 mg p.o., or on the order offrem 0.5 to 250 mg i.v" pptionally 2.5 to 125 mg i.v, and optionally 2.5 to 50 mg i.v.
Dosaging may also be arranged in a patient specific manner to provide a predetermined concentration of an agonist or antagonist in thu blood, as determined by tire ltlA technique. Thus patient dosaging may be adjusted to achieve regular otrgoing trough blood levels, as measured by RIA, optionally on the order of et least from 50 to 1004 ng/ml, preferably I50 to 500 nglml.
From about, pharmaceutical compositions are provided comprising an agonist or antagonist and a pharmaceutically acceptable carrier or excipient, which may be administered orally, reetaily, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray. Dy "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parertreral" as used herein refers to modes of administration which include intravenous, intranwecular, intraperitontal, invasternal, subeutaacous and intra8rlicul2r injection and infusion.
Optionally a composition for for parenteral injection can comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for rtconstitution into sttrilt injectable solutions or dispersions j ust prior to use. Examples of suitablt aqutous and nonaqueous carriers, diluents, solveuts or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylceuulose and suitable mixtures thereof, vegetable pill (such as alive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
Some compositions herein descibed may also contain adjuvants such as preservatives, wetting agents, emulsifyit~ agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents sllch as sugars, sod;um chloride, and the Like. Prolonged absorption of the injectRblb pharmdcauClcal form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearatc and gelatin.

Tn snme eases, in order to prolong the effect of one or therapeutic components herein described, it is desirable to slow the absorption from subcutaneous or intrarnuscular injection_ This may be accomplished by the use of a iiquid suspension of crystalline or amorphous material with poor water solublllry. The rate of absorption of she drug then depends upon ita rate of dissolution which, in rum, may depend upon orystal size and crystalline form. Alteraaavely, delayed absorption of a parcaterally adtnlnisteted drug form 1s accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of thu drug in biodegradable polymers such as polylactide-polyglycolJde. Depending upon the ratio of drug to polymer and the nature of the particular polymer ~npl0yed, the rate of drug release can be controlled.
Examples of other biodegradable polymers include poly(olthocsters) and poly(anhydrides). Depot injectable fonnulations are also prepared by entrappi~ the drug In liposamcs or mlcroemulsions which are compatible with body tissues.
The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorparatlng sterilizing apeats in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
The multifunctional ligand eaa also be administered in the form of liposomcs.
As is known in the art, liposomes arc generally derived ~-om phospholipids or other lipid substances.
Liposomes are formed by mono- ar multi-lameflar hydrattd liquid Crystals that ors dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and tnetabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to the agonist or antagonist, stabilizers, preservatives, excipiems, and the like. The preferred lipids are the phospholipids and the phosphatidyi choacs (leclthirrs), bath natural and synthetic. Methods to form liposomes era known in the art. Sea, for example, Frescott, Ed., Methods in Cell Biology, Voltune X1V, Academic Press, New York, N_Y. (1976), p. 33 et seq_ The present invention also contemplates a method oftreatment in which immunomodulaiors era administered to prevent, mitigate or reverse radiatiotrinduced or drug-induced toxicity of normal cells, and especially hemat4poietic cells. Adjunct imrourtomoduIator therapy allows the administration of higher doses ofcytotoxic agents due ca irtereased tolerance oftlio recip9entmatntnad.
Moreover, adjunct immttnomodulator therapy cmt prevent, palliate, or reverse dose-Ihniting marrow toxicity. Examples of suitable hnmunomodttlators for adjunct therapy Include G-CSF, GM-CSF.
thrombopoietin, IL-1, IL-3, IL-12, cad the like. The method of adjunct iulmunomodulator therapy is disclosed by Goldenberg, U.S. Fat.
No. 5,120,5? 5.
Far example, recombinant 1L-2 may be administered intravenously as a bolus at 6 x IO' lUlkg or as a contLmous infusion at a dose of 18 x 106 IUIm~ Id. Weiss et al., J. Ctin.
Qncpl. 1 Q:275 (1992).
Alternatively, recombinant li.-2 may be administered subcutaneously at a dose of 12 x 10610. Vogolzang et al., J. Clip. Onoal. 11:1809 (I993). Moreovtr, INF-.gamma. may be administered subcutaneously at a dose of 1.5 x10 U. Lienard et al., J. Clin. Oncol. 10:52 (1992). Furlhcrmoro, Nadesu et al., J. Phatmacol.
Exp. Then. 274:78 (1995), have showy that a single intravenous dose of recombinant 1Jr12 (42.5 .mu_glkilogram) elevated 1FN-_gamma. revels in rhesus monkeys.
Suitable 1L-Z formulations include PROLEUKIN (Chiron Gorp./Cetus Oncology Corp.; Emeryville, Calif ) and TECELEUTUN (Noffmenn-La Rache, lnc.; Nutley, N.J.). ACT>MMUI'1E
(Genentech, lnc.;
South San Francisco, Calif'.) is a suitable 1NF-.gamma preparation.
In the preceding detailed description, reference was made to various methodologies latown to those of skill in ti,e art pf molecular biology ~d immunology. Publicatipns ~d other materials sting forth such lmawtt methodologies to which reference was made or is made below are lncorparated herein by reference in their entireties along with refCrenCZS titCd 2htreira as lhOtigb Bet forth in Full.
Standard reference works setting forth the general principles ofrocombinatlt DNA technology include Watson, J. D. et al. Molecular Biology ofthe Gene, Volumes T and 11, the Benjamin/Cummings Publishing Company, Tnc., publisher, Mettle Fark, Calif. (1987), Darnell, J. E. et al., Molecular Cell Bioloe7r, Scientific American Bovks, Tne_, Publisher, New York, N.Y. (198b); Lewin, B.
M. Genes II, John Wiley & Sons, publishers, New York, N.Y. (19$5); 41d, R. W., et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2d edition, UniversIry of CaliforNa Press, publisher, l3erkeley, Calif.
(1981); Maniatis, T., et sd_, Molecular Cloning: A Laboratory Manual, 2nd Ed_ Cold Spring PIarbor Laboratory, publisher, Cold Spring Harbor, N.Y. ( 1989), Gad Current Protocols in Molecul<~r Biology, Ausubel et al., Wiley Press, New York, N.Y. (1989). 5taadard refereacc works setting forth general principles and techniques of immunology include Handbook 4f Experimental Jnttnunology BlaCkwell Science, Incorporated, rS~N:063200975b; Antibody Engineering Dlackwell Science, lncorporattd, ISBN:0632009756; Therapeutic lromunology 1SBN: 086542375JC BlaCkwtll Science, Incorporated ;
Encyclopedia of Immunology (1998) M4rgan Kaufmann Publishers, ISBN:0122267656;
Immunology Mosby, Incorporated, ISBN:0723429189; Abbas AK. et al. Cellular & Molecular immunology 4'~ Ed.
2000 LSBN 0721650023; Breitling F. et al. Recombinant Antibodies 1999 ISBN 0-471-17847-0;
Masseyeff R_ et al. Methods of Immunological Analysis Wiley-VGH T58N 3-537-27906-7, 1992;
Mountain et al. Eds, Eiotechnolosy 2"d ad. Vol 5A 1998 ISBN 3-527-28315-3 Wiley-VCH; Campbell, A, , "Monoclonal Antibody Technology," in, Burdon, R., et a1.> ads, Laboratory Techniques in BioGhtlriistYy and MoleCUl3Y Biology, VolunYt 13, ElsCvier, PublishCr, Amsterdam [1984);
Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is not so limited. It will occur to those of ordinary skill in the art that venous modifications may be made to the disclosed embodiments and that such modtiications are Intended to bt within the scope of rl5t prtstnt invention.
AlI publications referred to herein are indicative of the level of skill of chose in the art to which the invention perrains_ A11 publications are herein (as well as references cited therein)are incorporated by reference to the semi extent as if tech individual publications were specifically and individdually indicated to be incorporated by reference in its endrety_ The present invention, thus generally deserlbtd, will be understood more readily by reference to the preceding and following examples, which ate provided by way of illustFatiun and are not intended to be lImithtg of the present invention.
With respect to making applications, methods of using, target cell-associated markers ate. ofmultispecific li$ands and blspecitlc antibodies see also Methods Mod Bio! 2001; 166:177-92;
USP 6,071,517; USP
5,897,861; USP 6,096,311; USP 5,932,845, Journal of hnmtmological Methods February 200 I Vol.
248(1-2) page 1-Z00; Mot lnununol 1999 May; 36(7):433-45; US6051227 US06143297 US03977318 US05968510 US05885796 US05885579 T.TSo5849050 Methods of blocking T-cell aCtlvat19t1 U51I1~ anti-B7 monoclonal antibodies; US05$51795 0505747034 US06 J

B7-f targeted ribozymes; USD5844095 US06090914 US05718883 Transgcnic animal model for autoimmunc diseases; US05855887 US05811097 0505770197 Methods for regulating the immune response using B7 binding molecules and ~4-binding molecules; US06084067 $P01073741A2 US06130316 0506068984 Antibodies to lymphocyte activation antigens uses therefor;
US057bb570 Lymphocyte activat;on antigens and thereto; US05434131 US05316920 Lymphocyte activation antigen HB15, s member of the immunoglobulin superfamily; US06111090 Mammalian cell surface antigens; US06083751 Chimeric receptors for the Generation of selecrively-activatable TH-independent cytotoxic T cells;
US05977303 Mammalian cell surface antigens; US05738852 Methods of enhancing antigen-specific T
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20(3A):1551-5_ Yoshida H, Katayose Y, Suzuki M, Macsuno 5, Kudo T, Kumagai I. Functional Fv fragment of en antibody specific for 01728: Fv-mediated co-stimulation of T cells. FEES Lett. 2000 Jul 7;
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Recombinant antibody constructs in cancer therapy. Curr Opin Tmmunol. 1999 Oct; 11(5):548-57 Alt M, et al Novel tetravalent and bispecific 1gG-like antibody molecules combiaittg shtgle-chain diabodies with the immunoglobulia gammal Fc or CH3 region. FEJ35 LeLt. 1999 Ju12; 454(1-2):90-4.
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Robert H, et at Tumor targeting with nearly de8igled blparatopiC antibodies direCG:d against two differed epitapes of the carcinoembryonic atulgen (CEA). Tut J Cancer. 1999 Apr 12; 81 (2):285-91 _ Kreutz FT, et al Efficient bispecific monoclonal antibody purification aging gradient thiophlllC affinity chromatography. J Chromatogr B Biomed Sci Appl. 199$ Sep 4; 714(2):161-70.
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84:121-34. Merchant AM, et prateln, lVUCleotIde An ef6ciant route to human bispecific IgG. Nat Biotechnol. 1998 1u1; 16(7):677-81. ' Zhu 2, et aI High level secretion of a humanized bispccific diabody from Eschericltia coli.
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With respect to anti-CCRS antibodies used to kill CCRS-expressing cells, with for example, bi-specific antibody chemokine fusions see $ruhl H. et al. J lmmunol. 2001 Feb 15 166(4):
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With rsspect to targeting IIC,AP proteins see for.examplc US 6172195.
With respect to pertinent diseased cells, disease causing cells and other suitable targets for immttnotoxltts, as weri as optional toxins and methods of maldllg and using immuziotoxius and related technologies see for example US05980895 Immunotoxin containing a disulfide-stabilized antibody fragment joined to a Pseudomonas exotoxin that does not require proteolytic activation; US05686D72 ?<pitope-specific monoclonal antibodies and immunetoxins and uses thereof; US04956453 Antihuman ovarian cancer immunotoxfns and methods of thereof, 0506146631 Immunotoxins comprising ribosome-inactivating proteitl5; US05756699 Immunotoxins comprising ribosome-inactivating Proteins;

Immunotoxios comprising ribosome-inactivating Proteins; US0614683D Ptvteins encoding gelonin sequences; 0505837491 Polynucleotides encoding gelonin sequences; 0$05578796 Methods and compositions concerning homogenous immunptpxin preparations; US05185434 Prolonged-action immvnptoxius containing a glycopeptide constituent which inactivates ribosomes, modified on its polysaccharide units; US04958009 Anti-human ovarian cancer immunotoxins and methods of use thereof; US05980896 Antibodies reactive with human carcinomas; U506D74(s44 Nucleic acids encoding itnmunotoxins containing a disulfide-stabilized antibody fragment replacing half or more of domain IB of pseudomonas exotoxia, and methods of use of the encoded immtuotoxins;
US04981953 lmmunotoxins, process for their preparation and pharmaceutical compositions in which they are present; US04980457 Cytotoxic conjugates whip can be used in therapy and process or their preparation; >,~S04g459$5 Pseudomonas exotoxin conjugate inrmunotoxins;
US06020145 Methods for determining tht: preseoee of carcinoma using the antigen binding region of monoclonal antibody BR96;
US05792458 Mutant diphtheria toxin conjugates; 0503338542 0506051230 Compositions for targeting the vasculature of solid tumors; B3 antibody fusion proteins and their uses; 0505990275 Linker and linked fii5ipn polypeptide5; US059$1726 Cbimeric and mutationally stabilized tumor-specific 131, B3 add B5 antibody fragments;
immunotoxic fusion proteins;
and uses thereof; US05965132 Methods and compositions for targeting the vasculature of solid tumors;
US05889157 Humanized B3 antibody fragments, fusion proteins, and uses thereof Multivalent antigen-binding proteins;

FR.AGMEN'TUS05776427 Methods for targeting the vasculature of solid tumors;

Monoclonal antibodies directed to the I-IER2 receptorUS05665357 Antibodies recognizing tumpr associated antigen CA 55.1; 0505660827 Friedrich K, et al A two-step selection approach for the identification of ligand-binding determinants in eytokine receptorsAnal Biocbem. 1999 Mar 15; 268(3):179-86.

Krcbs S, et alRecombinmt human single chwn Fv antibodies recognizing hutnaninterteukin-fi_ Speei6e targeting of cytokine-secreting cells. J Biol C'.hem. 1998 Jan 30; 273(5):2858-65Wilbur DS,et alRelatedArticles Biotin reagents for antibody pretargeting. 2. Synthesis and in vitroevaluation of biotin dimers and trimer$ for cross-linking ofstreptxvidin. Hioconjug Chem_ 1997 Nov-Dec; $(6):$19-3Z.
Ring DB, et alAntigen fbrks: 6ispecifie reagtxtts th8t inhibit cell growth by bindingselected paiTS of tumor antigens. Cancer Immuaol Immunother. 1994 Jul; 39(1 ):41-8.
W00994z597A1 MONOVALENT, MULTTVALENT, AND MUL'T)ItrIERIC MIdCBIND1NG DOMAIN
FUSION PROTELNS AND CONJUGATES, AND USES TI-~REFOR; EP00935G07A2 SC1L1;JBLE
MONOVALIrNT' ANl7 MULTIVALENT MHC CLASSIT F1JSTON PlEZ41'EINS, AND USES
7-HEREFOR;

ANThIRAJC TOXIN FUSION PROTB1NW009728191A1 MIdC COMPLEXES AND USES THEREOF;
TJS05580?5G B7IG fusion protein; US06143298 Soluble truncated form$ pf 1CAM-1;
US058S2175 F-selectin glycoprutein ligand blocking antibodiesUS05800815Antibodies to F-selectin aad their uses;
US06037454 I~iumanized anti-CDI la antibodies; US06020152 Lymphocyte-associated cell surface protein1.1S05$07734 Monoclonal antibodies and FV specific for CD2atttiaenUS45622701 Crass-reacting monoclonal antibodies speciftcfor !r and P-soloctin 0505622700 Method for treating a LEA-I-mediated disorder; 3P06209788A2 IMMUNOASSAY OF
HUMAN SOLUBLE ICAM-1, ANTIBODYAND 1C1T FOR MEASUR,>:~NT THEREOF;
JF03072430A2 ANT1VIR.A1, AGENT B'Y' US1NG FUNCTIONALDERIVATtYE OF
INTERCELLULAR
ADHESIVE MOLECULE; JPOI 13S724A2 TREATMENT FOR NONSPEC1F1C 1NFLAMMATTON;
US06123915 Methods For using agents that bind to VCAM-1; W009929706A2 ANALOG BASED S1ALYL LEWISxN[1MFTICS; W009918442A1 DIAGN05tS OF THROM80TIC
EVENTS BY DETECTINGP-SELECT1N; US05877295 Antibodies which bind a subpvpulation ofMac-1(CDIIb/CD18) molecules which mediate neutrophil adhesion to ICAM-land fibrinogen; 1:TSQ5$69444 Sulfated and phosphated sacclu~tride derlvatives,proccss for tile preparation of the same and use thtrcof L1S05858994 Carbohydrate conjugates as inhibitors of eelladbeaion; 0805811405 Multiply fucosylated dicSYboxylic acidspossessing antiadhesive properties; US05654282 Selectin binding glycopeptides;
US05632991 Antibodies specific for >rselectin and the usesthereof; US05599676 Method for isolating a novel receptor for_alpha_4 integrins; US05580862 Sulfate llgands for L-selectins and methods ofpreventing sulfate addition; 0505508387 Selectin >iinding glycopept[des;
US06177547 Antibodies to P-selectin glycaprotein ligandUS05827670 Methods of isolating and detecting bone marrow stromal cells with VCA1V1-1-speeifte antibodies; 0505756095 Antibodies with specificity for a common epitope on H-selectin and L-selectin; US05565550 Antibodies to ICAM-?, and fragments thereof US0609J$38 Phtsrmaceutical cOmpasitions comprising anti-CI)45RH antibodies for the inhibition ofT-cell mediated immune responses;
US05595737 Methods for using monoclonal antibodies specific for cell-surface bound LAM-1;
US05324510, 0506183988 Leukocyto-specific protein snd gene, andmethods of use thereof-, 0505997865 Agoniat antibodies again$t the flld/flC3 rtceptor and uses illereoE
0505993816 Methods to inhibit humoral immune responses, immunoglobulin production and B cell activation with 5c8-speoific antibodies;
US05869453 Cytotoxic T-cell epitapes; 0805861131 Soluble fttsion molecules with binding speci$cfty for cell adhesion molecules; US03843441 Use of endothelial-leukocyte adhesion, ntoleGtalo-1 speClflc antibodies in the treatment of asrhmaUS05821332 Receptor on the sutfaoe of activated CD4+ T-calls:
ACT-4; EP00$68 I 97A 1 ANTI-S>~L.>~GTI1V ANTI$OD1ES FOR PREVENTION OFMULTIPLE
ORGAN
FAILURE AND ACUTE OIZCAN DAMAGE; U5p58175I5 Human B3 inte~rin alpha subunit antibodies;
EP00528931 B 1 HUMANIZED CIiIMERIC ANTI-ICAM-1 ANTiHODIES, METHODS OF
PItEPARAfION AND USE; 0505776775 Anti-LAM 1-3 antibody and hybridoma;
US06063906 Antibodies to lntegrin alpha subunit; 0505997865 Agoni$t antibodies against the flk2ltlt3 receptor and uses thereof; US05993816 Metltod5 to inhibit htunoral ittutauye responses, immunoglobulin production and B cell auivatlon with Sc8-specific autibvdiesU505951982 Methods to suppress eon immune response with variant CD44-specific anttbodiasUS05843441 Use of endothelial-leutcocyte adhesion molecule-1 specific antibodies in the treatment of asthma US03821332 Receptor on the aur~ace of activated CD4+T-cells_ AGT-4; USDS&21123 ModiSod antibody variable domains;

AN'rl-S$LECTIN ANTIBODIES FOR FREVENTION OF MULT iPLE ORGAN FAILURE AND
ACUTE ORGAN DAMAGE; 0505817515 Human B2 integrin alpha subunit antibpdie9;
EP00528931$1 HUMANIZED CHIMERIC ANTI-1CAM-1 ANTi$ODIES, METHODS OF PTtEpARATION AND USE;
US05776775 Anti-LAM 1-3 antibody and hybridvma; US05776755 FLT4, a receptor tyrosine kinnse;
0505730978 Inhibition of lymphocyte adherence with alpha.4ࢷ -specific antibodies;
Examples of tumor specific antigens are numerous and ~e refernd to in the hertlnabove cited references endes well as the in the following referencesUS0613298010/1712000 Antibodies specific for TRP-2 a human tumor antigen recognized by eytatoxlc T lymphocytes U506165464Monoclonal antibodies directed to the lihR2 receptor US058243I l7;<eatment oftumors with monoctpnal antibodiesagslnst oucogtne antigens.
0S0614005010131 /2000 Methods for determining breast cancer and melanoma by assaying for a plurality of antigens associated herewith; US0605122G MN-specific antibodies and their use in cancer treatment;
US06020145Methvds for determining the presence ofoareinoma using the antigen binding region of monoclonal antibody B1t96 ; 0505980896 Anh~bodies reactive with human carcinomasUS05955075Method of inhl6iting tumor growth using 2latibodles to MN
protein US05917124'transgenic mouse modal of prostate cryncar; US059143890613211999 E6 associated proteinUS0591214306/1511999 PolynueleotSdes encoding a human mage proteinhomologUS0591062606/0811999 Acetyl-CoA carboxylase compositions and methodsof useU80587456002/2311949 Melanoma antigens and their use in diagnostic and therapeutic merhodsU5058722170211611999 Anribodies which specifically bind a eaneerrelated antigenU50586963602/09/1999 lmmunareactive peptide sequence from a 43 kDhuman cancer antigenUS0586904502/09/1999 Antibody conjugates reactive with human carcinomas US0586612402/0211999 Antiidiotypic antibodies for high moleCUlarweight-melanoma associated by sameUS0584708312/4S/1998 Modified p53 US05844075 Melanoma antigens and their use in diagnnsticsnd therapeutic methods US0584368512I01I1998 Production of chimerie mouse-human antibodies with specificity to human ttunor antigensUS05843648 P15 and tyrosinase melanoma antigens and their use in dia~ostic and therapeutic methods US0384085405058304701110311998 Humanized antibodies to ganglioslde GM2U50583046411/03/1998 U505808005Hurnan carcinoma Bispeci8c n,alecules recognizing lymphocyte antigen CD2 and tumor antigensUS05792456 Mutant BR96 antibodies reactive with hurnanCareinomas US0578368U505773579 Lung cancer marker US05772997 Monoclonal antt'bodies directed to the I3ER2 receptorUS05770374; 0505705157 Methods oftreadrll; cancerous cells with and-receptor antibodies U5D569599412/0911997 Isolated cytolytic T cells speclflc foreomplexes of MAGE related peptides and HI,A moLeculesUSO5d9376312/D2/1997 Anubvdies to human carcinoma satigea Tumor rejection antigens which correspond toamino aoid sequences in tumor rejection antigen precursor bage, and uses thereofU5D568170I lmmortaiized human fetal osteoblastlc cellsUS056815~210/2$/1997 US4567717110/14/1997 Monoclonal antibodies directed to the HER2reeeptorUS05674486t0107It997I,150566535749/09/1997 Antibodies recognizing tumor associated antigen CA 55.1 Fonsatti E, et al F,energing role of proteetin (CD59) in humoral immunothenrpy of solid malignancies.Clln Ter. 2000 May-Jun; I S l (3): I 87-93 Knuth A, et aICancer inununotherapy in clWcal oncology. Cancer Chemother 1'hartnacol. 2000; 46 5upp1:S46-51 : Slavers EL.Targeted therapy of ecutie myeloid leukemia with monoclonal antibodies andinvnunoconjugntes. Cancer Chemother Phatmacol.
2000; 46 Suppl:Sl8 22 .I72 van Spriel AB,et allmrnunotherapeutic perspective for bispecific antibodies.Immunol Today. 2DD0 Aug; 21 (8):391-7 273: Green MC, et alMonoclonal anfbody therapy for solid tumors. Cancer Treat Rev.
2000 Aug; 26(4):369-86 ~Claag, J 'fargetlng cytokInes to tutnors to induoe active antitumor immune respouses by recombinant fusion proteins. l:Ium Antibodies. 1999; 9(1):23-Engberg .T, et alRecombinant antibodies with tire antigen-speeil9e, MHC restricted specificity of T cells: novel reagents for basic and clinical investigations and lmtnunotherapy. Immunotechnology. 1999 Mar; 4(3-4):273-$. 0')3rien TJ, Tet alMore than 15 years of CA
125: what is l;nowrr about the antigen, its sttuctute audits function.Int J
Biol Markers. 1998 Oct-Dec;
13(4):188-95.
5harifi J, et alImproving monoclonal antibody pharmacoklnetics via chemical modi~cation.ø J Nucl Med.
1998 Dec; 42(4). 242-9.
Ligands on immune or other cells which may be targoted with bispecifie ligands in which one ligattd of the pair dictates specificity for a population of cells or particular sub-population of those cells prtd a second lioand with reduced functiaeal affinity is used to effect a specific immune function include those referenced in the Following patents and pubIicati4rs thereltl CCferetlced: 0506132992 Expression voctors encoding bispeclflc fusionproteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell; Antibody heteroconjugates and bispecificantibodies for use in regulation of lymphocyte activity;
wOD9942077 COMPOSITIONS AND MET1~IODS FOR REC3ULATIhIGLYMPHOCYTE ACTNAT10N;
05059165600 Methods for inhibiting art immune response byblvclciag the GP39/CD40 and CTLA4/CD2$/B7 pathways and cornpositionafOr use therewith; US05876718 Methods of inducing T cell non-responsiveness to transplanted tissues end of treating graft-versus-host-disease with anti-gp39 antibodiesEP00445228BIMMUNOTHERAPY INVOLVING CD28 ST1MUI,.pTTON;
0505709859 Mixed specificity fusion pmtcins;1JS05637481 Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispeei6e fusion proteins in a mammalian cell; W009720048 MODIFIED SFV MOLBCULBS WHICH MJiDIATE ADHESIONBETWEEN CELLS
AND USES THERBOF;
EP00336379 Antibody hcteroconju~atos for use in regulation of lytnphacyte activity;
EP005372y3 L1GAND 1~0R CD28 RECEPTOR ON B CELLS ANDME'rI~ODS; CIS05182368 Li,gands and methods for augmenting B-cell proliferation; WOD9300431 CTL4A RECEPTOR, FUSION

PROTE1N$ CQNTA1NTNG ITAN17 USES THEREOF; EP(1044522$ IMM'UNdTHERAPY INVOLVING
C)a2$ STIMULATION;
Role of celhder adhesion molecules in HJV type 1 infection and their impact onvinrs neutralization.
AIDE Res Hum Retroviruses. 1998 Oct; I4 Suppl 3:5247-54 Cavenagh JD,et alAdhesion mplecple9 in clinical medicine. Crit Rev Clin I..ab Sci. 1998 Sep; 35(5):415-59 Viney IL, Fang S_ Beta 7 inteQrlns and their ligartds la lymphocyte migration to the gut. Cheaa lmmunal_ 199$; 71.64-76 Apl3n AE, et alSlgnal transduction and s;gnal modulation by cell adhe9toa receptors: the ralCOf integtltls, cadhlxins, itnmunoglobuIln-cell adhesion molecules, and selecNaf. Pharmacot Rev. 1998 Jun; 50(2):197-263 With respect to ascertaining important amino acid residues for receptor activation or binding see also 7ang, Q., Springer, T. A_ (2001). Amino Acid Residues in the PSI Domain and Cystelne-rich Repeats of the Integral beta 2 Subunit That Restrain Activation of the Integrin alpha abets 2, J, i3iol. chem. 276: 6922-6929; Biadine site on the marine 1FN-gamma receptor for 1FN-gamma has been identified using the synthetic peptide approach,Thc Journal of Immunology, Vol 151, Issue 11 6206-6213; The loutnal of Immunology, Vol 143, issue 113368-3579 The main immvuogenic region of the nicatlnic acetylcholine rtceptor. Identifrcatirnt of amino acid residues ink with different antt'bodics, M Bellone et al.;
Arend, W. P., Malyak, M., Guthridge, C. J., Gabay, G. (1998). INTERLELiICTIV-1 RECEFTpR
ANTAGONIST: Role in Biology. Anent. Rev. lmmunol. 16. 27.55; The Journal of Immunology, Vol 155, issue 10 4719-4725, Mapping, of receptor binding sites on iL-1 beta by recvnstructioo of ILr I re-like domains; The Journal of Immunology, 2000, 165: 6966-6974 Identification of Fetal Liver Tyrosine Kinase 3 (Flt3) Ligand Domain Required for Receptor Biadiog and Function Usiltg Naturally Qceurring Lirand Isoforms Waithaka Mwangis, Wendy C. Brown and Guy H. Palmer.
The invention also contemplates multifunctional ligands comprising various cooibinations and permutations of such ligands including pairs and three different such ligands including multifunctional ligands includlitg such combinations and a llgand which binding to a lymphatic vessel associated ligand.Additional pertinent references pertahtlng to formation of antibody dimer5, micrOarlaya Of (end tissue nticroarrays) proteins including hetmofunetiana[ proteins and recombinant, ligands having application to the iuventioa, and phage or ribosome display strategies having relevance herein include Z.hu H. et al. Protein arrays and microarrays,Curr Opin Chem Biol. 2001 Feb;
5(1):A0-5, references in IBC's conference an Protein Microarray Technology March 19.21 Santiaj;o California;
WO 99/06834, WO
99/19506; Wp 97/02342, WO 04163701; WO 99140434 ; US 6,127,127; US6146830, WO
ODl07S298, US61657t79 US0620402303/2012001 Modular assembly of antibody genes, anribodies prepared thereby and use; US0584681812108/1998 Pectate lyase slg~tal sequence; US0569843512I16I1997 Modular assembly of antibody genes, antibodies prepared thereby and use; US0569841712/16/1997 Modular assembly of antibody genes, antibodies prepared thereby and use; US0569349312/0211997 Modular assembly of antibody genes, antibodies prepared thereby and use; US055145480510711996 Method for in vivo selection of ligand-binding proteins; 05056482370711511997 Lxpressioa of functional antibody $'ElgmeuTS;
US0618034 In vitro scanning saturation muruganesis v~prpteias; US06027933 Surface expression libraries o~fheterQmerle receptors; US059105730610$/1999 Monomeric and dimeric antibody-fragment fusion proteins; US0615058311Transgcnic 8nimals expressing artificial epitope-tagged proteins; US06132992 Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell; US0612752413iuding molecules add computer-based methods of increasing the binding amity thereof;; USOd07151 S Dirner and multimer forms of singlt chain potypeptides; US46054297 Htnnaaized antibodies and methods for makingtham;

O!; PROTEINS AND METHODS OF USE THEREOF; US06008023 Cytuplasmic expression of antibodies, antibody fragments and ant-body fitagment fhslon proteins in E.
colt; Phagemid for antibody screening; 05059808951 I/09/1999 Tmmumotoxin cpntalning a disulfide-5tabilited antibody fiagntent joined to a Pseudomonas exotoxin that does not require protealytie activation;
0505962255 Methods for producing recombinant vectors; US05955341Heterodimerie receptor libraries using phagemids:

COMPOSIT'lONS THERjv,l OR; W009931267 METHODS FOR THE SIMtJLTANEQT,J$
1DENTTFICATTQNQF NAVEL BTpLOGTCAt~ TARGETS AN17 LEAl7 S'fIZUCTURES FOR
riRUGri)sVBLOPMEN1; US05$69619 Modlflcd antibody variable domains;
US0585588515olativn and production ofcatalytic antibodies using phage technology; US058518011212?/1998 Method of preparing polypeptlde binding compositions derived from immunoglobutin variable regions;
US05$49500 Phagentid fqr antibody scree»ng;binding composition; US05$37$4b111171199$ l3iosynthetlc binding proteins for immuno-targEting; US0582133710113/1998 Immunoglobulin variants;
US05821123ModitSed antibody variable domains; US0578965508104/1998 'Transgenic animals expressing artificialepitope-tagged proteins; USDS783384 Selection of binding-molecules; US05780225 Method for generating Libarics oC
antibodygenes comprisirto ampliftcation ofdiverse antibody DNAs and methodsfor using these libraries for the production ofdiverse antigen combining molecules; US057703561'hagetnids coexpresslng a surface receptor and a surface heterologous pmtein; W409$08b03 lsoLATION of IMMUNOGLOBUL1NS;
0505716805 Methods of preparing solubiE, oligvmeric proteins; US05595898 Modular assembly of antibody genes, antibodiesprepared thereby and use; US05582996 Bifunctional antibodies and method of preparing same; US05580717Recombinant library screening methods; ETaab BB, Dttnham MJ, Brown PO
.h'rotein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.Genome Biol. 2001; 2(2):: Moch H, Kononen T, Kalliprliemi OP, Sauter G. Tissue microarrayS
Borrtbaeck CA.Antibodics in di$geostics - from immunoassays to protein chips.Immunol Today. 3000 Aug; 21 (8):379-82 Mendoza LG, et alHigh-throughput microarray-based enzyme-linked imtnunosorbent assay (ELISA).
Biotechniques. 1999 Oct; 27(4):778-80, 782-6, 788. Morozov VN, Morozova TYa Electrospray deposition as a method for mass fabrication of mono- amdmulticomponent microarays of biological and biologically active substances_Anal Chem_ 1999 Aug I ; 71 ( 15);3 l 14-7.619: Lueking A. et alProlein microarrays for gene expression and antibody screening. Anal Biochem. 1999 May 15; 270(1):103-11. Silzel JW, et alMass-sensing, multianalyte microarray immunoassay with imaging detecrion.
Clin Chem. 1998 $cp;
44(9):20363 Elcins RP.Ligand assays: from elecupphoresis to miniaturized microarrays.Clin Chem. I998 Sep; 44(9):2015-30.
All publications referred to herein are indicative of the level of skill pf those in the art to which the invention pertains, With respect to lymphatic vessel associated ligands see also US 5, 776,759 (flt4), Mod Path412000 Feb;l3(2):180-5; EMBO J 2001 Mar 13;20(6):1323-1231, Nat Med 2001 Feb;7{2):199-205 inhibition of lymphangiogenesis with resulting lymphedema is tranggenic mice expressing soluble VEGF receptor-3 (VEGFIt-3), J Parhol 2001 Fcb;193(2):147-54 Localization of vascular endothetial growth factor-b in r,~aligtrant melanoma suggests a role in tumour angiogenesis.
With respect to technologies having application herein see also lmmuniry 2001 Apr; !4(4);437-46 The immunological barrier to xe~norransplantationCascalho M, Platt 1L.; W4 01/43779; WO 0!/42285; WO
98/10795; WO 41/40803; WO 40!14212; Gastroenterology 2001 May;120(6):1330-8 An engineered human antibody to TNF (CDP571) for active Crohn's disease: a randomized double-blind plaeebo.eontroiled fial.
8andborn WJ; Wo 01144282; w0 011403D9; WO 01140274; W4 01144340; Ann Rhatun Dis zoo!
May;60(5):433 Cancer and autoimmunity: autoimmune and rheumatic features in patients with malignancits, Abu-Shakra M, et al.; WO 01/40468; WO 01/40307; WO 01/42297; WO
01/42294; WO
01/43296; Wp 01/40456; WO DI/40308; WO 01142306; Curr Opin lmrnunoi ?Qpl Apr;l3(2):134-ll4 hrununity against cancer: lessons learned from melarxoma. Houghton AN, Gold JS, Blachere NE.; WO
01/422$$; w0 01/422$8; WO 41/43771; Wb 01/42308; WO 01/41804; WO 01/39722; WO
011448D8;
WO 01/43770; WO 01/16166; WO 01/41803; WO 01113110; WO ODl32752; WO 98/33528;
WO
01/43595; J Am Pharm Assoc (Wash) 2001 May-Jun;41(3):383-91 Magic bullets finally find their mark.;
Leukemia2001 Apr;lS(4):675-6; W001144301; AnticancerRes2441 Jan-Feb;21(IB):621-lmmunotherapy fpr rtCprre~r GQlprectal C&tlCeYS with human monoclonal antibody SIC-1. ICoda IC et al.;
WO 01/10911; WO 01/42546; Int J Clin lyract 2001 Apr;55(3):211-6 Tumour necrosis factor as a therapeutic tSrgCt in rheumatoid arflltiLis and othBr chronic inflammatory diseases: the clinical experience with infliximab; WO O1 J44472; WO 01140302; WO 01140305) With respect to surface plasmon resonance measurements of afFnity see US6111652:J-ligh throughput surface plasmon resonance analysis system U506?084~ Surface plasnen scnspr >~POt p$0365 SURFACE
PLASMON RESONANCE SENSOR FOR THE SIMULTANEOUS MEASUI~MENT OF A

I'LURALfTY OF SAMJ'LES 1N FLUID FORM WOOO10623bA HIGH THROUGHPUT ANALYSIS OF
MOLECULAR TNTERACTTON USING SURFACE PLASMON RESONANCE High throughput surface plasmon resonance analysis system as well as DImensions of antigen recognition and levels of immunological specificity_ Adv Cancer lees. 2001;80:147-87. Use of optical blosensors for the study eyf mec6anlstically concerted surface adsorption proeesses_ Anal Bioehem_ 2pp1 tan 15;288(2):109-25 Experimental design for analysis of complex kinetics ping st,rface plasmon resonance. Methods. 2000 NIar;20(3):310-8. , and references cited in the foregoing references.
27: Skeie GO, Lunde PIC, Sejsrstad OM, Mygland A, Aarli JA, Gilhus NE.
Autoimmunity against the ryanodine receptor in myasthenia pravis, Acts Physiol Soand_ 2001 Mar,171(3):379-84_ 28: Haufs Mf3, Haneke E. Epidermolysis bullosa aequisita treated with basiliximab, an interleul:In-2 receptor antibody.
Acta Derut Venereol. 2001 Jan-Fab;81(1):72. Woo J;Y, et al Regulatory CD4(+)CIY25(+) T pelts in tumors from patienta with early-stage non-small cell tong cancer end late-stage ovarian Cancer. CarlCCf Rts. 2001 tun 15;61(12):4766-72. Batrera P, Joosten LA, den Breeder AA, van Dc Putts LB, van Riel PL, van Deu Berg WB. Effects of treatment with a Fully human anti-ttunour necrosis factor alpha monoclonal antibody on the local and systemic homeostasis of interleukin 1 and TNFalpha in patients with rheumatoid arthritis.
Ann Rheum Dis. 2001 Ju1;60(7):660-9. Nlcholson JK, Browning $W, I-Iengel RL, Lew E, Gallagher 1.)r, Rimland D, Mcl7ougatl JS. CCRS and CXCR4 txprrssion on memory and naive T
cells in HIV-1 infection 8nd respousc to highly active antiretroviral therapy. J Acquit Immune Defic Syndr. 2001 tun 1;27(2):I05-15. Kung SK, Su RC, 5hattnon J, Miller RC3. Characterization of four new monoclpnal antibodies that recognize mouse natural killer activation receptprs. Hybridama. 2001 Apr;20(2):91-101. 43: )Jaak I, Dardik R, Levy V, Goldsttin I, Shoham J. Differential expression and regulation of CD6 on T-cell subsets revealed by monvclvn~l anhbvdy (MAb) CH11. Hybridoma. 2001 Apr;20(2)=75-84.
With respect to making multifunctional Uganda see also i,T$P 5,731, J 68 and 5,821,333.
With respect to TNF end TNFR variants, and functional fragments thereof, for use as antibody targets end binding moieties with respect to various aspects of the invention herein see WO 00/67793, WO 011303 D0, WO 01/44321, WO 00/62790, WO 01/03720, WO 00160079, WO 97/46686, Wp 01/41803, WO 01/38526, WO 01/37874, W0 01112812, WO 01/12671, WO 01/05834, WO 01103'720, WO 00/77191 WO 00/73321, WO 00/71150, WO 00/6'1793, WO 00/67034, WO 00166b08, WO 00166156., WO Ol /24811, as well as reFcrcncts cited therein. Many other TNFR variants and TNF analogs are known In the art.
With respect to cytoklnes and cytokine receptors see also the latest editions of Cytakine Reference: A
Compendium of Gytokines and Othtr lVttdiators of Hast Defense by Joost J.
Oppenheim (Editor), Jan Vilcek, Nicas A. Nicole (Editor); Cytokine Molecular Biology : A Practical Approach by Frances R.
Balkwill (Editor}, Fran Balkwill (Editor); Guidebook to Cytokines and Their Receptors by Nicos Nicole (Editor); The Cytokine Network and Immune Functions by Jacques '/haze; Navel Cytokine Inhibitors by Gerry A. T-lis,~s (Editor), Brian Henderson (Editor); Homology Folding of Proteins : Application to Cytokine Engineering by Subhashini Srinivasan; Cytokines and Cytpjdne Receptors (2001); lnterrtational Review of Experimental Pathoipgy : Cytokine-Induced Pathology, Part B :
Tnflammatory Cytokines, Receptprs, and Disease by G.'W. Richter, Icim Sol~z (Editor).
With respect to antibodies that bind to CCRS sa: Mot Biol CeU 2002 Feb; 13(2) : 723-737.
With respect to variations in chemokine receptors, cytokine and other receptors that can ht exploited according to one or more aspects of the invention herein sec 1: C5aSZar A, Abel T.Receptor polymotphisms and diseaSes,Bur J PharlnacoI. 2001 Feb 23;414(1):9-2z. 2:
GibejovaA.Chemol:ine receptors.Acta Univ Palacki Olomuc Fac Med. 2000;143;9-18. 3: Nishimoto N, Kishimoto T, Yoshlzaki K.Antl-interleukin b receptor antibody treatment in rheumatic discasc_Ann Rheutn 015. 3000 Nov;59 Suppl 1:i.21-7. 4: Aggarwal $B.Tumour necrosis factors receptor associated signalling molecules and their rolein activation of apoptosis, INK and NF-kappaB.Ann Rheum Dis. 2000 Nov;59 Suppl l:iG-l6. 3:
Qrignani G, Maiolo A.Cytokines and hemostasis.Haematologica. 2000 Sep;83(9):967-72. 6: Idriss HT, Naismith 1H.7-~' alpha and the TNF receptor yuperfamily: structure-action relationship(s).Microsc Lies Tteh. 2000 Aug 1;50(3):184-95. 7: van Doventtr Sl.Cytokine and cytokine receptor pvlymnrphisms in infectious disease.Intansive Care Med. 2000;26 Suppl I:S98-102. 8: Gessner A, Rollinghoff M.Biologic functions and signaling of the interleukin-0 receptor complexes.lmmunobiology_ 2000 dan;201(3-4}:285-307_ 9. Plaianias LC, Fish EN.Signaling pathways activated by interferons.Exp Hematol. 1999 Nov;27(11):1583-92. 10:
Schwertschlag US, Trepicchio WL, Dykstra KH, Keith JC, Turner ICJ, Dorner AJ.Hematpppietic, ilnxtluoo~OdulaOOry and epithelial effects of interleukin-I l.Leukemia_ 1999 Srp;l3(9):1307-15_ 11: 731asi F.~'he uroklnase receptor. A cell surface, regulated chernoklnc.APMIS. 1999 Jan;107(1):96-101.12:
Izuhara K, Shirakawa T.Sigrtal transduction via the intarleukin-4 receptor and its correlation with atopy.Int J Mol Med. 1999 Tan;3(I):3-10. 13: Tsokos GC, Liossis SN.Lymphocytcs, cytokines, intlamrnation, and immune traftlcking.Curr Opfn RheumatoL 1998 Sep;lO(5):417-25. 14: Morishita R, Nakamura S, Hayashi $, Aoki M, MatsuSl~ita H, Tomita N, YamemotoK, Moriguchi A, Higaki 1, Qgihara T,COiltr(butioll of ti vascular modulator, hepatocyte growth fhctor (HGF), to tbepad~ogenesis of cardiovascular disease.) Atheroxler Thromb. 1998;4(3):128-34. 15: Kashiwamura S, Okamura H.[IL-18 and IL-l8 receptor].Nippon Rinsho. 1998 Ju1;56(7):1798-806. Japanese.l6: Paxion WA, Kang S.Chemokine receptor allelic polymorphisms= relationships to HIV resistance anddisease progreg5ion.Semin Itnntunol. 1998 Jun;lO(3):187-94. 17: Arend WP, Malyak M, Guthridge Cl, Gabay C.Interleukin-1 receptor antagonist:
role in biology.Annu Rev lmmutwl. 1998;16:27-55. 18. Camussi G, Lupin E_The future rale of and-tt,mot,r necrosis factor (TNF) products is the trcs~nentofrheumataid arthritis.zhvgs_ 1998 May;55(5):613-20. 19:
Tags T, Klshimota T.Gp134 and the bttarleukin-6 fatally of oytokines.Altnu Rev lmmunot. 1997;15:797-819. .~.0: Paul WE.lnterleukin 4: signa111Jtg meehaoisms and control of T cell differentiatiou.Ciba Pound Symp. 1997;204:208-16; discussion 2l 6-9; 1.(V)0 01/49321) TNF INHIBIT ORS FOR
THE
TREATMENT OF NEUROLOGICAL, RETINAL AND MUSCULAR DISORDERS2.(WO OI146261 }
METHOD FOR TREATING INFLAMMATIQN3_(w0 01144464) INTERLEUKIN-1-RLCEPTOR
ASSOCIATED KINASE-3 (IRAIG3) AND ITS USE 1N PROMOTION OR 1NHT13JTION OF
ANGI0C3ENESIS AND CARDTOVASCULARIZATION4,(WO 01/40464) INTERLE1JK1N-1-RECEPTOR ASSOCIATED KINASE-3 (IRAK3) AND JTS USE IN PROMOTION OR INHIBITION OF
ANGIOGENESIS AND CARDIOVASCULARIZATtpNS.(wp 01/30850) UMLR
POLYPEPTlI7ES6.(W4 00/77195) NUCLEIC AG117 ENCODTNG NOVEL EGF-LIKE GROWTH
FACTORS7.(WO 00174719) METHOD OF TREATING CARCINOMA U51NG ANTTBODY THERAPY
AND AMELIORATING 51DE EFFECTS ASSOCTATED WITH SUCH'fFILRAPY8.(WO 001025$2) TREATMENT OF CELIAC DISEASE WITI41NTERLEUK1N-) g ArffAGONIST99.(WO 9914717D) PREVENTIVES OR REMEDIES FOR INFLAMMATORY INTESTINAL DISEASES CONTAINING
AS T» ACTIVE INGREDIENT IL-6 ANTAGONISTS10.(WO 99/46376) RECEPTOR FROM THE
SUPERFAMILY OF TNT-RECEPTORS FROM THI; HUMAN LLl'NGl !.(WO 99143809) PROTEAS>J-ACTIVATED RECEPTOR 4 AND U5ly$'fTiIrREOFI2.(WO 98/48017) hAMI:E.Y OF
IMMUNOREGCJLATORS bESIGNATED LEUKOCYTE INIMUNOaLOBULIN-LIKE RECEPTORS
(LIR)13.(WO 98/47923) IL-SR ANTAGONISTS FOR TREATMENT OF INFLAMMATION, ASTHMA
AND OTHER ALLERGIC DISEASES14.(WO 98/46620) A NpVET ~JM~,,N G-PR4TEI1V COUPLED
RECEPTOR15.(WO 98146285) METHQIaS FOR USING ANTAGONISTIC ANTI-AVB3 INTEGR1N
ANTT$OJaIESlb.(WO 9$/36767) MODL1LAT14N OF THE HYPOTHALAMIC-FITUITARY-ADRENAL-ADIPOSE AXlS WITH LEPTIN REC>~PTOR LIGANDS17,(W O 98131809) HUMAN CC
CHEMOKINE SLC18.(WO 98/30706) COMPOUNDS, COMPOSITIONS AND METHODS I~OR THE
ENDOCYTJC PRESENTATION OF IlvlTTUNOSUPPRESS1VE FACTORS19.(w0 9$/24$17) NOVEL
DNA, NOVEL PRQTE1N, AND NOVEI:. ANTIT3ODY20.(WO 98/22499) NEURON AND NEURAL
TUMOUR CiRO'~VTH RI3GULA'Y'ORY SYSTEM, ANTIBODIES THERETO AND USES
THEREOFZ1.(WO 98/19706) IDENTIFICATION OF UNTQTJE BINDING INTERACTIONS BETWEEN
CERTAIN ANTIBODIES AND TAE HUMAN 87.1 AND 137.2 CQ.STIMULATORY
ANT1GENS22.(W4 98118456) PROTEASE-ACTIVATED RECEPTOR 3 AND USES
THEREOI~'23.(WO
98114480) G PROTEIN-COUPLED RECEPTOR ANTAGONISTS 24.(W0 98!02341 } GAMMA-HERFGULIN25.(WO 97/49818) G-BETA-GAMMA REGULATED PHOSPHATfpY~.T~IQSI'fOL.3' KINA$E2(5.(WO 97/4$8(14) TIE-2 RECEPTOR LIGANDS (TIE LIGANI)-3; TIE L1GAND-4) AND
TIiEIR USE527.(WO 97/41225) MAMMALIAN MIXED LYMPHOCYTE RECET' TORS, CHEMOKINE
RECEPTORS [MMLR-CCR]28.(WO 97/24373) MONOCLONAL ANTIBODY ANTAGONISTS TO
HAEMOPOIETIC GROWTI4 FACTORS29.(WO 97121732) DESIGN OF HpRMOrTE-LJKE
ANTIBODIES WITH A(30NIST1C AND ANTAGONISTIC FUNCTIONS, 6,235,880 Htnnan sulfonylurea t~coptor 6,221,660 DNA eneosll,tg SNORFZS receptor 6,214,797 Urocottin peptides, nucleic scid encoding same methods for usingsame6,314,344 Hepatocyte g,-owth Factor receptor antagonists and uses thereof 6,210,904 Anticoagulant test6,207,1521-lepatocyte growth factor receptor antagonists and uses thereof6,204,017 Palynucleotide encoding a histamine receptor 6,197,541 Recombinant thrombin receptors and assays using them6,184,358 lp-101Mig receptor designated CXCR3, antibodies, nucleic acids,and methods of use therefor6,177,079 Antagonists of interleukin-156,177,078 Monoclonal antibody autagatusts to JL-36,177,077 TNT inhlitara for the treatment of neumlogical disorders 6, T 7I ,815 I~uman sulfonylurea receptvr6,168,783 Antagonists of interleukin-I56,166,185 Antibodies to human TIE-2 ligands6,165,466 Antagonists of interleukin-156,162,431 Serine/threonine protein kinase6,143,870 Thrombin rectptor homolog6,136,957 Antibodies which bind ~anulocyte-macrophane eolony-stimulatingfactor receptor6.124,101 Recombinant thrombin reocptor and related pharmaceutieals6,11 L,075 Pratese-activated receptor PAR4 (ZCH.6NIRZ) 6,103,874 Human iGDEL receptor 6,096,873 Gamma-heregulin 6,086,874 Antitumor agent effect enhancer containing 1L-6 antaganist56,084,07s Agpttist and antagonist antibodies to the chetnokine receptor-2(CClt2) 6,063,596 Gprutein couplod I'eceptor9 associated with immune responsa6,054,292 T-cell recepppr protein6,043,212 l~tCOmbinabt C 140 receptor, its agonists and antagonists, andnucleic acids encoding the receptvr6,033,869 poIynucleotide encoding a rtovCl human cytokinelsteroid receptor 6,024,936 Antibody-based method of localizing activated thrombitmceptors 6,017,763 G-beta-gamins regulated phosphatidylinositol-3' kinase6,0I3,480 Antagonists of interleukin-156,013,479 Human Emrl-ltlce G protein coupled receptor 5,994,097 Polynucleotide encoding human G-protein coupled reoeptor5,985,828 Mammalian receptprs for interluukin-10 (IL-10) 5,985,583 Cloning and expression of gOnadotrOpht-releasing hormonereceptor5,977,072 F~igh affinity tr»,tnun0i;lobt11111 E rtCeptor-like protein5,976,852 K.kappa./µ-like protciu tyrosine phosphatase, pTP .lambda5,976,815 Bioassay using ALK-7, a novel serine threonine kinasc receptor 5,972,62 l Methods of iderrtifying compounds that modulate body weightusing the OB
receptor 5,965,709 IgE antagoniats5,965,365 Serine/tltreoniue protein kinlse5,935,303 Human chemokine receptor-hke protein 5,952,173 DNA encoding a human prose:terone receptor camplox p23-1'keprotein5,945,308 ITuman oxidized LDL roceptor5,942,606 Viral receptor protein 5,928,887 .kappa./mu.-Like protein tyrosine phosphatase, FTP .lambda.5,912,144 Edg-1-receptor homolog3,903,383 Methods of inducing T cell ~nreaponsivaness to donor tissue ororgan In a recipient with GP39 ants;onists 3,892,014 DNA encoding a protease-ttct3vated receptor 35,891,720 Isolated DNA
tncodlng a novel human G-protein coupled receptor5,891,674 Insulin receptor tyrosine kinase substrate 5,891,638 Serine ihreoninc kliussa YaGaptor, elk-75,888,811 Cortieotropin-releasing hormono receptor5,888,51D Chronic rheumatoid arthritis therapy containing 1L-6 antagonistas efkctivo component 3,886,148 Parathyroid hermone receptor5,874,40Q Recombinant C L40 receptor, its agonists and antagonists, andaucleic acids encoding the receptor 5,874,273 G-taro-gamma regulabvd phosphaddylinositob3' kinase5,874,224 Growth factor receptor binding protein 5,871,930 High affinity itmnunoglobulin ); receptor-like protein 5,869,633 Thrvntbin receptor homolog polynucleotide5,869,609 G protein coupled glutamate reeeptors5,869,2? I G~
beta-gamma regulated phusphatidylinositol-3' kittase3,869,049 Methods of inducing T Cell um~esponsiveness to bone marrow withgp39 antagonists 5,863,796 Antibodies which specifically bind mammalian receptors forlnterleukln-10 (1L-10)5,863,766 Human sigma receptor 5,859,201 G-beta-gamma regulated phosphatidylinositol-3' kinase5,856,448 Antibodies specifically reactive with thrombin receptor and itscompononts 5,856,133 G-beta-gamma raattlated phosphatidylinositol-;'kina5e5,$36,132 G-btta-ganuna regulated phosphatidyllnositol-3' kina~$,851,797Tie ligand-3, mtthods aftnaking and uses thereof3,840,833 Paratliyroid hormottt receptor and DNA encoding same5,837,499 DNA encoding C3A
receptor ant<-tganists having substantially noagonist activity and methods of expressing same5,834,240 DNA encoding a tt'ansforming growth factor-.beta. receptor associated protein 5,$33,987 Treatment of T
cell mediated autoimmune disorders5,$31,047 Oligonucltotide probes to L-AP4 sensitive glutamate receptorstqutnees 5,830,678 Method for identifying a target peptide that modulates thabinding of epinectitt ligand to integrin receptors5,824,500 Nucleic acid encoding novel human ICD>rI, receptor 5,817,48017NA encoding a histamine H2 receptor5,814,507 _kappa,/,mu. like prortehl tyrosi>yt phvsphatase, PTP .lambda.5,814,464 Ntceteie acids etlCOding TIE-2 llgand-25,811,245 Antibodies that specifically bind tp ALK-7, a novel 5erinethreori$ie ldnase reeeptor5,807,$24C5 A receptor antagonists having subatantiaIly no ago>tistactivity5,795,966 Antagonists of ittterleukin-155,789,565 5erine threonine kinase t~ecsptor, ALK-7 5,789,192 Mammalian receptors for interleukin-10 (IL-10)5,763,575 Agonist and antagpn[st peptides of the 0140 receptor 3,759,994 Recombinant thrombin receptor and related pharmaceutica155,750,366 Cloning and expression of gonadonopiu-releasing hormoneraceptor 5,747,279 Nuclele acid molecules encoding kappa₃ opioid receptors,receptors encoded thereby, and uses thereof3,747,267 Method for identifying a G protein coupled glutamate receptoragonist and antaaonist3,738,999 L-AY4 sensitive glutamate t~eceptprs5,730,976 Method for h~atiug raacrophage pathogon infections by TGl~-$aatagon1st55,726,036 Granulocyte-macrophage colony-stimulating factor receptor andderivatives thereof5,721,107 Antibodies to G protein coupled glutamate receptors5,716,804 Mammalian interleukin-1 D (ll.-10) super~activating receptors;and variants5,716,789 Method to determine ligands, agonist and antagonist of C140receptor 5,707,632 Receptery fpr t~rpbltlst growth fectors5,688,768 Recombinant thrombin receptor and related pharmaeeutiea1s5,686,597 Thrombin receptor honwlog 5,686,292 Hepatocyte growth factor receptor antagonist antibodies anduses thareof5,683,884 Methods for identifying modulators of human calcitoninmediated metabolism5,683,693 Method for inducing T cell utuesponsiveness to a tissue nrorgan graft with anti-CD40 ligand antibody or soluble CD405,674,981 Eluman calcitonin receptor polypeptides3,674,689 Human calcitonin receptor polypeptides and methods of use5,646,036 Nucleic acids encoding hepatocyte growth factor reecptorantagonist antibcxties5,629,?83 Granulocyt~macrophaga colony-stImulatlng factor receptor andderivadves thoreof5,622,$39 Recombinant production ofhuman calcitotiln receptorpolypeptldCS5,614,609 Serine rhretmine kinase r~eptor5,356,780 CDNAS sncodlng mouse and rat type-2 angiotensin II reccptorsand tholr expresSioD in hOSL ce1155,543,143 MCthod for activating macrophames/monorytes5,516, 894 A.sub_2h -adCbosiriC rtceptors5,514,555 Assays and therapeutic tntthods based on lymphocytechemoattractants 5,505,107 Selecting li8and agonists and antagonists5,494,8D6 DNA and vectors encoding the parathyroid hormone receptor,transformed cells, and recombiuant production of PTHR
proteins andpeptides5,451,658 Antagonists of human gamma interfaron5,441,935 Growth factorreceptors5,385,831 Method for producing a mammalian G protein coupled glutamatereceptOT5,334,380 Anti-elldotoXill, idtCrleukin-1 receptor antagonist andanti tumor tleCroSis ftlctor Mdbody with arginine-free formulations forthe tt~eatment of hypaterasiot15,356,766 Itecotnbinatlt thrombid receptor and related pharmacetrticals5,177,190 Puritied C5a receptor from human polymorphonuclCar ICukoGytes4,857,63? Methods and compositions for inununologically modulating growthhvnnone receptor activity; and ref~r~ttecs aired therein. (sea also 1.(W0 01149744) MOUSE G-PROTEIN COUPLED RECEPTOR MAS 2.(W0 01/49726) A NOVEL
POLYPEPT1DE-HUMAN NATRNRETIC PEPTIDE RECEPTOR 18 AND TH>r POLYNUCLEOT1D)E
BNCObING SAID POLYPEPTIDE 3.(W0 01/49321) TNF INfIIBITORS FOR THE TREATMENT OF
NEUROLOGICAL, RETINAL AND MUSCULAR DI50RDER5 4.(W0 01/00657) NOVIJL INDOLE
PEPT1DOMIMETICS AS TI~OMBIN RECEPTOR ANTAGONTST5 S.(w0 00!62790) SOLUBLE
TUMOR NECR0515 FACTOR RECEPTOR TREATMENT OF MEDICAL DISORDERS 6.(W0 01/03720) PROMQT10N QR CNH1SITZQ1~1 OF ANGI4GENESIS AN17 CARD10VASCULART7AT10N
BY TUMOR NECROSIS FACTOR LIGANDIRECEPTOR HOMOLOGS 7.(W0 01146261 ) METHOD
FOR TREATING INFLAMMATION 8.(W0 01!46191) 4-[ARYL(8-AZA$ICYCLO[3.2.1]OCTAN-3-YL)]AMTNOBENZOIC ACID DERIVATIVES 9.(W0 01146176) NQN PEPTIDE TACI-IYI~ININ
RECEPTOR ANTAQONISTS 1 D.(WQ Q [/45730) TWEAK RLCE.P'TOR L 1.(W0 01/45703) NITROSATED AND NITRQSYLATED CYCLOOXYGENASE-2 )1VH113TfORS, COMP051T10N5 AND METHODS OF USE 12.(W0 01140464) INTERLEUICIN-I-RECEPTOR ASSOCIATED
IUNASE.3 (1RAK3) AND IT5 USE 1N PROMOTION OR INHIBITION OF ANGIQGENESIS AND
CARDIOVASCULAR1ZATION I3.(WQ 01/44213) NL~W 1y2X7 RECEPTOR ANTAGONISTS FOR USE
IN THE'fREATMENT OF INFLAMMATORY, IMMUN$ OR CARDIOVASCULAR DISEASES
14.(W0 01/42268) DOG OREXIN 1 RBCEPT'OR 15.(W0 01/42208) CYCLOAMINE CCRS
RECEPTOR
ANTAGONISTS 16.(W0 01/41752) 150FORM 5PECffIC INHIBITION FOR TREAT~(ENT OF
PAIN
AND REDUCTION OF ANESTHETIC THRESHOLD 17.(W4 01/03720) PROMOTION OR
INHIHITIQN OF ANGIOGENESIS AND CAIi171OVASCULARIZATION BY TUMDR NECROSIS
!;ACTOR L1GAND/RECEPTOR HOMOLOGS 18.(W0 01140464) 1NTERLEUKiN-1-RECEPTOR
ASSOCIATED K1NASE-3 (IRAK3) AND ITS USE IN PROMOTION OR INHIBITION QF
ANGIOGENESIS AND CARDIOVASCULAR1ZAT1QN 19,(WQ 01140259) MONKEY QREXitV 1 RECEPTpR 20.(W0 01/40252) MONKEY CALCIUM SENSI1VG RECEPTOR 2!.(W0 01104139) HUMAN A?COR29 RECEPTOR 22.(W0 O I 136480) MOUSE 7-TRANSMEMBRANE RECEPTOR, AXOR45 23.(W0 01/00656) NOVEL INDAZOLE PEPTIDOMIMETiCS AS THROMBIN RECEPTOR
ANTAGONISTS 24.(W0 00167793) DEATH DOMAW CONTAINING RECEPTOR 4 25.{WO
01/34645) MODULATING 1L-13 ACTIVITY USING MUTATED IL-13 MOL1:CULES THAT ARE
ANTAGONISTS OR AGON1S'fS Ol~ IL-13 26.(W0 0113413 B) COMPOSITIONS AND METHODS
FOR
TREATMENT OF N);UROLOGICAL DISORDERS AND NEURODEGENERAT1V)3 DISEASES
27.(W0 01/32656) POLYMORPI~C FORM Ola A TAQI-TYI;f,ININ RECEPTOR ANTAGONISE' 28,(W0 01!321 G6) NEW COMBINATION COMPRISING A β2-ADRENORECEPTOR AGONIST AND A
LEUKOTRIENE RECEPTOR ANTAGOMST 29.(W0 01132163) NEW GOM$TNATION COMPRISING
A BETA 2 (d;#946;)2 ADRENO RECEPTOR AGOMST AND A LENKOTRIENlr RIrCEpTOR
ANTAGONIST 30_(W0 01/01922) USE QF STJ$STANCE P ANTAGONISTS FOR THE TREATMENT
OF ADEN4CARC)rIOMA 31.(W0 01/30$50) UMLRPOLYPEPTIDES 32.(W0 01/27153) A MURINE
SEVEN-TRANSMEMBRANE RECEPTOR, MUS MUSCULUS MHNEAA81 33.(W0 D1/25269) NOVEL HUMAN G-PROTGIN COUPLED RECEPTOR 34.(W0 01124828) MODULATORS OF
CYTOKL1VI:: MEDIATED SIGNALLING PATHWAYS AND INTEGRIN αV~#946;3 RECEPTOR ANTAGONISTS FOR COMBINATION THERAPY 35.(W0 O1 /24?98) USE OF CENTRAL
CANNA13INO1D RECEPTOR ANTAGOMST FOR PRI3PARING N>EDlCI3JES 36.{WO 01/24797) LNTEGR1N RECEPTOR ANTAGONISTS 37.(W0 0016$250) 7TM RECEPTQR RAT APJ 3 $.(W0 01/16121) HETEROCYCL1C G4MPOUNDS AND METHODS OF USE THEREOF 39.(W0 01/14406) ANTIANDKOGEN At3ENTS 40.(W0 01112671 ) ):IUMAN TUMOR NECROSIS FACTOR RECEPTOR
TR1~ 41.(W0 01/10891) Ih-16 ANTAGONISTS 42.(W0 01/10889) RAT-G-PROTEIN COUPLED
$1 RECEPTOR BRS3 43.(W0 01/10423) USE OF 5-HT3 RECEPTOR ANTAQQNIS'1~S FOR THE
TREATMENT OF 1NFLAMMATiON$ OF THE RESPIRATORY TRACT 44.(W0 01/0702$) THE U SE

4S.(WO 01105834) HUMAN TUMOR NECROSIS FACTOR RECEPTORS TR13 AND TR14 46.(W0 0 L/05783) HRADYKINITf B1 RECEPTOR ANTAGONISTS 47.(W0 01104139) POLYNUCLEOTiDE

SCREENING FOR AGONISTS AND ANTACrONISTS OF THE INTERACTION BETWEEN I3UMAN
AXOR29 RECEPTQR AN'D TtS L1GANDS 48_(W0 01103720) PROMO'CION OR lNHiBITION OF
ANGI4GENESIS AND CARDIOVASCULARIZATlON !3Y TUMOR NECROSIS FACTOR
L1GANDlRECEPTOR HOMOLOGS 49.(W0 01101922) USE OF SUBSTANCE F ANTAGONISTS IN
THE TREATMENT OF THE ADENOCARCINOMAS 50.(W0 01/00659) BENZIMIDA20LONE
PEPTIDOMIMET1CS AS THROMBIN RECEPTOR ANTAGONISTS 51.(W0 01100657) NOVEL
INDOLE PP.ET)DOMIMETICS AS THROMBIN RECEPTOR ANTAGUN1STS 52,(W0 01/00656) NOVEL WDAZOLE PEPT1DOMlMETICS AS THROMBIN RECEPTOR ANTAQONJSTS S3.(WO
O I/00575) 1NDQ~,E AND 7NDAZOLE URk:A.PET'TOIDS AS TFIROM$IN REC$PT OR
AIV~'AGON1ST5 54.(W0 01/00198) COMPOSITIONS AND METHODS OF TREATING CANCER
USTNG COMPOSITIONS COMPRIS1N(3 AN BQIEBITOR OF ENDOTI~LIN RECEPTOR ACTIVITY
55.(W0 00/7831'7) INTEGRIN RECEPTOR ANTAGONTSTS 56.(W0 00/77195) NUCLEIC ACID
1~NCOD1NG NOVEL EGF-LIKE GROWTIi FACTORS 57.(W0 00/76502) METFIpDS AND
CpMPpSITTONS FOR TR.EATTN'G RAyNAUD'S PI~:NO1VIENON ANb SCLERODERM~158.(WO
00/74719) METHOD OF TREATING CARCINOMA USING ANTIBODY THERAPY AND
AMELIORATI1VG S1DB EFFECTS ASSOCIATED WITH SUCH THERAPY 59.(W0 00/73321) HUIviAN TUMOR NECILOSIS FACTOR RECEPTOR TRIO 60.(W0 00/72801) ALPHA V INTEGR1N
REC>~PTOR ANTAGOMSTS 61.(W0 00/71150) TUMOR NECROSIS FACTOR RECEPTOR 5 62.(W0 00/69831) SPIROtMTDAZOLIDTNE DERIVATIVES, THEIR PREPARATION, THEIR USE AND
PHARMACEUTICAL PREPARATTONS COMPRISING THEM 63.(W0 00/69820) CYCLIC AMIZVE
DERIVATIVES AND THEIR USES 64.(Wp 00/69463) COMPOSITIONS AND METHODS FOR
TREATING CELL PROLIPIrIL4TION DISORDERS 65.(W0 00/69459) TREATMENT OF
REFRACTORY HUMAN TUMORS WITIi EPIDERMAL GROWTH FACTOR Rk',CEPTOR
ANTAGONISTS 66.(WU 00/68250) 7TM RECEPTOR RAT APJ 67.(W0 00168244) 7TM
RECEPTOR
MOUSE APJ 68.(W0 00/67793) DEATH DOMAIN CQNTA)NING RECEPTOR 4 69.(W0 00/67034) METHODS OF USE OF THE TACIITACI L INTF~tACTION 70.(W0 00167024) CANCER
TREATMEN f WITfI ENDOTHELllY RECEPTOR ANTAGONISTS 71.(W0 00/66632) AGONISTS OR
ANTAGOMSTS FOR HAEMOFOIETIC GROWTH FACTORS 7?.(WO 00/66522) GLUCOCORTICOID RECEPTOR MODULATORS 73.(WQ 00/66156) DEATH DOMAIN
CQNTATNTNG RECEPTOR 5 74.(W0 00/64465) DEATH DOMAIN CONTAINING RECEPTORS
75.(W0 00/62790) SOLUBLE TUMOR NECROSIS FACTOR RECEPTOR TREATMENT OF
MEDICAL DESORDERS 76.(W0 00/5953?) THE USE OF DOMAINS OF TYPE 1V COLLAGEN T
INHIBIT ANGIOGENESIS AN TUMOUR GROWTH 77.(W0 00136862) HUMAN TUMOR
NECRpSIS FACTOR RI:CEPT4R TR9 ?8.(W0 00156405) HUMAN TUMOR NECROSIS FACTOR
REGEP'!'OR-LIKE 2 79.(W0 00154772) AMYOTROPIC LATERAL $CLER0515 TREATMENT WITH
A COMBINATION OF RILUZOLE AND AN AMPA RECEPTOR ANTAGONIST 80.(W0 00153596) IMiDAZOLE COMPOUNDS SUBSTITUTED WITH A Sl~: OR SEVEN MfiMHERED
HETEROCYCL1C RING CONTAINING TWO NITROGEN ATOMS 81.(W0 00/53175) COMPO1JNDS
AND MI~THODS $2.(W0 00/52x28) TUMOR NECROSIS /'ACTOR RECEPTORS 6&ag; and 6&bgr;
83.(W0 00/51974) ALFFIA~AMINOACETIC ACLD DERIVAT1VE5 USEFUL A5 ALPHA 4 BETA 7 -RECFPTOR ANTAGONISTS 84.(W0 00150459) HUMAN TUMOR NECRQSIS FACTOR RECEPTOR-LIKE PROTETN$ TR1 1, TR11 SV1, AND TR11SV2 85.(W0 00/49170) MUR1NE l lCby RECEPTOR
$6.(W0 00/48603) DIBENZO AZEPINE DERIVATIVES AS ~ag;V INTEGRIN RECEPTOR
ANTAGONISTS 87.(W0 00/48397) SYSTEMIC U5E OF 5-HT 3 RECEPTOR ANTAGONI51'S
AGAINST RHEUMATIC INFLAMMATORY PROCESSES 88_(WQ 00148581) USE 4F 5-IIT3 RECEPTOR ANTAGONISTS 89.(W0 00/d634$) $CREfiNINrs ASSAY FOR ANIAGONISTS OF
FGFR-MEDIATED MALIGNANT CELL TRANSFORMATION AND TUMOR FORMATION 90.(W0 00/46215) BENZAZEPINE DERIVATIVES AS ALPHA-V INTEGRIN RECEPTOR ANTAGON15TS
91.(W0 00146197) 1NDOLE DlSRIVAT1VES AND THEIR U5E A5 MCP-1 RECEPTOR
ANTAGONISTS 92.(WU 00/44763) COMPOSITIONS FOR TREATING INFLAMMATORY
RESPONSE 93_(W0 00143031) TUMOR NECROSIS FACTQR ANTAGONISTS AND THEjR USE TN
ENDOMETRIOSIS 94.(W0 00142852) COMPOUNDS AND METHODS 95.(W0 04140716) SOLUBLt RECEPTOR BR43xZ AND METHODS OF USING 96.(W0 00140239) COMPOUNDS AND METHODS

97.(VNO 00/39166) NOVEL HYA.LURONAN-BINDING PROTEINS AND >rNCODING GENES
98.(W0 00137462) NON-FEPTTDE NK 1 RECEPTORS ANTAGONISTS 99.(W0 00/35887) VITRONEGTIN
RIsCEPTORANTAGONIST PHARMACEUTICALS IQQ.(WO 00133492) VTTRONECTTN RECEPTOR
ANTAGONIST PHARMACEUTICALS 51.(W0 01100657) NOVEL INDOLE PEPTIDOMIMETICS AS
THROMBIN RECEPTOR ANTAGONI5T5 52_(W0 01100656) NOVEL INDAZOLE
PEPT1DOMTMET1C5 AS THROMBIN RECEPTOR ANTAGONISTS 53.(W0 01/00576) IrVDOLE
AND INDAZOLE UREA-PI'cPTOIDS A$ THROMBIN RECEPTOR ANTAGONISTS 54_(W0 01/00198) CoMP051T1UNS AN» METHt>DS OF TItEATIT1G CANCER USING C4MppSI'I'IONS
COMPRISING AN LNt3IEITOR OF ENDOTHELIN RECEPTOR ACTIVITa' S5.(WO OUI7S317) INTEQRIN RECEPTOR ANTAGONISTS 56.(W0 00177195) NUCLEIC ACID ENCODllVG NOVEL
EGF-LIKE GROWTH FACTORS 57.(W0 00176502) METHODS AND COMPOSTT10NS FOR
TREATING RAYNAUD'S PHENOMENON AND SCLERODERMA 58.(W0 00/74719) METHOD OF
TREATING CARCINOMA USING ANTIBODY THERAPY AND AMELIORATING SIDE EFFECTS
ASSOCIATED WITH SUCH THERAPY 39.(W0 ODJ73321) I-IUMAN TUMOR NECROSIS FACTOR
RZrCIJPTOR TItlO 60.(W0 00/'12801) ALPHA V 1NTEGRIN RECEPTOR ANTAGONISTS
61.(W0 00/71150) TUMOR NECROSIS FACTOR RECEPTOR S 62.(W0 00169831 ) SPTROTMTDAZOLTDINE
DERTVATTVES, THEIR PREPARATION, THEIR USE AND PHARMACEUTICAL PREPARATIONS
COMPR1S1'NG THEM 63.(W0 00/69820) CYCLIC AMINE DERIVATIVES AND TIiEIIt USES
64.(W0 00/69463) COMP4STTTONS AND METHODS FpR TREATING CELL PROLTFERATTON
DISORDERS 65.(W0 00/59459) TlUrA1"MENT Of REFRACTORY HUMAN T'UMOkS WITH
fiPIDERMAL GROWT1~I FACTOR RECEPTOR ANTAGONISTS 66.[W0 D0168250) 7TM RECEPTOR
RAT APJ 67.(W0 00/68244) 7TM RECEPTOR MOUSE APl 68.(W0 00/67793) DEATH DOMAIN
CONTAINING RECEPTOR 4 69.(W0 00167034) METAObS OF USE OF THE TACI/TACI-L
INTERACTION 70.(W0 40/67024) CANCER TREATMENT WTl"H ENDOTHELIN RECEPTOR
ANTAGOMSTS 71.(W0 OD166632) AGONISTS OR ANTAGONISTS FOR HAEMOPO1ET1C
GROWTH FACTORS 73.(W0 00/66522) GLUCOCORTICOID RECEP'fDR MODULATORS 73.(W0 00/66156) DEATH DOMAlN CONTAINING RECEPTOR 5 74.(W0 00164465) DEATH DOMAIN
CONTAINING RECEPTORS 75-(WO 00!b2790) SOLUELE TUMOR NECROSIS FACTOR
RECEPTOR TREATMENT OF MIrbICAL DESORDERS 76.(W0 00/59532) THE USE OF DOMAINS
OF TY')?>TV COLLAGEN T 1NHlBIT ANGIOGL'NESIS AN TUMOUR GROWTH 77.(W0 ODI56863) HUMAN TUMOR NECROSIS FACTOR RECEPTOR TR9 78.(W0 00!36405) HUMAN TUMOR
NECROSIS FACTOR RECEPTOR-LTKE 2 79.(W0 00154772) AMYOTROPIC LATERAL SCLEROSIS
TREATMENT WITH A COM$1NAT1ON OF R1LUZOLE AND AN AMPA RECEPTOR
ANTAGONIST 80.(W0 00153596) IMIDAZOLE COMPOUNDS SUBSTITUTED WITH A 51X OR
SEVEN MEM$ERED 1~IETEROCYCLIC RING CONTAINING TWO NITROGEN ATOMS 81 _(Wp 041531'75) COMPOUNDS AND METHODS 82.(W0 00/52428) TUMORNECROSI9 I"AC'TOR
RECEPTORS G&agr; and 6&bgr; 83.(W0 00/51974) ALPHA-AMTNOACET1G ACTD
DER.TVATIVES
USEFUL AS ALPHA 4 BETA 7 - RECEI''fOR ANTAGON1S1'S 84.(W0 00150459) HUMAN
TUMOR
NECROSIS FACTOR RECEPTOK LIKE PROTEINS TR11, TR11SV1, AND TR11SV2 85.(W0 40149174) NIURiNE llcby R>rCEPTpR 86.(W0 0014$403) IaIBE'~T7.0-AZ'EPTNE
DERIVATIVES AS
&$gr;V 1NTEGRIN RECEPTOR ANTAGON1S1 S $7.(W0 00/48597) SYSTEMIC USE OF 5-HT 3 RECEPTOR ANTAGONISTS AGAINST RHEUMATIC INFLAMMATORY PROCESSES 88.(W0 p0/48581 ) IJSE OF S-HT3 RECEPTOR ANTAGONISTS 89_(W0 00146343) SCREENINC3 ASSAY FOR
fINTAC,QNI$TS QF FGFR-MEDIATED MALIGNANT CELL TRANSFORMATION AND TUMOR
POTtMATION 90.(W0 00146215) BENZAZF,PINE DERIVATIVES AS ALPIiA-V INTEGRIN
RECEPTOR ANTAGONISTS 91.(W0 00/46197) INDOLE DERIVATIVES AND THEIR USE AS
MCP-I RECEPTOR ANTAGONI5T5 92.(W0 00/44763) COMP051TION5 FOR TREATING
INFLAMMATQRY RESPONSE 93.(W0 00/43031) TUMOR NECROSIS FACTOR ANTAGONISTS
AND 3'IIEIR USE IN ENDOMETRIOSIS 94.(GVO 00/42852) COMPOUNDS AND METHODS
95.(W0 00/40716) SOLUBLE RECEPTOR HR43x3 AND METHODS OF USING 96.(W0 0014D299) COMPDUNDS AND METHODS 97.(W0 00139166) NOVEL HYALURONAN-BINDING PROTEINS
AND BNCODING GENES 98.(W0 00/3?462) NON-PEPTIDL NK 1 RECEPTORS ANTACyONISTS
99.(W0 00/35887) VITRONECT1N RECEPTOR ANTAGONIST PHARMACEUTICALS 100-(WO
00/35492) VITRONEC'f IN RECEPTOR ANTAGONTST PT-IARMACEUTICALS 10I.(WO
00135488) VITRONECTIN RECEPTOR ANTAGONIST PHARMACEUTICALS 102.(W0 40/35455) HETEROARYL-ARYL UREAS AS IGF-I RECEPTOR ANTAGONISTS 103.(W0 00/32578) BL~NLIMIDAZOLE CDMPOUNDS THAT ARE V1TRONECTIN RECEPTOR ANTAGONISTS
104.(W0 D0128988) N1TROSATED AND NITROSYLATED H2 RECEPTOR ANTAGONIST
COMPOUNDS, COMPOSITIONS AND M>~THODS QF TJSE 105_(W0 00!27421) LOCAL USE OF

50LUBLE TUMOR NECROSIS RECEPTOR 1 (sTNFRl) FOR PROPHYLAXIS AND TREATMENT
OF CORNEAL TRANSPLANT RF~'ECTION AND OTHER DISORDERS OF THE EYE 106.(w0 00125$05) VASCULAR ENDOTHELJIAL GROWTH FACTOR-LTKE PROT$IN FROM ORE VIRUS
NZ2 BINDS AND ACTIVATES MAMMALIAN VEGF RECEPTOR-2 107.(W0 00!25745) 1_TttxIGATTON SOLUTION AND METHOD FOR INHIBITION OF PAIN AND INFLAMMATION
108.(W0 00/24393) NEW U5E OF GLI,JTAMATE s~A~TAGO~STS FOIx T1-1'E TREATMENT OF
CANCJGR 109.(W0 OO/Z3471) USE OF A C'Yl~OKtIVE-PRODUCING LACTOCOCCUS STRAIN TO
TREAT COLITIS 1 !0.(W0 00/23469) FRAGMENTS OF 1NSULTN-LIKE GROWTH FACTOR
BINDING PROTEIN AND INSULIN-LIKE GROWTH FACTOR, AND USES THEREOF 111.(W0 00123438 ) N-(IMIDAZOLYLALKYL)SUBST1TUTED CYCLIC AMINES AS HISTAMINE-I3 3 AGONiSTS OR ANTAGONISTS 1 I2_(WO 00123 I 13) PEPTIDE-BASED CARRIER DLV1CE5 FOR
STEI,LATE CELLS 113.(W0 00123066 ) IRRIGATION SOLUTION AND METHOD FOR

OF PAIN AND INFLAMMATION I 14.(W0 00123062) IRRIGATION SOLTJ'C'TpN ANl? METHOD
FOR iNl-TTHITI4N OF PA,TN AND TN~TION 115.(DVO 00!20378) A METHOD OF
MODULATING CELL SURVIVAi. AND REAGENTS USEFUL FOR SAME 116.(W0 00120389) NAPNTHALEh'ECAR190JCAMIfDBS AS TAGHYKININ RECEPTOR ANTAGONISTS 117.(W0 00120371) PROSTAGLANDIN RECEPTOR L1GANDS 118.(W0 00/20003}
NAPHTHALENECARBOJi;AMIDES A5 TAGHYK1NIN RECEPTOR ANTAGONISTS 1 t 9.(WQ
00114109) HAS1C PRODUCTS HAVING ANTAGONISTIC ACTIVITY 4N THE NI~-1 RECEPTOR
AND THEIR USE lld PI3ARNIAC1:UTICAL COMPOS1TION5 120.(W0 00110391) TFIE USE OF
ADENOSIhTE A3 RECEPTOR ANTAGONISTS TO INHIBIT TUMOR GROWTH 121.(W0 00109303) INTEGR1N RECEPTOR ANTAGONISTS 122,(W0 00/09152) TH)GRAPEUT1C QHEMQ~NE
1ZECEPTOR ANTAGON1$T"a 123.(W0 00/08401) SUBSTITUTEb ISOXAZOLE AS ESTROGEN
RECEPTOR MODULATORS 124.(W0 00106169) INTfiGRIN RECEPTOR ANTAGONISTS 123.(W0 00103716) TOPICAL COMPOSITIDNS COMPRISING AN OPIOID ANALGESIC AND AN NMDA
ANTAGONIST 126.(W0 00102859) N-SUBSTITUTED NAPHTHALENE CARI343CAM1DES AS
NEiJRQKI'1.TIN-RECEPTOR ANTAGONIST'S 127.( W O 00/02582) TREATMENT OF CELIAC
DISEASE WITH INTERLEUK1N-15 ANTAGONISTS 128.(W0 00/01802) PEPTIDE ANTAGONTSTS
OF TI~ HUMAN UROKWASE RECEPTOR AND METHOD FOR S13LECTING THEivI I29.(WO
00/00 J 94) OPHTHAL,M1C USES OF PPARGAMMA AGONISTS AND PPARGAMMA
ANTAGONISTS L30.(Wp 99/65944) 1P$1P'J'IpE INHIBITORS OF &sgr;V&bgr;3 AND
dtagr;VBcbgr;5 131.(W4 99/62955) METFIOD O~' 17ESIGN1NG AGONISTS AND ANTAGONISTS TO EGF
RECEPTOR FAMILY 132.(W0 99/60015) IM1DAZOL1DINE DERTVATIVES, THE PRODUCTION
THEREOF, THEIR USE AND PPARMACEUTICAL PRLPARATION$ CONTAINING THL SAME
133.(W0 99/59635) USL, OF A COX-2 INHIBITpR AND A NK 1 IUvCI,PTOR ANTAGONIST
FOR
TREATING TNFI,AMNIATION 134.(W0 99158142) USE OF ANTI-PROLACTIN AGENTS TO
TREAT
PROLIFLRATIVE CONDITIONS 135.(W0 99158097) USE OF ANTI-PROLACTIN AGENTS TO
TREAT PROLIFERATNE CONDITIONS 136.(W0 991572x5) METHODS OF $C11;BENING FOR
AGONISTS AND ANTAGONISTS OF THE iNT>~RpCTION BETWEEN T'13E HUMAN KIAA0001 RECEPTOR AND LIGANDS THBRE4F 137.(W0 99!31245) NON-PEPTIDE HRADYK1N1N
RECEPTOR ANTAGONISTS FOR USE IN TREATING OPHTHALMIC DISEASES AND
DISORDERS 138.(w0 99150249) rNTEGRIN ANTAGOMSTS J!39_(Wp 99149856 ) ANTAGONISTS
P'OR'1~REATMENTOF CD11/CDIB ApT~F$lON RECEPTO1~M$J71.ATED DISORDERS 140.(W0 99/47170) PItEVENTI~IES OR REMEDIES FOR INFLAMMATORY INTESTINAL DISEASES
CONTATNTT~TG AS THE ACTIVE INGREDIENT 1L-6 ANTAGONISTS 141.(W0 99147158) TIiERAPEUTIC CHEMOKINE RECEPTOR ANTAGONISTS 142.(W0 99/46376) RECEPTOR FROM
THE SUPERFAMILY OF TNT-RL~CEPTORS FROM THE HUMAN LUNG 143.(W0 99145927) VITRONECTIN RECEPTOR ANTAGONISTS 1a4.(wo 99145903) FROFHYLAXIS AND
TREATMENT OF MIGRATNE HEADACHES WITI~I THROMBOXANE SYNTHETASE INHIBITORS
AND/OR RLCFPTOR ANTAGONISTS 145.(W0 99/44612) SUBSTTtUTED QUINAZOLJNES AND
ANALOGS AND THE USE THEREOF 146.(W0 99143809) PROTEASE-ACTIVATED RECEPTOR 4 AND USES THLREOF I47_(WO 99/42464) STJHSTTCUTED IMII7AzO[1,2-2~;3,4-a']DIQUINOLINYLICiM INTERLEUI~IN-$ RECIrp'cOR ANTAGONISTS 145.(W0 99143463) 149_(W0 99/42461 ) SUBSTITUTED QUl!NOXAL1NE DbRIVATIVES AS INTERLEUICIN-8 R'EC1:;P1'OR ANTAGONISTS 150.(W0 99141257) GLUCOCORT1COID-SELECTIVE
ANTaINFI,.A,N~iATORY AGENTS t 51 _(w4 99/41236) GLUCOCORTIC4ID-SELECTIVE ANTI-1NFLAlVIMPvTORY AGENTS 152.(W0 99!44192 ) HUMAN RECEPTOR GPRl4, AND A METHOD
OF F1NUING AGONIST AN1~ ANTAGONIST TO I~IUMAN AND RAT GPRI4 153.(W0 99140091) HICYCLIC PYRIDINE AND PYRZMmINE DERIVATIVES AS NEUROPEPT117E Y RECEPTOR
ANTAGONISTS i54,(WO 99138532) METHODS FOR THE PREVENTJON AND TREATMENT OF
FIBROSIS AND SGLER05IS 155.(WO 99136541) 1NTERLEUKTN-1 RLCEPTOR ANTAGONIST
BETA (ILrIRA&bg~;) 156.(WO 99133806) 4-[ARYL(P1PER1D1N-4-YL)] AM1NOBENZAMTDES
WHICH 13TND TO THE DELTA-0PIOID RECEPTOR 157.(WO 99!31099) INTEC~RIN REGEPTOR
AN fAGONISTS 15$,(WO 99131061) 1NTEGRIN RECEPTOR ANTAGUN15T5 159.(WO 99/30713) TNfEGRIN RECEPTOR ANTAGONIS'>~S 168.(OVO 49130709) INTEGRIN RECEPTOR
ANTAGONISTS 161.(WO 99129729) ANTAGON)STS OF NEUROPILIN RECEPTOR FUNCTIONAL
AND USE TH EREOF 162.(WO 99127962) USE OF A FIBRINOGEN RECEPTOR-ANTAGONIST FOR
PREVENTING DISSEMINATED 1NTRAVASCULAR COAGULATION 163.(WO 99/26945) 1,3,4-'FHTADIAZOLES AND 1.,3,4-OXAD1A7.O1,ES AS &agr; v ~bgr; 3 ANTAGONISTS 164.(WO
99/26943) THR4M81N RECEPTORANTAGON1STS 165.(WO 99125857) TRANSGENIC MODELS OF
INFLAMMATORY DISEASE 166.(w0 994471 ) OPIaTB, CaNNAHINOro, AND ESTROGEN
RECEPTORS 167.(WO 99/24423) P1P$R1DIN$ DEkIVA"tiVES AND T'IIEIR USL AS
TACIiYKININ
ANTAGONISTS 168.(WO 99/24421) >NfIDAZOYLALKYL SUBSTITUTED WITH A FIVE, SIX OR
SEVEN MEMBERED I~TEROCYCLIC RING CONTAINING ON6 NITROGEN ATOM 169.(WO
99/24406) PHENYL-ALKYL-1MIDAZOLES A5 H3 RECEPTOR ANTAGONISTS 170-(WO 99/24405) Ii 3 RECEFTORLIGANDS OF THE PHENYL~ALKYL-IMIDAZOLES TYPE 171.(WO 99/21555) ADEN051NE A3 RECEPTOR ANTAGONISTS 172.(WO 99r10758) FIUMAN TUMOR NECROSIS
FACTOR RECEPTOR-LIKE PROTEINS TR11, TRl 15V 1, AND TRI 15V2 173-(WO 99/19462) ANTAGONISTS 174.(WO 99117773) COMPOUNDS AND METHODS 175.(WO 99/16455) METHOD
FOR TNHTBITTNG TUMOR ANG10GENESIS LN A LIVING SUBJEC 176.(WO 99/11790) TUMOR
NECROSIS FACTOR Rl'sCi'sPTOR ZZ7~TFR-6 177.(WO 99/06049) INTEGRIN RECEPTOR
ANTAGONISTS 178-(WO 99104001) TUMOR NECROSIS FACTOR RECEPTOR ZTNFR-5 179.(WO
99/02499) (ZU1NOLINL COMPOUNDS AND MEDICINAL USES THEREOF 180.(WO 99101764) METHOD I~OR RECOGNIZING AND DETERMINING GNRH RECEPTORS AND THE USE OF
GNRiI AGONISTS AND GNRH ANTAGONISTS AND OTHER GNRI-1 RECEPTOR LIGANDS FDR
THE TREATMENT WITH GNRH RECEPTORS OF TUMOURS ORIGINATING IN THE BRAIN
ANIaIOR NEIIVO~JS SYSTEM AND/OR MEN1NGES AND/OR OF KAPOSI SARCOMA 1$1-(WO
99/01444) POLYMORPHIC FOTLM OF Tl~ E TACHYKIN11V RECEPTOR ANTAGONIST 2-(R)-(1 ~(R) -(3,5-BI5(TRTFLUOROMETHYL) PHENYL)ETHOXY}3-(S)-(4-FLUORO) PHENYL-4-(3-S (-oxo-1H,4H-1,2,4; TRIAZOLO) METHYL,MORPI~IOLINE 182.(WO 99!01127) COMPOUNDS AND
METHODS 183,(W4 99100406) CYCLIC AGONISTS AND ANTAGONISTS OF C6a RECCpTORS
AND G PROTEIN-COUPLED RECEPTORS I84.(WO 98/58674) ANTI-TUMOUR
PHARMACEUTICAL COMPOSITIONS CAPABLE OF REDUCING DRUG RESISTANCE IN
TUMOUR CELL5 185.(WO 98/57647) COUP-TFll: AN ORPHAN NUCLEAR RECEPTOR REQUIRED
FOR ANGIOGENESIS 186-(WO 98156892) HUMAN TUMOR NECROSIS FACTOR RECEP1 OR Tf~9 187.(WO 98!56779) 4-9ULFiNYL J3J:NZAMIDES A5 GALCITON1N GENE-RELATED PEPTIDE
RECEPTOR ANTAGONISTS 188,(WO 98/55153} NON-STERO)DAL RADIOLAHELED

189.(WO 98!54325) HUMAN FRP AND FRAGMENTS THEREOF INCLUDING ME"fHODS FOR
USING TFl'.EM 190,(WO 98!54202? HUMAN TUM4RNECROSIS FACTOR RECEPTOR TR10 191.(WO 98/54201) HUMAN TUMOR NECROSIS FACTOR RECEPTOR-LTKB PROTEIN 8 192.(WO
98/54187) SPIRO-A7.ACYCLIC DERIVATIVES AND THEIR USE AS THERAPEUTIC AGENTS
I93.(WO 9$/53049) GDNF RECEPTO1RS 194.(WO 98149170) SPIRO-AZACYCLIC
DERIVATIVES
AND THELR USE AS TIdERAlyEUTTC AGETIT'S 195.(WO 9814$017) FAMILY OF
IMMUNOREGULATORS DESIGNATED LEUKOCYTE IMHfUNOGLOHULIN-LIKE RECEPTORS
(LIR) 196.(WO 98/47923) IL-5R ANTAGONISTS FOR TREATMENT OF INFLAMMATION, ASTHMA AND OTHER ALLERGIC DISEASES 197.(WO 98146751) OSTEOPROTEGERIN BINDING
PROTEINS AND RECEPTORS 19$.(VVO 9$146620) A NOVEL HUMAN G-PROTEIN COUPLED
RECEPTOR 199.(WO 98/462b5) METHODS FOR USING ANTAGONISTIC ANTI-AYB3 INTEGRIN
ANTIBODIES 2D0,(WO 98143962 } HETEROCYCLIC TNTEGRIN INHIBITOR PRODRUGS 251.(WO
97/44333) 1,2,4-OXADIAZOLE5 AS ADHESION-RECEPTOR ANTAGONISTS 252.(WO 97/44329) DIARYLALKYL CYCLIC DlAMINE DiJIxIRIVATIVES AS CI3EMOKINE RECEPTQR ANTAGONISTS
253.(WO 97/4)22S) MAMMALIAN MIXED LYMPHOCYTE REpEPTQRE, CHEMOKINE
RECEPTORS [MMLR-CCR] 254.(WO 97!37655) &agr;vBcbgr;3 ANTAGONISTS Z55.(WO
97/35969) PEPTIDE LIGANDS OF TT3E UROK1NASE R.ECF_PTOR 256,(WO 97134$78) SU'ESTITUTED
2,3-HENZODIAZEP1N-4-ONES AND THE USE THEREOF 257,(WO 97/33904) DEATH DOMAIN

CONTAINING RECEPTORS 258.(W0 97133$87) SPIROCYCLE INTEGRIN INHIBITORS 259.(W0 97!33613) PARASITE-DERIVED ANTI-INFLAMMATORY 1MMUNOMODULATORY PROTEIN
260.(W0 97/30991 ) NOVEL SUBSTIUTED N-METHYL-N-(4-(4-(1H-B81VZ1MIDAZOL-Z-YL)[1,4]DIAZEPAN-1-YL)-2-(ARYL)HUTYL)BEN2;AMIDES USEFUL FOR THE TREATMENT OF
ALLERGIC DISEASES 261.(W0 97!30990) NOVEL $U$$TTTUTED N~METHYL-N~.(4.(PIPER1D1N-1-YL)-2-(ARYL)BUTYL)BENZAM1DES USEFUL F

Claims

1. A composition comprising a multispecific ligand comprising at least a first ligand binding moiety which specifically binds to a first ligand having a first biodistribution and a second ligand bluding moiety which specifically binds to a second ligand having a second biodistribution different from that of the first ligand, and wherein the affinity of the first and second ligand binding moieties are different and selected to blas the biodistribution of the multispecific ligand.

2. The composition according to claim 1, further comprising a physiologically acceptable excipient.

3. The composition according to claim 2, wherein the multispecific ligand comprises a bispecific antibody.

4. The composition according to claim 3, wherein the affinity of said first ligand binding moiety for the first ligand is higher than the unity of the second ligand binding moiety for the second ligand and wherein the biodistribution of the multispecific ligand favours the first ligand.

5. The composition according to claim 4, wherein the first and second ligands have overlapping biodistributions.

6. The composition according to claim 5, wherein the first ligand is present on a first target cell population and wherein said second ligand is present on a second target cell population comprising the first target cell population and wherein the biodistribution of the multispecific ligand favours the first target cell population.

7. The composition according to claim 1 or 3, wherein said first ligand is a cell surface marker associated with one or more specific cell populations, infectious or parasitic agents, diseased cells, or disease-associated cells.

8. The composition according to claim 7, wherein said marker is an antigen.

9. The composition according to claim 7, wherein said marker is an epitope.

10. The composition according to claim 7, wherein said marker is a CD marker.

11. The composition according to claim 10, wherein said marker is CD4.

12. The composition according to claim 7, wherein said marker is specifically associated with a cancer cell or pre-cancerous cell.

13. The composition according to claim 11 or 12, wherein said second ligand is a CCR5 or CXCR4 receptor.

14. The composition according to claim 7, wherein said marker is associated with an immune cell that is susceptible to viral infection.

15. The composition according to claim 7, wherein said marker is specifically associated with an autoimmune disorder or rheumatic disease.

16. The composition according to claim 7, wherein said marker is associated with a specific tissue type.

17. The composition according to claim 7, wherein said marker is associated with a specific organ.

18. The composition according to claim 7, wherein said marker is associated with a cell or tissue of specific origin or class.

19. The composition according to claim 7, wherein said marker is an MHC-peptide complex.

20. The composition according to claim 7, wherein said marker is s cell surface immunoglobulin.

21. The composition according to claim 6 or 7, wherein said second ligand is a cell surface receptor, a family of cell surface receptors or one or more particular call surface receptor family members.

22. The composition according to claim 21, wherein said second ligand is a cell surface receptor.

23. The composition according to claim 22, wherein said second ligand is a marker associated with a lymphatic endothelial cell.

24. The composition according to claim 22, wherein said second ligand is a cell surface receptor is selected from the soup consisting of tyrosine kinase type receptors, series kinase type receptors, heterotrimeric G-protein coupled receptors, receptors bound to tyrosine kinase, TNF family receptors, notch family receptors, guanylate cyclase types, tyrosine phosphatase types, decoy receptors, and adhesion receptors.

25. The composition according to claim 22, wherein said second ligand is an IL-8 receptor, a CCR7 receptor, a FAS receptor, or a CXCR4 receptor,

26. The composition according to claim 22, wherein said receptor requires cross-linking for biological activity,

27. The composition according to claim 22, wherein binding of said second ligand binding moiety to said cell surface receptor blocks said receptor.

28. The composition d according to claim 22, wherein binding of said second ligand binding moiety to said cell surface receptor activates said receptor.

29. The composition according to claim 22, wherein said cell surface receptor initiates a signal transduction and wherein binding of said second ligand binding moiety to said cell surface receptor effects a signal transduction.

30. The composition according to claim 6, 7 or 22, wherein said antibody comprises a first VH which specifically recognizes said first ligand and a second VH which specifically recognizes said second ligand.

31. The composition according to claim 30, wherein at least one of said first and second VHs require a VL for binding to its ligand.

32. The composition according to claim 31, comprising a first VL in functional association with said first VH and a second VL in functional association with said second VH and wherein both said first and second functional associations are required for binding to the first and second ligands, respectively, and wherein said first and second VLs are the same or functionally interchangeable.

33. The composition according to claim 31 or 32, wherein said antibody is a four chain antibody.

34. The composition according to claim 33, wherein said antibody is a minibody or antibody lacking a CH3 domain.

35. The Composition according to claim 33, wherein said antibody is a diabody.

36. The composition according to claim 31 or 32, wherein said antibody lacks antibody light chains.

37. The composition according to claim 32, wherein said antibody comprises a pair of disulfide linked heavy chains or heavy chain portions each comprising at least a VH region, a hinge region and at least a portion of an the region at the carboxy terminus of the hinge region.

38. The composition according to Claim 37, wherein said bispecific antibody comprises a pair of VHs linked through a flexible linker.

39. The composition according to claim 4, 6, 7 or 22 wherein the affinity of the first ligand binding moiety for the first ligand is at least approximately, one, two, three. four, five, six, seven or eight orders of magnitude heater than the amity of said second ligand binding moiety for the second ligand.

40. A composition comprising a multispecific ligand comprising a first ligand binding moiety which specifically binds with a pre-selected first affinity to a first ligand having a first biodistribution and a second ligand binding moiety which specifically binds with a pro-selected affinity to a second ligand having a second biodistribution, and wherein the affinity of first and second ligand binding moieties are selected to bias the biodistribution of the multispecific ligand.

41. The composition according to claim 40, further comprising a physiologically acceptable excipient.

42. The composition according to claim 1 or 40, wherein the biodistributions of said first and second ligands overlap and wherein the affinities of the first and second ligand binding moieties are selected to bias the biodistrisbution of the multispecific ligand in favour of a target cell population on which both first and second biodistributions occur relative to one or more non-target cell populations.

43. The composition according to claim 42, wherein the affinities of said first and second ligand binding moieties ate both selected to limit their individual ability to bind to the first and second ligands, respectively, and wherein their combined functional affinity biases the distribution of the multispecific ligand towards said target cell population.

44. The composition according to claim 42, wherein the affinity of first ligand binding moiety for the first ligand is at least, approximately, one, two, three, four, five, six, seven or eight orders of magnitude greater than the affinity of the second ligand binding moiety for the second ligand.

45. The composition according to claim 42 or 44, wherein first and second ligands are recognized contemporaneously by the first and second ligand binding moieties.

46. A composition comprising a multispecific ligand which specifically binds to a target ligand on a selected sub-population of a heterogeneous cell population bearing the target ligand, the multispecific ligand comprising a first ligand binding moiety which specifically binds to a cell sub-population associated ligand and a second ligand binding moiety which binds to the target ligand, said first ligand binding moiety having an affinity for the sub-population associated ligand that is higher than the affinity of the second ligand binding moiety for the target ligand.

47. The composition according to claim 46, further comprising a physiologically acceptable excipient.

48. The composition according to claim 46, wherein the affinity of said first ligand binding moiety for the cell sub-population associated ligand is approximately, one, two, three, four, five, six, seven or eight orders of magnitude greater than the affinity of said second ligand binding moiety for said target ligand.

49. The composition according to claim 48, wherein said target ligand is a receptor.

50. The composition according to claim 46, wherein at least one of said fast or second ligand binding moieties comprises an antibody heavy chain or functional portion(s) thereof including a VH or fragment thereof and an antibody light chain or functional portion(s) thereof including a VH or fragment thereof.

51. A composition comprising an antibody which specifically binds to an epitope on a ligand wherein said ligand exerts a biologic effect by binding to a target site on a target ligand through an affinity for said target ligand, said epitope being proximal to the binding site of said ligand for the target ligand, such that the antibody reduces but does not prevent the affinity of the ligand for its target ligand.

52. The composition according to claim 51, further comprising a physiologically acceptable excipient.

53. A composition comprising a multispecific ligand comprising a first ligand binding moiety which specifically binds to a lymphatic endothelial cell associated marker and a second moiety comprising a therapeutic moiety.

54. The composition according to claim 53, further comprising a physiologically acceptable excipient.

55. The composition according to claim 33, wherein the therapeutic moiety provides an immune function.

56. The composition according to claim 53, wherein the marker is selected to limit the ability of said endothelial cell to internalize said multispecific ligand.

57. The composition according to claim 53, wherein said first portion is an antibody.

58. The composition according to claim 54, wherein said second portion moiety binds to a target ligand.

59. The composition according to claim 53, wherein said therapeutic moiety comprises an antibody moiety.

60. The composition according to claim 53, wherein said ligand is selected from the group consisting of CCR5, CTLA-4, LFA-1, ICAM-1, CD2, CD3, CD4, CD22, CD40, CD44;
CD84, CD86, CD134 and CD154.

61. The composition according to claim 53, wherein said first portion binds to LYVE-1 or podoplantin.

62. The composition according to claim 53, wherein said second portion comprises an anti-idiotypic antibody.

63. The composition according to claim 62, wherein said anti-idiotypic antibody binds to an autoimmune antibody.

64. The composition according to claim 63, wherein said anti-idiotypic antibody mimics a cell surface expressed tumour antigen.

65. The composition according to claim 53, wherein said second portion binds to a diseased cell.

66. The composition according to claim 65, wherein said diseased cell is a cancer cell.

67. The composition according to claim 53, wherein said second portion binds to an infectious agent or parasite.

68. The composition according to claim 67, wherein said diseased cell is a virally infected cell.

69. The composition according to claim 53, wherein said second portion binds to a cell of the immune system.

70. The composition according to claim 69, wherein immune cell is associated with an autoimmune reaction.

71. The composition according to claim 69, wherein said immune cell is a CCR5-expressing cell.

72. The composition according to claim 64 or 69, wherein said second portion binds with greater functional affinity to its target ligand than said first portion binds to its target ligand.

73. The composition according to claim 64 or 69, wherein said second portion binds with greater affinity to its target ligand than said first portion binds to its target ligand.

74. The composition according to claim 53, wherein said second portion binds with greater avidity to its target ligand than said first portion binds to its target ligand.

75. The composition according to Claim 33, wherein said second portion comprises an internalizing antibody and a cytotoxic component.

76. The composition according to claim 53, wherein said multispecific ligand is a bispecific antibody having a monovalent first portion and a monovalent second portion.

77. The composition according to claim 53, wherein said multispecific ligand is a bispecific antibody having a divalent first portion and a divalent second portion.

78. The composition according to claim 33, wherein said multispecific ligand is a trispecific antibody hooting a monovalent first portion arid a second portion comprising a divalent immune function exerting moiety which binds to one or more target ligands ore a target diseased cell or immune cell and a monovalent anti-CD3 or anti-CD28 antibody.

79. The composition according to claim 53, wherein said multispecific ligand is a trivalent trispecific antibody having a monovalent first portion and a second portion comprising a monovalent immune function exerting moiety which binds to a target ligand on a target diseased or immune cell and a monovalent anti-CD3 or anti-CD28 antibody.

80. The composition according to claim 53, wherein said multispecific ligand is a trivalent trispecific antibody having a monovalent first portion and a second portion comprising s divalent immune function exerting moiety which binds to a target ligand on a target diseased or immune cell.

81. The composition according to claim 53, wherein said second portion comprise a cytokine component.

82. The composition according to claim 53, wherein said second portion comprises a cytotoxic component.

83. The composition according to claim 53, wherein said second portion comprises a ligand capable of binding to T cells.

84. The composition according to claim 83, wherein said ligand is an antibody which binds to T cells.

85. The composition according to claim 53, wherein said second portion comprises an anti-CD3 antibody or anti-CD28 antibody.

86. The composition according to claim 53, wherein second portion is a cytokine component.

87. The composition according to claim 53, wherein second portion is an anti-CD3 antibody or an anti-CD28 antibody.

88. The composition according to claim 53, wherein said second portion further comprises one or more components selected from the group consisting of a cytokine component, a cytotoxic component and an anti-CD3/CD28 component.

89. A composition comprising an immunocytokine having an anti-idiotypic antibody component which recognizes the paratope of an antibody which binds to a lymphatic vessel associated ligand and a cytokine component.

90. The composition according to claim 89, wherein the cytokine component is fused with or conjugated to the lymphatic vessel associated ligand.

91. An immunocytokine as claimed is claim 89, wherein said cytokine component comprises IL-2 or a functional fragment thereof and/or IL-12 or et functional fragment thereof.

92. An immunocytokine as claimed in claim 89, wherein said cytokine component comprises TNF-.alpha. or a functional fragment thereof.

93. A composition comprising a bispecific antibody having an anti-idiotypic antibody component which recognizes the paratope of an antibody which binds specifically to a lymphatic vessel associated ligand and an anti-CD3 antibody or an anti-CD38 antibody component.

94. The composition according to claim 90 or 91, wherein said anti-idiotypic antibody component has a lower functional affinity for the paratope of the antibody which binds specifically to the lymphatic vessel associated ligand than the latter antibody has for the lymphatic vessel associated ligand.

95. A composition comprising a bispecific ligand comprising a first ligand which binds to a first target ligand and a second ligand which binds to a second target ligand, and wherein the affinity of said first ligand is selected to enable binding to the first target ligand independently of the ability of said second ligand to bind to the second target ligand and wherein the affinity of said second ligand is selected to substantially reduce the probability of its binding to the second target ligand without the first ligand binding first or substantially contemporaneously to the first target ligand.

96. A composition comprising a bispecific antibody comprising a first antibody component which binds to a first target ligand and a second antibody component which binds to a second target ligand, and wherein the affinity or avidity or both the affinity and avidity of said first antibody component are selected to enable binding to the fast target ligand independently of the ability of said second antibody component to bind to the second target ligand and wherein the avidity or affinity or both the affinity and avidity of said second ligand are selected to substantially reduce the probability of its binding to the second target ligand without the first ligand binding first or substantially contemporaneously to the first target ligand.

97. A composition comprising a multispecific ligand comprising a first moiety which binds to a first target ligand and a second moiety which binds to a second target ligand, and wherein the affinity or avidity or both the affinity and avidity of said first moiety are selected to enable the first moiety to bind to the first target ligand independently of the ability of said second moiety to bind to the second target ligand and wherein the avidity or affinity or both the affinity and avidity of said second moiety are selected to substantially reduce the probability of its binding to the second target ligand without the first moiety, first or substantially contemporaneously, binding to the first target ligand.

98. The composition according to claim 97, wherein both moieties bind to different target ligands on the same cell.

99. A composition comprising a multispecific ligand comprising a first moiety which binds to a first target ligand and a second moiety which binds to a second target ligand, and wherein the affinity or avidity or both the affinity and avidity of said first moiety are selected to enable the first moiety to bind to the first target ligand independently of the ability of said second moiety to bind to the second target ligand and wherein the avidity or affinity or both the affinity and avidity of said second moiety are selected to substantially reduce the probability of either moiety binding for a sufficient duration or series of durations to its respective target ligand to accomplish a therapeutic function without the other moiety, first or substantially contemporaneously, binding to its respective target ligand.

100. The composition according to claim 99, wherein both moieties bind to different target ligands on the same cell.

101. A composition comprising a multispecific ligand comprising a first moiety which binds to a first target ligand and a second moiety which binds to a second target ligand, and wherein the affinity or avidity or both the affinity and avidity of said first moiety are selected to enable the first moiety to bind to the first target ligand independently of the ability of said second moiety to bind to the second target ligand and wherein the avidity or affinity or both the affinity and avidity of said second moiety are selected to enable the second moiety to bind to the second entity in preference to the first moiety binding to the first entity when both first and second moieties are substantially contemporaneously bound to the respective first and second entities.

102. The composition according to claim 101, wherein the first moiety comprises at least one antibody component which binds to a first cell and the second moiety comprises at least one antibody component which binds to a second different cell.

103. A composition comprising a multispecific ligand comprising a first moiety which binds to a first target ligand and a second moiety which binds to a second target ligand, and wherein the affinity or avidity or both the affinity and avidity of said first moiety are selected to enable the first moiety to bind to the first target ligand independently of the ability of said second moiety to bind to the second target ligand and wherein the avidity or affinity or both the affinity and avidity of said second moiety to bind to the second target ligand and wherein the avidity or affinity or both the affinity and avidity of said first moiety are selected to enable the first moiety to bind to the first entity in preference to the second moiety binding to the second entity when both first and second moieties are substantially contemporaneously bound to the respective first and second entities, and wherein the avidity or affinity or both the affinity and avidity of said second moiety are selected to enable the third target ligand to bind to the second entity in preference to the second moiety binding to the second entity when both said third target ligand and the second moiety are substantially contemporaneously bound to the second entity.

104. A composition comprising a multispecific ligand comprising at least a first ligand binding moiety which specifically binds with a pre-selected first affinity to at least a first ligand having a first biodistribution and a second ligand binding moiety which specifically binds with a pre-selected affinity to at least a second ligand having a second biodistribution, and wherein the affinity of first and second ligand binding moieties are selected to bias the biodistribution of the multispecific ligand in favour of a selected location of one or both of the ligands.

105. A composition comprising a multispecific ligand comprising at least a first ligand binding moiety which specifically binds to a first ligand having a first biodistribution and a second ligand binding moiety which specifically binds to a second ligand having a second biodistribution, and wherein the affinity of the first and second ligand binding moieties are different and selected in bias the biodistribution of the multispecific ligand, and wherein the affinity of the first ligand binding moiety for the first ligand is at least, approximately, one order of magnitude greater than that of the second ligand binding moiety for the second ligand.

106. A composition comprising a multispecific ligand comprising at least a first ligand binding moiety which specifically binds to a first ligand having a first biodistribution and a second ligand binding moiety which specifically binds to a second ligand having a second biodistribution, end wherein the affinity of the first and second ligand binding moieties are different and selected to bias the biodistribution of the multispecific ligand, and wherein the affinity of the first ligand binding moiety for the first ligand is at least, approximately, two orders of magnitude greater than that of the second ligand bending moiety for the second ligand.

107. A composition comprising a multispecific ligand comprising at least a first ligand binding moiety which specifically binds to a first ligand having a first biodistribution and a second ligand binding moiety which specifically binds to a second ligand having a second biodistribution, and wherein the affinity of the first and second ligand binding moieties are different and selected to bias the biodistribution of the multispecific ligand, and wherein the affinity of the first ligand binding moiety for the first ligand is at least, approximately, three orders of magnitude greater than that of the second ligand binding moiety for the second ligand.

108. A composition comprising a multispecific ligand comprising at least a first ligand binding moiety which specifically binds to a first ligand having a first biodistribution and a second ligand binding moiety which specifically binds to a second ligand having a second biodistribution, and wherein the affinity of the first and second ligand binding moieties are different and selected to bias the biodistribution of the multispecific ligand, and wherein the affinity of the first ligand binding moiety for the first ligand is at least, approximately, four orders of magnitude greater than that of the second ligand binding moiety for the second ligand.

109. A composition comprising a multispecific ligand comprising at least a first ligand binding moiety which specifically binds to a first ligand having a first biodistribution and a second ligand binding moiety which specifically binds to a second ligand having a second biodistribution, and wherein the affinity of the first and second ligand binding moieties are different and selected to bias the biodistribution of the multispecific ligand, and wherein the affinity of the first ligand binding moiety for the first ligand is at least, approximately, five orders of magnitude greater than that of the second ligand binding moiety for the second ligand.

110. A composition comprising a multispecific ligand comprising at least a first ligand binding moiety which specifically binds to a first ligand having a first biodistribution and a second ligand binding moiety which specifically binds to a second ligand having a second biodistribution, and wherein the affinity of the first and second ligand binding moieties are different and selected to bias the biodistribution of the multispecific ligand, and wherein the affinity of the first ligand binding moiety for the first ligand is at least, approximately, six orders of magnitude greater than that of the second ligand binding moiety for the second ligand.

111. A composition according to any one of claims 105 to 110, wherein the biodistributions of said first and second ligands comprise a target population of cells and at least one non-target population of cells and wherein said first and second ligands are present only on said target population and wherein the biodistribution of the multispecific ligand is biased in favor of the target population of cells.

112. A composition according to claim 111, wherein said multispecific ligand is adapted to bind to two ligands on the same cell.

113. A composition according to claim 112, wherein said multispecific ligand comprises at least two full length heavy chains or heavy chain fragments having differing specificities, or is chosen from a F(ab')2, a minibody, a diabody, a four chain immunoglobulin having a truncated Fc portion, a tetravalent antibody having a four chain framework and a divalent Fab.

114. A host cell or cell free expression medium comprising one or more polynucleotides, said one or more polynucleotides comprising one or more DNA sequences, said one or more DNA sequences comprising one or more polypeptides which are sufficient to constitute a multispecific ligand as defined in any of the preceding claims

115. A kit comprising one or more polynucleotides, said one or more polynucleotides comprising one or more DNA sequences, said one or more DNA sequences encoding one or more polypeptides which are sufficient to constitute a multispecific ligand as defined in any of the preceding claims.

116. A liquid medium comprising comprising one or more polypeptides which are sufficient to constitute a multispecific ligand as defined in any of the preceding claims.

117. A liquid medium comprising one or more host cells, and one or more host cells comprising one or more polynucleotides, said one or more polynucleotides comprising one or more DNA sequences, said one or more DNA sequences encoding one or more polypeptides which are sufficient to constitute a multispecific ligand as defined in any of the preceding claims.

118. A substantially isolated polynucelotide comprising one or more DNA
sequences, said , said one or more DNA sequences encoding one or more polypeptides which are sufficient to constitute a multispecific ligand as defined in any of the preceding claims

119. A substantially isolated polynuceotide comprising a DNA sequence encoding a polypeptide portion of a second ligand binding moiety as defined in any of the preceding claims, said polypeptide portion comprising a VH or VL, said second ligand binding moiety having a low affinity for said second ligand.

120. A substantially isolated polynucleotide according to paragraph 119, wherein said polynucleotide is a substantially isolated expression or cloning vector.

121. A method of making a multispecific ligand as defined in any of the preceding paragraphs comprising expressing at least one polynucleotide as defined in claim 115, 118, 119 or 120.

122. A pharmaceutical composition comprising a multispecific ligand as defined in any of the preceding claims and a pharmaceutically acceptable excipient.

123. A therapeutic composition comprising a multispecific ligand as defined in any of the preceding paragraphs and a pharmaceutically acceptable excipient.

124. A method of treating a disease in s mammal comprising administering a therapeutically effective amount of a multispecific ligand according to any of the preceding claims.

125. A kit comprising a plurality of different multispecific ligands as defined herein.

126. A composition according to claim 1, 111 or 112, wherein at least one of said first and second ligand binding moieties comprises human sequences.

127. A composition according to claim 1 or 111, wherein at least one of said first and second ligand binding moieties comprises human framework sequences.

128. A combinatorial library comprising a diverse population of multispecific ligands according to claim 1, 111 or 112, characterized by members of said population having a diversity of affinities for at least one of said first and second ligands.

129. A diverse population of nucleic acids which encode a combinatorial library as defined in claim 28.