CA2402930A1 - Targeted ligands - Google Patents

Abstract

The invention contemplates a composition containing a multispecific ligand containing at least a tirst 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 aftinity of tirst and second ligand binding moieties are selected to bias the biodistribution of the multispecitic ligand in favour of a selected location of one or both of the ligands.

Description

Tareeted Ligands Field of The Invention The present invention relates to multispecific ligands, for example a heterofunctional ligand comprising at least first and second binding moieties which have cooperating functional affinities including a multispecific ligand, for example, a bispecific antibody, having at least a tirst portion which binds to a 'lymphatic vessel associated' antigenlreceptor and a second portion having at least one immune-affecting functionality including, without limitation, functions related to antigen presentation, immune signaling, suppression or enhancement of immune tolerance or immune stimulation, or binding to a target molecule, for example a cell surface antigen, receptor etc.
Background of the Invention Immunotherapy has gained wide acceptance as a promising measure to address several disease states including autoimmune disease, transplant rejection, infectious disease and cancer. Despite rapid and exciting progress in approaches to treatment, 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 immunotherapy in human subjects, continue to be some of the obstacles to successful advances in treatment.
Successful approaches to immunotherapy are predicated on the ability of the immunotherapeutic 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 target population. In the case of an injectable preparation and especially an intravenous mode of delivery, in contrast to readily self administered modes of delivery, the optimal dosing frequency for therapeutic purposes could impose an undesirable burden on the patient and care-giver, assuming that such optimal frequency is to begin with deemed convenient fur clinical trials.
Numerous research efforts are underway to identify and test ligands including antibodies, biologic effector ligands (e.g. cyrtokines, chemokines, growth factors colony stimulating factors) receptor agonists orantagonists etc. which will bind to or otherwise interact with or trigger responses in or towards target entities, including pathogenic organisms, tissue specific cells,diseased cells, immune cells etc. A recent example is a renewed interest to tind molecules and methods of triggering an interaction with CD45 (see for example Nature (?001 ) Vol. 409 p. 349-354). Evaluating the biological effect of interactions with such target ligands is often obfuscated and retarded by the biodistribution of such ligands on cells other than the target population which results in undesired and/or conftrsing pleiotropic effects.
The present invention facilitates scientific assessment, development, role evaluation, therapeutic evaluation, and delivery, particularly targeted delivery of molecules that exert biologic functions and particularly immune relatedfunctions. In particular, the targeting agents and methods which are the subject of the invention herein facilitate scientific evaluation of the biological effects of a more targeted biodistribution of such targeting agents, by limiting undesired or confusing side effects. In preferred aspects the invention contemplates compositions of matter and methods of~delivery, in some cases using ligands that but for the tar~~etin~~ methods herein defined would be ineffective or have a broader effect than is desirable; or similarly, but for the severity of the disease or the absence of other therapeutic alternatives for which such ligands are useful, they would otherwise be inappropriate for therapeutic use. The present invention accommodates evaluation of the biological role and!or effects of such ligands for therapeutic or other scientific purposes using such targeting strategies. In particular, the present invention provides a vehicle to preferentially target, on a sub-population of cells for which there is a cell-associated marker, a receptor or receptor ligand which is present on a more heterogeneous population of cells.

Summary of The Invention 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 multispeciflc ligand in favour of a selected location of one or both ofthe ligands.
The invention contemplates a composition containing a multispecific ligand.
The multispecific li~and contains at least a first li~~and binding moiety and a second ligand binding moiety. The first ligand binding moiety specifically binds to a first ligand having a first biodistribution.
The second li<~and binding moiety specifically binds to a second ligand having a second biodistribution. The second biodistribution is different from that of the first biodistribution, and the affinity 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 multispecitic ligand. 'rhe multispecific ligand contains 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 a first ligand. The first ligand has a first biodistribution. The second ligand binding moiety specifically binds with a pre-selected affinity to a second ligand. The second ligand has a second biodistribution. In this embodiment of the multispecific ligand, the affinity of first and second ligand binding moieties are selected to bias the biodistribution of the multispecific ligand.
The invention further contemplates a composition containing a multispecific ligand. The multispecific ligand specifically binds to a target ligand. The target ligand is specific to a selected sub-population of a heterogeneous cell population. This embodiment of the multispecitic ligand contains a first ligand binding moiety and a second ligand binding moiety. The first ligand binding moiety specifically binds to a cell sub-population associated ligand. The second ligand binding moiety binds to the target ligand. In this embodiment, the first li~~and binding moiety has an affinity for the sub-population associated ligand higher than the affinity of the second li~~and bindings moiety for the tartlet ligand.
The invention further contemplates a composition containing a bispecific ligand containing a first li~~and and a second li<~and. The first ligand binds to a first target ligand and the second li<~and binds to a second target ligand. In this embodiment of the bispecific ligand. the affinity of the first liaand 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 bindin<~ first or substantially contemporaneously to the first target liy~and.
The invention further contemplates a composition containing a bispecific antibody containing a first antibody component and a second antibody component. The first antibody component binds to a first target ligand and the second antibody component binds to a second target li~and. 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 tartlet ligand independently of the ability of the second antibody component to bind to the second target ligand. The avidity or affinity or both the affinity and avidity of the second ligand are selected to substantially reduce the probability of its bindin~~ to the second target ligand without the first ligand binding first or substantially contemporaneously to the first target iigand.
The invention further contemplates a multispecitic ligand containing a first moiety and a second moiety.
The first moiety binds to a first target ligand. The second moiety binds to a second target li~.;and. The affinity or avidity or both the affinity and avidity of the first moiety are selected to enable the first moiety to bind to the first tartlet li~.;and 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 are selected to substantially reduce the probability of its bindings to the second target ligand without the first moiety, first or substantially contemporaneously, binding to the first target ligand.

The invention further contemplates a multispecific li;,~and containing a first moiety and a second moiety.
The first moiety binds to a first target ligand. The second moiety binds to a second target liganct. 'fhe affinity or avidity or both the affinity and 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 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 tartlet li~~and.
The invention further contemplates a composition containing a multispecific ligand containing a first moiety and a second moiety. 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 and avidity of the first moiety are selected to enable the first moiety to bind to the first target liganct independently ofthe 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 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.
The invention contemplates a composition containing a multispecitic ligand containing a first moiety, a second moiety and a third ligand bindings moiety. The first moiety binds to a first target ligand and the second moiety binds to a second target li~~and. In this embodiment, the affinity or avidity or both the affinity and avidity of the first moiety are selected to enable the first moiety to bind to the first target ligand 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 the avidity or affinity or both the affinity and avidity of the second moiety are selected to enable the third target li~and to bind to the second entity in preference to the second moiety binding to the second 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 ligand. The ligand recognized by the antibody exerts a biologic effect by binding to a target site on a target ligand. The epitope bound by the antibody is proximal to the binding site of the ligand for the target ligand. so that binding of the antibody reduces but does not prevent the affinity of the ligand for its target lid=and.
The invention further contemplates a composition containing a muitispecific ligand containing a first ligand binding moiety and a second moiety. The first ligand binding moiety specifically binds to a lymphatic endothelial cell associated marker. The second moiety contains an independent therapeutic function.
The invention further contemplates a composition containing an immunocytokine containing an anti-idiotypic antibody component and a cytokine component. The anti-idiotypic antibody component recognizes the paratope of an antibody which binds to a lymphatic vessel associated ligand.
The invention further contemplates a composition containing a bispecific antibody containin« an anti-idiotypic antibody component and an anti-CD3 antibody or an anti-CD?8 antibody component. The anti-idiotypic antibody reco<~nizes the paratope ofan antibody which binds specifically to a lymphatic vessel associated lieand.
The invention additionally contemplates physiologically acceptable compositions of the compositions encompassed by the invention.
The invention likewise contemplates methods of use of the compositions encompassed by the invention.
A composition comprisin~~ a multispecitic ligand comprising at least a first IiV~and binding moiety which specifically binds to a first li~~and having a first biodistribution and a second li~and binding moiety which specifically binds to a second li~~and having a second biodistribution different from that of the first ligand, and wherein the affinity of the first and second ligand bindings moieties are different and selected to bias the biodistribution ofthe multispecific li~~and.

Detailed Description of Preferred Embodiments As exemplified above, the dual "aftinity" based targeting strategy of the invention, may be understood in one aspect, in terms of a strategic allocation of the respective affinity properties of the multispecific ligand to at least one "targetin~~'' function and at least one "effector: " function.
Accordingly, with respect to some embodiments ofthe invention, the term "multifunctional" ligand is used interchangeably.
Thus according to one preferred embodiment, at (east 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 e~~. cell populations, and the other ligand binding moiety is an "effector" arm which binds with relatively less affinity or functional aftinity to a target ligand which has a more diverse biodistribution. In this case, the biodistribution of the multispecific ligand is biased in favour of the locations) of both ligands relative to the location(s'? of the target ligand 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 "effector" is used to refer to the ability to effect a biological consequence through binding, for example effecting a signal transduction event by activating a receptor, or blocking the target ligand from associating with a complementary ligand, for example blocking a receptor from associating w kith a complementary ligand (eg. its natural li~~and) and thereby, for example, preventing a signal transduction, or for example in thevcase of a decoy receptor preventing the biological consequence (e~~. protective effect) associated with the function of such receptor, or blocking a li'~and from associating with a complementary ligand eg. receptor on another entity eg. a cancer cell, infectious agent or immune cell.
A biased biodistribution is preferably accomplished by the multispecitic ligand contemporaneously recognizing both ligands on the same entity e~~ cell, and may be accomplished by such contemporanous recognition occurring on adjacent entities or by increasing the propensity of the multispecfic li~land to locate in proximity to a target entity in virtue ofthe relatively high aftinity targeting arm. The targeting arm may itself be an effector .
In another embodiment, the biological consequence accomplished by the effector arm is at feast minimally retargeting, for example wherein the lesser affinity or functional affinity of the first binding moiety is selected to permit the multispecific ligand to preferentially bind to an adjacent entity, for example. a circulating entity which circulates in proximity to a lymphatic endothelial cell to which the multifunctional ligand is bound with lesser affinity. Again, the relatively high aftinity first bindin<~ moiety may itself be an effector.
In another embodiment the biological consequence accomplished by the effector arm is minimally cooperative targeting, for example where the biodistributions of at least one of the first and second li~ands extends to a diverse population of cells other than target cell population and where binding is only possible or consequential if both ligands are available for contemporaneous binding, in this case due to the affinities of the first and second ligand binding being individually insufficient for effective targeting (eg. insufficient for other than ephemeral binding). In the context of this embodiment of the invention, the "cooperative targeting" is not simply ameliorated by the effector am, it is predicated and reliant on this arm. One or both ligand binding moieties may exert additional eflector properties.
It will also be appreciated that any multispecitic ligand of the invention or any component thereof may be fused or conjugated to a separate effector as exemplified below, including toxins, cytokines, adhesion molecules etc.
The ligand bindings moiety is preferably an antibody or a sequence or sequences of amino acids etc.which are the natural ligand for the target ligand, for example where the ligand is a cytokine or lymphokine receptor, such as IL-? receptor, the li<,Tand binding moiety may comprise a sequence of amino acids which is IL.-2. 'The li;and binding moiety may also be a mutated or a newly developed form of the natural ligand (eg. developed through combinatorial libraries) or a natural or synthetic chemical lieand (developed through combinatorial chemistry).
In one aspect. the invention contemplates a composition containing a multispecific li~~and containing at least a first ligand binding moiety and a second ligand binding moiety, the first ligand binding moiety specifically binding with a pre-selected first affinity to at least a first ligand, having a first biodistribution and the second ligand binding moiety specifically binding with a pre-selected affinity to at least a second ligand with a second biodistribution and wherein the aftinity of fast and second ligand binding moieties are selected to bias the biological site of biologic activity of the multispecitic ligand; and wherein the first ligand binding moiety preferably binds with hi~~h affinity (preferably nanamolar affinity or greater) to a specific cell associated marker (e.g. a CD marker, a marker associated with diseased cells, a a marker associated cells in a particular physiological state (e.g. activated T cells, B cells) etc. (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, such as epithelial cells. endothelial cells, immune cells (lymphocytes, memory cells, effector cells) monocytes,Tcells (CD4+, CD8+, CD45R0+).
hepatocytes, stem cells, etc.(expand) and wherein said second ii~and bindings binding moiety binds with relatively low, or medium affinity (preferably 0.1 micromolar or less) to a receptor leg. chemokine, growth factor, cytokine) involved in cell signaling or a decoy receptor, a cell surface receptor ligand eg. the ligand for such receptor which effects a signal or inhibits a si~~nal (e~~. CTLA4). a li<.;and involved in cell adhesion.
a receptor or channel (ion channel) for a molecule involved in cell regulation or homeostasis etc.
The invention contemplates that the difference in affinity will in most cases be an essential element in biasin~~ the location of action of the multispecitic ligand to yield an acceptable or desired safety profile and that the high affinity of the first ligand binding moiety for the cell associated marker will be optimized for this purpose insofar as the safety profile of the muftispecifie ligand dictates maximizing its affinity characteristics. The invention also recognizes that choosin<~ the relatively lower affinity of the second ligand bindings moiety may assist in this regard up to a point where its effectiveness to bind to the second ligand is significantly comprimised. In this regard. the invention also contemplates that factors other than the choice of aftinityof 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-target cell) may be taken into consideration or optimized to balance the safety and effectiveness profiles of the multispecific ligand, especially if such careful balance is required.
Examples of such factors, one or more of which can be employed in various combinations, are described hereafter:
1') the selection of cell associated marker, in terms of its cell surface density relative to cell density of the second ligand. The number of tirst and second ligand can readily be assessed by radiolabelling studies or approximated by tlow cytometric methods relative to a standard.The selection of the cell surface marker in this respect will depend on the function of the relatively low aftinity binding moiety (whether it causes a signal transduction (directly or indirectly eg. through binding to a receptor (where less emphasis on relative cell density may be warranted)(agonist antibodies are well known in the art and include those described in US 6,342,220, l1S 6,331,302, US >.635,177, lJS 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 antibodies capable of cross-linking receptor components (see references below) including antibodies in which the Vtl and VL, are capable of bindings individually to different receptor components), binding to a decoy receptor. binding to an inhibitory receptor etc.) or prevents a signal transduction (directly or indirectly, e.g. binding to a receptor, bindiny~ to receptor ligand) and in the final analysis how many binding events per cell are required to cause or prevent the sou~~ht-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 cytokines, chemokines. growth factors, colony stimulating factors using various immunostaining techniques including flow cytometry (e~~r. to measure apoptosis (e.g. annexin V' binding assay) signal transduction (e.g. using phosphospecific antibodies that detect phosphorylarion of serine, tyrosine, threonine), differential gene expression etc.) depending on the type of effect that is being measured (see for example Biosource Method Booklets at Ettt~_ vs,_w w_hin"~uty c~isr~.r ~ofrtent ttchC;,'.<>tnt;r~(_cy,itentirytpo~F?(..)F~:'isply ,isn.: see also Amersham Bioscience catalogues, and those of other well known suppliers etc.) or via animal studies. For example, some Growth factors, lymphokines or molecules/ions required for homeostasis are in more delicate balance and can more easily disrupted. IL-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 specitic marker to approximate (preferably the the number of cell specific markers on the target cell population is no less in number than SO% more preferably no less than 90°%o in number relative to the second li'~and - as stated above, which will depend on what degree of causation or prevention ofthe signaling/interaction will cause the desired biologic effect) or preferably out-number (by greater than 50°,'°, preferably b_v ~'.a,reater that 100%
(greater than two fold), preferably by greater that 200°~% (greater than 3 fold), by greater than 300°~0 (~~reater than 4 fold)) the target li~~and for the relatively low aftinity bindings moiety especially. for example, if the goal is to block interaction of a receptor with a high affinity ligand that exerts a biological effect in low concentration (eg. a cytokine).
2) Furthermore, in the latter case the affinity ofthe first binding moiety will preferably be selected to approximate (preferably no less than one order of magnitude, more preferably no less than 5 fold less, more preferably no less three fold less, more preferably no less two fold less, more preferably no less one fold (100%) less). and will preferably equal or exceed the affinity ofthe natural ligand.
3) Furthermore, the concentration (in virtue of the choice of administered dose) of the multispecitic ligand in the target cell microenvironment may also be selected to exceed that ofthe natural ligand (MTD
permiting).
4) the choice of construct will maximize the steric blocking of the target (IgG or F(ab')' vs diabody)_ Furthermore, in some mammalian systems (eg. mice) the hinge region is naturally longer and this effect can be mimicked for human antibodies through a hin~~e extension on the N-terminal side of the hinge region using well known neutral linkers (gly4ser) or a repeat of all or a portion of the natural hinge sequence. 'this extension will also permit a greater span between first and second ligands to be bridged.

5) the choice of construct will include an Fc portion or partial Fe portion (eg. CH2 or minibody-CH3) or weighted Fc eg. by pegylation (site specific pegylation is well known in the art) or 1gG subtype naturally having additional Fc domains (e.g. an I~~E) (which Fc if it includes the CH3 is preferably mutated to preclude its binding andior increase its half life as is known in the art see USP 6,131, 022) so as to maximize the shear effects on the multispecific ligand wfiich will be most consequential in the case of univalent binding in order to minimize the duration of such bindings (maximun shear force is also preferred where there is an excess in the total number of bioavailable targets of the second ligand binding moiety relative to the total number of bioavailable tartlets of first ligand binding moiety(greater number of cells and/or greater number of targets per cell and!or increased bioavailablity of such targets eg. on normal cells relative to cancer cells).

6) Optionally, the multispecitic ligand will include a 3''~ binding moiety which binds to and neutralizes the natural ligand for the receptor sought to be blocked. Such formats are well known in the art (see for example particularly Schoonjans R et al. A new model for intermediate molecular wei~~ht recombinant bispecitic and trispecitic antibodies by efficient heterodimerization of single chain variable domains throu~~h fusion to a Fab-chain. Biomol Eng. 2001 Jun;17(6):19_i-202.
Schoonjans R et al. Fab chains as an efficient heterodimerization scaffold for the production of recombinant bispecitic and trispecific antibody derivatives.) Immunol. 2000 Dec 15;165(12):7(IS(t-7. Schoonjans R. et al.
Efficient heterodimerization of recombinant bi- and trispecific antibodies. Bioseparation. 2000;9(p):179-8 i.
see also French RR.
Production of bispecitic and trispecitic F(ab)2 and F(ab)~ antibody derivatives. Methods Mol Biol.
1998;80:121-34; US Patent Application No. 20020004587; Kortt AA, Dinreric and trimeric antibodies:
high avidity scFvs for cancer targetin~~. Biomol En<~. 2001 Oct 1 ~: I 8(p):95-108).

7) Optionally, two multispecitic ligands each binding to different cell specific markers and each having a second ligand binding moiety which binds to the same second ligand e~~. a receptor, optionally to a different polypeptide.%component of the receptor, may be employed to achieve the desired biologic effect. One or both may also be trispeeitic as discussed above..Accordin~~ to another embodiment the multispecifc liaand binds is used to protect a first target cell population in virtue of its hi<~h affinity first ligand binding moiety from the effects of a therapeutic entity which desirably binds to a second tartlet cell population via the second ligand but also undesirably binds to the first tar~,et cell population.
'therefore the second ligand binding moiety can be used to selectively block the binding of the therapeutic entity e.g. an interleukin, interferon. innnunotoxin, etc. to the tirst target cell population in virtue of the relatively low ~ aftinity second ligand binding moiety. In this case, the multispecific ligand may also comprise a third ligand binding moiety which binds to the therapeutic entity, particularly where the multispecific li~~and is first administered tirst, optionally an anti-idiotypic binding moiety component where the therapeutic comprises an antibody component. Some sample targets are listed immediately below, while others are listed later.
Greater targeting usin~~ a hi~~h aftinity tirst ligand bindings moiety which binds to a cell associated or specific marker may be imparted to a variety of existing antibodies with suitably diminished affinity including those marketed or in clinical trials or listed below which are the subject of the patent and scientific literature, including those listed in PharmaBusiness June 2002 No.>l Category Functional li~"and (:ell localizing ligand for high MEode of action Comments for low affinity affinity arm arm -(locatiou of ti and) Growth factor 11.-?. CD 4 T cells Growth Ittctor blockade for a IL-2 required fur naive CD4 <>r CDR
blockade (soluble) specifically tbr CD4'f cell and memory CD=1'f cell responses:.
subset: i c. sclcctiv a liinditt~ of C'Dq or CD8 would also innnunosuppression block interaction with anti_en _ resenting cells.
I).-1 i C D8 T cells Growth factor blockade rite a - It.-I, needed for memory C'D8' T cell specifically Ibr('I)8'I'ecll responses subset: i.e. selective inununo~tultprcssion Chcmokine MC'P-I C'I)I lc (monocyte!macrophage) Monocyte%macrophage AI tints ofpercutaneous coronary ' blockade ~ (soluble) chcmokines; c.=. anti- intervention (PC'I) to limit restenosis:
inllammatorv a'~utt arthritis i C'ell activation ' 1 hrombin CD 31 (endothelial cells) I'ruvent thrombin binding to I Limit thrombosis and endothelial blockade (soluble) or i thrombin receptors on ' activation . e.g at time of PC'f P-selectitt Ithrottthin aUivatcd ' Icukocyms or endothelial cells endothelial cells) at endothelial surface i Cell activation CD80l8Ec Cp83 (dendritie cells) F3lock interaction with ('1)28: At tints ofallografting to induce blockade (dendritic antigen immuno~,uppression ~ tolerance presenting cells) i Inhibitory receptor Fc ganmta RII Fc epsilon I21 lnhance association of R of acute of allergic disease.
activation (Mast cells) I (Mast cells) avtivaung and inhibitory Advantages rner bispecific Fe fusion rcce:ptors, reagents because of more specific cell i tar~;eting . Sec Ihu D et aLNat ly1ed ?0U2 May:8(~lal8-21 ) Inhibitory receptor CTI.A-4 , C D8 Block C D80/86 interaction ~ Enhance specifically CTI, mediated blockade I v ith C l 1.,r1-~t ~ anti-tumor responses tvithout global 'I' I ' cell activation. Anti-C"fI-A4 ahs now in therapeutic trials to enhance tumor _ imnntnitv Adhesion ntolecule V("AM-I C'D31 (endothelial cells) or L:- Block VI,A-=4-dependent f Acute Itx of MS flare-up. Anti-Vf.A-4 blockade (activated selectin (ttctivafid endothelium) ? cell and monocyle adhesion to is in trial as 12x tar fy1S.
I i endothelial cell) VCAM-I c>n endothelial cells.
Adhesion molecule ~ IC':1M-I CDl l (endothelial cells) or E:- Block neutrophil adhesion to i ~lcute Rx to reduce repwfusion injury blockade )activated sclectin (activated endothelium activated ~ (myocardial infarction, bowel endothelial cell) . ischcmia~sur~y y) Cell death ligand CI)9s1. l l as-ligand) C'D2.s (activated T cells) Block activation induced cell Both ligands on same cell; CD95L
blockade ~ ( I cell ) death of I' i;ells: enhance anti- expressed on activated . not restin5'f tumor irnnnmitv crll_a Protect bout IFN ;~ CDR I Block ll-'N-y toxicity towards ('ell selective block of IFNy etlects;
another therapeutic: (soluble) endothelial cells during II -?
i.c. immunotoxins thertt v liu tuutors Protect from 1FN-yR ~ CD31 ~ Block IFN-y toxicity towards Both ligands on same cell. Cell another therapeutic: (endothelial cell) (endothelial ml)) ~ endothelial cells during II? selective block of iFNy effects;
therapy tier tumctn Inhibitory receptor . I <iF-(~ -. CD4 or CDR Block imutunosuppressim I'C1 -(?, ntodv of innnuuosupprcssion blockade ~ eflvcts of tumors; enhance not clear anti-tunu,r innnunity I
_ -~-Ccll type specific CD3 CD4sR() Trivalent ab so C'D3 can be I Anti-tunu>r Rx;
selectively entrance activation ' (mcntory;'el7ector-f cells) cross-linked memory f cells: reduce nonspecific activation of irrclvent T cells Category Functional (.'ell localizingMode of action Possible therapeutic ligand ligand for high uses for low affinity arm affinity arm (location of ti and) Growth 11.-i s Growth factor I or Rx of alloerall factor hlockadu fur rejection; 'f cull CD8 T cell, blockade ~ suhset specific I specifically suppression will ti,r C'U8 'h limit cell ~

svlectim inluctious complications subset: i.e. or unintended innnuncrsup rcssion inhibiticm of ('D~l regulators T cells Cell activationCD80/86 C'I)83 (dcudritic At time ofallot?ralling cells) t3lock to induce interaction with C'D2R:

blockade (dendritic immunosupprussion tctlurancu_ I ar~etine antigun to dundritic cells resenting will enhance etlictencv/
culls) Adhesion VCAM-1 C'D31 (endothelial molecule cells) or I_'-~
clock V LA-4-dependent T Acute Rx of MS flare-up.
Anti-VL.A-4 blockade (activated I
snleetin (activated undotlwlium) ~ cell and utonocytu adhesion to ~ is in trial as Rs fitt MS.

endotheliali V(:'a\M-1 on cull) endothelial ' culls.

Cell deathCD9sL (fas-ligand)CD?5 tactivatud ligand T cellsl ; Block activation induced cell ! Enhance anti-tumor immunity blockade ( f cell) _ ~, death of I cells;

Protect IFN-yR CI)31 ' Cell Block IFN-y to.vicity from selective block tosvttrds :,f IFNy another (endothelial(endothelial endothelial culls therapeutic:cell) , cell) durine 11.-? therapy i for tumors svfthout impairin=other _ useful IFN-v effects l InhibitoryFc ~amnrr E:c epsilon RI R x of acute receptor RII ; Enhance association of allergic disease.
of activation(Mast culls)(Mast cells) Advantages over activating and hispecitie 1u fusion inhibitory i receptors. reagents because of more specific cull i tar~etin4_ . See !he f) et aLNat Iv1ed 2002 May:B(s la 18-? I ) The invention also contemplates that FAS can be selectively blocked on various different types of cells such as pancreas beta cells using markers such as GAD65, lA-2, IA2-B, ICA-12.
Type 1 Diabetes is characterized by the destruction of insulin producing Beta Cells in the pancreas. One method in which Beta cells are destroyed is thou~~ht to be through apoptosis mediated by CD95 receptors on Beta cells. CD-9s seems to be upregulated in Beta cells of those with Type 1 diabetes (see Ann N
Y Acad Sci 200' Apr 958 297-304; J Clin Itnmunol '?00 t Jan;? 1 ( I ): I S-8'). Similarly, usin~~
'I°g, TPO ligands as cell associared markers CD95,TRAILR1, TRAILR2 can be blocked on thyroid cells.
Hashimoto's'fhyroiditis (liT) is characterized by the destruction of thyroid hormone producing cells and therefore hypothyroidism. It has been observed that some of this cell destruction is due to apoptosis. The CD9s receptor which is responsible for apoptosis is up regulated in thyroids affected by HT. Blocking the CD95 receptor by the RLAA may reduce the amount of apoptosis. The HAA can target either Tg or TPO
which are unique to thyroid tissue. There are also 2 other receptors suspected to be involved with apoptosis in thyroid cells:
TRAILR1 and TRAILR2 (see Nat Rev Immunol 3002 Mar;2(.i):195-204) Fas can also be selectively activated on distinct subsets of disease mediating immune cells associated with autoimmune and inflammatory disorders such as activated T cells, regulatory T
cells, CD4-~ cells, CD8+
cells etc.
Amon;,~ other embodiments hereinafter enumerated, the invention is also directed to multifunctional li~~ands which comprise antibodies which recognize cell type specific markers (hereinbefore or hereinafter exemplified), including those available commercially or published in the art, in any combination with those antibodies (hereinbefore or hereinafter exemplified, includin<, those available conunercially or published in the art) that recognize ligands on a broader population of cells including the cell population bearing the cell type specific marker, wherein the instrinsic affinity difference is maintained or adjusted to one, tv~o. three, four, five, six or seven orders of magnitude. The invention contemplates that affinity differences (increases or decreases in affinity) can be readily generate(1 by modifying amino acid:;
that are expected a piori to result in changes in affinity (see for example Chowdhury P. et al. Improving antibody aftinitv by mimickin<~r somatic hypermutation in vitro ( 1999) Nature Biotechnol(tgy Jun:
17 p.568-572'), or by a variety of other well-known high throughput methods that can readily be applied tt~
this task, for example by light chain shuttling, CDR grafting, parsimonious mutagenesis, shotgun scanning mutagenesis etc. Methods of affinity maturation are well kwown in the art. The invention also contemplates that two hybrisoma derived antibodies can be digested e;~. with pepsin to create F(ab')? and can be chemically recombined to create bispecific antibodies. Hybridoma fusion technolo~~y can also be used to create to tetromas for this purposes.
In this regard, the invention contemplates that hvbridoma derived I~,G
antibodies can be used for the relatively hi~~h affinity cell targeting moiety and that hybridoma derived I~~Ms can be used for the relatively low affinity binding arm. ~l'he invention also contemplates that the repective high and low affinity arms can be conjugated. fused etcaccording to well known methods to respective complementary ligands such as fasljun strepavidin/biotin (making it possible to pre-target the high affinity arm independently, particularly where the aftinity of the complementary li~~ands for each other is ~~reater than that of the second ligand binding moiety for its target ligand.
According to one embodiment of the invention, the first liv'and binding moiety can bind to a cell specific marker and and said second ligand binding moiety binds to the extrracellular portion of a li<~and involved involved in membrane transport across the cell membrane, for example an ion channel. vitamin recepetor etc. to inhibit uptake or export. F'or example the first ligand binding moiety may bind to a cancer cell specific marker and the second ligand binsin~~ moiety can bind to p-glycoprotein. Hi<~h affinity tumor specific antibodies are well known and anti-PGP antibodies are well known (see for exarnple Chen Y et al.
J Cell Biol Mar 6; l48('5):863-70: Mickisch GFI et al. C.';tncer Res. 1992 5?:
3768-3775). Some anti-PGP
antibodies function by by interfering with antibody utilization.
It will also be appreciated that a cell specific marker need not differentiate between sub-populations of cells that do not express the second target ligand eventhough those cells are not the targets of action of the multispecitic ligand.
Examples of antibodies that bind to cell specitic ligands, receptors. ete. are abundant and well known in the art (see for example Biosource International 3002 Research Products Catalog e.g. pages 178-19;, Upstate Cell SiSnalling Solutions 2002 Cataio~T and other catalogs of well known to those skilled in the art) Some examples are appended to the end of the disclosure and others are mentioned or referenced throughout the disclosure.
Definitions The term "associated" in relation to markers that are dominantly distributed on one or more particular entities is used to mean exclusively expressed, primarily expressed, or over-expressed to advantage from a tar~~eting standpoint.
The term "receptor li~~and" means a target ligand which is a li~~and for a receptor, for example, a receptor on a cell or infectious absent or a receptor which circulates independently of another entity.
The term affinity is contrasted to functional affinity which may result from avidity.
The term epitope though technically understood to be specitic for a given antibody. is used in a preterred embodiments to refer to antigenic determinants that are situated proximally to one another so that two antibodies will be considered to bind to the same epitope if one competively inhibits the binding of the other throu~~h any' probative competitive inhibition experiment known to those skilled in the art.
The invention contemplates that two antibodies with the same epitope specificity may have substantially the same amino acid composition ie. with possible exception of one or more additions, deletions or substitutions including conservative amino acid substitutions which do not substantially affect the specificity and an tino acid composition ofthe paratope The teen approximately in the context of orders of magnitude variations in affinity refers a variability that is up to a half an order or magnitude.
Without limiting the scope of the claims it is generally understood that biodistribution ofa multispecific ligand in contrast to that of a ligand will be predicated on the bioavailability of its target ligand.
The term "overlap'' and related terms connote that notwithstanding the difference in distributions of the first and second li~ands the first and second ligands are bioavailable for recognition on the same entity.
This term and related terms, exemplified below, are intended to exclude a situation where boat ligands are preferentially expressed on substantially the same entity, for example two different tumor associated antigens associated differentially with a differentiated population of cells within a tumor , most particularly in the case where they are individually suitable tartlets for delivery of a toxic payload. Thu sthe terms "different" in regard to biodistributions and "heterogeneous" and "diverse" in reference to populations of entities are similarly understood to exclude such a common distribution, in the appreciation that the invention primarily represents an improved strategy for tar~~eting two different ligands, in which one li~and has a broader distribution than the other or both have distributions that may overlap but are different from that of the target population. It will also be appreciated that the invention has particular application to a situation in which at least one of the non-target populations is one on w which one of said first and second ligands is substantially represented (in contrast to one on which it simply enjoys limited expression).
The term "receptor ligand" means a target ligand w which is a 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 "antigen binding fragment" refers to a polypeptide or a plurality of associated polypeptides comprising one or more portions of an antibody including at least one VH or VL
or a functional fragment 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 biological consequence.
The tern neutralizing in regard to an an immune function is used broadly to refer to any interposition, interference or impediment which affects the function of the target entity The terms modulating" mediating, neutralizin~~ function etc. are not intended to be mutully exclusive and are each used broadly, for example, without limitin~~ the generality of the scope accorder herein or by those skilled in the art the term modulating preferably refers to effecting a change, and the term mediating preferably connotes an indirect effect achieved through the instrumentality of another entity, for example a cell. cvtokine. chemokine etc..
The term "preferentially binds" recognizes that a given ligand binding moiety might have some non-defeating cross-reactivities.
The term biologic effector ligands is used to refer to any li~.:and for which there is a complementary target ligand on a target entity, and wfierein binding of the biologic et~ector ligand to the tartlet ligand exerts a biologic effect. For example the target ligand is typically a receptor and thf: biologic effector ligand may be any complementary ligand such as a cytokine, chemokine, hormone, colony stimulating factor, growth factor. receptor inhibitor, a~~onistor antagonist, which binds to the receptor with resultin~~ biolo~~ic effect.
The term "pre-selected" in reference to the affinity of ligand binding moiety refers to any selection or choice of differential or cooperative affinities relative to a second li«and bindings moiety w 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 second affinities and/or more preferably throuv~h an empirical evaluation of different choices for at least one of the first and second affinities, and preferably both.
The term multi means at least two and the term li~and is used broadly to refer to any entity or patrt thereof which can participate in an intermolecular interaction that can result in specific binding of suitable affinity for the interaction in question.
The term entity includes without limitation any molecule including without limitation. antibodies, complex or association of molecules, drugs, drug carriers (eg.vesicles eg. liposomes, nanoparticles,ete.) or any cell as well as any infectious a'rent or parasite (includinw, without limitation, spores, viruses. baceria, fungi ) as well as any other immune or therapeutic target.
The term "low affinity' means an affinity of approximately (this term is defined herein) 10 -'' molar to micromolar affinity, preferably (subject to safety considerations), approximately, !0-' molar affinity, more preferably (subject to safety considerations). approximately micromolar affinity, the term "medium affinity" means approximately l0-'to nanomolar affinity. preferably approximately l0-'' molar affinity, more preferably approxim<rtely nanomolar affinity, and the term "high affinity" means approximately 10-"' affinity or greater. Thus is one embodiment the invention contemplates that the multispecific ligand comprises a "target-ligand" bindin'T moiety which binds with low ~ or medium affinity to a target li~~and present on a diverse population of cells (preferably this moiety is an effector moiety ie_ one w ~hich~exerts a biological effect attributable to its binding eg. blocking or activatir~~ a receptor or blocking a cell membrane channel) and a "targeting", ligand binding moiety, which binds with medium or high affinity to a ligand associated with a sub-population of those cells so as to bias the biodistribution of the multifunctional li~and in favor of said sub-population. Preferably the multispecific ligand is adapted to be bound contemporaneously to the same cell. In another embodiment the tirst and second li~~ands 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 arc adapted to be bound contemporaneously and to both bind individually with low affinity, so as to bias the distribution ofthe multispecific ligand to the population of cells bearing both ligands.
As discussed elsewhere the teen approximately. in reference to "order of magnitude" increments in affinity, refers to up to a halt order of magnitude in affinity.
According to another embodiment, the invention is directed to an antibody termed a "coybody". A
"coybody" 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 affinity of up to several orders of magnitude, preferably a reference antibody of approximately une to three orders of magnitude greater affinity, preferably a reference antibody of medium affinity or preferably hi~~h affinity. As discussed above the reference antibody preferably comprises cooperating light and heavy variable regions in which at least at least one of the CDRs of at least one of the chains, preferably at least the CDR3, preferably that of the heavy chain. is exclusively or primarily responsible for the binding affinity of the coybody preferably in conjunction with the contribution of <tt 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-contributin~~ CDRs can be leveraged to diminish the on-rate, for example due to steric and/or electrostatic hindrance. In one embodiment the on-rate is reduced by a factor of 3 to 100x.
In one embodiment the coybody binds to a ligand which is over-expressed on a tartlet population of entities (eb. cells) relative to a non-target population of entities such that the biodistribution of the coybody to the non-tar~~et population (and target population) is diminished in a <given increment of time following administration. This targeting strategy is understandably adapted to situations where the resultin~~ delay in biodistribution is preferable for diminished toxicity attributable to reduced non-target entity binding in a <riven unit of time especially where the effectiveness threshold in that same amount oftime is not significantly if at all compromised or is preferable due to a sustained release effect (for example using a larger antibody format that is not readily cleared) As discussed below, advantages accrue particularly when this antibody is coupled to a hi_~~her affinity antibody (in the form of a multifunctional ligand) which binds to a different ligand associated with the target population. The invention contemplates that coybodies have multiple independent applications, includin~~ tempering the effects through antibody mediated neutralization of an over-production or sensivity to biologic effector ligands (eg. cytokines eg. ~i~NF2,r,,,~, chemokines e_~.
IL-~16 (crohns disease) etc. which are over-produced and!or mediate or aggravate eg. a chronic medical condition (which for example is not an acute phase) by bindings to such li~~ands, over a prolon~~ed periods, preferably using lar<~er antibody formats which are not readily cleared, especially where such tempering has side effects which are better spread over time and.~or where effectiveness is not a limiting factor and!or where a second therapeutic with different non-cumulative side-effects shares 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 reunions or domains (eg. FRs and CDRs) of dit~ferent origins or species eg. humanized, any combination of one or more antibody fragments or recombinant reconstructions (scFvs) of antibodies including dimers, diabodies, triabodies, a myriad of known bispecific, trispecific, tetraspecific antibody formats or monovalent, divalent, trivalent, tetravalent or other multivalent antibody formats (see for example review in Krian'~kum J, et al.
Bispecific and bifunetional sin~~le chain recombinant antibodies. Biomol En~~
2001 Sep;18(2):31-40 and others herein directly or otherwise referenced) or any fragment. portion, or reconstruction of one or more portions of an antibody (scFv) or any truncated form a li~,and binding entity, such antibody typically comprising at least a VN or VL portion or both or a functional portion of same (eg microbodies). including single domain antibodies, F(ab'),_ Fab, Fab', Facb, Fc, etc. The term antibody alsov includes fusions of such an antibody so defined and other functional moieties (eg. toxins, cytokines, chemokines. streptavidin, adhesion molecules).
According to one aspect, the invention is directed to a multispecific ligand with at least two different bindings speciticities for different tartlet ligands on the same target entity eg. a cell and which is preferably adapted to bind contemporaneously to (ie. there are no geometric or other constraints which preclude both moieties from functionally interactin~~ with their respective target li<~ands at the same tirne)the different target ligands, said multispecific ligand comprising a first target binding moiety which preferentially(some cross-reactivity(s) does not preclude the utility of the invention) recognizes a first target ligand and a second target binding moiety which preferentially recognizes a second target li~~and. 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 bindin ~; 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 independently of the second target bindin~,~ moiety binding to the second target ligand and an off rate (with respect to the first target ligand) which at least sufficiently exceeds the on-rate of the second target bindin~T moiety for the second tartlet ligand to at least provide opportunity for the second target moiety to bind the second tar~7et li~~and when the first target binding moiety is bound to first target li~~and, the second target binding moiety havin<~ a relatively diminished ability to bind and; or stay bound to the second target ligand independently of the binding of the first target binding moiety to the first target ligand (such that a plurality of the multispecific ligand will bind to a population of cells bearing both target ligands in preference to a population of cells bearing only the second target li~~and (ie. <rt least in part duev to the first target bindings moiety assisting (ie. providing opportunity) the second tar<~et binding moiety to bind to the second tartlet ligand and preferably out of proportion to what could be statistically attributed to the presence of two targets ligands on the target cell e~,. the binding of the first target bindint=, moiety providing necessary assistance for the second target moiety to bind is relatively increased (ie. relative to the situation where both of are of comparable affinity).
It will be appreciated that relative number of bioavailable second target ligands relative to the number of the bioavailable first target ligands will influence the selection of affinities of the first and second target binding moieties. For example, from the standpoint of safety. the affinity of the first target binding moiety for the first target ligand may well be sufficient if initially approximatingnanomolar affinity and the affinity of the second target binding moiety for the second target ligand will be selected to limit the number of effective binding events on the population of cells bearing only the second tarp>et moiety: an affinity which is inversely proportional to the number of bioavailable second tartlet ligan<ls on the population oi~cells bearing only the second target ligand ie. the non-target population (relative to the number of first target ligands on the target population of cells). For example, this may be assessed by determining the amount of labelled multispecific li~~and on the target and non-target populations of cells in vivo (or in vitro where the number of bioavailable first and second target ligands can be roughly estimated). This selected affinity, from a effectiveness point of view, will then be assessed as to whether it is sufficient for the second ligand binding moiety to bind to the second tartlet ligand on the tartlet population of cells, with the benefit of the first ligand bindings moiety bound or having been bound to first tar«et (igand. For example, where the binding of the second target binding moiety may be assessed through an in vitro assay (cg. an assay in which the blockin~~ or activatin~~ of a receptor is rl~easurable eg. through inhibition of binding of the natural ligand for a target receptor or through some measurable parameter associated with effective binding for example the release of cytokines or other biologic effector li«and. The effect of binding may be also be assessed by comparing the effects over time relative to a hi«her affinity second bindings moiety v~ hich is not associated with a first ligand bindings moiety. It will be appreciated that a more ubiquitous second target ligand may require selecting a higher initial affinity of the first target binding moiety for the first target ligand eg. picomolar affinity, and selectin;~ an affinity of the second target li'~and which may for example be of micromolar aftinity pluslminus approximately one order of magnitude. It will also be appreciated that 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 tar<,et population of cells. In the tinal analysis a suitable difference in affinity between the two binding affinities may well be at least, approximately, one, two, three. four, five, 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 af~frnity multispecific ligand may be assessed in a high throu~~hput screen or series ofsuch screens with a view to selecting a variant that has one or more predefined properties. alluded to above such as a) the ability to mediate a biologic effect on a target population relative to a negative control; b) the ability to mediate an improved or diminished biologic effect on a target population relative to a positive control. 'l his 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 diminished ability may be also assessed in a competition experiment of any probative type well known to those skilled in the art:~d) the ability 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. The invention also contemplates that the multispecific ligand may bind to a ligand which is cell specific in the sense that it binds to cells to which it has been delivered by prior administration (eg an antibody or fusion protein thereof which only binds to the target cells or at least to cells which do not have the ligand recognized by the second ligand binding moiety present in any significant amount), akin to the pre-targetting strategies well known in the art. For example, this strategy could be used to increase:
the number of first ligands relative to second li~~ands, where indicated.
In one embodiment, said first target binding moiety recognizes an entity-associated ligand eg. a target cell-associated* 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 recognizes 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 TRAIL. The multispecific ligand is thereby adapted to block or activate the receptor primarily on the target population of cells. In this connection, the invention is also directed to methods of evaluating or implementing the effects of this enhanced selectivity for the receptor on the target cell population and can be employed to diminish the adverse consequences and evaluate the benefits associated with using a ligand binding 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 permutations to dififerentiate between target cells and/or 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 directions) of this 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 is/are most effective and the importance of the size of the molecule for reaching the target as well as bioavailablility, and the importance of creating immunoconjugates and immunofusions with other molecules (insofar as this affects the size and distribution of weight in the molecule). The invention contemplates Chat employing more than one than one type of construct may be desirable and the invention is therefore directed to the various combinations and permutation of constructs according to the invention, in combination with each other and other therapeutic molecules or modalities.
One of constructs contemplated by the invention, is a multispecific antibody, for example a bispecific antibody havin<> a configuration which allows for binding to two antigens on the same cell. for example a traditional four chain immunoglobulin configuration having a hinge region (including F(ab')~
minibodies etc.), a diabody configuration (depending on the relative positions of the target ligands) and others herein referenced and, known to those skilled in the ar-t. It will also be appreciated that the mode of action of the multifunctional ligand may be contributed to by fusing or conjugating the multifunctional ligand to another functional moiety, for example, as described in the literature referenced below. These supplementary strategies are set forth below:
Additional Strategies For Modifying Targeting Capabilities According to one embodiment, the intrinsic affinity of the first target binding moiety for the first target is greater than the intrinsic affinity of the second target binding moiety for the second target. The term ~
"intrinsic" affinity connotes a measure of the affinity of a given target binding moiety for its target ligand which is independent of the affinity of the at least one other target binding moiety for its target ligand and as used herein could theoretically be evaluated in the context of the multispecificJ li<=and as a whole, if the other target binding moiety had an irrelevant specificity and therefore could not bind to its target ligand.
The invention contemplates that at least approximately onc, 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 the first target binding moiety is greater than the relative on-rate of the second target binding moiety. 'The term relative on rate is used to connote an effective difference in on-rate that may be instrinsic to the individual target binding ligand or may attributable to its configuration or relationship vis-a-vis other parts of the molecule.

Where the intrinsic on-rates of the first target binding moiety is greater than the intrinsic on-rate of the second target binding moiety, the invention contemplates that the off-rate contribution to the affinity of the second target bindings moiety may be proportionally greater than the off rate contribution to the affinity of the first target binding moiety. The invention contemplates that the binding of the second target ligand binding moiety to its target ligand may be more effective if its lower affinity is attributable in part due its reduced on-rate. The invention contemplates methods for reducing the affinity a target binding moiety by reducing its on rate for example by mutating or adding amino acid residues in reunions of the VI-i or VL that don't directly contribute to the off-rate (of a relatively high aftinity binder for the target. for example, as determined by modeling and structural analysis, for example, by evaluating, x-ray crystal structure and evaluating NMR data of the binding, or by mutagenesis, preferably by introducing a diversity of changes in a high-throughput manner (eg. phage display, ribsome display,microarray or other expression library) including substitutions, additions and deletions within various regions of the VH or VL and determining 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 red=ions of V H
or Vl.. including particularly the CDRI and CDR?, for example the CDR1 of the VH or CDR2 ofthe VL, is shortened andior mutated in a manner to reduce the probability of its having any direct contribution to the affinity of the selected molecule (throu~~h 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 are important for electrostatic interactions and optionally also hydrogen, bindings, generating a binder whose affinity will be postulated to be independent of the contribution of the modified CDR, and then optionally evaluating the success of this latter step through further mutagenesis (this step is most revealing if the CDR
is shortened but not. mutated or mutated to introduce amino acids important for intermolecular interactions) and then using.: the library to incrementally lengthen the reunion and%or introduce amino acids important for intermolecular interaction at a distance (eg. electrostatic interactions and 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 invention also contemplates modifying the amino acid composition of an existing binder by introducing or one or amino acids or mutations into a framework region at a location which is proximal to the binding region or a region which borders the interface of approach to the binding rf,~~ion or any interface between the target binding moiety and the target ligand. The invention conternplate~s that the on-rate and off-rate can be routinely measured using various technologies (e~~. Biacore) known to those skilled in the art, including various techniques of measurin~~ these rates in real-tune. for example those that rneasurc the de;tlection pattern of an incident form of radiation (e~~. 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 there to their DNA eg a phage (as per techniques known to those skilled artisans or published in the art) and the antibodies are evaluated independently of a phage (eg. they may even be cleavable from the pha;~e) or other expression system linkage which allows a more accurate measure of their true on rates and off-rates. The invention also contemplates that FR I could be lengthened in a relatively high affinity second target binding moiety to reduce its on rate. The cleanable peptide could be a unique identifying CDR.
In another aspect the invention contemplates that the multispecitic liV~and may comprise an Fe portion and a hinge portion and that one or both of a) the length, amino acid composition or* molecular weight (or various combinations of these interrelated factors) of the Fab or Fc portion;
and b) the amino acid composition (including len~~th) of the hinge portion (eg. any polypeptide segment that provides means for linking two typically heavy chains, eg. through one or more disulfide bonds, leucine zipper fos-jun, optionally a flexible hin~eJtypical of an IgG 1 or having one to several more disulfide bonds eg. 1V~G3) are selected to reduce the circurnstantial(shear rate. presence of de<~rading enzymes) affinity of the second ligand bindings moiety where the firsC ligand binding moiety is unbound relative to the circumstantial affinity of the second ligand binding moiety where the first ligand bindin;~
moiety is bound. The term circumstantial affinity broadly contemplates that the length and molecular weight of the Fc and the flexibility ofthe hinge re<~ion will individually and collectively contribute to the affinity ofthe molecule in proportion the shear rate of the fluid environment to a degree dependinv~ on whether the target is stationary or moving, once the multispeciCc ligand is bound. (fbound via the second tartlet binding moiety, any increase in the molecular w~i~~ht 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 The actual on-rate if the on-rate was to be measured independently of the on-rate of the other binding moiety binding especially since the off-rate of this binding arm is relatively low 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 an extent depending on the context in which binding occurs, the invention also contemplates that the high affinity ligand binding moiety will draw the multispecitic ligand from the circulation into a desired target tissue and that the low affinity binding arnr will then have greater opportunity to bind even if it does not bind simultaneously with the high affinity binding arm. Where the hinge region is extra flexible or has several regions of flexibility (for example where the heavy chains are finked through several disulfide bonds with regions of flexibiie linker therebetween) the disenga~~ing effect on the individual and paired binding of both the first and second ligand binding moieties will be less Similarly, using a truncated Fc porrtion (CH3 deleted, flab'), or minibody format) will assist the tirst ligand binding moiety to remain bound or foster binding of the second ligand moiety and will assist the second ligand binding moiety to remain bound. This construct rnay be preferred from an effectiveness standpoint (getting both ligand binding moieties bound), where the affinity of the second ligand binding moiety is low to begin with.On the other hand, decreasing the flexibility of the hinge region by alteration to its length and.'or amino acid composition and increasin~~ the molecular weight distribution towards the wfree'' end of the Fc will affect all binding, scenarios to a greater extent. The latter strategy may be less desirable where the Fab of the first ligand binding moiety is lengthened leg. has a longer hinge reunion at the N-terminus of the disulphide bond linking the heavy chains. than the: low affinity binding arm) to increase its propensity for individual binding. For example, in a conventional four chain or heavy chain antibody (two heavy chains but no light chains) the hin~~e reunion could be len«thened or shortened on the amino terminus side of the disulfide bond linking the heavy chains to an extent that does interfere with the simultaneous binding to both the first and second target binding moieties. ~fhe invention also contemplates that the target cell environment, naturally or through intervention, is a fluid environment (low shear rate) 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 which a cleavage site has been introduced so that disengagement due to the lever effect will primarily impinge on binding of the second ligand moiety to the non-target cell population (e~~. low shear rate or presence of MMP type enzymes in a tar~~eted solid tumor environment).
The invention also contemplates that second ligand bindin<~ moiety may be selected in an environment in which there is a selective pressure (moderate fluid flow eg~usin~~ lyive cells or tissue, candidate ligand binding molecules or pairs of the tar~Tet ligands on latex beads, where the substrate to which they are bound is on an incline or otherwise subject to fluid flow (optionally with rigid or high r710I. weight Fe), for simultaneous binding so that the affinity of the second ligand binding moiety is selected on the basis of its ability to augment the binding affinity of a first ligand binding moiety of preselected aftinity for the first target ligand (after or optionally before its aftinity maturation, depending on the shear force and affinity in question) and thereby augment the affinity of the multispecitic binding ligand as a wfiole, while the tirst ligand binding moiety is bound. (n this way, the strength of the binding affinity ofthe second ligand may be predicated on the tirst li~~and moiety bein;~ bound. 'fhe foregoin~~ strate~~y may have accentuated or at least equal application where the first ligand binding moiety has a longer Fab or for example where both the first and second li;_,~and binding_ moiety are devoid of a li~~ht chain ie. where havin~~ the correct bindings interface for the second target binding moiety might be more acute. The invention contemplates that the individual affinity of second Iigand birtdiry~ moiety selected in the above manner would be tested to ensure that its individual affinity was not sufficient for substantial independent targeting.
The invention also contemplates that engineering a suitable affinity antibody for solid tumor targeting in which the on-rate contribution to affinity is reduced (according to the strategy suggested above) may assist a dose of such antibody in achieving better tumor penetration. An antibods havin~~ a reduced on rate could be fused to a toxin such as a truncated version of PE or conju~.;ated to a radionuclide, etc. the reduced on-rate contribution ensuring that the antibody will be less likely 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 that the strategies decribed above will better permit the affinity to be more suitably apportioned between the on-rate and the off rate. The invention contemplated that a higher on-rate lower off rate Ab could be delievered in alternating days or other cycles of treatment. Thus the invention is directed to an antibody conjugated or fused to a functional moiety, wherein the on-rate contribution to the affinity of the antibody is anywhere between 3x and two order of magnitudes less than typical molecules havin~~ suitable properties fbr tumor penetration through diffusion. for example molecules having anywhere (any increments) between I O~ and 10-"' molar attinities leg. ~x 10 '', 3x 10~") preferably increments between 10-R to 10~'° (molecules where the on rate is norrttally approx. 10-') molar affinities. more preferably increments betty been 5x 10-'~ and Sx I 0-' It will be appreciated that the foregoing strategies could be employed for designing a multispecific ligand which will primarily tary~et cells which have both the tirst 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 cell population, by employing a multispecitic ligand in which neither target ligand is of sufficient affinity in the circumstances to effectively (with effect) bind or remain bound without the other target ligand being available for simultaneous binding. ~1s suggested above, it will be appreciated that a relatively higher af~tinity ligand could initially be employed on one of the ligand binding anus to select a second li;~and binding arm which improves the binding properties of the multispecific ligand under a suitable biologically relevant shear stress and which is selected or later modified so that it is individually insufficient for targeting its target on non-target cells in the circumstances in which it will be 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 receptors on the same cell, for example chemokine receptors cg.
CCR7 and CXCR4 on a breast cancer cell. In one embodiment. the off rate of one or optionally both li~and binding moities is sufficient in the circumstances to permit the mc»ety to remain bound for a sufficient duration for the other moiety to bind ie. it exceeds its effective or intrinsic on-rate. In one embodiment, both arms of such multispecitic ligand, bind to their respective ligands with low affinity. In one embodiment, one such arm is a "coybody...
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 as to permit greater flexibility in the binding of different anti«ens at different possible proximities to one another.
'fhe invention also contemplates that the two heavy chains of an IgG (with or without light chains an d,'or CHIiCL domains), minibody' F(ab')~ (with or without light chains and!or C111/CL domains), may be linked (whether they have a full size or fully truncated Fc or elon<~ated hinge regions) through a flexible peptide linker (such as used for makings seFvs l.c. multiples ofgly~ser) in order to ensure correct pairing of the heavy chains by expressing the linked heavy chains in E. Coli, for example, as inclusion bodies, which are refolded in refolding solution according to well established techniques in the art. In a construct employing light chains, the light chains may be linked through a disulphide bond linking according to well known methods of makin« disulphide stabilized Fvs (dsl=vs) and the same li~~ht chain may be employed for both the high and low affinity arms.
With respect to each of the preceding aspects of the invention, the invention also directed to a multispecific ligand comprising a tirst li~~and moiety which recognizes a tirst target ligand that is over-expressed on a disease associated entity for example a diseased or disease-causin~~ or mediating cell or infectious absent and a second ligand binding moiety that recognizes a target ligand and wherein the first tartlet ligand is characterized in that it does not lend itself to facilitating or permitting internalization of the second li~~and binding moiety.
The invention also contemplates that a target li<~and can be distributed in various concentrations for testing purposes on cell sized latex beads, columnar packing materials or flat substrates havin<'~ a high density dispersion of both target ligands.
The invention is also directed to combination therapies with the foregoing multispecitic ligands including, without limitation, immunotoxins, drugs, therapies with other multispecitic ligands herein described and particularly for cancer therapies directed at interfering with the inte~~rity of tumor cell vasculature.
Delivering Biologic Effector Ligands To A Target Entity W ith respect to each of the precedin~~ aspects of the invention, the invention also contemplates that the second ligand binding moiety may be constituted in whole or in part by a li~and which binds to a biologic effector ligand (such as a cytokine, colony stimulatin~~ factor, chernokine, growth factor etc. or related extracellularly expressed regulatory molecules that control their expression such as inhibitors, agonists, antagonists of same, which may have corresponding biological receptors), the ligand optionally having a higher affinity 'for the biolo;~ic effector ligand than the affinity of that biologic effector ligand for its receptor, and wherein the ligand. combined with the biologic eftector ligand (ie. bound thereto), has a relatively diminished ability to bind and~~or stay bound to the receptor (the second tar;: et li~~and) independently of the binding of the first target binding moiety to the first tar: et ligand eg. a lower affinity of approximately one, two, three, four, five, six, seven or eight orders of magnitude. The invention contemplates that the foregoing construct can be used to deliver the biologic effector ligand more selectively to the tarjet cell population recognized by the first ligand binding moiety. The second ligand bindings moiety may be an antibody portion of a multispecific ligand of the invention and the invention contemplates that a library of second ligand binding, moieties, recognizing multiple different epitopes on the biologic effector ligand, can be screened for their ability to bind to the biologic effector ligand, while it is bound in situ to its receptor, for example, using a microarrary of such antibodies, and the affinities of the binders can be evaluated. The invention also contemplates that suitable antibodies could be generated by "panning'' (with an expression library, eg. phase display, ribosome display.
or other similar display systems including yeast, bacterial, viral. cell based or cell-free display systems') or otherwise screening (eg. using antibody microarrays) against the biologic effeetor ligand while bound to its receptor and screening for their ability to bind to the biologic effector li;~and independently of its receptor. Again. the affinities of the antibody coupled to the-biologic effector ligand for the target receptor could be evaluated. More generally, the invention contemplates that an array of antibodies which recognize all different epitopes on a given biologic effector ligand could be generated and tested for their ability to accommodate binding ofa 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 effcctor ligand (BE L.) while bound to its receptor, and testing the identified members for their ability to bind to the second receptor, preferably by loading the biologic effector ligand onto an array of those members pre-bound with BEL and detecting those BEL, bound members for those which do and do not bind to the second receptor.
Therefore the invention is also directed to an antibody which accommodates binding of the BEL, 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 epitope on the BEL to the BEL's receptor binding site and optionally also the an Wino acid composition of the antibody at that interface.
The invention contemplates that fluid flow can be simulated in a purification or immunoaffinity column packed with one or more known packin~~ materials to simulate flow over a ligand coated substrate.
The invention also contemplates an apparatus and method for testing ligand binding in a circulatin~~ fluid environment in which the multispecitic ligands of the invention can be tested and wherein a continous flow of ligands, including target ligands, ligands ofthe invention and..~or ligand bearinLg entities (eg. cells or synthetic eg. latex spheres which can be adjusted to a cell size) to which one or types of li'gands have been affixedly associated accordingly to known'rnethods) can be generated. The fluid contact interface of the apparatus has a generally circular shape and is convex or otherwise capable of containing the fluid and thereby preferably permits fluid to flow around the surface 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 any air may optionally be displaced or evacuated.
The invention contemplates that the apparatus (at least the fluid contact vessel) can be rotated or oscillated (e~~. in an elliptical, oval or similar shape welt known to those skilled in the arts of fluid mechanics and related en'.;ineerin 'g arts) in a variety of different planes or with rocking-like motion in multiple planes cu-subject to peristaltic pressure (ie. where flexible tubing is used) to generate a continuous, optionally turf' ulence free fluid flow over the fluid contact surface at selected rates simulating: the various shear rates of arterial, venous, infra-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 linking ligands of the invention or target ligands or ligand bearin~Ve entities to permit fluid flow trcross the substrate in a plane suhstantially parallel or confonoing to the axis of flow.
In another aspect the invention is directed to methods of making a multispecific antibody in which:
a) the light chains are the same for both the Vl, domains . For example, the light chains (assuming the construct has two light chains) are generated for a first target binding moiety e«, in one aspect of the invention, the relatively high affinity binder, optionally ti-om a light chain ~.:ermline sequence, and this light chain is then coupled with a diversity of heavy chains to select a pair of chains which bind to the second target ligand, thereby constituting the second ligand binding moiety. which may be a relatively low affinity binder. An alternate or concomitant strategy to generate a lower affinity second ligand binding moiety would simply be to substitute the light chain of the first ligand binding moiety for that of the second ligand binding moiety and to test the affinity. In the case of a multispecitic which tartlet BELs to patrticular tar~'et cells, where for example, two high affinity binders are preferred, the heavy chain and light chain bindings to the BEL can be truncated correspondin~,ly at the Cf I1!CL, region so that the VH/VL interlaces and cysteines pairings these heavy and light chains are similarly spaced but spaced differently from the other VHiVL chains. By linking the heavy chains as explained above, all chains will pair correctly. It will be appreciated that the fore~oin~~ production strate~~ies could be applied to the production of heavy chain antibodies (two chains structures without associated li~~ht chains), wherein the heavy chains are from human or other species and that production in this case could be adapted to E. Coli. It will also be appreciated that deletion of a substantial part of the CH i and CL domains can be measured to provide a space for the BEL to sit in line with the other Fab which can be len<,thened in the linker or CLi 1 domain, as shown in Fi~~ure C.
fhe invention contemplates that evaluation of a diversity of the first tarv~et binding moiety can be accomplished with the BEL place to best accommodate selection in the context of the entire structure as a whole.
b) With respect to other methods to make bispecitic and bispecific fusions see Antibody Fusion Proteins Wiley-L.iss 1999 (infra) eg. particularly p 131 et 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 functional moieties fused or conjugated thereto, for example toxins, cytokines, enzymes, prodrugs, radionuclides etc.
In one preferred embodiment, the invention is directed to a multispecific ligand* with at least two different binding speciticities for different target ligands* on the same tarp=et cell*
and adapted to bind contemporaneously to the different target ligands, said multispecific ligand comprising a first target binding moiety which preferentially* recognizes a tirst target li~~antl and a second tar'et binding moiety which preferentially recognizes a second target ligand. and wherein said first tarl;et binding moiety recognizes a target cell-associated* tartlet ii<vand and said second target binding moiety recognizes a non-cell-associated target ligand w which is present on target cells and non-tary~et cells: and wherein the ability of the second target bindings moiety to bind to the second target is diminished relative the ability of the first target binding moiety to bind to the first target ligand, the first tartlet bindings moiety havin~~ an ability to bind to the tirst target ligand which is at least sufficient for the first tartlet moiety to bind to the first tartlet li~~and independently of the second target bindings moiety bindings to the second target li~and and an oft-rate which at least sufficiently exceeds the on-rate of the second target binding moiety for the second tartlet ligand to provide opportunity For the second target moiety to bind the second target li~~and when the first target binding moiety is bound to first target ligand, the second target binding moiety having a relatively diminished ability to bind or stay bound to the second target litand independently of the binding of the first target binding moiety to the first tartlet iigand, such that the multifunctional ligand will bind to the target population of cells in preference to the non-target population of cells. As su<~~~ested above, the strategy embodied in this preferred embodiment can also be employed in connection with any one or any combination of compatible strate~~ies reterred to above, to diminish in degree the requirement of using a low affinity second ligand binding's moiety.
In another aspect the invention is directed to heterofunctional li~~and comprisin~~ a first moiety which binds to a first tar~:et li~~and 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 tirst moiety to bind to the tirst target li«and independently of the ability of said second moiety to bind to the second target ligand and wherein the relative avidity or affinity or both the affinity and avidity of said second moiety are selected or adjusted to substantially reduce the probability of the second moiety bindings to the second tartlet Iigand without the first moiety, first or substantially contemporaneously. bindings to the first target ligand. For example, in one embodiment the first moiety is divalent and the second moiety is monovalent.
In one embodiment the affinity of the first moiety for its target ligand is for example up to several orders of magnitude greater than the affinity of the second moiety for its tartlet ligand, as discussed below. In a preferred embodiment both moieties are capable of binding to different tar<~et li<:,Tands on the same cell, for example as hereinafter specified. although in the case of tumor cell tar~~etin~, particularly with respect to cells that are ~rrow~in~~ adjacent to another the invention contemplates that.
tho first moiety may bind to one cell and the second moiety may bind to a neighbouring= cell. Accordingly, in the case of receptors requiring cross-linking for biolo'~icai activity the invention contemplates that such same cell interactions and adjacent cell interactions are optionally accomplished when the second moiety is bivalent. In one embodiment, at least one of said first and second moities comprise one or more antibody components. In another embodiment, said tirst moiety binds to at least one cell-surface ligand which differentiates between cells of the same population or sub-population, for example, at least one ligand which diffentiates which between populations or sub-populations of immune cells (eg. see WO 01/21641, US
6156878), for example, activated vs. non-activated, disease-associated or non-disease-associated (over-expressing or uniquely expressing certain receptors or other ligands rfor example cytokine or growth factor receptors, particular immuno~lobulin like molecules or MHC peptide complexes] or other differentiating markers hereinafter exemplified or apparent to those skilled in the art), anti said second moiety, in virtue of its binding to the second tartlet li~and, directly or indirectly exerts a biologic effect eg. a therapeutic effect. for example are immune modulatin<.~ effect. In a further preferred embodiment said second moiety has a broader target cell population than said first moiety E~~. see W'iley H. et al. Expression of CC
Chemokine Receptc>r-7 and Lymph Node Metastasis..., J. Natl. Cancer lnst. 91:1638-1643; Moore MA
Bioessays 2001 Aug:2 3(8):674-6. (The invention contemplates that by targeting CCR 7 receptor selectively on tumor cells, for example using a relatively hi~~h affinity bindings moiety for a tumor associated antigen and a relatively low attinity moiety which binds to and blocks CC R7 receptor, eg, when combined in therapy with a chemotherapeutic agent or an immunotixun 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 cell but also on a broader population of cells. In another embodiment said tirst moiety binds to an antigen associated with particular population of leukocytes and said second moiety binds to a receptor which is found on that population of cells but also on a broader population of cells (e~~. apoptosis mediating receptors Journal of Immunology 1998 1(10:3-1. Nat Med 2001 Aug; 7(8)954-960, WO 01:85782: 1CAM-R WO 00!29020: see also WO 01185768, WO
01185908: WO
01/83755, WO 01!83560, WO 01129020; Vitale et al. Prpc. Nat. Acad. Sci. 2001 May 8; 98(101:5754-5769;
CCR2 see also USP 6312689; USP 6,294,655 Anti-interleukin-1 receptor antagonist antibodies and uses thereof; USP 6.262,239; USP 6,268.477) . In another embodiment the second moiety does not necessarily bind with lower affinity to its tar~'et however it may bind to a first ligand which in turn binds to a second ligand on a tartlet cell (e~~. a receptor on the tartlet cell eg. a cytokine, chemokine or ~~row~th factor receptor), for example the receptor being on the same cell to which he first moiety binds, and it binds in a manner in which it partially interferes with the bindings of the tirst li~~and to the second li~~and and thereby directs or retargets that first li~,~and to the second li~~and in a manner which accomplishes the intended interaction of the the first with the second li'~and (eg a signal transduction or blockin~4 interaction ie. the second moiety causes the e~~. cytokine to bind to its receptor without en~~enderina the biological effects attributable to receptor bindings eg. si<~nal transduction, which may be assessed by assayin~~
for effects of eg. signal transduction according to well established techniques in the, art) but less competitively relative to the first moiety so that the fwst moiety exerts a targeting function ix. where the first ligand bound by the second moiety binds to a broader than desired population of cells. 'The bindings of the second moiety rnay also be compatible with the first ligand binding to one cell surface lip=and but not another e~z. see WO 00164946 the contents of which are hereby incorporated by reference. The ability to identify ligand residues of importance to binding or residues other these, the alteration of which rni~,~ht interfere with binding is well established in the art. The invention contemplates varying, by high throughput techniques e~~. phage display, residues of an antibody that are not involved in tirst li;~and bindings to create variants which can be tested for partial interference w°ith first ligand bindings to the second liganc! e~~, receptor binding.
Examples of receptors for blocking or activation by the targetin~~ methods described herein include tyrosine kinase type recptors. serine kinase type receptors, heterotrimeric G-protein coupled receptors, receptors bound to tyrosine kinase, fNF family receptors. notch family= receptors.
guanylate cyclase types, tyrosine phosphatase types, adhesion receptors etc. (for example receptors see those discussed in Cancer: Principles and Practice of Oncolo~~y 6't' Ed. De Vita et al. Eds Lippincott 2001.
includin~~ particularly Chapter ;. 7 and 18, The Autoimmune Diseases. Academic Press Third Edition, Rose;~Mackav ISBN:
0125969236, Immunology 6'~' Edition, Mosby 2001 Roitt et al. Eds; Molecular Mimicry.
Microbes & Autoimmunity by Madeleine W. Cunningham (Editor). Robert S. Fujinami (Editor) December 2000, amon~~ other references hereininbelow identified). Further mention rnay also be made of interleukin and interferon type receptors, HGF receptor (see for example LJSP 6,214,344), CD45, CXC family receptors including CXCRI and CXCR2 receptors including 1L.-8 receptor, EGFRs, receptors far molecules with functions in apoptosis or homeostasis. receptors such as FGF which sensitize tumor cells to chemotherapeutic agents, etc. It is known for example to modify receptor ligands in a way which does not int~°rfere with a si~~naling tunction (the residues important for siulnaling may be known or can be readly ascertained e~. see Retar<=eting interleukin 13 for radioimmunodetection and radioimmutrotherapy of human high-~~rade ~~liomas. Debinski W, Thompson JP.CIin Cancer Res 1999 Oct;S( 10 Suppl):3143s-3147s) but reduces the affinity of the ligand for this receptor (see also WO 0119861 ). Alternatively. the second moiety may be an antibody which is algonistic or antagonistic and used to block, activate, neutrali-re etc the receptor. With respect to EGFR family,TNF family and other receptor targeting antibodies which are capable of causing apoptosis directly or indirectly, see US 5,876, I 58, WO 00120576, W096, 08515, WO O I
~a4808 (P75AIRM 1 ), WO
00/29020 (ICAM-R). W'0 99r'12973, CA 2236913 etc. The invention also contemplates that the second moiety may also be targeted to a specific portion of a receptor which diffc;rentiates it from other receptors of its class and more generally contemplates that the second moiety may contribute to the targeting ability of the multifunctional li~~and.
In another aspect, the invention also contemplates that the first moiety binds to a target cell and said second moiety binds to a li<~rand, for example a natural ligand, (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 ligand (for example, the ligand may be retar~etted from circulation) to a targeted cell. For example the invention comtemplates that Il.-2 may be retargeted to I,AK cells or CTI,s via a high affinity l..cu-19 binding first moiety. For example, antibodies including fragments thereof which bind to cytokines or other natural li~~ands for retargeting purposes (eg.
single domain antibodies) can be made by phage display a~~ainst the cytokine or li'~and v\hile bound in situ to its receptor, The invention also contemplates that the affinity for the cytokine may he adjusted to re~~ulate the degree of tar~~eting and that serum samples may be evaluated to assess the degree of bound cytokine and the relative de~~ree of bound and unbound cytokine. Amon<~ other methods, for example, the invention contemplates that a radiolabelied multifunctional ligand may be used assess the amount of label associated with the multifunctional ligand when bound to the cytokine, by capturing the 'complex' with an antibody that r~;co;~nizes both <jnti'~enic determinants on both the cytokine and an adjacent portion of the ligand binding thereto ie. forming a composite epitope), such as may be generated by phase display and assessing the amount of label relative to the amount of captured complex.The invention also contemplates administration of supplemental amounts of natural ligand to compensate for the degree in which the ligand is retargeted insofar as such retargeting might impact negatively on immune or other physiological processes.
In another aspect the invention contemplates that patients treated with antibodies to a particular biologic effector ligand e~~. a natural ligand e'~. a cytokine, for example TNFa, may preferably be treated with a multifunctional ligand having a first moiety which binds to at least one cell type and a second moiety which binds to a natural li~~and such as a cytokine for retargeting that cytokine to that cell type, as in a preventative method for treatin~~ a disease, e'~. cancer. In this respect the invention contemplates that the antibody is capable of bindin'= to the cytokine but once bound the cytokin'°_, the cytc>kine is incapable andlor only weakly capable of binding to its receptor and-or that the multifunctional ligand also comprises a higher affinity receptor blocking moiety to minimize ret;trgeting of the primary disease site. In one embodiment, the fiirst moiety binds with relatively hi<~her functional aft7nity (ie. avidity. affinity. and!or relatively advantageous binding capacity in virtue of multiple ligand binding arms. each binding to different li~~ands on the target cell) to ensure bindin'~t to the retarget cell. In another embodiment the bound cytokine is capable of binding to the cytokine receptor at the retarget site but incapable of binding to the receptor at the disease site owing to differences in the receptors at the two sites. 'fhe nvention also contemplates using: antibodies which interfere but do not preclude binding of the biolo~~ic efftctor to provide a less toxic effect.
For example. patients with Crohn's disease that are treated with anti-TNFct (see for example. Expert Opin Pharmacother 2000 May:I (4):61 i-22 and references cited therein) may be treated according to the invention with a bispecitic antibody havin~~, in addition to an anti-TNFa binding moiety. which reduces the affinity of the bound TNF for the receptor, but also an antibody moiety which binds to tumor antigen which is expressed on many different tumor types or optionally a trispecitic antibody which additionally binds to a second multi-carcinomic antigen. preferably ane which broadens the ran;;e of targeting against prevalent cancers. With respect to tumor antigens mention may be made of EGFR. EPCAM, MU('INs, TAG-72, CEA, H 1 1 among other known multicarcinomic antigens (see also Cancer:
Principles and Practice of Oncology 6'r' Ed. De Vita et al. Eds Lippincott 3001 Chapters 18 and 20.5). In another embodiment, the second moiety differentially retargets a cytokine to one receptor in preference to another, for example, to a TNF receptor over-expressed or. tumor cells in preference to a TNF receptor associated with C'rohns disease. In a related but also independent aspect, the invention contemplates a method of screening for an antibody which preferentially binds to a ligand when bound to a first receptor relative to another second receptor by screening for antibodies (eg. by pha;~e display, ribosome display, etc.) which bind to the ligand e~~. a cytokine, when bound in situ to the first receptcar, and selecting among them those that bind to the ligand eg. cytokine but do not bind (substractive screening) or bind with lesser affinity to the cytokine when bound to the second receptor, as well as to antibodies and multifunctional ligands created by this method (see also USP 6,046,048 and WO 99:' 12973 and references cited therein with respect to TNF
family of receptors). Variations in the extracellular domains of 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 ligand which interferes with the bindings ot'the regular li~~and for this receptor. For example, the invention comtemplates a first ligand bindin;~ moiety which recognizes activated T-cells and a second li~~and binding moiety which blocks the It..-16 receptor for testin~~ the effect on Crohns disease (or alternatively activates an IL-16 receptor on those cells eg. by usin~~ a high affinity I1.-16 bound second moiety which becomes relatively low affinity 1L-16 receptor ligand when bound to the antibody, anain to test the effect on Crohn's disease (see Gut 2001 Dec. 49(6) 795-803) For example, in one embodiment. the invention contemplates that the second moiety hlocks a receptor that are found on cells other than the target cell, the blockage of w which leads to the apoptosis of or destruction of the cell eg. CD95 (eg. see June G.
et aL,Tar~~et cell-restricted triggering of the CD95 (,4 PO-l;Fas) death receptor with bispecific antibody fra~~ments C.'ancer Res 2001 Mar 1:(i1(5):1846-8). With respect to blocking insulin like growth factor receptor, insulin receptor etc. see The IGF system in thyroid cancer: new concepts. Vella V.. Mol Pathol 3001 Jun;54(3):121-4; Insulin receptor isoform A, a newly recognized, hi~~h-affinity insulin-like <growth factor II receptor in fetal and cancer cells. Mol Cell Biol 1999 May:l9(5):3278-88: Expression of the insulin-like growth factors and their receptors in adenocarcinoma of the colon. Freier S Cut 1999 May:44(5):704-8; Pandini G.. Insulin and insulin-like ~~rowth factor-I (IGF-l) receptor overexpression in breast cancers leads to insulin; lGF-f hybrid receptor overexpression: evidence for a second mechanism of IGF-1 signalingClin Cancer Res 1999 Jul;S(7):1935-44. With respect to tar~etin~~~ beta-1 inte~~rins see e~~.
Masurnoto A, et al Role of betal integrins in adhesion and invasion of hepatocellular carcinoma cells.
Hepatolo~~y. 1999 .lan;29( I ):68-74.
Arao S, et al. Betal integrins play an essential role in adhesion and invasion of pancreatic carcinoma cells.
Pancreas. 2000 Mar;20(2):129-37. Xie Y, Xie H. Characterization of a novel monoclonal antibody raised against human hepatocellular carcinoma. Hybridoma. 1 ~)98 Oct; l 7(5):43 7-44.
Peng H, et al Production and characterization of anti-human hepatocellular carcinoma monoclonal antibodies.
1-tux Xi Yi Ke Da Xue Xue Bao. 1990 Sep;21(3):259-62; W'hittard JD, Akiyama SK. Activation of betal inte~~rins induces cell-cell adhesion. Exp Cell Res. 3001 Feb 1:263( I ):65-76 Nejjari M, et al.
alpha6beta 1 integrin expression in hepatocarcinoma cells: re~:.ulation and role in cell adhesion and migration.
Int J Cancer. 1999 Nov 12:83(4):518-25: Yao M et al Expression of the inte'~rin alphas subunit and its mediated cell adloesion in hepatocellular carcinoma. .I Cancer Res Clin Oncol. 1997;1? 3(8):435-40.
The invention also contemplates a method of optimizing the cooperative affinities of respective binding ligands of a multifunctional li~~and described herein and the len~~th of a linker therebetween for the above and applications described below by pha~~e or ribosome display etc. in which the multifunctional ligand is a single polypeptide chain, for example, two sin~.:le chain Fvs or~sin~~le domain antibodies linked in sequence, or a diabody (see USP 5,837.242). by varying the DNA seduence correspondin;~
to amino acids that represent linker andlor for example CDR regions that are postulated to impact on affinity according, to methods and strategies that welt known in the art for affinity maturation.
These same strategies can be employed for engineering low°er affinity molecules. Accordingly, more generally the invention is directed to a phage display or similar library (e~=. a ribosome display library or a microarray) in which the population of variants is a multispecitic ligand. includin~~ a multispecific li~.:and accordin4~ to the invention herein defined.
The term ligand binding moiety includes any ligand that can be used as a targeting arm or to bind to the second ligand for example vytokines. chemokines. etc and optionally those that can be modified, preferably by high throu~~hput means to adjust its affinity and!or specificity, including scatTolds comprising a polypeptide, a peptide. a carbohydrate, a ribonucleic acid, a lipid and a small molecule. or a binding moiety consisting essentially of a combination of such types of molecules. E xample include non-immuno~lobulin protein scaffolds such affibodies (see LJS 5831012, US 5958736. EP 0486625) Trinectin domains (Phylos Inc.). TCRs, peptide mimetics. peptides located within a antibody variable reunion scaffold (eg. see WO 02. 44197, especially w ith respect to selectively targeting interferon alpha to specific cell populations) peptide fusion proteins and stabilized polypeptide loops. disulfide stabilized loops (eg. see W'O 99,'232?2):
all known in the art for such uses. These may be linked by peptide linkers of for example 10 to 50 amino acids. A variety of linkersincludin~~ flexible linkers such as multiples ofd=ty~lser are well known in the art.

In another embodiment blockage of a receptor does not necessarily lead to cell death but may lead only to decreased or increased release of certain cytokines etc. for example as mediated via the IL-6 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 multispecific ligand may also be fused or conjugated to a toxic moiety or other effector. In another or further preferred embodiment, said'rst moiety comprises two binding ligands (cg. one or both of which may be an antibody) which respectively bind to two different target li~ands each of which contributes to its total bindings capacity and neither of which are sutfiicient to efficiently target the the cell, for example a ligand which binds to a specit7c MHC peptide complex and a second reduced affinity ligand which binds to a li~~and on an APC. This approach also obviates the need to create hi~~h affinity ligand for a particular MHC 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 li~and which stimulates the activity of said immune cell. a ligand which inhibits the activity of said immune cell, a li'~and which causes one or more cytokines to be released, a li~~and which prevent one or more cytokines from bein;~ released, a ligand which causes or facilitates apoptosis of said immune cell or a ligand which permits internalization of said multispecitic ligand. In another preferred embodiment the heterofunctional ligand is fuses) or conjugated to a therapeutic agent or a moiety that binds to a therapeutuc agent (exemplified below) or a li~~and which effects bindin<~ to another immune cell, for example a T cell.
In another preferred embodiment, the multispecific ligand is a bispecific antibody. a trispecfic antibody or a tetraspecific antibody. In a further preferred embodiment the first moiety binds to but is incapable of modulating the activity of said immune cell and said second moiety modulates the activity of said immune cell independently of said first moiety. In another preferred embodiment the multispecitic li'~and further comprises a moiety that binds to at least one ligand located on the intraluminal surface of a lymphatic vessel, preferably a lymphatic vessel associated li~and, as hereinafter defined. In other aspects the invention is directed to a pharmaceutical composition comprising such a multispecific ligand and a pharmaceutically acceptable carrier, a method of usin~~ the heterofunctiona) li~~and 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.
'fhe invention is also directed to a multispecific ligand which comprises a first ligand binding moiety which neutralizes a ligand e'~. a natural li'~and such as a cytokine, chemokine, colony stimulating factor or grow=th factor and a second ligand binding moiety which binds ro a cell marker associated with a cell through which the natural li',and exerts a deleterious affect. Preferably the affinity for the first ligand bindin<~
moiety for the natural ligand will be greater than that ofthe second ligand binding moiety for the cell associated marker. Optionally the construct will be selected so that the bindinya interfaces are pointed in opposite directions and the lever effect is maximized, For example a bispecilic construct where the heavy chains are joined directly or through an inflexible linker and are optionally linked to their respective (optionally common respective li~,~ht chains) throu~~h a disulphide linker via framework residues as is well known in the art. Optionally, the molecular wei~~ht of the first ligand binding moiety is substantially less or mare (by at least I 0°io, preferably at least 30°,%. more preferably at least 2S°%) and preferably less than that of the second ligand bindin<~ moiety as may be effected or maximized through mutating from higher to lower (on the first ligand binding moiety) and lower to higher mot. wt residues (on the second li'and binding moiety), the native residues which are not exposed ( to avoid immuno'~enicity) and not essential for proper folding and Function of the VHIV 1. (as may be determined from the de~~ree of conservation of such residues among immunoglobulins of the species, through °~o frequency tables available throw: h the Kabat database and well known published determinations in this regard (see for example Vv'O 02f~0545).
Examples include neutralizing It.-2 via a marker on activated T cells, blockin~~ IL-15 via CD8-~- ~C' cells, blockin'~r TNFalpha on mast cells; binding thrombin via activated endothelial cells etc.
The term heterofunctional is used broadly to refer to a linand: 1 ) comprising at least tw ~o functional moieties that have different functions on different capacities to perform the same function and ?) which is typically and preferably heterospeeitic (having two binding speeificities)_ Unless the context dictates otherwise the term avidity when used in a comparative, quantifiable or controllable sense is used to refer the valency of the binding entity or moiety. The term functional affinity is used a composite term referrin~~ to a quantitative and contollable (thou~~h not necessarily quantifiable, especially when its consists of both avidity and affinity components) propensity to specific binding attributable to one or both cafavidity and affinity effects.

In another aspect, the invention contemplates that cells, particularly immune cells, that are expected to be present at or proximal to a disease site (eg. at the site where an immune cell crosses the vascular endothelial cell wall). 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.markers associated with activated immune cells or disease mediating immune cells eg. LEU-19, a marker associated with activated or killerT-cells, etc for example with an antibody, which is linked to a moiety that is capable of exerting a therapeutic effeca in relation to the disease. for example, an immunoliposome or an antibody linked to another therapeutic delivery system (for example example streptavidin or biotin fused, coated or conjugated entities or other payload carrying entities (see for example US patents 5439686, 6007845, 5879712, 5456917, 616550?, 5079005, _5888500, 5861 159, 6193970, 6190692, 6,077, 499, WO OOG9413, WO 01;07084, etc.). For example, an immunoiiposome may carry one of or a combination of cytokines, chemokines, toxins or other therapeutic molecules suitable for treating the disease directly or indirectly, for example by attracting or preventing the attraction, activating, aner~~izing or otherwise modulating the activity of immune cells for therapeutic or related purposes. 'thus accordin~~ to another aspect, the invention is directed to a multifunctional ligand characterized in that it exerts an independent hiologic function said multifunctional ligand comprising a ligand which binds to a non-diseased disease associated cell and: a) a therapeutic entity; b) a ligand which binds to a therapeutic entity; or c) a li~~and which binds to a disease mediating entity eg. a biologic effector molecul which is released by the disease mediating entity or the diseased cellve~, a cytokine or other BEL
which mediates or ag~~ravates a disease process. Preferably said multifunctional li<,and comprises at least two of a), b) or c) and preferably all three.
The term "independent" refers to a function which is primarily exerted in relation to an entity other than the entity that is targeted (save for possible entity associated side effects).
The invention contemplates that targeting a cell which localizes to a disease site will better localize the independent effect of~the targetin<~
ligand to that locale. For example, an antibody which binds to and neutralizes a cytokind or other BEL
associated with Crohn's disease eg. TNF alpha at the disease locale if tar_~eted to an activated CD4't T-cell using a marker which identifies activated T-cells.
In another aspect the invention is directed to a heterofunctional ligand comprising a first moiety which specifically binds to at least a first target ligand on a first entity and a second moiety which specifically binds to at least a second tartlet ligand on a second entity, 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 at least orte first target ligand independently of the ability of said second moiety to bind to the at least one second tar<~et 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 seeond 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. In one embodiment the first moiety comprises at least one li~_and preferably at least one antibody which binds to a first cell, for example an intraluminal 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 cells or disease causing,_.. mediating (ie, having a role whim 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). In other aspects the invention is directed to a pharmaceutical composition comprising such a heterofunctional liwand and a pharmaceutically acceptable carrier, a method of using the heterofunctional li~~and 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.
In another aspect the invention is directed to a multispecitic ligand comprising a first moiety which specifically binds to at least one first target ligand on a first entity (eg.
a lymphatic endothelial cell, a diseased cell or a cell proximal to a site of disease) and a second moiety which specifically binds to a second target ligand or site on a second entity, and wherein the second entity binds to a third target ligand, and wherein the first and third target ligands may be on the same or different entities eg. the same or different cells, and wherein preferably the aftrnity 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 independently of the ability of the second moiety to bind to the third target ligand (the first moiety optionally comprising more than one li~:and (which may be the same ligand or a different ligand) one or more of which are necessary for binding and optionally each of which is sufficient for specific binding) to corresponding first target libands) and preferably wherein I ) the avidity or affinity or both the affinity and avidity of said first moiety is/are selected to enable it to bind to the at least first target ligand in preference to the second moiety binding to the third target li~and when both said first and second moieties and the second entity are substantially contemporaneously bound to their respective target ligands eg. to effect a tranfer or ?) wherein the avidity or affinity 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 and,'or 3) w herein the avidity or affinity or both the affinity and avidity of said second moiety for the second entity are selected to enable the second moiety to bind to the third target ligand in preference to the second moiety binding the second entity when both first and second moieties are substantially contemporaneously bound to the respective first and second entities and the second moiety is substantially comtemporaneousiy bound to the third target ligand), or 4) wherein both I ) and 2) above are both operative conditions. In one embodiment, the tirst entity is a diseased or disease causing, mediating or mitigating cell, for example an immune cell (the first moiety preferably binding to a particular population or sub-population of the tirst target entity e~~.
the immune cell, tear example activated T cells), the tirst moiety optionally comprising two or more ligands which may be the same or different and which bind to two or more respective first target cell surface ligands (though not necessarily to any particular effect (and in one embodiment to no effect at all) utker than to better bind to and thereby target the cell, preferably in competition with the second entity, which in a preferred embodiment tor~ets a broader population of cells), and the second entity e~. a biologic etfectore liband is an entity that binds to a third target ligand. the third target ligand preferably being expressed on the surface of a cell for example the same immune cell, for example a natural cell surface li~~and, to which binding yields a desired etTect. for example a therapeutic advantage, the second moiety being, fbr example, the natural ligand for the cell surface ligand or functional mimitope or antagonist or arsonist thereof, for example a cytokine, the third target ligand in this case bein<~ a cytokine receptor on the immune cell. The invention is also directed to a method of"targeted delivery' of a therapeutic entity to a cell in need of such therapy by administering, said heterofunctional li~~and. In this respect numerous therapeutic entities will be apparent to those skilled in the art, only some ofi which are mentioned herein by referring to the therapeutic entity itself or by referrin~~ to the third target lip=and for which such entity is known and available or readily made by routine skill in the art. Optionally the heterofunctional li<~and (and similarly in the case of other multispecitic ligands of thv invention described above which are adapted to deliver a BEL,) is delivered with the second entity, preferably in the same composition (preferably bound).
In the case where the second entity is a natural li~~and circulatin~~ in the path of delivery of the heterofunetional li~~and, some proportion (0-100%) of the heterofunctional li~.:and may be delivered without supplied second entity, particularly if the treatment or the disease generates an abundance of the natural li~and. In another embodiment the first moiety binds to a target li~~and on a stationary cell (for example a vascular endothelial cell or a lymphatic endothelial cell), preferably a tissue or cell type "associated" ligand (more abundantly expressed uniquely expressed on target cells relative to non-target cells). and the third target li~,and and the second moiety are cell-suface target and ligand therefor as stated above, for example the second moiety binds to a cytokine and the third target li~and is a cytokine receptar. for example on an immune cell. In one embodiment the first moiety binds to at leant one target ligand which differentiates between populations or sub-populations of immune cells and the second entityin virtue 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 further preferred embodiment the tirst moiety is incapable of modulating the activity of said immune cell and said second entity modulates the activity of said innmune cell independently of said first moiety. In another or further preferred embodiment the second entity binds to a molecule involved in cellular adhesion. a cy~tokine receptor, a ligand which stimulates the activity of said immune cell, a li~and which inhibits the activity of said immune cell (eg. via anergy or tolerance mechanisms), a li~and 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 said immune celll, a ligand which permits internalization of said heterofunctional li«and. In another preferred embodiment the heterofunctional li~~and is fused or conjugated to a therapeutic agent or a moiety (eg. biotin, avidin) that binds to a th erapeutuc agent (exemplitied below) or a ligand which effects binding to another immune cell, for example a T
cell. In another preferred embodiment, the heterofunctional ligand is a bispecific antibody, a trispecfic antibody or a tetraspecit7c antibody. In another preferred embodiment the heterofunctional ligand further comprises a moiety that binds to at least one li~~and located on the intralurninal surface of a lymphatic vessel, preferably a lymphatic vessel associated ligand, as hereinafter deFned.
In other aspects the invention is directed to a pharmaceutical composition comprising such aforementioned heterofunctional li~~and and a pharmaceutically acceptable carrier, a method of usin~~ the heterofunctional ligand in the preparation of a pharmaceutical composition for treating a disease, and to a method of treating a subject by administerin~~ same in a therapeutically effective amount. As suggested below, the foregoing strategy could be used in combination with other targW ing strategies herein mentioned or known in the art.
The invention contemplates making antibodies to second entities, for example.
while bound to their natural receptor, by phage or ribosome display, by methods as hereinafter disclosed.
In another aspect the invention is directed to a heterofunctional ligand comprising at least a first moiety which specifically binds to a first tarr~et ligand on a cell and a second moiety which specifically binds to at least a second target ligand on the same cell, and wherein the affinity or avidity or both the affinity and avidity of said first moiety and the affinity or avidity or both the affinity and avidity of the second moiety are selected to substantially reduce the probability of tyre either moiety sin '~ly binding to its respective ligand for a suffiicient duration or series of durations to accomplish the function of said heteroi-unctional ligand unless both first and second moieties are substantially contemporaneously bound to the cell. In a preferred embodiment the first moiety binds to at least one target li~~and which differentiates between populations or sub-populations of immune cells and the second moiety in virtue of its binding to the second target ligand. directly or indirectly exerts a therapeutic effect, for example by modulatin~~ the activity of said immune cell. In another or further preferred embodiment the first moiety is incapable of modulating the activity of said immune cell and said second moiety modulates the activity of said immune cell independently of said first moiety. In another or further preferred embodiment the second moiety binds to a BEL, for example a molecule involved in cellular adhesion, a cytokine receptor, a li'~and which stimulates the activity of said immune cell, a ligand which mhrbus the activity of said immune cell (eg. via anergy or tolerance mechanisms). a li~_and which causes one or more cytokines to be released, a li~~and which prevent one or more cytokines from being released, a ligand which causes or facilitates apoptosis of said immune celll, a ligand which permits internalization of said hcterofunctional ligand.
In another preferred embodiment the heterofunctional ligand is fused or conjugated to a therapeutic a~~ent or a moiety (eg.
biotin, avidin) that binds to a therapeutuc agent (exemplified below) or a ligand which effects binding to another immune cell, for example a T cell. In another preferred embodiment, the heterofunctional ligand is a bispecific antibody, a trispecfic antibody or a tetraspecific antibody. In another preferred embodiment the heterofunctional li~~and further comprises a moiety that binds to at least one li~and located on the intraluminal surface of a lymphatic vessel, preferably a lymphatic vessel associated ligand, as hereinafter defined. In other aspects the invention is directed to a pharmaceutical composition comprising such a heterofunctional li~~and 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 administErin~~ same in a therapeutically effective amount.
In other aspects the invention is directed to a method of in vivo modeling or testing using one or more foregoing targeting strate~~ies by administering a heterofunctional ;
multifunctional li'~and as hereinbelow disclosed as well as a method of infra-lymphatic drug delivery employinz~ 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 in the form of such a heterofunctional ' multifunctional li~~and and compositions thereof In one aspect, the invention is directed to a heterofirnetional ligand, comprising a first moiety which specifically binds to at least one ligand located on the intraluminal surface of a lymphatic vessel and a second moiety which specifically binds to a disease associated cell and the use of such heterofunctional linand in treating or preparin~~ a pharmaceutical composition for treating disease associated cells, includin~a diseased cells or disease causing, mediating (ie. having a role whicn is known to be intermediary or indirectly facilitating e~~. antigen presenting cells) or mitigating cells (cells, typically immune cells, which directly or indirectly counteract the diseased or disease causing.; or mediatin~~ cells), w ithin a lymphatic vessel. Preferably, the ligand located on the intraluminal surface of a lymphatic vessel is a lymphatic vessel associated ligand.
In another aspect, the invention is directed to a pharmaceutical composition comprising <r pharmaceutically acceptable carrier and a heterofunctional ligand comprising a first moiety which specifically binds to a ligand located on the intraluminal surface of a lymphatic vessel and a second moiety which specifically binds to said disease associated cell and the use of such li~~and in treating try°atin<.~ disease associated cells, including diseased cells or disease causing or mediatin~~ cells. within a lymphatic vessel. Preferably. the ligand located on the intraluminal surface of a lymphatic vessel is a lymphatic vessel associated lieand.
In another aspect, the invention is directed to a method of treating disease associated cells, including diseased cells or disease causing or mediating cells, within a lymphatic vessel comprisin~~ administering to a subject a heterofunetional ligand comprising a first moiety which specifically binds to a li~~and located on the intraluminal surface of a lymphatic vessel and a second moiety which speciticall_v 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 to a heterofunctional lir~and, comprising a first moiety which specifically binds to at least one ligan d located on the intraluminai surtace of a lymphatic vessel and a second moiety which specifically binds to a therapeutic entity for example a cytotoxin or cytotoxin-linked-entity or a non-tonic entity which is present in toxic amounts and to a method of reducing the toxic effect of such entity in a subject by administering said heterofunctional ligand to said subject.
In another embodiment the invention is directed to a method of therapeutic evaluation and/or targetin~~ :' intervention in which such heterofunctional li~~and is administered substantially contemporaneously with a cytotoxic substance 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 both to exert their respective effects, preferably one or both exvrtin« their respective et~'ect optimally, or one exerting its effect dominantly. It 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: cooperatin;;
entities are delivered proximally to their respective target cells, for example by cannulating one or more blood vessels as proximally as possible to the sites) of a tumor and/or actual or anticipated site('s) of metastases (as discerned by using one or more tumor and vascular imaging agents. for example, one or a combination two or more absents selected from a vascular opaquin~ absent, a radionuclide conjugated anti-angiogenic antibody, and a radionuclide conjugated anti-vascular endothelial cell marker antibody. which cannulation may occur for example in the course of initial surgical intervention with respect to the primary tumor site) and-or at the same time cannulating one or more lymphatic vessels (which may optionally be located which the help of a radionuclide conjugated anti-lymphatic vessel marker antihodv) leading to or from such tumor sites or metastases. The invention contemplates that small sections of vascular prostheses, well known to those skilled the art. (eg. Dacron types) may be grafted into those locations to permit a prolonged and secure attachment of such prosthesis to an intra-vascular cannula for secure delivery to such vascular or lymphatic locations for repeated and;'or prolonged administration, optionally while the patient is mobile, optionally using one or more portable infusion devices, including micropumps desi~~ned for such purpose (see for example J Neurosci Methods 1997 Mar;72( 1 ):35-8, US ~ 180 i65:lmplantable infusion device. See also Cancer: Principles and Practice of Oncology (infra). Numerous embodiments and improvements in vascular prosthesis and in such portable infusion devices and micopumps are described in the relevant scientific and patent literature known to thaw skilled in the art. The invention also contemplates deliverin~~ anv multifunctional li~~and herein disclosed in the above manner.
It is to be understood that targetin~~ strategies employing the cooperative action of ligands with different affinities for their targets exemplified above, may preferably have affinities which differ, depending on the application and their avidity, by a factor of 30°% up to a number of orders of ma~~nitude which nr<ty one, two, three, four. five, six and even seven or ei~~ht order of magnitude, in order to achieve substantial advanta~~le, as hercfter detailed in connection with one such strategy.
In another aspect the invention is directed to a heterospecitic ligand comprising a first moiety- which specifically binds to at leant a first disease associated ligand located on a diseased or disease causing, mediating or mitigating cell for example a cancer cell or an immune cell, as well as on non- diseased or disease causing, mediating or miti~~ating cells (non-target cells) and at least a second moiety which specifically binds to a second difTerent disease associated li'~and on the carne cell and wherein each ligand is expressed on a substantially (see detinition below) different, n-overlappin~~, subset of non-tar~~et cells, so that functional binding to a non-target tissue is substantially (see definition below) precluded. In another embodiment the functional affinities of the respective liv~ands may be selected in accordance with the strategies suggested above, to further facilitate targetin~~. In another embodiment, both different li~~ands are required for internalization. In other related embodiments. the heterofunetional ligand comprises at least two different pairs of binding moieties (eg. a trispecific or tetraspecific antibody which depending on its construction will permit 2. 3 or ~ such different pairs e~~. a tetraspecitic sinUle domain type antibody (ie.
consistin« primarily of the heavy or li4~ht chain variable region or a functional fragment thereon (see discussion below regardin~~ its construction) allowing the _~rreatest variation in such ~~eometries and preferably simultaneous binding of more than one pair), wherein 1 ) at least three such ligands are expressed on a substantially (see detinition below) different, preterahly non-overlapping, subset of non-target cells, so as to further limit binding to non-target cells and; or 2) wherein at least two different pairs of ligands target a substantially different subset of cells within the same target population eg. different cells within the same tumor (eg. proliferating vs. non-proliferating cell -- the respective amounts of the ditizrent types of cells will dictate the perecenta~~e of the dose that will be targeted to one population or another). In other aspects the invention is directed to a pharmaceutical composition comprising such a heterospecific li~and and a pharmaceutically acceptable carrier, a method of usin~~ the heterospecitic li;~and 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. It will be appreciated that the foregoing general strategy can be accomplished with two or more different antibodies have differiny~ and preferably non-overlapping normal ie. non-tar<~eted cell distributions, preferably administered in the same composition and preferably cross-linked by biotin-avidin like complementary pairs to facilitate cross-linking for internalization or targetingr of therapeutic agents. In a preferred embodiment each such independent antibody can'ies a different complimentary aspect of a toxic payload e;. a different liposome (or other payload carryinU entity for example a micro or nano particle or sphere or albumin) which complement each other in virtue of their respective contents (eg. one carries the prodrug and the other the necessary convertin~~ enzyme).
In another aspect, the invention is directed to <t multifunctional ("multi"
meanings at least two) li~and having, at least, a first portion which binds to a lymphatic vessel associated ligand and a second portion comprising an immune function-exerting moiety.
'fhe tens lymphatic vessel is used to facilitate broader reference to ligands (eg. antigens I receptors) present on cells bordering the infra-luminal pathway throu~~h the lymphatic 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-luminal cell surfaces (not necessarily to the exclusion of non-luminal surfaces) on the infra-luminal endothelial cells (not necessarily to the exclusion of non-luminai lymphatic endothelial cells) of those vessels.
The term 'associated' with reference to lymphatic vessels, is used to mean differentially expressed on the surface of endothelial cells of those vessels for targetin' purposes. such as to accomplish an object of the invention, but unless otherwise expressly indicated in a particular instance, it is used limitatively, to reference li~~ands that are predominantly, if not exlusiveiy, found on the aforementioned endothelial cell surface (as well as in lymph nodes), such that the first portion of the multifunctional li~~and is for all intents and purposes functionally targeted to the infra-luminal surface of the lymphatic system. for instance, it is appreciated that the ligand in question may be targetted to a limited extent elsewhere eg. in the case of preferred LYVE-1 ligand discussed below. to parts of the spleen (which also provides a venue for immune cell interactions).
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 li~.;and of the invention is intended to exclude only, unless otherwise specifically stated in the claims, only those embadiments disclosed in WO 98!06$_x9 or other references describing li~,ands. antagonists or antibodies which bind to a lymphatic vessel associated li4~and or receptor (see examples ofsuch references below), insofar as such embodiments comprise lymphatic vessel associated ligands as hereinabove limitatively defined, and to this limited extent only, the term therapeutic function exerting moiety or immune function exertin~~ moiety preferably excludes: 1 ) an antibody f~. recwptnr, insofar as such limitation excludes from the scope of the multifunctional li~~and (per se) aspects of the invention, substantially intact naked antibodies which simply bind to a lymphatic vessel associated ligands, as well as preferably excluding?) cW ntoxin.s or drugs, insofar as this excludes from the scope of the mutifunctional ligands of the invention an antibody or fragment thereof w which is flrsed or eonju'ated etc. exclusively to a cytotoxic molecule (including an atom) or drug (ie. an antibody linked to a cytotoxin or drug only, which is not per se an or is not inte~~rated with an ir~dep~rzdent biulo~ric or immune function exerting component) so as to accomplish a function in relation to cells or other entities (including other multifunctional li~ands) within the lymphatic system other than the cell or li;and to which the multifunctional li~and is anchored. Similarly, the invention is not concerned with multifunctional ligands which are adapted to be internalized into a lymphatic endothelial cell and the invention is specifically concerned w-ith targeting a lymphatic vessel associated marker which does not promote internalization andlor in which the first portion has an af~tinity (hi~.r,h or medium) w which limits this effect (ie. to a side effect) In the same vein, the term immune function is broad in intent (as discussed below, and includes particularly any function, including binding. capable of being exerted by an ligand preferably an antibody (e~~.

multifunctional ligands which are bispecific antibodies) however it is to be understood that the invention and particularly the immune function exerting moiety does not have as an object (despite possible incidental effects) evaluating or exertin~~ a disease responsive or immune function vis-a-vis ligands / cells lining the intra-luminal surface of the lymphatic system ie. insofar as such ligands have a role in disease (other than simple binding exclusively for anchoring purposes which is attributable not the immune funtion exerting moiety but to the first portion) hut rather, as evident in preferred aspects of the invention, preferably an independent biologic or immune function which is not predicated on blockin4~ the lymphatic endothelial receptor or treating cells bearing the receptor ie. exerted vis~-a-vis 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, inhibition etc. in the lymphatic system); and 2) in the case of non-stationary diseased or disease causing cells or molecules, at the site of those cells' molecules including, preferably, within the lymphatic system, for example by binding to or signaling those cells in the lymphatic system.
In one embodiment, the first portion of the multifunctional ligand is an antibody fn another embodiment, the immune function exerting moiety binds to a target ligand and thereby directly or indirectly accomplishes its effect (in wtrole or part).
For example, the target ligand may be a cytokine, for example in order to target immune cells to the lymphatic system to assist in, diseased, disease causin<~ or other target cell ablation or phagocytic type activity (eg. by the cytokine in turn binding to a ligand, for example on an immune cell having pha'~ocytic activity) or exertin~~ a chemotactic 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 immunotherapy (such as anti-CD3 therapy; see for example USP
6193969. Kummer U. et al., Immunol Lett 3001 Jan 1: 75(2):153-I 58) (with respect to removing disease associated antibodies from circulation see for example a bispecific dsDNAx monoclonal antibody construct for clearance of anti-dsDNA IgG in systemic lupus erythematosus. J Immunol Methods. 2001 Feb l; 248(1-2):125-138). (see also, for example, US 5,9b8,510 with respect to antibody-CTLA-4 fusion proteins for use in bindings to various tartlet li~lands).
In another embodiment, said immune function exerting moiety comprises an antibody and optionally both the first portion and the immune function exertin~~ moiey are antibodies (with respect to bispecitic antibodies, and a recent review of some of the technologies referred to or applicable to various aspects of the invention (see particularly, Journal of Immunological Methods February 2001 Vol. 2480-2) pa~.:e 1-300) In another embodiment, said immune function exerting moiety binds to an immune cell, a diseased host cell 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 to a cancerous (in the broadest sense of that term) cell and a virally infected cell (these examples are given inasmuch as the invention in a preferred embodiment involves tar~.~etin~~ such cells for destruction) and the term disease causin~~ cell includes but is not limited to a virus or other infectious agent and as w ell as immune cell which is directly or indirectly involved in mediatin~~ or causing a undesired, deleterious or patholo~.;ic consequence. including but not limited to autoimmune disorders, transplant rejectiun, 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 etc. that is directly or indirectly involved or associated in mediatinwT or causing a disease or disease causing event includin~~ an antibody. Such binding to the entity may be effected throu~~h the instrumentality of one or mare (same or different) multifunctional li<~ands and through bindings to any ligand or set of li~ands, including receptors, multi-component epitopes etc, including_ for example, tumor "as.s~ocriuted" (ie. differentially expressed to advantage for targeting purposes) epitopes which may or may not or may only be partially present on tumor associated antigens, or commonly. for example anti~~ens ;' epitopes l ligands ' receptors etc.
w which are over-expressed in association with cancer culls: or for example, antigens % epitopes ligands / receptors etc. involved in immune signaling, stimulatory, co-stimulatory.
inhibitory, adhesion or other interactions, including without limitation, cytokine receptors, ligands associated with immune cell adhesion (see For example L1S 5,747.()35), li,ands to which binding results in stimulation, activation, apoptosis, aner~~y or costimulation, or ligands which differentiate between different populations or subpopuiations or immune cells.
including sub-populations of B cells and '1 cells, activated versus non-activated lympocytes, diseased or disease-causing cells versus non-diseased disease causing lymphocytes and specific immune cell clones for example those having specific Ig type and MHC-peptide type ligands and correlative ligands. Examples of such li'~ands include CCRS.
C'fl.A-4, LFA-1, LFA-3. ICAMs eg. ICAM-I, ELAM-1, CD3, CD3. CD4 (eg see US 6.136,310), C'D5, CD6, GD18, CD?2, CD40, CD44: CD80, CD86, CDI 34 and CD154, to name only a few (see also US6087475: Pf4A receptor. US6135941, WO 01i 13946 Such ligand may also selectively be tar~~eted usin~~ any dual affinity strategy accordin~~ to the invention..
5. The invention is also directed to a multifunctional ligand and a method which comprises using the multifunctional ligand to assess the toxicity of directly or indirectly targeting, for example.
primarily within the lymphatic vessel system (see discussion below), cells having well known markers that are associated with immune cells, for example, those exclusively associated with activated immune cells, in-so-tar as such targeting has a role in prolongin~~
or counteractin~~
the activated state, destroyin<~ the cell (eg. where the multifunctional ligand is a immunotoxin) causinv~ the cell to be destroyed (e~~. through apoptosis (eg. see WO O
119861, fas - fast, U.S
6,046,048) or assistin~~ another molecule or cell for example a T-cell or other killing or immune modulating cell to do the modulation or killing (markers such as CD?3.
CD25, CD26, CD28. CD30, CD38. CD49a, CD69, CD70, are just some ofthe markers associated with activated immune cells) ete. (for a complete listing of marker associated e~ ith activated immune cells see for example Roitt t et al. Immunology, sixth edition, Mosby 20() I
referenced below and Encyclopedia of Immunology ( i 998). .Abbas et al.
Cellular and Molecular Immunology ?000, Harcourt & Brace, the contents of w which are incorporated by reference herein). Antibodies for many such iigands are known or could be readily made by eg. phage display (see references herein including .l lntnnrnol A9ethods 1999 Dec 1 (1;?3 I ( 1-2):65-81 ), and natural ligands for such markers or functional analogues thereof are in some cases known or could be made by recombinant DNA technolo~~ies referenced herein (see also Cellular & Molecular Inununolo~w ~1'~' Edition, Abbas Ak et al. V1~'B Saunders and Company X000. Antibody Fusion Proteins, Steven M Chamow ~ , Avi Ashkenazi Eds. ISBN

May 1999 V'iley: Kontermann, R.. et al.(Eds.) Antibody Engineering, Sprin~~er 2001. ISBN 3-540-41354-5; Antibody Engineerin~~. Carl A. Borrebaeck Oxford CJniversity Press, 199:1:
Antibody En~.eineerin<~:A Practical Approach David J. Chiswell, Hennie R.
Floo~~enboom, John McCafferty Oxford University Press,l996: Antibody E.n~ineerin~~
Protocols. Sudhir Paul ( 1995) Humana Press; Antibody Expression & En~~ineering ( 1998) Henry' Y. Wang, Tadayuki Imanaka, American Chemical Society). ~'he term modulation is used broadly to refer to any change, directly or indirectly, in an immune function or effect, as broadly understood. Many such forms of modulation are well know ~n vn the art (some are excmplitied herein), and therefore these need not be specifically recited (fir a review of such effects see for example Roitt I et al. Immunology, sixth Edition, Mosby '.001:
Encyclopedia of Immunology ; ( 1998) Morgan Kaufinann Publishers, ISBN:O 123267656).
In one aspect the invention contemplates that the multifunctional ligand exerts its function substantially (ie. 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 cells and'or tnoiectrle.c circtrlatin~ through the !v=tttpl~crlic .st:mertt, for example with respect to some embodiments, for ;4reatest effect, to avoid an undesired degree of immunosuppression (for example, embodiments where immune cells are targeted for ablation and: or apoptosis). Preferably, such effect, is exerted at least in part, and preferably substantially to the exclusion of reunions within lymphatic system that house at the time of administration non-circulating 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 trr antibodies) preferably, non-activated cells. This specificity of tar~~eting can be accomplished in part to the natural distribution of the lymphatic endothelium associated marker of choice, the mode of administration and various tar~~etin~~ strategics herein described.
For example, the invention contemplates modes of delivery that to wrt°in~ deb,>rce,s ensure a greater degree of lymphatic system targetin~~, for example administration directly within the lymphatics, adminstration in tissues that drain to the lymphatics or parts thereof, intravenous delivery, as are well know ~n to those skilled in the art, preferably in each individual case at strategic sites of administration that are most pertinent or selective for the disease in question, to the extent that selectivity is desired. The invention contemplates a variety of different size multifunctional ligands (MRU, sin~~le domain, scFv, Fab, minibodies, F(ab)~.
F(ab')~.
substantially w hole antibodies etc. and known or obvious multimers thereof referenced herein and in the referenced literature) that are most suitable (e~=. for small enou~~h or, for example, having lon~~est half life in circulation) for par-ticlar modes of administration to the extent that this is a limitation (eg. size, where drainage into the lymphatic system is sou~~ht to be increased or optimized).
In a preferred embodiment the invention contemplates that the immune function exerting moiety of the multifunctional ligand comprises (eg. by way of recombinant fusion, conjugation etc.), or binds to (such antibodies are known or may be made by phage, ribosome 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, ICAMs (eg. ICAM-1. ICAM-?) V-CAM, M.AdCAM-I or functional analo~~ues or portions thereof (see for example USP6143?98, 5512660, 5861 I51, 54895;3, 5.5;8,725, 6037454, 5565550. C'ir~culatio~a 2001 Feb 27; 103(8):1 128-1 134, and specific examples,!referenees recited below) in order to control cell traffic including facilitating cell anchorin~~ within the lymphatic system, including for example to facilitate interaction with another "arm" (functional moiety) of the multifunctional listand or a second etc.multifunetional ligand or an immune cell (or a cell-sized latex sphere as described herein -for this pupose the adhesion molecule may be on the surface of another, preferably multifunctional-ligand-anchored latex sphere or on a similarly anchored cell) as well as combination therapies, for example, with therapeutic entities that enhance or inhibit leucocyte adhesion, or multifunctional ligands or antibodies that bind to one of their correspondin~~
ligands on immune cells (eg. intergrins) or other ligands eg. CD44, to facilitate control and/or some selectivity of cell entry into the lymphatic system, for example. for reactivity with the multifunctional ligands of the invention. The invention also contemplates that such adhesion molecules may be the subject of targetin~~ with dual affinity ligands of the invention and that such ligands may include a moiety which binds to a lymphatic endothelial cell.
he invention also contemplates that one or more multifunctional li'~ands in which the immune function exertin~~ moiety comprises an antibody type molecule tar<~eted to a particular cell surface li~,~and may be able to mimic effect of such adhesion molecules, as discussed below (any such discussion of an antibody mimickin~~ this function is unless otherwise stated not intended to limit the broader concept of utilizin~~ any class of molecule that would facilitate anchoring or controlling, a<~. slowing the passage of cells throu~~h the lymphatic vessels). It is to be understood that there may be limitations in the number of cells that can be targeted for ablation in the lymphatic system by ~slowin~~ the passable of cells, particularly for the purpose herein specified of allowing thorn the requisite period of residence within the lymphatic system for immune cell targeting or interaction or prolonged interaction with multifunctional li~ands ofthe invention for binding purposes while bound to the lymphatic system endothelium, for example, certain end stage lyrnphomas'leukemias. In this particular context it is to be understood that: 1 ) the invention may ham greatest application when the multifunctional ligand is administered so as to primarily tar~.;et 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 invention contemplates that an effective amount of hyaluronic acid is pre-administered to tissues drainin~~ to the lymphatic system so as to initially occupy bindings sites on l_YVf-1 primarily in the smallest lymphatic vessels and thereby minimize excessive binding within the narrowest vessels.
10. In a preferred embodiment said first portion binds to LYVF- I or podoplantin described below ~.
1 I . In a preferred embodiment, said tirst portion is fused, conjugated or otherwise linked directly or indirectly to an immunizing moiety, for example an antigen, epitope, mimotope or peptide etc. presenting/incorporating entityiscaffold that generates by itself or with the help of one or more cytokines, costimulatory molecules and,'or adjuvants etc. an immune response to a desired antigenic determinant (this term is used broadly to correspond at least in scope to the overlapping groupings: antigen, epitope, mimotope or peptade), for example an anti-idiotypic antibody. an antibody component which is capable of binding to a T cell activatin~~ entity for example a cell (cg. an APC see Int Inrman-ml ?000 Jan; 12( I ):>7-66 or other cell having cg.
immune modulating activity cg. see USP 6,00=1,81 1 ) which is for example genetically en~~ineered to express relevant ligands for activating (or with respect to functions not necessarily related to immunizing, aner~.:izing, tolerizin~~ or otherwise Inodulatin~ the activity ot), an immune cell for example a B cell or T-cell, for example an MHC-peptide and B7 co-stimulatory molecules for activation of '1'-eel Is ( see for example Proc ,W
nl .4c«ct Sci (S A
?001 Jan 2; 98(1):241-246 see also ~fham EL et al. J of Immunological Methods ~'ol. ?49(1-2)(2001 ) p I I 1-I l9 with respect to latex spheres that can be used for this purpose). or for example a CTLA-4 scaffold, a peptide fused to an Fc domain (see WO 01!18?03) a HSP-peptide cornplex/conjugate, an MEiC protein or peptide complex etc. Antibody-MHC'' complex fusions and antibody-B? costimulatory 'fusion molecules are known and the invention contemplates that fusion molecules with anti-lymphatic marker antibodies could be made and used to~~ether for immunization purposes. For example the invention contemplates that two Fabs (or linked Fvs, with linker extensions of suitable len '.;ths comparable (l.c. in a rankle of up to two fold shorter%longer when compared to extensions used in the art see Park et al. A divalent recombinant immunotoxin formed by a disulfide bond between the extension peptide chains. Mol Cells. 2001 Dec .'> 1:12(3): 398-402) which reco~>nize a lymphatic endolethelial marker can be disulttde linked and fused respectively to an MEtC
petides and a B7 molecule) It is also contemplated that the absence of costimulatory molecules for presentation in a co-stimulatory fashion with an MI~C peptide complex will cause a tolerizing effect. Accordingly the invention is also directed to a multifunctional li~~and comprising an immune function exerting moiety which comprises an MHC'.. preferably complexed or otherwise linked to a peptide. Peptide linking may for example be effected independently, naturally or for example through CausIn~T release of peptides from an MH(.' peptide or HSP
peptide complex by injecting a weak acidic solution into tumor cg. ,just prior to excision.
Suitable such solutions which may for example be combined with a cytokine ' e~~.ll,-12 and-or adjuvant are known in the art.
12. In a preferred embodiment said immune function exerting moiety comprises an anti-idiotypic antibody, for example an antibody' that a) mimics, for example, a cell surface expressed tumor associated epitope, a virus or other infectious agent associated surface epitope. a toxin, an immune stimulatory, costimulatory, inhibitory, or otherwise interactive ligand; or b) serves to bind to the idiotype (lc, paratope) bearing antibody to which it binds as an anti-idiotype, for example an autoimmune antibody. etc. or an antibody bearing a toxic moiety for removing such antibody from passage into the circulation.
13. In a preferred embodiment, the invention contemplates that the first multifunctional ligand is used for development, therapeutic evaluation or combination therapy in conjunction with a second different multifimetional ligand of the invention, to achieve a cooperative effect (for example. in the same composition or substantially contemporaneously administered (lc. to reach the same or an interrelated destination in a cooperative time frame) or in necessary or desired sequence,'interval, etc.). An example of such cooperative effect is an interaction (not necessarily simultaneously) with two different immune cell surface ligands (for example via an antibody binding interaction), or to deliver different payloads cg. toxins.
to a diseased cell see (USP 6,077,499). 'fhe invention also contemplates a method of effecting substantially coordinated interactions of ditfering temporal and spatial complexities, ran~~in~~ from a somewhat proximal and contemporaneous delivery (cg. in the same composition) of a first multifunctional ligand having, for example, a cancer cell binding second portion, and a second multit'unctional ligand havin~~, for example, a cytokine bindim~ Ab, e~;. to reduce any toxic effects associated with toxic levels of cytokine release. a cytokine component ( far example to harness the effect of such component as a means to attract one or more immune cells to kill a diseased cell or to harness the inhibitory effect of such component (e~~. using one or more cytokines employed by cancer cells to evade innnune eel( targeting) cg. on undesired immune cell elimination or immune cell elimination ofthe multifunctional ligand, or a 1-cell bindings component (cg. anti-CD3) t.o harness the effects of such component on cancer cell killing optionally with a concomitant object of assessing> possible counterproductive immune cell elimination (cg. as would be enabled by using a radiolabelled multifunctional ligand and determinin~~ the disposistion of the label over time) of the multifunctional ligand.
l4. Also contemplated are methods to implement more spatially and.'or temporally sensitive interactions. For example, when administered in empirically determined suitable proportions and in empirically determined sufficient total amounts for, at least, partial and;'or local lymphatic-vessel-associated-ligand saturation or partial saturation to achieve proximal bindin<~ of a tirst to second multifunctional ligand (having reheard to the route of administration e~~. local saturation can be more readily accomplished by administration into the lumen of the lymphatic vessel). Two different such multifunctional li~~ands may be used, for example, to deliver two different immune function exerting moieties in substantial proximity to one another for contemporaneous interaction with two different li'~ands on an immune cell (ie. when it approaches the luminal wall of a I~~mphatic vessel).
For example, this approach may be used to implement one or more effects including increased avidity to the cell for proton<~ed cell anchoring, which may positively impact on desired (in some embodiments) transfer of the multifunctional ligand from the lymphatic vessel wall to the target cell cg. for achieving an inhibitory etfect via ligand binding (e~T. assessed via duration of multifunctional ligand binding e~. quantitative or radioima<,e approximated label elimination)(N.B. this effect may be assessed with multiple copies of the same multifunctional li~Tand), delivery of a cooperative payload cg. 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 interaction, tier example an enzyme-substrate interaction to quantitatively assay the amount of an enzyme converted substrate (cg. usin« a conjugated prodru« and pro-drug conversion akin to ADEPT and assessing the extent of prodru~ conversion e~ by labeled anti-drtr~~ specific antibody). For example, the invention contemplates the use of a respectively linked catalytic antibody component (see for example US5658753:Catalytic antibody components) and labeledy substrate or RNAase and labeled RNA etc.for this purpose. Another example, discussed in more detail below is the use of one multifunctional ligand for targetin~~
(selectivity) purposes and another for implementing directly or indirectly a desired therapeutic effect, both ligands optionally b~in~~T required to to ~~ive rise to a substantial probability of binding (the invention also contemplates that this strate~w could be used with a sin=le multifunctional li~and having two infra-luminally directed bindings moietios).
15. The invention contemplates that such interactive entities may be conjugated fused or otherwise linked to a respective first and second multifunctional ligand for achievin~~ 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 a f;. a) luminal ligand saturation for dosing" b) multiple simultaneous binding interactions, and c) perhaps most spatially sensitive, development ofa process to achieve cross-linked binding with multiple cg.
immune cell li~~ands cg, a eostimulatory innnune effect (ie. the effect of different simultaneous interactions cg. on stimulation, inhibition etc.of cg. an immune cell for example combining a first multifunctional ligand capable of selectively binding to.
conjugated to or fused to a B7component (see .l Jmnrar~aothc 2001 Jan-Feb; 24( 1 ):27-36; J
Jntn~trnol 2001 Feb 1 >; 166(4):2505-2513; Chalitta PM et al. J. Immunol. 160:3419-3426) and a second multifunctional li~~and capable of selectively bindin;~ to, conju~~ated to or fused to an MHC
molecule delivered initially with Yor without peptide. For example, the invention contemplates using.; various amounts.~proportions of multifunctional ligands having antibody components fused or conjugated to or capable of binding selectively to, for example an MHC
class I or II peptide complex and recombinant B7-I-Fc and'or B7-2-Fc respectively (see Eur J
Immunol 2001 ,Ian; 31( I ):32-38; E;aar J JnrnrurmJ 2001 Feb; 31 (2):440-449) (for tumor reactive peptides see forexample,/Irnmnnother2001 Jan-Feb; ~4(1):I-9). In this latter connection (cross-linking type interaction). am d:-odor permanence of binding or ease of attachin;~ other cooperative entities (for example biotin coated or conju~~ated radionuclides, liposornes or other payload carryings entities (cg. see for example US'patwts 5439686, 6007845. 587971?, 5456917, 6165502, 5079005. 5888500. 5861 159. 6193070.
6190692, WO
00!69413, WO O 1:'07084) the invention contemplates biotinylatin~~ the two multifunctional ligands and linkin~~ the two biotinylated cooperative mulfunctional ligands with avidin, streptavidin (or other modified forms thereof e~l. de~~lycosysylated avidin or using other complementary linkin~~ components- see a<~. US Patent (USP) 6,077, 499).
17. The invention also contemplates enhancin~~ the cross-linking ofthe multifunctional ligands of the invention through complementary components such as biotin and avidin.
18. Preferably. with respect to, for example. increasing selectivity of targeting certain cells (e~~.
to induce immune tolerance), the invention also contemplates that a first multifunctional ligand is used to bind to a marker specific to a particular kind of cell (eg.
activated immune cells) and a second multifunctional ligand (which may not be specilic for activated immune cells) is used to modulate the activity of the immune cell (for example inactivate it or reduce its disease causin<~ capability directly or indirectly by binding to it ). For example, where the marker is used to determine the selectivity of the tar~~eting but cannot be used for modulating 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 cooperatin<, multifunctional ligands can be selected to at least partially control the selective modrrlatin~, effect of the pain, for example both interactions would be required for the second multifunctional ligand to have an optimal opportunity to bind. For example, the functional affinity for the tar~,~et cell is relatively weak for the purpose of attachin<~ to the eg. immune cell for a sufficient duration (cg. so as to yield the effect of becoming attached to the immune cell in preference to the lymphatic vessel), compared with that of the first multifunctional li~~and (ie the one that accomplishes the selective recognition through binding) to rectzzce the likelihood that the second moiety will bind in the absence of binding of the first moiety (notably a similar type of coordinated interaction ie. two binding interactions, is naturally used for fell adhesion). (NB. this type of coordination has application ie. both specificities are optimally required for bindings. to a single multifunctional ligand, having a divalent immune function exerting moiety eg a triabody or tetrabody or for cross-linking and other types of coordinated interactions). In a preferred embodiment, if transfer of binding of the first multifunctional ligand to the in nnune cell is not desired its functional affinity of the first portion to the lymph vessel can be greater than that of its second portion.
while the reverse could be true for the second multifunctional li~~and. It will also be appreciated that antibodies which cross-link for example an integrin and a marker of immune cell activation could be used to limit the number of activated innnune cells that migrate through the lymphatic system. For example bispecitic d Abs, diabodies, etc. in which the functional affinity of each specitic bindin<v portion individually does not sron~~ly favour bindingJ. could be used to selectively tarv~et specific sub-populations of immune cells or even specifically activated immune cells (for example antibodies that recoy~nize particular antigen l peptide specitic T cell or B cells).
l9. Accordingly. more generally speaking.:. the invention is directed a bispecitic li~~and, preferably a bispecitic antibody, having a tirst portion which binds to a ligand which differentiates between members of the same immune cell population (eL~ a particular type of T
cell) and a second portion which binds to a second ligand on the same cell, which binding exerts directly or indirectly a desired et~fect_ wherein the functional affinity 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 ligands relative to those which a single such portion is bound to a single li~~and and preferably wherein the amount of immune cells to which the bispecific ligand is not bound is substantially greater than the number of immune cells that are not bound when compared to using a bispecifie ligand having the same specificity and for example a 10' to 10~' (preferably I 0' to 10'', preferably 10' to P 0'', preferably 10' to 10') increase in aflinity of one or both portions. This invention also contemplates that binding to the li~~and which differentiates between members of the same population (a particular type of T cell) does not have a negative consequence other than to cause the molecule to be ineffectual unless both of its portions are bound and that its binding is itself sufficient for binding and'or stron~~er relative to the second portion by two fold to ~
orders of magnitude ,preferably I to 3 orders ofma~~nitude. The term substantially greater imports medical significance and rnay preferably be 1 >°ro - 10000°'0 ~,reater.
The foregoin~~ examples are not meant to be limitative.
20. In a preferred embodiment, the invention more broadly speaking contemplates a two lioand interaction (using one or more multifuncaional ligands) wherein for example both are required or increase the likelihood of interaction and w herein the interaction of at least one contributes 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 ligand and the lymphatic system venue, would be toxic in the desired therapeutic dose.
Examples of markers that could assist in selectivity include those are unique to, for example, activated B cells or f cells or those having particular speciticites in virtue of unique I~~ type receptors. C:xamples of li<~ands on, for example immune cells, through which modulation'inhibition.'stimulation etc. (including, for example apoptosis), for example by antibody binduy~ or supply of a natural interactive ligand, are well known.
Some examples are provided herein. Combinations and permutations of markers and li~ands for selectivity and exerting an immune effect such as modulation; inhibition,~stimulation referred to herein or in the literature incorporated herein by reference or well known in the art are contemplated to be within the scope of the invention.
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, route of administration leg within tissue that best drain to lymphatic vesssels or a portion thereof. use of cytokines, etc. and irnrnune rnodulatin~~ drug's, as well combination therapies with known entities, can be employed in various combinations for strategies of harnessing the unique properrties of the multifunctional ligand of the invention, to achieve a selectivity enhancing and or modulatorylinhibitory,'stimulatory etc or otherwise cooperating effects with respect to the desired target population of cells. Unless their function are self-evidently contlictin~~ the invention contemplates all permutations ofthe multifunctional ligands disclosed herein or in the literature incorporated by reference hererin as well as those evident to persons skilled in the art whose mention is omited.
32. In a preferred embodiment, the immune function exerting moiety binds with greater functional affinity to its target ligand than said first portion binds to its target ligand. For example said immune function exerting moiety may bind with ~~reater avidity (preferably at least 2 times ~~reater (divalent vs. monovalent) and lesser or greater affinity (e~~. within a range of 1 ~ 10-' to I x 10' fold) or with the same avidity and greater affinity leg, up to 1 x 10'' fold). In applicable aspects, the invention contemplates that this increased functional affinity can be employed to effect transfer of a lymphatic vessel bound multifunctional ligand leg. a bispecific antibody) to a cell passin~~ through lymphatic system. The invention also contemplates a method comprising radiolabelling the mutifunctional ligand to assess, for example. the degree to which immune cells at a disease site have passed throu;h the lymphatic system. Certain aspects of the invention, discussed herein, relate to a multifunctional ligand based system of tar4~etin~~ a particular immune cell li<~and for stimulation, inhibition etc. predominantly within select portions of the lymphatic system that contain mi<~rating cells (although some ~~eneral tars~etin~~ can controllably occur before the multifunctional ligand binds to the lymphatic system or when the multifunctional li~and releases from the lymphatic system without havin~~ found its target within the lymphatic system) will have at least a partially selective effect on tar~~etin~~ disease causincrimediating immune cell> leg. activated with a specificity that effuses the disease) as opposed to non-disease causin'~lmediating cells, in the case where such ligand is also expressed on such other immune cells eg. of the same type eg. T cells. This pennies tar~~etin'~ of immune cells primarily within the portions of the lymphatic system that contain migrating cells particularly disease causing;'mediating cells while minimizing immune system dysfunction.
This effect can be even more selectively 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 of binding of the multifunctional lit.;and deterrninin~, the degree to which the multifunctional li~~and is bound to such diseased related cells at the disease site and similarly the degree to which it is bound to the cells unrelated to the same disease e~~. via radiolabel. As discussed more fully below, the invention also contemplates a multifunctional ligand based system of assessing the effects of certain types of immune stimulation e~~.
how~ stimulatin~~
enhanced migration or adhesion, will differentially affect disease activated cell migration through the lymphatic system to enhance such disease cell targeting within the lymphatic system. For example, for tumor cell targetin<,~ and stimulation ofdisease-activated immune cells the invention contemplates evaluatin~~ cytokine leg. 'fNFoe) linked anti-angiogenic marker antibodies, optionally, preferably in combination with anti-tumor vaccination strate~~ies, to direct disease activated immune cells to tumor site and the lymphatic system for further immune stimulation. Based on a "bait and trap" type approach, ligands such as OX40L
and CD44 may also be assessed for this purpose.
23. In this connection and more generally the invention also contemplates using a bi-specific antibody, for example having a lymphatic endothelial binding first portion and for example a cytokine binding second portion, wherein the cytokine binding portion has a lower functional affinity for the cytokine (for example I x 10 -6 to 0.9 fold) compared with that of its natural inceptor on an immune cell. It is contemplated that a multifunctional ligand of the invention could be used optionally in conjunction with a multifunctional ligand which displays a functional adhesion molecule (a selectin, ICAM, etc.) to assess the optimal parameters for transfer of the cytokine. for example, as is known to occur by monitoring the effects of cvtokine release attributable to such cytokine transfer. It will be appreciated that this information or approach could be used to optimize the bindings parameters for other li~aands as w ell (eye, anti CD3) and could be employed not only in lymphatic system but to locally deliver inhibitory or stimulatory cytokines or other ligands to certain tissue targets. for example new- blood vessels formin~~ within tumors or other Tissue specific markers.
24. The foregoing strategies could be used as part of a primary, adjunct or low ~ disease burden therapy.
25. In a preferred embodiment, the second portion comprises a ligand which is capable of binding to an immune cell for example B cells. T cells etc. preferabl~Y in one embodiment to assist in cell killings or immune modulation of a target cell (re NK cells see for example I.IS
5770387)(see also US6071517:Bispecific heteroantibodies with dual effector functions;
Bispecific antibody-mediated destruction of Hodgkin's lymphoma cells.
.llmnurnnl .h-9crhuds 2001 Feb I; 248(1-2):l 13-123; Bispecific antibody-targeted phagocytosis of HER-2meu expressing tumor cells by myeloid cells activated in vivo..l Immunol Methods.
2001 Feb 1;
348(1-3):167-182 as well asJ lmrnur~ol a-fethucls 2001 Feb 1:248(1-?1:103-1 I
1 ).
36. W ith respect to avidity. affinity and other elements of design includin~~
size, blood clearance, additional functionality etc.the multifunctional li~~and may be. for example, a bispecitic antibody having a monovalent tirsc portion and a monovalem second portion, a bispecific antibody havin<~ a divalent first portion and a divalent second portion, a trivalent trispecitic antibody havin~~ a monovalent first portion and a second portion comprisirt~~
a monovalent immune function exerting moiety which binds, for example, to a target li~~and on a tartlet diseased. disease causin~~ or immune cell, and for example, a monovalent portion which binds to an immune cell which assists in killing or modulation tbr example anti-CD3 or anti-CD28 antibody component, a tetravalent trispecific antibody having. a monovalent first portion and a second portion comprising a divalent immune function exertin~~ rnoietv which binds, for example, to a target li~and on a target diseased, disease causing or immune cell, and for example, a monovalent anti-C D3 or- anti-CD28 antibody component (it is contemplated that this orientation might advantageously position the anti-CD3 component for interaction with a T-cell almost exclusively when the first portion is not bound To the luminal wall of a lymphatic vessel). a trivalent bispecific antibody havin~~ a monovalent first portion and a second portion comprisiny~ a divalent innnune function exertin~~ moiety, for example, one which binds, for example, to a tar~~et ligand on a tartlet diseased, disease causin~~ or innnune cell. fhe antibody' subunit may be for example, a Fab, a substantially intact antibody, a single:
domain antibody (see also Hutton SE. Dis Markers 2000;16( 1,2):37 Single domain 11Ur11ar1 immuno~~lobulin fold-based biomolecules; Antigen specificity and high affinity binding provided by one single loop of a camel sin~~le-domain antibody. J Biol Chem.
?001 Jut 13;276(28):26285-90. Optimal Desi~~n features of Carnelized Human Single-domain Antibody Libraries. J Biol Chem. ?001 Jul 6;276(27):24774-?4780; Recognition of antigens by sin~~le-domain antibody fra~~ments: the superfluous luxury of paired domains.'Urends Biochem Sci. 2001 Apr;26(4):230->; l..lama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features. Mot Immunol. 2000 Au<~:37(10):579-90) a minibodv an scFv or a minimal recognition unit (MRU cg see US6174691:Mirtimum recognition unit of a PEM mucin tandem repeat specific monoclonal antibody).
27. In a preferred embodiment, the multifunctional liy~and binds to an immune cell which is associated with an autoimmune reaction, for example a CCRS-expressing cell.
(see also Apoptosis <genes and autoimmunity. Curr Opin Immunol. '000 Dec; 12(6):719-24.
for application herein) 28. In a preferred embodiment, the second portion comprises a cytokine component.
?9. In a preferred embadiment, the second portion comprises a cytotoxic component.
30. In a preferred embodiment, the second portion of the multifunctional li~~and comprises an internalizing antibody and a cytotoxic component.
31. In a preferred embodiment, the second pardon consists of an antibody which binds to T cells, for example, an anti-CD, antibody or an anti-CD28 antibody.
32. In a preferred embodiment, the second portion consists of a cytokine component.
33. In a preferred embodiment, the second portion comprises an antibody which binds to a target diseased. disease causing or immune cell and further comprises one or more cornpanents selected from the group consistin~~ of a cytokine componem, a cytotoxic component and an anti-CD.i:'C'D28 component.
34. In another aspect the invention is directed to a composition comprisin~~ a multifunctional ligand and a pharmaceutically acceptable excipient.
;5. In another aspect the invention is directed to a composition comprising a plurality of different multifunctional li~~ands.
36. In another aspect the invention is directed to methods and compositions for develaping and evaluating the therapeutic value of stimulators, mediators, inhibitors etc. of immune cell signaling (~~~. stimuVatory, inhibitoy, costimulatory), adhesian, rnigration,ete. including the effects of li~aand~'receptor blockin<~ and supply of specific cooperative ligands, using the multifunctional ligands of the invention.
s7. 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 ttrat migrates into lymphatic vessels. In particular, the invention is directed to assessing the expectation that some disease causing, mediating or otherwise disease active immune cells have an enhanced ability%opportunity (and, or can be enhanced in their abilitylopportunity to make their way into the lymphatic system) so that targeting of relevant li~~ands on that sub-population of cells within the lymphatic system will cause at least a parrtial selective tarwetin~~ effect, preferably with positive effect onythe dosing capability and choice of lif;ands ie. in terms of limiting more universal and: or deleterious consequences. fhe invention is also directed to a method of reduein~~ the toxic side effects of a pharmaceutical composition comprisin~~ a multifunctional li~~and in which the immune function exerting moiety is tar~~eted to a ligand that is not found exclusively on disease causing, mediatin~,~ or otherwise disease active inunune cells, by administerin~~ said composition in a manner in which it enter more directly into the lumen of a lymphatic vessel. (ft contemplated that immunization within the lymphatic system can also be enhanced in virtue of such selective tar~~eting.) In particular, the invention is directed to a multifunctianal li~and. a pharmaceutically acceptable composition therof and methad of using same for assessing enhanced migration or enhancing migration ofdisease-active immune cells, said multifunctional ligand comprising an immune function effecting moiety which has an immune effect on an immune cell surface li~~and ie. effects including signaling (e~.:.
stimulatory, inhibitory, costimulatory. antagonistic, agonistic), includin~~
tlr adhesion and mi~~ration eftects,etc. This may be accomplised practically, tc~r example throu~~h ligandreceptor blockin~~ e~~, via antibody. or by antibody fusions;~con.jugates etc. that supply the natural li'aand or a functional fi-a~~ment c>r chemical%biolo~;ical mimotope thereof: In a preferred embodiment the invention 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 ~~roup consisting of CTLA-4. ll.-? receptor, GCRS. CD44, CD 134. CD3, C'D?8, CD2.

38. In another aspect the invention is directed to a composition con uprising a plurality ofdifferent multifunctianal ligands which exert a potentially cooperative immune effect with respect to an immune cell. for example bindin;.: to two or more different li~~ands on an immune cell, wfierein said ligands are selected, for example from the group consisting of CTLA-4, ll.-2 receptor, CC'R>, CD44, C'D134. from any of the ligands herein mentioned or referenced or preferably ('D3, CD28, CD3.
39. The invention is also directed to a method of inhibiting metastasis durin~~ the course of surgical removal of a tumor comprising administering to a patient prior to sur~~ical treatment of the tumor site, a pharmacetical composition comprising a multifunctional ligand in which the immune 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 an anti-idiotypic antibody component which reco~~nizes the paratope of an antibody which binds to a lymphatic vessel associated ligand and a cyiokine component fused therewith or conjugated thereto. For example the cytokine component comprises IL-? or a functional fragment thereof and.~or II.-13 or a functional fragment thereof. In addition to their individual use in fusion proteins for tumor cell killing, combinations of 11-3 and lL-13 have been used successfully for this purpose. It is contemplated that such cytokine fusion could be used to tartlet 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 endothelium in preference for binding its target. In this connection it is contemplated that the functional affinity of the anti-idiotypic Ab for the first portion would he 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 of the immunocytokine occur substantially contemporaneously but separately and after that of the multifunctional li~~and. optionally by a different route of administration.
41. Similarly the invention contemplates for the same purpose, a bispecific antibody having an anti-idiotypic antibody component which recognizes the paratope of an antibody which binds specitically to a lymphatic vessel associated ligand (preferably with lower affinity than that of the Ab forJits target) and for example an immune cell bindin;~ portion eg. an anti-C D3 antibody or an anti-CD28 antibody component.
42. Thus the invention is directed to a method of targeting a diseased or disease causing cell for destruction by the immune system comprising administering separately but substantially contemporaneously to a subject frostings the diseased or disease causing cell.
preferably in sequence with an interposed interval and'or by different routes of administration, first a multifunctional ligand in which the immune function effecting moiety binds to a diseased or disease causing cell surface associated epitope. and an innnunoeytokine or bispecitrc antibody as decribed in the immediately preceding two paragraphs.
43. In a preferred embodiment the invention contemplates modification of the mufti-functional ligand to substitute one or more amino acids which reduce without functional impact on the molecule the number of immunogenic MHC II class peptide sequences within the molecule.
This can be accomplished through procedures available to those skilled in the art, for example through the I:)elmmunisation services of Biovation Limited (see also US 5821 1?~ and related Xoma patents).
44. Inasmuch as the invention is predicated on intraluminal lymphatic system targeting such lymph associaton may be alternatively implemented, in suitable circumstances by the method of delivering the multifunctional ligand, for example into the lumen of a lymphatic system vessel or ('where the multifunctional li_~and is not of an unsuitable size (see for example Ikorni, F. et al. 1_ymphology 32 ( 1999) 90-122, within a portion of body that drains to the lymphatic system (ie a portion ofthe lymphatic system), for eventual mi~~ration to the lymphatic system. Particularly. with respect to embodiments of the invention in which the immune function exerting moiety is targeted with greater functional affinity to a therapeutic target (ie, not the lymphatic system target), such lymphatic system oriented modes of deliven~
coupled with preferred tarrgeting to the therapeutic target may combine, absent saturated binding to the therapeutic target, to better accomplish functional lymphatic tar<~eting.
Accordingly, in a broader aspect the invention is directed a lymphatic system tar~~eted multifunctional ligand in which the second portion is as described herein and in which the specificity of the first portion exclusively for a lymphatic system is inessential. In this connection, the invention contemplates tar~~etin<~ markers on lymphatic vessels that are also present. for example on blood vessel endothelial cells leg. VI~CiF2). (with respect to lymph specific markers see also Birner P. et al. Clin Cancer Res ?001 Jan; 7(1):93-7 "Selective immunohistochemical staining of blood and lymphatic vessels reveals independent prognostic influence of hlood and lymphatic vessel invasion in early-stage cervical cancer'' and published references to the markers therein mentioned.) 4~. In the case of purely sustained release aspects of the invention where the tirst portion is temporarily anchoring a second portion for eventual release Luck into the circulation, the use of term immune function affecting moiety with reference to the role of the second portion does not adequately accommodate the breadth ofthe invention since any form ofdisease palliating active moiety or entity which exerts its effect elsewhere than at the lymphatic endothelial cell may gain advanta<~e from this form of delayed delivery (depot effect) or anchorin~~.
46. Furthermore, in another preferred aspect, the second portion is capable of binding directly or indirectly (e'.=. binding to an entity which in turn binds to a target entity) to a target entity, fbr example a therapeutic entity (for example to mop up excess such entity that does not imtnediatelv reach its target leg. 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 tune or in particular amount or concentration leg. a cytokine, for example when released as a result of anti-CD3 therapy), to rediy~ec~t an an entity to a tartlet, for example a therapeutic entity, for example through the instrumentality of an antibody portion that is directed to that target leg. a multifunctional ligand in which the second portion comprises an anti-tumor antibody portion that is conjugated to streptavidin. to retarget biotin conjugated radionuclide back to the tumor (see Martin J. et al. ( 1997) Cancer Chemother, Pharmacol.
40:189-301 ). to temporarily anchor liposomes or other carriers of entities leg. drugs) having an direct or indirect beneficial effect elsewhere.
In a preferred embodiment, the invention provides a multifunctional li~and having, at least, a first portion which binds to a lymphatic vessel associated antigen 'receptor (and thereby exerts, not necessarily to the exclusion of other effects) at least an anchoring_ function, and a second portion having at least one independent immune function. The term "immune function" is broad in intent including but noc limited to direct or indirect and primary or corollary effects related to simple tar~~eting, tolerance, immunization, stimulation, inhibition, modulation or various other immune related effects (other than simply forming pan of the entity which blocks the lymphatic endothelial associated ligand). The term independent is used to exclude only an effect specifically targeted tow bards the ligand (bloc-kingi or cell bearing the ligand to which the first portion of the multifunctional li;~and is bound, which is not contemplated as an object of the invention. The invention contemplates rather that the immune function is exerted. for example, vis-a-vis immune cells or molecules or a~~ainst cancer or infected cells to affect an immune function that relates to assessment, diagnosis, therapeutic modelins:. or treatment of various disease states such as autoimmune disease, transplant rejection, cancer and infectious disease. In a preferred embodiment, the invention contemplates that the independent immune function is exerted throu<~h a physical ligand-ligand interaction.
In a preferred embodiment the multifunctional ligand has an ability to bind in the manner of an antibody in virtue of at least one of the tirst or second portions, and preferably at least the first portion. The lymphatic system directed first portion may in some embodiments (LYVE-1 ) be hyaluronic acid or analogues thereof that have the appropriate bindings capacity. In a further preferred embodiment the second portion binds to a target ligand on a cell or molecule leg. a cytokine or autoimmune antibody) which passes through the lymphatic system. In a more preferred embodiment the multifunctional ligand is a bispecitc antibody. The term antibody is used to refer to any antigen binding ti-a'~ment of an antibody that substantially has the binding capability of an antibody including anti-idiotypic antibodies, and therefore the term hispecific antibody is used (unless the context implies a more specific usage) in a functional sense to refer to at least two different speciticities (including trispecifc antibodies etc.) and includes well known entities wfiich are diabodies, triabodies, tetrabodies, minibodies, scFv dimers, etc., and entities in which one or both binding moieties are seFv or single domain type antibody fragments or dimers etc of such fragments (with respect to single domain antibodies see for example Carrel single-domain antibodies as modular building units in J
Biol Chem. 2000 Oct 2>, R Mulligan-Kehoe U.S. patents ).
The term "anchoring function" is used hroadly to refer to physical attachment for a period which renders the second portion of the mufti-functional ligand capable of exerting its immune function. For example where the function ofthe 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 specitically interacting moiety includin~~ binding moieties (eg antibodies, receptors etc.) and bound moieties (e~~
antigens. epitopes etc) and;'ine;luding otherwise interacting moieties (e'~, chemotactic interactions or interactions that require multiple points of interface eg. cross-linkin~~ or mufti-component epitopes). In other words, the term li~~and is used broadly to refer to any entity or part thereof which can be subject to an intermolecular interaction that can result in bindin<.;. The term moiety is used broadly and non-limitatively to refer primarily to a functional part of an entity, and may refer to even the whole of the entity depending on the context in light ofthe broadest concept of the invention.
Optionally, dependin~~ on the mode of delivery and the relative functional aftinity of the respective first and second portions, the mufti-functional ligands of the present invention. may exert their immune function primarily in lymphatic system and also significantly bet>re and optionally after entry into the lymphatic system. In a preferred embodiment the multifunctional ligand is capable of simulatin~~ a depot effect by bindin<~ for a prolonged period to the intra-luminal lymphatic endothelium for later release over time back into the circulation. 'fhe choice (avidity effect resulting ti-om multiple binding "arms") and affinity of the binding molecule as well as various, preferably controllable factors impacting on any "undulating"
movements of the lymphaticvessels (eg. water consumption)or competitive bindings is contemplated to impact the binding time.
With respect to the depot and delivery aspects of the invention discussed herein, it is contemplated the second portion of the mufti-functional 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 (.% ) means the broader of "or'' or "and.~or" unless to the context dictates otherwise.
Some innnune interactions require. prefer or are capable of being enhanced via coordinated ligand interactions, for example for optimal immune stimulation, for example, specific costimulatory li~~and interactions eg. CD80/CD86 interactions with CD28, or for example.
interactions aimed at tolerizing or otherwise inhibiting or reducin~~ immune effects or preventing such inhibition (for etample usin<~ anti-CTLA-4.!CD152 see related U.S. patents, for example 6,051.327, 5.844,095) (see also Hodge JW et al.
Ernst Schering Res Found Workshop 2000 (30): 23-53 and Immunological Reviews Vol 172 Dee 1999.
Entire Issue).
The invention contemplates modeling, evaluating and-or effectin~~ these interactions for therapeutic intervention within the lymphatic system through the substantially contemporaneous use of different multifunctional ligands of the invention. Furthermore, control of the relative proportion of each of the different li~~ands permits different spatial interspersion of these li~~=ands on the intraluminal suface of the lymphatic system (primarily) so as to provide controlled variability of spatial configurations appropriate for optimizing the coordinate interaction with multiple li~~ands on another entity, tUr example immune cells or cancer cells. This strategy also permits controls on avidity that extend beyond the choice of valency for a given single multifunctional ligand for controlling 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, for example. cytokines (through cytokine antibody fusions), superantigens etc. to the site of interaction. Such coordinate interactions may be substantially contemporaneous or sequential, for example the effect of a tirst interaction with a first multifunctional li~~and slowing the progression of a cell or infectious agent throu~~'h the lymphatic system for eventual rection with another first multifunctional ligand (ie ofthe same type) or reaction with a second type of multifunctional li~~and. The invention also contemplates as a strategy. alone or in combination witft other strategies: I
) delivery of a multifunctional ligand of the invention to a particular site of action f<>r the purpose of e~x~rting, for example a local effect, with the result of causing the multifunctional ligand (whether or not it has exerted its effect, provided that or to the extent that it remains functional in at least one aspect) to subsequently be targeted to the lymphatic system for exerting a second effect (be it the same or a different disease counteracting effect) including simply elimination, or return back to the circulation lie. where the ligand is selected leg. based on size, immunogenicity etc.) to be preferably minimally eliminated (at least not maximally eliminated) by the body in the course of its circulation, havin5 regard to competin~~ desi~~n considerations) for example, in the case of multifunctional ligand which is an anti-tumor li<7and that has some residual binding to normal tissues, to set up, in effect, a site of competitive binding that advantageously impacts ( ie_reduces) undesired binding more than desired target binding; 2) delivery of a multifunctional ligand of the invention or an entity that binds to a multifunctional ligand of the invention to a particular site of action eg. local disease mediating immune cells, for the purpose 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 different multi-functional ligands of the invention. 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. intradernnal etc which (for present purposes) are preferably not incompatible with delivering a multifunctional ligand ofthe invention to the lymphatic system. 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 effect of a particular therapeutic:
entity against a particular target with reduced effect on undesired targets; 2) facilitating elimination a therapeutic entity; -- by administering the therapeutic entity as part of or in circumstances which permit interaction with, a multifunctional ligand of the invention.
The invention also contemplates cannulating particular portions of the lymphatic system to localize the delivery of a multifunctional ligand (see United States Patent 4,911,690 ) , for example 1) to accommodate or further accommodate the treatment of conditions in which the immune affectin<, molecule has an undesirable systemic or localized side-effect if delivered otherwise; 2) for the localized delivery, as required, of larger molecules, complexes leg. for temporarily anchoring MHC-peptide complexes) or otherwise associated (at bast temporarily) entities lie. associated other than through complex formation) etc. andior 3) for the localized delivery of additional compositional elements e';. adjuvants. cytokines (see Immunological Reviews 2000 Vol 177 p. 5-246: Nature Innnunology Feb 2001 Vol 2 No. 3 page 89), or for affecting only subsets of populations of cells or molecules that pass through the lymphatic system or a desired portion of the lymphatic system or are found with ~~reater concentration within the lymphatic system. The invention also contemplates methods of selective, enhanced or localized, targeting! delivery by administering multifunctional ligands ofthe invention as well as methods (includin<~ methods directly or indirectly employing the multifunctional ligands of the invention) of er~hancin~>:' inc~trciy= entry of cells or molecules, particularly immune cells lie. cells having an immune system function as broadly understood) or subsets thereof, to the lymphatic system or a portion of the lymphatic system, for example for the purpose of direct or indirect interaction with the multifunctional ligands of the invention (in order to be acted on directly or indirectly, by multifunctional li<~ands of the invention) or for recruitin<~ cells that will for example kill or modulate the activity of other cells, for example kill cancer cells or infected cells that will have, are havin' or have had direct or indirect interaction with the multifunctional li~ands of the invention, as discussed further below ~. for example in the case of cancer. by tar~~etin~~ immunocytokines to the disease affected tissue e~~. using cytokines eg. 'fNFa fused to antibody that binds specitic;ally to tumor cell markers or markers for an~~io~~enesis. Similarly tissue targetted as opposed to disease targeted immunocytokines could be used selectively recruit immune cells within that tissue for example a diseased tissue to enter the lymphatic system for such purposes including for example interaction with a multifunctional ligand of the invention.
It is also contemplated that a single multifunctional li~~and can have multiple requisite interactive functionalities for example to stimulate, attract, anergize (or otherwise inactivate) sub-populations of B-cells of T cells via the use, for example, of trivalent or tetravalent antibodies and antibody conjugates.-fusions thereof having multiple li_and interactive capabilities (see also for example technologies being developed for selection of successful binders by phage or ribosome display (see for example WO
01/00866; Acfv Protein C'herrr 2000; 5>: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 Manzke O. et al. Int. J. Cancer 83. 700-708 (' 1999); Br~ ,l C'urrcer 2000 Jan:
82(3):472-9; J C'oratrol Relc~u.se 2000 Feb 14: 64( l-3):229-39 as well as related relerences, cited therein or citing these publications, The present invention accommodates such technology through multispecific antibodies or alternatively obviates the need for combining a T-cell receptor type molecule with the primary immune function effecting moiety leg, a cancer cell binding moiety) by using a separate multifunctional ligand which combines, for example, a first portion and a second portion comprising a T-cell interacting moiety leg. anti-CD3). This is accomplished by administering in the same composition or substantially contemporaneously an amount of the second multifunctional ligand that provides, as may empirically predicted by assessing the dispersion ofthe marker on the endothelial cell, a strong probability leg.
.001-100°'°, optionally I-100%.
optionally 5-100°%. optionally 10-100°~0, etc) that the T cell will be targeted in the vicinity of a <,iven lymphatic endothelial cell that happens be proximal to the cell sought be targeted eg the cancer cell. It is self=evident that a 50.!50 proportion of the first and second multifunctional li~and will yield a stronC~ chance that a second multifunctional ligand will be immediately adjacent on a particular given side (assuming for the sake of argument that there are sides when in reality the dispersion of the lymphatic endothelial marker is governing). It is also contemplated that adjacent multifunctional ligands may be linked for example through linkage effective pairs of li<~ands (avidin-biotin), the second portions having an antibody component which binds to a common ligand (e'~ on a iiposome (see US 6197333 and refs. therein cited) or other pharmaceutically acceptable micro/nano partiele/sphere of preferably 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 multi-functional li~~ands of the present invention provide for a method and preferably a means for evaluating and-or inducing immune tolerance (with respect to B cells see strategies discussed in Immunolo~~ical Reviews ?000 Vol. 176 pp. 5-247).
It is believed that immune tolerance is enhanced or prolonged through prolonged /strategic exposure to tolerance inducing and!or enhancing molecules for example prolonged antigen exposure (see Wand Y et al.
Eur. J. Immunol. 2000; 30( 18):2226-2234; Encyclopedia of Immunology : ( 1998) Morgan Kaufirann Publishers, ISBN:0122267656: Hoyne GF et al. lmmurzol~y 2000 Jul; 100(3):281-8: Lerner CG et al. J
Immunol. 2000 Apr. I 5: 164(81: 3996-4002: Grossman I. et al. .Scrrir lnununo!
2000 Jun; l 2(3):197-203; discussion 357-344 Textbook of the Autoimmune Diseases by L..ahita R. et al. ISBN: 0781715059 Lippincott Vv'illiams K, Vfilkins; Multi-Systemic Auto-immune Diseases : An Integrated Approach Dermatological & Internal Aspects ISBN: 0444818960 Elsevier Science :
Arthritis and Allied Conditions - A Textbook of Rheumatolo~~y, Thirteenth and Fourteenth Editions, William J.
Koopman. MD 14'~':1SBN:
0-7817-2240-3, November 2000; Principles of Dru~~ Development in Transplantation cY Autoinununity Landes Bioscience, ISBN:0412100614; Cancer c~, Autoimmunity by Ciershwin M. et aI.,ISBN:
0444503315 Elsevier Science : J.9utoi~nmun 200(1 Jun: 14(4):278-82; The multi-functional ligands of the present invention, depending on their mode of administration (direct application by cannulatin~~ a lymphatic vessel or conventionally e~~. intradermally or intravenously), can be advantageously employed to provide prolonged.%strate~~ic exposure to tolerance enhancin~~ molecules (for example by employing a multivalent eg. bi-specific Ab fragment or diabody which hs a first portion which binds to a lymph associated antigen and second portion which optionally comprises anti-idiotypic Ab portion mimicking the desired A~~ or the antigen itself or a suitable portion thereof fused or conjugated to the first portion) on the intra-luminal surface of the lymphatic vessels, optionally. in addition to its conventional effects, when administered intradermally or intravenously, etc.. It is anticipated that the multi-functional li~~ands of the present invention would be useful to assess and/or effect toler<rnce induction (see Bassadona GP et al. Proc Natl .4enct Sci U S.a 1998 Mar 31; 95(7):3821-6; USP 6,106,834; USP 6,099,838;
US6010902: Antibody heteroconjugates and bispecific antibodies for use in re~yulation of lymphocyte activity: as well as additional examples cited below with reference to examples of suitable anti-idiotvpic antibodies).
It is also contemplated that a multispecitic contruct as described in WO99/37791 could be used with respect to various aspects aspects of the invention..
Additional Applications of Various Aspects of the Invention It is contemplated that the present invention could be used to strategically mediate, CD45 (or variants/other PTPs) related ''cell signalin~'~. for example through signaling molecules leg. inhibitors) using multifunctional li~~ands ofthe invention including but not limited to bispecific antibodies, antibody fusions/conjugates e~~. where the immune affectin~~ antibody portion or other moiety is conjugated, fused etc. to an antibody or fi~a~~ment that binds to an entity associatied marker e~~. LYVE-1 ( 1999) Journal of Cell Biology Vol 144 No 4 p. 789-801 ) (see for example ISP 5,914,1 I I
Sievers EL., Cancer Chemother Pharmacol 2000 46 Suppl s18 ?2 W09946268, Neel BG Curr Opin Inmunol 1997 Jan 9(3) 405-420:
Front Biosci 1998 Nov I 3:D-1060-96, Slifl:a MK et al. J. Mol. Med 2000 78(2) 74-80 Goodnow CC Ciba Found Symp 1997 204: 190-202; Mustelin T'. et al. Front Biosci. 1998 Nov l; 3:
D 1060-96; Gaya A.
Leuk Lymphoma, 1999 Oct 35 (3-4): 237-43; Sievers EL, Curr Opin Oncol. 2000 Jan 12( I ): 30->;
Thomas ML, et al. Imnrul. Today 1999 Sep 20(9): 406-41 l; Appelbaum FR, Semin.
Hematol. 1999 Oct;
36 (4 suppl. 6): 2-8; Ulyanova T; Immul. Res 1997 Feb: 16(1 ): 101-13; re PP32 for example USP
5,846,822 and Brody JR, et al. J Biol Chem. 1999 Jul l 1i; 374(29):20053-~
regarding the functional moiety of PP32 which is necessary for interaction with CD45, and for example IISP
6,981,351 with respect to methods of~identifying such molecules).
In preferred embodiments the invention is directed to multifunctional ligands that comprise immune function exertiny~ moieties havin~~ funetionalities of molecules currently in clinical trials or proposed for clinical trials (see for example G lennie MJ et al. Aug 2000, Immunology Today 408 Vc>I 21 (8); sce also Journal of Immunological Methods 237 (2000) 13 I-145; Mol Immunol 2000 Jun;
37(9) 515-526; Annu Rev Med 2001; 52:125-145: Annu Rev Med 2001 52:63-78: Q J Nucl Med 2000 Sep;
44(3) 268-83) including those that have an anti-CD2 functionality (see USP 5,795,572) anti-CD4 functionality (see for example USP 6.136,310 Herzyk D, J Infect Immun 2000 Feb; 69(2): 1(132-1043) anti-CD3 functionality (for example WO 00/41474; WO 9813936 3; USP 6, I I 3,901; Transplantation 2000 Dec 27 70 ( 12') 1707-12); Anti-CD44 functionality see for example Weiss L, et al., Proc Nat Acad Sci IJSA 2000; ,Ian 4 97( I ) 285-290; Sugiyama K. Immunol Invest ( 1999) Mar-May 28(2-3) I 85-200;
Brocke S. et al. Proc Nat Acad Sci USA 1999 Jun 8 96( 12) 6896; Mickeez K et al. Nat MecJ I '1995 Jun; 1(6); 558-63;
Ahrens T et al., J Invest Dermatol. 2001 Janl 16(1) 93-101); with respect to control of migration ofT-cell lymphocytes see Nohara C, et al. J Immunol. 2001 Feb I ; 166(3) 2108-21 15), anti-CD20 functionality (see Crit Rev Oncol Hematol 2001 Jan 37(1):13-25) etc. anti-CD22 functionality see for example Newton DL, et al. Blood 2001 Jan I 5: 97 (?): 538-5_i 5. (JSP 5. I 84,892; Anti-CD4U!CTLA-4 see for example J
Immttnol 2000 Oct 1; 165(7):3612-9. Microsur~~ery 2000: 2c (8); 448-452; USP
5874082: USP
6056959; USP 5,801,227; USP 6004552; USP 5677165; USP 6087329; IJSP 5961974:
USP
6051228; White CA,et al. Annu Rev Med. 2001; 52: 63-78 (see also reviews and specific applications referred to in Ditzel et al., Immunol Res. 2000; 21(2-3):185-93; USP
6,010,902, IJSP 5876950; USP
5876718; CJSf 5.601,819, USf 5981251, USP 5885579 and 5885796: C'crncc>r Immtttzol Imnutttothc>r 2000 Jun; 49(3):173-80: Oman K, J Neuroimmunol 3001 Feb I, I 13(1) 129-141;
Bellido M, Fur J.
Haematol 2001 Feb. 66(2) 100-106: Broeren et al. J Immunol (2000) DeclS
165(12) (i908-14:
Alexandroff AB et al Mol Immunol 2000 June 37(9) 515-526; Werkerle T ,I
lmmunol. 2001 i,eb I S
166(4) 231 1-2316; Howard LM J Immunol 2001 Feb; 1 16(3) 1547-53 anti-CD154:
.I I'hurntac'okinet Biophurnt 1999 Au_~; 37(4):397-420,./('liza Ut~cn12000 Apr; 18(8):1622-36, Leukemia 2000 Mar;
14(3):474-5, Clin C'czncv>r Re.s 2000 Feb; 6(2'):372-80, Lezrkemiu 2000 .Ian:
14( 1'):129-35, ,I ,Nucl rLlc:ct 1999 Nov; 40(1 I ):1935-46, f3loocl 1999 Nov 15; 94(10):3340-8, Blood 1999 Aug l5;
94(4):1237-47. C'cznc;er Res 1999 May 1; 59(9):3096-101, 1'crccira~ 1999 Apr 9; 17( 15-16):1837-45, l3loocl 1998 Dec I; 92( 1 1 ):4066-71, J Rltezrrzzutol 1998 Nov; 25( 1 1 ):2065-76, C'!in Phnrnurcol Ther 1998 Sep:
64(3):339-46, ;lltrh .Scler 1996 Jul: 1(6):339-42. C.'ctnc'er lttatnuzanl hratrttenotltcrr 1997 Jul: 44(5):265-72. 7rctn.splcrnt Pnoc 1996 Dec;
28(6):3210-1, Arthritis Rheum. 1996.1u1; 39(7):1 102-8, Itttttmnodu~~t' 1996 May; 88(1):1 3-9 and USf 5,876,7 I 8).
The invention contemplates assessment and therapeutic benefit of lymphatic localization in the case of antibodies and multispecitic 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 potrtion e~~. antibody for its desired target with an undesired target ('see eg. l,cancer 1999 Nov 13: 354(9191 ):1691-5). It is contemplated that the toxic effect of a given effector rnoietv of a multifunctional ligand of the invention could be alternated usin~~ an additional binding arm for a lymphatic marker.
Antibody Structure and Function Antibody' structure and function has bee extensively described in the literatue. For example see Antibody Engineering 2"'~ ed. Carl A.K. Borrebaeck, Oxford University Press 1995 p 3-44.
Production of Bispecific Antibodies A variety of different constructs have been developed tbr the production of bispecific antibodies including conventional four chain antibodies (including truncated version thereof such minibodies (see IJSP

5,837,821), F('ab')~_ (see ,Antibody Fusion Proteins, Steven M Chamow , Avi Ashkenazi Eds. ISBN
0471 18358X May 1999 Wiley p.136-144; or using CH3-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 creatin~~ a fusion protein with the CL and CH 1 immunoglobulin constant domains (see WO 02:02781 ).
In recent years, a variety of chemical and recombinant methods have been developed for the production of bispecific and/or multivalent antibody fragments. For review, see: Kriangkum J, et, al. Bispecific and bifunctional simile chain recombinant antibodies. Biornol Eng 2001 Sep;18(2):31-4(1, Holli~~er P. and W inter, G., C'urr. Opin. f3iotecvhnol. 4. 446-499 ( 1993 ); Carter, P. et n1., J. Hemulotherapv 4, 463-470 (1995); Pluckthan, A. and Pack, P._ Inaurrrraotec~hraolos~o 3, 83-105 (1997).
Bispecifieity and/or bivalency has been accomplished by fusing two scfv molecules via tlexible linkers, leucine zipper motifs. C"C,,--heterodimerization, and by association of scFv molecules to form bivalent monospecitic diabodies and related structures. Multivalency has been achieved by the addition of multimerization sequences at the carboxy or amino terminus of the scFv or Fab fragments, by using for example, p53, streptavidin and helix-turn-helix motifs. For example, by dimerization via the helix-turn-helix motif of an scfv fusion protein of the form (scFv I )-hinge-helix-turn-helix-(scFv2), a tetravelent bispecitic is produced having two scFv bindings sites for each of two target antigens.
Production of IgG type bispecitic antibodies, which resemble IgG antibodies in that they posses a more or less complete IgG constant domain structure. has been achieved by chemical cross-linkin;~ of two different IgG molecules or by co-expression of two antibodies from the same cell. Both methods result in production of significant amounts of undesired and non-functional species due to mispairin~~ among the component heavy and light chains. Methods have been employed to reduce or eliminate mispairing.
One strategy developed to overcome unwanted pairing's between two different sets of IgG heavy abd light chains co-expressed in transfected cells in modification of the C,,3 domains of two heavy chains to reduce homodimerization between like antibody heavy chains. Merchant, A. M., et crl..
( 1998) h'cu. l3iotcchnolo~v 16, 677-681. In that method, light chain rnispairin~~ was eliminated b;' requiring the use of identical light chains for each binding site of those bispecific antibodies.
To produce bispecific antibodies, Kostelny et al (J. Immunology 148:1547 ( 1992)) fused Fab fra~~rments of antibodies to the leucine zipper portions of fos and jun proteins in the absence of a sin<~le chain construct for the anti~~en combinin~~ region. These methods are well described in the literature and summarized with references in Antibody Fusion Proteins, Steven M Chamow . Avi Ashkenazi Fds.
ISBN 0471 18358X May 1999 Wiley; Kontermann, R., et al.(Fds.) particularly at pages 139-145. Pack and Pluckthun, fused a single chain antibody to amphipathic helices froth a four helix bundle or from leucine zipper proteins, Bispecific antibodies that are in a conventional I~~G-like and Fab-like format have been developed by Ihu as tetravalent or bivalent molecules, respectively with each of the chains serving to anchor a binding moiety (see WO 01/90192 and Figure 1 therein'), preferably consistin~~ of a scF'v. In the bispecific I~~G-like construct, each side of the molecule comprises a Cli l domain and a Cl, domain and each CH and CL
domain is linked through its N-terminus to a scFv of different specificity.
The invention herein contemplates that this construct can readily be adapted to have each each halt of the molecule associated with a polypeptide e~~. a sefv of the same specificity so that each half of the molecule is monospecific (or to have each half of the molecule associated with different pairings of scFvs) so that each half of the molecule is effectively monospecilic. The invention herein contemplates that a bivalent relatively low aftinity second ligand binding moiety is used to activate receptors that require cross-linkin~u for activity.
The invention also contemplates that numerous permutations in which the functional affinity of the first ligand binding moiety v'hether monospecitic or bispecitic can be accentuated relative the functional affinity of the second ligand bindin<~ moiety including ernployin'~ a first ligand high affinity seFvs for a sin~~le antinstances in which the second li~;and binding moiety is effectively monovalent (has one. or one useful binding moiety). The invention also contemplates that this construct can have a truncated Fc portion and various known methods in the ar-t for improving the pairing efficiency of the heavy chains. The invention also contemplates that the C'. H I and CL domains of the second li~~and birtdin~~ moiety can be truncated as in camelid antibodies for efticient delivery eg. of biolo~~ic effector ligands.

Methods of Generating Antibodies That Bind To Selected Target Ligands A variety of technologies for generating antibodies with desired specificity have been extensively developed and become well known to and routinely practiced by those skilled in the art including phage display (see review in Basic Methods in Antibody Production R Characteriztion G.C. Howard et al. eds.
CRC Press 2001 p. 105) and other display systems (ribosome display, display on the surface of various cells), immunizin~~ mice, including particularly mice having human Ig genes, and antibody microarray technologies. These methods have also been extended to making antibodies with dual specificites such as diabodies (USP 5,837,2-F2 Multivalent and multispecitic bindin~,~ proteins, their manufacture and use) and are the subject of extensive scientific and patent literature. For example, see US patents of Winter et al.
6,29 I ,650; 6,291,161; 6,39 I , I 58; 6,017,732; 6,225,447: 6. I 72.197:
6,140,47 I . 6.0 I 0.884 5,969,108, 5,871,907. 5,858,657; 5°733,743, 5,723,287and those of Dyax, Morphosys, and Cambridge Antibody Technology .
Affinity Maturation Methods of codon based mutagenesis have been extensively developed t'or engineering the antibody binding site. For example, the use of such methods in a filarnentous pha;~e display system is described in Antibody Engineering 2"~ ed. Carl A.K. Borrebaeck, Oxford University Press 1995 p I 17-128 see also pp.53-84 with respect to techniques of phage display of antibodies (see also !~-rnrl~~r ru.rrm-R l~iilnrl .5J.
~fds.~ ~'f>t!li~\otibacfv I n~inc~rin;~ ISBN: 3-540-4J1354-5. --_ _ __ -Methods of Generating Single Domain Ligands The ability of a single variable fragment of an antibody to bind with specificity and suitable selected affinities in the nanomolar- range has been extensively demonstrated using camelid and human VH
fragments. Methods of generating VHs with the desired specificity have been extensively described (see USP 6,248,516 Single domain ligands, 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 Antibodies In E. Coli The expression of recombinant antibodies, including diabodies in E. Coli has become routine. General precepts, and methods are discussed in Antibody Engineerin~~ 2"'~ ed. Carl A.K. Borrebaeck, Oxford University Press 1995 p229-266 see also Antibody Therapeutics WJ Flarris et al. eds. CRC Press 1997 p.
221: see also review in tzis~l~~hr~cslc~";e, 1'r~lrrn~c 5.~, I~cc°c~r~ri~inant I'r°ot~in~< ~~-l~scraaitcanarl antiFsss~ticH, zsrrd 'F~trer.rl~~aata~ i~erris ~. '~'1,.;,t~t~;:i~3. 1=. '~ ; .~~~=,r._w wtU,c~zz_L,~.~~ 1.'° ft',. . _ ?"-'~ ~' "-' tf~~t~sa:~ Issg Arstit~od~ I'rocitrsli<3rs:la~~r~tiarl ies~hrriq~rc~('~c~°, l I=)°'~<.~1~~3~.t:E.l;? c3,i)'y"4','il~clrri;~ !t>z3"a:rr_I
Antibody Therapeutics Production. Clinical Trials, and Strategic Issues, By Rathin C. Das, Ph.D., M.B.A. & K. John Morrow. Jr., Ph.D., D&MD Publications October 2001 Chapter 3.
Eukaryotic & Other Expression & Production Systems Approaches for the eukaryotic expression of antibodies and antibody fusion proteins and the preparation of vectors for use in such methods are well known and extensively described in the literature. General precepts, and methods are discussed is Antibody F_ngineerin~; 2°'r ed.
Carl A.K. Borrebaeck, Oxford University Press 1995 p267-293 (see also Antibody Therapeutics WJ Harris et al. eds. CRC Press 1997 p.
183-220; see also review in F3is=tec°Irr~r~le3gi, 't t>lurt'c~ 5;4, (~c~c}rnl~iraarrt i'r°c~t~ins. ylixnc~rlto~:~f :la~lit~oclit~;, <~rr~t fl-l~~~r.~t~errlis° (~er3cs :y. '~-It, . ...~
.~. ( - ~c;. t3ie~tffit:t °~c:oc;.nhms, I'~t°:~: _ ~?~-~~ z ( ~-_s.
1-~iie°,..J,~~E~ra:u 1;3a~cj,~~, ~~ntal>ckcl~
#'~c~<iustic~c~:las~s~ti.ill'c.°cl~r~i~lrse~!':s~Y.l..)G? ~5"~ti'v:t)-~?I-~)7~t~i>-~ ~tU~:;v 1~.~~~ l~u> % a;r~l Antibody Therapeutics Production, Clinical Trials. and Strategic Issues, By Rathin C. Das. Ph.D.. M.B.A. & K. John Morrow, Jr.. Ph.D., D&MD
Publications October 2001 Chapter 3.
With respect to a review of immunotoxins set also Antibody Therapeutics WJ
Harris et al. eds. CRC Press 1997 p 33 With respect to Methods for producing recombinant vectors see also ~.S>62,25s Methods for producing recombinant vectors Formulation, purification and analytic methods involving antibodies are well knoen to those skilled in the art and have been extensively reviewed. With respect to formulation, purification and analytic methods see for example, reviews in Antibody Therapeutics Production, Clinical 'Trials, and Strate<1ic Issues, By Rathin C. Das. Ph.D., M.B.A. & K. John Morrow, Jr.. Ph.D., D&MD Publications October 2001, Chapter -1.
W ith respect to methods of generating antibodies against self antibodies see USP x.885.793 Production of anti-self antibodies from antibody segment repertoires and displayed on phage Antibody Conjugates Methods of chemical manipulation of antibodies for attachment of ligands (eg.biotin), radionuclides etc. are well known in the art and have been extensively reviewed (for example see review in Basic Methods in Antibody Production & Characteriztion G.C. Howard et al. eds. CRC Press 2001, p. I 99; with respect to therapeutic principles see for example, Antibody Therapeutics WJ Harris et al.
eds. CRC Press 1997 p >3-88).
The applications of bispecific antibodies, including methods of making and using them have been extensively reviewed (ee for example van Spriel AB, van Ojik HH, van De Winkel J(i. Immunotherapeutic perspective for bispecific antibodies. Immunol Today. 2000 Aug; 21(8):391-7;
Weiner LM. Bispecific antibodies in cancer therapy. Cancer J Sci Am. 2000 May; 6 Suppl 3:5265-7l.
Barbet .l, et al. Pretar~,~eting with the affinity enhancement system for radioimmunotherapy. Cancer l3iother Radiopharm. 1999 Jun;
14(3):153-66. de Wolf I=A, Brett GM. Li~~and-binding proteins: their potential for application in systems for controlled delivery and uptake of ligands. Pharmacol Rev. 2000 Jun; ~2~(?):207-36.: Wang F-t, Liu Y, Wei L, Guo Y. Bi-specific antibodies in cancer therapyAdv Exp Med Biol. 2000;
46:369-80; Staerz UD, Lee DS, Qi Y. Induction of specific immune tolerance with hybrid antibodies. In nnunol Today. 2000 Apr;
2((4):172-6: 1999 Dec; 43(4):336-43. Elsasser D, Stadick H, van de Winkel JG.
Valerius T. GM-CSF as adjuvant for immunotherapy with bispecific antibodies. Eur J Cancer. 1999 Aug;
35 Suppl 3:S2>-8.
Molema G. Kroesen BJ, Helfrich W. Meijer DK, de Lei,j LF. The use of bispecific antibodies in tumor cell and tumor vasculature directed immunotherapy. J Control Release. 3000 Feb 14_ 64(1-3):229-.,9. Bodey B, Bodey B, Siegel SE.
Kaiser HE. Genetically engineered monoclonal antibodies for direct anti-neoplastic treatment and cancer cell specific delivery of chemotherapeutic a;~ents. Curr Pharm Des. 2000 Feb;
6(3):261-76. Kudo T, Suzuki M, Katayose Y, Shinoda M, Sakurai N, Kodama H. Ichiyama M. Takemura S, Yoshida H, Saeki 11, Sai,jyo S, Takahashi J. Toimina«a T. Matsuno S. Specific targeting immunotherapy of cancer with bispeciiic antibodies. Tohoku J Exp Med. (999 Aug; 188(4):376-88. Koelemij R, et al.
Bispecitic antibodies in cancer therapy, from the laboratory to the clinic. J Immunother. 1999 Nov;
22(6):514-34. Se«al DM, Weiner GJ, Weiner LM Bispecific antibodies in cancer therapy Curr Opin Immunol. 1999 Oct; I 1 (~):558-62. Hudson PJ. Recombinant antibody constructs in cancer therapy. Curr Opin Immunol. 1999 Oct;
1 1(5):548-~7. Barth RF et al, Boron neutron capture therapy of brain tumors:
an emergin~~ therapeutic modality. Neurosurgerv. 1999 Mar: 44(3):433-50; Fleckenstein G, Osmers R.
Puchta J. Monoclonal antibodies in solid tumours: approaches to therapy with emphasis on gynaecological cancer. Med Oncol.
1998 Dec; 15(4):212-21. Guyre CA, Fanger MW. Macrophage-targeted killings and vaccines. Res Immunol. 1998 Sep-Oct: 149(7-8):655-60 Cao Y, Suresh MR. Bispecific antibodies as novel bioconjugates. Bioconjug Chem. 1998 Nov-Dec: 9(6):635-44. Farah RA, et al, The development of monoclonal antibodies for the therapy of cancer. Crit Rev Eukaryot Gene Expr.
1998; 8(3-4):321-56.:
VOItII M. Multidrug resistance and its reversal.Anticancer Res. 1998 Jul-Au~;
18(4C):290~-17. Rouard IH, et al, Fc receptors as tartlets for immunotherapy.lnt Rev tmmunol. 1997; 16(1-2):147-8~. Fan Z et al.

Therapeutic application of anti-growth factor receptor antibodies; Curr Opin Oncol. 1998 Jane 10(1):67-73. de Gast GC, et al,Clinical perspectives of bispecitic antibodies in cancer. Cancer Immunol lmmunother. 1997 Nov-Dec; 45(3-4):121-3. Carter P, Merchant AM. Engineering antibodies for imaging and therapy.Curr Opin Biotechnol. 1997 Aug; 8(4):449-54. Pluckthun A. et al, New protein engineerin<>
approaches to multivalent and bispecific antibody fragments. Immunotechnology.
1997 .lun; 3(2):83-105.
Rihova B. Targeting of drugs to cell surface receptors. Crit Rev Biotechnol.
1997; 17(2):149-69. Molema G
et al,Tumor vascular endothelium: barrier or target in tumor directed drug delivery and immunotherapy.
Pharm Res. 1997 Jan; 14( 1):2-10. Bodey B, et al, Human cancer detection and immunotherapy with conjugated and non-conjugated monoclonal antibodies. Anticancer Res. 1996 Mar-Apr; 16(2):661-74 Hartmann F et al, Treatment of Hodgkin's disease with bispecitic antibodies.
Ann Oncol. 1996: 7 Suppl 4:143-6. Wels W, et al, Intervention in receptor tyrosine kinase-mediated pathways: recombinant antibody fusion proteins targeted to ErbB2. Curr Top Microbiol hnmunol. 1996; ~ 13 ( Pt 3):1 13-28.: Kairemo KJ.
Radioimmunotherapy 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):1067-73.
Haagen IA. Performance ofCD3xCDl9 bispecitic monoclonal antibodies in B cell malignancy. l~euk Lymphoma. 1995 Nov; 19(5-6):381-93.
In another aspect the invention is directed 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. Activatin~~ cytokines for example as specified above, can also be employed to enhance the immune response. Examples of antibodies having an anti-idiotypic counterpart or for which an anti-idiotypic counterpart could made by well known techniques in the art (and that are capable of exerting the desired anti-idiotypic effect) are numerous and numerous such antiidiotypic antibodies have application to immunization as well as applications relating to tolerance (see for example US patents: 6,146,627 Method for reducin~~ T cell-mediated cytotoxicity in HIV
using anti-idiotypic antibody; 6,063,679 Anti-idiotypic monoclonal antibodies and compositions includin~~ the anti-idiotypic monoclonal antibodies; 6,060,049 Surrogate tolerogenesis for the development of tolerance to xenografts; 6,042,827Anti-idiotypic antibody induction of anti-tumor response;
6,007,815 Anti-idiotype vaccination against diseases resulting from pathogenic responses by specific T
cell populations; 5,981,502 Methods and compositions for inducing apoptosis in tumor cells; 5,766,588 Tumor immunotherapy using anti-idiotypic antibodies: 5.728,813 Anti-idiotypic antibody composition for inhibitin~~ acute complement-mediated cytotoxicity.
According to another aspect of the invention the multi-functional ligand comprises a first portion which binds to a lymph associated antigen and a second portion which binds to a tumor 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, fi>r example, the portion which binds to a lymph associated antigen has a lower affinity and'or 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 the invention in which the second portion is for example targeted to an immune cell.
Optionally, multiple such muti-functional ligands may permit sufticient tumor cell anchoring to permit the tumor cell to be killed within the lymphatic system via a toxic payload carried by the multifunctional ligand or through the recruitment of immune cells which accomplish this end (eg using the same or a different multifunctic'mal ligand fused or conjugated to a suitable cytokine (eg IL-2, IL-12). The prolonged presence of these cells could be advantageously used to assess methods of immunization directly against the tumor cell using, for example, cytokines including cytokines fused or conjugated in whole or functional part to a lymph targeted Ab on the same, or a different multifunctional ligand'delivered in a suitable dose (with respect to ~Teneration of anti-tumor antibodies and other antibody fragments for application herein as well as important related technologies see also WO 00/50008: WO 01!01 137; WO 97/37791; WO 99/37791; WO
97/10003;
Hoogenboom et al. Nat. Biotechnology 15(2) Feb 1997 p125-126: Fell H. et al.
Journal Of Immunolgy Vol 146(7) Apr 1991 p2446-2452; Anderson D. et al Bioconjugate Chemistry 14(1) Jan 1993 p10-18;
USP 6, 172,197; USP 6,171.782; Imrnunological Investigations 2000 29(2) entire issue). Optionally the tumor binding portion internalizes and!or delivers a toxic payload, for example a radionuclide, or other toxin, or a cytokine to the tumor cell (with respect to selection of tumor internalizing human antibodies see for example Pool M et al. J Mol Biol. 2000 Scp l; 301(5):1 149-61, see also Kohl MD et al. J Mol. Biol.
Biotechniques (2000) Vol 28( 1 ) p 162 In this way the rnulti-functional ligands of the invention, for example, when provided in a sufficient dose to both target the tumor and line a portion of the lymphatic system to which the tar<~et tumor is likely to drain, acts as a cancer treatment as well as a sentry system for assessing / augmenting (for example as an adjunct therapy) the ability of the tumor binding portion with/without payload to inhibit metastasis. There are numerous examples of functional cytokine 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 99/37791: W099 WO00;'06605 ; WO
99/52562W0 99!37791 MULTIPURPOSE ANTIBODY; Proceeding ofthe IBC's 1 1'r' Annual International Conference on Antibody Engineering State of the Art Science, Technology and Applications, December 3-6, 2000; Amplification of T cell-mediated immune responsca by antibody-cytokine fusion proteins. Irnmunol Invest. 2000 May; 29(2): I 17-20: Cancer Res.1999 May l ;
59(9):2159-66.;
Pharmacokinetics and stability of the ch 14.18-interleukin-2 fusion protein in mice. Cancer Immunol Immunother. 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 Immunother.
2000 Jan; 23( I ):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 fusion proteins. Hum Antibodies. 1999, 9( 1):23-36: Lode 14N, et al. Tumor-tar~~eted IL-2 amplifies T cell-mediated immune response induced by y~ene therapy with single-chain IL-12. froc Natl Acad Sci U S A. 1999 Jul 20: 96(15):8591-6: CancerlVaccines and Immunotherapy 2000 (textbook) ;
Immunotherapy W ith Intravenous Immunoglobulins P. Imbach ( 1991 ) ,A~~ademic Press: Molecular Approaches to 'humor Immunotherapy (1997) World Scientific Publishing Company, Incorporated;
Vaccines & Immunotherapy S. J. Crvz ( 1991 ) McGraw-Hill Ryerson, Limited With respect to internalizing antibodies see eg Biological Effects of Anti-ErbB2 Single Chain Antibodies Selected for lnternalizin,4 Function.; Biochem Biophys Res Commun. 2001 Jan 12; 280( 1 ):27~t-279 and references cited therein, Immunoconjugates ofgeldanarnycin and anti-HER2 monoclonal antibodies:
antiproliferative activity on human breast carcinoma cell lines J Natl Cancer Inst. 200() Oct 4; 92( 19):1573-81; Foulon CF, et al., Radioiodination via D-amino acid peptide enhances cellular retention and tumor xenograft targeting of an internalizing anti-epidermal growth factor receptor variant I II monoclonal antibody. Cancer Res. 2000 Aug 15; 60(16):4453-60. Poul MA, Becerril B, Nielsen 11B, Morisson P, Marks Selection oftumor-specific internalizing human antibodies from phage libraries J Mol Biol. 2000 Sep I; 301(5):1 149-6l.Vrouenraets MB, et al..'fargeting of a hydrophilic photosensitizes by use of internalizing monoclonal antibodies: A new possibility for use in photodynamic therapy. Int J Cancer.
2000 Oct 1; 88( I ):108-14.
In yet another aspect, the invention contemplates that the passage of tumor cells can be inhibited within the tumor vasculature using a bispecific ligand, optionally a bispecitic antibody, which targets on the one hand a well known vascular endothelial marker and one the other hand binds to a ligand 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 vasculature can be simultaneously targeted with bispecitic ligands of the invention to inhibit tumor metastasis and or immunize a subject a~~ainst tumor cells.
It is contemplated that the multifunctional ligands of the invention when used to inhibit metastasis, for example, in the manner described above, could be advantageously employed in combination with other well known therapies foe example cytoxic drugs, other tumor targeted antibodies and conjugate/fusions therewith used or currently being evaluate for immunotherapies, angiogenesis targeted dru;~s etc. (re angiogenesis sec for example Ar~giogenesis in cancer and other diseases.
Nature. 2000 Sep 14:
407(6801 ):249-57).
Similarly, a bi-specific antibody of the invention could be used to bind to antigens,'ligands on Ivmphocytes which are known or become known to inhibit or enhance immune function or mediate a disease e~~. CD45.
With respect to target receptors related to the inventions defined herein see also USP 6,277,962.
As discussed above, as used herein the term "lymph associated antigen" refers to antigens that are 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 analo~~ue which hinds to HA (February 22, 1999, Banerji et. al., Journal ofCell Biology Vol. 144. ~4, p789-801) and which is expressed primarilyy on lymphatic endothelial cells. LYVE-I specific antisera have been shown to inhibit binding of HA. The invention contemplates research and treatments using multi-functional lis;ands ofthe invention with respect to non-human mammals. including preferably agricultural animals. canine species. primates and mice having similar receptors;'antigens. For example, a murine counterpart to LYVE-I (published in Prevo R.
et al. 2001 Feb 20, J. Biol. Chem.; Manuscript MO I 1004200) can be employed to implement the various methods and embodiments described herein in a mouse model, for example to assess the extent of inhibition of metastasis effected by a multifunctional ligand (optionally comprising for example to a toxin, cytokine T cell receptor etc) which has a first portion which binds to LYVE-I
and a second portion which binds to, for example to Cil-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 6107540 and references cited in these patents, particularly with respect to other metastatic models and methods of evaluating anticancer drugs in mice). LYVE-1 counterparts in other mammals can be identified in the manner described by Prevo R. et al. (see also Skobe M. et al. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis Nat. Med. Feb; 7(2) 192-8.) Other models of metastasis in animals are well known in the art (see for example Chirgwin JM, Guise TA.Molecular mechanisms of tumor-bone interactions in osteolytic metastases.
Crit Rev Eukaryot Gene Expr. 2000;10(2):159-78. 3: Kobaek-Larsen M, et alReview of colorectal cancer and its metastases in rodent models: comparative aspects with those in humans. Comp Med. 2000 Feb;50(I):16-26. 5: Magnano M, et al.A physical-based model for the simulation of neoplastic growth and metastasis. J Sur<= Oncol. 2000 Jun;74(2):122-9. 6: Hol~fman RM. Orthotopic metastatic mouse models for anticancer dru~~ d scovery and evaluation:a bride to The clinic. Invest New Drugs. 1999:17(4):343-59.
Russo J, Russo IH.The pathway of neoplastic transformation of human breast epithelial ceIIs.Radiat Res.
3001 Jan; 155( 1 Pt 2):15 I-154. Duffy MJ, McCarihy K.Matrix metalloproteinases in cancer: prognostic markers and targets for therapy(review).fnt J Oncol. 1998 Jun:12(6):1343-8.
22: Banerjee A, Quirke P.Experimental models of colorectal cancer. Dis Colon Rectum. 1998 Apr;41(4):490-505.
Wu TT et aLEstablishin~~ human prostate cancer cell xenografts in bone:
induction of osteoblastic reaction by prostate-specitic antigen-producin~~ tumors in athymic and SCID/b'~ rnice using LNCaP and lineage-derived metastatic sublines. Int J Cancer. 1998 Sep 1 1;77(6):887-94.61:
Molpus KL, et alCharacterization of a xenograft model of human ovarian carcinoma which produces intraperitoneal carcinomatosis and metastases in mice. Int J Cancer. 1996 Nov 27;68(5):588-95.65: Pages JC, Sordat B. Bautista D, Costa J, Benhattar J. Detection of rare circulating human colon tumor cells in a nude mouse xenograft model.
Cancer Lett. 1996 Au~~ 2p:106( I ):139-44.66: Sakakibara T, et al.Doxorubicin encapsulated in sterically stabilized liposomes is superior to tree drug or drub containing conventional liposomes at suppressin~~
growth and metastases of human lung tumor xenografts. C ancer Res. 1996 Aug 15;56( 16):3743-6.
With respect to modifying an antibody to increase its affinity see also Crystal structure of Fab 198, an efficient protector of the acetylcholine receptor against myasthenogenic antibodies. Eur J Biochem. 2001 Ju1;268( 13):3685-3693.
For example, in one embodiment the invention contemplates a bispecific antibody comprisinv~ an antigen binding component specific for a tumor cell associated antigen and a relatively low affinity anti-IL-6 receptor antibody component. With respect to the anti-tumor role of IL-6 see Wei LH et al. Interleukin-6 in cervical cancer: the relationship with vascular endothelial growth factor.
Gynecol Oncol. 2001 Ju1;82( 1 ):49-56.
'The invention contemplates that TCRs and modified TCRs (see for example, WO O
1/48145) may be used as ligands, in place of antibody fragments for bindin<~ to target ligands such as peptide~MHC li~~ands.
Techniques for generating antibodies, and methods, for example of subtractive screenin<~ useful to identify other lymphatic vessel associated antibodies, including; those optionally having smaller scFv, Fab and dAb (single domain antibody or functional fragment thereot~ component (more easily passa~Tin~~ to lymphatic vessels from tissues particularly when constructed in the form of bispecitic antibodies a«. diabodies etc.) by phage or ribosome display are well known in the art (see for example Hoogenbom HR et al. Immunol.
Today (Aug. 2000) Vol 8 p 371: Schaffitzel C. et al. J Imrnunol. Methods (Dec.

10, I 999) 331 ( 1-2) p.
1 19; Roberts RW et al. Curr Opin Chem Biol. 1999 Jun; 3(3):268-73; Winter G.
et al. Annu Rev Immunol 1994 12:433-55; Kontermann RE et al. Nat Biotechnol. 1997 Jul; I
5(7):629-31: Phage Display of Peptides and Proteins, A Laboratory Manual Kay BK et al. Eds 1996 Academic Press;
Immunology Methods Manual Lefhovits, I ed. 1997 Academic Press;Hoogenboom et al.
Immunotechnolo~~y 4 (1998)1-20;

**With respect to making single domain antibodies see for example USP
5,824,520, USP 5622836, USP
5,702,892, USP 5,959,087, Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies.J Mol Recognit. 1999 Mar-Apr; 12(2):131-40. An antibody single-domain phage display library of a native heavy chain variable region:
isolation of Functional single-domain VH molecules with a unique interface. J Mol Biol. 1999 Jul 16; 290(3):685-98 and reterences cited in these references.
Methods for making antibody fusion proteins and bi-specific antibodies including diabodies etc. and fusion proteins thereof are well established in the art (for reviews and particular applications see for example Adams GP et al. Journal of Immunological Methods 2 3 I ( 1999) 249-260; USP
6,121,424, 6,027,725 and 6,025.165; EP 0654085: Hudson P. Exp. Opin. Invest. Drugs (2000) 9(6): 1231-1242; Antibody Fusion Proteins Steven M Chamuvv , Avi Ashkenazi F:ds. ISBN 0471 18358X May 1999 Wiley; Antibody Engineering, Carl A. Borrebaeck Oxford University Press. 1995; Antibody Engineering:A Practical Approach David J. Chiswell, Hennie R. Hoogenboom. John McCafferty OxfordUniversity Press,1996;
Antibody Engineering Protocols, Sudhir Paul ( 1990 Humana Press; Antibody Expression R Engineering ( 1998) Henry Y. Wang, Tadayuki Imanaka, American Chemical Society: Zhu Z.
Biotechnology (NY) 1996 Feb.; 14(2): 192-6: Nielsen UB et al. Cancer Res. 2000 Nov 15;
60(22):6434-40; Lawrence LJ.
Et al Febs Lett. 1998 Apr. 3; 425(3 ) 479-84; Hollin~~er et al., Cancer Irnmunol Immunother 1997 Nov-Decc 45 (3-4) 128-30; Immunotar~~etin~ of tumors: state of the art and prospects in 2000 Bull Cancer.
2000 Nov; 87( 1 l ):777-91; Hellfrich Wet al Int. J. cancer 1998 Apr 13 76(2):
232-9; Wu AM, Q J Nuc Med. 2000 Sep.; 44(3):?68-8 3 KrebsB. Et al. J Interferon cytokine Res 1998Sep 18(9): 783-9 l ; 'fakemura Si, et al. Protein Eng, 2000 Aug.; 13(8): 583-8; Cochlovius B et al. J
Immunol. 2000 Jul I 5;
165(2):888-95; Atwelll Jl. et al. Protein En;~. 1999 Jul; 12(7) : 597-604;
kiprivanov SM et al. ,I. Mol Biol. 1999 Oct 15, 293 ( I ): 41-56; Alt M. et al FEBS Lef-f. Jul 2 454 ()1-2) 90-4. Hudson P.I et al. J
Immunol Methods 1999 Dec 10; 231( 1-2):177-89 Ardnt MA et al. Blood 1999 Oct 15 94(8): 2662-8;
Lu D. et al. J Immunol. Methods 1999 Nov. l9; 230( 1-2):159-171; Santos AD et al, Clin Cancer Res 1999 Oct 5 ( 10 supply: 3 I 185-31235 Kontermann RE et al. Nat Biotechnol.
1997 Jul: 15(7):629-31;
Dolez et al. Protein en's. (2000) Aug 13 (8): 565-74; Adams GP et al. Nucl.
Med. Biol (2000) May 27 (4); 339-46; Williams LE et al. Med phys 2000 may 27(5) 988-94; fitzgerald K.
Protein En~~ 1997 oct 10(10): 1221-5 and the various references cited therein) as are various methods for identifying internalizing antibodies and creating toxin, radionuclide and cytokine fusions ! conjugates (see go Y et al Bioconj. Chem 1998 Nov-Dec; 9(6): 635-44) for fully exploiting various aspects ofthe invention herein detmed (see for example Becerril B et al. Biochem Biophys Res Comm 1999 Feb 16; 2'_~5(?):386-93 see also additional references blow.
Triabodies and other known multivalent antibodies etc. (see for example Iliades P et al. fEBS I,ett. 1997 June 16; 409(3):437-41 ) etc. could advantageously be employed to provide additional functionalities, as well as variation in avidity etc. for the purposes of variously exploitin<~
the invention herein.
Methods of expressing and identifying new molecules like LYVE-I are also well known in the art (see WO
98/06839) Technologies for rendering the multifunctional ligands of the invention less immunogenic (eg such as employed by Biovation) are preferably applied to the multifunctional li~~ands ofth a invention.
For recent progress in the treatment of lupus nephritis see Zimmerman R. Annu Rev. Med. 2001; 52:63-78.
With respect to targeting Fas-L see US6068841:Antibodies to Fas-L for treatment of hepatitis.
The invention also contemplates usin~~ chemokines 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. l.,ymphology 32 ( 1999}
166-170; and Gerard C. et al.
Nature Immunology (20(11, Feb.) 2(2): p108; ImmunoloL=ical Reviews I~)99 Vol 170 p 5-197) to attract immune cells into target tissues for eventual penetration into the lymphatic vessels for activation, signalling, binding to, inhibition, etc.. For example, for cancer treatment antibodies that bind to angiogenesis markers fused to such type such molecules a<~. 'FNF-a can be advanta~~eously employed optionally in conjunction with various vaccination strategies (including the use of the muti-functional ligands ofthe present invention) to attract innnune cells includin~~.
optionally, vaccination-activated tumor targeting lymphocytes to the tumor site. In an indirectly related aspect (havin~~ independent applications as well as for combination therapy with a multifuctional ligand, the invention is also directed to an antibody that targets an angiogenesis marker fused/con,jugated to a cytokine or antibody (ie a bispecific antibody) which binds to a cytokine, which cytokine augments adhesion of immune cells to blood vessels and method of using same (by administration to a subject), alone, in combination with multifunctional ligands of the invention or with other vaccination strategies to increase immune cell targeting to a solid tumor. In the case of a bispecitic antibody it is contemplated that the cytokine binding portion has a relatively low functional affinity to the cytokine so as to compete unfavourably fc>r its bindings to its natural receptor.
With reference to modulating binding of leucocytes to endothelial adhesion molecules see for example US
Patent No. 6.123.915 and the references therein cited.
It is well known to those in the art to make bispecitic antibodies which are adapted to bind two different ligands on the same cell. for example so called antigen-forks as disclosed in USP 6,705,614 (see also Shi '1' et al. Murine bispecific antibody 1 A l0 directed to human transferrin receptor and a 42-kDa tumor-associated glycoprotein also Clin Immunol Immunopathol 1996 Feb;78(2):188-95;
Amoroso AR et al., Binding characteristics and antitumor properties of I A 10 bispecific antibody recognizin~~ gp40 and human transferrin receptor Cancer Res i 996 Jan 1;56( 1 ): l I 3-20; Ring DB et al., Antigen forks: bispecific reagents that inhibit cell ~~rowth by binding selected pairs of tumor antigens, Cancer Immunol Immunother 1994 Ju1;39( 1'):41-8; l~u D et al., Complete inhibition of vascular endothelial growrth factor (VEGF) activities with a bifunctional diabody directed against both VEGF kinase receptors. fms-like tyrosine kinase receptor and kinase insert domain-containing receptor. Cancer Res 2001 Oct 1;61(19):7002-8; Schmicdl A, Breitlin'~ F. Dubel S. Expression of a bispecitic dsFv-dsFv' antibody fragment in Escherichia call. Protein En~~ 2000 Oct; I 3( 10):725-34 see also Park SS, et al.. Generation and characterization <>f a novel tetravalent bispecific antibody that binds to hepatitis B virus surface antigens Mol Irnmunol 2000 Dec;37( 18'): I 123-30;
Kriangkum J et al., Bispecific and bifunctional single chain recombinant antibodies Biomol Eng 2001 Sep; l 8(2):31-40; LJSPs 4.17-J,893, 5,989.830; VVO OOi2943 l ).
With respect to antibodies to autoantigens, ADEPT, use of anti-eotaxin antibodies, Delmmunization, antibody-cytokine fusions. ribosome display. xenomouse technology: cuttin;~
edge phage display techniques. construction of human antibody fragment based pha~~e display libraries, selection of internalizin~~ antibodies by phage-display. cancer targeting antibodies.
antibody arrays, plantibodies, design of mutant IGSF domains of CD2, CD58 and TCR; oligopeptide e~, paratope mimetics, diabodies, minibodies, triabodies, tetrabodies and related size:-kinetics issues, caspase activatable pro-druts, delivery of Bismuth-213 via scFv <rnd diabodies,anti-an_~ionenesis marker strategies, immunoenzype therapy of cancer (eg. with Rnases) pancarcinomic antigens like CEA (~~AG")-72; and related technolo~~ies set the papers and references in Proceedings of IBC's 1 I'~' Annual International Conference on Antibody Engineerin~~. State ofthe Ar-t. Science. Technology and Applications Dec :>-6 2000 f_a Jolla. CA.
With respect to biology of the lymphatic system havin~~ practical application herein see Ikomi, F
lymphology (1999) 32:90-103; Shield JW. Lymphology 1999 32: 1 18-122 and L.ymphology 33 (2000) 144-147, as well as the reterences cited therein.
The invention also contemplates control of such migration by inhibition of receptors that mediate such mibration (see far example Sun J. et a(. Eympholo~y 32 ( 1999) 166-170) for controlled application of the multitunctional ligands of the invention.
With respect to recent developments with respect to target ligands and~'or immunotherapy having application herein see also WO 01!12224, WO 01'14550, WO 01/11059, V'O Ol%
10205_ V1''O 01;00679, W0029445 WO 01 / 14885, WO14564, WO 0114558. WO 01,'' 14234, WO 01;' 13945, WO
01:' 12840, WO
01 % 12781, Vv'o Oli 12674, w'0 0 1126?0, WO 0 112224. WO 0112646; VI!O
01'12223. WO 0I % 12218, WOOI~'12217, WO01; 12216, WO01,'12154, WOOIi14557, VJOOli11059. WOOli10912, WO01/11040.
WO 0 1!10888, Vv'O OI. 10=t60, WO O1 ' 10205. VfO 01'0961 I, WO 01!09328, WO
01 1'09186, WO 01 '0919?, Vv'O Ol 108635. W'0 0('07481. V1~'O 011'07082. WO 01,''07084, Vv'O 01'07081, WO 01!07484, WO 01107466, 'Triggering Fc alpha-receptor 1 (CD89) recruits neutrophils as etfeetar cells for CD20-directed antibody therapy. J Immunol. 2000 Nov 15; 165( 10):5954-61. CD47 engagement inhibits cytokine production and maturation of human dendritic cells. J Immunol. 2000 Feb 15; 164(4):2193-9.
The invention also contemplates that a multifunctional li~Tand 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 example, in virtue of its second portion comprising such toxic component fused or conjugated thereto. The invention also contemplates attracting or supplying other immune cells or molecules to kill, or otherwise inactivate the tartlet immune cell (eV~. lymphocytes eg. by TH cell modulation or CD4 cell modulation or usin~~
antibodies including anti-idiotypic antibodies. The invention therefore contemplates that treatment of such immune cells can be accomplished by a combination of different mechanisms or drugs depending on the disease so as to reduce immunosuppression due to immune cell ablation where this is the dominant consideration. Such interactions may require interaction with one or more ligands on the surface of the targeted immune cell, as facilitated via anchoring interactions of varying affinity/avidity/duration. The invention also contemplates using multifunctional ligands comprising or bound to selectins and ICAMs etc.
to facilitate such targetin«, for example co-adminstering same in a proportion which is for example 0.01%
to 25°,% of the tar;~etin~~ multifunctional ligand. The relative amounts of the selectin!ICAM etc. (including antibody mimics) bearin<,~ multifunctional ligand as compared with the targeting multifunctional ligand can be determined empirically by varying the proportions and assessing any objective indicator of successful targeting in a disease related or purely experimental context. For example successful targeting leg. antibody binding to eg. CD3, C D28, CD2) using multifunctional ligands of the invention could be monitored by evaluating levels of cytokines normally attributable to such binding (see for example CD8 T cell activation after intravenous administration of CD3 x CD19 bispecific antibody in patients with non-Hodgkin lymphoma. Cancer lmmunol lmmunother. 1995 Jun; 40(6):390-6. Definition of a lamina propria T cell responsive state. Enhanced cytokine responsiveness of T cells stimulated throu~~h the CD2 pathway. J
lmrrrunol. 1995 ,Ian I 5; 154(2):664-75.
With respect to multifunctional ligands that are used to directly or indirectly exert an immunization function, other examples of disease associated peptides that can be presented as imrnunogens or inhibitor,%modulators of immune activity or disease pro~~ression in one of the fashions su<~~~ested above include. examples as well as technologies referenced in, for example, Knuth A, Cancer Chemother Pharmacol (2000): 46 suppl: 546-51; Engelhard VH, CancerJ Sci Arn 2000 May; 6 Suppl 3: 5272-80;
Pietersz GA et al, Cell Mol Life Sci. 2000 Feb; 57(2): 290-310; Algarra I et al, Hum Immuno). 2000 Jan;
61 ( 1 ): 65-73; Tumour vaccines: a new immunotherapeutic approach in oncolo~~y.Ann Hematol. ?000 Dec;
79(12):651-9; Human tumor-rejection antigens and peptides from genes to clinical research Nippon Geka Gakkai Zasshi. 2000 Sep; 1 Ol (9):612-7. Pinilla-Ibarz J, et al CML vaccines as a paradi~Tm of the specific immunotherapy ofcancer. Blood Rev. 2000 Jun; 14(2):1 I I-20).
In order to present an MHC-peptide complex in proximity to a B7 co-stimulatory molecule. the invention contemplates using, in addition to varying amounts (varying from a 50'50 proportion) of adjacent multifunctional ligands (which may be a dAb, diabody etc.) preferably cross-linked by an avidin component. -- as a different strate~~y -- cross-linking with avidin or the like adjacent arms of a single diabody, triabody or tetrabody etc. which binds to or has been fused or conjugated individually to respective B7 and MHC peptide components (with respect to recombinant B7 and MHC molecules and fusion proteins thereof including antibody fusions and related technologies see references above and EP
99/97477 WO 9y '42597. WO 97 28191. US 6, 197, 302, US6015884 US61401 13, US
(i,045,796, US
5580756, EP0935607, WO 9806749 W()9803552. EP I (154984. US 5869270, Construction and characterization of bispecitic costimulatory molecules containing a minimized CD86 (B7-2) domain and single-chain antibody fra~arnents for tumor targeting; method is useful for cancer therapy Rohrbach F et al., CIin.Cancer Res.; (2000) 6. 1 I, 4314-22 WO 00!008057 17 Feb 2000; V~10 9921572 (i May 1999: WO
9913095 18 Mar 1999: VI~'O 9742329 13 Nov 1997; WO 9720048 5 Jun 1997; WO
9640915 19 Dec 1996; WO 00'023087: E:I' 610046 10 Au~r 1994, IJSP 6056952 as well as references therein cited).
In a related aspect, the invention similarly contemplates using or more antibodies (optionally biotinylated and cross-linked by an avidin component) that bind to the same or different epitopes on a tumor including, where two such antibodies are used different proportions of MHC and B7 linked lie fused, conjugated or capable of bindings to) antibodies as well as different proportions of different epitope-specific antibodies to optimize the distribution of such cross-linked B7 and MF1C peptide complexes for T-cell recognition. In this way any strongly imrnunogenic peptide may be used in conjunction with suitable vaccination strategies to create a universal cancer antigen. Using a tumor unrelated peptide is advantageous to avoid any tolerization effects resulting front 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 li~~and or pair of multifunctional li~~ands optionally biotinylated 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 trispecific antibody with monovalent linkage to both the cancer cell and lymphatic endothelial cell and a third antibody component having respective fusions try one of MHC-peptide and B7 on heavy and light chain, or a trispecific or tetraspecific tetrabody having an antibody component devoted to each or the B7 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. However, It will be appreciated that.
presentation of MHC-peptide complex on a tumor does not necessarily require costimulatory B 7 presentation to induce a cytotoxic T cell response which is specific for the peptide and that multiple such presentations, preferably in a cross-linkable fashion may be preferable.
Accordin~~ly, strategies herein for costimulatory presentation of MHC-peptide and B7 may be differently applied to a lymphatic endothelial cell surface for immunization purposes and and a tumor cell surface (primarily for recognition purposes), for example by using avidin facilitated cross-linking of in the former but not the latter (tumor) 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 B'J
component.
Subject to the latter proviso, in preferred embodiments, the invention contemplates using as separate counterparts 1) separate trispecific Abs, each including for example, one antibody component which binds to the each of the respective B7 and MHC molecules which are preferably together, separately administered. Such multifunctional ligands are preferably biotinylated for cross-linking - both between adjacent trispeeific Abs and adjacent T-cell stimulatorylco-stimulatory arms:
or ?) separate bispecific pairs of Abs each respectively havin~~ I ) either <t B7 and lymphatic vessel or B7 and tumor binding portion or 2) a MHC peptide complex and a lymphatic vessel or MHC complex and tumor binding, portions which again are prefereably cross-linked by an avidin.streptavidin or a variant (ie.
using biotinylated antibodys) This latter embodiment permits smaller size antibody molecules to be used for better tumor targeting.
Antibody components which recognize the non-T cell interactive portion of the B7 or MHC molecule can be readily generated by phage display. for example in the case of a known peptide specific antibody to an MHC peptide complex (sec Chames et al, Proc Natl Acad Sci USA 97, 7969 and Chames et. al. "Attinity Maturation of TCR-Like MHC-peptide specific antibody: peptide specificity is possible over a wide affinity range" Proceedin~~s of IBC Conference on Antibody Engineeerin<~ Dec.
2000) eg. by first causing binding of the "peptide specific" antibody and then doing the phage display e~~. usin~~ an array of multiple (eg. repeats of the same antibody) such peptide specific MHC antibodies.
applying the MHC peptide complex to effect binding and then performing the phage or ribosome display.
Alternatively a TCR (eg cell bound) or analo~,ue.-'mimotope could be used for the orientation.
Similarly antibodies could be generated which in effect do not compete with CD28 or a mimotope thereof to create suitable anti-B7 type antibodies. Anti-B7 antibodies are known in the art. The invention also contemplates that the MHC-peptide binding function may be supplied using a linked superantigen (US 619729~a, WO
9601650 25 Jan 1996:
Proc.Natl.Acad.Sci.U.S.A.; (1994) 91, 19, 8945-49) in both the tumor and lymphatic system binding sites.
Optionally, the tumor anti~:en or one or both of the antigens are a pan-carcinomic antigen like TAG-72, CEA, Ei I 1 (WO 97!44461 ). The invention also contemplates using one or more phage display libraries to optimize the development of MHC; B7 costimulatory bispecific antibodies, by usin~~ cell sized latex spheres coated with an antigen eg. C:EA in various surface dispersions (or a cell) and usinG~ a array ofpreferably biotinylated antibodies which recognize the antigen and have a "oppositely located"portion fused, conjugated or capable of binding to one or both of MHC and B7, the library optionally also presenting also variations and combinations of lengths (truncations) of one or more constant reunions or for example the CDR2 generated by phage display, dependin~~ on the choice of antibody', and with microarray technology, using a signalling means to detect T-cell recognition and evaluating cytotoxicity with for example a CrSI release assay. (with respect to protein chip or microarray technology see WO 00.-'63701 references, for example in the Proceedings of IBC's conference on Protein Microarray Technology March 19-21 Santiago California The invention also contemplates use of recently published antibodies in the context of the invention (see WO Ol! 19861, WO OI % 19990, WO Ol % 19860, WO 0119987, WO 01:'19990, WO
99.58678, WO 00.''i994 3, WO 01/18014, WO 01/18016, WO 01:'18204, WO 01;'18043, WO 01/18021, WO
01/18014, WO 01/18046, WO01-16166, W00115731, W00115728, WO01'16183. W001%16171), WO01!1573?.
The invention is also directed to a method of evaluating dosing, ligand saturation, avidity effects of simultaneous ligand bindin'a on prolonged anchoring and associated benefits (eg. to delay a cancer cell for targeted killing or facilitate transfer of the multifunctional ligand to the targeted cell), cooperative interactions, cross-linking interactions (see J lntnnnau! 2001 Mar 1; I 66(5):
3356-3265; Nippon Rinsho.
(999 Dec; 57 Suppl:4?8-3?: Harefuah. ?000 Jun 15: 138(12):1046-50. Leuk Lymphoma. 1998 Mar; 29(1-2):1-l~ ) and costimulatory interactions by administering to a test subject tw ~o different multifunctional ligands of the invention with cooperating second portions.

With respect to the display of functional peptides on an antibody type scaffold see Nuttal SD; et al.., Proteins (1999) 36: 217-227; see also Skerra A., J. Mol. Recognition 2000 July-Auk 13(4): 167-187. The invention also contemplates bispecific multifunctional ligands in which the immune function exerting moiety exerts its function throw<~,h binding to an immunc'rgenic component or carrier for such eornponent as discussed above, for example an Fc domain fused to a peptide, a heat shock protein (see for example Wany XY, Immunol Invest 2000 May 29(2): 1 3l-7 and references cited therein as well as US6168793:
US6071956 ; US05981706; US05948646 Methods for preparation of vaccines against cancer comprising heat shock protein-peptide complexes; US05830464 Compositions and methods for the treatment and growth inhibition of cancer using heat shock/stress protein-peptide complexes in combination with adoptive immunotherapy as well as patents. scientitic atricles and patent applications referenced in these patenta: with respect to MHC peptide complexes( see for example WO 99/64597, WO
98103552, WO 98r06749 and references cited therein).
As described above, the invention also contemplates that the lower affinity ligand binding arm of the aforementioned multifunctional li~,and (1e. having a high aftinity targeting arm and a lower affinity effector arm) is constituted by a high affinity ligand, for example an high affinity antibody or functional fra~,ment thereof, which binds to a target biological effector (eg. a cytokine, chemokine, growth factor. hormone or other biological response modifier or drug) with high af~iinity, in a manner which permits the effector to continue to bind to its desired target receptor while bound to the antibody (1e. the antibody binds to a portion of the effector which is not critically involved in the effector binding to its receptor) provided that when bound to the effector the antibody or fragment thereof has. when cornbined with the eflector, a suitably lower affinity for the receptor than the li~~and bindin<,g arm which functions as the hi<~h affinity binder has for its target cell marker. In one embodiment the bindings moiet_d which binds to the biological effector binds to it with hi'=her affinity than the atfinity that the effector has for the effector receptor. The invention also contemplates that this bindings ann can bind to biolo~~ical eFfector in a manner which permits it to bind to one receptor but not a related receptor to which the effector would otherwise bind (see examples below). The invention also contemplates that antibody arrays are used to screen for antibodies which are capable of bindings to such biological eftectors, while bind in situ to their receptors. The invention also contemplates that such binders, when bound to the biolo<~ica1 effector, can be used to test their ability to bind to related receptors. such as those within the same family a<,. within the same family of TNF like receptors. With respect to antibody microarrays see for example Cahill DJ.Protein and antibody arrays and their medical applications.) Immunol Methods. 2001 Apr:250( 1-2):81-91. MacBeath G.
Proteomics comes to the surface.Nat Biotechnol. 3001 Sep:19(9):828-9. flewley JP. Recombinant protein arrays.Commun Dis Public Health. 2000 Dec:3(4):31 I-2; Holt LJ, Enever f, de Wildt RM, Tomlinson IM.
The use of recombinant antibodies in proteomics.Curr Opin Biotechnol. 2000 Oct; l I (5):445-9. Walter G, et al.Protein arrays for gene expression and molecular interaction sereening.furr Opin Microbiol. 2000 Jun;3(3):298-302. de ~'ildt RM, Mundy CR, Gorick BD, Tornlinson IM.Antibody arrays for hi~~h-throughput screening of antibody-antigen interactions.Nat Biotechnol. 200,) Sep; 18(9):989-94.FIolt LJ, et aLBy-passin~~ selection: direct screening for antibody-anti~~en interactions r.rsin~ protein arrays. Nucleic Acids Res. 2000 Au'~ 1;28( 15):E72 and the references cited therein. Th a term receptor as used herein for greater certainty includes decoy receptors. Examples of decoy receptors inc;ludeTRAIL decoy receptors (APO-2L), CD44 decoy like receptors (hyaluronan), interleukin receptor like protein (1l.-17) ( see J Biol Chem 2001 Nov 12), CD~75-Fe decay receptor, -fRAMf. IL.-1 RII receptor, osteoprotegerin (OPG), IL 13 Ralpha2.
**Affinity Maturation Techniques for affinity maturation using hi~~h throughput screening techniques to evaluate mutants are well known in the art. Femtomolar affinities have been achieved and it is quite common to obtain nanomolar to picomolar affinities as a result of an affinity maturation process. For example it well known to use techniques of parsimonious mutagenesis to engineer amino acid change at selected whotspots".With respect to affinity maturation see fbr example Coia G. Hudson PJ. lrvin~~ RA. Protein aftinity maturation in vivo using E. coli rnutator cells. J Immunol Methods. 3001 May 1;251(1-2):187-93.
Manivel V, Sahoo NC, Salunke DM, Rao KV. Maturation of an antibody response is governed by' modulations in tlexibility of the antigen-combining site. Immunity. 2000 Nov; 13(5):61 1-20. Boder LT, Midelfort KS, W'ittrup KD.
Directed evolution of antibody fragments with monovalent femtomolar antigen-bindin~~ affinity.Proc Natl Acad Sci U S A. 2000 Sep 26:97(20):10701-5. holler 1D, Holman PO, Shasta EV.
O'Herrin S, ~'ittrup KD. Kranz DM.In vitro evolution of a T cell receptor with high affinity for peptide 'MHf. Proc Natl Acad Sci U S A. 2000 May 9:97( 10):5387-92. Daugherty PS, Chen G, lverson BL, Georgiou G.Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies.

Proc Natl Acad Sci U S A. 2000 Feb 29;97(5):2029-34. VanAntwerp JJ, Wittrup KD. Fine affinity discrimination by yeast surface display' and flow cy'tometry.Biotechnol Prog.
2000 Jan-Feb; i 6( 1 ): 31-7.
Adams GP, Schier R.Generatin~~ improved sin~~le-chairs Fv molecules for tumor tar~~eting. J Immunol Methods. 1999 Dec 10;231(1-2):249-60. Dau'~herty PS. Chen G, Olsen MJ, Iverson BL, Georgiou G.
Antibody affinity maturation using bacterial surface display. Protein En~~..
1998 Sep;1 1 (9):825-32. VVons1 YW, Kussie PH, Parhami-Seren B, Margolies MN. Modulation ofantibody affinity by an engineered y amino acid substitution. J Immunol. 1991 Apr 1;154(7):3351-8. Balint RF.
Larrick JVv.'.Antibodv ensineerin~~ by parsimonious rnuta'~enesis. Gene. 993 Dec 27;137(1):1()9-18.
Schillbach JF, Near Rl, Bruccoleri RE, Haber E, Jeffrey PD, Novotny J.Sheriff S, Mar~~rolies MN.
Modulation of antibody affinity by a non-contact residue. Protein Sci. 1993 Feb:2(2):206-l4.Chantes P, Baty D.
Engineering ofan anti-steroid antibody: amino acid substitutions chan~.;eantibody fine specificity from cortisol to estradiol.Clin Chem Lab Med. 1998 Jun;36(6):355-9. Kussie PH, Parhami-Seren B, Wysocki L.J, Margolies MICA single en~~ineered amino acid substitution changes antibody tine specificity. J
Irnmunol. 1994 Jan I ;152( I ):146-52, as well as references cited therein.
With respect to generation of high affinity antibodies and affinity rttaturation of antibodies see also Hanes J.
Nat. Biotechnol. 2000 Dec; 18(12): 1287-92; references in Hudson PJ Exp. Opin.
Invest. Drugs (2000) 9(6) 1231-1242; Toran JL et al Evr. J. Immunol. 2001 Jan; 31(1') 128-137.
Nieison VB et al. Cancer Res 2000 Nov l5: 60 (22) 6434-40 Adams Gp, Journal of Innnunological Methods ( 1999) 249-260;
Chowdhury PS et al (June 1999) Nature Biotechnology Vol 17 p. 568With respect to strategies and recent technologies which have application to the invention see references in Hudson PJ Exp. Opin. Invest. Drugs (2000) 9(6) 1231-1242 and in particular references relating to strategies to achieve multivalency and multispeciticity; recruitment of viruses, ADF,P'F, photoactivation of cytcrtoxic radionuclides; surface receptor cross-linking: (see also Eur. J. Immunol 2000 30( 10) 3006), use of anti-B antibodies:
imrnunocytokines (see also Lode HN lmmul. Res. 2000, ? I (2-3) 279-88; Gillies SD Cancer Research 59 2159-2166 May 1999; (,ode HN et al Drugs of Today 2000 36(5) 3221-.i36).
With respect to practicial size limitations and pharmacokinetics of various types of antibodies and fragments see Colcher D. et al. G.J. Nucl. Med (1999) 43: 132-139: Wu AM et al G.J. Nucl. Med 2000 Sep; 44(3): 268:83; Williams 1_E et al Med Phys 2000 May 37(5): 988-941 lkomi F L_vrnphology 32 (1999)90-102.
With respect to the construction of diabodies see also Takemura SI et al.
Protein hng. 2000 Aug; l 3(8) 583-8; Biomol. Eng. 2001 Sept;l8(2):31-40.
With respect to anti-cancer antibodies see also 6,180,357.
With respect to technolo~~ies to produce multivalent and'or multispecific antibodies see also USI' 6, 172, 197; WO 92!01047; WO 93!1 1 161: WO 94!07921: WO 94.'13804: Helfrieh W. et al.
Journal of Immunological Methods ?37 (2000) 131-14>. Proceeding's of 1 1'r' IBC
Conference on Antibody Engineering; WO01%8579i Monoclonal antibodies rnay be routinely produced as taught by Harlow, E. and D. Lane, ( 1988) ANTIBODIES: A Luhormor_v :l~antral, Cold Sprin~~ Harbor Laboratory, Cold Spring Harbor N.Y.
Humanized antibodies ma_v be routinely produced as taught, for example, by U.S. Pat. No. 5,585,089 and U.S. Pat. No. 5,530,101. l echniques for engineering antibodies are well known and described in Winter and Millstein ( 1991 ) Nature 349:293, and Larrich and Fry ( 1991 ) llum.
Antibod. and Hybridorttas 2:17.
One having ordinary skill in the art may use well known techniques and startin~~ materials and'or commercially available expression vectors and systems that are readily available and known in the art. See e.g., Sambrook et al., Molecular Cloning a Lcrhururorw !Llanucrl, Second Ed.
Cold Sprin4~ Harbor Press ( 1989).
Examples of radionuclides useful as toxins in radiation therapy are well known. Some examples are referred to below. Auger emitters rnay be preferred for internalizing antibodies. As sugy~ested above, the teen antibody is used interchangeably with antibody fragment and antigen bindings fra~~ment and includes a whole antibody: antibody ti~agment a portion of an antibody such as a scFV
F(ab') ~ Flab) ,. Fab', Fab, dAb, microbodies ( WO00 ?9004) or the fike or multivalent such 1-ra~~ments., including those itemized or referenced herein. Regardless of structure, an antibody ti~agment can be made to bind with the same antigen that is recognized by the intact antibody. More particularly, in addition to fragments formed by enzymaic digestion of an intact Ah the term antibody or "antibody tragment" 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 including%as applicable, cysteine noose peptides and minimal recognition units consistin~~
ofthe amino acid residues that mimic the hypervariable region. Although fully human antibodies, for example, antibodies generated via human-human hybridomas or through pha~,t:
display usin~~ human antibody based libraries, are preferred, the invention does not preclude other strategies to avoid a HAMA
type response.
A chimeric antibody is a recombinant protein that contains the variable domains and complementary determinin~~ regions derived from, for example, a rodent antibody, while the remainder of ttte antibody molecule is derived from a human antibody.
With respect to stability engineering of scFv fragments for enhanced mulfunctional ligands comprising scFvs see.l:h-Col L3io12001 Feb 3; 305(5):989-1010.
Humanized antibodies are recombinant proteins in which murine LDR's of a monoclonal antibody have been transferred from heavy and light variable chains of the murinc immunoglobulin into a human variable domain.
The term therapeutic agent as used herein, is a molecule or atom which is conjugated etc. to an antibody moiety to produce combination including a conjugate which is useful for therapy. Examples of therapeutic agents include drugs, toxins, immunomodulators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes.
The term "a naked antibody" may be used to refer specitically 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 humanized antibodies.
The term immunoconjugate may be used to refer a conjugate 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 went. Examples of therapeutic agents suitable for such fusion proteins include immunomodulators ("antibody-immunomodulator fusion protein") and toxins ("antibody-toxin fusion protein").
Production of Antigen - Specitic Monoclonal Antibodies, Rodent monoclonal antibodies to antigen can be obtained by methods known to those skilled in the art. See ~~enerally, for example, Kohler and Milstein, Nature 256:495 ( 1975), and Coli~~an et al. (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991 ) ["Coligan"]. Briefly. monoclonal antibodies can be obtained by injectin~~
mice with a composition comprising the antigen in a question (Ag), verifying the presence of antibody production by removing a serum sample, removin~~ the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas.
selecting positive clones which produce anti-Ag antibodies, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma 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 and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A
Sepharose, size-exclusion chromatography, and ion-exchange chromatography.
See. for rxample, Coligan at pages 2.7. I-2.7.12 and pages 2.9.1-?.9.3. Also, see Baines et al..
"Purification of Immunoglobulin G
(IgG) ," in Methods in Molecular Biology. Vol. 10, pages 79-104 ( Che Humana Press, lnc. 1992).
With respect to relevant molecular biology techniques See also, for example, Ausubel et al., (eds.).
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages 8.2.8 to 8.2.13 ( 1990) ["Ausubel"]. Also, see Wosnick et al., Gene 60:1 15 (1987); and Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3rd Edition. pages 8--8 to 8-t) (John Wilev & Sons, Inc. 1995). Established techniques using the polymerase chain reaction provide the ability to synthesize ~~t:nes as large as I .8 kilobases in len<~th. Adam et al., Plant Molec. Biol. 21:1 131 (1993) Bambot et al., PC.'R Methods and Applications 2:26(i ( 199;);
Dillon et al., "l)se of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White led.), pages 263-268, (Humana Press. lnc. 1993).
Techniques for constructing chimeric antibodies are well-known to those of skill in the art. As an example, Leung et al., Hybridoma 13:469 ( 1994).
In yet another embodiment, an antibody of the present invention is a "humanized" monoclonal antibody.
That is. mouse complementarity determining regions are transferred from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, followed by the replacement of some human residues in the framework regions of their murine counterparts. Humanizc;d monoclonal antibodies in accordance with this invention are suitable for use in therapeutic methods, General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Oriandi et al., Proc. Nat'l Acad. Sci. USA 86: 383 3 ( 1989). Techniques for producin;~
humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522 (1986), Riechmann et al., Nature 332:323 (1988), Verhoeyen et al., Science 239:1 X34 (1988), Carter et al., Proc. Nat'1 Acad. Sci. USA 89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), and Singer et al., J.
Immun. 10:28=14 (1993). T'he publication of Leung et al., Mol. Immunol. 32:1413 ( 1990, describes the construction of humanized LL2 antibody.
In a preferred embodiment of the invention the multifunctional li~~and has a unique portion which differentiates it from other antibodies and preferably other co-administered different multifunctional ligands, which unique portion, allows the multifunctional ligand to be efficiently segregated on an immunoaffinity column. In the case of differentiating a sin~,le multifunctional ligand an anti-idiotype (assuming the first portion consists of an antibody) or other antibody uniquely recognizing the first portion could be employed. Modifying a portion of the tirst portion. for example in the case where it is antibody component and creatin~~ a antibody thereto, for example by pha~~e display, is a matter of routine skill in the arts of antibody engineering and phage display.
In another embodiment, an antibody of the present invention is a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "en~tineered" to produce specific human antibodies in response to 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 trans<~enic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antihodies from transgenic mice are described by Green et al,.
Nature Genet. 7:13 ( 1994), Lonberg et al., Nature 368:86 ( 1994), and Taylor et al., Int. lmmun. 6:579 1994).
Examples of Production of Antibody Fragments Antibody fi-a~~ments can be prepared, for example, by proteolytic hydrolysis of an antibody or by expression in E. coli of the DNA coding for the fragment.
Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can he produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fra~.:ment denoted F(ab')~. This fra~~ment can be further cleaved using a thiol reducing agent, and optionally a blocking ~~roup for the sulfhydryl <groups resulting from cleava~~e of disulfide linkages, to produce 3.5 S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directhy.
These methods are described, for example, by Goldenberg, tJ.S. Pat. Nos. 4,036,94 and 4,331,647 and references contained therein.
Also, see Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:1 19 ( 1969), Edelman et al., in Methods in Enzymology Vol I, page 422 (Academic Press 1967), and Coli';an at pages 2.8. I -2.8.10 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 cleava~~e of fragments, or other enzymatic, chemical or genetic 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 V,~ and V,. chains. 'this association can be noncovalent, as described in mbar et al., Proc. Nat'I Acad. Sci. USA 69:2659 (1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, for example, Sandhu, supra.
Preferably, the Fv fragments comprise V,r and V, chains which are connf:cted by a peptide linker. 'these single-chain antigen binding proteins (scFv) are prepared by constructing. a structural gene comprising DNA sequences encodin4.: the V" and V,_ domains which are connected by an oli~~onucleotide. 'fhe structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methuds for producing scFvs are described, 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), Ladner et al., U.S. Pat. No. 4,946,778, Pack et al., Bio~'Technology 1 I :1271 ( 1993), and Sandhu, supra.
Another form of an antibody fragment is a peptide coding for a single cornplementarity-determining region (CDR). CDR peptides ("minimal reco~~nition units") can be obtained by constructing genes encodin~~ the CDR of an antibody of interest. Such ;genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region trom RNA of antibody-producing cells. See. for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106 ( 1991 );
Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Bitter et al. leds.), pages 166-179 ('Cambridge University Prf~ss 1995): acrd Ward et al., "Genetic Manipulation and Expression of Antibodies." in Monoclonal Antibodies:
Principles and Applications, Birch et al.. (eds.), pages 137-185 (Wiley-Liss, Inc. (995).
**Preparation of lmmunoconjugates The present invention contemplates immunoconjugates to assess and effect treatment of various disease conditions. Such immunoconjugates can be prepared by indirectly conjugating a therapeutic agent to an antibody component. For example, ~~eneral techniques are described in Shih et al.. Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1 101-1 106 (1990); and Shih et al., U.S. Pat. No. 5,057,313. 'the general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function and that is loaded with a plurality of drug, toxin, chelator, boron addends, or other therapeutic ag.=ent. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
The carrier polymer is preferably an aminodextran or polypeptide of at least 50 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 for use in therapy. 'Thus, solubilizing functions on the carrier polymer will enhance the serum solubility of the: tinal immunoconjugate. In particular, an aminodextran w ill be preferred.
The process for preparing an immunoconjugate with an aminodextran carrier typically begins with a dextran polymer, advantageously a dextran of averay.:e molecular weight of about 10,000-100,000. The dextran is reacted with an oxidizing agent to effect a controlled oxidation of a portion of its carbohydrate rings to generate aldehyde groups. The oxidation is conveniently effected with ~~lycolytic chemical reagents such as NalO₄, accordin~~ to conventional procedures.
The oxidized dextran is then reacted with a polyamine, preferably a diamine, and more preferably, a monu-or polyhydroxy diamine. Suitable amines include ethylene diamine, propylene diamine, or other like polymethylene diamines, diethylene triamine or like polyamines. 1.3-diamino-2-hydroxypropane, or other like hydroxylated diamines or polyamines, and the like. An excess of the amine relative to the aldehyde ~~roups of the dextran is used to insure substantially complete; conversion of the aldehyde functions to Schiff base groups.
A reducing agent, such as NaBH,, NaBHCN or the like, is used to effect reductive stabilization of the resultant Schiff base intermediate. The resultant adduct can be purified by passage throu~~h a conventional sizing column to remove cross-linked dextrans.
Other conventional methods ofderivatizing a dextran to introduce amine functions 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-activated derivative, prepared by conventional means, e.g., using dicyclohexylcarbodiimide (DCC') or a water soluble variant thereof, to form an intermediate adduct.
Alternatively, polypeptide toxins such as pokeweed antiviral protein or ricin A-chain, and the like, can be coupled to aminodextran by glutaraldehyde condensation or by reaction of activated carboxyl groups on the protein with amines on the aminodextran.
Chelators for radiometals or magnetic resonance enhancers are well-known in the art, Typical are derivatives of ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA).
These chelators typically have groups on the side chain by which the chelator can be attached to a carrier.
Such groups include, e.v~.. benzylisothiocyanate, by which the D'TPA or FDTA
can be coupled to the amine group of a carrier. Alternatively, carboxyl groups or amine groups on a clnelator can be coupled to a carrier by activation or prior derivatization and then couplings, all by well-known means.
Boron addends, such as carboranes, can be attached to antibody components by conventional methods. For example, carboranes can be prepared with carboxyl funcaions on pendant side chains, as is well known in the art. Attachment of such carboranes to a carrier, e.~~., aminodextran, can be achieved by activation of the carboxyl groups of the carboranes and condensation with amines on the carrier to produce an intermediate conjugate. Such intermediate conjugates are then attached to antibody components to produce therapeutically useful immunoconjugates, as described below.
A polypeptide carrier can be used instead of aminodextran, but the polypeptide carrier must have at least 50 amino acid residues in the chain, preferably 100-5000 amino acid residues. At least some. of the amino acids should be lysine residues or glutamate or aspartate residues. The pendant amines of lysine residues and pendant carboxylates of glutamine and aspartate are convenient for attaching a dru~~, toxin, immunomodulator, chelator, boron addend or other therapeutic absent. Examples of suitable polypeptide carriers include polylysine_ polyglutamic acid. polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded carrier and immunocon,jugate.
Conjugation of the intermediate conjugate with the antibody component is effected by oxidizing the carbohydrate portion of the antibody component and reacting the resulting aldehyde (and ketonel carbonyls with amine groups remaining on the carrier after loadin;~ with a drug, toxin, chelator, innnunomodulator.
boron addend, or other therapeutic agent. Alternatively. an intermediate conjugate can be attached to an oxidized antibody component via amine groups that have been introduced in the intermediate conjugate after loadin~~ With the therapeutic agent. Oxidation is conveniently effected either chemically, e.~~.. w ~ith NalOr or other glycolytic reagent, or enzymatically, t.~~., with neuralninidase and ~~alactose oxidase. In the case of an aminodextran carrier, not all of the amines of the aminodextran ;are typically used for loading a therapeutic agent. 'The remaining: amines of aminodextran condense with the oxidized antibody component to form Schiffbase adducts, which are then reductively stabilized, normally with a borohydride reducing agent.
Analogous procedures are used to produce other immunoconjugates according to the invention. Loaded polypeptide 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 immunoconjugatc is puritied using conventional techniques. such as sizing chrornato~graphy on Sephacryl S-300.
Alternatively. immunoconju~,~ates can be prepared by directly conjugating an antibody' component with a therapeutic agent. The L~eneral procedure is analo<Vous to the indirect method of conjugation except that a therapeutic agent is directly attached to an oxidized antibody component.
For application to linking MHC 1'1l peptide~~B7 molecules to a latex which has previously conjugated to biotin, for avidin assisted linking to a multifunctional li~and, it will be appreciated that biotin can be conju~~ated to a part of a latex sphere which is then linked to MHC peptide and B7 molecules by placing the spheres in a confluent layer or in the spheres in a microwells such that only part of the sphere is exposed for conjugation and then coatings the spheres onto avidin coated plates for the B7 and MI-1C linkage.

It will be appreciated that other therapeutic absents can he substituted for the chelators described herein.
Those ofskill in the art will be able to devise conjugation schemes without undue experimentation.
As a further illustration, a therapeutic agent 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 to an antibody component. As an alternative, such peptides can be attached co the antibody component using.: a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al~. Inf. J. Cancer 56:244 ( 1994). General techniques for such conju<~ation are well-known in the art. See, for example, Wong, (.
HEMISTRY OF PROTEIN
CONJUGATION AND CROSS-LINKING (CRC Press 1991 ); Upeslacis et al., "Modification of Antibodies by Chemical Methods," in MONOC.'t.ONAL ANTIBODIES: PRINCIPLES AND
APPLICATIONS. Birch ct al. (eds.). pages 187-230 (Wiley-Liss, Inc. 1995);
Price, "Production and Characterization of Synthetic Peptide-Derived .Antibodies," in MONOCLONAL
ANTIBODIES:
PRODUCTION, ENGINEERING AND CLINICAL. APPLICATION, Ritter et al. (eds.), pages (Cambridge LJniversity Press 1995).
As described above, carbohydrate moieties in the Fc reunion of an antibody can be used to conju~~ate a therapeutic agent. However. the Fc region is absent if an antibody fragment is used as the antibody component of the immunoconjugate. Nevertheless, it is possible to introduce a carbohydrate moiety into the light chain variable re~_ion of an antibody or antibody fra=ment. See, for example, Leung et al., J. Immunol.
154:5919 (1995); Hansen et al., U.S. Pat. No. 5,443,95 3 ( 1995). The engineered carbohydrate moiety is then used to attach a therapeutic agent.
In addition, those of skill in the art will recognize numerous possible variations of the conjugation methods.
For example, the carbohydrate moiety can be used to attach polyethyleneglycol in order to extend the half-life of an intact antibody, or anti~~en-binding fragment thereof, in blood.
lymph, or other extracellular fluids. Moreover, it is possible to construct a "divalent immunoconjugate" by attaching therapeutic agents to a carbohydrate moiety and to a free sulthydryl ~~roup. Such a free sulfhydryl ~~roup may be located in the hin~~e rev~ion 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 Mapping." in METHODS IN
MOLECULAR BIOLOGI'. VOLIIME 1(): 1MMUNOCLIEMICAL PROTOCOLS, Manson led.), pages 105-1 16 (The Humana Press, lnc. 1992). More specifically, competitive blocking assays for example to determine CD22 epitope specificity are described by Stein et al., Cancer Immunol, lmmunother. 37:293 ( 1993), and by Tedder et al., U.S, Pat. No. 5,484,892 ( 1996).
In another aspect the invention is directed to a bispecitic ligand, preferably a bispecifc antibody, comprising at least a first ligand, preferably an antibody component, which binds specifically to a first cell surface associated ii~~and and at least a second ligand, preferably a second antibody component which binds specifically to a second cell surface associated ii~~and on the same cell, and wherein the functional at4inity of at least one and preferably both of said antibody components is selected so as to substantially limit functional bindin<~ unless both of said first and second antibody components are substantially contemporaneously bound to said cell. It is known to provide bifunctional ligands wherein functional binding, for example. to accomplish si~~nal transduction, is predicated on both ligands being bound or cross-linking. However this effect is not contemplated to be predicated on differentially controlling the functional affinity of the respective li~~ands. Accordin~~ 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 bispecitic ligands which inherently have a suitable differential functional affinity.
Such bispecitic ligand are mentioned herein. By controlling the at4inity of at least one of said li<~ands, for example where the functional affinity of one said ligands is substantially less than that of the other li~~and the invention contemplates that a substantially greater percenta;.;e of the administered dose of the bispecitic ligand will affect cells in which only both ligands are present, and;'or that a reduced percentage ofthe dose administered will functionally bind to the cells in virtue only of the reduced functional affinity ligand. The invention also contemplates that functional affinity of one ligand is greatly increased to establish the functional affinity differential and that the functional affinity of both ligands is reduced relative to that of a standard, for example relative to that of a comparable li~~ands 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 microarray (or library) of bispecitic li~~ands~in which for esamp(v, the bispecitic ligand is "tethered" (ie. immobilized) directly or indirectly in virtue of one or more amino acid residues which are positioned within the molecule to preferably minimally interfere with any binding, and in which the signal (e~~ its intensity) associated with a single li~~and bindiny~
interaction can be differentiated from a two or more li'~and interactions, for example cell surface binding (alternatively the ligands or cell may be immobilized) and that ribsome and phage display could be adapted to bispecitic single domain antibodies constitutin~~ a single chain (see references herein) by elon~,ating the end of the chain from which the molecule is tethered. The invention contemplates that the affinity of one such li~.;and 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 assessing sin'=le ligand binding capability of successful bi-ligand binders for example by blocking the other (non-assessed at that time) ligand (cg. with correlative ligand or a mirnotope thereon and for example determining limited or non-existent such binding to as well as using inclined ligand testing surfaces fbr washing over the correlative ligand, for example of defined surface area, including preferably defined lengths and widths and concentrations % distributions :' amounts of the bound li'~and, where the de~~ree of incline is selected to rou~~hly sunulate the micro-environment of the comparable in viva target, he it a stationary cell with a rou4;hly defined avera~~e shear force of bathing fluids a<.;. within a tumor or in the lymphatic system, or a nubile cell within a vein, artery, or lymphatic vessel,~including those of different sizes. The invention is also directed to a mthod of generating a target ligand or improving:, the tar;~et specificity of any ligand by using a population of variants of that ligand within a micro-environment simulated microarrav system in w which the at least one of the follw~in~, factors is sirnulat~d: concentration or amount or distribution of correlative ligand, shear force and shape using ie~n~~th and width parameters to simulates intraluminal diameter and Iength.The invention also contemplates in the case of a multifunctional li~.:and or ire the case of a hispecitic or rnultispecfic li~,?and (as herein described) that the affinity of its component binding ligands may be selected for venous or arterial tagetin~~ or to accommodate lymphatic system targeting or targeting within or through tissues or combinations of the aforementioned cg. median, average or or wei<~hted compromises to improve desired tarwetin~~. In a preferred embodiment the first ligand is selected on the basis of its ability to at least partially discriminate between a target population of cells (cg. a li~and 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 other effect other than binding for tar~~eting purposes) and the second ligand is selected for its ability to modulate the activity ofthe targeted cell, optionally in virtue of binding alone cg. without delivering a payload )the term modulate referring broadly to any desired effect on the cell or its functionality) In this case the functional affinity for the ligand which is tarv~eted for modulating the activity of the cell is selected so as to reduce the likelihood of binding unless bindings has first or contemporaneously occurred to the first ligand targeted for selectivity (cg, the second ligand~worrld have rnonovalent as opposed to divalent binding to the ligand required for selectivity andior from 0.20 to 10-~' fold reduction in affinity (for example as measured by Biacore) relative to the bindings affinity for the first ligand. This reduction in affinity is preferably ~~reater than a 100°,~ reduction in affinity (multiply by O.l ), preferably greater than a 200°% reduction in affinity, preferably ~~reater than a 300°o reduction in affinity, preferably greater than a 400% reduction in affinity, preferably ~~reater than a S00°~o reduction in affinity, preferably greater than a 600% reduction in affinity, preferably greater than a 700°-~i, reduction in affinity, preferably ~~reater than a 800°ro reduction in affinity, preferably grceater than a 900°o reduction in affinity, preferably greater than a 1000°io reduction in affinity, preferably ~~reater than a ?000°~ reduction in affinity, preferably greater than a 3000°'o reduction in affinity.
preferably greater than a 4000° o reduction in affinity, preferably greater than a 5000°io reduction in affinity.
preferably <greater than a (i000°,!° reduction in affinity, preferably ~~reater than a 7000% reduction in affinity, preferably greater than a 8000% reduction in affinity, preferably greater than a 9000% reduction in affinity, preferably ~~reater than a 10000°'o reduction in affinity, preferably- greater than a ?0000°o reduction in aftmity.
preferably ~~reater than a 30000°io reduction in affinity. preferably greater than a ~t0000°ro reduction in affinity, preferably greater than a 50000% reduction in affinity, preferably ~_reater than a 60000°r reduction in affinity, preferably greater than a 70000% reduction in affinity, preferably greater than a 80000°%
reduction in affinity, preferably greater than a 90000°,% reduction in affinity, preferably greater than a 100000°ir reduction in affinity, preferably ~~reater than a 500000°%b reduction in atlinity, preferably greater than a 10000()0°~o reduction in affiinity, preferably greater than a 10000000~~o reduction in affinity, preferably greater than a ?0000000%
reduction in affinity, preferably a ;greater than 3000000° o reduction in affinity, preferably a greater than 40,000.000% reduction in affinity, preferably a ~_reater than 50000000°,~u reduction in affinity. preferably a greater than 60000000% reduction in aff7nity, preferably ar greater than 70000000% reduction in affinity_ preferably a ~~reater than 80000000°-~ reduction in affinity, preferably a greater than 90000000°ro reduction in affinity preferably a greater than 100,000,000°r reduction in affinity, preferably a reduction in affinity of between one and two orders of magnitude, preferably a reduction in affinity of between two and three orders of magnitude, preferably a reduction in affinity of between three and tour orders of magnitude, preferably a reduction in affinity of between four and five orders of magnitude, preferably a reduction in affinity of between five and six orders of magnitude, preferably a reduction in affinity of between six and seven orders of magnitude preferably a reduction in affinity of between seven and eight orders of magnitude, preferably a reduction in affinity of between eight and nine orders of magnitude, preferably a reduction in affinity of between nine and ten orders of magnitude.
It will be appreciated that a suitable reduction in affinity, if any, will depend on the valency of the respective first and second ligands and the selected affinity of the tirst ligand, which for example may have been augmented. The invention also contemplates a trispecific (and triavalent) ligand in which two ligands differently define its speciticity to reduce the likelihood of an undesired effect attributable to the function exerting moiety binding alone. In terms of the physical constitution of a ligand having a trispecitie 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 Vv'O
99'42077, (JS 6174691, W00029004. Camel single-domain antibodies as modular building units in J Biol Chem. 2000 Oct 25, & Mulligan-Kehoe U.S. patents including US 5702892, US
5824120; 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 99/42077, Celltech's TFM, leucine zippers, US 5,910,573.
US5892020, f~,P 0654085B, see also EP 0318554B). The teen functional binding is used to refer to binding which yields the desired effect, for example a therapeutic et"tect 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 immune cells, and the second liaand may be different and may upon being bound to, for example result in inactivation, anergy, apoptosis or reduced capacity for endothelial adhesion of the immune cell. In this case, the invention contemplates that the functional affinity of the antibody component which binds 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 I, preferably approximately 1.1 > to I, more preferably approximately 1.20 to I, more preferably approximately 1.25 to I, more preferably approximately 1.30 to 1. more preferably approximately 1.35 to I, more preferably approximately 1.40 to 1, more preferably approximately 1.45 to I, more preferably approximately 1.50 to l, more preferably approximately 1.55 to l, more preterably approximately 1.60 to 1, more preferably approximately 1.60 to l, more preferably approximately 1.65 to l, more preferably approximately 1.70 to l, more preferably approximately 1.75 to 1, mare preferably approximately 1.80 to I, more preferably approximately 1.85 to l, more preferably approximately 1.90 to I, more preferably approximately 1.95 to 1, more preferably approximately 2 to I, more preferably greater than 2 to I, more preferably approximately greater than 3 to 1, more preferably approximately greater than 4 to l, more preferably greater than 5 to 1, more preferably greater than 6 to 1, more preferably greater than 7 to 1, more preferably greater than 8 to 1 . more preferably greater than 9 to 1 , more preferahl~~ greater than 10 to I , more preferably greater than 20 to I , more preferably greater than 30 to I , more preferably greater than 40 to I , more preferably ~~reater than 50 to 1 , more preferably ;greater than 60 to I , more preferably greater than 70 to I, more preferably ~~reater than 80 to l, mare preferably greater than 90 to I, more preferably <greater than 100 to l, more preferably greater than 500 to 1 . more preferably ~~reater than 1000 to 1, more preferably ~~reater than 10,000 to I, more preferably greater than 100,000 to l, more preferably ~~reater than 500,000 to 1 more preferably greater than 1,000.000 to 1.
It will be appreciated by persons skilled in the art that the foregoin~~
aspects of the invention apply to a variety of different combinations of immune function or other therapeutic function exerting ligands and specificity dictating ligands includin~~ those involved in immune signaling;, stimulatory, eo-stimulatory.
inhibitory, adhesion or other interactions, including without limitation, cytokine receptors, ligands associated with immune cell adhesion. ligands to which bindings results in stimulation, activation, apoptosis, anergy or costimulation, or li<~ands which differentiate between different populations or subpopr.rlations or immune cells (see eg. US 6135941, WO 00/63251. WO 00/61 132, US 61:?0767).
including sub-populations of B cells and T cells (see for example US 6197524) activated versus non-activated lympocytes, diseased or disease-causing cells versus non-diseased 1 disease causing lymphocytes (see for exampleW'O
Ol/13945A1, US 6132980, ) and specific immune cell clones for example those having specific Ig type and MHC-peptide type ligands and correlative ligands. Examples of such iigands include CC R5, CTLA-4, LFA-1. LFA-.>. ICAMs e~~. /CAM-1. CD2, CD3, CD4 (e~~ see US 6,136,310), CD18, CD22. CD40, CD44;
CD80, CD86, CD134 and CD154, to name only a few (see also US60874'75: PF4A
receptor) (see also Glennie MJ et al. Clinical Trial of Antibody Therapy. Inumunology Today Aug 2000. Vol. 2 ! (no. 8) p.406).
The invention also contemplates that the therapeutic function or immune flmction effecting ligand is also a specificity imparting ligand, which in the case of for example, an antigen presenting cell may be an antibody which recognizes and binds to a specific MHC peptide complex, as is established in the art (see pertinent Chames et al. references herein, see also WO 97!02342 , Direct selection of <r human antibody fragment directed against the tumor T-cell epitope HLA-A 1-MACE-A I from a nonimmunized phage-Fab library. Proc Natl Acad Sci U S A. 2000 Jul 5: 97( 14):7969-74). In this case it will be appreciated that the APC targeting ligand assist the particular MHC peptide bindings antibody to bind to its target.
See also WO 97107819 which is hereby disclaimed with respect to all relevant aspects of the invention herein insofar as inherently disclosed therein. See also US 5,770,403 with respect Co antibodies which bind to cytokines.
In one embodiment, the respective antibody components of the multispecific ligand 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 1e. non-targeted tissue distributions. In a preferred embodiment the target cell is a cancer cell and the respective first and second cell surface associated ligands are expressed on different subsets of normal cells, which are non-overlapping subsets, so as to minimize deleterious normal cell targeting and distibute the undesired effects or normal call targeting (eg. with a toxin), to different cell populations. For example in the case of tumor cell targeting one or both ligands may be expressed exclusively on a single tumor type (e~~. a human sarcoma or carcinor~~a, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma. sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Vv~ilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma. epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendrov~lioma, meningioma, melanoma. neuroblastoma, retinoblastoma;
leukemias, e.'~., acute lymphocytic leukemia and acute myelocytic leukemia (mycloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia): and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease). multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease) or a particular category of tumor types (eg. adenocarcinomas, tumors of neuroectodennal origin, or on multiple different 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. for example both may be scFvs and the resulting product may be a diabody, triabody, or tetrabody. For example in a preferred embodiment the bispecific antibody comprises two dAb components comprisin~~ 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 CH2 domain (optionally also at least a partial CH3 domain). In another embodiment, a trispecific antibody or tevtraspecific antibody with at least two different and preferably 3 or 4 subsets (preferably at least one or more of such subsets beings non-overlapping subsets) of non-targeted cell reactivities may be employed in the form of a trispecitic or tetraspecific antibody respectively whereby up to three or four different pairs of ligands are targeted, so as further minimize normal cell targeting and also preferably tartlet a heterogenous population of cells within the same tumor. Ligands with distributions on normal tissues are well known, some being referenced herein, for example CEA. CD-20, P53, epidermal growth factor, including known multicarcinomic and pancarcinomic ligands (eg. see USS, 171.665, US 4349528.
The teen functional binding is used to mean binding for the purpose of accomplishing the object of the binding, for example binding for a sufficient duration to inhibit or enhance a particular effect, such as cell killing, for example in the case where one both antibody components arc' selected for their ability to internalize, binding for :~ sufficient duration to permit internalization, for example to deliver a toxic payload. As discussed above. the term substantially in reference to therapeutic advantage is used to refer to a degree which provides a si'~niticant benefit from a therapeutic standpoint.
Examples of tumor associated antigens (eg. WO O 1:21828) and targets a.nd related antibodies <tre referenced throughout the disclosure and the foregoing aspect of the invention is for greater certainty directed to bispecific antibodies (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 permuations of the target cell (diseased, disease causing or immune) associated antigens, ligands, epitopes or receptors well known to those skilled in the art, herein directly or indirectly 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 ~) groups of pairs including for example pairs in which one member is used for targetin_~ and the second is used for modulation puposes such modulation ineludiny= without limitation, simple binding eg. to deliver a payload, apoptosis inducin'1 (eg. anti-fas), modified vascular adhesion properties (eg. anti-CU44'), modified eytokine binding (anti-CCRS) etc.(re: relevant ligands,!markers see also USP 6,010,902 and the references cited therein. Samter's Immunologic Diseases, f=fifth and Sixth Edition, Lippincott, Frank Austen, MD
Michael M. Frank, MD
John P. Atkinson, MD Harvey I. Cantor, MD (6'~'-ISBN: 0-7817-2120-.'.):
Fundamental Virology, 'fitird and Fourth Edition, Lippincott Uavid M. Knipe. PhD Peter M. Howley, MU Uiane t-;. Griftin, MD, PhD
Robert A. Lamb, PhD, ScD Malcolm A. Martin, MD Bernard Roizman, ScD Stephen E.
Straus. MD (4'h-ISBN: 0-7817-I 833-3 ): Arthritis and Allied Conditions - A Textbook of Rheumatology, 'thirteenth and Fourteenth Editions, William J. Kooprnan, MD 14'~':ISBN: 0-7817-2240-3.
November2000; Cancer-Principles and Practice of Oncology, Fifth and Sixth Editions, Lippincott, Vincent T. DeVita. .1r.. MU
Samuel Hellman, MD Steven A. Rosenber~~, MD, PhD ISBN: 0-7817-2229-2; Dubois' Lupus Erythematosus. Fifth Edition. Daniel J. Wallace, MD ISBN: 0-683-086Ei~-0, December 1996: Cytokine Therapeutics in Infectious Diseases, Steven M. Ftolland, MD, PhD, Lippincott, ISBN: 0-7817-1625-X, US
6054561 ), in each of their permuatations of size;'valency (ie. dabs, scFv, diabodies etc herein referenced) as applied to each of the applicable disease conditions herein referenced or otherw rise known to those skilled in the art.
With respect to recombinant techniques for producing Fv fragments see also WO
88!01649, ~'O 88.06630, WO 88'07085, WO 88;07086, and WO 88!09344.
With respect to preparing ligands for specific MHC peptide complexes see also WO 01 /22083; Direct selection of a human antibody ti~agment directed against the tumor T-cell epitope HL..A-A I-MAGE-A 1 from a nonimmunized phage-Fab library. Proc Natl Acad Sci U S A. 2000 Jul 5:97( 14):7969-74.
With respect to bispecific antigen binding constructs that are suitable for for binding to more than one antigen on the same cell see also Schmiedl A et al. Protein Eng 2000 Oct 13(10):725-34.
Preferred immunoconjugates include radiolabeled antibody components and conjugates of an anti-Lyve-1 antibody component and an antbody component which comprises an immunomodulator.
A radiolabeled immunoconjugate may comprise an .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 ~''~
Au, '. p,. ~-5 l, ~;~ 1, ''n' Y. n~ Re, ~~~ Re,"Cu, -n At. and the like.
As discussed above, a radioisotope can be attached to an antibody component directly or indirectly, via a chelating agent. for example, ~'' Cu. considered one ofthe more promising radioisotopes for radioimmunotherapy due to its 61.5 hour half=life and abundant supply of beta particles and gamma rays.
can be conjugated to an antibody component usin~~ the chelating a;~ent, p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA) . Chase, "Medical Applications of Radioisotopes," in Remington's Pharmaceutical Sciences, 18th Edition. Gennaro et al. (eds.), pages 624-6~2 (Mack Publishing. Co. 1990) (see also 19'x' edition of Reminton's). Alternatively. "' Y, which emits an ener~~etic beta particle, can be coupled to an antibody component usin~~ diethylenetriaminepentaacetic acid (DTPA). Moreover, a method for the direct radiolabeling of the antibody component with ~'' I is described by Stein et al., Antibody lmmunoconj. Radiopharn. 4: 703 ( 1991 ) (see also LJSP 6, 080, 384).
Alternatively, boron addends such as carboranes can be attached to antibody components, as discussed above.
In addition, therapeutic immunoconjugates can comprise an immunomodulator moiety suitable for application for the purposes herein. Broadly speaking, the term "immunomodulator" includes cytokines.
stem cell growth factors, lymphotoxins, such as tumor necrosis factor (1'NF), and hematopoietic factors, such as interleukins (e.~~., interleukin-1 (1L-1 ), 1L-2, Il.-3, IL6, II~-10 and I1.-12), colony stitnulatin'T factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.<~., interferonsalpha, -beta and gamma.), the stem cell growth factor designated "S I factor," erythropoietin and thrombopoietin. Examples of suitable immunomodulator moieties include 11.-2, Il.-6. IL-10, IL12, interferon-gamma., TNF-alpha., and the like.
A related form of therapeutic protein is a fusion protein comprising an antibody moiety and an immunomodulator moiety.
Methods of making antibody-immunomodulator fusion proteins are known to those of skill in the art as discussed herein. For example, antibody fusion proteins comprising an interleukin-? moiety are described by Boleti et al., Ann. Oncol. 6:945 ( 1995), Nicolet et al., Cancer Gene ~
her. 2:161 ( 1995), Becker et al., Proc. Nat'I Acad. Sci. USA 93:7826 ( 1996). Hank et al., Clin. Cancer Rca.
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')=fragment and a tumor necrosis factor alpha moiety.
Such immunoconjugates and antibody-immunomodulator fusion proteins provide a means to deliver an immunomodulator to a target cell and are particularly useful against tumor cells. The cytotoxic effects of immunomodulators are well known to those of skill in the art. See, for example. Kle et al.. "t_~~mphokines and Monokines," in Biotechnology and Pharmacy, Pessuto et al. (eds.), pages 5 3-70 (Chapman R Hall 1993) as well as other references herein cited. As an illustration, interferons can inhibit cell proliferation by inducing increased expression of class I histocompatibility antigens on the surface of various cells and thus, enhance the rate of destruction of cells by cytotoxic T lymphocytes.
Furthermore, tumor necrosis factors, such as TNF-alpha., are believed to produce cytotoxic effects by inducing DNA
fragmentation.
Moreover, therapeutically useful immunocon_jugates can be prepared in which an antibody component is conjugated to a toxin or a chemotherapeutic drug. Illustrative of toxins which are suitably employed in the preparation of such conjugates are ricin, abrin, ribonuclease. DNase 1, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pserrdomonas exotoxin, and Pseudomonas endotoxin.
See, references herein as well as for example, Pastan et al., Cell 47:641 ( 1986), and Golden berg, CA-A
Cancer Journal for Clinicians 44:43 (1994). Other suitable toxins are known to those of skill in the art.
With to respect to bispecific antibody constructs which are capable of biding simultaneously to two ligands on the same cell see also W096!32841. Various such constructs are known in the art.An alternative approach to introducin~~ 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 exotoxin A fusion proteins have been described by Chaudhary et al., Nature 339:394 ( 1989), Brinkrmann et al.. froc. Nat'1 Acad.
Sci. USA 88:8616 (1991), Batra et al.. Proc. Nat'I Acad. Sci. USA 89:5867 ( 1992). Friedman et al., J.
Immunol. I 50:3054 ( 1993), Wels et al., lnt. J. Can. 60:137 ( 1995), fominaya et al., J. Biol. Chen.
271:10560 ( 1996), Kuan 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., .l. Biol. Chem. 268:5302 (1993), Thompson et al., J.
Biol. Chem. 270:28037 (1995), and Vallera et al., Blood 88:2342 ( I 996). Deonarain et al., Tumor Tar~etin~~
1:177 ( ( 995), have described an antibody-toxin fusion protein having an RNase moiety, while l_.inardou et al., Cell Biophys. 24-25:243 ( 1994), produced an antibody-toxin fusion protein comprising a DNase I
component. Gelonin was used as the toxin moiety in the antibody-toxin fusion protein of Wane et al., Abstracts of the 209th ACS National Meeting, Anaheim, Calif., Apr. 2-6, 1995, Part I, BIOT005. As a further example, Dohlsten et al., Proc.
Nat'I Acad. Sci. USA 91:8945 ( 1994), reported an antibody-toxin fusion protein comprising Staphylococcal enterotoxin-A. Numerous other examples have been reported in the literature.
Useful cancer chemotherapeutic drugs for the preparation of immunoconjugates include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, antibiotics, epipodoph_yllotoxins, platinum coordination complexes. hormones. and the like.
Suitable chemotherapeutic agents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995), and in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 7th Ed.
(MacMillan Publishing Co.
1985). Other suitable chemotherapeutic agents, such as experimental drugs, are known to those of skill in the art.
In addition, therapeutically useful immunoconju;ates can be obtained by conjugating photoactive agents or dyes to an antibody composite. Fluorescent and other chromogens, or dyes, such as porphyrins sensitive to visible light, have been used to detect and to treat lesions by directing the suitable light to the lesion. In therapy. this has been termed photoradiation, phototherapy. or photodynamic therapy (Jori et al. (eds.), Photodynamic Therapy of Tumors and Other Diseases (L.ibreria Progetto 1985);
van den Bergh, Chem.
Britain 22:430 (1986)). Moreover, monoclonal antibodies have been coupled with photoactivated dyes for achieving phototherapy. Mew et al.. J. Immunol. 130:1473 ( 1983); idern., Cancer Res. 45:4380 ( 1985);
Oseroffet al., Proc. Natl. Acad. Sci. USA 83:8744 (1986); idem., Photochem.
Photobiol. 46:83 (1987);
Hasan et al., Prog. Clin. Biol. Res. 288:471 (1989); Tatsuta et al., Lasers Surd. Med. 9:422 (1989);
Pelegrin et al., Cancer 67:2529 ( 1991 ). 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 in nnunoconjugates comprising photoactive agents or dyes.
With respect to a multifunctional ligand having a first portion that binds to both lymphatic endothelial cells and tumor vasculature, the invention contemplates using phage display or ribosome display to generate an antibody that binds to vef'~r-3 as well as one or both of of vegfr-2 or vegfr-L, having regard to the sequences of those respective receptors (see USPs 5,776.755, 5877020, 5952199, 6107046, 6130071, 6221839, 62357(3, 6245530; see also WO 00!21560, WO 95;'33772. WO 97!05250, WO
98133917).
Preferably 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, fusin~~ the bindint,~
domain of VEGF-C or VEGF-D to antitumor antibody. The invention also contemplates that the risk of retargeting cancer cells to non-tumor sites of an;;iogenesis, 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 function, such as a toxic moiety, cytokine or antibody component which retargets immune cells capable of killing such cancer cells. The invention also contemplates using in combination or alone a multifunctional ligand having a second portion that comprises an anti-VEGF
antibody portion which hinds to one or more of the VE:GF family of li~~ands in order to inhibit lyrnphangiogenesis and;'or angiogenesis. (see also for example, WO 00/.>7025, WO 98/33917, USP
6130071, WO Oli 12669). With respect to angio~~enesis and particularly lymphangiogenesis see also:l:
Shibuya M. Structure and function of VEGE%VEGF-receptor system involved in angiogenesis. Cell Struct Funct. 2001 Feb;26( 1 ):25-35: Yonemura Y, et al.Lymphan~~iogenesis and the vascular endothelial growth factor receptor (VEGFR)-Sin gastric cancer Eur J Cancer. 2001 May;37(7):918-23.: Iljin K, et aIVEGFR3 gene structure, regulatory region, and sequence polymorphisms FASEB J. 2001 Apr;lS(6):1028-36.: Tang RF, et alOverexpression of lymphangiogenic growth factor VEGF-C in human pancreatic cancer. Pancreas.
2001 Apr:22(3):285-92: Kadambi A, Carreira CM, Yun CO, Padera TP, Dolmans DE', Carmeliet I', FukumuraD, Jain RK.Vascular endothelial growth factor (VEGF)-C differentially affects tumorvascular function and leukocyte recruitment: role of VEGF-receptor 2 and hostVEGF-A.Cancer Res. 2()01 Mar I 5;61 (6):2404-8. Karpanen T, et alVascular endothelial growth factor C
promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res. 2001 Mar 1;61 (5):1786-90: Baldwin ME, et al The Specificity of Receptor Binding by Vascular Endothelial Growth Factor-D Is Different in Mouse and Man. J Biol Chem. 2001 Jun I?76(22):19166-19171: Niki T, et al J
Pathol. 2001 Apr; 193(4):450-7: Veikkola T, et al Signallin~~ via vascular endothelial ~~rowth factor receptor-3 is sufficient forlymphangiogenesis in transgenic mice. EMBO J. 2001 Mar 15;20(6):1223-3 I Achen MG, et al Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis. J Pathol. 2001 Feb; 193(2):147-54 Stacker SA, et aIVEGF-D
promotes the metastatic spread of tumor cells via the lymphatics. Nat Med. 2001 Feb;7(2):186-91 Plate K. From an~~iogenesis to lymphangiogenesis. Nat Med. 2001 Feb;7(2):151-2. . Joukov V, et al; A novel vascular endothelial growth factor, VEGF-C. is a ligand for the FIt4 (VECiFR-3) and KDR (VEGI=R-2) receptor tyrosine kinases.EMBO J. 1996 Apr 1;15(7):1751. Lee J. et al. Proc Natl Acad Sci USA.
1996 Mar 5;93(5):1988-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 of multimodal therapy, subjects receive the multifunctional ligands of the present inventionand standard cancer chemotherapy. For example, "CVB" ( 1.5 ;~~m² cyclophosphamide, ?00-400 mg/mv etoposide, and I 50-200 mg/m- carmustinel is a regimen used to treat non-Hodgkin's lymphoma.
Patti et al., Eur. J. Haematol. 5 I :18 ( 1993). Other suitable combination chemotherapeutic regimens are well-known to those of skill in the art. See, for example, Freedman et al..
"Non-I lodgkin's Lymphomas," in Cancer Medicine, Volume 2, 3rd Edition. Holland et al. (eds.), pages 2038-2068 (Lea & Febiger 1993). As an illustration, first generation chemotherapeutic regimens for treatment of intermediate-grade non-Hodgkin's lymphoma include C-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). A useful second generation chemotherapeutic regimen is m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone and leucovorin), while a suitable third generation regimen is MACOP-B
(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin and leucovorin).
Additional useful drugs include phenyl butyrate and brostatin-1.
In general, the dosage of administered multifunctional ligands, immunoconjugates, and fusion proteins will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desir<rble to provide the recipient with a dosage of antibody component, immunocon,jugate or fusion protein which is generally at least in the range of from about I
pg/kg to 10 mg,~kg (amount of agent%body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate, particularly to take advantage of the depot effect of the invention.
Administration of the invention including, immunoconjugates or fusion proteins to a patient can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection.
When administering therapeutic proteins by injection. the administration may be by continuous infusion 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 of the circulation in rapidly distributing antibodies.
Intravenous administration, however, is subject to limitation by a vascular bawier comprising endothelial cells of the vasculature and the subendothelial 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, contributin~~ to effective antibody delivery. Intralymphatic routes of administratyion, such as subcutaneous or intramuscular injection, or by catherization of lymphatic vessels, also provide a useful means of treating lymphomas.
With regard to "low doses" of''' I-labeled immunoconjugates, the invention includes a dosage is in the range of 15 to 40 mCi, 20 to 30 mCi. In contrast, a preferred dosage of''° Y-labeled immunoconjugates is in the range from 10 to 30 mCi, while the more preferable range is 10 to 20 mCi.
Immunoconjugates having a boron addend-loaded carrier for thermal neutron activation therapy will normally be effected in similar ways. However, it will be advantageous to wait until non-targeted immunocon_jugate clears before neutron irradiation is performed. Clearance can be accelerated using an antibody that binds to the immunoconjugate. See U.S. fat. No. 4,624,84Ei for a 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. whereby 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 administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known to those in the art. See, for example, REMING'I"ON'S PHARMACELIT1CAL SCIENCES, 19th Ed. ( 1995).
For purposes of therapy. antibody components (or immunoconjugates/fusion proteins) and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount. A
combination of an antibody component, optionally with an immunoconjugate/fusion protein, and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An absent is physiologically significant if its presence 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.
Yet another therapeutic method included in the invention is a method of treating cancer by administering to an animal suffering from cancer a pharmaceutically effective amount of one or more multifunctional ligands capable of binding to cancer cells, wherein the compound is associated with a substance capable of damaging cancer cells.
Pharmaceutical compositions herein described or alluded to include multifunctional ligands of the invention or therapeutics used in combination therapy which may be administered by a variety of routes of adminstration.
By administration of an "effective amount" is intended an amount of the compound that is sufficient to enhance or inhibit a response, is some embodiments particularly an immune response or cellular response to a multifunctional ligand. One of ordinary skill will appreciate that efitective amounts of a multifunctional ligand can be determined empirically and may be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt, ester or prodrug form. The multifunctional ligand rnay 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 compositions of the present invention will be decided by the attending physician within the scope of sound medical jud~~ement. The specitic therapeutically etfective dose level for any particular patient will depend upon a variety of factors including the type and de<~ree of the cellular response to be achieved; activity of the specific multifunctional ligand employed: the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the agonist or antagonist; the duration of the treatment; drugs used in combination or coincidental with the specific monist or anta~xonist; and like factors well known in the medical arts.
On administration parenterally, for example by i.v. drip or infusion, dosages optionally at leasr on the order of from 0.01 to 5 mg/k~~'day. optionally 0.05 to 1.0 mg/kg/day and more preferably 0. I to 1.0 rn~= kg/day can be used. Suitable daily dosages for patients are thus on the order of from 2.5 to 500 mg p.o., optionally to 250 m~~ p.o., optionally 5 to 100 mj p.o., or on the order of from 0.5 to 250 mg i.v., optionally 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 the blood, as determined by the RIA technique. Thus patient dosaging may be adjusted to achieve regular on-going trough blood levels, as measured b_~ RIA, optionally on tyre order of at least from SO to 1000 n«'ml, preferably 150 to 500 n~~,'ml.
From above, pharmaceutical compositions are provided comprising an agonist or antagonist and a pharmaceutically acceptable carrier or excipient, which may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The teen "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular 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 reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers. diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like). carboxymethylceuulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper tluidity can be maintained, for example, by the use of coatings materials such as lecithin, by the maintenance ofthe 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, emulsifying 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 such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical fornn may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of one or therapeutic components herein described, it is desirable to slow the absorption from subcutaneous or intramuscular injection.
This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drub= then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of the drug in biode~~radable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled.
Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents 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 can also be administered in the form of liposomes.
As is known in the art, liposomes are generally derived from phospholipids or other lipid substances.
Liposomes are formed by mono- or multi-lameflar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in (iposome form can contain, in addition to the agonist or antagonist, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and the phosphatidyl choaes (lecithins). both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed.. Methods in Cell Biology, Volume XIV. Academic Press.
New York. N.Y. ( 1976).
p. 33 et seq.
The present invention also contemplates a method of treatment in which immunomodulators are administered to prevent, mitigate or reverse radiation-induced or drug-induced toxicity of normal cells, and especially hematopoietic cells. Adjunct immunomodulator therapy allows the administration of higher doses of cytotoxic agents due to increased tolerance of the recipient mammal.
Moreover, adjunct immunomodulator therapy can prevent, palliate, or reverse dose-limiting marrow toxicity. Examples of suitable immunomodulators for adjunct therapy include G-CSF. GM-CSF, thrombopoietin, IL-I, IL-3, IL-13, and the like. The method of adjunct immunomodulator therapy is disclosed by Goldenberg, U.S. Pat.
No. 5,120,525.
For example, recombinant IL-2 may be administered intravenously as a bolus at 6 x 10' IU!kg or as a continuous infusion at a dose of I 8 x 10G IU/m' /d. Weiss et al., J. Clin.
Oncol. 10:275 ( 1992).
Alternatively, recombinant 1l.-2 may be administered subcutaneously at a dose of 12 x 10'' 111. Vogelzang et al., J. Clin. Oncol. 1 1:1809 ( 1993). Moreover, INF-.'~amrna. may be administered subcutaneously at a dose of 1.5 x10' U. Lienard et al., J. Clin. Oncol. 10:52 ( 1992).
Furthermore, Nadeau et al., J. fharmacol.
Exp. Ther. 274:78 ( 1995), have shown that a single intravenous dose of recombinant 1L-12 (42.5 µg/kilogram) elevated IFN-.gamma. levels in rhesus monkeys.
Suitable IL-3 formulations include PROLEUK1N (Chiron Corp./Cetus Oncology Corp.; Emeryville, Calif) and TECELEUK1N (Hoffmann-La Roche. Inc.; Nutley, N.J.). ACTIMMUNE
(Genentech, Inc.;
South San Francisco, Calif.) is a suitable INF-.gamma. preparation.
In the precedinu detailed description, reference was made to various methodologies known to those of skill in the art of molecular biology and irnmunolo~;y. Publications and other materials setting forth such known methodologies to which reference was made or is made below are incorporated herein by reference in their entireties along with references cited therein as though set forth in full.
Standard reference works setting forth the general principles of recombinant DNA technology include Watson, J. D. et al. Molecular Biology of the Gene, Volumes I and II, the Benjamin/Cummings Publishing Company, lnc., publisher. Menlo Park. Calif ( 1987), Darnell. J. E. et al., Molecular Cell Biology.
Scientific American Books, Ine.. Publisher, New York. N.Y. (1986); I~t~win. B.
M. Genes Il, .lohn Wiley & Sons, publishers, New- York, N.Y. ( 1985); Old. R. W., et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering. 2d edition. University of California.
Press, publisher, Berkeley, Calif.
(1981); Maniatis. T., et al.. Molecular Cloning: A Laboratory Manual, 2nd Ed.
Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, N.Y. ( 1989), and Current Protocols in Molecular Biology, Ausubel et al.. Wiley Press, New York, N.Y. ( 1989). Standard reference works setting forth general principles and techniques of immunology include Handbook of Experimental Immunology Blackwell Science, Incorporated, ISBN:0632009756; Antibody I~ngineering Black well Science. Incorporated, ISBN:0632009756; Therapeutic Immunology ISBN: 086542375X Blac:kwell Science.
Incorporated ;
Encyclopedia of Immunology (1998) Morgan Kaufmann Publishers, ISBN:0122267656;
Immunology Mosby, Incorporated, ISBN:0723429189; Abbas AK. et al. Cellular & Molecular Immunology 4'~' Ed.
2000 ISBN 0721650026: Breitling F. et al. Recombinant Antibodies 1999 ISBN 0-471-17847-0;
Masseyeff R. et al. Methods of Immunological Analysis Wiley-VCI-i ISBN 3-527-27906-7, 1992;
Mountain et al. Eds, Biotechnology 2°'' ed. Vol 5A 1998 ISBN 3-527-28315-3 Wiley-VCIi; Campbell, A. , "Monoclonal Antibody Technology," in, Burdon, R., et al., eds, Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13, Elsevier, Publisher, 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 various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention.
All publications referred to herein are indicative of the level of skill of those in the art to which the invention pertains. All publications are herein (as well as references cited therein)are incorporated by reference to the same extent as if each individual publications were specifically and individdually indicated to be incorporated by reference in its entirety.
The present invention, thus generally described, will be understood more readily by reference to the preceding and following examples, which are provided by way of illustr<rtion and are not intended to be limiting of the present invention.
With respect to making applications, methods of using. target cell-associated markers etc. of multispecific ligands and bispecitic antibodies see also ,~-Tethocls Wol l3iol 2001; 166: t 77-92; USP 6.071,517; USP
5,897,861; USP 6,096.31 l; USP 5,922,845. Journal of Immunological Methods February 2001 Vol.
248(1-2) page 1-200; Mol Immunol 1999 May; 36(7):433-45; US6051227 US06143297 US05977318 US05968510 US05885796 US05885579 US05869050 Methods of blockinU T-cell activation using anti-B7 monoclonal antibodies: US05851795 US05747034 US061 B7-1 targeted ribozvmes; US05844095 US06090914 US05718883 Trans~~enic animal model for autoimmune diseases; US05855887 US0581 1097 US05770197 Methods for regulating the immune response using B7 binding molecules and IL4-binding molecules; US06084067 EP01073741 A2 US06130316 US06068984 Antibodies to lymphocyte activation antigens uses therefor;
US05766570 Lymphocyte activation anti~~ens and thereto; US05434131 US05316920 Lymphocyte activation antigen HB 15, a member of the immunoglobulin superfamily; US061 1 1090 Mammalian cell surface antigens: US0608375 I Chirneric receptors for the generation of selectively-activatable TH-independent cytotoxic T cells;
US05977303 Mammalian cell surface antigens; US05738852 Methods of enhancing antigen-specific T
cell responses US05714667 Bruhl H, et alDepletion of CCRS-Expressing Cells with Bispecific Antibodies and Chemokine Toxins: A
New Strategy in the Treatment of Chronic Intlammatory Diseases and 1-IfV. J
Innnunol. 2001 Feb 15;
166(4):2420-2426; Tesch H, et alTreatment of patients with malignant lymphomas with monoclonal antibodies. Bone Marrow Transplant. 2000 May: 26 Suppl 2:S50-3; Jung G, Brandl M, Eisner W.
Fraunberger P, Reifenber;;er G.Schlegel U, Wiestlcr OD. Reulen HJ_ Wilmanns W.
local imrnunotherapy of glioma patients with a combination of 2 bispecific antibody fragments and restin<~ autolo~~ous lymphocytes: Evidence For in situ t-cell activation and therapeutic efficacy.
Int J Cancer. 2001 Jan 15;
91(2):225-230; Kan KS, Anderson VA, Leong WS, Smith AM, Worth .AT, Stevenson G7 . Thioether-Bonded Constructs of Fab'gamma and Fcgamma Modules Utilizing Differential Reduction of Interchain Disulfide Bonds J Innnunol. 2001 Jan 15: 166(2):1320-1326: Cao Y, Suresh MR.
Bispecific MAb aided liposomal drug delivery. J Drug Target. 2000; 8(4):257-66; Schoonjans R, et al Mertens N. Fab chains As an efficient heterodimerization scaffold for the production of recombinant bispecific and trispecific antibody derivatives J Immunol. 2000 Dec 15; 165(12):7050-7. ; Schmic;dl A, et al Expression of a bispecific dsFv-dsFv' antibody tragment in escherichia coli Protein Eng. 2000 Oct; 13( 10):725-34.
Rohrbach F, et al Construction and characterization of bispecific costimulatory molecules containing a minimized CD86 (B7-2) domain and single-chain antibody fragments for tumor targetin~~ Clin Cancer Res. 2000 Nov; 6( I 1 ):4 314-22. Schoonjans R, Efficient heterodimerization of recombinant bi- and trispecitic antibodies Bioseparation. 2000; 9(3):179-83. Wallace PK, etal Production of macrophage-activated killer cells for targeting of glioblastoma cells with bispecific antibody to Fc~~amrnaRl and the epidermal growth factor receptor Cancer Immunol Immunother. 2000 TJov;
49(9):493-503.Bioconjug Chem. 2000 Nov; I I (6):842-54. Conrath KE. et al Camel single-domain antibodies as modular building units in. J Biol Chem. 2000 Oct 25 [Cancer Immunol Immunother. 20(10 Oct;
49(8):441-8. Talac R, Nelson H. Current perspectives of bispecitic antibody-based immunotherapy J
Biol Regul Homeost Agents. 2000 Jul; 14(3):175-81. Tomlinson I, Holliger P. Methods for generating multivalent and bispecific antibody fragments Methods Enzymol. 20()0; 326:461-79 Morimoto K, Inouye K. Application of bispecific F(ab') fragments prepared from IgMs a~~ainst carcinoembryonic antigen and alkaline phosphatase Clin Chem. 2000 Sep: 46(9):1492-3. Cochlovius B, et al Cure of Burkitt's lymphoma in severe combined immunodeficiency mice by T cells, tetravalent CD3 >; CD19 tandem diabody, and CD28 costimulation. Cancer Res. 2000 Aug 15; 60( 16):4336-41. Takemura S.i, et al.
Construction of a diabody (small recombinant bispecific antibody) using a refoldin~~ system Protein Eng.
?000 Au«: 13(8):583-8. y Katzenwadel A, et alConstruction and in viva evaluation of an anti-PSA a anti-CD 3 bispecific antibody for the immunotherapy of prostate cancer. Anticancer Res. 2000 May-.lun;
20(3A): I 551-5. Yoshida H, Katayose Y. Suzuki M. Matsuno S. Kudo 'f, Kumagai 1. Functional Fv fragment of an antibody specific for CD28: Fv-mediated co-stimulation of T cells. FEBS Lett. 2000 Ju,l 7: 476( 3):266-71. 6 38: Zeidler R, et The Fc-region of a new class of intact bispecific antibody mediates activation of accessory cells and NK
cells and induces direct phagocytosis of tumour cells. Br J Cancer. 2000 Jul:
83(2):261-6. 0 39: Hillairet et al Enhanced targeting specificity to tumor cells by simultaneous recognition of two antigens.
Bioconjug Chem. 2000 Jul-Au~7; I 1 (4):452-60. Weiner LM. Bispecitic antibodies in cancer therapy.
Cancer J Sci Am. 2000 May: 6 Suppl 3:S265-71 Zangemeister-Wittke U. Leech SH.
Olie RA, Zuo Z. et al An efficient route to the production of an IgG-like bispecific antibody.
Protein En~~. 2000 May; 13(5):361-7 Helfrich W, et al A rapid and versatile method for harnessing scFv amibody fragments with various biological effector functions. J Immunol Methods. 2000 Apr 3: 237(1-2):131-45 Lu D, et al Acquired antagonistic activity of a bispecific diabody directed against two different epitopes on vascular endothelial growth factor receptor 2. J Immunol Methods. 1999 Nov 19; 230(1-2):159-71.
Ohmi Y, et al Tumor-specific targeting of'h helper type 1 (Th I ) cells by anti-CD3 x anti-c-ErbB-2 bispecific antibody. Cancer Immunol Immunother. 1999 Nov; 48(8):456-62.
Kipriyanov SM, et al Ci. Schuhmacher J, Cochlovius B. Von der Lieth CW, Matys ER, Little M.
Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics. J
Mol Biol. 1999 Oct 15: 393(1):41-56.Segal DM, Weiner GJ, Weiner LM. Bispecific antibodies in cancer therapy. Curr Opin Immunol. 1999 Oct; I I (5):558-62 4 88: Hudson I'J.
Recombinant antibody constructs in cancer therapy. Curr Opin Immunol. 1999 Oct: 1 1 (5):548-57 Alt M, et al Novel tetravalent and bispecific IgG-like antibody molecules combining single-chain diabodies with the immunoglobulin gammal Fc or CH3 region. FEBS L.ett. 1999 Jul 2; 454(1-2):90-4.
Kontermann RE. Muller R. Intracellular and cell surface displayed sin~~le-chain diabodies. .I Immunol Methods. 1999 Jun 34; 226(1-2):179-88.
Morimoto K, Inouye K. Method for the preparation of bispecitic F(ab')2mu fragments from mouse monoclonal antibodies of the immunoglobulin M class and characterization of the ti~agments. J Immunol Methods. 1999 Apr22: 224(1-3):43-50.
Chaudri ZN, et al Dual specificity antibodies using a double-stranded oligonucleotide bridge. FEBS
Lett. 1999 Apr 30; 450( I-2):23-6. : Somasundaram C, et al Development of a trispecific antibody conjugate that directs two distinct tumor-associated antigens to CD64 on myeloid effector cells. Hum Antibodies. 1999; 9( I ):47-54.
Robert B, et al Tumor targeting with newly designed biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen (CEA). Int J Cancer. f 999 Apr 12: 81 (2):285-91.
Kreutz FT, et al Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography. J Chromatogr B Biomed Sci Appl. 1998 Sep 4; 714(2):161-70.
Muller KM, et al A dimeric bispecific miniantibody combines two specificities with avidity. FEBS
Lett. 1998 Ju1 31; 432(1-2):45-9. Kreutz FT, et al A new method to f;enerate quadromas by electrofusion and FACS sorting. Hvbridoma. 1998 Jun: 17(3):267-73.

Production of bispecific and trispecific F(ab)2 and F(ab)3 antibody derivatives. Methods Mol Biol. 1998;
80:121-34. Merchant AM, et Protein, Nucleotide An efficient route to human bispecific IgG. Nat Biotechnol. 1998 Jul: 16(7):677-81.
Zhu Z, et al High level secretion of a humanized bispecitic diabody from Escherichia coli.
Biotechnology (N Y). 1996 Feb; 14(2):192-6. Rapid assay of phage-derived recombinant human fobs as bispecific antibodies. Biotechnology (N Y). 1995 Nov; 13( 1 1 ):1321-4.
McGuinness BT, et Phage diabody repertoires for selection of large numbers of bispecific antibody fragments. Nat Biotechnol.
1996 Sep; 14(9):1 149-54Helfrich W, et al Construction and characterization of a bispecitic diabody for retargeting:,= T cells to human carcinomas. Int J Cancer. 1998 Apr 13;
76(2):232-9. Mullet KM, et al The first constant domain CH I and CL of an antibody used as heterodimerization domain for bispecific miniantibodies. FEES Lets. 1998 Jan 30; 422(2):259-64. 223: FitzGerald K, et al Improved tumour targeting by disulphide stabilized diabodies expressed in Pichia pastoris.
Protein Eng. 1997 Oct;
l 0( I 0):1221-5.
Coloma MJ, Morrison SL_. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. 1997 Feb; 15(2):159-63. Hoogenboom HR, Mix and match: building manifold binding sites.
Nat Biotechnol. 1997 Feb; 15(2):125-6.
Ridgway JB, et al 'Knobs-into-holes' en<~ineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 1996 Jul; 9(7):617-21.
Reinartz HW, et al Bispecific multivalent antibody studied by real-time interaction analysis for the development of an antigen-inhibition enzyme-linked immunosorbent assay.
Analyst. 1996 Jun;
121(6):767-71. Tibben JG, et al Pharmacokinetics, biodistribution and biological eflecis of intravenously administered bispecific monoclonal antibody OC'TR F(ab')? in ovarian carcinoma patients.
Int J Cancer. 1996 May 16: 66('4):477-83.
de Kruif J, Logtenberg T. Leucine zipper dimerized bivalent and bispecific seFv antibodies fi-om a semi-synthetic antibody phage display library. J Biol Chem. 1996 Mar 39; 271( 13):7Ei30-4.
Ruf J, et al The molecular recognition theory applied to bispecific antibodies... Nat Med. 1995 Dec:
1 ( 12):1222.
447: Grant SD, et al Expression of monovalent and bivalent antibody fragments in facherichia coli. J
Hematother. 1995 Oct; 4(5):383-8.
Dubel S, et al Protein, Nucleotide Bifunctional and multimeric complexes of streptavidin fused to single chain antibodies (scFv). J Immunol Methods. 1995 Jan 27; 178(2):201-~>.
Kostelny SA. et al Protein. Nucleotide Formation of a bispecific antibody by the use of leucine zippers.
J Immunol. 1992 Mar I; 148(5):1547-53 Pack P, Pluckthun A. Miniantibodies: use ofamphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli.
Biochemistry. 1992 Feb 18; 31(6):1579-84 Smith W, et allmmunoglobulins secreted by a hybrid-hybridorna: analysis of chain assemblies, hlybridoma. I 992 Feb: I 1 ( 1 ):87-98.
Suresh MR.et al Bispecific monoclonal antibodies from hybrid hybridomas.
Methods Enzymol. 1986;
121:210-28. Paulus H. Preparation and biomedical applications of bispecific antibodies. Behring Inst Mitt. 1985 Dec; (78):1 18-32. Brennan M, et al Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G I fragments. Science. 1985 Jul 5;
229(4708):81-3.

With respect to anti-CCRS antibodies used to kill CCRS-expressing cells, with for example. bi-specific antibody chemokine fusions see Bruhl H. et al. J Imrnunol. 2001 Feb 15 166(4):
2420-2426.
With respect to targeting IKAP proteins see for example US 6172195.
With respect to pertinent diseased cells, disease causing cells and other suitable targets for immunotoxins, as well as optional toxins and methods of making and using immunotoxins and related technolo~~ies see for example US05980895 Immunotoxin containing a disulfide-stabilized antibody fragment joined to a Pseudomonas exotoxin that does not require proteolytic activation: US05686072 Epitope-specific monoclonal antibodies and immunotoxins and uses thereof: US04956453 Antihuman ovarian cancer immunotoxins and methods of thereof; US0614663 I Innnunotoxins comprising ribosome-inactivating proteins; US05756699 Imrnunotoxins comprisin;~ ribosome-inactivating Proteins;

Immunotoxins comprising ribosome-inactivating Proteins; US06146850 Proteins encoding gelonin sequences; US05837491 Polynucleotides encoding gelonin sequences; LJS05578706 Methods and compositions concerning homogenous immunotoxin preparations; US05185434 Prolony~ed-action immunotoxins containin~~ a glycopeptide constituent which inactivates rih~osomes, modified on its polysaccharide units; US04958009 Anti-human ovarian cancer immunotoxins and methods of use thereof; US05980896 Antibodies reactive with human carcinomas; US06074644 Nucleic acids encoding immunotoxins containing a disulfide-stabilized antibody fragment replacing half or more of domain IB of pseudomonas exotoxin. and methods of use of the encoded immunotoxins;
US04981953 Immunotoxins, process for their preparation and pharmaceutical compositions in which they are present; US04980457 Cytotoxic conjugates which can be used in therapy and process or their preparation; US04545985 Pseudomonas exotoxin conjugate immunotoxins;
US06020145 Methods for determining the presence of carcinoma using the antigen binding region of monoclonal antibody BR96;
US05792458 Mutant diphtheria toxin conjugates: US05338542 US06051230 Compositions for targeting the vasculature of solid tumors; B3 antibody fusion proteins and their uses; US05990275 Linker and linked fusion polypeptides; US05981726 Chimeric and mutationally stabilized tumor-specific Bl, B3 and B5 antibody fragments; in ~munotoxic fusion proteins;
and uses thereof: US05965132 Methods and compositions for targetinf; the vasculature of solid tumors:
US05889157 Humanized B3 antibody fragments, fusion proteins, and uses thereof Multivalent antigen-binding proteins;

FRAGMENTUS05776427 Methods for targetinV~ the vasculature of solid tumors;

Monoclonal antibodies directed to the HER2 receptorUS05665357 Antibodies recognizin~~ tumor associated antigen CA 55.1; US05660827 Friedrich K, et al A two-step selection approach for the identification of li'~and-binding determinants in cytokine receptors.Anal Biochem. 1999 Mar 15; 268(2):179-86.
Krebs B, et al Recombinant human single chain Fv antibodies recognizin~~
humaninterleukin-6. Specific targeting of cytokine-secreting cells. J Biol Chem. 1998 Jan 30; 273(5):2858-65 Wilbur DS,et alRelatedArticles Biotin reagents for antibody pretargeting. 2. Synthesis and in vitroevaluation of biotin dimers and trimers for cross-linkin« ofstreptavidin. Bioconjug Chem. 1907 Nov-Dec; 8(6):819-32.
Ring DB. et alAntigen forks: bispecific reagents that inhibit cell growth by bindingselected pairs of tumor antigens. Cancer Immunol Immunother. 1994 Jul; 39( l ):41-8.
W009942597A1 MONOVALENT, MULTIVALENT, AND MULTIMERIC MHCB1NDING DOMAIN
FUSION PROTEINS AND CONJUGATES, AND USES THEREFOR; EP00935607A2 SOLUBLE
MONOVALENT AND MULTIVALENT MHC CLASSII FUSION PROTEINS, AND USES
THEREFOR;

W009811914A1 TARGETING ANTIGENS TO THE MHC CLASS IPKOCESSING PATHw'AY WITH
ANTHRAX TOXIN FUSION PROTEINW009728191A1 MHC COMPLEXES AND USES THEREOF;
US05580756 B71G fusion protein; US06143298 Soluble truncated forms of ICAM-l;
US05852175 P-selectin glycoprotein ligand blocking antibodiesLJS05800815Antibodies to P-selectin and their uses;
US06037454 Humanized anti-CD1 la antibodies; US06020152 Lymphocyte-associated cell surface proteinUS05807734 Monoclonal antibodies and FV specitic for CD2antigenUS05622701 Cross-reacting monoclonal antibodies specificfor E- and P-selectin US05622700 Method for treating a LFA-1-mediated disorder; JP06209'788A2 IMMUNOASSAY OF
HUMAN SOLUBLE ICAM-1. ANTIBODYAND KIT FOR MEASUREMENT THEREOF;
JP03072430A2 ANTIV1RAL AGENT BY USING FU'~ICTIONAI_DERIVATIVE OF INTERCELLULAR
ADHESIVE MOLECULE; JP01135724A2 TREATMENT FOR NONSPECIFIC INFLAMMATION;
US06123915 Methods for using agents that bind to VCAM-l; W009929706A2 DISAt_1CYLATE
ANALOG BASED SIAI_YL LEWISxMIME~I~ICS; W'009918442A1 DIAGNOSIS OF'THROMBOTIC
EVENTS BY DETEC'TINGP-SEL.ECTIN; US05877295 Antibodies which bind a subpopulation of Mac-1(CDI lb/CD18) molecules which mediate neutrophil adhesion to ICAM-land fibrinogen; LJS05869460 Sulfated and phosphated saccharide derivatives,process for the preparation of the same and use thereof;
US05858994 Carbohydrate conjugates as inhibitors of celladhesion; US0581 1405 Multiply fu cosylated dicarboxylic acidspossessing antiadhesive properties; US05654282 Selectin binding glycopeptides;
US05632991 Antibodies specitic for E-selectin and the usesthereof; US05599676 Method for isolating a novel receptor for.alpha.4 integrins: US05580862 Sulfate ligands for L,-selectins and methods ofpreventing sulfate addition: US05508387 Selectin binding glycopeptides;
US06177547 Antibodies to P-selectin glycoprotein ligandUS05827670 Methods of isolating and detecting bone marrow stromal cells with VCAM-1-specific antibodies; US05756095 Antibodies with specificity for a common epitope on E-selectin and L-selectin; US05565550 Antibodies to ICAM-3, and fragments thereof;
US06099838 Pharmaceutical compositions comprising anti-CD45RB antibodies for the inhibition of 1-cell mediated immune responses;
US05595737 Methods for using monoclonal antibodies specific for cell-surface bound LAM-1;
US05324510, US06183988 Leukocyte-specific protein and gene, andmethods of use thereof;

US05997865 Agonist antibodies against the tlk2/flt3 receptor and uses thereof;
1JS05993816 Methods to inhibit humoral immune responses, immunoglobulin production and B cell activation with 5c8-specific antibodies:
US05869453 Cytotoxic T-cell epitopes; US05861 15l Soluble fusion molecules with binding specificity for cell adhesion molecules; US05843441 Use of endothelial-leukocyte adhesion molecule-1 specific antibodies in the treatment of asthmaUS05821332 Receptor on the surface of activated CD4+ T-cells:
ACT-4; EP00868197A I ANTI-SELECTIN ANTIBODIES FOR PREVENTION OFMUL,T11'1_E
ORCi.AN
FAILURE AND ACUTE ORGAN DAMAGE; US05817515 Human B2 integrin alpha subunir antibodies;
EP00528931B1 HUMANIZED CHIMERIC ANTI-ICAM-1 ANTIBODIES, METHODS OF
PREPARATION AND USE; US05776775 Anti-LAM 1-3 antibody and hybridoma;
US06063906 Antibodies to integrin alpha subunit; US05997865 Agonist antibodies against the flk2/flt3 receptor and uses thereof; US05993816 Methods to inhibit humoral immune responses, immunoglobulin production and B cell activation with 5c8-specific antibodiesUS059519fi2 Methods to suppress an immune response with variant CD44-specific antibodiesUS05843441 Use of endothelial-leukocyte adhesion molecule-1 specific antibodies in the treatment of asthma US05821332 Receptor on the surface of activated CD4+T-cells: ACT-4; US05821 123 Modified antibody variable domains;

ANTI-SELECTIN ANTIBODIES FOR PREVENTION OF MULTIPLE ORGAN FAILURE .AND
ACUTE ORGAN DAMAGE; US05817515 Human B2 integrin alpha subunit antibodies;
EF'00528931 B I
HUMANIZED CHIMERIC ANTI-(CAM-l ANTIBODIES, ME'T'HODS OF PREPARATION AND USE;
US05776775 Anti-LAM I-3 antibody and hybridoma; US05776755 FI_T4, a receptor tyrosine kinase;
US05730978 Inhibition of lymphocyte adherence with alpha.4&tt2231; -specific antibodies:
Examples of tumor specif7c antigens are numerous and are referred to in the hereinabove cited references andas well as the in the following referencesUS0613298010/17/2000 Antibodies specific for TRP-2 a human tumor antigen recognized by cytotoxic T lymphocytes US06165464Monoclonal antibodies directed to the HE:R2 receptor US0582431 (Treatment of tumors with monoclonal antibodiesagainst oncogene antigens.
US0614005010/31/2000 Methods for determining breast cancer and melanoma by assaying for a plurality of antigens associated herewith; US06051226 MN-spexific antibodies and their use in cancer treatment;
US06020145Methods for determining the presence ofcarcinoma using the antigen binding region of monoclonal antibody BR96.; US05980896 Antibodies reactive with human carcinomasUS05955075Method of inhibitin«, tumor growth using antibodies to MN
protein US05917124Transgenic mouse model of prostate cancer; US0591438906,'22/1999 E6 associated proteinUS0591214306;' 15i i 999 Polynucleotides encodin<~ a human ma<~e proteinhomologUS0591062606,''08..% 1999 Acetyl-CoA carboxylase compositions and methodsof useUS0587456002/23/1999 Melanoma antigens and their use in diagnostic and therapeutic methodsUS058722170' 16/1999 Antibodies which specifically bind a cancerrelated antigenUS0586963602:09/1999 Immunoreactive peptide sequence from a 43 kDhuman cancer antigenUS0586904502(19;1999 Antibody conjugates reactive with human carcinomas US0586612402/02/1999 Antiidiotypic antibodies for high molecularweight-melanoma associated by sameUS0584708312/08'1998 Modified p53 US05844075 Melanoma antigens and their use in diagnosticand therapeutic methods US0584368512/O l / 1998 Production of chimeric mouse-human antibodies with specificity to human tumor antigensUS05843648 Pl _5 and tyrosinase melanoma antigens and their use in diagnostic and therapeutic methods US05840854US05R~304701 1/03/1998 Humanized antibodies to ganglioside GM2US0583046411/03/ 1998 US0580800SHuman carcinoma Bispecific molecules reco~~nizin<g lymphocyte anti~~en CD2 and tumor anti'~ensUS05792456 Mutant BR96 antibodies reactive with humancarcinomas US0578368US05773579 Lung cancer marker US05772997 Monoclonal antibodies directed to the HER2 receptor 1JS05770374; US05705157 Methods of treating cancerous cells with anti-receptor antibodies US0569599412'09/1997 Isolated cytolytic T cells specific forcomplexes of MACE related peptides and HL.A moleculesUS0569376312021997 .Antibodies to human carcinoma antigen Tumor rejection antigens which correspond toamino acid sequences in tumor rejection antigen precursor ba~~e, and uses thereof US05681701 Immortalized human fetal osteoblastic ceIIsUSOS68156210;'28: 1997 US05(i7717110.'I:L,'1997 Monoclonal antibodies directed to the FlER2receptorlJS0567448610/07~1997US0566535709/09!1997 Antibodies recognizing tumor associated antigen CA 55.1 Fonsatti E, et al En~er~~in~~ role of protectin (CD59) in humoral irnmunotherapy of solid malignancies.Clin Ter. 2000 May-Jun; 151(3):187-93 Knuth A, etalCancer immunotherapy in clinical oncology. Cancer Chemother Pharmacol. 2000; 46 SuppI:S46-51 : Sievers EL.Targeted therapy of acute myeloid leukemia with monoclonal antibodies andimmunoconjugates. Cancer Chemother Pharmacol.
2000; 46 SuppI:Sl8-22 .172 van Spriel AB,et allnnnunotherapeutic perspective for bispecific antibodies.lmmunol Today. 2000 Aug; 21(8):391-7 273: Green MC, et alMonoclonal antibody therapy fur solid tumors. Cancer Treat Rev.
2000 Aug; 26(4):269-86 Xiang J Targeting cytokines to tumors to induce active antitumor immune responses by recombinant fusion proteins. Hum Antibodies. 1999; 9( 1 ):23-Engberg J, et al Recombinant antibodies with the antigen-specific, MHC restricted specificity of T cells: novel reagents for basic and clinical investigations and immunotherapy. Immunotechnology. 1999 Mar; 4(3-4):373-8. O'Brien ~~J. 'fet alMore than I 5 years of CA
125: what is known about the antigen, its structure andits function.lnt J Biol Markers. 1998 Oct-Dec;
13(4):188-95.
Shariti J, et allmprovin~T monoclonal antibody pharmacokinetics via chemical modification.Q J Nucl Med.
1998 Dec: 42(4): 242-9.
Ligands on immune or other cells which may be targeted with bispecific ligands in which one ligand of the pair dictates specificity for a population of cells or particular sub-population of those cells and a second ligand with reduced functional affinity is used to effect a specific immune function include those referenced in the following patents and publications therein referenced: US06132992 Expression vectors encoding bispecific fusionproteins and methods of producing biologically active h~ispecitrc fusion proteins in a mammalian cell: Antibody heteroconjugates and bispecificantibodies for use in regulation of lymphocyte activity;

COMPOSITIONS AND METHODS FOR REGULATINGLYMPHOCYTE ACTIVATION;
US059165600 Methods For inhibiting an immune response byblocking the GP39/CD40 and CTLA4/CD28iB7 pathways and compositionsfor use therewith; US058~76718 Methods of inducing T cell non-responsiveness to transplanted tissues and of treating graft-versus-host-disease with anti-gp39 antibodiesEP00445228BIMMUNO~fI-iERAPY INVOLVING CD28 STIMULATION;
US05709859 Mixed specificity fusion proteins; US05637481 Expression vectors encoding bispecific fusion proteins and methods of producing biologically active bispecific fusion proteins in a mammalian cell; W009720048 MODIFIED SFV MOLECULES WHICH MEDIATE ADHESIONBETWEEN CELLS
AND USES THEREOF;
EP00336379 Antibody heteroconjugates for use in regulation of lymphocyte activity;
EP00537293 LIGAND FOR CD28 RECEPTOR ON B CELLS ANDMETHODS; US05182368 I_igands and methods for augmenting B-cell proliferation; W009300431 CTL4A RECEPTOR.
FUSION
PROTEINS CONTAINING ITAND USES THEREOF; EP00445228 IMMUNOTHERAPY INVOLVING
CD28 STIMULATION:
Role of cellular adhesion molecules in HIV type 1 infection and their impact onvirus neutralization.
AIDS Res Hum Retroviruses. 1998 Oct; 14 Suppl 3:S247-54 Cavenagh JD,et al Adhesion molecules in clinical medicine. Crit Rev Clin Lab Sci. 1998 Sep: 35(5):415-59 Vine:y JL, Fong S. Beta 7 integrins and their ligands in lymphocyte mi~~ration to the gut. Chem Inununol. 1998; 71:64-76 Aplin AE, et alSi~~nal transduction and signal modulation by cell adhesion receptors: the roleof integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins. Pharmacol Rev. 1998 Jun; 50(2):197-263 With respect to ascertaining important amino acid residues for receptor activation or binding see also Zang, Q., Sprin~~er. T. A. (2001 ). Amino Acid Residues in the PSI Domain and Cysteine-rich Repeats of the Integrin beta 2 Subunit That Restrain Activation of the Integrin alpha xbeta 3. .l. Biol. C'hena. :?76: 6922-6929; Bir~etinh site on the murine IFN-gamma reevptarr for IFN-gamma has been identifiied using the synthetic peptide approach,The Journal of Immunolo~~y, Vol 15 I. Issue 1 1 6206-6313; The Journal of Immunology, Vol 143, Issue 1 1 3568-3579'fhe main immunogenic region of the nicotinic acetylcholine receptor. Identification of am ino acid residues interactin~~ with different antibodies, M Bellone et al.;
Arend, W. P., Malyak, M., Guthridge. C. J., Gabay, C. (1998). INTERLEUKIN-1 RECf_P'fOR
ANTAGONIS'h: Role in Biology. .=lnnu. Rev. lmmtsnol. 16: 27-55: The Journal of Immunology, Vol 155, Issue 10 4719-4725, Mapping of receptor binding sites on IL-1 beta by reconstruction of IL- Ira-like domains; The Jourrmrl o/ lrrmnnoloy, 2000. 165: 6966-6974 Identification of Fetal Liver Tyrosine Kinase 3 (FIt3) Ligand Domain Required for fZeceptor Binding and Function Using Naturally Occurring Ligand Isoforms Waithaka Mwangi'', Wendy C. Brown and Guy' H. Palmer.
The invention also contemplates multifunctional li~~ands comprising various combinations and permutations of such li,~ands including pairs and three different such ligands including multifunctional ligands including such combinations and a li~~and which binding to a lymphatic vessel associated Iigand.Additional pertinent references pertaining to formation of antibody dimers, microarrays of (and tissue microarrays) proteins including heterohunctional proteins and recombinant, ligands having application to the invention, and phage or ribosome display strate~~ies havin<~ relevance herein include Zhu H. et al. Protein arrays and microarrays.Curr Opin Chem Biol. 2001 Feb:
5(1):40-5, reterences in IBC's conference on Protein Microarray Technology March 19-21 Santiago California;
WO 99/06834, WO
99/19506; WO 97!02343, WO 00/63701; WO 99%40434 ; US 6,127,127; US6146830, WO
00!075298, US6165709 US0620402303!20i2001 Modular assembly of antibody genes, antibodies prepared thereby and use; US0584681812;08%1998 Pectate lyase si~~nal sequence; US0569843513/16;
1997 Modular assembly of antibody genes, antibodies prepared thereby and use; US0569841712/ Ifiil997 Modular assembly of antibody genes, antibodies prepared thereby and use: US0569349312/02!1997 Modular assembly of antibody genes, antibodies prepared thereby and use; US055 t454805/07i 1996 Method for in viva selection of ligand-binding proteins; US0564823707;'15/1997 Expression of functional antibody fragments;
US0618034 In vitro scanning saturation mutanenesis of proteins; US06027933 Surface expression libraries of heteromeric receptor; US0591057306i08i 1999 Monomeric and dimeric antibody-fragment fusion proteins; US061505831 1 Transgenic animals expressing artificial epitope-tagged proteins; US06132992 Expression vectors encoding bispecific fusion proteins and methods of producin<,~ biolo~~ically active bispecific fusion proteins in a mammalian cell; US06127524Binding molecules and computer-based methods of increasing the binding affinity thereof;; US06071515 Dirner and multimer forms of single chain polypeptides_ L1S06054297 Humanized antibodies and methods for makingthem;

OF PROTEINS AND METHODS OF USE THEREOF; US06008023 C'ytoplasmic expression of antibodies. antibody fra~~ments and antibody fragment fusion proteins in E.
coli: Pha'.:emid for antibody screening; US059808951 1!09/1999 Immunotoxin containing a disulfide-stabilized antibody fragment joined to a Pseudomonas exotoxin that does not require proteolytic activation:
IJS05962255 Methods for producin~~ recombinant vectors; US05955341 Ileterodimeric receptor libraries using phagemids;
W009939210A1 HIGLi DENSITY ARRAYS FOR PRO ~EOME ANALYSIS AND ME~I LiODS AND
COMPOSITIONS THEREFOR: W009931267 METHODS FOR THE SIMULTANEOUS
IDENTIFICA hIONOF NOVEL. BIOLOGICAL TARGETS AND LEAD STRUCTURES FOR
DRUGDEVELOPMEN f; US05869619 Modified antibody variable domains;
US058558851solation and production of catalytic antibodies using phage technology; US05851801 12/22;
1998 Method of preparing polypeptide binding compositions derived from immuno~~lobulin variable regions; US05849500 Phagemid for antibody screenin~~;binding composition; US058378461 1/17/1998 Biosynthetic binding proteins for immuno-targeting; US0582133710/13% 1998 lmmunoglobulin variants: IJS05821 123Modilied antibody variable domains; US0578965508/04/ 1998 'hransgenic animals expressin~~
artificialepitope-tagged proteins; US05783384 Selection of bindinV~-molecules; US05780225 Method for generating libaries of antibodygenes comprising amplification of diverse antibody DNAs and methodsfor using these libraries for the production of diverge antigen combinin~~ molecules; US05770356 Phagemids coexpressing a surface receptor and a surface heterologous protein; WO09808603 ISOLATION OF
1MMUNOGI_OBULINS:
US05716805 Methods of preparing soluble, oli~omeric proteins: US05;s95898 Modular assembly of antibody genes, antibodiesprepared thereby and use; US05582996 Bifunctional antibodies and method of preparing same; US05580717Recombinant library screenings methods; Haab BB.
Dunham MJ, Brown PO
.Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions.Genome Biol. 2001; 2(2):: Moch 1-l. Kononen T, Kallioniemi OP, Sauter G. Tissue m i c roarrays Borrebaeck CA.Antibodies in diagnostics - from immunoassays to protein chips.Immunol Today. 2000 Aug: 21 (8):379-82 Mendoza LG, et all-ligh-throughput microarray-based enzyme-linked imrnunosorbent assay (EL,ISA).
Biotechniques. 1999 Oct; 27(4):778-80. 782-6, 788. Morozov VN, Morozova TYa Electrospray deposition as a method for mass fabrication of mono- andmulticomponent microarravs of biological and biologically active substances.Anal Chem. 1999 Aug 1; 71 ( 15):3 I l0-7.619: Lucking A, et alProtein microarrays for gene expression and antibody screening. Anal Biochem. 1999 May 15; 270( 1 ):103-1 I . Silzel JW, et alMass-sensing, multianalyte microarray immunoassay with imaging detection.
Clin Chem. 1998 Sep;
44(9):2036-43 Ekins RP.Ligand assays: from electrophoresis to miniaturized microarrays.Clin C.'hem. 1998 Sep; 44(9):2015-30.
All publications referred to herein are indicative of the level of skill of those in the art to which the invention pertains.
With respect to lymphatic vessel associated li~~ands see also US 5, 776,755 (17t4), Mod Pathol 2000 Feb; 13(2):180-5; EMBO J 2001 Mar 15;20(6):1223-1231, Nat Med 2001 Feb;7(2):199-205 Inhibition of lymphangiogenesis with resulting lymphedema in trans~~enic mice expressing soluble VEGF receptor-3 (VEGFR-3). J Pathol 2001 Feb; 193(2):147-54 Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angio~~enesis.
With respect to technolo~~ies having application herein see also Immunity 2001 Apr; 14(4):437-46 The immunological barrier to xenotransplantation.Cascalho M, Platt JL.; WO
01!43779; WO 01/42285; WO
98/10795: WO 01!40803: WO 00/14212; Gastroenterology 2001 May;l20(6):1330-8 An engineered human antibody to TNF (CDP571 ) for active Crohn's disease: a randomized double-blind placebo-controlled trial.
Sandborn WJ: WO 01!44282: WO 01140309; WO O1i40274; WO 01/44300; Ann Rheum Dis May;60(5):433 Cancer and autoimmunity: autoimmune and rheumatic features in patients with mali~~nancies, Abu-Shakra M, et al.; WO 01;'40468; WO 01/40307; WO 01;42297;
WO 01!42294; WO
01/42296; WO 01!40456; WO 01;40308; WO 01'42306: Curr Opin Immunol 2001 Apr:l3(2):134-40 Immunity against cancer: lessons learned from melanoma. Houghton AN. Gold JS, Blachere NEB.; WO
01!42288; WO 01/42288; WO 01!43771; WO 01/42308; WO 01!41804; 'vV0 01,'39722;
WO 01;44808;
WO 01/43770; WO 01'16166; WO 01/41803; WO O1/ 131 10; WO 00/32 7 52; WO
98'33528; WO
01/43695; J Am Pharm Assoe (Wash) 2001 May-Jun;41 (3):383-91 Magic: bullets finally find their mark.;
Leukemia 2001 Apr; I 5(4 ):675-6; WO O 1''44301; Anticancer Res 2001 Jan-Feb;2 I ( 1 B):621-7 Immunotherapy for recurrent colorectal cancers with human monoclonal antibody SK-I. Koda K et al.;
WO 01!1091 l; WO O1i42506: Int J Clin Pract 2001 Apr;55(3):211-6'Tumour necrosis factor as a therapeutic target in rheumatoid arthritis and other chronic inflammatory diseases: the clinical experience with infliximab; WO Ol /44472_ WO O 1/40302: WO 01 40305) With respect to surface plasmon resonance measurements ofaftmity see LJS611 1652:High throughput surface plasmon resonance analysis system 1JS06208422 Surface plasmon sensor Ef01080365 SURFACE
PLASMON RESONAN(,E SENS012 FOR THE SIMULTANEOUS MEASUREMEN f OF A

MOLECULAR INTERACTION USING SURFACE PLASMON RESO1JANCE High throughput surface plasmon resonance analysis system as well as Dimensions of antigen recognition and levels of immunological specificity. Adv Cancer Res. 2001;80:147-87. Use of optical biosensors for the study of mechanistically concerted surface adsorption processes. Anal Biochem. 2001 Jan 15;288(2):109-25 Experimental design for analysis of complex kinetics using surface plasrr~on resonance. Methods. 2000 Mar;20(3):310-8. , and references cited in the foregoing references.
27: Skeie GO, Lunde PK, Sejersted OM, Mygland A, Aarli JA, Gilhus NE.
Autoimmunity against the ryanodine receptor in myasthenia gravis. Acta Physiol Scand. 2001 Mar;171 (3):379-84. 28: Haufs MG.
Haneke E. Epidermolysis bullosa acquisita treated with basiliximab, an interleukin-2 receptor antibody.
Acta Derm Venereol. 2001 Jan-Feb;81 ( I ):72. Woo EY, et al Regulatory CD4(~)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer. Cancer Res. 2001 Jtrn 15;61(12):4766-72. Barrera P, Joosten l~A, den Broeder AA, van De Putte LB, van Riel Fl., van Den Berg WB. Effects of treatment with a fully human anti-tumour necrosis factor alpha monoclonal antibody on the local and systemic homeostasis of interleukin I and TNFalpha in patients with rheumatoid arthritis.
Ann Rheum Dis. 2001 Ju1;60(7):660-9. Nicholson JK, Browning SW. Hengel R1_.
Lew E, Gallagher 1..E, Rimland D, McDougal JS. CCRS and CXCR4 expression on memory and naive T cells in HIV-I infection and response to highly active antiretroviral therapy. J Acquir Immune Defic Syndr. 2001 Jun 1:27(2):105-15. Kung SK. Su RC, Shannon J. Miller RG. Characterization of four new monoclonal antibodies that recognize mouse natural killer activation receptors. Hybridoma. 2001 Apr;20(2):91-101. 43: Bank I, Dardik R, Levy V, Goldstein I, Shoham J. Differential expression and regulation of CD6 on T-cell subsets revealed by monoclonal antibody (MAb) CH I I . Hybridoma. 2001 Apr;20(2):75-84.
With respect to making multifunctional ligands see also USP 5,731,168 and 5,821,333.
With respect to TN E' and TNFR variants, and functional fragments thereof, for use as antibody targets and binding moieties with respect to various aspects of the invention herein see WO 00/67793, WO O l /30300, WO 01/49321, WO 00/62790, WO 01!03720, WO 00/60079, WO 97/46686, WO 01/41803, w'0 01/38526, WO 01/37874, WO 01/12812, WO 01!12671, WO 01!05834, WO 01/03720, WO 00/77191 WO 00/73321, WO 00/71 150, WO 00/67793, WO 00!67034, WO 00/66608. WO 00/66156., WO 012481 l, as well as references cited therein. Many other TNFR variants and TNF analogs are known in the art.
With respect to cytokines and cytokine receptors see also the latest editions of Cytokine Reference: A
Compendium of Cytokines and Other Mediators of Host Defense by Joo,t J.
OppE:nhe im (F_ditor), J;nn Vileck, Moos ~A. Nicoln (Editor); Cytokine Molecular Biology : A Practical Approach by I=rarrcc~> R.
Balk4vill (Editor), ("ran I3alkwill (Editor); Guidebook to Cytokines and Their Receptors by Nicos Nicola (Editor); The Cytokine Network and Immune Functions by Jacqr~os ThL~:e; Novel Cytokine inhibitors by Gerrv ,1. C-li<~«s (Editor). 3rian I-Ienderson (Editor); Homology Folding of Proteins : Application to Cytokine Engineering by Sublr~rstrioi Srinivas~io; Cytokines and Cytokine Receptors (2001 ); International Review of Experimental Pathology : Cytokine-Induced Pathology, Pan E3 :
Inflammatory Cytokines, Receptors, and Disease by C~.~~~. Richter, Kio~ Soiez (Editor).
With respect to antibodies that bind to CCRS see Mol Biol Cell 2002 Feb; 13(2) : 723-737 With respect to variations in chemokine receptors, cytokine and other receptors that can be exploited according to one or more aspects of the invention herein see I : Csaszar A, Abel T. Receptor polymorphisms and diseases.Eur J Pharmacol. 2001 Feb 23;414(1):9-22. 2: Gibejova A.(~hemokine receptors.Acta Univ Palacki Olomuc Fac Med. 2000;143:9-18. 3: Nishimoto N, Kishimoto T, Yoshizaki K.Anti-interleukin 6 receptor antibody treatment in rheumatic disease.Ann Rheum Dis. 2000 Nov;59 Suppl I:i21-7. 4: Aggarwal BB.Tumour necrosis factors receptor associated signalling molecules an<i their rolein activation of apoptosis, JNK and NF-kappaB.Ann Rheum Dis. 2000 Nov;59 Suppl l :i6-16. 5:
Grignani G, Maiolo A.Cytokines and hemostasis.Haematologica. 2000 Sep;85(9):967-72. 6: Idriss HT, Naismith JH.TNF alpha and the TNF receptor superfamily: structure-function relationship(s).Microsc Res Tech. 2000 Aug 1;50(3):184-95. 7: van Deventer SJ.Cytokine and cytokine receptor polyvmorphisms in infectious disease. Intensive Care Med. 2000;26 Suppl 1:S98-102. 8: Gessner A, Rollinghoff M.Biolo~~ic functions and signaling of the interleukin-4 receptor complexes.lmmunobiology. 2000 Jan;201 (3-4):285-307 9: Platanias LC, Fish EN.Signaling pathways activated by interferons.Exp Hematol. 1999 Nov;27( 1 1):1583-92. 10:
Schwertschlag US, Trepicchio WL, Dykstra KH, Keith JC, Turner KJ, Dorner AJ.Hematopoietic, immunomodulatory and epithelial effects of interleukin-1 l.Leukemia. 1999 Sep;
13(9):1307-15. 1 I: Blasi F.The urokinase receptor. A cell surface, regulated chemokine.APMIS. 1999 Jan;107( 1):96-101. 12:
Izuhara K, Shirakawa T.Signal transduction via the interleukin-4 receptor and its correlation with atopy.lnt J Mol Med. 1999 Jan;3( I ):3-10. 13: Tsokos GC, Liossis SN.Lymphocytes, cytokines, inflammation, and immune trafficking.Curr Opin Rheumatol. 1998 Sep;lO(5):417-25. 14: Morishita R, Nakamura S, Hayashi S, Aoki M, Matsushita I I, Tomita N, YamamotoK, Mori~uchi A, Higaki J, Ogihara T.Contribution of a vascular modulator, hepatocyte growth factor (EIGF), to thepathogenesis of cardiovascular disease.) Atheroscler Thromb. 1998;4(3):128-34. 15: Kashiwamura S, Okamura Hf.[IL-18 and receptor].Nippon Rinsho. 1998 Ju1;56(7):1798-806. Japanese.l6: Paxton WA, Kan<~ S.Chemokine receptor allelic polymorphisms: relationships to HIV resistance anddisease progression.Semin Immunol. 1998 Jun;lO(3):187-94. 17: Arend WP, Malyak M. Guthridge CJ, Gabay C.Interleukin-I
receptor anta;~onist:
role in biology.Annu Rev Immunol. 1998;16:27-55. 18: Camussi G, Lupia E.The future role of anti-tumour necrosis factor (TN F) products in the treatmentof rheumatoid arthritis.
Drugs. 1998 May;55(5):613-20. 19:
Taga T, Kishimoto T.Gp 130 and the interleukin-6 family of cytokines.Annu Rev Immunol. 19~>7;15:797-819. 20: Paul WE.fnterleukin 4: signalling mechanisms and control of T cell differentiation.Ciba Found Symp. 1997;204:208-16: discussion 216-9; 1.(W0 01!49321) TNF INHIBITORS FOR
THE
TREATMENT OF NEUROLOGICAL, RETINAL AND MUSCULAR DISORDERS2.(WO 01/46261) METHOD FOR TREATING INFI_AMMATION3.(WO O1i40464) INTIRLEEJK1N-I-RECEPTOR
ASSOCIATED KINASI?-3 (IRAK3) AND IT S USE IN PROMOTION OR INHIBITION OF
ANGIOGENESIS AND CARDIOVASCULARI7ATION4.(WO 01/40464) INTERLEUKIN-I-RECEPTOR ASSOCIATED KINASE-3 (IRAK3) AND ITS USE: IN PROMOTION OR INHIBITION
OF

ANGIOGENESIS AND CARDIOVASCULARIZATIONS.(WO 01!30850) UMLR
POI_YPEPTIDES6.(WO 00!77195) NUCLEIC ACID ENCODING NOVEL EGF-LIKE GROWTI-I
FACTORS7.(WO 00/74719) METHOD OF TREATING CARCINOMA USING ANTIBODY' THERAPY
AND AMELIORATING SIDE EFFECTS ASSOCIATED WITH SUCH THERAPYB.(WO 00/02582) TREATMENT OF CELIAC. DISEASE WITH IN~TERLEUKIN-15 ANTAGONISTS9.(WO 99/47170) PREVENTIVES OR REMEDIES FOR INFLAMMATORY INTESTINAL DISE?ASES CONTAINING
AS THE ACTIVE INGREDIENT IL-6 ANTAGONISTS 10.( WO 99/46376) RECEP~'OR FROM THE
SUPERFAMILY OF TNT-RECEPTORS FROM THE. HUMAN LUNG l 1.(W0 99/43809) PROTEASE-AC;TIVATED RECEPTOR 4 AND USES THEREOF 12.( WO 98,148017) FAMIf.Y OF
IMMUNOREGULA'TORS DESIGNATED LEUKOCYTE IMMUNOCiI_OBULIN-LIKE RECEPTORS
(LIR)13.(WO 98/47923) II_-5R ANTAGONISTS FOR TREATMEN~h OF INFLAMMATION', ASTHMA
AND OTHER ALLERGIC DISEASES14.(Vv'O 98/46620) A NOVEL HUMAN G-PROTEIN COUPLED
RECEPTOR15.(WO 98-46265) METHODS FOR USING ANTAGONISTIC ANTI-AVB3 INTEGRIN
ANTIBODIES16.(WO 98136767) MODULATION OF THE HYPOTHALAMIC-PITUI'TARY-ADRENAL-ADIPOSE AXIS WITH LEPT1N RECEPTOR LIGANDS 17.(W0 98/31809) HC1MAN CC
CHEMOKINE SLC18.( WO 98;30706) COMPOUNDS. COMPOSITIONS AND METHODS FOR THE
ENDOCYTIC PRESENTATION OF IMMIJNOSUPPRESSIVE FACTORS19.(WO 98/24817) NOVEL
DNA, NOVEL PROTE IN, AND NOVEL ANTIBODY20.(WO 98/22499) NEURON AND NEURAL
TUMOUR GROWTH REGULATORY SYSTEM, ANTIBODIES THERETO AND USfS
THEREOF21.(W0 98/19706) IDENTIFICATION OF UNIQUE BINDING IN fERACfI'IONS
BETWEEN
CERTAIN ANTIBODIES AND THE HUMAN B7.1 .AND B7.2 CO-S~~IMULATORY
ANTIGENS22.(WO 98' 18456) PROTEASE-ACTIVATED RECEPTOR 3 AND USES THEREOF23.(WO
98!14480) G PROTEIN-COUPLED RECEPTOR ANTAGONISTS 24.( WO 98/02541 ) GAMMA-HEREGULIN25.(WO 97/49818) G-BETA-GAMMA REGULATED PHOSPHATIDYLINOSIT'OL-3' KINASE26.(WO 97/48804) TlE-2 RECEPTOR L1GANDS (TIE LIGAND-3; TIE LIGAND-4) AND
THEIR USES27.(WO 97/41225) MAMMALIAN MIXED I..YMPHOCYTE REC'EP fORS, CHEMOKINE
RECEPTORS [M.MLR-CCR]28.(WO 97/24373) MONOCLONAL ANTIBODY ANTAGONISTS TO
HAEMOPOIETIC GROWTH FACTORS29.(WO 97'21732) DESIGN OF HORMONE-L,IKI?
ANTIBODIES WITH AGONISTIC AND ANTAGONISTIC FUNCTIONS, 6,235,880 Human sulfonylurea receptor 6.221,660 DNA encoding SNORF25 receptor 6, 214,797 Urocortin peptides, nucleic acid encoding same methods for usingsame6,214,344 Hepatocyte growth factor receptor antae.onists and uses thereof 6,210,904 Anticoagulant test6,207, 152 Hepatocyte ~~rowth factor receptor antagonists and uses thereof6,204,017 folynucleotide encoding a histamine receptor 6,197,541 Recombinant thrombin receptors and assays using them6, l 84,358 IP-10/Mig receptor designated CXCR3, antibodies, nucleic acids,and methods of use therefor6,177,079 Antagonists of interleukin-156,177,078 Monoclonal antibody antagonists to IL-36,177,077 TNT inhibitors for the treatment of neurological disorders 6,171,815 Human sulfonylurea receptor6,168,783 Antagonists of interleukin-156.166,185 Antibodies to human TIE-2 ligands6,165,466 Anta~~onists of interleukin-156,162,431 Serinelthreonine protein kinase6,143,870 Thrombin receptor homolog6,136.957 Antibodies which bind granulocyte-macrophane colony-stimulatingfactor receptor6,124,101 Recombinant thrombin receptor and related phannaceuticals6, I I 1,075 Protese-activated receptor PAR4 (ZCHEMR2) 6.103,874 Human KDEL receptor 6,096,873 Gamrna-here'~ulin 6,086,874 Antitumor agent effect enhancer containing IL-6 anta~Tonist56,084.075 Agonist and antagonist antibodies to the chemokine receptor-2(CCR2) 6,063,596 G-protein coupled receptors associated with immune response6,054,292 T-cell receptor protein6,04~~,212 Recombinant C140 receptor, its agonists and antagonists, andnucleic acids encoding the receptor6,033.869 Polynucleotide encoding a novel human cytokine; steroid receptor 6,024,936 Antibody-based method of localizin~~ activated thrornbinreceptors 6,017,763 G-beta-gamma regulated phosphatidylinositol-3' kinase6.013,480 Antagonists of ir~terleukin-156,013,479 Human Emrl-like G protein coupled receptor 5.994,097 Polynucleotide encoding human G-protein coupled receptor5,985,828 Mammalian receptors for interleukin-10 (IL-10) 5.985,583 Cloning and expression of gonadotropin-releasin«
hormonereceptor5,977,072 High affinity immunoglobulin E receptor-like protein5,976,852 K.kappa./µ-like protein tyrosine phosphatase. PTP .lambda.5,976,815 Bioassay using ALK-7, a novel serine threonine kinase receptor 5,972,621 Methods of identifying compounds that modulate body weightusing the OB
receptor 5.965,709 IgE antagonists5,965,365 Serine!threonine protein kinase5,955,303 Human chemokine receptor-like protein 5.952,175 DNA encoding a human progesterone receptor complex p23-Iikeprotein5,945,308 Human oxidized LDL receptor5.942,606 Viral receptor protein 5.928.887 .kappa.!µ-Like protein tyrosine phosphatase, PTP .lambda.5,912, 144 Edb I-receptor homolog5,902,585 Methods of inducing T cell unresponsiveness to donor tissue ororgan in a recipient with GP39 antagonists 5,892,014 DNA encoding a protease-activated receptor 35,891,720 Isolated DNA
encoding a novel human G-protein coupled receptor5,891,674 Insulin receptor tyrosine kinase substrate 5.891,638 Serine threonine kinase receptor, alk-75,888,81 1 Corticotropin-releasin« hormone receptor5,888,510 Chronic rheumatoid arthritis therapy containing IL-6 antagonistas effective component 5,886,148 Parathyroid hormone receptor5.874,400 Recombinant C 140 receptor, its agonists and antagonists, andnucleic acids encoding the receptor 5,874,273 G-beta-gamma regulated phosphatidylinositol-3' kinase5,874,224 Growth factor receptor binding protein 5,871,930 High affinity immunoglobulin E receptor-like protein 5.869,633 Thrombin receptor homology polynucleotide5,869,609 G protein coupled glutamate receptors5,869,271 G-beta-gamma regulated phosphatidylinositol-3' kinase5,869,049 Methods of inducin~~ T cell unresponsiveness to bone marrow withgp39 antagonists 5,863,796 Antibodies which specifically bind mammalian receptors forinterleukin-10 (IL-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 itscomponents 5,856.133 G-beta-gamma regulated phosphatidylinositol-3'kinase5,856, l3:>_ G-beta-gamma regulated phosphatidylinositol-3' kinase5,851,797Tie ligand-3, methods of making and uses thereof5,840,853 Parathyroid hormone receptor and DNA encoding same5,837,499 DNA encoding C5A
receptor antagonists having substantially noagonist activity and methods of expressing sarne5,834,240 DNA encoding a transforming growth factor-.beta. receptor associated protein 5,833,987 Treatment of T
cell mediated autoimmune disorders5,831,047 Oli~~onucleotide probes to L-AP4 sensitive glutamate receptorsequences 5,830,678 Method for identifying a target peptide that modulates thebinding of epinectin ligand to inte'~rin receptors5,824,500 Nucleic acid encodin~~ novel human KDEI_ receptor 5,817,480 DNA encoding a histamine H2 receptor5.814,507 .kappaJµ-like protein tyrosine phosphatase, PTP .Iambda.5,814,464 Nucleic acids encodin~~ TIE-2 li'~and-25.81 1,24:>
Antibodies that specifically bind to ALK-7, a novel serinethreonine kinase receptor5,807.824C5 A receptor anta~~onists having substantially no agonistactivity5,795.966 Antagonists of interleukin-155,789,565 Serine threonine kinase receptor, ALK-7 5,789,192 Mammalian receptors for interleukin-10 (IL-10)5,763,575 Agonist and antagonist peptides of the C140 receptor 5,759,994 Recombinant thrombin receptor and related pharmaceuticals5.750,366 Cloniny~ and expression of y~~onadotropin-releasing hormonereceptor 5,747,279 Nucleic acid molecules encoding kappa₃ opioid receptors,receptors encoded thereby, and uses thereof5,747,267 Method for identifying a G protein coupled glutamate receptoragonist and antagonist5,738,999 L-AP4 sensitive glutamate receptors5,730,976 Method for treatin~~ macrophage pathogen infections by ( GF-Bantagonists5.726,036 Granulocyte-macrophage colony-stimulating factor receptor andderivatives thereof5,72 I ,107 Antibodies to G protein coupled glutamate receptors5,716,804 Mammalian interleukin-10 (1I,-10) super-activating receptors:and variants5,716,789 Method to determine ligands, agonist and antagonist of C 140receptor 5,707,632 Receptors far tibroblast growth factors5,688,768 Recombinant thrombin receptor and related pharmaceuticals5,686,597 Thrombin receptor homology 5,686,292 Hepatocyte growth factor receptor antagonist antibodies anduses thereof5,683,884 Methods for identifying modulators of human calcitoninmediated metabolism5,683,693 Method for inducing T cell unresponsiveness to a tissue ororgan graft with anti-CD=t0 li«and antibody or soluble CD405,674.981 Human calcitonin receptor polypeptides5,674,689 Human calcitonin receptor polypeptides and methods of use5,646,036 Nucleic acids encoding hepatocyte growth factor receptorantagonist antibodies5,629,283 Granulocyte-macrophage colony-stimulating factor receptor andderivatives thereof5,622,839 Recombinant production of human calcitonin receptorpolypeptides5,614,609 Serine threonine kinase receptor5,556,780 CDNAS encoding: mouse and rat type-2 an~~iotensin II receptorsand their expression in host cells5,543, 143 Method for activating macropha~;es-monocytes5.516,894 A_2b -adenosine receptors5,514,555 Assays and therapeutic methods based on lymphocytechemoattractants 5,506,107 Selecting ligand agonists and antagonists5,494,806 DNA and vectors encodin~~ the parathyroid hormone receptor,transfbrmed cells, and recombinant production of PTHR
proteins andpeptides5.451,658 Antagonists of human gamma interferon5,441,935 Growth factor receptors5,385,831 Method for producing a mammalian G protein coupled glutamatereceptor5,334,380 Anti-endotoxin, interleukin-I receptor antagonist andanti-tumor necrosis factor antibody with arginine-free formulations forthe treatment of hypotension5.256,766 Recombinant thrombin receptor and related pharnaceuticals5,177,190 Purified C5a receptor from human polymorphonuclear leukocytes4.857,637 Methods and compositions for immunologically modulating growthhormone receptor activity; and references cited therein. (see also 1.(W0 01149744) MOUSE G-PROTEIN COUPLED RECEI''fOR MAS 2.(W0 01/49726) A NOVEL
POLYPEPTIDE-HUMAN NATRIURETIC PEPTIDE RECEPTOR 18 AND THE POI_YNUCLEOTIDE
ENCODING SAID POL,YPEPI~IDE 3.(W0 Ol~'49321) TNF INHIBI TGRS FOR TH1:
TREATMENT OF
NEUROLOGICAL,RF:fINAL ANDMUSCULARDISORDERS4.(WO01!00657)NOVEL.INDOLE
PEPTIDOMIMETICS AS THROMBIN REC.'EPTOR ANTAGONISTS 5.(W0 00!62790) SOLUBLE
'TUMOR NECROSIS FACTOR RECEPTOR TREA I'MI:NT OF MEDICAL DISORDERS 6.(W0 01/03720) PROMOTION OR INHIBITION OF ANGIOGENESIS AND CARDIOVASCUI,ARIZATION
BY TUMOR NECROSIS FACTOR LIGAND;'RECEI'TOR HOMOLOGS 7.(W0 01/46261) METHOD
FOR TREATING INFLAMMATION 8.(WOOL/46191)4-[ARYL(8-AZ.ABICYCLO[3.2.I~OCTAN-3-YL)]AMINOBENZOIC .ACID DERIVATIVES 9.(W0 01;46176) NON PEPTIDE TACHYKININ
RECEPTOR ANTAGONISTS 10.(W0 01;45730) TWE?AK RECEPTOR I I.(WO 01;45703) NITROSATED AND NITROSYLATED CYCLOOXYGENASE-2 INHIBITORS, COMPOSITIONS
AND METHODS OF USE 12.(W0 01!40464) 1NTERl,EUKIN-1-RECEPTOR ASSOCIA'T'ED

(IRAK3) AND ITS USE IN PROMOTION OR INHIBITION OF ANGIOGENESIS AND
CARDIOVASCULAR1ZA'TION 13.(W0 01;'44213) NEW P2X7 RECE:F''fOR ANTAGONISTS FOR
USE
IN THE TREATMENT OF INFLAMMATORY. IMMUNE OR CARDIOVASCULAR DISEASES
14.(W0 01;'42268) DOG OREXIN 1 RECEPTOR IS.(WO 01;42208) Cl'CLOAMINE CCRS
RECEPTOR
ANTAGONISTS 16.(W0 01/41752) ISOFORM SPECIFIC INHIBITI01~I FOR TREATMENT OF
PAIN
AND REDUCTION OF ANESTHETIC THRESEiOI_f) 17.(W0 01!03730) PROMOTION OR
INHIBITION OF ANGIOGENESIS AND CARDlOV.4SCULARIZATION BY TUMOR NECROSIS
FACTOR LIGAND/REC'EPTOR HOMOLOGS 18.(W001''40464) INTI?RLEUKIN-I-RECEPTOR
ASSOCIATED KINASE=-3 (IRAK3) AND ITS tJSE IN PROMOTION OR INHIBI'T'ION OF
ANGIOGENESIS AND CARDIOVASCULARIZATION 19.(W0 01;4(1259) MONKEY OREXIN I
RECEPTOR 20.(W0 01.'40252) MONKEY CAL,CIUNI SENSING RECEPTOR 21.(W0 OIi04139) HUMAN AXOR29 RECI?PTOR 22.(W0 01;36480) MOUSE 7-TRANSMEMBRANI: RECEPTOR, AXOR45 23.(W0 01!00656) NOVEL INDAZOL.E PEPTIDOMIMET'ICS AS THROMBIN RECEPTOR
ANTAGONISTS 24.(W0 00;'67793) DEATH DOMAIN CONTAINING RECEPTOR 4 25.(W0 01/34645) MODULATING IL-13 ACTIVITY USING MUTATED II_-I 3 MOI_ECULE:S T'EiAT
ARE
ANTAGONISTS OR AGONISTS OF IL-13 26.(W0 O1%34138) COMPOSITIONS AND METHODS FOR
TREATMENT OF NEUROLOGICAL DISORDERS AND NE1.JRODEGENERATIVE DISE,~SES
27.(W0 01/32656) POLYMORPH1C FORM OF' A TACHYKININ RECEPTOR ANTAGONIST" 28.(W0 0(132166) NEW COMBINATION COMPRISING A 8#946;2-ADRENORECEPTOR AGONIST AND A
LEUKOTRIENE RECEPTOR ANTAGONIST 29.(W0 01!32163) NEVI'' COMBINA'flON
COMPRISING
A BETA ? (&#946;)2 ADRENO RECEPTOR AGONIST AND A LENI<OTRIENE RECEPTOR
ANTAGONIST 30.(W0 01/01922) USE OF SUBSTANCE P AN'TAGON1STS FOR THE
TREATMEN'I~
OF ADENOCARCINOMA 31.(V1,'0 0('30850) IJMI.R POE,YPEPTIDES 32.(V1'0 01'27153) A MURINE
SEVEN-TRANSMEMBRANE RECEPTOR, MUS MIJSCULLJS MHNEAA81 33.(WO01/25~69) NOVEL HUMAN G-PROTEIN COUPLED RECEPTOR 34.(W0 01/24828) MODULA'T'ORS OF
CYTOKINE MEDIATED SIGNALLING PATHWAYS AND INTEGRIN &#945;V&946;3 RECEPTOR ANTAGONISTS FOR COMBINATION T"FiERAPY 35.(W0 Ol/?4798) USE: OF
CENTRAL.
CANNABINOID RECEPTOR ANTAGONIST FOR PREPARING MEDICINES 36.(W0 O1i24797) INTEGRIN RECEPTOR ANTAGONISTS 37.(V1'0 00,'68250) 7TM RECEPTOR RA r APJ
38.(V4'0 01/16121) I IETEROCYCLIC COMPOUNDS AND METHODS OF USE THEREOF 39.(W0 01!14406) ANTIANDROGEN AGENTS 40.(W0 01; 12Ci71) HUMAN TUMOR NI~CROSIS FACTOR RECEPTOR
TRl6 41.(W0 01:'10891 ) IL-16 ANTAGONISTS 42.(Vv'O 01'10889) RAT-G-PROTEIN
COUPLED
RECEPTOR BRS3 43.(W0 01/10423) USE OF 5-HT3 RECEPTOR ANTAGONISTS FOR THE
TREATMENT OF INFLAMMATIONS OF 'fHE RESPIRATORY 'TRACT 44.(W0 01/07028) THE USE
OF RETINOID RECEPTOR ANTAGONISTS IN THI? TREATMENT OF PROSTATE CARCINOMA
45.(W0 01/05834) HUMAN TUMOR NECROSIS FACTOR RECEPTORS TR13 AND TR14 46.(W0 01/05783) BRADYKININ B I RECEPTOR ANTAGONISTS 47.(W0 01!04139) POI.,YNUCL,EOTIDE

SCREENING FOR AGON(STS AND ANTAGONISTS OF TFiE INTERACTION BETVI-'EEN HUMAN
AXOR29 RECEPTOR AND ITS LIGANDS 48.(W0 01/03720) PROMOTION OR INHIBITION OF
ANGIOGENESIS AND CARDIOVASCULARIZATION BY TUMOR NECROSIS FACTOR
LIGAND%RECEPTOR HOMOLOGS 49.(W0 01'01922) USE OF SUBSTANCE P ANTAGONISTS IN
THE TREATMI~NT OF TEIE ADENOCARC.'INOMAS 50.(W0 01''00659) BENZIM1DAZOLONE
PEPT1DOMIMETTCS AS THROMBIN RECEPTOR ANTAGONISTS 51.(VVO 01!00657) NOVEL
INDOLE PEPT1DOMIMETICS AS THROMBIN REC.'EPTOR ANTAGONISTS 52.(W0 01:00656) NOVEL INDAZOLE PI~PTIDOMIMET(CS .AS THROMBIN RECEPTOR ANT'AGONIS'TS 53.(W0 01!00576) INDOLE AND 1NDAZOLE UREA-PEPT'C)IDS AS TFIROMBIN RE:CEP'fOR
ANTAGONISTS 54.(V1'0 Ol %00198) COMPOSITIONS AND METHODS OF TREATING CANCER
USING COMPOSITIONS COMPRISING AN 1NHIB1'fOR OF ENDOTHELIN RECEI' fOR ACTIVITY
55.(W0 00/78317) INTEGRIN RECEPTOR AN TAG(7N1ST'S 56.(W0 00/77195) NUCLEIC
ACID
ENCODING NOVEL EGF-LIKE GROWTH FACTORS 57.(W0 00.'76502) ME'T'HODS AND
COMPOSITIONS FOR TREATING RAYNAUD'S PHENOME?NON AND SC'LERODERM.4 58.(W0 00/74719) METETOD OF TREATING CARCINOMA USING ANTIBODY TIiERAPY AND
AMELIORATING SIDE EFFECTS ASSOCIATED WITH SUCH THERAPY 59.(W000!73321) HUMAN TUMOR NECROSIS FACTOR RECEPTOR TR10 60.(W0 00;'72801 ) ALPHA V INTEGRIN
RECEPTOR ANTAGONISTS 61.(W0 00/71150) TUMOR NECROSIS FACTOR RECEI''fOR 5 62.(W0 00.!69831) SPIROIMIDAZOLIDINE DERIV,A'fIVES. THEIR PREPARATION, TIiEIR USE:
AND
PHARMACEUTICAL I'REPARA'fIONS COMPRISING THEM 63.(W0 00/69820) CYCLIC' AMINE
DERIVATIVES AND THEIR USES 64.(W0 00!69463) COMPOSITIONS AND METHODS FOR
TREATING CELL PROLIF'ERA'I ION DISORDERS 65.(W0 00/69459) TREATMENT OF
REFRACTORY HUMAN -TUMORS WITI~ EPIDERMAL GROWTH FACTOR RECEPTOR
ANTAGONISTS 66.(W'0 00/68250) 7TM RECEPTOR RAT APJ 67.(W0 00!68244) 7'fM
RI?CEPTOR
MOUSE APJ 68.(W0 00/67793) DEATH DOMAfN CONTAINING RE?CEPTOR 4 69.(W0 00/67034) METHODS OF USE OF THE TACI/TACI-1. INTERACTION 70.(W0 00/67024) CANCER
TREATMENT WITFI ENDOTHELIN RECEPTOR ANTAGONISTS 71.(W0 00/66632) AGONISTS OR
ANTAGONISTS FOR IiAEMOPOIE'TIC GROWTH FACTORS 72.(W0 00/66522) GLUCOCORTICOID RECEPTOR MODUC,ATORS 73.(W0 00/66156) DEATH DOMAIN
CONTAINING RECEPTOR 5 74.(W0 00/64465) DEATH DOMAIN CONTAINING RECEPTORS
75.(W0 00;'62790) SOLUBLE TUMOR NECROSIS FAC~I"OR RECEPTOR TREATMENT OF
MEDICAL DESORDERS 76.(W0 00/59532) THE USE OF DOMAINS OF 'TYPE IV COLLAGEN T
INHIBIT ANGIOGENESIS AN TUMOUR GROWTH 77.(W0 00!56862) HUMAN TUMOR
NECROSIS FACTOR RECEPTOR TR9 78.(W0 00/56405) HUMAN TUMOR NECROSIS FACTOR
RECEPTOR-LIKE 2 79.(W0 00/54772) AMYOTROPIC LATERAL SCLEROSIS TREATMENT WITH
A COMBINATION Of R1LUZOLE AND AN AMPA RECEPTOR ANTAGONIST- 80.(W0 00/53596) IMIDAZOLE COMPOUNDS SUBS T(TUT'ED WI'fH A SIX OR SEVEN MEMBERED
HETE:ROCYCLIC RING CON'I AIMING TWO NI'I ROGEN ATOMS 81.(W0 00/5 3175) COMPOUNDS
AND METHODS 82.(W'0 00/52028) TUMOR NECROSIS FACTOR RECEPTORS 6&agr; and 6&bgr;
83.(W0 00/51974) ALPHA-AMINOACETIC ACID DERIVA (IVES USEFUL AS ALPHA 4 BETA 7 -RECEPTOR ANTAGONISTS 84.(W0 00/50459) HUMAN TUMOR NECROSIS FACTOR RECEPTOR-L1KE PROTEINS TRI I, TRl 1SV1, AND TRl ISV2 85.(W0 00/49170) MURINE I lcby RECEPTOR
86.(W0 00/48603) DIBE.NZO-AZEPINE DERIVATIVES AS &abr;V INTEGRIN RECEf''I~OR
ANTAGONISTS 87.(W0 00!48597) SYSTEMIC USE OF 5-f-I~I~ 3 RECEPTOR ANTAGONISTS
AGAINST RHEUMATIC INFLAMMATORY PROCESSES 88.(W0 00148581) USE OF 5-HT3 RECEPTOR ANTAGONISTS 89.(W0 00/46343) SCREENING ASSAY FOR ANTAGONISTS OF
FGFR-MEDfATED MALIGNANT CELL TRANSFORMATION AND TUMOR FORMATION 90.(W0 00/46215) BENZAZEPINE DERIVATIVES AS ALPHA-V INTEGRIl\i RECEPTOR ANTAGONISTS
91.(W0 00/46197) INDOLE DERIVATIVES AND THEIR USE AS MCP-I RECEPTOR
AN~fAGONISTS 92.(W0 00!44763) COMPOSITIONS FOR TREATII'lG INFLAMMATORY
RESPONSE 93.(W0 00143031 ) TUMOR NECROSIS FACTOR AN~I~AGONIS TS AND THEIR USE
IN
ENDOMETRIOSIS 94.(W0 00%43852) COMPOUNDS AND ME'fIiODS 95.(W0 00/40716) SOLUBLE
RECEPTOR BR43x2 AND METFIODS OF USING 96.(W0 00/40239) COMPOUNDS AND METHODS
97.(W0 00/39166) NOVEL FIYAI_URONAN-BINDING PROTF~INS AND ENCODING GENES
98.(W0 00/37462) NON-PEPTIDE NK I RECEPTORS ANTAGONISTS 99.(W0 00;35887) VITRONEC'TIN
RECEPTOR ANTAGONIST PF-IARMACELJTICALS 100.(W0 00/354'72) VITRONECTIN RECEPTOR
ANTAGONIST PHARMACEUTICALS 51.(W0 01'00657) NOVEL II'JDOLE PEPTIDOMIMETICS AS
THROMBIN RECEPTOR ANTAGONISTS 52.(W0 01!00656) NOVEL INDAZOI.E
PEPTIDOMIMETICS r1S TI-IROMBIN KECEPTOR AN'TAGONIS'TS 53.(W0 01/00576) INDOLE
AND INDAZOLE UREA-PEP'fOIDS AS THROMBIN RECEPTOR ANTAGONISTS 54.(W0 01100198) COMPOSITIONS AND METI-IODS OF TREA fING CANCER USING COMPOSITIONS
COMPRISING AN INE-IIBITOR OF ENDOTHELIN RECEPTOR ACTIVITY 55.(W0 00/78317) INTEGR1N RECEPTOR ANTAGONISTS 56.(W0 00/77195) NUCLEIC ACID ENCODING NOVEL
EGF-LIKE GROWTFI I~AC fORS 57.(W0 00/76502) METHODS AND COMPOSITIONS FOR
TREATING RAYNAUD'S PHENOMENON AND SCI.ERODERMA 58.(W0 00!74719) MI?THOD OF
TREATING CARCINOMA USING ANTIBODY THERAPY AND AMELIORATING SIDE; EFFECTS
ASSOCIATED WITFI SUCH THERAPY 59.(W0 00 73331 ) HUMAN 'TUMOR NECROSIS FACTOR
RECEPTOR TRIO 60.(W0 00;72801 ) ALPHA V 1NTEGRIN RECEPTOR ANTAGONISTS 61.(W0 00/71 150) TUMOR NECROSIS FACTOR RECEPTOR 5 62.(W0 00169831) SPIROIMIDAZOLIDINE
DERIVATIVES, THEIR PREPARATION, 'THEIR USE AND PHARMACEUTICAL PREPARATIONS
COMPRISING THEM fi3.(WO 00/69820) CYCLIC AMINE DERIVATfVES AND 'THEIR F:SES
64.(W0 00.'69463) COMPOSITIONS AND METHODS I~OR TREAT11~1G CELL, PROLIFERATION
DISORDERS 65.(W0 00/69459) TREATMENT OF KEFRAC'TORY I-IUMAN TUMORS WITFI
EPIDERMAL GROW'TI1 FACTOR RECEPTOR ANTAGONISTS 66.(W0 00/68350) 7TM RECEPTOR
RAT APJ 67.(W0 00~'68?44) 7TM RECEPTOR MOUSE APJ 68.(W0 00%67793) DE-:ATH
DOMAIN
CONTAINING RECEPTOR 4 69.(W0 00!67034) METHODS OF USE OF THE 'TACI/TACI-I.
INTERACTION 70.(W0 00;'67024) CANCER TREATMENT WITFI ENDOTHELIN RECEPTOR
ANTAGONISTS 71.(W000%66632) AGON1SI'S OR ANTAGONISTS FOR HAEMOPOIETIC

GROWTH FACTORS 72.(W0 00/66522) GLUCOCORTICOID RECEPTOR MODULATORS 73.(W0 00/66156) DEATH DOMAIN CONTAINING 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 0059532) THE USE OF DOMAINS
OF TYPE IV COLLAGEN T INHIBIT ANGIOGENESIS AN TUMOUR GROWTH 77.( WO 00/56862) HUMAN TUMOR NE('ROSIS FACTOR RECEPTOR TR9 78.(W0 0(1/56405) HUMAN TUMOR
NECROSIS FACTOR RECEPTOR-LIKE 2 79.(W0 00/54772) AMYOTROPIC LATERAL SCLEROSIS
TREATMENT WITH A COMB1NA'TION OF RILUZOLE AND AN AMPA RECEPTOR
ANTAGONIST 80.(W0 00/53596) IMIDAZOLE COMPOUNDS SUBSTITUTED WITH A SIX OR
SEVEN MEMBERED HETEROCYCLIC RING CONTAINING TWG NITROGEN ATOMS 81.(W0 00/53175) COMPOUNDS AND METHODS 82.(W0 00/52028) TUMOR NECROSIS FACTOR
RECEPTORS 6&a~r; and 6&:b~r; 83.(W0 00/51974) ALPHA-AMINO.ACETIC ACiD
DERIVATIVES
USEFUL AS ALPHA 4 BETA 7 - RECEPTOR ANTAGONISTS 84.(W0 00/50459) I-IUMAN TUMOR
NECROSIS FACTOR RECEPTOR-LIKE PROTEINS TRI I, TR11SV1, AND TRl ISV2 85.(W0 00/49170) MURINE I lcby RECEPTOR 86.(W0 00/48603) DIBENZO-AZ.EPINE DERIVATIVES
AS
&agr;V 1NTEGRIN RECEPTOR ANTAGONISTS 87.(W0 00/48597) SYSTEMIC USE OF 5-HT 3 RECEPTOR ANTAGONIST"S AGAINST RHEUMATIC INFLAMMATORY PROCESSES 88.(W0 00/48581) USE OF 5-H'I'3 RECEPTOR ANTAGONIS'T'S 89.(W0 00/46343) SCREENING
ASSAY FOR
ANTAGONISTS OF F(uFR-MEDIATED MALIGNANT" CELL TRAI'lSFORMATION AND TUMOR
FORMATION 90.(W0 00/46215) BENZAZEPINE DERIVATIVES AS ALPHA-V (NTEGRIN
RECEPTOR ANTAGONISTS 91.(W0 00/46197) IN DOLE DERIVATIVES AND THEIR USE AS
MCP-1 RECEPTOR ANTAGONISTS 92.(W0 00/44763) COMPOSITIONS FOR TREAT"ING
INFLAMMATORY RESPONSE 93.(W0 00/43031 ) TUMOR NECROSIS FACTOR ANTAGONISTS
AND THEIR USE IN F~NDOMETRIOSIS 94.(W0 00/42852) COMPOUNDS AND METH(>DS 95.(W0 00/40716) SOLUBLE RECEPTOR BR43x2 AND METHODS OF USING 96.(W0 00/40239) COMPOUNDS AND METHODS 97.(W0 00/39166) NOVEL HYALCRONAN-BINDING PROTEINS
AND ENCODING GENES 98.(W0 00/37462) NON-PEPTIDE NK 1 RECEPTORS ANTAGONISTS
99.(W0 00/35887) VITRONECTIN RECEPTOR ANTAGONIST PHARMACEUTICALS 100.(W0 00/35492) VITRONECT IN RECEPTOR ANTAGONIST PHARMACEUTICALS 101.(W0 00/35488) VITRONECTIN RECEI''TOR ANTAGONIST PHARMACEUTICALS 102.(W0 00.-35455) HETEROARYL-ARYL UREAS AS IGF-l RECEPTOR ANTAGONISTS 103.(W0 00/32578) BEN7.IMIDAZOLE COMPOUNDS THAT ARE VITKONEC1'IN RECEPTOR AN fAGONIS'F'S
104.(W0 00/28988) NI I ROSATED AND NITROSYLATED 1-12 RECEPTOR ANTAGONIST
COMPOUNDS, COMPOSITIONS AND METHODS OF USE 105.(W0 00/27421) FOCAL USE OF
SOLUBLE TUMOR NECROSIS RECEPTOR I (sTNFRI) FOR PROI'HYLAXIS AND TREATMENT
OF CORNEAL TRANSPLANT REJECTION AND OTHER DISORDERS OF'I"HE EYE 106.(W0 00/25805) VASCULAR ENDOTHELIAL GROWTH FACTOR-LIKE PROTEIN FROM ORE VIRUS
NZ2 BINDS AND ACT"IVATES MAMMALIAN VEGF RECEPTOR-2 107.(W0 00/25745]
IRRIGATION SOLUTION AND METHOD FOR INHIBITION OF PAIN AND INFLAMMATION
108.(W0 00/24395) NEW USE OF GLUTAMATE ANTAGONISTS FOR THE TREATMENT OF
CANCER 109.(W0 0023471 ) USE OF A CYTOKINE-PRODUCING LACT"OCOCCUS STRAIN TO
TREAT COLITIS I 10.(W0 00!23469) FRAGMENTS OF INSULIN-LIKE GROWTH FACTOR
BINDING PROTEIN AND INSULIN-LIKE GROW FFI FACTOR. AND USES THEREOF 111.(W0 00/23438 ) N-(IMIDAZOLYLALKYL)SUBSTI~I"U'1'ED CYCLIC AMINES AS HISTAMINE-H 3 AGONISTS OR ANTAGONISM"S I 12.(W0 00/231 13) PEPTIDE-BASED CARRIER DEV1C'ES
FOR
S'TELLATE CELLS 1 13.(W0 00/23066 ) IRRIGATION SOLUTION AND METHOD FOR
INHIBITION
OF PAIN AND INFLAMMATION I 14.(W0 00/23062) IRRIGATION SOLUTION AND METHOD
FOR INHIBITION OF PAIN AND INFLAMMATION I 15.(W0 00/2()578) A METHOD OF' MODULATING CELL SURVIVAL AND REAGENTS USEFUL FOE: SAME I 16.(W0 00/20389) NAPHTHALENECARBOXAMIDES AS TACHYKININ RECEPTOR ANTAGONISTS I 17.(W0 00/20371) PROSTAGLANDIN RECEPTOR LIGANDS 1 18.(W0 00!20003) NAPHTHALENECARBOXAMIDES AS'TACHYKININ RECEPTOR ANTAGONISTS 119.(W0 00/14109) BASIC PRODUCTS HAVING ANTAGONISTIC ACTIVITY ON THE NK-I RECEPTOR
AND THEIR USE IN PHARMACEUTICAL COMPOSITIONS 120.(W0 00/10391) THE USE OF
ADENOSINE A3 RECI~PTOR ANTAGONISTS TO INHIBIT TUMOR GROWTI-1 121.(W0 00/09503) INTEGRIN RECEPTOR ANTAGONISTS 122.(W0 00!09152) TFIERAPEUTIC CHEMOKINE
RECEPTOR ANTAGONISTS 123.(W0 00/(18001 ) SUBSTITUTED ISOXAZOLE AS ESTROGEN
RECEPTOR MODULA'I~ORS 124.(W0 00/06169) INTEGR1N RECEPTOR ANTAGONISTS 125.(W0 00/03716)'TOP1CAL COMPOSITIONS COMPRISING AN OPIOID ANALGESIC AND AN NMDA
ANTAGONIST 126.(W0 00/02859) N-SUBSTITUTED NAPHTHALENE CARBOXAMIDI~S AS

NEUROKININ-RECEPTOR ANTAGONISTS 127.(W0 00/02582) TRI~ATMENT OF CELIAC
DISEASE WITH INTERLEUKIN-15 ANTAGONISTS 128.(W0 00!01802) PEPTIDE ANTAGONISTS
OF THE HUMAN L)ROKINASE REC.'EPTOR AND METHOD FOR SELECTING THEM 129.(W0 00/00194) OPHTHALMIC USES OF PPARGAMMA AGONISTS AND PPARGAMMA
ANTAGONISTS 130.(W0 99/65944) PEPTIDE INHIBITORS OF &agr;V&bgr;3 AND
&agr;V&bgr:5 131.(W0 99/62955) METHOD OF DESIGNING AGONISTS AND ANTAGONISTS TO EGF
RECEPTOR FAMILY 1 32.(W0 99!60015) IMIDAZOLIDINE DERIVATIVES, THE PRODUCTION
THEREOF, THF_IR USE. AND PHARMACEUTICAL PREPARATIONS CONTAINING ~'HE SAME
133.(WO 99/59635) USE OF A COX-2 INHIBITOR AND A NK-I REC.'EPTOR ANTAGONIST
FOR
TREATING INFLAMMATION 134.(W0 99'58142) IJSE OF ANTI-PR_OLACTIN AGENTS TO
'TREAT
PROL1FERATIVE CONDITIONS 135.(W0 99/58097) USE OF ANTI-PROI_ACTIN AGEN'TS'TO
TREAT PROLIFERATIVE CONDITIONS 136.(W0 99157245) METHODS OF SCREENING FOR

RECEPTOR AND L1GANDS THEREOF 137.(W0 99!51245) NON-PEPTIDE BRADYKININ
RECEPTOR ANTAGONISTS FOR I1SE~ IN TREA fINCi OPHTFiAl.MIC DISEASES AND
DISORDERS 138.(W0 99/50249) INTEGRIN ANTAGONISTS 139.(~1~0 99/49856 ) AN'FAGON1STS
FOR TREATMENT OF CDI 1lC.D18 ADHESION RECEPTOR MEDIATED DISORDERS 140.(W0 99/47170) PREVEN'LIVES OR REMEDIES FOR INFLAMMATORY INTESTINAL. DISEASES
CONTAINING AS THE ACTIVE INGREDIENT IL-6 ANTAGONIST S 141.(W0 99/47158) THERAPEUTIC CHEMOK1NE RECEPTOR ANTAGONISTS 142.(W0 99/46376) RECEPTOR FROM
THE SUPERFAMILY OF TNT-RECEPTORS FROM THE HUMAN LUNG 143.(W0 99!45927) VITRONECTIN RECEPTOR ANTAGONISTS 144.(W0 99/45905) PROPHYLAXIS AND
TREATMENT OF MIGRAINE I-iEADACHES W1TL1 THROMBOXA1~E SYNTHETASE INHIBITORS
AND/OR RECEPTOR ANTAGONISTS 145.(W0 99/44612) SUBSTITUTED QUINAZOLINES AND
ANALOGS AND THE L'SE THEREOF 146.(W0 99/43809) PROTEASE-ACTIVATED RECEPTOR 4 AND USES THEREOF 147.(WO 99/42464) SUBSTITUTED IMIDAZO[1,2-a:3,4-a'~DIQUINOLINYLIUM INTERLEUKIN-8 RECEPTOR ANTAGONISTS 148.(WO 99/424(13) SUBSTITUTED QUINOXAI,INE DERIVATIVES AS INTERLEUKIN-8 RECEPTOR ANTAGONISTS
149.(W0 99/42461) SUI3ST1TUTED QUINOXALINE DERIVATIVES AS INTE;RL.E1JKIN-8 RECEPTOR ANTAGONISTS 150.(W0 99/41257) GL.UC'OCORTICOID-SELECTIVE
ANTI INFLAMMATORY AGEN 1~S 151.(WO 99!41256) GI,UCOCORTICOID-SELEC~1~1VE ANT(-INFLAMMATORY AGENTS 152.(W0 99/40192 ) EIUMAN RECEPTOR GPR14, AND A METHOD
OF FINDING AGONIST AND ANTAGONIST TO HUMAN AND RAT GPR14 153.(W0 99!40091) ANTAGONISTS 154.(W0 99138532) METHODS FOR THE PREVENTION AND TREATMENT OF
FIBROSIS AND SCLEROSIS 155.(W0 99;36541) INTERLEUKIN-I RECEPTOR ANTAGONIST
BETA (IL-1 RA&bgr;) I 56.(W0 99%33806) 4-[ARYL(1'IPERIDIN-4-YL)]

WHICH BIND TO THI; DELTA-OPIOID RIJCEPTOR 157.(W0 99/31099) INTEGRIN R6'CEPTOR
ANTAGONISTS 158.(W0 99/3 1061) INTEGRIN RECEPTOR ANTAGONISTS 159.(WO 99/30713) INTEGR1N RECEPTOR ANTAGONISTS 160.(W0 99!30709) INTEGRIN RECEPTOR
ANTAGONISTS 161.(WO 99129729) ANTAGONISTS OF NEUROPILIN RECEPTOR FUNCTIONAL
AND USE THEREOF 162.(W0 99.'27962) USE OF A FIBRINOGEN F;ECEPTOR-ANTAGONIST
FOR
PREVENTING DISSEMINATED INTRAVASCULAR COAGULATION 163.(W0 99/26945) 1,;,4-THIADIAZOLES AND 1,3,4-OXADIAZOLES AS &agr; v &bgr; 3 A''dTAGONISTS 164.(V1'0 99'26943) THROMBIN RECEPTOR ANTAGONISTS 165.(W0 99/25857) TRANSGENIC MODELS OF
INFLAMMATORY DISL:ASE 166.(V1'0 99;24471) OPIATE, CANNABINOID. AND ESTROGEN
RECEPTORS 167.(W0 99,!24423) PIPERIDINE DERIVATIVES AND THEIR USE AS TACT-IYKININ
ANTAGONISTS 168.(W0 99/24421) IMIDAZOYLALKYL SUBSTITUTED WITH A FIVE, SIX OR
SEVEN MEMBERED HETEROCYCLIC RING CONTAINING ONE NITROGEN ATOM 169.(W0 99/24406) PHENYL-ALKYL-1MIDAZOLES AS H3 RECEI'T'OR ANTAGONISTS 170.( WO
99/24405) H 3 RECEPTOR LIGANDS OF THE PHENYL-ALKYL-IMIDAZOLES TYPE 171.(W0 99/21555) ADENOSINE A3 RECEPTOR ANTAGONISTS 172.(W0 99/20758) HUMAN TUMOR NECROSIS
FACTOR RECEPTOR-LIKE PROTF;INS TR I I, TRI I SV1, AND TRI I SV2 173.(W0 99/19462) ENHANCED IMMUNOCiENIC CELL. 1'OPUI.ATIONS PREPARED USING H2 RE:CEPTOR
ANTAGONISTS 174.(W0 99/17773) COMPOUNDS AND METHODS. 175.(W0 99!16465) METHOD
FOR INHfBfTING TUMOR ANGIOGENESIS IN A LIVING SUBJEC f 176.(W0 991 I 1790) TUMOR
NECROSIS FACTOR RECEPTOR ZTNFR-6 177.(W0 99.%06049) fN T E:GR1N RECEPTOR
ANTAGONISTS 178.(WO 99,%04001 ) TUMOR NECROSIS FACTOR RECEPTOR ZTNFR-5 179.(W0 99/02499) QUINOLINE COMPOUNDS AND MEDICINAL USES THEREOF 180.(W0 99!01764) METHOD FOR RECOGNIZING AND DETE:RMINING GNRIi RECEPTORS AND THE USE OF

GNRH AGONIS'TS AND GNRH ANTAGONISTS AND O-ft-lER GNF;H RECEPTOR LIGANDS FOR
THE TREATMENT WITH GNRH RECEPTORS OF TUMOURS ORIGINA~'ING IN THE BRAIN
AND/OR NERVOUS SYSTEM AND/OR MENINGE:S AND/OR OF KAPOSI SARCOMA 181.(W0 99/01444) POLYMORPHIC FORM OF THE; TACH~'KININ RECEI'~TOR ANTAGONIST 2-(R)-( I-(R) -(3,5-BIS(TRIFLUOROMETHYL) PHENYL)ETHOXY)-3-(S)-(4-FLUORO) PHENYL.-4-(3-5 (-0X0-1 H.4H-1,2,4,-TRIAZOI-O) METHYLMORPHOLINE, 182.(W0 99-O1 127) COMPOUNDS AND
METHODS 183.(W0 99/00406) CYCLIC AGONISTS AND ANTAGONISTS OF C5a RECI~PT'ORS
AND G PROTEIN-COUPLED RECEPTORS 184.(W0 98/58674) ANTI-TUMOUR
PHARMACEUTICAL. COMPOSITIONS CAPABLE OF REDUCING DRUG RESISTANCE IN
TUMOUR CELLS 185.( WO 98'57647) COUP-TFII: AN ORPHAN NUCLEAR RECEPTOR REQUIRED
FOR ANGIOGENESIS 186.(W'0 98156892) HUMAN TUMOR NECROSIS FACTOR RECEPTOR TR9 187.(W0 98!56779) 4-SUL,FINYL BENZAMIDES AS CALCITONIN GENE-RELATED PEPTIDE
RECEPTOR ANTAGONISTS 188.(W0 98155153) NON-STEROIDAL RADIOI_ABELED
AGONIST/ANTAGONIST COMPOUNDS AND THEIR USE IN PROSTATE CANCER IMAGING
189.(WO 98!54325) Hl~MAN FRP AND FRAGMENTS THEREOF INCLUDING METHODS FOR
USING THEM 190.(WO 98/54202) HUMAN TUMOR NECROSIS FACTOR RECE:P'TOR TR10 191.(W0 98/54201) LIUMAN TUMOR NECROSIS FACTOR RECEPTOR-LIKE PROTEIN 8 192.(W0 98/54187) SPIRO-AZACYCLIC DERIVATIVES AND THEIR L1SE AS THERAPEUTIC AGENTS
193.(W0 98!53069) GDNF RECEPTORS 194.(W0 98!49170) SPlRO-.AZACYCLIC' DERIVATIVES
AND THEIR USE AS 1 HERAPEIJTIC AGENTS 195.(W0 98/48017) FAMILY OF
IMMUNOREGULATORS DESIGNATED LEUKOCYTE: 1MMLJNO~iLOBUL,IN-LIKE RECEPTORS
(L.IR) 196.(W0 98/47923) IL-5R ANTAGONISTS FOR TREATMENT OF INFLAMMATION, ASTHMA AND OTIlE:R ALLERGIC DISEASES 197.(W0 98!46751 ) OSTEOI'ROTEGERIN
BINDING
PRO'fEINS AND RECI-:PTORS 198.(W0 98'46620) .A NOVEL HUMAN G-PROTEIN COUPLED
RECEPTOR 199.(W0 98/46265) METHODS FOR USING ANTAGOTJISTIC ANTI-AVB3 1NTEGRIN
ANTIBODIES 200.(WO 98/43962 ) HETEROCYCLIC INTEGRIN INHIBITOR PRODRUGS 251.(W0 97/44333) 1,2,4-OXADIAZOLE:S AS ADHESION-RECEPTOR ANTA.GONIS'TS 252.(W0 97/44329) DIARYL,ALKYL CYCLIC DIAMINE DERIVATIVES AS CHEMOKINE RECEI'I OR ANTAGONISTS
253.(W0 97141225) MAMMALIAN MIXED LYMPHOCYTE RECEPTORS. CHEMOKINE
RECEPTORS [MMLR-C'CR] 254.(W0 97/37655) &agr;v&b~r;3 ANTAGONISTS 255.(W0 97/35969) PEPTIDE LIGANDS OF THE UROKINASE RECEPTOR 256.(W0 97/34878) SUBS'1-ITUTED 2,3-BENZODIAZEPIN-4-0NES AND THE LJSE T'EIEREOF 257.(W0 9733904) DEATH DOMAIN
CONTAINING RECEPTORS 258.(W0 97!33887) SPIROCYCLE IN'TE:GRIN INHIBITORS
259.(W0 97/33613) PARASITE-DERIVED ANTI-INFLAMMATORY IMMUNOMODULA~I'ORY PROTEIN
260.(W0 97%30991 ) NOVEL SUBSTITUTED N-METHYL-N-(4-(4-( I li-BENZIMIDAZOL-2-YL)[1,4]DIAZEPAM-I-YL)-2-(ARYL)BUTYL)BENZAMII)ES USEFUL FOR THE TREATMENT OF
ALLERGIC DISEASES 261.(W0 97130990) NOVEL SUBSTITUTED N-MEl-HYI,-N-(4-(P1PERIDIN-1-YL)-2-(ARYL)BUTYL)BENZAMIDES USEFUL FOR THE ~fREA~fMEN f QF ALLERGIC
DISEASES 262.(W0 97'30989) NOVEL SUBSTITU fED N-METHYL-N-(4-(4-( I II-BENZIMIDAZOL-2-YL-AMINO)P IPER1DIN-1-YL)-2-(ARYI.)BUTYL.)BENZAMIDE'S USEFIII_ FOR THE
TREATMENT OF ALLERGIC DISEASES 263.(W0 97-30079) PEPTIDE ANTAGONISTS OF
CELLULAR MITOGENESIS AND MOTOGENESIS AND TIiEIR THERAPEUTIC USE 264.(W0 97130069) 17-BETA-Cl'CL.OPROPYL(AMINO/OXY) 4-AZA STEROIDS AS ACTIVE
INLTIBITORS
OF TESTOSTERONE 5-ALPHA-REDUCTASE AND C17-20-LYASE 265.(W0 97'29775) COMfOSI'TIONS COMPRISING A CYCLOOXYGENASE-2 INHIBI fOR AND A LEUKOTRIENE B
4 RECEPTOR ANTAGONIST 266.(W0 97/29079) NOVEL COMPOUNDS AND PHARMACEUTICAL
USE THEREOF 267.(W0 97/28190) CYTOKINE ANTAGONISTS AI'JD AGONISTS 268.(W0 97/24373) MONOCLONAL ANTIBODY AN-fAGONISTS'fO HAEMOPOIETIC GROWTH FACTORS
269.(W0 97/23480) NOVEL INTEGRIN RECEPTOR ANTAGONISTS 270.(W0 97,'2260=I) NOVEL.
SUBS'fITUTED 4-(lE1-BENZIMIDAZOL-2-YL)[1,4]DIAZEPANES USEFUL FOR THE TREATMENT
OF ALLERGIC DISEASES 271.(WO 97/21732) DESIGN OF HORMONE-LIKE ANTIBODIES WITH
AGONISTIC AND ANTAGONISTIC FUNCTIONS 272.(W0 97!21702) 3-ANTAGONISTS 273.(W0 97!21445 ) VASCULAR IRRIGATION SOLUTION AND ME HOD FOR
INHIBITION OF PAIN, INFLAMMATION. SPASM AND RESTENOSIS 274.(WO 97/20062) IL-12 P40 SUBUNIT FUSION POLYPEPTIDES AND USES THEREOF 275.(W0 97/19074 ) SUBSTfTUTED
4-(I H-BENZIMIDAZOI.-2-YL-AMINO)PIPERIDINI?S USEFUL FOF; THE TREATMENT OF
ALLERGIC DISEASES 276.(W0 97% 19059) NOVEL SUBSTITUTED ARYL COMPOUNDS USEFUL
AS MODULATORS Of ACETYLCHOLINE RECEPTORS 277.(W0 97/ 16442) SUBSTITUTED
PYRIDYL PYRROLES. COMPOSITIONS CONTAlNINCi SUCH Ci:)MPOUNDS AND METIiODS OF

USE 278.(W0 97/16302) CYTOKINES AND THEIR LJSE IN TREATMENT AND.'OR
PRC)PHYLAXIS
OF BREAST CANCER 279.(W0 97'1619) ENHANCED ANTI-INFLAMMATORY ORAL
COMPOSITION CONTAINING H 2 RECEPTOR AN'FAGONIS f APJD ANTIMIC ROBIAI. OILS
280.(W0 97/15298) COMBINATION OF LTD, RECEPTOR ANTAGONISMS WI'H
GLUCOCORTICOSTE:KOIDS 281.(W0 97114671 ) CYCLOPENTYL TACIIYKININ RECEPTOR
ANTAGONISTS 282.(W0 97% 137 I) 1NDOLE CARBAMATES AS I_EUKOTRIENE ANTAGONISTS
283.(W0 97!l3~ 14) NK-1 RECEPTOR ANTAGONISTS FOR PREVENTION OF NEUROGENIC
INFLAMMATION IN GENE THERAPY 284.(W0 97!09046) COMPOUNDS AND METHODS
285.(W0 97/07130 BINDING OF OSTEOGEN1C PROTEIN-1 (OI'-1 ) AND ANALOGS THEREOF
TO
THE CELL SURFACE RECEPTOR ALK-I AND ANALOGS THEREOF) With respect to clinical and pre-clinical trial development see Antibody Therapeutics Producaion, Clinical Trials, and Strategic Issues, By Rathin C. Das, Ph.D.. M.B.A. & K. John Morrow, Jr., Ph.D., D&MD
Publications October 2001, Chapter 6.
Entity Associated and Entity Specific Markers The term marker is used broadly to refer to any' ligand or binding site for a "targeting" or an "effector" moiety of a multispecific ligand or antibody of the invention and is primarily used herein to refer to ligands which are the target of a "targeting" moiety (most often though not exclusively referred to herein as a first ligand binding moiety).
The literature is replete with examples of such markers, as well as antibodies which recognize them.
With respect to cancer markers and immune cell markers, etc. many of these are referred to in Cancer: Principles and Practice of Oncology 6'~' Ed. De Vita et al. Eds Lippincott 2001 and some are summarized at pages 309-311, 3197.
With respect to markers for osteoclasts and antibodies that bind thereto see for example Endocrinology 1989 Aug; 125(2):630-7; Endocrinology 1990 Dec: 127(6): 3215-21;
Lab Invest Apr;
60(4):532-8; Calcif Tissue Int 1998 Aug; 63(2): 148-53. Such markers could be used for example to target RANK (associated with bone resorption etc.) on osteoclasts using a relatively low affinity second ligand binding moiety.
Other target applications of multispecific ligands of the invention include particularly receptors associated with angiogencsis (eg.VEGFRs 1,2,3) such as KDR, FLK-1 and FLT-1, and various cancers cell types eg. HER-2 and EGF-R, including FGF-R, PDGF-.R, Tek and Tie2. Numerous other examples are referred to specifically and through references to the literature herein cited. Suitable markers for many types of target entities eg. cells bearing such receptors are referred to or referenced herein or described in various subject reviews and texts herein cited, in connection with one or more aspects arid embodiments of the invention described in this application, and many others are known to those skilled in the art and desribed in the literature including antibodies.
With respect to binding to biologic effector ligands the multispecific ligand may comprise a recombinatly produced receptor for such ligand.
As the invention contemplates that the multispectic li~~,ands herein may be used for cancer, it is contemplated that combination therapies with chemotherapeutic and biotherapeutic agents may be used to advantage. Such agents are well known to those skilled in the art and include for example, alkvlating agents, cisplatin anVd its analogues, antimetabolites, topoisomerase interactive agents, antimicrotubule agents, interferon s, interleukins, hormonal therapeutics, differentiation agents, antiangiogenesis agents (see Cancer: Principles and Practice of Oncology 6'" Ed. De Vita et al. Eds Lippincott 3001 pp.335-~ 17) With respect to ligands involved in mediating apoptosis see also W001~I4808 METHODS OF
DIAGNOSIS AND TREATMENT BY BINDING P73iAIRM 1 WOOl~I4282 BCL-G POLYPEPTIDES, ENCODING NUCLEIC' ACIDS AND METHODS OF USE US6242S69 Regulators of apoptosis EP1106183 Antibodies to erbB2 and their therapeutic uses WOO 13659~I Mcl-I
GENE REGULATORY
ELEMENTS AND A PRO-APOPTOTIC Mcl-1 VARIANT US2001001712 Monoclonal antibodies having property of causing apoptosis W00134798 CLONING AND CHARACTERIZA-fION
OF VIRAL
IAP ASSOCIATED FACTOR (VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducingapoptosis HU0003513 MONOCLONAL ANTIBODY INDUCING APOP'1'OSIS EP1094316 Method for the detection of DNA replicating cells US62074~2 Antibody of the anti-proliferation domain of human Bcl-2 W00123568 NOVEL MEMBERS OF THE LAP GENE FAMILY US6190661 Methods and compositions for the use of apurinic/apyrimidinic endonucleases EP 1087993 FAS PEPTIDES AND
ANTIBODIES FOR MODULATING APOP'FOSIS WO01 19861 APO-.', RECEPTOR ANTIBODIES
US6184034 Deoxyribonuclease 1l proteins and cDNAS US617221 1 Nucleic acid encoding tag?
polypeptide WO01 18042 APOPTOSIS PROTEINS Vv'001 16180 CD40 LIGAND AND CD40 AGONIST

CELL DEATH REGULATION W00144808 ME'T'HODS OF DIAGNOSIS AND TRC:ATMENT BY
BINDING P7~-AIRM 1 W00144282 BCL-G POI_YPEPTIDES, ENCODING NUCLEIC ACIDS AND
METHODS OF USE US6242569 Regulators of apoptosis EPl 106183 Antibodies to erbB2 and their therapeutic uses W00136594 Mcl-I GENE REGULATORY ELEMENTS AND A PRO-APOPTOTIC
Mcl-1 VARIANT US2001001712 Monoclonal antibodies having property of causing apoptosis (VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducing apoptosis MONOCLONAL ANTIBODY INDUCING APOPTOSIS EP1094316 Method for the detection of DNA
replicating cells US6207452 Antibody of the anti-proliferation domain of human Bcl-2 W00123~68 NOVEL MEMBERS OF 'THE LAP GENE FAMILY US6190661 Methods and compositions for the use of apurinic,%apyrimidinic endonucleases EP1087993 FAS PEPTIDES AND ANTIBODIES FOR
MODULATING APOPTOSIS W00119861 APO-3 RECEPTOR ANTIE30DIES US(i184034 Deoxyribonuclease II proteins and eDNAS US617221 I Nucleic acid encodin~~
ta~~7 polypeptide WO01 18042 APOPTOSIS PROTEINS WO01 16180 CD40 L.IGAND AND CD40 AGONIST

CELL DEATH REGULrI fION WO01=14808 METI-iODS OF DIAGNOSIS AND fRE:ATMENT BY
BINDING P75!AIRM1 WO0144282 BCL-G POLYPEPTIDES, ENCODING NUCLEIC ACIDS AND
METHODS OF USE US(i242569 Regulators ofapoptosis EP1 106183 Antibodies to erbB2 and their therapeutic uses W00136594 Mcl-1 GENE REGULA'I ORY ELEMENTS AND A PRO-APOI'TOTIC
Mcl-1 VARIANT US2001001712 Monoclonal antibodies having property of causin~~
apoptosis W00134798 CLONING AND CHARACTERIZATION Of VIRAL LAP ASSOCIA'T1D FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducing apoptosis MONOCLONAL ANTIBODY INDUCING APOPTOS1S EP1094316 Method for the detection of DNA
replicating cells US6207452 Antibody of the anti-proliferation domain of human Bcl-'? W00123568 NOVEL MEMBERS OF -CHE IAI' GENE FAMILY US6190661 Methods and compositions for the use of apurinic,!apyrimidinic endonucleases EP1087993 FAS 1'EP'FIDES AND ANTIBODIES
FOR
MODULATING APOPT(:)SIS WO01 19861 APO-2 RECEPTOR ANTIBODIES US6184034 Deoxyribonuclease 1I proteins and eDNAS US617221 t Nucleic acid enc~~ding tag?
polypeptide CELL DEATH REGULA CION W00144808 METI-fODS OF DIAGNOSIS AND 'TREATMENT BY
BINDING P7~lAIRM I W'00144282 BCL-G I'OLYPEPTIDES, ENCODING NUCLEIC ACIDS AND
METHODS OF USE US(i242569 Regulators of apoptosis EP1 106183 Antibodies to erbB2 and their therapeutic uses W00136594 Mcl-I GENE REGULATORY ELEMENTS AND A PRO-APOf'TOTIC
Mcl-1 VARIANT US2001001712 Monoclonal antibodies havin<~ property of causing apoptosis W00134798 CLONING AND CHARACTERIZATION OF V1RAI. LAP ASSOCIATI:v:D FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducin~~ apoptosis FiU0003513 MONOCLONAL ANTIBODY INDUCING APOP'TOSIS EP 1094316 Method for the detection of DNA
replicating cells IJS6207452 Antibody of the anti-proliferation domain of human Bcl-2 WO01:?3568 NOVEL MEMBERS OF TE-IE LAP GENE FAMILY (JS6190661 Methods and compositions for the use of apurinic;apyrimidinic endonucleases EP1087993 FAS PEPTIDES AND .ANTIBODIES FOR
MODULATING APOPT()SIS WO01 19861 APO-3 RECEPTOR AN'FIEtODII=,S US6184034 Deoxyribonuclease 1l proteins and eDNAS US617221 l Nucleic acid encoding ta~~7 polypeptide WO01 18042 APOPTOSIS PROTEINS WO01 16180 CD40 LIGAND AND CD40 AGONIST' COMPOSITIONS AND ME'hHODS OF USE WO01 16170 NOVEL, CARD PROTEINS INVOLVED IN
CELL DEATH REGULATION WO0144808 METHODS OF DIAGNOSIS AND'FREATMENT BY
BINDING P7~%AIRMI W'00144282 BCL-G POLYPEPTIDES, ENCODING NUCLEIC ACIDS AND
METHODS OF USE US6242569 Regulators of apoptosis EPl 106183 Antibodies to erbB2 and their therapeutic uses W00136~94 Mel-1 GENE REGULATORY ELEMENTS AND A PRO-APOPTOTIC
Mcl-1 VARIANT US2001001712 Monoclonal antibodies having property of causin~~
apoptosis W00134798 CLONING AND CHARAC1'ERIZA'I ION OF VIRAL, LAP ASSOCIATED FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducing apoptosis MONOCLONAL ANTIBODY INDUCING APOPTOSIS EP 10943 l6 Method for the detection of DNA
replicating cells US62074~2 Antibody of the anti-proliferation domain of human Bcl-2 WO01:'3568 NOVEL MEMBERS OF THE IAP GENE FAMILY US6190661 Methods and compositions for the use of apurinic/apyrimidinic endonucleases EP1087993 FAS PEPTIDES AND ANTIBODIES FOR
MODULATING APOPTOStS W00119861 APO-? RECEPTOR ANTIBODIES US6184034 Deoxyribonuclease II proteins and cDNAS U5617221 I Nucleic acid encoding tag?
polypeptide CELL DEA'fli REGULATION W00144808 METHODS OF DIAGNOSIS AND'TRfATMENT BY
BINDING P75!AIRMI Vv'0014428? BCL-G 1'OL.YPEPTIDES, ENCODING NUCI_f:IC ACIDS
AND
METHODS OF USE US6242569 Regulators of apoptosis EP 1 106183 Antibodies to erbB2 and their therapeutic uses W00136594 Mcl-1 GENE REGULATORY ELEMENT'S AND A PRO-APOPTOTIC
Mcl-1 VARIANT US?001001712 Monoclonal antibodies having property of causin~~
apoptosis WOOi34798 CLONING AND CHARACTERIZATION OF VIRAI_ IAP ASSOCIATf:D FACTOR
(VIAF) EN SEVERAL, ORGANISMS CZ20000907 Monoclonal antibody inducing apoptosis MONOCLONAL ANTlE3ODY INDUCING APOPTOSIS EP1094316 Method tbr the detection of DNA
replicating cells US6207452 Antibody ofthe anti-proliferation domain ofhuman Bcl ? W00123s68 NOVEL MEMBERS OF 'THE IAf GENE FAMILY US6190661 Methods and compositions for the use of apurinicapyrimidinic endonucleases EP1087993 FAS PEPTIDES AND.4NTIBODEES FOR
MODULA'hING APOPTOSIS Vl'001 19861 APO-2 RL:CEPTOR AN'fIBODIES US6184034 Deoxyribonuclease 1l proteins and cDNAS USC 17221 1 Nucleic acid encoding tag?
polypeptide WOOL 18042 APOPTOS1S PROTEINS U1~'001 16180 CD40 LIGAND AND CD40 AGONIST
COMPOSITIONS AND METHODS OF USE W'001 Ifi170 NOVEL. CARD fRO'I EINS INVOLVED

CELL. DEATH REGULATION W00144808 METHODS OF DIAGNOSIS AND T Rf:ATMENT BY
BINDING P75~'AIRMI W00144282 BCL-G POLY PEPTIDES. ENCODING NUCLEIC ACIDS AND
METI-IODS OF USE US6242569 Regulators of apoptosis EP 1 106183 Antibodies to erbB2 and their therapeutic uses W00131i594 Mcl-1 GENE RE:GUL.ATORY ELEMENTS AND A PRO-APOP'fOTIC
Mcl-1 VARIANT' US2001001713 Monoclonal antibodies having property of causing apoptosis W00134798 CLONING .AND CHARACTERIZATION OF VIRAL IAP ASSOCIATED FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducin~~ apoptosis MONOCLONAL ANTIBODY INDUCING AfOPTOSIS EP 1094316 Method for the detection of DNA
replicating cells US6207452 Antibody of the anti-proliferation domain of human Bcl-' W00123568 NOVEL_ MEMBERS OF THE IAP GENE FAMILY 1JS6190661 Methods and compositions for the use of apurinic.%apyrimidinic endonucleases EP1087993 FAS PEPTIDES AND ANTIBODIES FOR
MODULATING APOPTC)SIS W'001 19861 APO-2 RECEPTOR AN TI E3ODIES US6184034 Deoxyribonuclease 1l proteins and cDNAS US617221 I Nucleic acid encoding tag?
polypeptide WO01 18042 APOfTOSIS PROTEINS WO01 16180 CD40 L1GAND AND CD40 AGON1ST
COMPOSITIONS AND METHODS OF LJSE WO01 16170 NOVEL CARD PRO~fEINS INVOLVED IN
CELL DEATH REGULATION V~'OO144808 METHODS OF DIAGNOSIS AND 'fREA'rMENT BY
BINDING P75/AIRM I WOO144282 BCL-G POLYPEfTIDES. ENCODING NLJCI.EIC ACIDS AND
METHODS OF USE US6242569 Re~~ulators of apoptosis EPI 106183 .Antibodies to erbB2 and their therapeutic uses W0013(i594 Mcl-1 GENE R.E:GLJLATORY ELEMENTS AND A I'RO-AfOPTOTIC
Mcl-I VARIANT LJS200100171? Monoclonal antibodies having property of causing apoptosis W00134798 CLONING AND CHARACTERIZATION OF VIRAL. IAP~ASSOCIATED FACTOR
(VIAF) 1N SEVERAL ORGANISMS CZ?0000907 Monoclonal antibody induciny~ apoptosis MONOCLONAL ANTIBODY INDUCING APOPTOSIS EP1094316 Method for the detection of DNA
replicating cells US6207=453 Antibody of the anti-proliferation domain of human Bcl-2 W001~3568 NOVEL MEMBERS OF'fHE IAP GENE FAMILY LJS6190661 Methods and compositions for the use of apurinic;'apyrimidinic endonucleases EP1087993 FAS PEPTIDES AND ANTIBODIES FOR
MODULATING APOPTOSIS WO01 19861 APO-? RECEPTOR ANTIBODIES 1JS6184034 Deoxyribonuclease 11 proteins and cDNAS US617221 1 Nucleic acid encoding tag?
polypeptide WO01 18042 APOPTOSIS PROTEINS W001 1618() CD40 L,IGAND AND CD40 AGON1ST' COMPOSITIONS AND ME'ft-IODS Of USE WOOI 16170 NOVEL CARD PROTEINS INVOLVED IN
CELL DEATH REGULATION Vf00144808 METHODS OF DIAGNOSES AND'fRE.ATMENT BY
BINDING P75/AIRMI W00144282 BCL-G POLYPEPTIDES, ENCODING NUCLEIC,' ACIDS AND
METHODS OF USE US6?42569 Regulators of apoptosis EP I 106183 Antibodies to erbB2 and their therapeutic uses W00136594 Mcl-1 GENE REGULATORY ELEMENTS AND A PRO-APOPTO'TIC
Mcl-1 VARIANT US200t001712 Monoclonal antibodies havin~~ property of causin~~
apoptosis W00134798 CLONING AND CHARACTERIZATION OF VIRAL, IAP ,ASSOC.'IATED FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducing apoptosis MONOCLONAL ANTIBODY INDUCING APOPTOStS EP1094 316 Method for the detection of DNA
replicating cells US6?07452 Antibody of the anti-proliferation domain of human Bcl-2 W00123568 NOVEL MEMBERS OF THE IAP GENE FAMILY US6190661 Methods and compositions for the use of apurinic/apyrimidinic endonucleases EP1087993 FAS PEPTIDES AND ANTIBODIES FOR

Deoxyribonuclease II proteins and cDNAS LJS617221 1 Nucleic acid encoding tag?
polypeptide WO01 18042 APOPTOSIS PROTEINS WO01 16180 CD40 LIGAND AND CD40 AGONIS-f CELL DEATH REGULATION W00144808 METHODS OF DIAGNOSIS AND TREATMf:N1' BY
BINDING P75!AIRMI W00144282 BCL-G POLYPEPTIDES, ENCODING NUCLEIC ACIDS AND
METHODS OF USE US6242569 Re;.,~ulators ofapoptosis EPl 106183 Antibodies to erbB2 and their therapeutic uses W00136594 Mcl-1 GENE REGULATORY ELEMENTS AND A PRO-APOPTOTIC
Mcl-1 VARIANT US2001001712 Monoclonal antibodies having propc;rty of causing apoptosis W00134798 CLONING AND CHARACTERIZATION OF VIRAL IAP ASSOCIA~CED FACTOR
(VIAF) IN SEVERAL ORGANISMS CZ20000907 Monoclonal antibody inducing apoptosis MONOCLONAL., ANTIBODY INDUCING APOPTOSIS EP1094316 Method for the detection of DNA
replicating cells US6207452 Antibody of the anti-proliferation domain of human Bcl-? W00123568 NOVEL MEMBERS Of THE IAP GC:NE FAMILY US6190661 Methods and compositions for the use of apurinie/apyrimidinic endonueleases EP1087993 FAS PEPTIDES AND ANTIBODIES FOR
MODULATING APOP'fOSIS Vl-'001 19861 APO-2 RECEPTOR ANTIBODIES US6184034 Deoxyribonuclease II proteins and cDNAS US617221 I Nucleic acid encoding tag?
laolypeptide WO01 18042 APOPTOSIS PROTEINS WO01 16180 CD40 LIGAND AND CD40 AGONIS'f CELL, DEATH REGULATION
As stated above, in a related but also independent aspect, the invention contemplates a method of screening for an antibody which preferentially binds to a ligand when bound to a first receptor relative to another second receptor by screening for antibodies leg. by phage display, ribosome display, etc.) which bind to the ligand eg. a cytokine, when bound in situ to the first receptor, and selecting among them those that bind to the ligand eg. cytokine but do not bind (substractive screening) or bind with lesser affinity when bound to the cytokine to the second receptor, as well as to antibodies and multifunctional li~~ands created by this method (see also tJSP 6.046,048 and WO 99/ 12973 and references cited therein with respect to TNF
family of receptorsj. Variations in the extracellular domains of such rect:ptors are known and can be ascertained by methods known to those skilled in the art. Accordingly the invention is directed to an antibody characterized in that it binds to an epitope on the ligand which permits the li~~and to bind, while the antibody is bound to it. to a first receptor but not a second receptor. Yn a preferred embodiment both are cell surface receptors. In a preferred embodiment the li~~and is a natural ligand, preferably a growth factor, cytokine or chemokine. In another embodiment one of the receptors is a soluble receptor. The invention is also directed to a method of evaluating the pleitropic eti'ects of a natural ligand by administering the said antibody Including antigen binding fratiments thereof and MRUs) and monitoring its etfiects. 1"he invention contemplates that this antibody is a first or second moiety of a multifunctional ligand disclosed herein.
Examples of receptors include the classes of VEGF receptors (see also l :Sheppard D.Integrin-mediated activation of transforming ~~rowth factor-beta( 1 ) in pulmonary fibrosis.C'hest. 2001 Jul;120( I Suppl):549-53. Chow D, Ho J, N~~uyen Pham Tl., Rose-John S, Garcia KC.In vitro reconstitution of recognition and activation complexes between interleukin-6 and gp130.Biochemistry. 2(101 .lun 2G;40('25):759i-603.
Kotenko SV, Izotova LS, Mirochnitchenko OV. Esterova E, Dickensheets H.
Donnelly RI', Pestka S.Identification, cloning, and characterization of a novel soluble receptor that binds I1.-22 and neutralizes its activity.J lmmunol. 2001 Jun 15;166(12):7096-103. Gustin SE, Church AP, Ford SC., Mann DA, Carr PD, Ollis DL, Young IG.Expression, crystallization and derivatization of the complete extracellular domain ofthe beta(c) subunit of"the human IL-5, IL-3 and CiM-CSF receptors.Eur.l Bioehem. 2001 May;268( 10):2905-1 1. MeCall AM. Shahied L, Amoroso AR, tJorak EM, Simmons IiH, Nielson U, Adams GP, Schier R, Marks JD, Weiner LM.Increasing the aftinity for tumor antigen enhances bispecitic antibody cytotoxicity.J Immunol. 2001 May 15;166( 10):61 12-7. Piehler J, Roisman LC, Schreiber G.New structural and functional aspects of the type I interferon-receptor interacrion revealed by comprehensive mutational analysis ofthe binding interface.) Biol Chem. 2000 Dec 22;27501):40425-33.DLINE]7:
Wiesmann C, Muller YA, de Vos AM.Ligand-binding sites in I~ like domains of receptor tyrosine kinases.J Mol Med. 2000;78(5):247-60. Review.DLINE)8: Born TL" Sn nith DE:, Garka KE, Renshaw BR, Bertles JS, Sims JE., Protein, Nucleotide Identification and characterization of two members of a novel class of the interleukin-I receptor (IL.-1 R) family. Delineation of a new e:lass of IL-I R-related proteins based on signaling.) Biol Chem. 2000 Sep 29;275('39):29946-54.DLINE)9: Xia XZ, T'reanor J. Senaldi G, Khare SD, Boone T, Kelley M, Theill LE, Colombero A, Solovyev l, Lee F, McCabe S, Elliott R, Miner K, Hawkins N, Guo J, Stolina M, Yu G, Wang J, Delaney J, Meng SY, Boyle WJ, Hsu LI.. Protein, Nucleotide TACI is a TRAF-interacting receptor for TALL-l, a tumor necrosis factor family member involved in B
cell regulation.) Exp Med. 2000 Jul 3;192(1):137-43.DLINE] 10: Kumaran J, Colamonici OR, Fish EN.Structure-function study of the extracellular domain of the human type l interferon receptor (IFNAR)-1 subunit.J Interferon Cytokine Res. 2000 May;30(5):479-8s.DLINEJ l I: I_u D, Kussie P, Pytowski B, Persaud K, Bohlen P, Witte L, Zhu Z.Identitication of the residues in the extracellular region of KDR
important for interaction with vascular endothelial growth factor and neutralizing anti-KDR antibodies.) Biol Chem. 2000 May 13:275(19):14321-30.DLINE]12: Bowie A, O'Neill LA.T'he interleukin-1 receptor/Toll-like receptor superfamily: signal generators for pro-inflammatory interleukins and microbial products.) Leukoc Biol. 3000 Apr;67(4):~08-14. Review. DL IN E] 13: Kumar S, McDonnell PC, Lehr R, Tierney L, Tzimas MN, Griswold DE, Copper EA, 'fal-Singer R, Wells Cil, Doyle Ml., Young PR.. Protein, Nucleotide. OMIM Identification and initial characterization of four novel members of the interleukin-1 family.) Biol Chem. 2000 Apr 7;275(14):10308-14.DLINE]14: Fujio K, Nosaka T, Kojima T, Kawashima T, Yahata T, Copeland NG. Gilbert DJ, Jerkins NA, Yamamoto K, Nishimura T, Kitamura T., Protein, Nucleotide Molecular clcrnin~ of a novel type I cytokine receptor similar to the common gamma chain.Blood. 2000 Apr 1:95(7):2204-IO.DLINE] 15: Touw IP, De Koning; JP, Ward AC. tiermans MH.Signaling mechanisms of cytokine receptors and their perturbances in disease.Mol Cell Endocrinol.
3000 Feb 2;160( 1-2); I-9. Review.DLINE] 11i: Chritton SL" Shen~, M.C'r'RL. a novel cytokine receptor-like protein expressed in testis, lung, and spleen.Biochem Biophys Res C~~mmun. 2000 .lan 27;367(3):697-703.DLINE] 17: 1.i H. Chen J, Huang A, Stinson J, lieldens S, Foster J, Dovvd P. Gurney AL, Wood W1.
Free in PMC , , Protein, Nucleotide. OMIM Clonin~~ and characterization of IL.-1713 and Il,-17C', two new members ofthe IL-17 cytokine family.Proc Natl Acad Sci U S A. 2000 Jan 18;97(3):773-B.DLINEJ18:
Gary-Gouy H, Bruhns P, Schmitt C, Dallaul A, Daeron M, Bismuth G.The pseudo-immunoreceptor tyrosine-based activation motif of CDS mediates its inhibitory action on E3-cell receptor signalin~'~.J Biol Chem. 2000 Jan 7:27x( 1 ):~48-i6.DLINE] 19: Wiesmann C, LJltsch MH, E3ass SH, de Vos AM., Protein, Structure Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TrkA
receptor.Nature. 1999 Sep 9:401(6749):184-8.DLINEJ20: Liu Y, Cruikshank W W, O'l,ou;,~hlin T, O'Reilly P, Center DM. Kornfeld Il.ldentification of a CD4 domain required for interleukin-16 binding and lymphocyte activation.) Biol Chem. 1999 Aug 13;274(33):23387-95.DL.tNE]21:
Donnelly RP, Dickensheets H, Finbloom DS.The interleukin-10 signal transduction pathway and regulation of gene expression in mononuclear phagocytes.) Interferon Cytokine Res. 1999 Jun;
19(6):563-7 3.
Review.DLINE]32: Kim H. Baumann H.Dual signaling role of the protein tyrosine phosphatase SHP-2 in regulating expression of acute-phase plasma proteins by interleukin-6 cytokine receptors in hepatic cells.Mol Cell Biol. 1999 Aug:19(8):~326-38.DLINE]2:i: Kernebeck T, Ptlanz S, Muller-Newer G, Kurapkat G. Scheek RM, Dijkstra K. Heinrich PC, Wollmer A, Grzesiek S, Grotzinger J.. Protein, Structure. The signal transducer ~p130: solution structure of the carboxy-terminal domain of the cytokine receptor homology region.Protein Sci. 1999 Jan:B(I ):5-12.DL,INE]24: Ernst M, Novak LJ, Nicholson SE, Layton JE. Dunn AR.The carboxyl-terminal domains of gp I _i0-related cytokine receptors are necessary for suppressing embryonic stem cell differentiation. Involvement of STAT3.J Biol Chem. 1999 Apr 2;274(14):9729-37.DLINE]25: Hibi M, Hirano T'.Signal transduction through cytokine rc:ceptors.lnt Rev Immunol. 1998:17(1-4):7s-103. Review.26: Staunton D. Hudson KR, Heath JK.The interactions of the cytokine-binding homolo~,y region and immunoglobulin-like domains of gp130 with oncostatin M:
implications for receptor complex formation. Protein En~~. 1998 Nov; I 1( I I
):1093-102).
With respect to markers that are useful for differentiatin« between various populations and sub-populations of cells. see also F-luman 11.-I 8 Receptor and ST2L Are Stable and Selective Markers for the Respective 'type 1 and Type 2 Circulating Lymphocytes, Woon Ling C,'han, Nada Pejnovic, C:hristinc A. Lee, and Nadia A. AI-Ali, J lnnnunol 2001;167 1238-124=1; CD4+CD25 high Regulatory Cells in Human Peripheral Blood, Clare Baecher-Allan, Julia A. Brown, Gordon J. Freeman, and David A.
Ilatler. J Irnmunol 3001;167 12~t~- I 353.
In another aspect the invention is directed to multifunctional li~and comprising at least a first moiety which specifically binds to a ligand on the surface ofa virus particle that is capable of infecting a mammalian and particularly a human cell including a cancer cell (excludin<~ viruses which are known for use in gene therapy) and is preferably selected from the ~~roup consisting viruses which infect substantial populations of individuals including for example influenza virus and at least a second moiety which speciticallv reco~~nizes a cancer cell, in one embodiment preferably a marker present on multiple different cancer types, especially cancer types that are individually or collectively most prevalent in the general population. In one embodiment such multifunctional ligand is a bispecitic, trispecitic or tetraspecitic antibody. The invention contemplates that such a multifunctional ligand may be used to target such viruses to tumors in a manner which preferentially kills the cancer cells either through the action of the virus and,'or by causing, the immune response to the virus or virus infected cell to preferentially (relative to non-cancer cells f target the cancer cell for ablation. T'he invention is also directed to a method of treating cancer by retar~~eting virus with which an idividual is otherwise infected to the cancer cell eg.
int7uenza.~The invention also contemplates that the multifunctional ligand includes one or more effector moieties which assist in killing the virus and/or cancer cell or directing immune cells to the virus and/or cancer cell, if and when present in the individual, for example a moiety which specifically binds to such immune cell eg. a T cell. as discussed above. Accordingly, the invention also contemplates that such multifunctional ligand may be used to treat infiluenza virus infections and secondarily to act prophylactically as a sentinel against any cancer cells which might develop during the course of the viral infection or a period of immune suppression or increased succeptibility to infection or cancer, including for example, as e-rperienced by individuals with a particular immune suppressive disorder or condition or under treatment with immune suppressive drugs, individuals at risk for cancer or recurrence of a cancer, individuals of a particular ale group, individuals experiencing a period of unusual stress which increases their succeptibility to disease or infection. The invention also contemplates that such a moiety is used in concert with prior immunization a~~ainst the virus.
so that the augemented immune response to the virus benefits the treatment of the cancer cells (see for example USP 6169175 and the art cited therein). The invention also directed to such a virus (excluding viruses known for use in gene therapy applications e~,. adenovirus) which is engineered to expresses on its surface a cancer targeting moiety such as a scfv (see for example EP 1038967, WO 94i (0323 and the art cited therein). The invention is also directed to a method of identifying the expression or over-expression of cell surface markers associated with infection by such a virus, by substractive screenin« relative to markers also expressed on non-infected such cells, for example usin~~ pha~Te display or the like. Such markers may be used for vaccine-type or other imrnunotherapeutic strategies. Anti-virus, markers includin~~ influenza virus markers and methods of identitying new such markers are well known in the art (see for example USP 5589174) (see also The role of the antibody response in influenza virus infection., Gerhard W.,Curr Top Microbiol Immunol 2001;260:171-90, Fernandez-Sesma A, Schulman JL, Moran TM.A bispecific antibody recognizing influenza A virus M2 protein redirects effector cells to inhibit virus replication in vitro.) Virol. 1996 Ju1;70(7):4800-4; Todorovska A, Roovers RC. Dolezal O, Kor-tt AA. Hoo~=enboom HR, hfudson PJ. Design and application of diabodies. triabodies and tetrabodies for cancer targeting. .l lmmunol Methods. 2001 Feb 1;248( I-2):47-66.. Staerz UD, Yewdell .1W, Bevan MJ. Hybrid antibody-mediated lysis of virus-infected cells. Eur J Immunol. 1987 Apr; 17(4):571-4; Fernandez-Sesma A, Schulman JL, Moran TM.A bispecific antibody recognizing influenza A virus M2 protein redirects effector culls to inhibit virus replication in vitro. J Virol. 1996 Ju1;70(7):4800-4.) The invention is also directed to a multifunctional ligand having at least a tumor cell targeting moiety and a moiety which binds to a tumor antigen which is shed from a cancer cell. In a preferred embodiment, the tumor antigen binding moiety preferably does not reco~~nize the portion of the antigen which is most imrnunogenic and leaves that portion exposed for reco~~nition by the immune system. The invention contemplates generating such preferred antibody or fragment thereof by using an an immune complex between an antibody that hinds to such immunogenic portion and the anti«en as a target for phage display or generation or polyclonal sera. The invention also contemplates identifying antibodies which recognize immunogenic portions of the antigen by screening patient sera for antibodies which reco<rnize the antigen.
The invention also contemplates that such multifunctional ligand includes one or more effector moieties which assist in killing the cancer cell or directing immune cells to the cancer cell, for example a moiety which specifically binds to such immune cell egva T cell receptor, as discussed above.
Without limiting the generality of or applicability of the foregoing, and without being limited by or limiting the scope of the claims, various embodiments of the invention may be summarized for ease of reference as follows: I. A multispecific li~~and comprising at least a first ligand binding moiety which preferentially binds with a first affinity to a first ligand having a first biodistribution*
and at least a second ligand binding moiety which preferentially binds with a second affinity to a second liaand having a second biodistribution which is different' from that of the fwst ligand, and wherein the affinity of first and second ligand binding moieties are selected to bias the biodistribution of the multispecific li~~and. 2. A
multispecific ligand according to paragraph I. wherein said multifunctional ligand comprises one or more ligand binding moieties which are antibodies. 3. A multispecfic ligand according to paragraph I or 2, wherein the affinity of said first li~~and binding moiety for the first ligand is higher than the affinity of the second ligand binding moiety for the second ligand so as to bias the biodistribution of the multispecific ligand in favor of the the first ligand. 4. A multispecific ligand accordin~~
to paragraph 3. wherein the first '' Contrasted to functional affinity which may result from avidity ' see fn 8 and second ligands have overlapping biodistributions.' ~. A multispecfic ligand according to paragraph 4, wherein the first ligand is a target cell population associated ligand and wherein said second ligand is present on a broader population of cells and wherein the biodistribution of the multispecific ligand is skewed in favour of the target cell population. 6. A multispecflc ligand according to paragraph I or >, wherein said first ligand is a marker associated with* one or more specific cell populations, infectious or parasitic agents, diseased cells, or disease associated* cells, optionally one of specific ligands herein mentioned or referenced or known to those skilled in the art. 7. A
multispecific ligand according to para~=raph 6, wherein said marker is a specific biological structure. 8. A
multispecific ligand according to paragraph 6, wherein said marker is a specific receptor or receptor li~and. 9.
A multispecitic ligand according to paragraph 6, wherein said marker is a specific antigen. 10. A
multispecific li~and accordin~~ to paragraph 6, wherein said marker is a specific epitope. 1 1. A multispecific ligand according to paragraph 6, wherein said marker is a CD marker. 12. A multispecific ligand according to paragraph 6, wherein said marker is associated with a cancer cell or pre-cancerous cell. 13. A
multispecitic ligand according to paragraph 6, wherein said marker is associated with an autoimmune disorder or rheumatic disease. 14. A
multispecitic li~and according to paragraph 6, wherein said marker is associated with a specific tissue type.
15. A multispecific ligand according to paragraph 6, wherein said mark.°r is associated with a specific organ. l6. A multispecific ligand accordin<~ to paragraph 6, wherein said marker is associated with a cell or tissue of specific origin or class. 17. A multispecitic li«and according to paragraph 6, wherein said marker is an MHC-peptide complex. 18. A multispecific ligand according to paragraph 6, wherein said marker is associated with a cell surface immunoglobulin. 19. A multispecitic li~and according to paragraph S or 6, wherein said second li~~and is a receptor, family of receptors or one or more particular receptor family members, optionally one of those specific receptors herein mentioned or referenced or known to those skilled in the art. The invention contemplates targeting any receptor present on any population of entities for which there is an entity associated marker. 20. A multispecific ligand according to para~~raph 19, wherein said second ligand is a cell surface receptor chosen from a group comprising tyrosine kinase type receptors, serine 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, optionally one of the specific receptors herein mentioned or referenced or known to those skilled in the art. 21. A
multispecific ligand according to paragraph 19, wherein said receptor requires cross-linking for biological activity. 22. A multispecific ligand according to paragraph s or 6, wherein said second li'~and is a cell surface receptor and wherein said second ligand binding moiety blocks said receptor. 23. A multispecitic ligand accordin<, to paragraph I, 5 or 6.
wherein said second ligand is a receptor ligand and wherein said second li~~and binding moiet~~ blocks interaction with the corresponding receptor. 24. A multispecific ligand according to paragraph 5 or 6, wherein said second ligand is a cell surface receptor which initiates a signal transduction and wherein said second ligand binding moiety effects a signal transduction. 3~. A
multispecific li~and according to para<~raph >. 6 or 19, wherein said antibody comprises a first Vh* which preferentially reco~~nizes said first ligand and a second VH which preferentially reco~~nizes said second ligand.
26. A multispecific ligand according to paragraph 25, wherein at least one of said first and second VHs require the cooperation of a VL for binding to their respective ligands. 27. A multispecitic ligand according to paragraph .?5, comprising a first VL associated with said first VI-1 and a second Vl, associated with said second VH and wherein both said first and second V Hs require the cooperation of a VL for binding to the first and second ligands, respectively, and wherein said first and second VLs are the same' or functionally interchangeable*.
28. A multispecific ligand according to paragraph 2~ or 27, wherein said bispecitic antibody is a tour chain antibody. 29. A multispecific ligand according to paragraph 28, wherein said bispecfic antibody is a minibody, F(ab')= or antibody devoid of a CH3 domain. 30. A multispecifie ligand according to paragraph 25, wherein said bispecitic antibody is a diabody. 31. A multispecific ligand according to paragraph 25 or 27, wherein said bispecific antibody is devoid of light chains. 32. A
multispecitic ligand according to paragraph 31. wherein said bispecific antibody comprises a pair of disulfide linked heavy chains or heavy chain portions each comprising at least a VH re~,~ion, a hinge region and preferably. at least a portion of an Fc region at the carboxy terminus ofthe hinge region. 33. A multispecific liaand accordin~~ to paragraph 31, wherein said bispecitic antibody comprises a pair of VHs linked via a polypeptidc linker. 34. A
' The term epitope though technically understood to be specific for a given antibody, is used to refer to antigenic determinants that are situated proximally to one another so that two antibodies will be considered to bind to the same epitope if one competivelv inhibits the binding of the other throu~~h any probative competitive inhibition experiment known to those skilled in the art.
have substantially the same amino acid composition ie. with possible exception of one or more additions, deletions or substitutions including conservative amino acid substitutions which do not substantially affect the specificity and amino acid composition of the paratope multispecitic ligand according to paragraph 3, 5, 6 or 19 wherein the affinity of the first ligand binding moiety for the first ligand is at least approximately c', one, two, three, four, five, six, seven or eight orders of magnitude greater than the affinity of said second ligand binding moiety for the second li~~and ~3~. A
composition comprising a multispecific ligand in a pre-determined dosage, said multispecitic ligand comprisinvt a first ligand binding moiety which preferentially binds with a pre-selected first affinity to a first ligand having a first biodistribution and a second ligand binding moiety which preferentially binds with a pre-selected affinity to a second ligand having a second biodistribution, which is different from that of the first ligand, and vaherein the affinity of the first and second ligand binding moieties are selected to bias the biodistribution of the multispecific ligand in favor of a selected location of one or both of the ligands' such that a desired proportion of the dosage is delivered to the selected location. 36. A
multispecific ligand according to paragraph 1 or 35, wherein the biodistributions of said first and second ligands overlapx and wherein the affinities of the first and second ligand binding moieties are selected to bias the biodistribution of the multispecific ligand in favour of a target cell population on which both first and second ligands are bioavailable for recognition by the first and second ligand binding moieties, relative to one or more non-target cell populations. 37. A multispecitic ligand according to paragraph 3ti, 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 bindings moiety for the second li~~and. 38. A multispecific ligand according to paragraph 3(i or p7, wherein first and second ligands are bioavailable fbr contemporanous* recognition by the first and second ligand binding moieties.
39. A method of controlling the biodistribution of a ligand which interacts with a tar~~tt li~~and present on a hetero<~enous population of ligand bearing entities, said method comprising using a multispecitic ligand comprising at least a first ligand binding moiety which preferentially* binds with a pre-selected* first af~inity9 to at least a first ligand associated with a target sub-population of said hetero~~eneous population on which said first ligand and target (second) li'~and are bioavaible for contemporaneous recognition and a second ligand binding moiety which preferentially binds with a pre-selected lesser af~tinity to said target ligand, and wherein the affinity of first and second ligand binding moieties are selected to bias the biodistribution of the multispecific ligand in favor of said target sub-population of ligand bearings entities.
40. A method of testing the biological effects of lirnitin'; the biodistribution ofa ligand which interacts with a target ligand present on a heterogenous population of lli~~and bearings entities, said method comprising the step of administering a multispecitic li<~and comprising at least a first ligand binding moiety which preferentially* binds with a pre-selected* first affinity "' to at (east a first ligand associated with a target sub-population of said population of ligand bearing entities on which said first li~~and and target (second) ligand are bioavaible for contemporanous recognition and a second ligand binding moiety which preferentially binds with a pre-selected lesser affinity to said tartlet ligand, and wherein the affinity of first and second li~~and bindiny_ moieties are selected to bias the biodistribution of the multispecitic li~~and in favor of said target sub-population of ligand bearing entities. 41. A
multispecitic li'.~and which preferentially binds to a target li~~and on a selected sub-population of a heterogeneous population of cells bearing the target-ligand, the multispecific ligand comprising a first ligand binding moiety which preferentially binds to a cell sub-population associated ligand and a second ligand binding moiety which binds to the tar~~et-ligand. said first li~~and 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 tartlet ligand.
G the term approximately in the context of orders of ma~~nitude variations in affinity refers a variability that is up to a half an order or magnitude.
' having regard to their respective bioavailabilities R The term "overlap" connotes that notwithstanding the difference in distributions of the first and second ligands the first and second ligands are bioavailable for recognition on the same entity. this term and related terms, exemplified below, are intended to exclude a situation where both ligands are preferentially expressed on substantially the same entity, for example mvo different tumor associated antigens associated differentially with a differentiated population of cells within a tumor , most paoicularly in the case where they are individually suitable tartlets for delivery of a toxic payload, and the terms wditterent" distributions and "heterogeneous" population are similarly understood to exclude such a common distribution, in the appreciation that the invention primarily represents an improved strategy for targeting two different ligands, in which one li~~and has a broader distribution than the other or both have distributions that may overlap but are different from that of the target population. It will also be appreciated that the invention has particular application to a situation in which at least one of the non-target populations is one on which one of said first and second ligands is substantially represented (in contrast to one on which it simply enjoys limited expression).
'' Contrasted to functional affinity which may result from avidity '° Contrasted to functional affinity which may result from avidity 42. A multispecific ligand according to paragraph 41, wherein the affinity of said first ligand binding moiety for the cell sub-population associated li<~and is approximately, one, two, three. tour, five, six, seven or eight orders of magnitude greater than the affinity of said second ligand binding moiety for said target ligand. 43. A multispecitic ligand according to para<graph 42, wherein sai<t target ligand is a receptor or a receptor ligand'r . 44. A rnultispecitic ligand according to paragraph 42.
wherein at least one of said first or second ligand binding moieties comprises an antibody heavy chain or functional portions) thereof includin<~ a VH or fra~~ment thereof and an antibody light chain or functional portions) thereof including a VH or fragment thereof. 4s. A method of selectively exerting a biological effect mediated through binding a tar~~et-ligand on a selected sub-population of <r population of cells bearings the target-ligand, the method comprising the step of exposing the cells to a multispecitic ligand comprisin<T a first ligand binding moiety which preferentially binds to a cell sub-population associated ligand and a second ligand bindin~7, moiety which binds to the target ligand, said first ligand moiety having an affinity for the sub-population associated ligand that is hi;~her than the affinity of the second ligand binding moiety for the target ligand.
46. A method accordin~~ to paragraph 4S, wherein the affinity of said first ligand binding moiety for the cell sub-population associated li~~and is at least approximately, one. two, three, four, five, six, seven or eight orders of magnitude ;greater than the affinity of said second li<~and binding moiety for said target ligand. 47.
A method accordin~~ to para~~raph 4>, wherein said tar<~et ligand is a receptor or a receptor ligand. 48. A
method according to para~,raph 4~ wherein at least one~of said first or second ligand binding moieties is an antigen binding firagment'v of an antibody . 49. A method of testing or controllin~~ the biological effects of a ligand binding molecule by circumscribin~~ its ability to bind to a diverse population of cells bearing a complementary target ligand, said method comprising usin<~ said ligand binding molecule together with a different li~~and binding,, molecule which preferentially binds to another target li~,~and which is exclusively or preferentially associated with one or more sub-populations) of said population of cells, and wherein the biological effects of said ligan d bindings molecule are controlled through prior association of said ligand binding molecule with said other li~~and bindin' molecule to form a multispecitic li~.:and and through the affinity of at least one of said li~~and binding molecules being pre-selected to limit the propensity of said ligand binding molecule to bind to cells within said population of cells which do not preferentially express said other target ligand. sU. A method according to paragraph 49, wherein the affinity of said ligand binding molecule for said complementary target ligand is less than the affinity of the other ligand binding molecule for the other target figand. 51. A method according to paragraph 49 or 50, wherein at least one of said first and second ligand binding molecules is an antibody*. 52. A method according to paragraph 49 wherein said first ligand bindings molecule is an entity which exerts a biologic effect and said second ligand binding molecule is a multispecitic ligand comprisin~~ a first li«and bindin;:
moiety that binds to a cell sub-population associated li~~and and a second ligand bindings moiety which binds to said entity. >3. A method according to paragraph 52 wherein said second ligand bin<~in<~ moiety is an antibody which binds to a pre-selected epitope on said entity and wherein the epitope of said second ligand bindings moiety is selected on the basis of its proximity to a ligand binding porrtion of said entity such that the entity when bound to said multifunctional ligand has an affinity for said ligand which is less than the affinity of said first li~:and binding moiety for said cell sub-population associated ligand. ~4. A
multispecific ligand according to paragraph 12, wherein said second ligand is a Il_,-8 receptor, a CCR7 receptor, a I=AS receptor, or a CXCR4 receptor. 55. A multispecific ligand according to para<~raph 6, wherein said marker is associated with an immune cell that is succeptible to viral infection. ~6. A multispecitic ligand accordin~~ to paragraph 5>, wherein said marker is CD4. ~7. A multispecific ligand accordin~~ to para~:raph ~5 or 56, wherein said second ligand is a CCRS or C XCR4 receptor. s8. A multifunctional ligand accordin~~ to paragraph 52, wherein said entity is a biologic effector li~~and. 59. A multispecific ligand, according to paragraph 36, Wherein the affinities of said first and second li~and bindings moieties are both selected to limit their individual ability to bind to the first and second li'~ands, respectively. and wherein their combined functional affinity biases the distribution of the multifunctional in favour of said target cell population. 60.
An antibody which binds to an epitope on an entity which exerts a biologic effect via a binding interaction with a target ligand, said epitope being proximal to the binding site of said entity for the target ligand, such that the antibody bound to the entity reduces the affinity of the entity for its ligand without precluding its functional binding activity vis-a-vis said li'~and. 61. A multispecitic ligand, comprisin~~ a first lig<md binding moiety which binds with a pre-selected affinity to a target entity associated ligand and a second ligand bindings moiety which binds with pre-selected affinity to an epitope on a biologic effector ligand "The term "receptor ligand" means a target ligand which is a ligand for a receptor, for example, ;r receptor on a cell or infectious agent or a receptor which circulates independently of another entity.
~' The term "antigen binding fra<'~rnent' refers to a polypeptide or a pluralitw of associated polypeptides comprising one or more portions of an antibody includin~~ at least one VI-1 or VL or a functional fragment thereof.

which exerts a biologic effect via a binding interaction with a target ligand , said epitope being proximal to the binding site of said biologic effector ligand for the target ligand, such that the second ligand moiety bound to the biologic effector ligand reduces the affinity of the molecule for the target ligand without precluding its functional binding activity vis-a-vis said ligand and wherein said target ligand is present on a diverse population of entities consisting of the target entity and one or more non-target entities and wherein the affinity of the first ligand binding moiety for the target entity associated ligand is greater than that of the biologic effoctor ligand for the target ligand when bound to second ligand molecule, and wherein the affinity of the first ligand binding moiety is selected to bias the biodistribution of said biologic effector ligand in favour of the target entity relative to the non-target entit('ies). 62. A multispecific ligand according to paragraph 61, wherein first ligand binding moiety comprises a light chain linked to an antibody heavy chain portion comprising at least a VH, CH 1 domain, hinge region and preferably at least a truncated Fc portion and said second ligand binding moiety comprises at least a VL linked to a heavy chain portion, optionally through a disultide bond'' and wherein the heavy chain portion of said second ligand binding moiety is devoid of CH I domain, and comprises a hinge region and preferably at least a truncated Fc portion, and wherein said heavy chain portions are linked via their respective hinge regions and optionally wherein the respective hinge regions are wholly or partially substituted or supplemented by a another linkage pair" eg. a leucine zipper. 63. A multispecitic ligand according to paragraph 12, wherein said second ligand is a marker associated with a lymphatic endothelial cell.
64. A multispecific li~~and comprising a first ligand bindin<:,~ moiety which preferentially binds to a lymphatic endothelial cell associated marker and a second moiety which exerts a biologic function", optionally a therapeutic function, optionally an immune function*, optionally at least one of an immunizing"' function, a tolerizin~~ function, a neutralizing" function, an immune mediating function, and immune modulating funetion'R, in relation*
to an independent "entity, preferably within the lymphatic system. 65. A
multispecific ligand accordin'= to paragraph 62, wherein the marker is selected to limit the ability of said endothelial cell to internalize said multispecific ligand. 66. A multifunctional liaand havinvg, at least, a first portion which binds to a lymphatic vessel associated ligand and a second portion comprising an immune function exerting moiety.
67. A multifunctional ligand as defined in paragraph 66, wherein said tirst portion is an antibody. 68. A
multifunctional ligand as detined in para;raph 66, wherein said immune function exerting moiety binds to a target ligand. 69. A multifunctional ligand as defined in paragraph 67, wherein said immune function exerting moiety comprises an antibody. 70. A multifunctional ligand as defined in para~~raph 68, wherein said immune function exerting moiety comprises an antibody. 71. A
multifunctional ligand as defined in paragraph 68, wherein said immune function exerting moiety binds to a ligand selected form the ~~roup consisting of CC RS, CTLA-4, LFA-l, ICAM-I. CD2, CD3. CD4, CD22, CD40, CD44:
CD80, CD86, CD134 and CD1~4. 72. A multifunctional ligand as defined in paragraph 70, wherein said first portion binds to LYVE-1 or podoplantin. 73. A multifunctional ligand as defined in paragraph 70, wherein said immune function exertin<~ moiety comprises an anti-idiotypic antibody. ','~. A
multifunctional li~and as defined in paragraph 73, wherein said anti-idiotypic antibody binds to an autoimmune antibody~75. A
multifunctional ligand as defined in paragraph 73, wherein said anti-idiorypic antibody mimics a cell surface expressed tumor antigen or a viral antivgen. 76. A multifunctional ligand as def fined in paragraph 70, wherein said immune function exertin~~ moiety binds to a diseased cell. 77. A
multifunctional ligand as defined in paragraph 70. wherein said immune function exerting moiety binds to an infectious ;agent or parasite. 78. A multifunctional ligand as defined in paragraph 76, wherein said diseased cell is a cancer cell.
79. A multifunctional li~gand as defined in paragraph 76. wherein said diseased cell is a virally infected cell.
'' For example of the disulfide stabilized type developed by the NCI
''' eg. fos-jun 's The maiety 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 biological consequence.
'~' For example using a toxin or immunogen fused or conjugated to (or having a corresponding li~gand on the second binding moiety to which it binds) to an antibody which recognizes a lymphatic endothelial marker, for example an anthrax toxin fusion " The tern neutralizing is used broadly to refer to any interposition, interference or impediment which affects the function of the target entity 'k the terms modulating, mediating, neutralizing function etc. are not intended to be mutully exclusive and are each used broadly, for example the term modulating referring to effecting a change, and the term mediating preferably connoting an indirect effect achieved through the instrumentality of another entity, for example a cell, cytokine, chemokine etc..
''' 1e. an entity other than the lymphatic endothelial cell and other than any cell to which the first moiety is anchored.

80. A multifunctional ligand as defined in paragraph 68, wherein said immune function exerting moiety binds to an immune cell. 81. A multifunctional ligand as detined in paragraph 69, 76 or 80 wherein said immune function exerting moiety binds with greater functional affinity co its target li«and than said first portion binds to its tartlet ligand. 82. A multifunctional ligand as detine<t in paragraph 69, 76 or 80 wherein said immune function exerting moiety binds with greater affinity to its target ligand than said first portion binds to its target ligand. 83. A multifunctional li~~and as defined in paragraph 69. 76 or 80, wherein said binds with greater avidity to its tartlet ligand than said first portion binds to its tar~,et li~~and. 8~. A
multifunctional ligand as defined in paragraph 80, wherein immune cell is associated with an autoimmune reaction. 8~. A multifunctional ligand as defined in paragraph 80, wherein said immune cell is a CC.'R5-expressing cell. 86. A multifunctional ligand as defined in paragraph 78 or 80, wherein said second portion comprises an internalizin~~ antibody and a cytotoxic component. 87. A
multifunctional li~and as detined in paragraph 78 or 80, which is a bispecitic antibody having a monovalent first portion and a monovalent second portion. 88. A multifunctional ligand as detined in paragraph 78 or 80, which is a bispecific antibody havin« a divalent first portion and a divalent second portion. 89. A
multifunctional lit;and as defined in paragraph 78 or 80, which is a trispecific antibody having a monovalent tirst portion and a second portion comprisin~~ a divalent immune function exerting moiety which binds to one or more target ligands on a target diseased cell or immune cell and a movovalent anti-('D3 or anti-CD?8 antibody. 90. A
multifunctional li~~and adefined in para~~raph 78 or 80, which is a trivalent trispecific antibody having a monovalent first portion and a second portion comprising a divalent immune function exerting moiety which binds to a target ligand on a tartlet diseased or immune cell. 91. A
multifunctional ligand as defined in paragraph 78, wherein said second portion comprises a cytokine component.
92. A multifunctional li~~and as defined in para~~raph 78, wherein said second portion comprises a cytotoxic component. 93. A
multifunctional ligand as defined in paragraph 78, w herein said second portion comprises a ligand which is capable of binding to T cells. 94. A multifunctional ligand as defined in paragraph 87, wherein said ligand is an antibody which binds to T cells. 9>. A multifunctional ligand as delfined in paragraph 78, wherein said second portion comprises an anti-CD3 antibody or anti-CD?8 antibody. 96. A
multifunctional ligand as deFned in para~~raph 67. wherein second portion is a cytokine component. 97. A
multifunctional li~and as defined in paragraph 67. wherein second portion is an anti-CD3 antibody or an anti-CD28 antihody. 98. A
multifunctional ligand as defined in paragraph 1 3, wherein said second portion further comprises one or more components selected from the group consisting of a cytokine component, a cytotoxic ec>mponent and an anti-CD3'CD?8 component. 99. A multifunctional li~and as detined in para~~raph 14, wherein said second portion further comprises one or more components selected from the group consistin<~ of a cytokine component, a cytotoxic component and an anti-CD3.'CD28 component. 100. A
pharmaceutical composition comprisin~u a multifunctional ligand as detined in para~~raph I OI. A
pharmaceutical composition comprising a plurality of different multifunctional ligands. 102. A
pharnoaceutica) composition as defined in paragraph 10i. wherein said plurality of different multifunctional ligands exert a cooperative immune effect. 103. A pharmaceutical composition as detined in para~~raph 101.
wherein said plurality of different multifunctional ligands ecnnprise a multifunctional ligand as described in para~~raph 76 and at feast one or bath of the multifunctional ligands described in paragraph 95 or 96. 104. A
method of inhibiting the formation of metastasis during the course of surgical removal of a tumor comprising administering to a patient prior to sur~~ieal treatment of the tumor site, a pharmacetical composition comprisin~~ a multifunctional IiYand as described in paragraph 78. 10~. An immunocytokine comprisin« an anti-idiotypic antibody which recognizes the paratope ofan antibody which binds to a lymphatic vessel associated ligand and a cytokine fused therewith or conjugated thereto. 106. An immunocytokine as detined in paragraph i O5, wherein said cytokine component comprises lL-2 or a functional fra;~ment thereof and'or 11_-12 or a functional fragment thereof 107. An immunocytokine as defined in paragraph 42, wherein said cytokine component comprises TNF-a or a functional fragment thereof 108. A bis,pecific antibody comprising an anti-idiotypic antibody which recognizes the paratope of an antibody which binds specifically to a lymphatic vessel associated li~and and an anti-CD3 antibody or an anti-CD28 antibody. 109. A
multifunctional ligand accordin~~ to paragraph 66 comprising one or more amino acids that are substituted for amino acids that contribute to an immunogenic epitope. I 10. A
multifunctional ligand having, at least, a first portion which binds to a lymphatic vessel associated ligand and a second porrtion comprising an independent therapeutic function exerting moiety. 1 1 1. ~4 bispecitic ligand comprising a tirst ligand which binds to a tirst tarvuet ligand and a second li~,and which binds to a second tartlet lir~and. and wherein the affinity of said first li~~and is selected to enable binding to the first tar~,ret ligand independently of the ability of said second liaand to bind to the second target ligand and wherein the e~ffinity of said second ligand is selected to substantially reduce the probability of its bindings to the second target ligand without the first ligand bindin~a first or substantially contemporaneously to the first target liv~and. I 12. A bispecific antibody comprisin<,~ 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 first target ligand independently of the ability of said second antibody component to bind to the second target li~and and wherein the avidity or affinity or bout 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. 1 13. A
multispecific ligand comprising a first moiety which binds to a first target li<~and 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 tartlet li~~and 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. 1 14. A
multispecitic ligand according to paragraph 1 13, wherein both moieties bind to different target ligands on the same cell. I I ~. A multispecitic ligand comprising a first moiety whi<:h 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 and 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 sut~icient duration or series of durations to its respective target ligand to a accomplish a therapeutic function without the other moiety. first or substantially contemporaneously, bindin<~ to its respective tartlet ligand I I (i. A
multispecific ligand comprising a first moiety which specifically binds to a first tartlet ligand on a first entity and a second moiety which specifically binds to a second target ligand on a second entity, and wherein the affinity or avidity or both the affinity and avidity of said first moiety are selected to enable the tlrst moiety to bind to the first target ligand independently of the ability oi'said second moiety to bind to the second target li~~and 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. I 17. A multispecitic li<~and comprising a first moiety which specifically binds to a first target li~and on a first entity and a second moicay which specifically binds to a second target ligand on a second entity, and wherein the second entity binds to a third target li~~and, and wherein the affinity or avidity or both the affinity and avidity of said first ;~~oiety 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 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 hoth first and second moieties are substantially <;ontemporaneously bound to the respective first and second entities, and wherein the avidity or affinity or both the ai~inity 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 bindings to the second entity when both said third target ligand and the second moiety are substantially contemporaneously bound to the second entity. I 18. A
multispecitic li~~and comprisin<~ at least a first li~;and binding moiety which specifically binds to a first ligand havin~~ a first biodistribution and a second ligand binding moiety which specifically binds to a second li<~and 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, one order of magnitude greater than that of the second ligand binding moiety for the second ligand. The affinity of the first ligand bindin<~ moiety for the first ligand is optionally at least, approximately, two orders of magnitude ~~reater than that of the second li~~and binding moiety for the second ligand. The affinity of the first ligand binding moiety for the first ligand is optionally at least, approximately, three orders of magnitude ~~reater than that of the second li,~and binding moiety for the second ligand. The affinity of the first ligand binding' moiety for the first ligand is optionally at least, approximately, four orders of magnitude greater than that of the second li~~and bindings moiety for the second Iigand. The affinity of the first ligand binding moiety for the first ligand is optionally at least, approximately, five orders of magnitude greater than that of the second ligand bindings moiety for the second li~~and.'i'he affinity of the first ligand binding moiety for the first lit;and is optionally at least, approximately, six orders of magnitude ;realer than that of the second ligand binding moiety for the second ligand. The affinity of the first ligand binding moiety for the first ligand is optionally at least, approximately, seven orders of magnitude ~,~reater than that of the second ligand binding moiety for the second ligand The affiinity cof the first ligand binding moiety for the first ligand is optionally at least.
approximately, eight orders of ma~~nitude greater than that of the second li~~and binding moiety for the second ligand. I 19. A multispecific ligand according to paragraph I 18, wh~°rein 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. 120. A multispecitis ligand according to paragraph I 19, wherein said multispecific li~~and is capable of contemporaneously binding the first and second ligands on said target population. 121. 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 para<.;raphs . 122. A kit comprisin~~ one or more polynucleotides. said one or more polynucleotides comprisin~~ one or more DNA sequences, said one or more DNA sequences encoding one or more polypeptides which are sufficient to constitute a multispecitic ligand as defined in any of the preceding paragraphs. 123. A liquid medium comprising comprising one or more poly peptides which are sufficeint to constitute a multispecific li~~and as defined in any of the preceding paragraphs. 12~. A liquid medium comprising one or more host cells, said 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 sutfc;eint to constitute a multispecific ligand as defined in any of the preceding paragraphs. 125. A substantially isolated polynuceotide comprising a DNA sequence encodin<~ a polypeptide porrtion of a second ligand binding moiety as defined in any of the preceding claims. said polypeptide portion comprising a VH or VL, said second li~~and binding moiety havin<~ a low affinity for said second ligand. 126. A
substantially isolated polynucleotide according to paragraph 125, wherein said polynucleotide is a substantially isolated expression or cloning vector. 137. A method of making a multispecific ligand as defined in any of the preceding para<=raphs comprising expressin~~ at least one polynucleotide as defined in paragraph 122 or 135. 128. A y pharmaceutical composition comprisin~~ a multispecitic li~~and as defined in any of the preceding paragraphs and a pharmaceutically acceptable excipient. 129. A therapeutic composition comprisin~~ a multispecific ligand as defined in any of the preceding para<~raphs and a pharmaceutically acceptable excipient. 130. A method of treating a disease in a mammal comprising administerin~~ a therapeutically effective amount of a multispecitic ligand according to any of the preceding claims. 13 I . A kit comprising a plurality of different multispecitic ligands as defined herein.
Notwithstanding any indication to the contrary, it will be appreciated that the references herein cited have application to multiple different subjects and any qualifying remarks as to the applicability of the references is to be understood as relating to each of the subjects tar which references are herein provided, as limited only by the title and subject matter of the reference.
All publications and references therein cited are herein incorporated by reterence to the same extent as if each ofthe individual publications were specifically and individually indicated to be incorporated by reference in its entirety.

Claims (13)

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 binding 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 bias 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 affinity 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.

13. 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 a 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 cell 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 group consisting of tyrosine kinase type receptors, serine 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 Fc 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 greater than the affinity 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 pre-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 biodistribution 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 are 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 first 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 53, 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;
CD80, 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 53, 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 53, wherein said multispecific ligand is a trispecific antibody having a monovalent first portion and a second portion comprising a divalent immune function exerting moiety which binds to one or more target ligands on 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 a 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 in claim 89, wherein said cytokine component comprises IL-2 or a functional fragment thereof and/or IL-12 or a 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-CD28 antibody component.

94. The composition according to claim 90 or 91, wherein paid 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 first 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 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. 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, 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, two orders of magnitude greater than that of the second ligand binding 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, said 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 snore 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 i 15, 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 a 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, chararacterized 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.

130. A composition according to any one of claim 1 to 10, wherein said first ligand is located on a cell which circulates in the vascular system.