CA2379586A1 - Fluid targeted ligands - Google Patents

Description

Fluid Targtted Ligands Field ofthe l.nvention The present irvcnt:ion relates to imPrnved iiimands, including, itnmunaligands, and methods of making them, to new immunoli$and constructs, to methods of evaluating a) the effect of fluid dynamic eonsidedttions on the design of ligand and 3n>itrunoligand constructs arid improve,nents in the selection of 6uCh ConstrUCis foT particular applicntivrn; and b) the cfFcotr~ of u: ing such constructs on the targeting of cells and on immunoligand. neutralizatiott where the immttnoligand has an immuttogenic component, for example a functionxt moiety which is immunogenic Such as a t4xin.
8aclcgroundofthe Invention Combinatorial methods of drug design have ushered in a new era in the developrna,t of di.egnostic and therapeutic entities. 'This remarkable development has focut~d on the chemical suitability of cOmbJnations and permutations of con 3tituent elements (eg, organic subatituants including amino as.ids) in thr. active or binding silo of the molecule. Small molecules have relatively ftw impediments in getting to the tatgst site.
Larger entities, such as immunoglobulins have wifiessCd the development of many different truncated formats to ovtrcolrie any size limitations, subject to balancing pharnacodynamlc and other evnsiderations.
gave for site related eonxrns for some purposes, ltowc:ver it is the binding sites of thtsC molecules that have understandably been the atmoat ex~lnaive fnous of the combinatorial methods applied to tlltir development or improvement.
diologtcal liigtrnds, comprising a. tai g~cting li$Aed moiety, for example, the entirety of or a firnrstlonal portion of a receptor, growth factor, cyto&ine, chemokine or other entity having a correlative target .ligttnd of interest, including imrnuno$lobulin type molecules and functional fragments thereof (including Fcs, scFvs, Fobs, k'(ab'}z, single domai» antibodies - srlAbs etc.) have revolutionized the Held of 'biulherapy and particularly iromunothorapy. lmmunoligands including antibodies ofatry kind, immunoconjugates, immunnfunions and other biologic affentnr f igand5 ef arty kind, whether or not fused or conjugated to a tbnctional moiety, are gaining increasing importanee Jn tkte arsenal of biological thetaptttdc agerlta that are employed to treat cancers, infectious disea~re and various immune disotdet's including autoimmune disorders and grait/transplant rcja~l~t~n.
Techniques for irr'tprovlng tltC affinity of such imtnunoligands htivo resulted in improvements in their targeting properties. However underlying at least one aspect of the i0vention herein 1t has been duwvcr ~i that various advantageous propertitB ofiFert:d by such size-reduced imrnunollgands, particularly their penetration ~Hnraeteristica and aasm of manflttnro. in F.. Coli (whether naked. fused or lacer corljugeted) might have otcasionally fostered their indiscriminate use itt contexts in whioh the masterlitl desilrn Of natural immunoglobulins has been unnecessarily glossed over.
Summary oftkte Invtntlon As will be dcacribod below, the invmtiott recognias rhrrt another oonsideratiori should be irttplfed to the design of ligands; ensuring that the binding rtgion together with any relatively invariant, non-functional or target-insensitive pert of any molecule c>r any associated entity or complex which is carrying the active part of the molecule to the target site, iS 9eleeted so that tire binding site ofthe entity is positioned in a binding orientation with respect to the direction of flow of the fluid in whioh it is tarried ancUor with respect to the location ofit5 targets (which may lee tangentially positioned relative to the direction 0f flow in a given Location), In vac nsp~.t the invention is directed. to a lisand including an organic mnle:nnls having at least OTIe weighting moilty Conjugated (eg. through covalent bonding, or other stable binding) thereto. The weighting moiety is linked to the ligand at 3 location which shifts the center oFmass ofthe t'notecule to a location which OTiCtIGR the molecule in a binBing orientation relative to its target in n mnvurg fluid environment. For example in the cast of an entity targGtcd to a t~eceptor on 8 coil within a tissue, whemc the entity has to moot:
through the interstitial fluid to reach the tafget, ifthe entity is assumed to have it, binding site eentered at zero dCgfees within a circle roughly clrcum;eriblng the mulcwla (iv. at least the regions of tho moleeulo where the vast majority of its weight is located), the entity can be weighked at a location between 90 and I80 degrees (or similnriy 1 RD and Z70 defies) for example 13S degrees ~1I- 25 degrees, to better orient the binding interface between the side and $ont ctf the molecule. 'fhe ttt7rf weighting moiety is used broadly to refer to an entity which i3 ftisod, conaugatod, obvalently linked or otherwise linked in a stable fashion to another ontity conskiluGiu~r" eomprisiztg or carrying nn asti,,.e or binding site, or simply part nfthe orlglnal template $uucmre, for oxample, the relatively invarianC part ofamolecule which is the subject of combinatorial variation at the active or binding site (which is soma cases is tested on a rotational basis).
In another septet, the present invention recognizCS that the properties of natural immunoglobulins, inotuding those of hnma»s and othor species (eg, camelids) quite apart from considerations of affinity and avidity, increase the frequency of successful eolllsions that oPFcr npportunitits for targeting events ~Nhile a#
the same time preferably minimizing the opportunities, if any, far interactions resulting in immunoli$attd nt:utraliZatttm anti olr~umaoe atriifutablc to imh~unogenicity of the antibody r,.r"rpnnrnt err a litlke~d (fused Or conjugated cr otherwise stably associated eg. through charge interactions) functional moiety (FM). The term functional moiety (f: M) is used to refer to a moiety that exerts any useful biolo f,ic effect and includes without limitation labeling irioieties, toxins (pseudomonas exotoxin yr functimral fra.;ments thereof, dipt)xria toxin, rlcin A chain, reStrictocin, I;elonin etc.), therapeutic radionuclides, cyiokines, chomokinea, growkh factors, drugs, annpling ligands such as biotin, avidln etc.
Accordingly, in one aspect the present invention is directed to harnessing the ta>~ing properkics of natural entibo~dy construcka in moving fhud environments by using a .fvrrnat or rCe4mbinant tton3ttvet in which the center of mass of the molecule permits or at least does rot interfere movem~t with the molecule in a binding orientation. In anoL#ter astxet the invention is directed to novel antibody constructs and immunoligands having laterally projecting binding moieties, which favor movement of the antibody fn a binding o~antation_ The invention is especially important for targeking circulating cancer cells, where any allcgad uiJwantages essociatcd with their dra~»atically reduced size. arF t)~
firlly at play, The invention contemplates Chat the features of natural antibody conshvets are, optimized 'for mulfiip)c diffbrent such moving fluid environmdnCS and that one of more of thane features can be acocntuated in particular such enviTOnmcnts, far example in the treatment ofhematologlc malignanciex xutsf od,cr condition atfcoting or resulting from circulating immune or cancer cells. circulating infectious asserts and circulating target lignnric of various types. The discovery also recognizes, tl5at natural tuttlbodies, are the ultimate targeting molecules, as their fluid responsive shape, including their an$ularly projecting current-engaging binding atTrls and hinge region (features which permit the molecule tv be carded in a Y 'Forward binding orienhdtion, adoptiun of a more strcamlincd ahnpc, n~ndlor forward displacement of the c.~ntwr of moat), as well as their stabilizing Fc region, respond and can be adapted to difl''orent current environments favor~bly,:fnr~xamlrle.
within rapidly moving and viscous tluid cnvironmr3ttCS (eg. blood) with a relatively forward ditqslaced binding, mterf#tces, and in slower or les s. viscous environments, with r>rlatiwely laterally displaced bindsng interfaces, in harmony with the Location of their targets. Accordingly, the corm binding orientation, in its broprteat sense, is used herein to refer to a preferred orientation with respect to the location of kite target.
However. unless otherwise specified, end especially with respect to a circulating (moving) target it refers to the binding situ being oriented in a Y func~ard direction in the direction of flow of rite liquid, which is postulated to be espesvially important in targeting circulating diseased or distass musing or marliating cells or infectious agents, for example for hematologic malignancies, In the case of an antibodyx which has a gonerally "Y" shape configuration, the binding oriofitltion rafors tC the "Y"
forward orientation.
' Thcsc ttdvantagca ore undaratood as best suited for impmverl dfffuRian or penetration.
i Tree tarot antibody, unless the context necessarily dictates otherwise (for example, where a particular fluid dynamic shape is imphcsted) is used broadly, unless the context dictaDes otherwise, to refer without limitation, to a whole antibody of any class or biologic origin, or chimeric uutnbitratim>3 of m>tibody regions or domains (cg. FRs and CDRs) of different origins or species eg.
humanized, any combination of one or more antibot~y fragtttcnts or recombinant reconstructions (seFvs) of antibodies including dlmers, diabodies, triabodies, a myriad oflcnown'bispecific, trispt:eiFc, tetraspecific antibody formats or The Inventions also contemplates situations in which more khan one type of constnrCt may requite to optimize the treatment of a particular condition and tot invention Is accordingly Qii rxcted tv various combinations of the constructs of the invention as a group or combined with heretofore known typos of eoncdwot5 fofthe type that err, fnnnd in nature as well as those that not found it nature, including those that have not beC» hel'etofhre selected for development), in any compatible penttutation5, 2utd to methods of therapeutic evaluation or trcatrr,cnt empl~,ying individual oanstmcts ar multiple constructs of the invention end the various permuLativna of such emnbination3. For example, the invention eonkemplatex that an fluid targtttd tigand in a format that is trot to natural antibody desisn as herein disclosed, may be used to treat circulating cehs of a solid tumor (ce113 possibly destined to farm metastasis) attd. that ores ofthe smaller known fOTmatx (eg. sCFv, Fab, disulNde stttbzlioed Fvs j ar formats of invcnliun wu<,id be used in combination for tergoting tin associated tumor mass.
The invention also recognizes fleet fluid tatgered antibody formats that arc larger than those conventibnally employed to target a solid tumor may penetrate non-distaSed tissues or tissue areas in proximity to a diseased tissue, 'for eXamplc a soils tun,c>r, and may not need to be substantially reduced. in si~F tn penetrate the diseased area from adjacent nornTal tissue areas, thus taking advantage of the fluid dynamic properties and longer half lives of a larger molecule to targcx metastasizing cells while they are circulating" as well as targeting the periphery ofthe tumor where access firom. relatively penetrable non-dtse~riaed tissue is possible.
In particular the invention contemplates that such antibodies and antibody conjus,,atcs ratty 6t u4cd to treat pain, far example pain sttnbutablc en an increase in voldmetl'ic prC9Sure, and that controlling, pEln due t0 volume expansion is a sufFcitntly important endpoint for approved therapy.
Accordingly, according to another aspect the Invention is directed to the use of a macrrnnoiccule that kills czlncer eclls, preferably an antrbody for example an tmmurrotLsiun or iuununoconjugatc, For axatnpl~, with a toxin, ~ytokine, radionuclidc. or an tnzytnc (eg. fvr ADEP"f}, ribozyme tic. with fluid dynamic properties as het'~at~cr discussed, by reduce pain associated with the accumulation of growth Of cancer cells in a confined spats, within a tissue. Too invention contemplates that the dose it optionally a fhaetlon of the maxiututn talcratod dose (for example 0.01 °J° to 90%, optional ly 0.1 % to 80'°10, optionally 4. I l0 70%, optionally 0.1 to 60%, optionally t to 50°0, optionally l o!° en 4ll°J°
nrtlonally 5 to 35~!°, optionally 5 to 30%, optionally 5°/n to 25~/0, optionally 14% - 25%) for improved safety with limited reduction in efficacy. Accordingly, in one aspect the invention is directed to a composition for treating pain associated with a tumor comprising a Fluid targeted l)gand cg. an inrmNw>fusian or immunoconjugnto (preferably adapted for moving in a binding orientation born within the blood and in the interstitial fluid, preferably an lgG type molecule or truncated version thereof having similar fluid dynamic properties), the composition (in one embodiment} preferably comprising the fluid targeted ligand in a dose amount nvhich is a fraction otthe maximum tolerated dvx.
Aeoording to one aspect the invention it rtirPCteri tn an immunoligan<1 which compristB & ErSt polypeptide or polypeptidc region comprising a N-terminal VL and a second polypeptidt oc polypaptidc region cOmprisittg a N-tcrnrinal VH, wherein both polypeptides preferably compri9c (at the Carboxy terttllttel ends ofthe variable dan,ains} a flexible linking Yo~ian through they are fttnctionally linked to one another, for exAntplc through a disulfide bond, at least one polypcptide pt'tferably also includiril~ an Fc regiod, FM (eg. a toxin, enzyme, eytokine, growth factor ate.) or a combined Fe-functiOna.) moiety region (FC-FM region) at the carboxy terminal end ofthe linking regipn, and wherein the CelSter Of mass of the molecule is in a Iocatibn vehicle at least does not preclude and optionally assists fluid..responsive movtrnent of too malocul~ in s binding oriontatsan, the center nFmess optionally located within the Fab t'egion or wlth.iti the Fc, FM or the Fc-FM region. Preferably, the center of mass is also substantially laterally centered between the VH and the Vl., far syrnmetry_ Alternatively, the cestter of mass may be monovalsnt, divalent, trivalent, tetravalent or other multivalent Atltlh~ody formists (see for example roview in lCrit~gkum 1, ct al. Bfspvcific and bifitnctionirl single chain reenmhirtant atttibodies. Biontol Ene 2001.
Sap; l13(Z):31-44 and Others herein directiy~ or otherwise referenced) or any fragment, portion, Or reconstruction of one ar more poztions of an antibody (scFv) or any truncated form a lipttnd binding entity, such antibody typically comprising at least a v'H or vL potrlan or both or a futctimal pNrtion of same (eg microbddies), including single domain antibodies, F(ab')2, Fab, Fab'. facb, Fc, etc. The term 8ntibody also includes fiisinns nftuch an antibody 9p defined and other functional moieties (Cg- toxins, cytokincs, chemokincs, streptavidin, adhesion molecules).

skewed to ont Side of the molecule with effect of advancing the positioning of that part of the molecule eg.
where the addikional mass 15 within only one of one of two laterally projecting binding arms so as to differentially facilitate harnessing ofthe motive force of tlm fluid. ruing t60.t extra mass, or on the other hand, causing that part of the molecule tc~ drag behind eg. where the extra weighting iri o»e one side of the Fc, FM or Fc-FM pnrtihn of the mOieCUle..
The Invantien also contemplates that a particular bindi»g ligand or a parental tigand (cg. the invariant part of an antibody, uxcd for creating a library of such m4tecules with a diversityr of hinriing reaiotls) Can be modified to optimize or shift the binding orientation by modifying the molecular weight of the constituent antibody chains or portions thetcof, by increasing or decreasing their molecular weight and the location of their weight in the final ligand COrthgeiration. For example a scFv muy Weight adjusted through amino ocid subsfiit~ttions, additions and dcletion3 which, for example equalize the weight of the heavy and light chains, anoint distribute the weight away from the bindin,~ intErFace. According to one embodiment 0'f the Invention this is aceompllshed in accordance with a cenecr of mass calculation, by modifying tho amino acid composition of the molecule, preferably in locations which arc known not be critical to the integrity of the muleculr (rutd/Cr preferably not at en extorior location which rendsrs thr~ molecule imritunogenic - such immttnogcnieiry may be also be limited through choice substitutions according to T.~elmmunization~
technologies which take into account overlapping MHG-peptide series of amino acids) regions which are well known to ihoSC skilled in the art, thratlgh addlllOns, deletluus xmd substitutions. The invcntian also contomplatea that the wtight of the various domains of a mulcispecitits molecule, for example, a. diabotly, can he mnAivert to give precedence to the binding ofone ofthe ligand binding rrioicties thereof, having regard to the appreciation that this form of antibody will move in an orientation iri which the heaviest part ofthe molecule will trail behind.
According to another aspect, the invention is directed to a method of ~,~enoratine a fluid targeted ligand and to a fluid targeted ligand generated by said method. said Hgand comprising at ive,~st one first ponian end a second portion, said flr3t portion comprising a primary binding srte and said second portion eharstcteriz~ in that it is not directly involved in bindinh, said method comprising at least one of the steps of: l) identifying a second portion or a portion thtrcof that bas alone or togethor with tht first porti0n(S) has a center of mass that permits or Favors generation of a fluid targttcd ligands; Z) identifying a first portions) or a portion thereof, wherein said fu $t portions) alone or togocher with, the second ponion has a cenrFr of matx that permits or favors generdtion of a fluid targeted ligend; 3) calculating the Center of mass of a fluid targeted ligand atKi~or a vrst anoint second portion thereof-, or a portion of either such first or second portions; and 4) obtaining data containing the result of a center of mass caleutatfon flit a thcrvpcMti.: yr diagnavtic fluid targeted ligand or a first or second portion therEOf or a portion of either such portion; and comprising at least nn~a of the sts.~:~s of 5) making or moclifyin~ a fluid targeted li~and, in accordanec with at least one mental step idantitlcd in sub-paragraphs l to 4 above ; 6) adjusting the molecular woight of at It;ast crag of the first and seconds portions or a fragment or domain of either such portion, in accordance with at least onC Of said )rt.'rttal steps G) using or making ~t tempi nta that i9 sujapted ~or making a fluid targeted ligand, for example a template for eornbi.natorial variants (tg. chemical or biological eg. protein) a DvA sequence or expression vector, enrfiding t ) a fluff d targeted ligand or a first Or second portion thereof or a portion (eg.
a domain) ofeither such first or second portion, or b) a parental ligand or a variant or invarinut uuanponent of a library of fluid targeted li~;ands; in accordanet with at least one mental stops identiftd. in sub-parryphs t to d above. The invrnrinn also eontcmplate6 that wei~hring domains can be linked onto a molecule, preferably cxteriorly to cxistung domains, far example a peptide or polypeptide, for example x peptide comprising a plurality of hydraphobie amino acids which self assOCiacc to form an intact (self Coalescing) domain or a scwnd uptionall~~ pharmacologically inort macromolecule leg. al6urrrin ar other natural macromolecules such a,~ globulins') that can be chemically conjugated or otherwiswe linked to a macromolecule having the binding site.
Most importantly with respect to antibodies, the invtntion is predicated on the recognition:1 ) that Rte laterally projecting binding arms are the primary focus nfiha force of th8 moVIriQ fluid and hence primary The terns fluid tat'Seted ligand refers t;o a ligand which i9 adapted or modified to mows in a binding orientation instrumentality for the molecule to bC transported in the binding orientation, that 2) where such projections are absent or minimal, that the center of mass Wil! dictate which end of the molecule will move ttsrward in the bindin~o oriantaeion, the principle being that thd heaW en4 of the moieculc will lag behind (thus center of mass calculations and adjustments ~n ensure than an scFv or single domain antibody (with of without a fused or conjugated funcriona) moiety) moves in a bindlrtg oticntation (Cg. by using or a trHl, CL domain or other weighting domain to ensure that the center is further from the tip of v$riablC domain than front the tip of the Fe, FM or Fo-FM pwlion}, 3) where such projections arc nor absent or minimal, that tile three of the fluid flow will be mVSt cffi~iantiy used to move the molecule if the center rrtasa is located within the projections (ie the fot~ce is most efficiEtttly appiied to the heaviest part of the molecule; d) that a hinge region pennies both forward and lateral displacement ofthcttindlng interface;
5} th$t the Fc, FM or Fc-FM
portion acts tts a stabiliser arm and provides anti-rotation stablliaation us the forward rnwCmcnt and s~ that the ov~ratl weight of the molecule wilt affect the rate of its movemcrtt.
According to another aspect the invention is directed to a fluid targeted ligand, for example, an improved immunoligand construct preferably comprising laterally projecting binding arms in which a fbnctional moiety (FM) is pYCfCrdbiy nssucitttcJ with or is subatitutcd far the Fo portion of molFCUle and whrreln the ratio ofthe molecular weight ofthe Fab portion ofthe molecule relative to the molecular tveight of FM, Fo or Fc-FM* portion of the molecule approximates that a natural immunoglohutin, fbr example that of a typical IgGI, provided that the cetteer of mass pi the immunoligdttd Is siritated in n Iuaation ofthe molecule, optionally proximal to or witFtin the Fab portion, nhat at least permits or causes the molecule to move in n binding, oriontatiort, in a moving. flnid_ The form "approximates" ag used herein preferably means any increments in variance (+/-) between Q and 6U%, relative to corrventional na6utal Ige, with the proviso that the confer of mass of the immtmnligand is situated in a location within the molecule, optionally even within the Fab , that at least permits ifnot causes the molecule to mow in a binding orientation, in a. moving fluid.'.
Accordingly the invention contempltlbes the weight of the the Fab porticm of the molecule when compared with the weight of the Frr-FM portion Of the molecule in question may be up to 59% different titan any conventional ratio within a natural immunogl4bulin preferably up to 5RQ/a r9ifferent, morC preferably up to 57°fo differCttt, etc. The preferred and permitted variance can be empirical ly tested and wilt did on the length of the Fob portion (a forger and more laterally projecting Fob will pick up the fluid force better Cspeoially In a vascular targeting Context) and the molecular weight of the Fab portion (which will correlEtE
tvith a greater mass to entrnin the fluid forces), recogttizing that the both ofthesc factors will influence the degreC to which the fluid targeting will be contributed by the laterally projecting arms relative to center of mass considerations, in another aspect the invention i3 directed to a fluid tar~;et~d Iigand for example an improved t-teombinant imrtunotigand construct in which an Fit rvnrtinn, is preferably associated With Or substituted for the Fc portion of molecules and wherein the ra~kio of the length or both the length andlor mot. wt. of the Fc, FM or Fc-PM portion of the molevulc relative to the length, or bath the length andlor molecular weight of the Fab portion approximatts~ that Of a nEttura.! lmrnunublobulin, wilt the proviso that the center of mass of the molecule is at a location in the molecule, optionally proximal to or within the Fab portion of the molecule, which at feast does not prevent the mole:cula from moving in a bittding orientation in a moving fluid. The term length in referCncC to the Fab portion of the moLFCUIC rCfeTS includes any incremental IbngtYt In the range between the length "a" and ICtrgth "b" shown in Figure 1. The length of the Fc-ligand. portion refers tC the length "e'' shown in Figure 1.
It is to be understood that the Fo portion of the molecule helps stabilize the molecule in the s8t19e of keeping it from spinning-out and trerefore keeps it alignesd in a binding orientation. Its optimal or acceptable size is depen.dant on the extent of the forward location of the cotter of mass. Since the location of the center of mass aftlte molecule may be of paramount nntponance to maintaining it in a binding orientation (depending on the extent to which the laterally projecting arms, if present (length and mass) The ligand may also he associated with the Fab portion, for CxamplC the light chain influence the molecule to move in a binding orientation) and the overall woiglrt of the molecule is important en determining the t~tte at which it is carried, relative to its target, the tetTrts "approximates" a9 used in connection with the properties of the. Fe, FM or >"'c~.h M recognizes that t) there is some latitude in the design of the molecule tn provide a stabilizing etyect as described above;
that there Tare both reasons far its length {etabilir,.lng effect) and curbing its length (overall weight and forward location bfthc Center of mass), 2) prefere»ce for the constant domains and especially the variable domains of the Fob portion to remain essentially intact in struoturc (except where the CIi 1 and CL srt substantially entirely deleted cg.
only a Critical Inter-eltaln linkage is runiu4ain~d) and taking into account that dsletionc within the constant regions cg. CH1 or CL cen be tested in a high throughput fa_ghion, and are not expetttd to interfere with the intrinsic binding properties ofthe VH pr Vl.., in this connection, the invention contemplates that 8ne or tttore pairs of linked beta strands are deleted togethC~r, optionally, those containing the fewest number of stratscgiC itrter~pair andlor inter~strand linka3es, and those that more interior to domain, and optionally by introducing or r~oone~tituting such linkages .n other parts of the domain, and preferably So a5 not to interfere with the primary disulfide band formation that stabiliT.es the domain)' 3) there is a large variance in the length and mot wt. of fMs afpotential utility, as wall ac overriding considerations, namely ensuring that the location of the cetttel of mass (v is-a-vis the divroctio~n of flog) does not prevent the molecule from moving in a binding orientation. One ofthe more important considtrations tray he the weight of the molecule relative to that of neutralising Abs. The invention contemplates as one consideratipn that the weight should be less than that ofthe neutralizing Ab, so as to minimize netrtralizing collisions fruur behind.
The invention also recognizes that large hinge regions such as those of an IgG3 do n4t model a typical lcngthlweightrelationship ofnatural antibodies. The invernion recognizxs that the location of the center of mass of the molecule (es a whole) may favor the binding ericntation, cvcnthau~h the ecntsr o~rnasc is not closest to the Fr Terminus ie it is closest to the Tab, llccordin~ly both the term Fab or Fc excludes unless the functional context sugge8ts otherwise, the hinge region, the Fab lying at the N terminus of the hinge region and the Fc or Fc-FM ref Crring herein to the porition of the binding moiety at carboxy end of tile hinge rebion.
The term natural immunoglobutin refets to any type of immunoglobulin molecule that is adapted to circulate in the vascular system, lymphatic system or circulating body fluid in the coarse afperformance of its furlCtlon, with the Vff am~Inr YG arJented irr Iha !~~~~dtyt orf~r:rattr~»
(lc. "Y'' fonrard relative to the direction of flow), including 1gs of mammalian apecles other than humans, and preferably humans.
Accordingly, in a general aspect the invent on is directed to an immun4ligand in which the location of the center ofmass within the FC portion, or proximal to or within the h~ pertion ofthe construct approxirnatw that of a circulating itmnunogloltulin or i5 more forward in the direction of flow for efficient catxiage but not to far forward t4 cease the molecule to flip out of a binding ntyentation and which has 211 Fc potties 0't Fe-FM portion acting as s stabilizer and pr°eferably a hinge rCgion.
The invention appreciates that a molecule in which the costar of mass is loCSted within the Fc will likely move in the binding orientation, because the heavletst part of flat molecule will tug brhiud, but that there must oleo b~ sufficient mass in thQ
laterally proJectinQ binding inns to cause the Fab portion of the molecule to be preferentially carried by the moving fluid (relative to other parts ofthe rnolecule),and that the most efficient movement of the weight will occur when more of the weight is in tire projections that art carried.
W;th respect to all precadin~ arpocts of the invention, thf invention contemplates in another aspect that at least part of the CH 1 domain (preferably in who lc or substantial part) of the Fab portion of the molecule is ahcent or removed (while maintaining a Fuffcient portion thereof or a surrogate thereof to permit linkage between the heavy and livttt ctlatnR, for example through a disulfide liukagc) (camclid hcevy chain antibodies arc instructive to model this construct), and optionally truncating a corcespondin$ part of CL
portion of the light chaitl while still rtlaintaininA a linkvage site to the CHl portion or surrogate thereof (the Construct also including a hinge portion to Fink the heavy chains) and. that the size and molecular weight of the F~-li3and portion of the molecule ir, Re lccted to maintain the center of mass within the Fab porrion and preferably a lcnpjth sufiicicnt to provide a stabilizing affect (For example a length that corresponds to the length proportion of an lgG1) , optionally so that Fob portion of the molecule enjoys the proportions relative to the Fc portion that approximatCS, that of a natural ankibody. This construct pelmit5 the design of smaller molecules that enjoy the advantat;ms ofthC invention herein.

The term center of mass has the meaning typically aserilsed in t_he.~.gLneering.sa,ieuces,.aS..HppllGd id the movement of a ma3R within a moving tluid, Accattlingly, the phrase "located wilhlu Ura Fob" refers to those Same engineering considerations as applied to the structure of an antibody that would not prevent the binding pfirtion of the molecule. being oriented in a binding orientation in a moving fluid. The Invention contemplates that the calculation may be reasonably carried out in more than crrtc way (eg. treating domains individually or ofhetwisa) and any such reasonable approach to the cn'Iculation and lacssting the center of mass is contemplated by the invcntim! provided thar it predictive oftha orientation ofthe blndin~ domain.
The term biologic effector ligand of biologic effectdr is used to refer to any tigand for which there is a complementary target liø~d on a target entity, and wherstn binding of the biologic effectur li~;mrd tv d,c target tigand exerts a biologic effect. Pdr example the t~~rgat ligand is tylaicnlly a receptor and the biologic cffcctor lignnd may be any eomplem~tary ligurd such as a cytokine, chemokine, hormone, colony stimulating faetnr, growth factor, receptor inhibitor, agonist or antagonist, which binds to the receptor with resulting biologic effect.
For example, the invention is directed to a 4 chain, 3 Chain (one of the heavy chains does not carty a light chain) or a z chain molecule in whlah the hinge region is maintained intact and the Fa or Fc-FM portion df tht malecu.le is ~rrepandingly smaller while malntain3ng an approximate weight ratkr with inspect tn an Fab which is in turn smalltr (eg. the CH 1 region is absent except for a pottion suitable t0 form eg. a disullldo lirkag~ to a truncated CL) the r~entr~r of rnar;s preferably being in the Fab portion of the molecule, so t~T to maintain proportions comparable to natural imrnunoglobulinS. With respect to the various embodiments of the invention, it is also contemplated that the VH may be linked to the VL through a FR
region eS i5 well-known In the art fin di~witidc 3tab~lizcd VH-'1lL molacuiAS
(Paatan at a1.}
The invention also contemplates an antibody comprising at least heavy Chains (light chains are optional) which comprise at Icest a VH (hr a functional ftagrrtent thereof as known to the art) and a hinge region (the CH 1 is optional) which are directly or indirectly linked through a linker connecting them, optionally through an Fo (off;, n C1r12 andlor C1-t3 andlor an FM). These embodiments of the invention are useful for making btspecific antibodies, including hispecific antibody fusiOns, as the heavy chains would nocestarily be cvrrcctly paired (for example for application with respect tn bispe<;ific moleauleS disClo3ed fn our cepcndlng t'4'i application tVa. PCT/CAOwl~003 l?, Fled Marsh I 1, 20D2).
TakinP acnter ofmass Into account, the invention also contcrnplatcs lighror cvnatrucrs that enjoy one or more properties of natural antibodies. For example a two cltaln antibody construct, ibr example a wrr~truat devoid o'f light chains eg, two fit.ll length heavy chains having "Fob portions" with or without a CH1 domain (in whale or part) (Figures 1 1 to 1 G aril Figure. 1 Ra end 3) arid that the size andlOr weight proportions relative to the Fc portion will permit the design of smaller rind more strearnlit7ed molecules that enjoy one or more advantages ofthe invention is, the Fc -FM portion may be correspondingly smaller artd lighter. Alternatively a VT. may be gtaPl'cd uruu Clit or hinge rogion of a conventional heavy chain (dEpending on whether the CHl is included in the design in whole or part), to fbfm a two Chain disulphide-linked hybrid VT -Fc..FM two chain construct.. Some of the Smaller Conatnu.ats contemplated by the imrention are shown in the Figures, and have feature Such as:
An immunaligand cornpri~ing a patypeptide c4mprising a VL and a nnlypcptide crn11pC1Bing a VIi. wherein bMh poiypeptidcs optim811y comprise at Icast a portion of a first constant domain region at carboxy terminal ends of the variable domains through they are: functionally linked to one another to feint a futtCtiOnal binding ligand, at lea$t One poiypeptlde atsu including oar Fc region or Pc-fM region at the carboxy terminal sod of the first constant domain region, and wherein the center of mass of the molecule is ;r, the Fah region. The term immunolistand is defined below. The term const~t~~rnta4t°ri~~~r-,~o:''rs-to a--polypeptide fragment which at Itast provides a linkage between the heavy and light chains eg. paired eysteines. According to one such embodiment each palypeptide comprises a Fc Or Fo-FM region and a hinge rcgivn. at iho N terminus ofthc Fc or Fo ligand region. Aceordlng to another Inch embodiment, tltC
center of mass is substantially laterally centered beiween the VH and the VL.

The invention also contemplates that one or both Fc 'fragment panions is attached to a FM. Alternatively, the invention contemplates that the carboxy terminus of each Fc portions (Fait-length or truncated) is linked to a single FM via a linker System, for example a pair of ilexlble linkers (eg. Figure 23).
The im~ontion oorttemplates that tits tnmcated Fc may be a minibodY having a Cli3 instead of a CN2 and the term Fc~ligand therefore includes an .Fc in which. the CH3 domain is used in place ofthe CH2 in whale or in patt.
The invention contemplates that both a light chain and a heavy chain are respectively fused to at least one or more constant domain or ctmstant dorttain surrogates (ilea functional fluid dynamic properties), fot~tting a surrogate Fc portihn wJtich optionally serves to provide a stabilizer srm, by itselfor together with vne or more associated (conjugated or fused moieties eg. polypeptides), fcx example fused polypeptides having a desired functionality (eg. toxin. onryme, rytnkine, avidin) and wherein said constant domain3 are associated togethsr through one or more linkages, optionally one or more disulfide Ilnka.ges, said surrogate Fc portion optionally including a hinge portion, which permits f4rward (in the direction of fluid flow) relocation of the centCi' Of ntaSS in respun5e to filuid flow, and wherein the center of mass is located in the Fv or Fab portion of the immunoligand and Is optionally substantially centered hetween to the two arltts to enable the molocu Ie to adopt a binding orientation which i.s results in a.
greater numher of collisions which foster binding.
Tlrc inrrntlon is al;,o dirxtod to improved ligatx3s having n targeting moiet<v, for' exant171e, a ey6okine~ Fc domain, growth Factor etc. fused t4 an cffector n,eicty, in whioh center of mass in the effector moiety.
In one entbodttrierit of the preceding aspects of the i».rcntion, the Fnb portion of the molecule Qpcciticafly targets a marker on a circulating diseased or disease mediating* cull or infectious agent, including a spore, a virus, a sinRlc.celled microorganism eg~ a bacterium, etc., For example, the veil maybe a cancer coil, for example a cell associated with a hema'taios c malignancy, or a non-t$ncerous immune cell, fcx esxauylG a cell targeted for' ablation, and. the ligand is a toxin, for example a truncated form a F PE compri.sing at least a functional tAxie domain (ace Kreitman R.1. Ft al F t~icacy of anti-CD-22 recombinant immunotoxiri BL 22 in chemotherapy resistant hairy cell leukemia N 8ng1 J Med l u1 y 26;.145(4)241-7: Kreitman TtJ et al. J Cliu.
Oncol. 200 Apr; 18(8).1522-36. Jn one embodiment, the invention is dircctedto a version offlaesc molecules in which the toxin i.s locxtcd un a truncntcd fc portion (cg.
prefornbly a Substantially intact CI12 or CH3 domain), which is designed according to one rrr mare principles of this invention, of is fused to both Fe's, for example, through flexible linkers via the tranlocation domain of PE or a part thereof (Figures 23, 23a, 23b). Optionally, the Fc is pegyltrted at a location on the rc-fM
which is proximal to the junutiwt between the Fc portion and the FM to a<s to reduce the irnmunopenicity ofthe ligand, in this ease the uuncatcd 1'L.
Brief Description of the Drawings Figure l is a diagram of a four chain antibody depicting force3 acting on the antibody;
Figure 2 depicts one embodiment of a fluid targeted ligand according to the invention;
Figure 3 depicts another embodiment of a fluid tar~etcd ligand according to the invention;
Figure 4 depiMS another embodiment of a fluid targeted ligand according to tire Inventiotl;
Fi4ure r depicts An4thcr embodiment of a fluid targ~t~td ligand arr,.prriinQ, to the Invention:
Figure b depicts anbther embodiment of a fluid targeted l.igand according to the invention;
figure 7 depicts another erxboditrent of a fluid targeted ligand according tb the Invention;
figure 8 del7icts another embodiment of a fluid targeted Iigand acwr~liy to tl,o invmtirnv;
Figure 9 depicts another embodiment of a fluid targeted llgand according to the inverrtion;
Fi~n~a 1 D depicts another embodiment of a fluid targeted ligand. according to the invention;
Figure 11 depicts another embodiment of a fluid targeted li&and according to the invention;
Figure 1. I a depicts another embodiment of a fluid targeted ligattd according to the invention;
Figure 12 depicts anvlhcr cmboditnent of a fluid tnrget~d ligartd according to the inventinrt;
Figure 12a depicts another dmbodimerrt of a fluid targeted ligand according in the invention;
Figure 12a' ~1 or 2) depicts another embodimdtt of a fluid targeted (igand according to the invention Figure I 2b depicts another embodiment of a fluid targeted ligand according to the invcntivu;

Figure 13 depicts another embodiment 4f a fluid targeted ligand according to the invention;
Fi>dure 13a depicts another embodiment of a fluid txttrgsted tigand according to the inventiotl;
Figurre ! 3a' depiCt3 another embodiment of a fluid ISr$eted llgand oceutdiclg tv IJrv invcntiCri Figttte 13b and relaked figltres depict other embodiments of a fluid targeted ligand according to the invantiott;
Figure I 3c deplCtS taflOlher embodiment Of a fluid targeted ligand according to the invention;
Figure 13d depicts another embodimcne of a fluid targeted ligand according to the invention;
Figure 14 depicts anOlher embullitna~lt c~f a fluid targeted ligand atCOrding to the invention;.
Figure LS depicts another embodiment of a fluid targeted ligand according to the invention;
Figure Ib depicts another embodiment ofa fluid targeted ligand according to the invention;
Figure 17 depict, another embodiment ofa fluid targeted ligand according to the invr;cdi~u;
f=igure 17a depicts another embodiment of a fluid targeted ligand according to the invention;
F;~ure 17b depicts another ernbnriiment of a flttld targrtcd IitJand according to the Invention;
Figure 17c depicts another embodiment of a fluid targeted ligand according to the invention;
Figure 17d depicts another embodinteot of a fluid tat'geted ligand according to the invention;
Nteuru l8 deptCts Cnoth~r mulxxllrnant afa fluid ta~ctcd ligand according to the inv~.minn;
Fi!3ure 18a depicts another eml,,odiment nf.a fluid targeted ligand according to the invention;
Fi>;u.re 1$b depicts another embodiment of a fluid targeted ligand according to the invention;
Figure 19 depicts another embodimCnt of a fluid targeted ligarid according to the invention;
Figure 19a depicts another embodiment of a fluid targeted ligand acCOrdina to the invention;
Figure 2t3 depicts another antbodiment of a fluid tsrgeted ligand eccor~ding to the invention;
Figure 21 depicts another embodiment of a fluid targeted ligttttd according to the invention;
higui~e Z2 deplCtg another Ccubudiu'wnt Of a fluid targeted 1 iGartd acsoYd.ing to the in~rntinn;
Figute 22a depicts another embodiment of a fluid tatglned ligand according t:o the invention;
Figure 22b depicts another embodiment of a fluid targeted ligand according to the invention;
Figure 32c depicts another embodiment of a fluid targeted ligand according to the inverctiurc, Figure 22d and related figures each depicts another embodiment of a fluid targeted iigand, according to the invention;
Figure 23 depicts an irther embodiment o,fa fluid targeted ligand according to the invention;
Figure 23a depicts another embodiment Qf a fluid targeted ligand according to the invention;
Figure 2:ib depicts another embvdiruent ~f a fluid targeted li~and according to the invention;
Figure 24 depicts another embodiment of a fluid targeted ligand according Lo the in~~ention;
Figure 25 depicts h»other embodiment of a fluid targeted ligand according to the invention;
Figure 25a depicts another embodiment of a fluid targeted ltgand according to the tnventiun, Detxilcd Dcsvription of Preferred Embodirn~errtR
Unless otherwise specified the tours field targeted ligand or itttmunoligand excludes a naturally occurring antibody construct.
The inva~tion is founded on the appreciation that a natural antibody is carried by the blood and other body fluids in the optimal orientation for its function, with its Fvs projecting in the direction of flow of tlla fluid (hereinafter referred to as a binding orientation) and somewhat laterally more or less depending on the viscosity and rdtc of fitow of the fluid. Without wishing fn he hound by a comprehensive theory, this pattern of movement is attributable primarily to the artgularly projecting binding attrts, to the ability ofthe antibody to readily align or re-align itself imo a binding orientation due to flexibility oftlte hinge region, permitting the center of mass to maul forward in the direction of IluiJ flaw in rsapOnwc to the foroos of fluid flow, and due to the Fc arm acting as stabilizer. T'hc symmetry of an antibody [ie, the symmetrical distribution of the mass) permits tl5e forces at play to orient the molecule into a preferred binding orientation, ASyttmctric constructs are useRtl for applications to bispecific antibodies, accordingly, the invention is directed to improvements in existing ligand nnnstrItCLS, such as ConStruCts in which: a) the center ofmass is nM located in the tab portton of the molecule;
or b) ifthorein located, is at the margin thereof andlor without the combined effect of a stabilizer acttt, with effect to increase the rate of disorientation from a binding orientation andlor diminish the rate of reUriGlItAtIVl1 t~7 R binding oricntntion, andlor 3) the center of mesa is not substantially Centered in between the two Fobs or the cane of a molecule in which the Fab portion comprises a single heavy and light chain, between the .heavy and light Chain to optimize the chance that a given collision v~nlt result in binding, as mare fully dess4riLcd below and/or ,s) the hinge acting component of the construct is absent or insufficient to permit significant forwgrd rclacation of the center of mass in re.FpnnRe tp the fOrCf Of the current, so as to ptfrttit reorl4ntadon to the binding aricntation aftw a disorienting collision: and/or 5) In which the overal l mass of the molecu lc is net optimized for more rapid r»ovcment relative to the target moleouleJael l or relative to neutralizing .
irnmunOQIobulins ak.. Exiling immunoligartd construct: with r~sptet to which the constmcts he:min mxy be selected as improvments may identified through search techniques well known throw those skilled in the art and more fully descrihed below. The invention also provides new irnmunoligand constructs possessing one or more of these features.
Are shown in Figure 1, ~~ v;rtue nfthe laterally projecting binding ernts, the forces of the fluid flow act to mop the molecule in a Y fotrovard binding otientatton to stabilize the Fc portion against lateral movement.
The ltngth "a", "b" end. "c" described above aro shown, as well as the general regions of the molecule described hCreln as the Fab purtim or region and the Fe portion or region.
'1"Ite invention contemplates that the various size-reduced and fluid dynamic features ofthe molecules exemplified herein can be combined in various pcrmutatians and comblnatlons.
~1a shown in Figure 2 the flmcti(,na1 moiety may be loCakcd in the )re portion. optionally, whet'e the rM is immunogenic a portion of the CH2 domain proximal to the FM may be pegylated, as shown in this Figure

2, t7epending on the size of the FM the total weight of the molecule may exceed chat of a natural antibody, WhtCtt may not prefbrretl frvrn an innnunokcnio stondpoint. That~fore, ahhougt~ the constmrt rlericted in Figure 3 is preferred fr'am a fluid targeting standpoint, prgylation may be required to avoid problems a.RSOCiated with the immunogenicity of the FM. Figures 4 and 3 are variations on Figure 2 showing different sites of pcgYylation 8f the molcoule. Figure 6 depocts two r M portions.
Ftgttre d in its roust essential aspect depicts a linkage via a flexible linker between the Fc portion of doe half of the molecule and the FM on I:he other half fif the molecule. Ctptianally, the linker which may vary in size cant<-tins One or mare ,pairs of cysteines to assist the molecule to fold. l7ptionally, the molecule may ire pegylated at shown in Figure 7 or in one ar more location shown in the prvar Figures. Optionally, the hinge region may have conventional or multiple dlsulhdc bonds twie akin to an I'~;G3. In its most ~cs9nt~al aspect.
Figure 8 shows that the linker connecting the Fc and Fc-FM portions of the molecule contains at Icast one enzyme cleavage site, particularly where this detacltrnettt from tile linker is required f<>r the functionality ofthe FM portion ofthe molecule. The Choice of amino acids for the cleavage site is sclecttad. for example, on the brtsis of a nntural ttxtr'aCehular enzyme prevalence within the tumor (eg MMPs) or intracellular enzymes which are presbnt at at antics~ated sites nftrafhcking of the li~~nrl or artificially introduced enzymes (C~. by means of a liposome sized to penetrate fenestrations in tits tumor capillary bed.
Optionally the molecule may be pegylated in one or morn locations, for t:xatmple as shown in Figure 8. Figure 9 depicts a truncated Fc portron akin to a mlntbOdy having a linker portion which is preferably flexible and consists of mnlti~.lrc of r~',ly4ser. Optionally the linker has one or more disulfide bonds as depicted in Figure 9 and one or more enzyme cleavage sites. The hinge region tray comprises one or mare disulfide bonds and tlt.e ~L and CH1 may he absent with linking being esktblished between residues in the V H and VL as is well known far disulfde sutbili7xd molecules. Light Chains are optional. As discussed herein the paired heavy chain construct may be produced in E sell, according to welt psrahllshed methods, including well established methods for refolding antibody constructs originally datived from insoluble inclusion bodies. As shown in Figure 10, ehc .FM is linked between the two heavy chains. This represence a lighter version of tire moleeuk;. Optional ly, thi5 ttlateeulc may be pegylatcc3, firr example i.~
sane of the lnentiono shown above. A,s shown in Figure I l, the invention is also directed to a two chain moh:cule devoid ar~light chains.
Optionahy, the FC portion is truncated as shown in Figure 1 I a, and all the variations discussed above with respect to the l0CAt10n of the FM, the substitution of the VL f~f the VI-I, linkage of the heavy Chains through a linker. a leueinc zipper (not shown with respect to most constructs but understood to apply to a.l) r:unsn u4t5, to optionally constitute part of tht Fc portion of the molec.rrln, nlttinnall5' In Cofibination with a flexible linker for additionally linking the heavy chains or linkage through the FM), ar through a FM (eg.
via one or mare tinkers), locations of one or more FMs, multiple disulfide bonds within the hinge andlor linker linking the heavy chains, pegylatipn sites, ttuncarion of the Fs; and clr~.y~no cleavage sites apply 1.Q

singly and all various compatible permutations to the basic constructs shown in Figures 11. 13a, 13b, 1.30, 13d, 11. 1 g, 22a, 22b. 22d some of which are shown in the remaining Figures.
Unless, othertvise expressly stated ouch ot' the Figut'~ may be seen to represent basic molecule plans (subject to variakions hot~in disclosed verbally or depicted in other Figures) that tray be employtd for a principle purpose of providing L) a fluid targeted ligand; or 2) a bispccific antibody (for example one in which a firstheaw Chain or binding portion thereof is forced to bo paired through an intervening sequence which intetveriing sequence may comprise a linker ur lG~kagc pair (tcucine nipper, foo jun) clod optiana113~ a functional moiety) and optionally one or more sacandary pttrposos of. 1 ) providing a fluid targeted ligand 2) forcing the pairing of heavy chains 3) skewing the weight of or length of one side of the molecule' 3) reducing tile overall size and/or weight of the molecule 4) protecting an immunogenlc pan of the mukcula from neutralization eg. by pagy.lation or outer form of gieric inhibition S) assisting folding through paired lhikates eg, cysteincs to form disul~,do bonds.
The construct shown in Figure 13a and 13d and 22b, 22c, and 22d can be made according the methods described in T,JSP 614'1:L03. The basic Construct shuwn in Figure 1 R can, depending an the length of the hinge region at the N.tertninus of the one or morn disulfide bonds, can havt tile binding arms ofthe Vl-!
and VL binding in ccxteert AS in a disulfide stabilir.,ed molecule or independently as separate binding arms.
It can have a leucine sipper as the Fc porticm or part of the f c pot'tion.
Mctltoda dl crrrtting librnrioo by combindtnrial cherniatry ore well known, a5 8rC met71od.9 of adding various ring struCttu'cs or other substituents including high mol. wt. substituents (lg. via iodination), through various reactions which arc well known to those skillod in the art of chemical synthesis (sec for example, Organic Chemistry Wade L.C't. Prentiee Flal1 1993 3'a eJ. And March's Advanced Organic Chemistra~;
Reactions, Mechanisms, and Stntcturc, 5th Edition by Michael B. Smith. Jetty Mturoh dth edition (January 1 S, 2001) Wiley-Interscienae). Examples of"such ring structures and other sub3titucnts ore depicted, for example, in the Chemical nomenclature t~cction of Remingtpn: The Scienee 1nd Nraettee of Pharmacy by Alfonso 2 Gennaro (Editor) 20s' Edition ~Aack Publishers. It will be appreciated that weighting a small ,ttalscule chemothcrupeutic could impact its pharmacn. logic pmpertie8 and that working with a template for making combinatorial libraries may be approached by adding weighting tnoietics to the template at substituont locations which ace preferably approxilriately l33 +~'- 25 degrees 8part from the 9ubstituent which is varied tilt pharlnaeologlc prop0rti~, pr~rctably to assess the impact of vnrintion of bath substituents, the wcightini moiety for example being pharmacologically inert (by itself] or having a known rnrnPatible phamtacolol;ic activity. For cx3tttple in the ca&e of 1,~4-Itenzodiazcpin-2,5-iliona librarios oaring substituents (Methods in Enzymolo,gy Val. 267 Academic Pre3s i~Ib p.
4>u) tcz tbrtherapeuttc activity and Rs fdr weil;hting the compound see also 5,962,337 Combinatorial 1,4-benEOdiazepin-2,5-dione library 3,70,577 Synthcai5 of qrrinarolinone librnries~ anti rirrivative4 thoreof; High Throug11pt12 Syltthe5i5 by Irvtng 5ucholeiki Marvel Dckker; ISBra; 0824702555; P'toteetive Groups in Organic Synthesis by POter G. M. Wets, Theodore W. Greene Wilcy-Inierscienee; ISBN: 04'711 40199 March's Advanced Organic Chemistry: Reactions. Mechanisms, and 5tru~~ure, Stir Edition by Michael 8.
Smith, Jerry March 5th edition (January 15, 2001) Wiley-Intenscience:1SBN:0471585890 Combinatorial Strategies in Biolo~~
and Chemistry by Annettc Beck-Siekingcr, Peter Weber, Mieltael Sodemtan (Translator), Allan ltVier (Translator) (Marclt 2002) John Wiley 8t Srnts; ISBN: 0471497274 Cvmhinatorial Library l7eelgn and Evaluation : Principles, Software Teals, and Applications in Drug Discovery by Antp K. Ghose (Fdltur), Vellarkad N. ~ixwanodhan (editor) (June 2001) rutarcet bekkcr;
lStiN: 0824704878 Optimization of Solid-Phase Combinatorial Synthesis by T3ing Yan (Editor), Anthony W. Czamik (Editor) (lanuary 15, 2002) Marvel Dekker; tS9N; 0$24706544 High Throughput Synthesis by Irving Sueholeiki (Editor) (Fcbntary 15, 2UU1) Marccl Llekl<er; t91f1~1: 0824702563 Typically, n fitnetirmal moiety is a label cafable of producing a dEtectsble signal. Such labels are useful, for oxample, in detection systems such as guantitation of tumor burden, and Imaging o(' metastatic toot and tumor imaginr~_ Such labels are known in the alt a,nd include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, cl~ou~ilumlnoscont compound3, hiolumirtos0ent compounds substrAtn cofactors 8n4 The lvrtath ofthe hinge regions of thc~ molecule Can be different, optionally On the amino terminus side of the linkage eg. paired cysteines to ensure that one Side of the molecule binds first inhibitors_ See, for examples ef patents teaching the use ofsuch labels. U.5.
Fat. Nos. 3,817,837;

3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,3bG,241. The moieties may be covalsntly linked to the immunoligand, reconlbinantly linked, or conjugated to the immunologand through a secondary reagent, such as a second artrihndy, protein A, or a biotin-avidin complex.
~thar functional moieties include signet peptides, agents that enhance inttrlunolagie reactivity, agents that feCilitate coupling to a solid support, vaccine carrsars. bioreRponse modifiers, paramagnetic labels and drugs. Signal pvplitla~ arc dcaerrbed abmrc and include prokcsryaeie and eukaryntia forms. ~l$Gnts that enhance fmmunologic reactivity include, but arc not limited to, ba~erial superantigerrs. Agents that facilitate coupling to a solid support include, but are not limited to, biotin or avidin, immut>agen carriers include, but are not limited to, any physiologscally acceprahle buffers.
Bivrrsponse modifiers include chemokines and cytokinc9, particularly tumor necrosis factor (TNF), IL-2, IL-12,1L-4, GM-CSF and gamma interferona.
Suitable drug tnoseties include antineoplastic agents. 'these include, but are not limited to, radioisotopes, vinca alkelolds Ouch as tl~e vinblaatinc, vincxi3t~~e and vindealne sulfates, adriamycfn, hleomyt:in sulfate, carbcplatin, cispTatin, cyelophosphamide, cytarabine, dacarbazine, dactinomyein, duanotubiein hydrochloride, doxorobiein hydrochloride, etaposide, fhtorouracil, lomustine, machlororethamune hydrochloride, tnelphelan, rttetcaptopunne, ntetttotrcxate, fTlitOrnycin, ts~itorane, pantastatin, pipVbromert, procarbaze hydrochloride, strel5tozotocin, taxol, taxotere, thioguanine, sod uracil mustard .
The inventkrn also contemplates an apparatus and method for testing ligand binding in a circulat(tt; fluid 611vitoritn~t in which the (nrpruvcd or new contruetu of the invention cxn he tested rod wherein H
continous flow of ligands, including ligand binding entities of the invention and/or ligand bearing entities (e;~. cells or synthetic eg. latex spheres which seat be adjusted m a cell sire) to which one or types of ligatsds have bean affxadly associated accordingly to known methods) can be gcnnralntl.
The fluid Contact interface of the apparatus has a ganerafty circular slsape and is sonvcx or otherwise capable Of Containing the fluid and thereby prefprahly permits fluid to flow around the suri"acc continuous[y, 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 andlor removing Sts Contents, or ik may Cbmpletely etlCioSed with the exception open access purl, f~'om which any air may optionally be displaced or evaruatexl. The invention contemplates that the apparatus (at least the: fluid contact vessel) can be rotated or o:acillated (eg. in an elliptical, oval or similar shape well known to those skilled in the arts of fluid mechanics and related engineerinlxZ arts) in a variety of different plants or with rocklnfi-like moticm in multiple planes or subject to peristaltic pressure (ie. where flexible tubing is used) to geneWre a continuous, preferably turbulence free tiuld flow ever the fluid cantfirt aw~face at selected rates simulstin~ the Various shear .rates of arterial, venous, infra-lymphatic flow (including diEforent diameters of such vessels) or interstitial fluicT flow. The invention also contemplates that the tluid contact surfarx may be provided with a 1) substrate for linking ligands bf the Invention or target ligands or ligand hCpring entities io pe~rrrrit fluid flow aCmsc the substrate in a plane substantially pafatlel or c0nf~>rrning to the axis o F flaw audlor 2) optionally with one or more micro-scrseCns or otherwise permeable substrates (eQ. a loose ncawork str matrix of bond-like materials or spheroids eg.letex spheres having associated ligands or purtficaticm column packing snaterialy, optionally buoyant materials, or composite materials including ~s smaller bead .serving as a spacer, or materials comprising proiectionc or int~rrconnertsona eervin~ as spacers, so as to dt least partially simulate flow around Cells within a tissue) which are arranged transversely across the fluid path and at least partially intersect that path (see Tigura ~, the micro-screens optionally being impervious to the ligand-bearing entities eg. Cells but t74t to the li~;ands ag. n wltispccifi4 ligands of the invention (being t~tQd for their binding ability). To its simplest embodiment, the invention contemplates a method of evaluating binding of more or more constructs according to the invention, optionally in cart115etition with conventional or ptiof art constnrcts, by introducing into the apparatus. prior to or in the course of Circulation of a physiologically compatible fluid, for example, target culls, immunoligands ofthc invention (optionally radiolabelted), the pi i~71~ art or convcntionnl eontrvcts (optionally labeled, prr~fcrsthly otherwise labeled eg. tloureSCEfli labeling, and/or Oetttralizing antibodies of the type that are naturally prflduced preferably antibodies which recogrslze an epitolrc on the Fc ligand portion of an immunoligand of the invention; and allowing a period Of targeting iz to transpire while the fluid is circulating a rate which is repr~cntative of the natural circulating venue of the target cell; and evaluating according routine methods of detection or more of the following;
l) The amount ofthc immunaligand ofthc invention hound to the target cells:
2) the amtrutlt of the prior art conttnact hC~und t0 the tat'get cells;
3) the amount of neutralizing antibody bound to free immunoliganda of the invention;

4) the amount of neutratiaing antibody bound to fi ~c prior art onnstrvcts;
3) the amount of neutrali~ciy aul.;denuly bound to dar,~at toll bound immunoligands oftha invantinn;

5) the amount of neutralirang antibody bound to target cell bound prior art constructs;
7) the amount of internalized immunolignnd of the invention (where the target ligand on the target cell fends itself tr2lciily to intennaii~.aiion of the imnsunQligand and prior art cor>struc;t) $) the amount of internalised prior art construct (where the target ligand on the target toll lends itfelf rcadilr to evaluating intematization) 9) the amount afneutralizing antibody in the target c~11 (assuming that there is no target 1i!_tand on the target calf that is recognized by th4 neutralizing Ab):
it)) the amount of free (not hound m .anything) neutralizing Ab, iatmunoligand oftha invention and prior art construct in the circulating fluid (in one embodiment the amount of the neutralizing antibody is selected to ensure float it is campl~cly bound when the fluid.
circulation is stopped, so as !:o minimize binding at the conclusion of rite eeperiment).
Definitions As used herein, fhe term inimunoligand includes one or more polypeptides which singly or together make up an irnmunoglobulin type molecule (incltulhy fuuctiunal fr-agmcnts of any nntural immuttoglobulins pplypeptides) end may include arty other t6nckional moieties iacludlng peptides, polypeptides, organic or inorganic chemical moieties (including palypetides composing functional domatns of toxins, enzymes fused, conjugated or otherwise functionally u!tsoeieted therewith).
The tcnn immunofusi4n refers to an antiborty nr fragment thereof fused as a fusion protein t0 a polypeptide, typically a non-antibody derived polypeptide used for immunothcrapy, for eXetnple a polypeptidC
comprising the functional domain of a toxin, eytokine, enryme, clotting factor, drug, prodt~tg ate.
'fhe term immunocanjugate refers to any therapeutically useful moiety (directly or indirectly uset'bl) conjugated to an antibody or fragment thereof.
With respect to making immunofusions and antibodieR in E. toll sec Antibody Fusion Proteins, Steven M
Chamow , Avi Aahlcenazi Eds. 1SBN 0471183 S83C May t 999 liViley; Kontermantl, R.. et a1.(Eds.) Antibody Engineering, Springer 2(101. TS8N 3-540-41354-5; Antibody >=ngintering, Carl A. Barrcboedc Oxford University Press, 1995; Antibody Etlgincerittg:I1 Practical Approach Iaavid J.
Chiawell, Hennie R.
Hcrogcnboont, John Mci:afferty Oxford UttiVeTSlry Prras,1996, Antibody l:nginccring Trotaools, Sudhir Paul (1495) fiumana Prtss; Antibody f?xpression & 8ngineering (1998) Henry Y.
Wang, Tadeyuki lmanaka, American Chemical Society). Roitt I et al. Irnmulogy. 4th )edition, Mosby 2441: Encyclopedia of immunology ; 1998) Morn itaufinantl Publishers, lSs1J;01~2267C56), Cellular ~c MoJeeular immunology 4' Edition, A.bbas Ak et a1. W8 Saunderx and Company w000.
With respect to making antibodies in eukaryotic systems see also Methods In Enzymology Vol. 30&
Expression of Recombinant Genes in Eukaryotic Systems Edir~d by .loseph C.
Glorioso and Martin C.
Schmidt 1999, ISBN: 01218~~Uny.
T7,r invPntinn ahn contemplatc9 that non-ptotcinacous drugs that have a ligand binding portion, for example they bind to certain receptors can be chemically or otherwise conjugated to weighting moieties, for example proteins, carbohydrates ate.
'With respect to well known tumor markets see for example, Cancer - Principles and Practice of Oncology.
Fifth and Sixth Editions, Lippincott, Vincent T. DoVita, ,1r , MD Samuel Hellman, MD Steven A.
RasenberQ, MD, PhD !S>3N: 0-781?-2229-2.

Standard reference works setting forth the general principles of recpmbinant pNA technology, methods of making antibodies and vectorslhost Cellslmicrorganisms usod in such methods atld pharmaceutical compositions comprising such antibtxlies and fusion proteins thereof include Wat,40n, J. D. et a1, Molecular Iliolagy ofthv Gone, Volamcs I and 11, the RenjaminlCummlngs Publishing Company, Inc., publisher, Menlo Park, Calif. {1987), D2rnell, J. E- of al., Mttlacular Call Biology, Scientific American Dooka, lhc., Publisher, NCw York, N.Y. (1986); Lewin, 9. N1 Genes II, .Tahn Wiley & Sons, pubFshers, New Ynrk, N.Y. (1985); Ui4 K. W., et al., Principles of C~Cno Manipulation: An Introduction to Genetic Engineering,.
2d edition, University of California Press, publisher, Derkelcy, Calif. ( 1981 ); Maniatis, T., et al"
Molecular Cloning: A Laboratory Manual, 2nd F.d. Cold Spring Harbor Laboratory, publisher, Cold Spring t-Ittrbvr, N.Y. (1989), and Current Protocols in Molecular Hiology, Ausubcl at at., Wiley Press, blew York, N.Y. (1989), Standard reference works settinø ftrrth general principles and techniques of immunology include Ilattdboak of Experimental Immunoln~y Rlackwell Science, Incorporated, iSBN:4632949756;
Antibody );nginecrlng Blackwell Scienec, Incorporated, .ISSN:(1632o09756;
Therapeutic Imutunology ISBN: 08b542375X Blaekweh Science, lnc:orporated ; Encyclopedia of Immunology (1998) Morgan Kattitnann Publishers, 1S$N:ot22267656, Immunology Mosby, tnanrporated, 15BN:07,'d3d29189; Ablyaa AK, et a1. Cellular 3r. Molecular ltnmunology 4'" Ld. 20U0 ISSN 17721650423;
Breitling F. ct al.
Recombinant Antibodies 1999 ISBN D-471-17847-Q; Masseyt:ff R. et al. Msthods of lmmunological Analysis Wiley-VCH ISHN 3-527-27900-7, 1992; Mnuntam et al. Eds, Biotechnology 2"" txl. Voi SA
1998 lSgN 3-527..28315-3 Wiley-VCII; f~ampbcll, A. . ''Monoclonal Antibody Tochnolo~," in, Burdon, R., ct al., cds, Laboratory Techniques nn Blochemisrry and Molecular BiolAgy, Volume 13. Elsevicr, Publisher, Amsterdam (1984).
The expression of rCCOmbtnanr antibodies. intmmw~uaivtra ok~- including dinbodiec in 6. Coli has becpme routine. General precepts, and methods era discussed in Antibody Engineering 2"~ ed_ Carl A.K.
BorrehaCCk. Oxford University Press 1995 p229-266 see also Antibody Therapeutics WJ f3arris et al. ads.
CRC Press 1997 p. 221; see also roviow in Eiotechnology, Volume 5A, Recombinant Proteins, Mtmucluual Antibodies, and Therapeutic Genes A. Mountain, U. Ney, Dietma.r Schomburg ISBN: 3-527-28315-3, 3muzrl'y 1999, Antibody I'roductio.n: Ea.sential Techniques Prtnr .T. I7eIVCS
IS$1~1.' p..471-97010-7 Wiley June 1997 and Antibody Therapeutics; YraduCtion,1~linical Trials, and Strategic Issues, By Rathin C. .Des, Ph.D., M.B.A. & 15., John Morrow, .Tr., Ph.D.,178cMD Publications f?etober 2001 Chapter 3. (with respect to prodttctiori of antibdd.ies and Imrnunofttsions in E. C~rli and rtfoid;ng of insoluble inclusion bodies and related protein purification steps see also reviews and methods in Park IH et al. Mol Cells 2001 I7ec 31;
121:3):398-402, Tsumoto K, Shinoki IC, Kondo EI, Uehikawa M, ,lujl T, Kumagai 1. Highly efficient reenvery of functional singl~chain Fv fras;mants from inclusion bodies overexpressed in 8gcharlchfa coil by Controlled introduction of oxidizing rcagont--application to a human single-Cilain Fv fragment. J
lmmunol Mcth,ad a. 1998 Oet I;Z19(1-2):119_29. Hanec 1, Jermutus L, Wcber-Bornhau9or S, BosS.itard I-tR, Pluckthun A. Ribosome display eflT~iently selects and Cvolves hiSh-affinity an ibodies in vitro from immune libraries. Proc'Natl Acad Sd U S A. 1998 Nov 24;95(24):1 d 130-5.
Suttnar J, Dyr Jl:, Hamsilcova E, Navak J, VOnl:a V. froCedttra for rC'IOldlDa and puriW ettion ofrecambinantprotaina floor Esoherichia coil inclusion bodies using a strong anion exchangtr. J Chromatogr B Bictmed Apph 1994 Jun 3;fi5f5(l):123-6. Buchner J, Pastan I, Brit~kmam~ U. A msthod for lncrsasmg the yield ofproperly folded recombinant fusion proteins: single-chain immunotoxins from rtnahtration of bacterial Inclusion bodies.
Ana1 6iochem. 1992 Sep;205(2):2b3-74. Kutvcz t, Titus JA, Jost CR, Sega1 DM.
Correct disulfide pairing and efficient refolding of detergani:.solNbiti2ed sin3lP-chain Pv prc,teins from bacterial inclusion bodies. Mol immunol. 1995 Dec;32(17-18):1443-52. Kipilyanov SM, Dubel S, Breitling F, Kontermann RE, Heyntann S, Little M. bacterial oxpression arid refolding of single.chain Fv Fragments with C-termir181 cysteines. CeII l3iophys. 1995 Jun;26(:ij: it;7-204. Worn A, Ptuckihun A.
Different cqviNbrium stability behavior of ScFv fragments: identification, classification, and improvement by protein engineering.
s3orhr..misiry 1999 ,1u16:38(27):8T39-50. .Kurucz I, Thus ,TA, Jost CR, Jacobus CSI, Segal DM, Retargeting ofCTL by an efficiontly refolded biqpccitjc single-chain Fv dimer produced in bacteria. J
Immunol. 1995 May 1;154(9):4576-82. Ramm K, Gehrig P, Pluckthun A. Removal of the Consa~ad disulfitJc ttriate9 from tllC acl"v fYttgmant ~rrFan antibody: effects on F~Idingkinatica and aregation. J Mol l3iol. 1999 Jul 9;290(2):535-46, t~nappik A, Pluckthun A. Engineered toms of a recombi»ant antibody improve its in vivo folding. protein Eng. 1995 Jan;B(I):81-9. Bettor M. 7'-cell~tarylCfed immunofusion proteins from )r, Call. Ann N Y Acad Sci. 1996 May 15; /tfi:544-54. 6,2D~1,023 M,udultw assc,nbly of antivedy genes, antibodies prepared thereby and use; b,146,631 immunptoxins comprising ribd5ome-i»activating proteins; 5,869,619 Modified antibody variable domai»a 1,846.$1$
Pcctate lysce signal sequence; 5,77(7,196 MOditted atllibOdy variable domains xna tlivrtspcutic uses thereof; 5,756,699 tmrnunotaxins Composing rlbasome..inacNvating proteins; 5,744,58D Immunotoxins comprising ribosome-inactivatinl; proteins; 5.ti9B.435 Modular assembly of antibody genes, antibodies prepared thereby and use;
5,49$,417 Modular assembly of ankibody genes, antibodies prepared thereby and use; 5,693,493 Modular assembly of antibody gores. antibodies prepared thereby and use; 5,621,083 Immunotoxins comprising ribosomo-inasiivdting proteins; 5,618,92D .Modular assembly a+ antibody ~Pnea, nntihodieS prepared thereby and use; 5,595,898 Modular assembly of antibody genes, antibodies prepared thereby and use;
5,576,195 Vectors with Acetate lyase signal sequence Ctur Opi» Imrnunol. 1993 Apr; 5(2) 256-fit; Anal Biochcm 2001; (let 1; 297(1) 79-85. EP U1 l:W~ ~, WtU 99!13091,U5 598985$, US
670'7420, WO 01173081;
1J5 5708148, US5410026, US 5288931,1:P 043460$, E'P 41b673, EP 0301835, WO
91087b2, b,323,326 Method nfsolubi.lizing, purifying, and refnlding protein 6,319,$96 Method of solubilizin(t, purif~in3, and refolding protein 6,207,420 Fusion protein systems designed to increase soluble cytoplasrnio expresssan of het~olagous proteins in );scherichia cell 5,866,362 Enhanced purification and expression of insoluble reeuntbinant proteins Cllis ItJ,1-tarel fU. Protein folding in tJx cell;
rnmreting medels of chaperonin function,FASE» .I. 1996 .lan;l0(1):20-6. fIoakncy RC.Reccnt develonmcnts in Iteterologous protein production in Eschetichia coli.Trends Biotechnol. 1494 Nov;12(11):456-63.
Chaudhuri ,1)x1. Refolding recombinant protein(: process strategies and novel apprdaChes,Ann'N '~' Acn4 9c1. 1994 May 2;721:37.x-85.
Nlshiyama M. Beppu T.[Preparation of mutant enzymes by means of rafolding of polypeptidc forming inclusion bodies and err~retary expression svstem)Tanpekushitsu l~ttkustin I~oso. 1994 May;39(7):1366-72.
9kerra A. Bacterial expression of immundglobulin fragtnents.Curr Opin lmmundl.
1993 A.pr;S(2):256-62.
Revicw.28: Fischer $. Sumner I. Gondcnough P.Isalatlon and rcnaxaration ofbio-aci;ive proteins expressed in Exherichia cell as inclusion bodias.Arzneimittslfors~hung. 1997. ricr-..;42(12):15 i'2-5. 29: TesChke C1~, King J.Folding and assembly o.f oligarne,ria proteins in Escherichia coli.Curr t7pin Biotcchnol. 1992 Cet;3(S):46R-73. Kane JF, Hartlay DL.Properties ofrecombinant protein-containing inclusion bodies in F.SCherichia coli.Bioprocess 'I echnbl, I y9 l: i Z: I z 1 ~45. Enfixs SO, Hallabust Ii, lCohlcr K, Strand'borg L, Veide A.Tmpact of genetic engineering on downstream processing of proteins prodtx;ed in E, coli.Adv Bioehem Eng Biatcchnnl. 1990:43:31-42. Kelley RF, Winkles ME.FoIding of eukaryotic proteins produced in Eschcrichia coli.Genet Eng (N Y), 1990;12:1-19. Mattes R.ThC productson ofimprovcd tissue-type plasmi»ogen activator in Escherichia, coli.Scmin Thromb 1-temost. ZOOI
Aug;27(4):325-36. Review. 4:
Pecyucur C, Yaoher b, Miroux l3.ExpreNsion and purl leafier of the mitowhrnxirial uncaupline proteins: a comparative study berween Escheriehia coil and Saccharomycos cerevisiae.Biachem 5oe T'rans. 1999 Dec;27(6):R88-93, Review. No abstract available. 5: Misawa S, Kumagai LRefolding oftheraptutie prptcins produced in Eacherichia cell as inclusion bodies.8ivpulyutms.
1994;51(4):97-307. Review. 7:
Buchanan SI<_Beta-barrel proteins from bacterial outer membranes: structure, ftmcdon and refolding.Curr Opin Stnrct Biol. lp9A An4;9(4):455-61. Review. 8: fuller RC,Micrtrbial inclusions with special reference 1p PHA inclusions and intrsctllular boundary envelopes.lnt d Hiol Macrpmol.
1999 .lun-Ju1;25(1 ~3):21-9.
Review. 11. Gumpert J, Hoischen C,Usc. of cell wall-less bacteria (L-forms) far efftcient expression and secrEdbn of h.etorVloguus gene products.Curr Upiri Bietechnal. 1998 Ort;9(S):546-9. R~~viow.l2: T.ilie H, 5chwarc E, Rudolph R.Advancos in refolding of prdtelns produced in E.
coli.Curr Gpin Hiotcchnol. 1998 Oct;9(5):497-SD 1. Review.l4: Thomas ,JCS, Ayling A, Baneyx F.M0lccular chap4rpnes, folding catalysts, and the recovery of active recombinant proteins ti~om is, coil. To fold or to refold.Appl Siuc1 wm Biotechnol. 1997 Jun :66(3):197-238, i~~view.l.5' Mukhopadhyay A.Inclusio»
bodies and purification of proteins in lslhlogicaliy arrive farms-Adv Biochem Etta Hi'oteChnal.
1997;56:61-109. ROview.l5: Guige AD, West SM:, Chaudhuri J.B.Protein folding in. viva and renaturatipn of recomhinartt proteins from inclusion bodies.Mal Biotechnol. 1h96 Aug:b(1):53-64. R.eview.l7: '.vtiroux H, Walker JB.Over-production of protCinJ in F_SClterichia vii: mut.xnt hosts that Allow .rynthesl3 of some membrane pxfitrins and e~lnlwtlnr proteins at high lcvels.J Mol Biol. 19911 Jul 19;260(3):289-9$. I~evicw.18.
Georgiou G, Valax P.Expression of correctly folded proteins in Escltaricloa coli.Cum Opin Biotachnol. 1996 Apr;7(2):190-7. 0824702565 Tmmunotoxin Methods a»d Protocols by Walter A. Hall (L:drtw), Waltar A.., MD
Hall (Lditor) (iarmaty 15, 2001) Humans Press; ISBN: 0896037'154 Ba3ic Methods in Antibody Production and Characterization by Gary C. Toward (Editor), Delis it- Ftethrll (Fditc~t'',I (5tptetriber 15, 2D00) CRC Frees; 1$13N:
0849394457 Monoclonal Antibodies - Preparation and Usc of Monoclonal Antibodies and Engineered Atttibpdy Derivatives (Basics: From Hadl~round to Eench) by Hcddy Zolx (becember 15, 2000) 8pringer Vorlag; ISBN; U3tcly ~ 3947 Monoclonal ~4~ntifrudicn . T'~'inciplas arc.
T'racticc : Production arc! Application of Monoclonal Antibodies in Cell Biology, Biochemistry and immunology by James W. Goding; 3rd edition f.>"'ebruarv 199b) Academic Pr; 15BT~1: 4122870239 Applications and Engineering ofMonoclonal Antibodies by D. J. King (February 15, 1999) Taylor ~ Francis; lStiN:
0748404a3d Manolclonal Antibody-Bawd Therapy of Cancer (Basic and Clinical Oncology) by Michatel 1,.
Crossbard (LditorxAugust 1, 1998) Mtacel Dekker: ISF3~1: OR2470196$ Protein Purit:acation: Principles and Practice by Robert K. Scopes, Rk Scopes 3rd edition {January 1994) SpningEr Verlag;15BN: 0387940723 Structure and Mcahanism in Protein Science : .~ Guide to >rnzyrno Catalysis and Prote;n Folding by Alan Fersht (January 199'0 W H Freeman dt C:o.; ISF3~1: 07 167926$8 Introduction to Protein $trvcture by Carl-lvar Brandon, 3ohn Tooze 2nd edition {January I 5, 1999) Garland Publishing;1SBN: 0815323050 introduction to Protein Architecture : 'Fhe Structural Biology of Proteins by Axdtur M. Lesk(2001) Oxford University Press; ISBN: Oi 9$544745 Protein Structure Prediction: I'rlethodc and Pmtocols by David M.
Wcbster {Editor) {August I5, 2000) l3umana Press;1SBN: 0896036375; Burgess RR.Purlfication of overproduced Esahorichist cola KNA polym,~rflce sigma Factors by &olubilizita~
inCIUSIOn bodies and refolding from Sarkosyl.Methods Grtzymol. 1996:273:145-9. Lorirnor Glrl.A
quantitative assessment of the role of the ehaperanin proteins so protein: folding in vivo.FASB>3 J. 1996 Jan;10(t):5-9; - and references cared therein) Approaches for the eukaryotie expression of antibcKlics and antibody fusion proteins and the preparation of vectors for use in such m~hods are watt known and extensively descrtbed in the literature. Genera) precepts, and methods am discussed is Antibody Engineering 2"$ ed. Carl A.K.
Bortebaeck, Oxford University l'rass 1 rMS p~67-393 {sat atxh Antibody Therapeutics VJJ Warris ~t 81. ads. CItC Press 1997 p.
183-220; sec also review in 8iotechnol4gy, Volume 5A, R.ecambinana Proteins, Mottoctonal Atttibodics, and Therapeutic Genes A. Mountain, U. Nay, Dietmar Schornbur~ ISBN: 3-527~28315-3, W'ilcy, January 1499 and Antibody Production- Essantin~l Techniqucx t'ctOY J. Delves ISBN: Q-X71-Q'70~10-7 Witey June 199'7 sad Antibody Therapeutics Production. Clinical 'trials, and Suategic Issues, By Ratliin C. Des, Fh.l3.. M.B.A. & K. John Marrow, Jr.,'fh.l~., D&MD Publications O~Ctober 2091 Chapter 3.
With respect to a review of immunotoxirrs ace also Antibody Therapeutics WJ
Harris et a1. t:ds. CRC ,Press 1997 p 33 With respect to methods for producing recombinant vectors see also USE
5,962,255.
Formulation, purification and analytic methods itwolving antibodies are well known m those skihed in the art and have been extensively reviewed. Witlt respect to formulation, purification and analytic methods sae for example, reviews in Antibody Therapeutics Pmdurtion, Clinical Trials, and Strategic issues, By .Ralbiu C. Des, Ph.D.,1HLB.A. & K. ,John Morrow, Jr., Pb.D., D&MD Publications October 2001, Chapter 4.
Methods of chemical manipulation of antibodies for attachment of ligands (eg.biotin), radianuelides etc.
are well known in the art and have been e,ntensivcly reviewed (for example sec review In Basic Methods in Antibody Production & Characterlztlon G.C-1-tuwzsrd Gr al, ads. CRC Trcsa 200L, p_ t~; with rampcct to therapeutic principles see for example, Antibody Therapeutics WJ Harris et al.
ads. CRC Press 1997 p 53-B8).
For example, immnnoconjngates can be prepared by indirectly conjugating a therapeutic agent to an :urti~ody comportcnt. For example, general techniques are described in Shih et &L, lot. J. CatlCCr 41:832-839 (1988); Shih etsl., lot. J. Cancer 46.1101-1106 (1990); and Shib et sl., U.S. Pat, No. 5,057,313. The general method involves reacting $» antibody component having an oxidised carboltydrata portion with a carfier polymer that has at ICast one free amine funCHQn and that is lutaltXl with a plurality 4'~drug, toxin, chelator, boron addends, Ox other therapeutic agent. This reaction results in an initial Schit~ base (amine) l;nkage, which can be stabilized by redr:ction to a secondary amine tp form the Rnal conjugate, A radiolaheled immunoconjugate may corttprise an .alpha.-emitting radioisotope, a .B-emitting radioisotope, a gattuna zmitting rad'roisotopc, au Auger ~lsctrOn emitter, a neutron capturing agent that ernlt$ alpha-particles or 2 radioisotope that deco s by electron capture.
Suitable radioisotopes include ~°"
Au, ~2 p,. t~s IT rst I 90 ~, tas Re5 tan Rer et yu~ 1t ~At, and the like.

As discussed above, a ~~edioisotopt cart bye nttaohed to an antibody component directly or indirectly, via a cholatin~ went. For example, 6' Cu, considered one of the more promising radioisampes for radioimmunothe~py due to its 5 l .5 hour half ii fc atxl abundant supply of beta particles and garnrna rays, can be conju?ated to an antibody component using the ~alating agent, p-l5romoacetamidp,benzyl-tc2racthylaminetotrnncetis Reid (TETA) - Chase., "?fledical AppliCtltiCulS
ofRadioisotopes," in Remingti7n's PharmtlCtu~cal Sciences, I 8th Edition, Gcttnaro et al. (ads.), pagts 624-b52 (Mack Publishing Co. 1990) (see also 1 fh tditiOn of Reminton's). Altemarlvcly, °" Y, which emits an ancrgttic beta particle, ca.n be Coupled to dtt antlb4dy componem using diethylcncLA iaminc cntaacetia acid (DTPA.). Moreover a method for the direct radiolabeling ofthe antibody component with ~31 I is described by Stein et al., Antibody Immunoconj. Radiopharm, 4: 703 ( 1941 ) (sec also U5P 6, 080, 3R4).
Alternatively, boron addends such as carboranes cdti. be attached to antibody cornponCnts, ns discussed above.
In ;tddition, therapeutic immunoccrttjugates can comprise an immunomodulator moiety suitable for application for the purposes herbM. BTOadly spetikin3, tlic term "immunomod<rlntor" includes cytokinen, stem cell growth factors, lymphotoxins, such as tumor necrosis factor (TNF), ilnd hematoXtoietic factors, ouch as intsrleukina (o.>z., intcrleukin-1 (TL-1),1L-2, IL~3,1L6, tL-i0 and IL-12), colony stimulating fbCtors (e.g., granulooyte-colony stimulating thrtor (G-CSF) and granuloeyte macrophage-colony Stimulating factor (GM-C5fi)), interferons (c.g., inte~feronsaipha, -Iteta and gamma.), the stem cell growth factor tlesignatcd "S1 factor," crythropaiotin and thrombopnirti» F,xamlrles of sttitabit immun4tl'IOdulator moieties include IL~~, IL-b, Il_.-10, IL12, intcrftron-ga~rma., TNF.alpha., and the liks.
A related form of thot2pCUtiC 'pt4tein Is a fUsiun prulain comprising nn nntibody moiety and an immunom4dulator moiety, Methods ofmaking antibody-immunomodulator fusion proteins are known to thost nfskil) in the art as discussed herein. For example, antibody fttsion prottins comprising an interieukin-2 moiety are described by Boleti et al., Ann. Onool. 6:9'15 (1005), Alicolc2 of s1., Cancer ("runs Thcr. Z: I $ l f 195), 138Cker et al,, Pros. Nxfl Acad. Sci. U9A 93:7$20 (1996;), Hank et al., Clin. Cancer Ices.
2:1951 ( 1996), and I-Tu ct al., Canctr Res. 5G:4998 (199G). In addition, 5'ang tt al., Hum, Antibodies Hybridomas 6:129 (1995), describe a fusion protein that mCludes an F(ab'~ frapmenr and x turuur nc:.rosis factor alpha moiety.
finch immuttocortiu8ates and antibody-irnmunomodulator fusirni proteins provide a means to deliver an immunomodulator to a target call and art particularly usefltl againat tumor ctll&. 1'he CytotOXic et'Chcts of immunamoduladors art will known to those of skill in the art. Sec, for example, lCle et al., "Lymphokine9 and Munukliics," In Dintcchnvlo~y and Pharmacy, Pessuto et al. (tads.), rmgec 53.70 (Chapman Ba Hall 1993) as well as other refinances herein Cited. As tin illustration, intcrfe~rons can inhibit call proliferation by inducing increased expression of class 1 histoeornplttibitity antigenA on the surface of various cells and thus, enhanct the rate of destruction of cells 't>y CSTOtoxle T lyrapl~w:yres.
Funhermorc, ttlmPr »ooroois factors, such as TNF-alphl., art bslieved to produrc cytatoxic tffects by inducing DNA
fragmentation.
Moreover, thtraptutically useful irnmunoconjttgates can be prepartd in which an alttibody component is conjugated to a toxin or a chemothet'tlpeutic drug. ISIu9tratlve of toxins which arc suitably employed in the.
preparation of yur;h wrijugatcs ai-c ricin, abri», ribonucleaso, DNase T, Staphylococcal rnterntrixin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas catdotoxin.
Sea references heroin as welt a9 for exa,nple, Pastan ct at., Cell 47:6n1 (1986), and i,;oldenberg, CA-A
Cancer,loumal for Clinicians 44.43 ( IySi4). Othef Suitable toxins arc knuwn lc those of skill in the art.
tlrith to raanec:t to hispecific antibody constructs which are capable oFbinding simuhaneously to two li$ands on the came cell set for example W094/32841. Various SUCIi conSttuCts are known in the art.
pg discussed above, an ahcrnttlivc approach to i»froducin)j the com'birsation of therapeutic antibody arid toxin i9 provided by antibody-toxin fission proteins. An antibody-toxin fusion protein is a fusion protein that comprises tin antibody moiety and a toxin moiety. Methods for making antibody-toxitl fuaiott prfteins are known to those of skill In the arc (see references cited ltertin);
antibody-Pseudomonas exvtoxiu A

fusion proteins have been described by Chaudhary et $1., Nature 339:394 {1989), Brinkmann et al., Pros.
NaCI Acad. Bei. USA 88:8616 (1991), Basra et al-, Proc. Naf1 Aead, Sei. USA
89:5867 (1992), Friedman et al., J. Immunol. 130:3054 (L993), Wels et al., Int. ,1. Cm. 60:13~ (1995), Pbminnyn of a1.,1. Biol. Chei,t.
271.10560 (1996), Kuan et al.. Biochemistry 35:2$72 (1996), and 5chmidt et al., Int. .1. Can. 65:538 (1996), Antibody~toxin fil~ainn proteins containing a diphtheria toxin moiety have been described by Kreitman et al,, Leukemia 7'553 (1993), Nicholls et al., J. Biol. Chem. 268:5302 (19931, Thompson et ah, J. Biol.
Chem_ 270:28037 (1995), and Vallcra ct al., I3lpod 88:2342 (1496). Deonarain et al., Tumor Targeting 1:177 (1993), have tlaa~ilbod an antibody-toxin fusion protein hsving an RNr,~a moiety. while Linat'dou et al., Cell 8iopltys, 24-25:243 (1994), pmduced an antibody~tctxin fiusion pmtein compri;:ing z DNase 1 component. Gelonin was used as the toxin moiety in the antibody-toxin fusion protein of Wano et al., Abstracts ofche 209th AC5 National Meeting, Anaheim, Calif., Apr. 2-4. 1495, Part 1, DIOTODS. As a further e~tample, Dohlsten et al., Proc. Naf1 Acad. SCi. USA 91.8945 ( 1994), reported an antibody-mxin fusion protein comprisinb Ctrtrhylncoccal enterotoxin~.A. hISJmerOUS other examples have been Tsptarted in the literature.
USefUI Cancer ehamVtherapeutic drugs fort tko preparation of immttnQCOrrjmaarea tncludc nitrogen mustards, alkyl sultionatES, nitm3oureas, triercrtes, folio acid analr?gs, pyrimidine analogs, purine analogs, antibiotics.
epipodophyllotoxins, platinum coordination complexes, honnanes, and the like.
Suitable chcirtothet'npcutic agents are described In Remington's Pharmaceutical Sciences, 14th Ftl. (Maok Publishing Co. 1995), and in Goodman and Gilrnan's Tha Pharmacological Basis of Therapeutics, 7th Fd, (MacMillan Publishing Co.
1985). Cnlwr suitable chemntheraneutic wants. such as experimental drugs, arc known to chose of skill in the art.
In addlti0n, therapeutically useful innnu»cxonjttgotc. ettn ba obtained by eanjvgstting phntnactive agents oY
dyes to an antibody composite. Fluorescent and other chromogens, or dyes. such as porphyrlns sensitive to visible light, have been used to detect and to treat lesions by directing the suitable light to the lesion. 1n therapy, this has beCn termed photoradiaNan, phototherapy, or photodynamic ttretapy (Jvri ct a1, (cda.), Photodynamic Therapy of Tumors and Other Diseases (Librcria Progetto 1985r van den Berp , Chem.
Brit:~in 22:430 (1986)). Moreover, monoclonal antibodies have been coupled with photoactivated dyes for achieving phatotherapy. Mew et al., J. lmmunol. 130:1473 (1983); idem" Cancer Rcs. 45:4380 (1985);
pseroff et aL, Proc. Natl. Aced. Sci. USA 83:8744 (1986); idern., photochem.
Fhotobiol. 46:83 (1987);
tlasan et al., Prog. Clin. l3iul, Ras. 288:471 (1989); Tatautn et al., Caaers Surg. Med_ 9:47.7. (1989);
pelegtin et at" Grocer 67:2529 ( 1991 ). I-lowever, these earlier studies did not include use of cndoscopic therapy applications, e5peeially with the use of antibody fragments or subfragmen~. Thus, the present invention cnntcrnplabCS the therapeutic use of immunoc4njugates comprising phutuactivc agwts or dyes.
wlrh r~spect to generation o~~nti-ttlmnr antibodies and other antibody fragments for application herein as well as important related. technologies see also WO OOISOODB; WO 01/01137; Wp 97137791; Wry 99137791; WO 97110003; Hoagenboam et a1. TTat. Biotechnology 15(2) Fob 1497 p125-126; Fell H. et al. Journal ()i" imtrlttrtolgy Vu1 146(7) Apr 1 "991 p244(~ 2452; Anderson D, et al L~.iaGb~jnppre Che~rslstty 14(1)Jan 1993 p10-l8; USP6, 172,197; USl'b,171,782; Tmmunological Investlgations200029(2) cntira is9uc), Optionally the tumor binding portion internalizes andlor deliverE a toxic payload, for example a radionuclide, ar other toxin, or a cytokine to the tumor sell (with respect to selecttan oftumur internalizing human antibodies See for example Pool M et al. J .Mol $iol. 2000 Sep 1; 301(5):1149$1, ace alts Kohl MD of el. J Mal. Diol. >Jictcchnic;ues (anon) Vnl 28t;,1) p162 Thero aro numerous examples of functional cytokine and toxin fusions used fax example in cancer therapy that may bans applicatiPn to the invcntiul 114rv"IIl (for c:~pmplcs and reviews oee references kt~'eln cited As wolf as WO 99137791; W099 W400I06605 ; WO 99/52562W0 99137791 MUf.TIPU13.POSB
ANTIHt3DY; Proceeding of the !BC's 11'~ Annual International Canfere~ncc on Antibody Engineering State of the Art Science, Technology and Applications, I?ECember 3-6, 2000;
Ampl iticatiori of T cell-mediatcd immune responses by antibody-r:ytokina fusion proteins. Immunnl invest. 2000 May; 2g(2):117-20; C:sl~a~r liC5.1999 May 1: 59(9):21 ~9-66.; pharntncokmetics nnrl stability Of the ch 14.18-interleukin-2 fusion protein in mice. Cancer Tmmunol Immunother. 1999 Aug; 48(5):219-29.
Phase T study ofsingle, escalating doses of a superatttigen-antibody fusion protein (PlflJ-2145b5) in patients with advanced colorectal or pancreatic carcinoma. ~ Immunother, 2000 .Tan; 23(1 ):1 øx~53.
Twgered toxin thrnapy ~or malignant astrocyroma.Neurosurgery, 2000 Mar; 46(3):544-5 t ; Targeting cytokines to tumors to induce active antitumor immune responses by res~bmbinant fusion proteins. Hum Antibodies. 1999; 9( I );23-36:
Lode I-IN, et al. Tumor-taTgetod IL-Z amplities 'T cell-tnadiatsd immuua tcap~x~sc induced by gene therapy with single-chain IL-12. Pros Natl Acad Sci U S A, 1999 Jul 20; 96((5):8591-6;
Cancer Vaccines and lmmunotherapy 3000 (textbook) : lmmu»athetapY with Tntravenous Intmuttoglobulins P. Imbach (1991) Academic Press; Molecular Approache8 to 'furnor Immunotherapy (1997) World Scientiitc Publishing Company, Incorporated; Vaccines 8c Tmn,unotherapy S. J. Cryz (1991) MeGraw-Hill Ryerscm, Limited f3'ilh respect to internalizing 8ntlbOdies see eg T3iological EfFects of .loll-6rb.B2 Single Chain Antibodies Selected far lntetnalizing Function.; Blochem Biophys Res Common. 2041 Jen l2;
280( 1 ):274-279 and reference8 cited therein, Immunoconjugates of geldanamyein and anti-HEKI
mpnoclr~nal antibodies:
antiproliforative activity on human breast carcinoma veil lines J Natl Cancer InBt. 2400 4~ 4; 92(19):.1573-81; Foulon Ci', ct al., Radioiodinat#on via D-amino acid prptide enhances cellular rebenti0» and tumor ttenograft targeting of an internalizing anti-apiderntal growth factor receptor variant ill monoclonal antibody. Cancer Tte4. 2000 Aug 15; 60((6):4433-G0. Poul MA, aecerril 6, Niel'en. UB, Morisson P, Marks SolCCtlon aftumor-specific internal)zlng human antiLr~dics from phagc librnries ~ Mo113ioi. 2000 Sep I; 30((5):1149-b:I,Vrouenraets M>3, et al.,Targeting of a hydrophilic photoscttsitizerby us~c of internalizing monoclonal antibodies: A new possibility for use in photadynatnic therapy. Int 1 Cancer.
2000 Oct 1; 88( 1 ):108 14.
The applicatiem of bispccific anti6od.ieE, inoiuding methods of nfeking and u.Sing them hSVe beCtt exten$ively reviewed (ee for example van Spric! AB, van Ojik 1-1H, van De Winkel JG. Immurtotherapeutic perspective forbLspecific antibodies. Immuttoi Today. 20Q0 Aug; 21(8):391-7;
Weiner LM. HispeCiflc antibodie8 in Cancer therapy. Cancer J ~a Arn. 200h May; d 9upp19.32G5-71.
Darb~t J, ct el. Protorgating with the affinity enhancement system far ra.dioitn,ttunothere~py. Cancer i3iother Radiopharm. 1999 Jun;
14(3)~ T S3-6B. do WoIfFA. J3rett GM. Liga.nd-binding proteins: their potential for application in systems for controlled delivery and uptake of tigandR Pharmacol Rev. 2000 Jun; 52(2);207-36.: Wang H, Llu ir, Wel L, Guo Y. Bi-specific antibodies fn cancer therapyAdv Exp Mcd $iol. 2000;
465:369-80; 5taerz (J17, Lee DS, Qi Y. Induction oFspcciFio immune tcsYcrance with hybrid antihndiefi.
Imrnunol Tpday. 2000 Apr;
21(4):172-6: 1999 T)ec; 43(4):336-43. Blsasser D, Sta.dick H, van de Wirtkel JG, Va.lerius T. GM-CSF as adjuvant for immunotherapy with bispecific antibodies. Eur ) Cancer. 1999 (lug; 35 Suppl 3:S25-8, Mplema C, lCroesen BJ, Hel'IT'tch W, Meijer DK, de Leij LF. The u5~ ~f bispecifio antibodies in tUmOr cell and tumor vasculature directed immunothernpy. J Control Reieaae. ?040 Feb 14;
6dC 1-3):229-39. Bodcy B, Borie:y R, SieQel 5E. Kaiser rlE. Gcnetica.lly engineered monoclonal antibodies for direct anti-neoplastic trmatrnent and cancer cell specific delivery of chemotlterapeutic agents. Curt Pharm l)es,1t1U0 p'eb;
b(3):261-7b. Kudo T, Suzuki M, Katayose Y, Shinoda M, Sakttrai N, Kodama H, Ichiyama M, "fakemttra ~, YusluJa H, Saeki I1, Saijyo s, 'Takaha:J,i J, Tominzgss T, Matsnnn :C.
Specific targeting immunotherepy of cancer with blspecific antibodies. Tohoku J Exp Med. 1999 Aug; 18$(4):275-88. Koelemij R, of al.
Bispecific antibodies in Cancer tkterapy, from the laboratdxy to the clinic. J
immunother. 1999 Nov;
22(6):514-24. Segal DM, Weiner CiJ, Wciner LM Blspeciflc antibmlies in cancertharapy Curt Opin Imrnunol. 1999 Oct; I 1 (5).558-62. Hudson PJ. Ttecombinant antibody constructs in cancer therapy. Curt Opin immune(, t $40 Clot: 11 (5):548-5?. Earth RF et al. Huron neutron capture therapy of brain turners: an emerging tlxrapcutic modality. Neurosurgery. 1999 h2ar; 44(3):433-5T);
Fleckenstein G, Osmers it, Puchta J. Monoclonal amibodies in solid tumours: approaches to therapy with emphasis on gynaecological cancer, Med Oncvl, 1998 De:c; 15(4);212..2 l . Guyre CA, Fangor M\~U.
Macrophage-rarosted killing and vaccines. Re3 immune(. 1998 Sap-Oat; 149(7-$)~t55-b0 Cao Y, Suresh MR.
Bispecific antibodies as naval bioconjugatos. Bioconjug Chem. 1998 Nov-Deo; 9(6):635-44. parch RA, at al, The developtnene of monoclonal antibodies for the therapy of ~ancar. l~rit Rev Bukaryot Gene Fxpr.
1998; 8(3-4):5ay-56.:
Volm M. Multidntg resistance and its reversal.Anticancer Res, 199$ Ju1-Aug;
18(4C):2905-17, Rouard H, et at, Ec re~rptors as targctR for immunotherady.lnt Rev lttmtunol. 1997; 16(1 ~2):147-$5. Fan Z et al.
Therapeutic 8.pplicatiolf of anti-growth factor toceptar antibodies; Curr Opin Gttcol. 1998 Jan; 1.0(1):67-73.
de Grit GC, et al,Clinical perspectives Qf' bispecife antibodids in cancer.
Cancer immune( Irnmunother.
1997 Nav-Uec; 45(3-4):121-3. (,ilftCr P, IulLr.;hant Atvl. Lnginccriag antibodies for imaging and therapy.Curr Opin l3iotechnol. 1997 Attg; 8(4):449-54, Pluckthun A, et al, New protein engineering apprrlaches to multivalent and bispecfiFe antibody fragments.
Tmmtmatechnolagy. 1997 Jun; 3(2):$3-145.
Rihova 8. Targeting of drugs to Cell 3urfaee receptors. Crit Rev Biotechnol.
1997; I 7(2):149-G9. MulauSa G

et al,Turtror vascular endothelium; barrier or target in tumor directed drug delivery and immunotherapy.
pharm Res. 1997 Jan; 14(1):2-~ 0. Bodoy B, et al, Human cancer detection end immunotherepy with conjugated and non-conjugated monoclonal arUibuelins. Anticancer Rcs. 1?96 Mnr Apr; IC~(2):561-74 Hartmann F et al, Treatment of Hodgkin's disease with bispecifrc antibodies.
Ann Oncol. 1996; 7 Supp1 d:l~i3-6 Well W, et al. Inficrvention in receptor tyrosine kinase-mediated pathways: recombinant antibody fusion proi~cins targeted to ErbB2. Curr Top Micmbiol Immundl. 1996; 213 ( Pt 3):1 l 3-28.: Kait'emo Icl.
Kadioimmunotherapy of solsd cancers= Acta Oncvl. 1996: 35(3):343-5:5.
~Jorhoeyen ME, et al, Antibody frngmr.uls far eontrotlcd. delivery of thsrape~utic agents. Biorhern Roc TYan9. 1995 NOv; 23(4):1 (K7-73.
Haagen IA. Performance of CI)3xCD19 hispecitic monoclonal antibodies in $ cel3 malignancy_ l.euk Lymphoma. 199,5 Nov; 19(5-6):381-93.
lmmultoconjugates having a boron addend-loaded carrier for thermal nsutron activation therapy will normally b~ Pi'Feeted in similar ways. However, it will be advantageous to wait until non-targeted immunoconjugate elear9 before neutrori irradiation is ,perfor~rned. Clearance cgn be accelerated using an anh-body that binds to the immunoconjugate. See U.S. Pat. No. 4,624,R4s for a description ofthis general principle.
The immunocanjugntcs, and fusion proteins of the present invention ca.n be formulated according to known methods tri prepare pharmaceutically useful Compositions, wlreroby the therapeutic pmtoins ors combined in a mixture with a pharmaceutically acceptable carrier. A composition is said to be a "pharmaceutically acceptebl' carrier" if its administration can be tolerated by a. recipient patient. Sterile phosphate-butilrrsd saline is one example of a pharmaceutically acceptable: carrier. Other suitable carriers are well-known to those in the art. See, for example, REMIhfGTON'S PHARMACEUTICAL sCiENCFS, 19th Fd. (i995) or 20* Fai.
For purposes of therapy, antibody components (or immunoconjugato5lfusson proteins) and a pharmaceutically acceptrtble catTiCt are administered her a patient in b thcropoutionlly effective amount. A
Combination of an antibody component, optipnally with an immunoeonjugttte/fuSion protein, and a phar7nz,ranticatly accoptabl~ carrier is said to 15e administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change ire the physiology of a recipient patient. Tn one aspect, an agent is physioir,rgically sl$nifiurnt if its prosance t~rsvtts in the inhibition of the grrrwth Of taT)tet ttn»or cells.
Yet another therapeutic method included in the invention 13 a method of ttvating cancer by 2rdtninistering to an animal 9ufferin~ from Cancer a pharmaceutically eitcceivc awvWr of one or mono fluid tnrgatsd ligand:<
capahie of bitrding to cancer cells, wherein the compound is ossociattd with a substance capable of dxrnas~irtg canrfr cella_ PhaxznaCeutical compositions herein described or alluded to include fluid targeted li~nds of the invention or LlleYapCUtics used in uumbination therapy which msy be administered by a Variety of mutts of adminstration.
)~y administration of an ''cffectivs nrnounf" is intended an emoullt of the wmpuuud tlutt is sufficient to enhance or inhibit a response, is some embodiments particularly an immune response or cellular rospon~
to o fluid targeted li~a,nri. One of ordinary skill will appreciate that effective amounts of a fluid targeted iigartd can be determined empirically and rnay be employed in pure form or, whore such forms exist, in pharmaceutically acceptable salt. ester or pmdnrg foma.1'he fluid targeted ligand may be administered in CoMposltlons in wnrviuation with one or atone pharmaceutically actepta6le excipientq. It will he understood that, when administered to a human patie>Lt, the total daily usage of the compounds and compositions of the present invenrion will be decided by the attending physician within the scope of sound medical judgement. The specific therapeutically cttbctrve Case level for any particular patia~rt will dcp~:nd upon a variety of factors including the type and degree of the cellular response to be achieved; activity of the specific fluid targeted ligand ernployea; the !specific c8mpodtion employed; thB age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion o1.'the agonise or antagonist; rite duration ofthe treatment; drugs used in combination or coincidental Wlth tire specific agonise yr autd~.4nist; and like fs~.etors wall known in the medical arM.

On administrarvn parentcrally, for Cxample by i.v. drip or infusion, dosages optionally at Icasr otl the order of from 0.01 to 5 r'n~kg/day, optionally 0.05 to 1 .0 mglk~day anJ ~ncr~'c preferably O.t to 1.0 m~kglday can be used. Suitable daily dosages for patients are thus vn the order of from 2.5 to 500 mg p.o., optionally to 250 mg p_o., optimally 5 to 104 lag p.o., or on the order of fmtr~n.5 to 250 mg i.v., optictnaliy 2.5 tv 125 mg l.v. and optionally 2,5 to 50 mg i.v.
l7oslng may else ba arranged in a patient specific rrrsnner to provide a prerlPtrrmined conCCntfati0n 4f an agonist or antagonist i» the blood, as determined by the iZIA technique. Thus patient dos2ging may be adjusted to achieve regular on-going troumlt blood levels, as measured by R1A, optionally on the order of at least from 54 to 1000 nglml, protcrably J >u to 500 nglml.
Frors~ above, phsrmac~~rical compositions are provided comprising an agonies or antagonist 8rid a pharmaceutically acceptable carrier or exeipient, which may be administered orally, roctally, paren~ral ly, iritracister»ally, intravaginally; iritrapetitancally, topically (as by powders, ointments, drops or trartsdermai patch), bucally, m as an oral or na9al'pray. By "pharmac~uticnlly acssptahle carrier" is meant a non-toxic solids semisolid yr liquid filler, diluent, encapsulating material or formulatiatt auxiliary of any type. The term "parcnteral° as used herein refers tn modes of administration which include intravenous, intramuseuiar, intt~peritoneai, Intrasternal, subcutaneous and intraarticular injection and infusion.
Optionally a soYnpositinn far far parenteral ittitcti0n can comprise pharmaceutically SCCeptable sterile aqueous or no»aqueous solutions, dispersions, Suspensions or emulsions as wall as stCtile powders for reconstitution into sterile injectable solutions or dispersions just prier tv use. Examples of suitable aqueous ffrid rionaqueous carrier, Jiluents, solvcntx or vchintoa include water, sthsnri~, polyol4 (!mch as glycerol.
propylene glycol, polyethylene glycol, arid tkie like), carboxymethylceuulose and suitable mixtures thereof, vegetable oils (such as olive oil), and irtjectable organic eaters such as ethyl oleato. Proper fluidity can be maintained, for example, by the use of coating matenal5 Such as lecithin, by tl~ maiWcnnnct df the required particle size in the Case of dispersions, and by the use of surfactants.
Some compositions herein descibed may else Contain adjuvants such as preservatives, wetting agents, emulsii~ing agents, and dispersing agents. Prevention of the aCtivn of microorganisms may be ensured by the lncluafon ofvatious:m4il~ctcriai and antitim~ol agents, for example, psraben, chlrnnhntsmnl, phtllol sorbic acid, and the like. It may assn be desirable to include isotonic agemts such as sugars, sodium chloride, and the like. Prolonged. absorption of the inf ectable pharmaceutical form may be brought about lty the inclusion of agents which delay ahsorptian such as aluminum monostaarate aua ~riatin.
In oome cases, in order rp rrnlnng the ctFect of one or theraacutic corilpcxtents herein described, it is desirable w slaw the absorption from Subcutaneous or intramuscular injection.
This may be accomplished by the u9e of a liquid suspension of crystalline ac amorphous material with poor water Solubility. The rate of absorption of the drug lien depends upon its talc of dis9olution which, in turn, may dopanrl nisnn ery9tal size and erystallini loan. Allternativety, delayed absorption of s parenterally administered drug farm is accomplished by dissolving or suspending the drug in an oil vehicle.
lnjactablo depot forms arc made by forming micros»c~psule matrices of the drug in biodegradable polymers such sa polylactide-polyglycnlirie. l~er~endin~ upon the .ratio of drug to polymer and the »etllre Of tlx particular polymer employed, the rete of drug release can be controlled.
>Jxamples of other biodegradable polymers include poly(oitliocsters) and pnly(anhydrides). Depot injectabie formulations are also prepared by entrapping the drug in lipusmnes or mi4r8~mulsians whinh era compatible with body tissues.
The injxta151e 'formulations can he sterilized, far Cxannple, by filtration thmu~,h a bacterial-retaining filter, or by incorporating ster'ilizin,g a.gettts in the farm of sterile solid coanpositions which can be dissolved ox dispersed in sterile water or other 8tcrile injectable medium just prior Co use.
Notwithstanding any indication to the contrary, it wil l be apprEciated that the references herein cited have application to multiplE dttl'erent Subjects and any qualifying rarltarks as to the applicubifity of the references is to ba understood ss relating to each of the subjects for which referancas arc herein provided, as limited only by the titia and subject matter of the referesace.
All pubiicaticn3 herein cited and ttforcnces therein cited and my copendin~
PST application No, PCTICAO.~,J003 ~ 7, frlec! March I 1, ~M?7.,. are hErein inoorC~oratecl by rcfaronca to the same extent as if each of the individual pubiicati0fi9 were specitiailly and individually indicated to bo incOt~orated by reference in its entirety.

Claims

Claims 1. A fluid targeted ligand comprising a targeting ligand (for example a VH
domain, VL domain, growth factor, cytokine, Fc etc) operatively linked to a functional moiety, said functional moiety having n molecular weight which is less than that of the VH. said fluid targeted ligand comprising a weighting moiety (for example a portion of a constant domain which may be a natural extension of said VH) which displaces the center of mass from the VH portion of the molecule.

2. A fluid targeted ligand according to claim 1, wherein tho functional moiety comprises a functional domain of a biologic effector ligand including a toxin, cytokine, chemokine, growth factor, labeling moiety.

3. A fluid targeted ligand comprising at least a pair of laterally projecting binding arms, a flexible hinge region comprising a pair of linkage elements~ and a stabilizer arm portion preferably comprising a functional moiety, said laterally projecting binding arms being responsive to an impinging moving fluid by being oriented in a binding orientation with respect to the direction of the fluid movement.

4. A fluid targeted ligand according to claim 3, wherein the center of mass of the ligand is positioned to assist the molecule to move in a binding orientation.

5. A fluid targeted ligand according to claim 4, wherein the center of mass is in the stabilizer arm portion.

6. A fluid targeted ligand according to claim 3, wherein the said pair of laterally projecting binding arms are part of a single polypeptide chain and are linked to one another by a polypeptide portion which is linked to said linkage elements.

7. A fluid targeted ligand according to claim 3, wherein said polypeptide portion comprises one or a plurality of one or more elements in the group comprising a flexible peptide linker10, a functional moiety, a CH2 domain and a CH3 domain.

8. A fluid targeted ligand according to claim 7, wherein said polypeptide portion comprises at least one enzyme cleavage site.

9. A fluid targeted ligand according to claim 3, wherein neither, one or bath of said binding arms is operatively linked to a light chain or a functional V~ portion or CL portion thereof.

10. A fluid targeted ligand according to claim 6, which is a bispecific antibody.

11. A fluid targeted ligand according to claim 3, wherein one or both of said laterally projecting11 binding arms comprises a CH~ and does not comprise a CL.

12. A fluid targeted ligand according to claim 3, wherein both of said laterally projecting binding arms comprises a VH and wherein one or both of said laterally projecting binding arms comprises a cooperating VL and does not comprise a CL or CH1.

13. A fluid targeted ligand according to claim 3, wherein said stabilizer arm comprises a CH2 and CH3 domain associated as par of a single polypeptide chain with one binding arm and a FM
associated as part of a second polypeptide chain with the other binding arm, optionally said second polypeptide chain being devoid of a CH2 or CN3 domain.

14. A method of improving the targeting efficiency of a therapeutic ligand by modifying its fluid dynamic properties so that it moves in a binding orientation with respect to the location of its target in a moving fluid12.

15. A method according to claim 14, wherein said therapeutic ligand comprises an antibody portion and a functional moiety.

16. A method according to claim 14, by modifying its center of mass l7, A method according t0 claim 16, by adding a weighting moiety.

18. A template molecule for combinatorial chemistry comprising a variant moiety and an invariant moiety13, wherein the variant moiety is positioned at angle of approximately14 45 degrees with respect to a vector drawn between the center of mass of the molecule and the front most edge of the molecule as it moves in a moving fluid.
19. A therapeutic ligand produced by the method of claim 14, 15 or 16.