Patent Application: US-29374707-A

Abstract:
the invention provides methods for stimulating the immune system using antibodies capable of activating immune cells , such as t cells , the antibodies having reversibly inhibited antigen binding sites . selective activation of the antigen binding sites at a site where an immune response is required leads to a corresponding activation of the immune response at that site . surprisingly good results are achieved even though the antibodies do not possess any targeting moiety which causes them to selectively accumulate at the site where immune cell activation is required .

Description:
the antibody used in the compositions and methods of the invention has an antigen binding site capable of binding to a cell of the immune system and consequently activating the cell of the immune system . thus , typically , the antibody is specific for a marker expressed on the surface of the immune cell . the marker is typically a protein . cells of the immune system are typically activated by receiving signals from the environment ( e . g . by binding of antigen to surface - expressed antibody molecules in b cells , or by binding of pro - inflammatory cytokines to specific receptors on the cell surface ) or by direct interaction with other cells ( e . g . by ligation of the t cell receptor by an mhc - antigen complex or homologue expressed on the surface of an antigen presenting cell ). such interactions may be mimicked by certain “ agonist ” antibodies , which are similarly capable of activating a cell of the immune system . activation of the cell of the immune system may cause it to secrete cytokines , proliferate , up - or downregulate expression of particular cell surface proteins ( often referred to as “ markers ”) or display other behaviour characteristic of participation in an immune response at a level above its resting or background level . thus the cell , once activated , will participate in mechanisms which initiate an immune response or enhance a pre - existing immune response at the relevant physiological site . for example , t cells are lymphocytes which respond to antigen presented on the surface of other cells of the body in the context of specialised antigen presenting molecules . these antigen presenting molecules include mhc class i and ii molecules , as well as others such as cd1 . most t cells ( normally expressing the αβ t cell receptor ) respond to peptide antigens presented by mhc molecules , but others ( often expressing the γδ t cell receptor ) respond to non - peptide antigens such as lipids . the t cell receptor is expressed on the surface of the t cell and forms part of a complex containing cd3 , which itself comprises at least 5 different polypeptide chains , designated γ , δ , ε , and ζ . one cd3 complex comprises one γ chain , one δ chain , and two of each of ε and ζ . engagement of the t cell receptor by a suitable antigen presenting complex on the surface of another cell induces signalling via the cd3 complex which results in activation of the t cell . a variety of different t cell subtypes exist . “ helper ” t cells ( which typically express cd4 and can themselves be divided into other subtypes including th1 and th2 ), when activated , proliferate and secrete cytokines to promote activation and proliferation of other immune cell types . “ cytotoxic ” t cells ( which typically express cd8 ), when activated , are capable of killing cells presenting the antigen to which they respond ( which are often cancer cells and / or cells infected by parasites such as viruses ). it is well - known that antibodies against cd3 are capable of activating and inducing proliferation of t cells . examples of anti - cd3 antibodies include okt3 and ucht - 1 , which are both directed against human cd3 . activation of t cells may also be achieved using antibodies against thy - 1 ( aab26353 . 2 gi : 13195770 ), tap , ly - 6c , cd2 ( np — 001756 . 1 gi : 4502653 ) and cd28 ( np — 006130 . 1 g1 : 5453611 ) ( takada , s , engleman , e g , j . immunol . 139 : 3231 ( 1987 ); langlet , c , guimezanes , a , kaldy , p , et al . cytotoxic t cells : biology and relevance to diseases . eds . battisto et al . ann , n . y . acad . sci . 532 : 33 ( 1988 ); gunther , k c , malek , t r , shenvach , e m , j . exp . med . 159 : 716 ( 1984 ); leo , o , foo , m , henkart , p a , et al . j . immunol . 139 : 3556 ( 1987 )). to induce optimal stimulation via cd2 , it may be desirable to use 2 or more different antibodies each binding to a different epitope on cd2 . known combinations include the t11 . 3 with either t11 . 1 or t11 . 2 antibodies ( meuer , s . c . et al . ( 1984 ) cell 36 : 897 - 906 ) and the 9 . 6 antibody ( which recognizes the same epitope as t11 . 1 ) in combination with the 9 - 1 antibody ( yang , s . y . et al . ( 1986 ) j . immunol . 137 : 1097 - 1100 ). alternatively , antibodies against one or more of these proteins may be used in conjunction with an antibody against cd3 to provide costimulatory signals to the t cell and thus enhance t cell activation . costimulatory antibodies may be reversibly inhibited or not , as desired . when reversibly inhibited , it may be desirable that they are inhibited by the same mechanism as the anti - cd3 antibodies , so that one stimulus can activate both antibodies . characteristics of t cell activation depend on the cell subtype , but may include increased cell proliferation , increased il - 2 receptor expression , enhanced proliferation in response to il - 2 and increased expression of cd69 . helper t cells may secrete increased amounts of pro - inflammatory cytokines ( e . g . il - 2 , ifn - γ , tnfβ , il - 4 , il - 5 , il - 6 , il - 10 , gm - csf , il - 10 and / or tnf - α , depending on the particular helper cell type ), while cytotoxic t cells will display enhanced cytotoxic ( cell - killing ) activity . thus antibody - induced activation of a t cell mimics antigen - specific activation of a t cell via the t cell receptor interaction , and induces qualitatively and / or quantitatively similar responses . antibodies against human cd3 include 7d6 , 12f6 , 38 . 1 , 89b1 , 131f26 , bl - a8 , bw239 / 347 , bw264 / 56 , cd3 - 4b5 , clb - t3 / 3 , cris - 7 , f111 - 409 , g19 - 4 . 1 , hit3a , ico - 90 , ip30 , leu - 4 , ly17 . 2g3 , m - t301 , m - t302 , mem - 57 , mem - 92 , nu - t3 , okt3 ( u . s . pat . no . 4 , 658 , 019 ), okt3d , smc2 , t3 , t3 ( 2ad2 ), t3 / 2ad2a2 , t3 / 2ad , t3 ( 2ada ), t3 / 2t8 - 2f4 , t3 / rw2 - 4b6 , t3 / rw2 - 8c8 , t10b9 , t101 - 01 , ucht1 , vit3 , vit3b , x35 - 3 , xxi11 . 46 , xxi11 . 87 , xxi11 . 141 , yth12 . 5 , and yth12 . 5 . see the human leucocyte differentiation antigens ( hlda ) antibody database for more details . it is believed that antibodies having igg2a isotype may be more effective at cell activation than antibodies of other igg isotypes . therefore the antibody may be igg2a , particularly if it is an anti - cd3 antibody such as okt3 or ucht1 . the antibody may have the complete native antibody structure , consisting of two complete heavy chains linked by disulphide bonds to two complete light chains , appropriately folded to form two fab regions and a fc region , optionally with glycosylation . however it is well known that fragments of a whole antibody can perform the function of binding antigens . the term “ antibody ” is therefore used herein to encompass any molecule comprising the antigen binding portion of an antibody . examples of binding fragments are ( i ) the fab fragment consisting of vl , vh , cl and ch1 domains ; ( ii ) the fd fragment consisting of the vh and ch1 domains ; ( iii ) the fv fragment consisting of the vl and vh domains of a single antibody ; ( iv ) the dab fragment ( ward , e . s . et al ., nature 341 , 544 - 546 ( 1989 )) which consists of a vh domain ; ( v ) isolated cdr regions ; ( vi ) f ( ab ′) 2 fragments , a bivalent fragment comprising two linked fab fragments ( vii ) single chain fv molecules ( scfv ), wherein a vh domain and a vl domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding member ( bird et al , science , 242 , 423 - 426 , 1988 ; huston et al , pnas usa , 85 , 5879 - 5883 , 1988 ). the antibodies described herein possess at least one antigen binding site capable of binding to a cell of the immune system . it may be desirable that they comprise two ( or more ) such antigen binding domains . this may facilitate cross - linking of the cognate antigen on the surface of the cell of the immune system , which may be necessary ( or optimal ) for cell activation . if the antibody possesses more than one antigen binding site , then all antigen binding sites are capable of binding to the same cell of the immune system , preferably to the same antigen on the cell of the immune system , and preferably to the same epitope on that antigen . for example , all antigen binding sites of the antibody may be identical . however , activation of some immune cell types may require ligation of more than one type of cell surface receptor . this may be achieved by co - administration of two different types of antibody as described in this specification , each antibody being specific for one of the receptors on the immune cell . alternatively , an antibody may be used which comprises two different antigen binding sites , each being specific for a different receptor on the surface of the immune cell ; i . e . the antibody may be bispecific . for example , it may bind to both cd3 and cd28 . the antibody does not possess an antigen binding site capable of binding to a cell type other than the cell of the immune system which is to be activated . furthermore the antibody has not been modified or engineered to carry any other targeting moiety which targets the antibody to another cell type ( i . e . causes the antibody to bind to that cell at a level significantly above background binding of the same antibody lacking the targeting moiety ). the antibody has not been modified or engineered to carry any non - antibody moiety , excluding a blocking moiety which may be present as discussed further below . thus it has not been engineered to carry ligands or receptors for molecules expressed on other cell types . it is , of course , appreciated that antibody fc regions are capable of binding to fc receptors expressed on various cell types . however , the fc region is not considered here to be a targeting moiety which has been deliberately and artificially introduced to the antibody in order to enable it to bind another cell type . neither is an antibody &# 39 ; s natural glycosylation considered to be such a targeting moiety . thus , preferably , the antibody binds above background levels only to cells expressing the cognate antigen for the antigen binding site , and ( if the antibody comprises an fc domain ) to cells expressing fc receptors . in particular , where the antibody is to be used to stimulate an immune response against a target antigen or a target cell type , the antibody is not capable of binding to that target antigen or target cell type , either via an antigen binding site or via a heterologous moiety introduced by artificial means ( e . g . genetic engineering or chemical modification ). the antibody is reversibly inhibited from binding to its cognate antigen . typically this is achieved by chemical conjugation of a blocking moiety , which can be selectively removed in order to activate the antibody . thus a blocking moiety may be linked to the antibody by a selectively cleavable group or bond . in preferred embodiments the selectively cleavable group or bond is cleavable by irradiation , e . g . by uv , infra - red , x - ray or visible irradiation . laser irradiation may be particularly suitable , especially for therapeutic methods , as its delivery can be very closely controlled . thus it can be used to irradiate only a site of diseased tissue ( e . g . a tumour ) without affecting surrounding healthy tissue . however irradiation from a uv lamp may be equally suitable . it may be possible to activate the antibody &# 39 ; s binding capability by irradiation through the recipient &# 39 ; s skin , so avoiding the need for surgical intervention . thus the antibody may be inhibited from binding to its cognate antigen by a photocleavable moiety . such photocleavable moieties are well known in the art . antibodies can be suitably derivatised by means of appropriate reagents which couple to hydroxy or amino residues . thus phosgene , diphosgene or dccl ( dicyclohexyl carbodiimide ) may be used to generate photocleavable esters , amides , carbonates and the like from a wide variety of alcohols . nitrophenyl derivatives may be used in this context . substituted arylalkanols may be used , such as nitrophenyl methyl alcohol , 1 - nitrophenylethan - 1 - ol , and substituted analogues . thompson et al . ( biochem . biophys . res . com . 201 , 1213 - 1219 ( 1994 ) and biochem . soc . trans . 225s , 23 ( 1995 )) describe reversible inhibition of protein function by addition of 1 -( 2 - nitrophenyl )- ethyl ( npe ) moieties . further photocleavable moieties will be well known to the skilled person , e . g . from “ biological applications of photochemical switches ”, h . morrison ( ed . ), bioorganic photochemistry series , volume 2 , j . wiley & amp ; sons . ( see especially chapter 1 , section 4 , pages 34 to 50 ). other suitable photocleavable moieties include 1 -( 2 - nitrophenyl ) diazoethane ( l . bédouet et al ., recovery of the oxidative activity of caged bovine haemoglobin after uv photolysis , bbrc , 320 ( 2004 ) 939 - 944 ), 2 - nitrophenylglycine ( m . endo et al , design and synthesis of photochemically controllable caspase - 3 , angew . chem . int . ed 2004 , 43 , 5643 - 5645 ), 6 - nitroveratryl ( m . endo et al , design and synthesis of photochemically controllable restriction endonumclease bamhi by manipulation of the salt - bridge network in the dimmer interface , j . org . chem ., 2004 , 69 , 4292 - 4289 ), o - nitrobenzyl and 4 - hydroxyphenacyl . the antibodies described in this specification will typically be administered to a recipient in the form of pharmaceutical compositions . these compositions may comprise , in addition to the antibody , a pharmaceutically acceptable excipient , carrier , buffer , stabiliser or other materials well known to those skilled in the art . such materials should be non - toxic and should not interfere with the efficacy of the active ingredient . the precise nature of the carrier or other material may depend on the route of administration , e . g . oral , intravenous , cutaneous or subcutaneous , nasal , intramuscular , intraperitoneal routes or topical application . intravenous , intramuscular or subcutaneous administration is likely to be appropriate in many instances , but other methods of administration are possible . pharmaceutical compositions for oral administration may be in tablet , capsule , powder or liquid form . a tablet may include a solid carrier such as gelatin or an adjuvant . liquid pharmaceutical compositions generally include a liquid carrier such as water , petroleum , animal or vegetable oils , mineral oil or synthetic oil . physiological saline solution , dextrose or other saccharide solution or glycols such as ethylene glycol , propylene glycol or polyethylene glycol may be included . for intravenous , cutaneous or subcutaneous injection , or injection ( which may or may not be at the site where immune stimulation is desired ), the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen - free and has suitable ph , isotonicity and stability . those of relevant skill in the art are well able to prepare suitable solutions using , for example , isotonic vehicles such as sodium chloride injection , ringer &# 39 ; s injection , lactated ringer &# 39 ; s injection . preservatives , stabililsers , buffers , antioxidants and / or other additives may be included , as required . administration is preferably in an “ effective amount ” sufficient to show benefit to the individual . the actual amount administered , and rate and time - course of administration , will depend on the nature and severity of what is being treated . prescription of treatment , e . g . decisions on dosage etc , is within the responsibility of general practitioners and other medical doctors , and typically takes account of the disorder to be treated , the condition of the individual patient , the site of delivery , the method of administration and other factors known to practitioners . suitable carriers , adjuvants , excipients , etc . can be found in standard pharmaceutical texts , for example remington &# 39 ; s pharmaceutical sciences , 20th edition , 2000 , pub . lippincott , williams & amp ; wilkins ; and handbook of pharmaceutical excipients , 2nd edition , 1994 . however the dose administered will be suitable to stimulate immune cells rather than suppress immune responses . in general lower doses of anti - cd3 are believed to stimulate t cells , while higher doses are more likely to suppress immune responses , possibly by depletion of t cells . see bluestone , j a , science 242 : 569 ( 1988 ), and u . s . pat . no . 6 , 406 , 696 . the skilled person will be capable of ascertaining a suitable dose for any given situation . the compositions and methods described herein are preferably used for treatment of mammals , more preferably primates ( e . g . humans , apes or monkeys ), domestic animals ( e . g . feline , canine , etc . ), laboratory animals ( e . g . rodents , lagomorphs etc .) or livestock animals ( e . g . bovine , equine , porcine , etc .). the cd3 + t - cell line h9 was obtained from etcc . the okt3 secreting hybridoma was obtained from atcc and okt3 was obtained from serum free media by ( nh 4 ) 2 so 4 precipitation . the human ovarian cell line , oaw42 , which was used to produce conditioned rpmi media was kindly provided by dr a wilson , derby general hospital . the cd3 + murine cell line el4 was stored in this laboratory . this cell line was grown in rpmi 1640 media containing 10 % fcs . the 145 - 2c11 monoclonal antibody secreting hybridoma was obtained from ecacc . the antibody was precipitated from serum free media using ( nh 4 ) 2 so 4 followed by extensive dialysis . okt3 ( 0 . 5 mg in 1 ml 0 . 1m bicarbonate ) was rendered inactive by the addition of 15 μl npe - carbonyl chloride ( in dioxan ) for 4 h at 20 ° c . followed by centrifugation and dialysis to remove excess reagents and insoluble reaction products 8 . when this was irradiated with uv - a light the npe groups cleaved , reactivating the antibody . in early experiments the npe - coated okt3 was irradiated in a quartz cuvette prior to its addition to the t - dells ( fig1 ). in later work the npe - okt3 complexes were irradiated in the presence of the t - cells ( fig2 ) in 96 well plates . likewise , 1 . 5 ml of 145 - 2c11 ( 0 . 5 mg / ml in 0 . 1m bicarbonate ) was inactived by the addition of 10 μl npe - carbonyl chloride ( in dioxan ) for 4 h at 20 ° c . followed by repeated dialysis in 10 mm phosphate buffer ph7 . 5 to remove uncoupled npe groups ( 1 ). on centrifugation at 13 , 000 g for 10 min the inactivated 145 - 2c11 was obtained as a pellet . this was resuspended in 1 ml of phosphate buffered saline to give a suspension containing 0 . 19 mg / ml inactivated antibody with an 01 ) 280 value of 1 . 4 . after subtracting the od 280 value for the antibody ( 0 . 26 ) the remaining od of 1 . 14 equates with approx . 300 npe residues being associated on each antibody molecule . this figure is almost certainly far too high due to the npe - coated antibody being a cloudy suspension and reflecting rather than absorbing the light beam . when this suspension was irradiated with uv - a light the npe groups cleaved , reactivating the antibody . the npe - coated okt3 samples were irradiated with a vl - 206bl uv - a lamp ( 2 × 6w tubes ) which has a total uv - a irradiance of approx . 16 mw / cm 2 at a distance of 1 cm . irradiations were carried out for six minutes in quartz cuvettes in preliminary experiments which demonstrated that & gt ; 90 % of the npe residues cleave in this time with no denaturing of antibody activity . longer periods of uv - irradiation ( 10 min ) were used when the cells and npe complexes were irradiated together to allow for absorption of the uv light by the plastic lid of the 96 well plates . 250 ul aliquots of h9 cells , 10 6 cells / ml in rpmi 1640 media containing 10 % fcs , had 30 ul of untreated or npe - coated okt3 ( 0 . 1 mg / ml ) added to them and were left for 1 h at 4 ° c . after washing the cells were resuspended in 100 ul of fitc - labelled goat anti - mouse ( bd pharmingen , 5 ul diluted to 1 ml in phosphate buffered saline ph 7 . 4 , pbs ) and left for 30 min at 4 ° c . after 3 further washes the cells were resuspended in pbs and okt3 binding was analysed by facs . the expression of the activation marker cd69 was analysed using facs . the h9 human t - cells were resuspended in oaw conditioned rpmi medium , 150 ul aliquots containing 150 , 000 t - cells were added to individual wells of a 96 well plate . 20 ul of untreated or npe - coated okt3 ( 0 . 2 mg / ml ) was added to the wells and the cells were irradiated in the presence of the antibody for 10 min through the lid of the plate . unirradiated npe - coated okt3 was then added to relevant wells and the cells were left for 3 h at 37 ° c . before the supernatant was removed ( for il2 analysis ). after washing the cells were resuspended in 50 ul pbs containing fitc - labelled anti - cd 69 ( 5 ul per 10 6 cells ) and were left for 1 hr at 4 ° c . after 3 further washes the cells were resuspended in pbs and analysed immediately using facs . il2 concentrations were measured using a bd biosciences human il2 elisa kit . oaw conditioned medium ( rpmi 1640 media containing 10 % fcs which had been left for 3 days in confluent cultures of oaw42 cells ) was required for the h9 cells to express cd69 and il2 . c57bl6 mice were purchased at 8 weeks old and were injected with tumour after they had been left for at least one week to acclimatise to their new surroundings . frozen pieces of m5076 tumour were thawed from liquid nitrogen storage . this was diced as finely as possible in 199 medium and 50 ul of packed diced tumour was injected subcutaneously into each animal using a fine guage needle . after approx 3 weeks the tumours were excised and freshly diced tumour ( 50 ul ) was simultaneously injected with 50 ul of medium containing 5 ug of 145 - 2c11 conjugates . controls contained medium alone . for in vivo photolysis a small area on the flank of the mice was shaved using hair clippers , the tumour and antibody were injected under the shaven area , and the shaven area was irradiated for 5 min with a hand held lamp ( see below ) from a distance of 2 - 3 cm above the mice . a coating of photocleavable 2 -( nitrophenyl ) ethanol ( npe ) groups was used to inhibit the biological activity of the anti - cd3 monoclonal antibody okt3 . the inhibition and reactivation of okt3 binding to the h9 human cd3 + cell line was then investigated using facs . native uncoated antibody was taken as representing 100 % binding ( fig1 b ). when the antibody was coated with npe its binding to the t - cell line reduced markedly with only 2 . 5 % of the cells fluorescing ( fig1 c ), a figure similar to that given by control cells incubated with only the second layer antibody ( fig1 a ). irradiation with uv - a light removed the npe groups and reactivated the antibody as shown by the antibody binding increasing to approximately 80 % of that given by untreated native okt3 ( fig1 d ). the light specific binding and subsequent activation of the h9 t - cell line was confirmed by the expression of early t - cell activation markers 3 h after the addition of the npe - okt3 complexes . in t - cells treated with un - illuminated npe - okt3 the levels of activation marker cd69 ( fig2 c ) were only slightly increased above background fluorescence ( fig2 a ). however the cd69 levels were significantly increased on the t - cells illuminated in the presence of npe - okt3 ( fig2 d ) to levels similar to those obtained with control illuminated uncoated antibody ( fig2 b ). this light controlled activation of the h9 cells was confirmed by the analysis of il - 2 levels in the cell culture supernatants . medium from control h9 cells which did not receive cloaked antibody contained 11 ± 3 pg / ml il2 . in medium from cells treated with npe - okt3 the concentration of il2 increased to 54 ± 13 pg / ml il2 . ( this was not unexpected and probably reflected the presence of a small residual fraction of uncloaked antibody ). however , illumination of the h9 cell npe - okt3 mixture increased the il2 concentration to 211 ± 30 pg / ml ( mean and variation of 3 separate experiments ). it is important here to point out that i ) reactivation was carried out in the presence of the h9 cells under physiological conditions without causing damage to the cells and ii ) the light from the hand held lamp was sufficient to reactivate the okt3 antibody even though the irradiation was carried out through the plastic lid of the 96 well plate . we have also been able to demonstrate the reversible inhibition of ucht - 1 , a second anti - human cd3 antibody , with very similar results to those reported above . this establishes the reproducibility of the procedure described . as we had successfully reversibly inactivated the mouse anti - human t - cell antibody , okt3 , we then decided to examine if we could reproduce this effect with the similar hamster anti - mouse cd3 antibody , 145 - 2c11 ( 20 ). this would enable us to investigate possible anti - cancer effects of our procedure in a syngeneic c57bl6 mouse system . the 145 - 2c11 antibody was therefore coated with npe . this antibody proved to be prone to coming out of solution during dialysis to remove excess npe . when resuspended to a suspension in isotonic saline the npe - coated 145 - 2c11 antibody could not bind to the cd3 expressing murine lymphoma cell line el4 . however after illumination with uv light for 10 min considerable binding of the antibody ( 70 % of that given by uncoated antibody ) to el4 cells could be detected ( table 1 ). this was an extremely important result , as this was carried out in the presence of the cells under physiological conditions . we then employed the npe - coated 145 - 2c11 antibody to carry out some initial studies to determine if we could regulate the growth of the m5076 ovarian tumour cell line in c57bl6 mice . initial toxicology studies demonstrated high doses of 145 - 2c11 and npe - coated 145 - 2c11 ( 30 ug / mouse ) had no deleterious effects on the mice . we grew up the m5076 ovarian tumour in c57bl6 mice , finely diced the resulting tumour and passaged 50 ul of the diced tumour into groups of 6 mice along with 50 ul of medium containing 5 ug of various 145 - 2c11 conjugates . after approximately 3 weeks the mice were humanely killed and the final tumour weights were measured . the results obtained from two separate experiments are given in table 2 . in the first experiment the control tumours had an average weight of 200 mg in the mice that only received media with the transplanted diced tumour . when control uncoated 145 - 2c11 was injected with the diced tumour , final tumour weights markedly reduced to around 20 mg . this is an interesting finding in itself , as it shows that if an anti - cd3 antibody is present in the area next to a tumour it will stimulate the immune response , even when it is not being specifically targeted to the tumour by a bi - specific antibody . when the npe - coated - inactivated antibody was injected with the diced tumour the average weight of the final tumours increased in size almost back to the level of control tumours ( as expected ). if the npe - coated 145 - 2c11 was irradiated in vitro for 5 min ( in a cuvette ) then injected with the transplanted tumour , then the final tumour weight decreased significantly , but not to the levels found with uncoated antibody . however when the tumour and npe - coated 145 - 2c11 was irradiated in vivo after the two components had been injected , there was a massive drop in subsequent tumour growth to the extent that no tumour was detectable in 5 of the 6 mice after 25 days . this was even more pronounced than the effect of uncoated antibody . this implied that irradiation of the npe - coated 145 - 2c11 in situ with the tumour conferred some additional potency . in order to confirm these results the experiment was repeated . tumour growth was more vigorous on this passage of tumour , control tumours and tumours treated with unirradiated inactivated 145 - 2c11 reaching 400 mg in size after only 22 days growth . treatment with uncoated antibody again markedly decreased tumour growth to around 25 % of control values whilst in vitro irradiated npe - coated antibody again reduced final tumour sizes to just over half control values . despite the more vigorous growth of the tumour in this experiment , very little tumour was again obtained in the animals that were irradiated in vivo to reactivate the 145 - 2c11 . a third experiment was then carried out in which control tumours were irradiated through a patch of shaved skin in addition to the animals treated with npe inactivated 145 - 2c11 ( table 3 ). here the tumours again grew more vigorously to a very similar size ( 365 mg ) obtained in the previous experiment . the irradiated control tumours were no smaller than un - irradiated control tumours confirming that reductions in tumour growth were caused by the reactivated 145 - 2c11 antibody . the tumours in the latter group , whose transplant had been irradiated in the presence of npe - coated antibody , were barely detectable . older mice had been used for this experiment and it was very hard to distinguish and dissect the tiny tumours from surrounding fat . figures given for the final tumour weights in this group are over - estimates due to fat contamination . 100 ul aliquots containing 200 , 000 el4 cells were added to individual wells of a 96 well plate . 10 ul of npe - coated 145 - 2c11 ( 0 . 19 mg / ml ) was added to the wells and the cells were irradiated in the presence of the antibody for 10 min through the lid of the plate . unirradiated npe - coated 145 - 2c11 was then added to relevant wells and the cells were left for 1 h at 4 ° c . before they were washed . after washing the cells were resuspended in 100 ul of a 1 / 100 dilution of biotinylated - anti hamster igg ( vector labs ) for 30 min , washed , and 100 ul of a 1 / 1000 dilution of strepavidin - fitc ( sigma ) was added , again for 30 min at 4 ° c . after 3 further washes the cells were resuspended in pbs and analysed immediately using facs . the value given is the mean fluorescence of the facs peak . groups of 6 mice were simultaneously injected ( subcutaneously ) with 50 ul of diced m5076 tumour and 50 ul of medium containing 145 - 2c11 conjugates . after approx . 3 weeks the resulting subcutaneous tumours were excised and weighed . values are given ( in grams ) for each animal . statistics analysis is not required as the differences in tumour weights are so large in the different groups . data is given for two separate sets of experiments . three groups of 4 mice were simultaneously injected with 50 ul of diced m5076 tumour and 50 ul of medium ( 2 groups ) or npe - coated 145 - 2c11 ( 1 group ). one of the control groups and the npe - coated antibody group were irradiated for 5 min through a shaved patch of skin . * two tumours were not completely excised . while the invention has been described in conjunction with the exemplary embodiments described above , many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure . accordingly , the exemplary embodiments of the invention set forth are considered to be illustrative and not limiting . various changes to the described embodiments may be made without departing from the spirit and scope of the invention . all references cited herein are expressly incorporated by reference . 1 . baeuerle p a , kufer p , lutterbuse r . bispecific antibodies for polyclonal t - cell engagement . curr opin mol therapeut 2003 ; 5 : 413 - 419 . 2 . lum l g , davol p a . retargeting t cells and immune effector cells with bispecific antibodies . cancer chemother and biological response modifiers 2005 ; 22 : 273 - 291 . 3 . van spriel a b , van ojik h h , van de winkel j g j . immunotherapeutic perspective for bispecific antibodies . immunology today 2000 ; 21 : 391 - 402 . 4 . withoff s , helfrich w , de leij l f , molema g . bi - specific antibody therapy for the treatment of cancer . curr opin mol therapeut 2001 ; 3 : 53 - 62 5 . morrow k j , jr . challenges remain for antibody products . genet engineering news 2005 ; 25 : no 19 pages 1 , 16 - 19 . 6 . brekke o h , sandlie i . therapeutic antibodies for human diseases at the dawn of the twenty - first century , nature reviews 2002 ; 2 : 52 - 62 . 7 . molema g , cohen tervaert j w , kroesen b j , helfrich w , meijer d k , de leij l f . cd3 directed bispecific antibodies induce increased lymphocyte - endothelial cell interactions in vitro . br j cancer 2000 ; 82 : 472 - 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beile u . construction and in vivo evaluation of an anti - psa x anti - cd3 bispecific antibody for the immunotherapy of prostate cancer . anticancer res 2000 ; 20 : 1551 - 1555 . 20 . alegre m - l , et al . cytokine release symdrome induced by the 145 - 2c11 anti - cd3 monoclonal antibody in mice : prevention by high doses of methylprednisolone . j . immunol . 146 , 1184 - 1191 , 1991 .