Patent Application: US-6982305-A

Abstract:
the present invention provides novel means and methods for treating unwanted side effects in transplantations , such as gvhd and allograft rejection . the invention provides immunotoxins comprising an antibody and a toxic substance , whereby cocktails of such conjugates are directed to different targets associated with one population of cells , wherein one target is chosen from cd3 or cd7 . the preferred combination is a cocktail directed against both cd3 and cd7 .

Description:
the rationale for its to treat gvhd is that these conjugates can be used for an efficient and specific eradication of immunocompetent t cells responsible for the disease . in this perspective , its might be more effective and may cause less side effects than broadly immunosuppressive reagents such as cyclosporine and corticosteroids . in 1990 , byers et al . reported a phase i clinical trial in which they intravenously administered an anti - cd5 rta - it ( xomazyme - cd5 ) to treat corticosteroid - resistant gvhd ( 39 ). the initial results were very promising with skin , gastrointestinal tract , and liver disease responding in 73 %, 45 %, and 28 % of cases , respectively ( 39 ). however , more recent clinical trials have shown that xomazyme cd5 is no more effective than alternative treatments ( 18 ). consequently , the further development of xomazyme - cd5 has been abandoned . encouraged by the initial success of the it - based treatment of gvhd , we set up to develop alternative its with superior anti - t cell activity . in order to achieve this , rta was conjugated to a panel of moabs that react with antigens that are expressed almost exclusively on t cells , namely the t cell differentiation - antigens cd3 , cd5 and cd7 , and each was assayed for its anti - t cell activity . from this preclinical study , it appears that a cocktail of spv - t3a - rta ( cd3 - it ) and wt1 - rta ( cd7 - it ) has the highest potential for treating patients with severe gvhd . this mixture affords : synergistic cytotoxicity in which the simultaneous incubation of half the effective individual dose of spv - t3a - rta and wt1 - rta is more effective in eliminating t cells than either it alone ( including the cd5 - it ). broad mechanism of action in which binding of spv - t3a to the t - cell receptor / cd3 complex results in an additive immunosuppressive effect by blocking the recognition by the donor t cells of the foreign patient antigens . this effect is independent of action of ricin a . moreover , the binding of this particular cd3 - moab does not stimulate t cells to produce cytokines which would otherwise augment the severity of gvhd . broad spectrum reactivity by which wt1 - rta is also reactive against nk cells . these lymphocytes are thought to aggravate the severity of gvhd , especially in the later phase of the disease . it has been previously described that the use of combinations of its can strongly enhance the efficacy of target cell elimination . the most obvious advantage over single - it - treatment is that fewer target cells will be multiple antigen - negative than single antigen - negative . in addition , those cells which do express substantial levels of all target - antigens might be loaded with it to a higher extent . when the respective its follow a different intracellular routing , the chance of escaping therapy might be further reduced . with respect to the use of anti - t cell it , reports addressing the combination or cocktail approach have thus far focused on in vitro applications , including the purging of bone marrow grafts . for one aspect of the present invention , we state that a mixture of murine mab spv - t3a ( cd3 , igg2b ) and wt1 ( cd7 , igg1 ), both conjugated to dga , forms a superior combination for the elimination or suppression of unwanted ( e . g ., over - reactive , misdirected , or malignant ) t cells and / or nk cells . this particular combination affords important benefits which surpass the “ common ” synergism as observed with the more or less “ random ” combinations of anti - t cell it . some aspects delineating the superior characteristics of this particular combination are described below . ( a ) antigen binding of a cd3 or a t cell receptor ( tcr ) mab results in at least partial blocking and modulation ( internalization or shedding ) of the cd3 / tcr - complex , thereby preventing alloactivation of the t lymphocytes . ( b ) binding of a cd3 or a tcr mab results in at least partial fas - mediated apoptosis of a significant fraction of activated t lymphocytes , according to a mechanism described as activation induced cell death ( aicd ). these two effector mechanisms , which are independent of a conjugated toxin , are of vital importance when intervening in an acute life - threatening situation such as refractory gvhd . the temporal ( blocking and modulation of cd3 / tcr ) as well as limited ( aicd ) nature of these effects only stresses the benefit of making a “ real killer ” of the mab by conjugating it to a toxin . one reason we selected spv - t3a as cd3 mab is that spv - t3a is an igg2b - isotype and out of the majority of the t cells isolated and examined from the human population spv - t3a has proven not to induce cytokine release . as a consequence , the risk of the so called “ cytokine release syndrome ,” which severely complicates the immunological disorder to be treated , is strongly reduced . ( c ) the presence of the cd7 - it in the it - cocktail is essential , apart from the above mentioned “ common ” synergism , in that it broadens the spectrum reactivity of the it - cocktail . the cd7 antigen is also expressed on nk cells which , accordingly , form a target for this particular it - cocktail as well . our changed insight regarding the processes underlying gvhd is that nk cells play a distinctive role in the pathophysiology of gvhd , particularly in the efferent phase of the disease . spv - t3a : spv - t3a is a mouse igg2b moab directed against the human t cell differentiation antigen cd3 ( 40 ). anti - cd3 antibody therapy is often associated with the cytokine release syndrome caused by the binding to the t cell receptor / cd3 complex ( 41 - 44 ). one of the important benefits of spv - t3a is that this particular moab does not induce cytokine release because it is an igg2b - isotype ( 45 , 46 ). at the department of hematology of the university hospital nijmegen , part of the bone marrow obtained from hla - matched unrelated donors is currently treated ex vivo with a cocktail of spv - t3a - rta and wt1 - rta in order to eliminate immunocompetent t cells . the patients transplanted with this marrow showed normal hematopoetic reconstitution without any signs of toxicity ( n = 3 , data not shown ). wt1 : moab wt1 is a mouse moab of igg2a isotype directed against the human t cell differentiation antigen cd7 ( 47 , 48 ). at the department of nephrology of the university hospital nijmegen , three patients who underwent a kidney - transplantation , have been treated with wt1 in order to treat an acute rejection . the administration of unconjugated wt1 appeared to be safe and did not result in either an allergic reaction or severe toxicities . no clinical efficacy could be noted . wt1 has been conjugated to dgrta and administered to rhesus monkeys to test its suitability for use in the therapy of leukemic meningitis ( 49 ). the major conclusion of this study was that wt1 - dgrta may be safely administered intrathecally to rhesus monkeys and could be a good candidate for the treatment of t - lymphoblastic cns leukemia . at the department of hematology of the university hospital nijmegen , wt1 - rta has been used since 1986 for the ex vivo purging of autologous bm of patients suffering from high - risk t cell leukemia / lymphoma in order to eliminate residual malignant cells . after purging , neither neutrophil engraftment nor immunological reconstitution was delayed ( n = 20 ) ( 25 ). smpt cross - linker : the moabs are conjugated to dgrta using the chemical cross - linker smpt ( fig3 ). the cross - linker contains a disulfide bond which is important for the intracellular dissociation of the moab and dgrta which is necessary for toxicity . ( see fig2 ). smpt is a so called “ second - generation cross - linker ,” characterized by having a hindered disulfide bond due to the presence of the phenyl ring . this renders the smpt - linker less susceptible to extracellular reduction by thiols present in the tissues and blood , and , therefore , results in a prolonged serum half - live of the it . thorpe et al . demonstrated in an in vivo mice tumor model that using smpt instead of the first - generation cross - linker spdp , strongly improves the anti - tumor effect of their dgrta - based its ( 20 ). amlot et al . performed a phase i trial in which they studied the treatment of malignant lymphoma by intravenous administration of a smpt - conjugated it ( rfb4 [ igg ]- dgrta ) ( 50 ). due to the long serum half - live of 7 . 8 hours , therapeutic serum concentrations could be maintained between the infusions given at 48 - hour intervals . dgrta : the earliest rta - based its consisted of a moab conjugated to native rta . the oligosaccharides present on the native rta resulted in rapid hepatic clearance and hepatotoxicity in vivo ( 20 , 51 ). this problem has been addressed in the second - generation its which make use of either deglycosylated rta ( dgrta ) or non - glycosylated recombinant ricin a ( rrta ) ( 52 , 53 ). vitetta and colleagues have reported the administration of dgrta - based its to patients with refractory b - cell non - hodgkin &# 39 ; s lymphoma . they tested two different constructs . in the first , the fab ′ fraction of moab rfb4 ( anti - cd22 ) was conjugated to dgrta ( 50 ). in the latter construct they used rfb4 whole molecule ( 54 ). the its were administered by 4 - hour intravenous infusions given at 48 - hour intervals . the phase i dose limiting toxicities included pulmonary edema , expressive aphasia , and rhabdomyolysis with acute renal failure . other side effects included hypoalbuminemia , weight gain , fever , tachycardia , decrease in electrocardiogram voltage , myalgias , anorexia , and nausea . the maximum tolerated dose ( mtd ) was 75 mg / m 2 for the fab ′- dgrta and 32 mg / m 2 for whole igg - dgrta . the mtd appeared to be inversely related to the serum half - life of 86 minutes and 7 . 8 hours , respectively . the two forms of the dgrta - it demonstrated no significant difference in clinical responses ( partial and complete responses in 45 % of the patients receiving greater than 50 % of the mtd ), in immunogenicity or in the toxic side effects . because of its lower costs , the igg - dgrta it was selected for further development . the major findings to be learned from these studies are : a ) the mtd of dgrta - its is dependent primarily on the size of an individual dose rather than the cumulative dose . when administering rfb4 ( igg )- dgrta at 48 - hour intervals at doses of 8 mg / m 2 or less , only grade i or ii toxicities were observed . total doses of 32 mg / m 2 rfb4 ( igg )- dgrta were consistently safe . as a consequence of the relatively long t 1 / 2 , therapeutic serum concentrations ( about 1 . 8 μg / ml ) could be maintained during and between infusions . b ) side effects of the dgrta - its administration were relatively modest and consisted predominantly of vls and myalgia . no hepatotoxicity and minimal bm toxicity was observed . c ) patients with underlying pulmonary disease should not be treated because of the danger of vls contributing to further pulmonary insufficiency . patients received four doses of it - cocktail administered intravenously in 4 - hour infusions at 48 - hour intervals . if no clinical response was observed and if no severe toxicities ( grade iii or iv ) occurred , the study continued with the next higher protein dose level . patients have received second - line high dose corticosteroid therapy ( methylprednisolone 1000 mg / d ) for at least three days without any decrease in the severity of gvhd . 1 . the patient has a significant history or current evidence of intrapulmonary disease . 2 . the patient has a history of allergy to mouse immunoglobulins or ricin . 3 . the patient has circulating high levels of human anti - mouse antibodies ( hama ). the it - cocktail has been prepared by the department of hematology under supervision of the department of clinical pharmacy of the university hospital nijmegen . the it - cocktail is stored at − 80 ° c . at 1 mg / ml in 0 . 15 m nacl , in lots of 5 and 20 mg . before infusion , the it - cocktail will be filtered through a 0 . 22 μm filter and diluted to a final volume of 100 ml in normal saline solution . the id 50 against the t cell line jurkat is taken as the standard for biological activity when evaluating the quality of different lots of it - cocktail . immunotoxins are administered via a central venous catheter . prior to therapy , patients are given an intravenous test done with 200 μg it - cocktail . therapy is only started in the absence of anaphylactoid reactions . the it - cocktail is administered in four doses at 48 - hour intervals . the rationale of this is to give all of the it - cocktail before any host antibody response is expected to arise ( usually not before 10 to 14 days after administration of xenogenic ig ). the patient is initially treated with two subsequent doses of 2 mg / m 2 . at this dose level no side effects are observed . in the absence of grade iii or iv toxicities , the dose will be enhanced to 4 mg / m 2 if necessary . toxicities related to the immunotoxin administration are graded as grade i ( mild ), ii ( moderate ), iii ( severe ) or iv ( life threatening ) based on world health organization ( who ) guidelines . special attention must be paid to the vascular leak syndrome ( vls ). the physical signs of vls are weight gain , peripheral edema , decrease in blood pressure , hypoalbuminemia , and small pleural effusions . consecutive doses given to the same patient : infusion of the second , third and fourth dose at any dose level is dependent upon the toxicity achieved after the previous infusion : grade i toxicity : no change in the scheduled dosage . grade ii toxicity : 24 hours - delay of the dosage , with the next dose given if toxicity improves . grade iii toxicity : the next dosage of immunotoxin will be withheld and only given if toxic parameters have improved ( halving of the dosage can be considered ). grade iv toxicity : no further dosage . dose escalation : progression from one dose level to the next should only occur after : the patient ( s ) of the group treated with the previous dose level have received four doses of it - cocktail and have been observed for at least 48 hours after the last dose ; and dose limiting toxicity has not been reached . dose limiting toxicity : dose limiting toxicity is defined as the occurrence of adverse reactions of grade iii or iv in an individual patient . if two patients experience a grade iii toxicity or if grade iv toxicity occurs in a single patient , three additional patients will be entered at this dose level . if none of these additional patients demonstrate toxicity of grade iii or iv , administration will again be continued to the next higher dose level . if grade iv toxicity occurs in two patients at a given dose , the next patients will be treated with the previous dose level which will be considered the maximum tolerable dose ( mtd ). immunosuppressive agents used for prophylaxis and initial treatment may maintain unchanged throughout immunotoxin therapy . before entry into the study , the patient undergoes a general examination consisting of medical history , physical examination with special emphasis on acute gvhd , measurement of oxygen saturation , electrocardiogram ( ecg ), and chest x - ray . the laboratory measurements will include na +, k +, cl —, hco 3 —, urea , creatine , bilirubin , glucose , ap , asat ( got ), alat ( gpt ), ggt , ldh , total protein plus electrophoresis , leukocytes plus differentiation , red cells , hemoglobin , hematocrit , and thrombocytes . the patient is assayed for human anti - mouse and anti - ricin responses ( hama / hara ), and serum levels of il - 2 , tnf - α and ifn - γ . furthermore a quantitative alloreactive t - helper / t - cytotoxic precursor assay will be performed . daily complete physical examinations are performed during it - cocktail administration until two days after the last dose and weekly thereafter . blood is analyzed daily for na +, k +, cl —, hco 3 —, urea , creatine , glucose , albumin , leukocytes plus differentiation , red cells , hemoglobin , hematocrit , and thrombocytes . blood is analyzed every two days for bilirubin , ap , asat ( got ), alat ( gpt ), ggt , ldh , total protein plus electrophoresis . vital signs ( blood pressure , pulse , respiration frequentation , and temperature ) are checked every 15 minutes during the first hour post infusion , every 30 minutes during the second up to fourth hour post infusion , and from then on every hour up to eight hours post infusion . in addition , vital signs are assessed daily during it - cocktail administration until two days after the last dose , and then weekly thereafter . pharmocokinetics and clearance of its : blood samples are collected at 0 , 1 , 3 , 4 , 8 , 12 , 24 , and 48 hours after each infusion . the serum concentrations of spv - t3a - dgrta and wt1 - dgrta are quantitatively determined in a sensitive and mab - isotype specific immuno - radiometric assay ( irma ). from these results the individual serum half - lives of the two are calculated using non - linear , least squares regression analysis . measurement of humoral responses : in order to examine hama and hara responses , serum samples are obtained one day pre - injection and weekly after the first infusion until the patient comes off the study . the concentration of hama and hara is determined in a sensitive radiometric assay . immunological monitoring : blood is sampled every other day during it - cocktail administration until two days after the last dose and then weekly thereafter for immunological monitoring . pbls are isolated and evaluated for composition by flow cytometry using antibodies reacting specifically with t cells subsets , b cells , monocytes / macrophages , and nk cells . serum is collected to determine levels of il - 2 , tnf - α and ifn - γ by commercial enzyme - linked immunosorbent assay ( elisa ) kits . the proliferative and cytotoxic activity of alloreactive pbls is tested two days following the last dose using standard t - helper and t - cytotoxic - precursor assays , respectively . staging and grading of gvhd and clinical responses : gvhd is scored daily during it - cocktail administration until two days after the last dose , and weekly thereafter until the patient comes off the study . each organ system is evaluated grade i through iv gvhd according to the criteria of glucksberg et al . : skin by amount of surface involved with rash , gastrointestinal tract by the volume of diarrhea , and liver by serum bilirubin levels . patients are also given an overall grade of gvhd based on severity of organ involvement . complete response ( cr ): the disappearance of symptoms in all organ systems ; partial response ( pr ): improvement of & gt ; 1 organ , with no worsening in other organs . mixed response ( mr ): improvement of & gt ; 1 organ , with worsening in & gt ; 1 other organ . stable disease : no significant change in any organ system . progressive disease ( pd ): progression in & gt ; 1 organ system without improvement in any organs . the duration of response is defined as the period from the date the response was first recorded to the date on which subsequent progressive disease is first noted . alloactivation was analyzed in a mixed lymphocyte culture ( mlc ). mlc were performed with “ responder ” peripheral blood lymphocytes ( pbl ) mixed in a one to one ratio with irradiated “ stimulator ” pbl . cultures were performed in triplicate ( 5 × 10 4 cells / well ) in u - bottomed microtiter plates in 150 μl culture medium at 37 ° c . and 5 % co 2 . prior to , or at different days following initiation of the mlc , spv - t3a ( 10 − 8 m ) or an irrelevant isotype - matched control antibody were added to the culture medium . following 72 hours of culture , plates were labeled with [ 3 h ] thymidine ( 0 . 4 μci / well ) for 4 hours . subsequently , the proliferation of responder cells was determined by collecting the dna using a cell harvester and counting the incorporated radioactivity . proliferation was expressed as a percentage of the untreated control . alloactivation was completely blocked when spv - t3a was added directly following the initiation of the mlc . when addition of spv - t3a was postponed to one or more days following initiation , this effect gradually ceased to exist . following four days , addition of spv - t3a no longer had an effect on proliferation . the irrelevant isotype - matched control antibody did not influence alloactivation at all time points . translated to the in vivo situation these results demonstrate that unconjugated spv - t3a is capable of delivering a direct and important immunosuppressive effect by preventing ongoing allostimulation of t lymphocytes . for the suppression or elimination of already stimulated t lymphocytes , spv - t3a is dependent on another effector mechanism , termed activation induced cell death ( aicd ), or needs to be conjugated to a toxin . the results are summarized in graphical form in fig4 . reduction of tcr - mediated cytotoxicity following it - treatment was assayed in vitro using a cytotoxic t cell clone ( ctl - clone ) recognizing ebv - peptide ebna3c presented in hla - b44 . clt activity was assayed by lysis of a loaded ebv - transformed lymphoblastoide cell line ( ebv - lcl ) originating from the same individual . the ctl - clone was treated for 24 hours with spv - t3a , washed and assessed either directly or following four days of additional incubation in culture medium . the extended four day incubation period was incorporated since during this time the ctl - clone restored its normal expression of the tcr / cds complex ( which is blocked and / or modulated directly following incubation with spv - t3a ). directly following treatment ( day 1 ), incubation with native mab spv - t3a ( 108 m ) resulted in a modest reduction of ctl - cytotoxicity . flow cytometric analysis revealed that this effect was predominantly caused by the blocking and modulation of the tcr / cd3 complex due to binding of spv - t3a . following four days of extended incubation , the ctl - clone regained its normal tcr / cd3 - expression , but ctl - cytotoxicity was further reduced to 18 % of the untreated control ( day 5 ). this time , flow cytometric analysis revealed that the majority of the ctl - cells had died due to apoptosis , according to the mechanism described as “ activation induced cell death ” ( aicd ). translated to the in vivo situation this means that unconjugated spv - t3a is capable of delivering an important immunosuppressive effect by eliminating a significant fraction of activated t lymphocytes . the efficacy of spv - t3a will be further enhanced when conjugated to a toxin like ricin a . the results are summarized in graphical form in fig5 . pha - stimulated pbl were treated with 10 8 m it for 24 hours at 37 ° c ., washed , and cultured for another four days at 37 ° c . in it - free culture medium ( to enable the it to display their full toxicity ). after this lag period , cells were incubated with 2 μg / ml propidium iodine ( pi ) ( molecular probes , junction city , oreg .) and 2 μg / ml calcein am ( calc ) ( molecular probes ) for 1 hour at rt . samples were then analyzed on a coulter epics elite ( coulter ) flow cytometer equipped with a 40 mw argon ion laser running at 15 mw . a longpass - filter of 610 nm was used for measurement of pi - fluorescence , a bandpass - filter of 525 / 30 nm for calc - fluorescence . overlap of the emission spectra of pi and calc could be adjusted by electronic compensation using single - labeled samples . samples were analyzed in triplicate using a minimum of 10 , 000 cells . viable cells were identified as being pi - negative and calc - positive . prior to fcm analysis , a fixed amount of inert beads ( dna - check , coulter ) was added ( 10 5 beads / ml ) to enable the calculation of the number of surviving cells . the reduction of pbl was related to the viable fraction of the untreated control . treatment factor of pbl reduction spv - t3a - dga 100 wt1 - dga 87 spv - t3a - dga and wt1 - dga 1770 ( half a dose each ) due to the “ common it - cocktail synergism ” it spv - t3a - dga and wt1 - dga appeared to be far more effective in combination ( half a dose each ) than either it alone . blood mononuclear cells were isolated from peripheral blood by ficoll centrifugation and incubated with 108 m mab or it in a concentration of 1 × 10 6 / ml for 24 hours . subsequently , cells were washed and analyzed for nk - activity after 4 additional days of incubation without it ( this lag period is essential for it to display their full efficacy ). during the experiment , 50 units / ml recombinant il2 was added to the culture medium to increase nk - activity . for analysis of nk - activity , cells were serial diluted and incubated with a fixed number of 51 cr - labeled k562 blasts ( 10 4 / 100 μl ) to yield an effector to target ratio of 10 : 1 , 3 . 3 : 1 , 1 . 1 : 1 , and 0 . 37 : 1 . after 3 . 5 hours of incubation at 37 ° c ., the cell mixtures were centrifuged and radioactivity was measured . nk - activity was expressed as a percentage maximum 51 cr - release as determined with saponin treated 51 cr - labeled k562 blasts . both were corrected for spontaneous 51 cr - release as determined with 51 cr - labeled k562 blasts incubated with culture medium only . incubation with saturating amounts of mab spv - t3a ( 10 μg / ml ) had no effect on the nk - activity , nor had treatment with spv - t3a - dga ( 10 − 8 m ). four days following incubation with wti - dga , in contrast , the nk - activity distinctively reduced to 8 % of the untreated control . neither unconjugated wt1 , nor the isotype - matched control it influenced the nk - activity . translated to the in vivo situation , this means that incorporation of wt1 - dga in the it - cocktail not only results in the common it - cocktail synergism , but also broadens the spectrum reactivity . this is of vital importance since , though initiated by ctl , gvhd is thought to be aggravated by less specific cytokine - stimulated bystander cells like monocytes and nkilak cells . the results are summarized in graphical form in fig6 . a . ld5o determination with balb / c mice ( see , fig7 ): b . administration to java - monkeys ( see , fig8 and 9 ): ongoing one center , non - randomized , open labeled , dose escalating study ( aim of treating 5 - 7 patients ) dose of it - cocktail ( mg / m 2 ) # patients d1 d3 d5 d7 total 1 2 2 4 4 12 2 4 4 4 4 16 2 8 8 8 8 32 2 10 10 10 10 40 evaluation : pharmacokinetics , toxicities , human - anti - mouse antibodies and human - anti - ricin antibodies ( hama and hara ), biological and clinical responses male 60 , multiple myeloma sibling transplantation gvhd of skin , gut and liver ( overall grade iv ) complication : multi - organ failure it - cocktail : 2 doses 2 mg / m 2 , 1 of 4 mg / m 2 died seven days following the first infusion mild capillary leakage , no weight gain no increase of ck - levels no further acute toxicities no hamas / haras skin : improvement starting at day 5 liver : stable ( poor condition ) gastrointestinal tract : not interpretable ( morphine ) impressive reduction of circulating t / nk cells ( cd2 + 5 + and cd2 +, respectively ) during first 4 hour infusion : decrease to 17 % gradually declines further to 1 % at day 7 dual mechanism : mab - based ( fast ) & amp ; dga - based ( lasting ) male 34 , cml matched unrelated donor grade 4 gvhd of the skin it - cocktail : 2 doses 2 mg / m 2 , 2 of 4 mg / m 2 dramatic ( complete ) response starting at day 5 lasting for — 1 . 5 month relapse of gvhd i - ii responding to low dose corticosteroids died 8 months following treatment due to an infection male 47 , mds hla - identical donor gvhd grade ¾ of the gut it - cocktail : 4 doses of 4 mg / m 2 rise in body temperature during infusions no further acute toxicities no hamas / haras reduction of lymphocytes ( see , fig1 ) decrease of stool volume endoscopy : strong improvement of gut - tissue it - cocktail is well tolerated , no acute severe toxicities extensive biological and clinical responses in the absence of acute severe toxicities it - cocktail forms effective tool for in vivo suppression or elimination of misdirected , over - reactive or malignant t cells and / or nk cells the monoclonal antibodies disclosed herein are prepared using hybridoma technology well known in the art . selection steps to select immunoglobulins having the properties disclosed hereinabove are also well known in the art and may include affinity chromatography and the like . f ( ab ′) 2 fragments of antibodies were prepared using a “ f ( ab ′) 2 preparation kit ” ( pierce , rockford , ill . ), according to the manufacturer &# 39 ; s protocol . briefly , antibodies were incubated with immobilized pepsin at ph 4 . 2 ( 20 mm sodium acetate buffer ) for four and sixteen hours , respectively . undigested igg molecules , and fc - fragments were removed by affinity chromatography with protein a - sepharose . remaining fragments smaller than 30 kd were removed by means of a centriprep - 30 concentrator ( amicon , beverly , mass .). the purity of f ( ab ′) 2 fragments was determined by sds - page , which revealed less then 1 % contamination with either intact igg or fc - fragrnents ( data not shown ). antibodies were conjugated on a 1 to 1 ratio ( m / m ) to deglycosylated ricin a ( dga , inland laboratories , austin , tex .) using the smpt - cross - linker ( pierce , rockford , ill . ), according to the method as described by ghetie et al . ( ghetie , v . et al ., the glp large scale preparation of immunotoxins containing deglycosylated ricin a chain and a hindered disulfide bond , j . immunol . methods 1991 , 142 : 223 - 30 ). antibodies were conjugated to ricin a ( kindly provided by dr . f . k . jansen ; centre de recherches clin . midy , montpellier , france ) using n - succinimydyl 3 -( 2 - pyridyldithio ) propionate ( spdp ; pharmacia ) or smpt , as described . ( the conjugation ratios of ricin a to mab were estimated by measurement of absorbance at 280 nm and ria , and were determined to be in the order of 0 . 8 to 1 . 2 .) preservation of antibody - binding activity following conjugation was assessed by fcm . table i dose levels : dosage of it - cocktail ( mg / m 2 ) patients d1 d3 d5 d7 total 1 2 2 4 4 12 2 4 4 4 4 16 2 8 8 8 8 32 2 10 10 10 10 40 1 vital signs are checked every 15 minutes during the first hour post - injection , every 30 minutes during the second up to the fourth hour , and from then on every hour up to eight hours post - injection . 2 the pre - study biochemistry panel includes na +, k +, cl −, hco 3 −, urea , creatine , bilirubin , glucose , ap , asat ( got ), alat ( gpt ), γgt , ldh , and total protein plus electrophoresis . during the follow - up study , blood will be analyzed daily for na +, k +, cl −, hco 3 −, # urea , creatine , glucose , and albumin . besides , every two days is added bilirubin , ap , asat ( got ), alat ( gpt ), γgt , ldh , and total protein plus electrophoresis . 3 the hematology panel includes leukocytes plus differentiation , red cells , hemoglobin , hematocrit , thrombocytes . 4 to be continued weekly until wbc numbers have returned to normal . 5 venous blood samples are obtained pre - injection and 1 , 3 , 4 , 8 , 12 , 24 , and 48 hours after each injection . besides , a sample is taken 72 hours following the last injection . 6 the flow cytometry panel includes the markers cd2 , cd3 , cd4 , cd5 , cd7 , cd8 , cd14 , cd19 and cd56 . 7 serum is assayed for levels of il - 2 , tnf - α and ifn - γ . 6 . snyder d . s ., findley d . o ., forman s . j ., et al . fractionated total body irradiation and high dose cyclophoshamide : a preparative regimen for bone marrow transplantation for patients with hematologic malignancies in first complete remission . blut . 1988 , 57 : 7 . 7 . martin p . j ., hansen j . a ., buckner c . d ., et al . effects of in vitro depletion of t cells in hla - identical allogeneic marrow grafts . blood 1985 , 66 : 664 . 8 . patterson j ., prentice h . g ., brenner m . k ., et al . graft rejection following hla matched t - lymphocyte depleted bone marrow transplantation . br . j . haematol . 1986 ; 63 : 221 . 9 . kernan n . a ., flomenberg n ., dupont b ., et al . graft rejection in recipients of t cell - depleted hla - nonidentical marrow transplants for leukemia . transplantation 1987 , 43 : 842 . 10 . vallera d . a ., blazar b . r . t cell depletion for graft - versus - host - disease prophylaxis . a perspective on engraftment in mice and humans . transplantation 1989 , 47 : 751 . 11 . horowitz m ., gale r . p ., sondel et al . graft - versus - leukemia reactions after bone marrow transplantation . blood 1990 , 75 : 555 . 12 . sullivan k . m ., weiden p . l ., storb r ., et al . influence of acute and chronic graft - versus - host disease on relapse and survival after bone marrow transplantation from hla - identical siblings as treatment of acute and chronic leukemia . blood 1989 , 73 : 1720 . 13 . truitt r . l ., lefevre a . v ., shih c . c . graft - vs - leukemia reactions : experimental models and clinical trials . in : gale r . p ., champlin r . 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