Phase I Study of Monoclonal Antibody-Ricin A Chain
`Immunotoxin XomaZyme-791 in Patients with Metastatic Colon
`Cancer
` 
`V. S. Byers, R. Rodvien, K. Grant, et al.
`Cancer Res  
`
`1989;49:6153-6160.
`
`Updated version
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`on October 30, 2014. © 1989 American Association for Cancercancerres.aacrjournals.org on October 30, 2014. © 1989 American Association for Cancercancerres.aacrjournals.org
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`Research. Research.
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`IMMUNOGEN 2294, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`(CANCER RESEARCH 49, 6153-6160, November I. 1989]
`
`Phase I Study of Monoclonal Antibody-Ricin A Chain Immunotoxin
`XomaZyme-791 in Patients with Metastatic Colon Cancer
`
`V. S. Byers, R. Rodvien, K. Grant, L. G. Durrani, K. H. Hudson, R. W. Baldwin, and P. J. Scannen
`XOMA Corporation, Berkeley, California [V. S. B., K. H. H., P. J. S.J; Cancer Research Campaign Laboratories, University of Nottingham, Nottingham, England
`[V. S. B., L. G. D., R. W. B.I; and Pacific Medical Center, San Francisco, California [R. R., K. G., K. H. H.]
`
`ABSTRACT
`
`recognizing a M, 72,000 antigen on
`Monoclonal antibody 791T/36,
`the surface of colon carcinoma cells, has been used to construct an
`immunotoxin by conjugating to it the ribosomal
`inhibitor protein, ricin
`toxin A chain. The antibody 791T/36 has been shown to bind to mem
`branes of freshly disaggregated tumor cells from human colon tumors,
`and to localize in tumors in vivo. Subacute toxicology testing in rats
`receiving immunotoxin i.v. showed, at highest doses, weight
`loss, de
`creased serum albumin, and hepatocyte vacuolization without elevation
`in liver function tests.
`A Phase I dose escalation study was carried out in which 17 patients
`with metastatic colorectal cancer were treated with doses of immunotoxin
`ranging from 0.02 to 0.2 mg/kg/day in 1-h i.v. infusions for a 5-day
`course. Side-effects included a composite of signs and symptoms thought
`to be generic to ricin A chain immunotoxins,
`including decreased serum
`albumin, mild fever, and flu-like symptoms, all being reversible. Two
`additional
`findings, reversible proteinuria and mental status changes,
`were also noted which may be characteristic of this immunotoxin.
`By 10-20 days after therapy, most patients developed IgM and IgG
`antibodies against both the ricin toxin A chain and the immunoglobulin
`portion of the immunotoxin, which were asymptomatic. A strong anticom-
`bining site antibody response was seen. Biological activity manifest as
`mixed tumor regression was seen in five patients.
`
`INTRODUCTION
`
`antigens
`MoAbs1 directed against human tumor-associated
`have allowed drug targeting to be explored as a therapeutic
`modality in cancer (1-3). Cytotoxic moieties, when coupled to
`MoAbs, can be directed specifically to the relevant
`target cell.
`One such moiety, RTA, has been used to construct
`several
`immunotoxins
`(1,4, 5). RTA is a ribosomal
`inhibiting protein
`which functions as an RNA /V-glycosidase specific for the 28-S
`ribosomal subunit
`(6). Since RTA alone is poorly internalized,
`it
`is functionally
`inactive as a free agent. When coupled to
`monoclonal
`antibodies, however,
`it can be targeted to tumor
`cells, internalized,
`and is cytotoxic to the cells.
`MoAb 79IT/36
`recognizes a M, 72,000 antigen present on
`tumor cells derived from ovarian, colorectal,
`and osteogenic
`sarcoma tissues (7-9). Flow cytometric analysis of tumor cells
`from colorectal and ovarian tumors, derived by enzymatic dis-
`aggregation of the tumors, demonstrated
`that 79IT/36 MoAb
`binds to the majority of cells from over 80% of both types of
`tumors
`(8, 9), indicating the antigen is expressed on the cell
`surface. In vivo, this MoAb localizes in tumors, as demonstrated
`by studies
`in which the antibody,
`labeled with 13II or
`'"In,
`
`revised 2/16/89, 5/17/89; accepted 8/4/89.
`Received 9/9/88;
`The costs of publication of this article were defrayed in part by the payment
`of page charges. This article must
`therefore be hereby marked advertisement
`in
`accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`ricin A
`' The abbreviations used are: MoAb, monoclonal
`antibody; RTA,
`chain; LDH,
`láclate dehydrogenase: SCOT,
`serum glutamic oxaloacetic
`acid
`transaminase; CT, computerized tomography; CEA, carcinoembryonic
`antigen;
`PBS, phosphate
`buffered saline: XomaZyme-791.
`immunotoxin made from
`791T/36 MoAb (clinical material); CPK, creatinine phosphokinase: BUN. blood
`urea nitrogen; SDS-PAGE,
`sodium dodecyl sulfate-polyacrylamide
`gel electro-
`phoresis; HPLC, high pressure liquid chromatography; EEC. electroencephalo
`gram: ELISA, enzyme-linked immunosorbent
`assay; FITC,
`fluorescein isothio-
`cyanate.
`
`images primary and metastatic ovarian and colorectal cancers
`(9-11).
`an immunotoxin,
`The MoAb has been used to construct
`XomaZyme-791,
`by conjugation with RTA chain (12, 13). In
`vitro studies indicate that
`the immunotoxin
`retains over 70%
`of its binding to cells carrying the M, 72,000 antigen, and when
`tested on 79IT target cells using a [75Se]methionine incorpo
`ration assay,
`there was more than a 1000-fold difference be
`tween the molarity of immunotoxin
`and free RTA necessary to
`attain 50% inhibition of tumor cell growth in vitro (12).
`It
`specifically and effectively inhibits growth of human tumor
`xenografts
`(13). On the basis of these findings, animal
`toxicol
`ogy studies were carried out, and XomaZyme-791 was then
`tested in a Phase I clinical
`trial for the treatment of metastatic
`colorectal cancer.
`
`MATERIALS AND METHODS
`
`Monoclonal Antibody. The generation of the hybridoma producing
`79IT/36 MoAb (IgG2b) has been previously described (12, 14). The
`MoAb used for these clinical trials was produced by XOMA Corpora
`tion from murine ascites and purified by affinity adsorption on Sepha-
`rose-protein A (13). The homogeneity of the IgG2b preparations, eval
`uated by SDS-PAGE, HPLC, and double immunodiffusion
`indicated
`preparations had purities of greater than 95%. Reactivity of the MoAb
`on normal tissues was assessed by immunoperoxidase
`staining on frozen
`sections of a range of normal adult and fetal tissue from five cadaveric
`donors using a modification of the ABC technique (15).
`To measure reactivity with hematopoietic progenitor cells, mature
`T-lymphocytes were removed from human bone marrow aspirates by
`first treating the cells with soybean lectin and removing the resultant
`agglutinates,
`then forming E rosettes and removing them. This tech
`nique produces a 4 logic depletion of T-lymphocytes
`(16). Binding to
`the remaining cells was assessed by indirect immunofluorescence, meas
`ured by flow cytometry.
`The purified MoAb is free of xenotropic and ecotropic viruses as
`well as the twelve murine viruses measured by the mouse antibody
`production test (a test in which contamination with 12 murine viruses
`is evaluated by injection of the test article into mice and determination
`of antibody production to the viruses of interest2).
`Ricin Toxin A Chain. RTA was purified from castor beans by a series
`of column based separations,
`including immunoaffinity chromatogra
`phy (17). The RTA was greater
`than 95% pure as judged by SDS-
`PAGE, and contained no detectable ricin, or ricin toxin B chain by any
`assay including immunoprecipitation.
`The IC!0 level of the purified
`RTA as measured by a reticulocyte lysate assay (17) was less than 10
`pM. This assay measures inhibition of protein synthesis in a cell free
`system. In a mouse toxicity assay, RTA injected into BALB/c mice at
`10 mg/kg produced no deaths.
`Immunotoxin. Immunotoxin XomaZyme-791 was prepared for clin
`ical trials by conjugating purified ricin A chain to the murine monoclo
`nal antibody 79IT/36
`by means of /V-succinionidyl-3-(2-pyridyldi-
`thio)propionate
`reagent, forming a disulfide bond (4,12). It was purified
`by gel filtration. Each lot was subjected to a series of tests prior
`to
`release. The free IgG level and amount of immunotoxin present
`in the
`immunotoxin
`preparation was determined by size exclusion HPLC.
`Binding of the conjugate to 79IT target cells was compared to that of
`
`2 Xoma Corporation,
`
`unpublished observations.
`
`6153
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`on October 30, 2014. © 1989 American Association for Cancercancerres.aacrjournals.org
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`
`Research.
`
`IMMUNOGEN 2294, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`MoAb-RlCIN A CHAIN IMMUNOTOXIN XomaZyme-791
`
`the native antibody by flow cytometry analysis as previously described
`(18). The cytotoxicity of the immunotoxin against relevant and irrele
`vant target cells was determined using an in vitro assay in which target
`cell survival is determined by [3HJthymidine incorporation. Target cells
`(791T) or the erythroleukemia
`cell line, Molt-4 (ATCC Bethesda, MD)
`which does not carry the M, 72,000 antigen recognized by this MoAb,
`were plated at 4 x 10" cells/well
`in 100 ml of RPMI 1640 (GIBCO,
`Grand Island, NY) with 10% fetal calf serum (Hyclone Labs, Logan,
`UT). After
`incubation at 37°Cfor 3 h, various concentrations
`of
`immunotoxin ranging from 20 to 2000 ng/ml were added in 100-/J
`aliquots to triplicate wells. After 48 h, 1 //Ci of [3H]thymidine was
`added to each well and 72 h later the wells were harvested and incor
`porated radioactivity determined. Results are expressed as amount of
`immunotoxin/ml
`producing 50% inhibition (IC50). These tests were
`repeated at monthly intervals after immunotoxin production with min
`imal change in reactivity. XomaZyme-791 was prepared for clinical use
`at a concentration of 1 mg/ml
`in phosphate buffered saline, pH 7.3. It
`was clear to visual inspection and was filtered through a low protein
`binding 0.22 /mi filter into about 100 ml of normal saline just prior to
`infusion.
`Animal Toxicology Studies. The LD50 level was assessed on BALB/c
`mice; subacute toxicology studies were performed in Sprague-Dawley
`rats, both from Charles River
`laboratories. Mice were injected i.v.
`through the tail vein and observed for 5 days. After at
`least 7 days
`quarantine,
`three groups of rats, 12 per group, were dosed i.v. with
`either saline, or 1 mg/kg or 5 mg/kg of 791T/36-RTA (RTA:MoAb
`ratio of 4.3:1; 3.7% free RTA) through the tail vein daily for 10 days
`(study days 1-10). Each group of animals was weighed daily, and three
`in each group were bled, sacrificed, and necropsied on days 6, 11, or
`17. Other Sprague-Dawley rats (three rats per group, Simonsen Labs)
`were given i.v. injections of 0.2, 1.0, or 5 mg/kg of RTA alone or of
`saline (2 ml/kg) for S sequential days. Animals were bled and necropsied
`on day 5. Serum chemistries
`included SCOT, serum glutamic pyruvate
`transaminase, bilirubin, BUN, creatinine,
`total protein, albumin, CPK,
`uric acid, LDH, glucose, and electrolytes. Hematology included indices
`and platelets were estimated.
`Patient Population and Treatment Plan. Seventeen patients were
`entered in the trial. All had at least one measurable lesion. No patient
`had received a murine monoclonal antibody prior to this therapy. No
`patient
`studied had significant organ dysfunction;
`i.e., neurological,
`cardiologica!,
`and pulmonary functions were within normal
`ranges.
`Signed informed consent was obtained from all patients prior to entry
`into the study which was conducted under a U. S. FDA investigational
`new drug exemption notice. All patients signed informed consent.
`XomaZyme-791 was given as 1-h daily i.v. infusions for 5 days, with
`the ability to postpone doses for up to 3 days if suspected side effects
`intervened.
`Immunotoxin
`doses,
`from 0.02 to 0.2 mg/kg/day, were
`infused over 1 h. Most patients were skin tested prior to the first dose
`with 100 /ig of unconjugated antibody; some received an i.v. challenge
`of the equivalent amount of diluted immunotoxin, with infusions pro
`ceeding 15 min later if no reaction was seen. No adverse reactions to
`the test dose were noted. Physical exams and laboratory evaluation,
`including hematological
`and serum chemistry panel and urinalyses,
`were done daily through study day 6, and then at study days 15, 28,
`and 60. Prothrombin
`time, partial
`thromboplastin
`time, complement
`levels and electrocardiograms were carried out on study days 0 and 6.
`Where indicated, EEC and CT examinations
`of the head were per
`formed. Patients were evaluated by sequential chest X-rays or CT scans
`of the abdomen, CEA levels, blood chemistries, and urinalyses for up
`to 6 months after completion of therapy.
`In most patients proteinuria
`was quantitated by dipstick where 1+ = 30 mg/dl and 4+ = 2000 mg/
`dl. One patient with 4+ proteinuria
`had quantitation
`of the urinary
`protein over a 24-h period and identification of the protein by urine
`electrophoresis.
`Assessment of Human Immune Response to Immunotoxin. The IgG
`and IgM antibody response to the immunoglobulin moiety or the RTA
`moiety of the immunotoxin were tested on days 0, 13-16, 21-23, 35-
`40, and 60 using an ELISA assay. ELISA microplates were coated with
`purified 79IT/36
`(250 ng/well
`in PBS) or RTA (2.5 Mg/well in PBS)
`or purified myeloma lgG2. or IgG2b(Sigma, Poole, UK, 250 ng/ml) in
`6154
`
`PBS. The plates were incubated for 1 h at room temperature with serial
`dilutions (1/10 to 1/10,000) of patient's serum diluted in 50 mM sodium
`citrate buffer, pH 4.5, containing 5% bovine serum albumin. Following
`extensive washing,
`the plates were incubated for 1 h at room tempera
`ture with a 1:1,000 dilution of alkaline phosphatase
`conjugated goat
`anti-human IgG or anti-human IgM (Sigma, Poole, UK) antiserum.
`After washing,
`the assay was developed with /7-nitrophenolphosphate
`(Sigma, Poole, UK) as the alkaline phosphatase substrate. The optical
`densities of each well were read at 405 nM and serum titers determined
`as the serum dilution producing 50% of the maximum ELISA value
`(19). Ami-combining site antibodies were detected using a flow cytom
`etry assay in which the capacity of patient's serum to block binding of
`FITC conjugated 79IT/36
`(791T/36-FITC)
`to target cells was deter
`mined (20). This was expressed as titer of serum which produced 50%
`inhibition of the maximum 791T/36-FITC binding to target cells.
`
`RESULTS
`
`Reactivity of MoAb and Immunotoxin
`
`from tumor cells, reactivity of the MoAb assessed by
`Apart
`immunoperoxidase
`staining is primarily with stromal
`(noncel-
`lular) tissue, although there is cytoplasmic staining in the region
`of the juxtaglomerular
`apparatus and occasional
`reactivity with
`pulmonary epithelium and isolated kidney glomeruli
`in some
`sections. There is no detectable binding to progenitor
`cells by
`flow cytometry.2 Other studies have found weak antigen binding
`mitogen stimulated
`(but not
`resting)
`lymphocytes
`(21). Two
`preparations of immunotoxin were used for clinical trials (Table
`1). Analysis by SDS-PAGE indicated several species of immu-
`noconjugate were present with antibody:RTA ratio of 1:1 to
`1:5. Less than 10% aggregates were present by weight. The
`intrinsic variation of the binding assay is 15% and that of the
`cytotoxicity assay is 50%; monthly analysis during the time the
`lots were in clinical use indicated all variations were within this
`range. No increase in free antibody or change MoAb:RTA ratio
`was noted.
`the first had
`While both lots fell within the accepted ranges,
`a higher MoAb:RTA ratio than the other and correspondingly
`less free antibody. The binding to target cells was decreased as
`compared to the lot with lower conjugation ratios but
`the in
`vitro cytotoxicity was similar.
`and sites of dis
`Patient Characteristics. The characteristics
`ease of the 17 cancer patients ( 10 females and 7 males) evaluated
`in the immunotoxin
`study are summarized in Table 2. The age
`range was 30-70 years. Sixteen patients had colorectal cancer;
`one patient
`(Patient 14) had the diagnosis of colorectal cancer
`later revised to ovarian cancer after
`laparotomy. Sixteen had
`liver métastasesdocumented by CT scan; one patient
`(Patient
`4) did not. Ten patients also had pulmonary métastases.All
`measurable lesions were less than 12 cm in size. Most patients
`had had the primary tumor
`removed. Some had received other
`therapy such as 5-fluorouracil
`chemotherapy
`of IL2-LAK cell
`immunotherapy
`no less than one month prior to immunotoxin
`
`Table 1 Characteristics of clinical lots of XomaZyme-791
`
`molar
`
`antibody9MoAb:RTAsubstitution
`ratio"1:3.3
`Lot12Free
`
`1:2Antibody
`
`against cell line'
`(IC50)791T
`
`Molt
`62 fig
`ND
`
`reactivity%*62
`
`41
`3 ng
`93Cytotoxicity
`12ng
`
`" Determined by SDS-PAGE.
`* Relative to antibody 79IT/36: determined by competitive inhibition of bind
`ing of MoAb 791T/36-FITC to 791T cells.
`for cultured target cells.
`c Determined by in vitro cytotoxicity of immunotoxin
`Cell survival determined by [3H]thymidine incorporation. Values expressed in
`terms of amount of immunotoxin/ml
`necessary to produce 50% inhibition.
`
`Downloaded from
`
`on October 30, 2014. © 1989 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2294, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`MoAb-RICIN A CHAIN IMMUNOTOXIN XomaZyme-791
`
`Table 2 Patient population treated with immunotoxin XomaZyme- 791
`
`since
`other
`(mg/dl)Site
`treatment
`ofdisease*Liver,
`
`(months)041None451NoneNone152None174NoneUnknown12NoneLiver
`
`function tests at entry
`
`W205618172248202233437208158386280519595482118199381SCOT12-45'40243221663561265858401205923303048
`
`lymph nodes, perito
`
`neum,lungLiver,
`
`lymph nodes,spine.lungLiver,
`
`lymph nodes,lung.periaortic
`
`massRight
`peritoneal mass, peri
`
`tonealmetsLiver,
`
`pelvic mass,lymphnodesLiver,
`
`PatientSex1
`
`F2
`
`F3
`
`F4
`
`F5
`
`F6
`
`M7
`M8
`M9
`F10
`M11
`M12
`F13
`
`M14
`
`lungsLiver,
`
`lymphnodesLiver,
`
`lymph nodes,lungsLiver,
`
`lung, lymphnodesLiver,
`lungLiver,
`
`lymph nodes,lungLiver,
`
`lymph nodes,lungs.presacrai
`massLiver,
`
`lungs,retroperitonealmass,
`boneLiver,
`periaortic nodes, pel
`vicmassLiver,
`
`peritoneum,lymphnodes,
`boneLiver,
`
`rectalrecurrenttumorLiverBili0.1-1.3'0.30.10.70.30.40.71.70.91.80.50.50.80.70.30.50.40.5LDH50-2
`MAge4960306032586967685345586236405070Time
`" Patients received no other therapy (none) or radiation, chemotherapy (principally 5-fluorouracil) or IL-2/LAC cells (Patient 5).
`* All except Patient 14 had diagnosis of coloréela]cancer made histologically. Patient 14 was subsequently rediagnosed as having ovarian cancer.
`' Normal range.
`
`F15
`
`F16
`
`F17
`
`
`
`than 70; all had
`scores were greater
`treatment. Karnofsky
`normal
`levels of blood urea nitrogen and serum creatinine; and
`no more than 1+ proteinuria
`by dipstick. Complement
`and
`coagulation parameters were within normal
`limits as were he
`moglobin and white blood cell counts. Of the 17 patients,
`five
`had normal
`liver function tests, 10 patients had mildly elevated
`LDH levels, and seven had elevation of SCOT. Most patients
`had values less than twice normal
`for each test; one had values
`less than three times normal, and two had mild bilirubin ele
`vations less than 30% above normal. Serum albumin was nor
`mal
`in all. None had other
`serious diseases or tumors apart
`from colon (or in one case ovarian) carcinoma. Fifteen patients
`received a full course of five doses of immunotoxin; doses were
`temporarily
`postponed
`in two patients
`(Patients
`9 and 13)
`because of neurological events (increased unilateral
`tremor and
`transient mental
`status change)
`thought possibly due to drug
`(Table 3). One patient
`(Patient 6) received only one infusion
`because of an anaphylactoid
`reaction consisting of periorbital
`edema. His skin test was negative prior to immunotoxin
`treat
`ment. Another patient
`(Patient
`17) received only four doses
`because of mental
`status change which required more than 3
`days to resolve.
`associated with
`Clinical Observations. Clinical observations
`immunotoxin
`therapy are summarized
`in Table 3. Patients
`generally tolerated the immunotoxin well. Decrease in serum
`albumin levels was noted in all patients, beginning during the
`5 days of infusion. This occurred with all doses of immunotoxin,
`and serum albumin levels fell to 12-48% of the starting level.
`There was no obvious correlation between dose of immunotoxin
`and degree of albumin drop.
`In all cases the albumin levels
`either stabilized or began to increase by day 15 (Fig. la). Weight
`gain of 5% or greater was noted in seven of 17 patients and was
`manifest as peripheral edema. Pulmonary edema was not seen.
`One patient developed fever of 39°Cduring or within 6 h of
`immunotoxin
`infusion. Asymptomatic
`proteinuria
`not associ
`
`ated with other renal abnormalities was first noted on days 5-
`10 of study and increased through day 15 (Fig.
`\b). Decreased
`serum albumin and weight gain occurred before onset of this
`delayed proteinuria.
`In all cases but one,
`the proteinuria
`re
`solved to 1+ or less after 30-45 days. The one patient
`(Patient
`3) in whom proteinuria persisted had an accompanying urinary
`tract
`infection. This delayed proteinuria was noted in 11/11
`patients
`treated with doses at or greater
`than 0.1 mg/kg/day,
`and in three of five patients
`treated with 0.05 mg/kg/day
`or
`less. Protein was quantitated
`in one of two patients with 4+
`proteinuria
`(receiving a dose of 0.1 mg/kg/day)
`and totalled
`2 g protein in a 24-h period. Urine protein electrophoresis
`demonstrated
`that
`the protein was primarily albumin. Urine
`sediment
`in all patients was unremarkable.
`In no case was there
`any decrease in serum complement
`levels (C3, C4, or CH50).
`No patient's
`serum albumin level decreased after the onset of
`proteinuria
`(Fig. 1). No increase in serum creatinine or BUN
`was seen in any patient.
`In most patients
`there was a slight
`decrease in platelet counts within the first 6 days of an average
`of 79,000 which was not
`related to dose. The lowest count
`reached was 146,000 in one patient. Counts returned to baseline
`or above within 15-20 days in all patients. There was no
`decrease in white blood cells or red cells and no change in
`prothrombin
`time, partial
`thromboplastin
`time, or fibrinogen
`levels.
`On study day 6, only one patient (Patient 12) had a significant
`increase in any liver function test;
`this was a twofold increase
`in the total bilirubin value which had returned to base line by
`day 15 occurring in a patient with liver métastases.Over the
`initial 28-day study period, one patient had SCOT and bilirubin
`values increasing by at least twofold.
`In one patient,
`there was worsening of a préexistanttremor
`of the left hand and four of 17 patients had reversible mental
`status changes; all had been treated at a dose of 0.1 mg/kg or
`higher (Table 3). In three patients, mild fatigue, slurred speech,
`6155
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`cancerres.aacrjournals.org
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`on October 30, 2014. © 1989 American Association for Cancer
`Research.
`
`IMMUNOGEN 2294, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`MoAb-RICIN A CHAIN IMMUNOTOXIN XomaZyme-791
`
`Table 3 Clinical observations in patients treated with XomaZyme-791
`
`gain
`drop
`in serum albu
`% (peak
`Patient
`dose
`eventsFevers*37.237.137.938.639.437.938.137.8+
`min(%)1213282445182640452629262228362748Weight
`
`
`number123456789IO11121314151617Dose(mg/kg)0.02e0.02e0.050.05e0.050.1"0.10.10.1'0.10.1e0.1e0.1e0.10.150.150.2Total(mg)5.05.013.011.014.56.842.537.525.038.040.026.527.727.445.061.452.8Maximumday)2(15)2
`
`(4)1
`(6)3
`(6)5
`(5)0
`(2)4(16)1
`
`(5)12(16)2(15)10(16)12(15)8
`37.938.4+
`
`38.338.3+
`
`(8)7(15)4(17)4
`
`38.137.637.8+
`
`(5)14(10)Neurological"
`
`37.8+
`
`38.5
`
`' Headache, dementia, or tremor.
`4 During therapy.
`c RTA:MoAb
`ratio of 2.0:1; other patients treated with lots with RTA: MoAb ratio of 3.5:1.
`* Received only one dose.
`' Postponed 4th and 5th dose study day 2-4.
`
`were most compatible with a mild toxic encephalopathy. The
`patient who developed dementia had a normal head CT scan
`and no other etiology for the dementia. Nausea with vomiting
`was noted in four other patients during therapy; headaches were
`seen in two.
`Biological Activity. Although this was a Phase I dose escala
`tion study, observations
`concerning
`antitumor
`activity were
`made. Of 16 patients with hepatic métastases(Table 4), two
`(Patients 10 and 16) had objective evidence by abdominal CT
`scans of decreasing size in large métastasesand disappearance
`of smaller lesions. These changes were seen at 3 months without
`additional
`intervening treatment.
`In another case (Patient 7),
`new calcification of the hepatic métastases,with stabilization
`of growth, was noted at 2 months, but there was increased size
`at 6 months. Three patients (Patients 8, 11 and 14) had fixed
`supraclavicular
`nodes which decreased in size or disappeared
`by study day 30. Three also had liver métastaseswhich increased
`in size or number over the 2-month follow-up period. One
`patient
`(Patient 6) had both liver and pulmonary métastases;
`the liver métastasesincreased in size but
`the lung métastases
`decreased in size 5 months after
`therapy. These patients
`re
`ceived no additional chemotherapy after the immunotoxin ther
`apy.
`Although there were transient decreases in the CEA values
`of some patients
`(Table 4), these could not be correlated with
`decreased tumor size. Of the three patients (Patients 7, 10, and
`14) who had calcification or decrease in size of the hepatic
`métastases,one had CEA values
`that decreased during the
`period of observation.
`patients were skin tested
`Immunological Studies. Thirteen
`with the unmodified antibody prior to initiation of therapy and
`all
`tests were negative. Between days 10 and 15, onset of
`erythema and induration at the skin test site was noted in 12
`of 13 patients. The exception was one patient
`(Patient
`4),
`treated at 0.05 mg/kg/day.
`In all cases the reaction lasted 3-5
`aphasia were noted. These events
`or expressive
`irritability,
`days and then resolved.
`usually began about study day 4 and were largely resolved within
`immuno-
`to murine 791T/36
`Humoral
`antibody responses
`2 days although complete resolution could take seven days. One
`of the immunotoxin were ob
`globulin and RTA components
`patient
`treated at the highest dose (0.2 mg/kg) became frankly
`served in all but one patient,
`including Patient 6 who received
`demented;
`the patient
`received steroids and this conditon re
`a single injection of immunotoxin
`(Table 5). Most patients
`versed after 3 days. EEGs done on the four patients with mental
`produced IgM and IgG responses to 79IT/36
`immunoglobulin;
`status changes after
`therapy revealed diffuse slowing and/or
`the one patient who did not (Patient 15) was only tested as late
`paroxysmal bursts, and both the clinical examination and EEC
`6156
`
`10
`Study Day
`treated with
`I. a, serum albumin levels in coloréela!cancer patients
`Fig.
`immunotoxin XomaZyme-791 (dose range, 0.02-0.2 mg/kg/day). Study days are
`plotted on a log scale to allow comparison between serum albumin and protein-
`uria, and patients are identified by dose in mg/kg; 0.02 (•),0.05 (D). 0.15 (•),
`0.1 (A), and 0.2 (O). A, proteinuria
`assayed by dipstick, scale 1-4 in colorectal
`patients treated with XomaZyme-791.
`
`100
`
`Downloaded from
`
`on October 30, 2014. © 1989 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2294, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`MoAb-RICINA CHAIN IMMUNOTOXIN XomaZyme-791
`
`Table 4 Biological responses in patients treated with immunotoxin XomaZyme-791
`
`(ng/ml)Change
`in
`liver
`Pa
`pulmonary
`tient
`métastases
`métastasesIncreased
`
`initials12345678910111213Dose0.020.020.050.050.050.1»0.10.10.10.10.10.10.1Changemos.StableIncreasednumberStableN/AIncreasednumberEnlargedStableat 2
`
`sizeIncreased
`numberStableN/AN/AIncreased
`
`in
`
`CEA
`
`numberat
`2monthsbut
`decreasedsize
`
`at5monthsN/AN/AStableNo
`
`withcalcificationIncreasednumberStableDecreasednumberIncreasednumberIncreased
`
`new,somedecreased
`
`insizeAppearance
`
`in other
`métastasesN/AN/AN/APeritoneal
`
`métastasesin
`
`creased insizeIncreased
`size,peritonealmassN/AN/ATransient
`
`
`sizeL
`decrease in
`
`supraclavicularnodeN/AN/AResolution
`
`supraclavicularnodeN/AIncreased
`
`ofnew
`lesions
`at2
`mos.Increased
`numberIncreased
`sizePre13419220181183285804522>10002256967TherapyPost"19315571901794425971024>100046521000Change
`retroperitoneal
`adenopathy
`Resolution supraclavicular
`mass
`
`14
`
`0.1
`
`15
`16
`17
`"Study day 15.
`* Received only one dose.
`
`0.15
`0.15
`0.2
`
`sizeIncreased
`sizeSerum
`
`Increased size
`
`N/D
`Decreased size
`Stable
`
`N/A
`
`N/A
`N/A
`N/A
`
`17
`
`142
`1000
`498
`
`18
`
`383
`1000
`374
`
`N/A
`N/A
`N/A
`
`79IT cells. Anti-combining site antibodies were detected in sera
`from 14 of 17 patients (Table 5 and Fig. 3). Peak serum titers
`were variable ranging from up to 1:5 in two patients
`to 1:100
`or greater
`in 12 patients. The kinetics of this
`response
`in
`patients is illustrated in Fig. 3 showing that the response begins
`about days 10 to 30. In most instances, antibody levels remained
`elevated up to day 70. In three patients there was a pronounced
`fall of the anti-combining site antibody titer.
`Antibody responses
`to RTA were detected in sera from 15
`patients (Table 5 and Fig. 4). One patient (Patient 15) receiving
`0.15 mg/kg/day
`of immunotoxin
`(total dose 45 mg) did not
`produce antibody to either RTA or 79IT/36
`immunoglobulin.
`A second patient
`(Patient 16) treated at the 0.15 mg/kg dose
`(total dose 61.4 mg) also produced only minimal
`responses
`to
`RTA (and also 79IT/36).
`Significant
`titers of IgM antibodies
`to RTA were observed in 12 patients,
`titer
`range 1/50 to
`1/16,600 (Table 5). IgG anti-RTA antibodies were detected in
`13 patients with titers ranging from 1/50 to 1/316,000. The
`kinetics of the anti-RTA IgG response (Fig. 4) indicates a rapid
`rise in antibody titer between study day 10 to 20, this being 5
`to 15 days after completion of immunotoxin
`treatment.
`In one
`of the patients
`(Patient 9) who generated a very pronounced
`response,
`the anti-RTA antibody levels decreased over a period
`of 70 days. In most patients, however,
`the IgG antibody level
`remained elevated throughout
`the 80-day period of investiga
`tion.
`
`as study day 19. The IgM response was first detected between
`days 5 and 20 of study, with maximum responses around day
`30. The overall pattern was that of a rapid rise in the IgM
`antibody response and then a fall in antibody levels up to day
`80 of study. Peak IgM responses against
`the MoAb portion of
`the immunotoxin
`ranged in titer from 0 to 1:500.
`The IgG response to 79IT/36 immunoglobulin was markedly
`greater
`than the IgM response, with peak antibody titers of
`1:7000 or greater observed in four patients.
`In comparison,
`the
`maximum IgM antibody titer was 1/500 (Patient
`12). The
`pattern of the IgG response was different from the IgM response
`with IgG antibodies continuing to rise from approximately days
`10 to 20 and remaining elevated for up to 80 days of study.
`(Fig. 2).
`im
`recognizing 79IT/36
`Sera were screened for antibodies
`munoglobulin
`(IgG2b) in comparison with nonspecific mouse
`IgG2b and IgG2a immunoglobulins
`to determine if the predom
`inant response was anti-mouse
`IgG, anti-subclass, or anti-vari
`able region (Table 5 and Fig. 2). The predominant
`response
`was specific for
`the 79IT/36
`immunoglobulin, where peak
`serum titers greater than 1:1000 were obtained in samples from
`11 patients.
`In 13 of 17 patients the anti-79IT/36
`response was
`at least twice as high as the response to mouse myeloma IgG2b
`or IgG2a.
`The pronounced antibody response to 79IT/36 immunoglob
`ulin (IgG2b), compared to normal mouse IgG2b and IgG2a sug
`gested that
`the patients generated antibody to the monoclonal
`antibody 79IT/36
`combining site. This was confirmed using a
`flow cytometry assay which measured the capacity of patients'
`The LD50 level of the immunotoxin assessed in BALB/c mice
`serum to block binding of fluorescein labeled 79IT/36
`(FITC-
`receiving a single i.p. injection of doses from 10 to 100 mg/kg,
`79IT/36) with the target antigen on tumor 79IT cells (18, 20).
`given in a volume of 0.5 ml, was calculated to be 81 mg/kg.
`In
`Titers were determined as the dilution of patients'
`serum which
`the subacute toxicology studies, rats receiving the highest dose
`produces a 50% inhibition of FITC 79IT/36
`binding to target
`of immunotoxin had a significant decrease in body weight from
`6157
`
`Animal Toxicology
`
`Downloaded from
`
`cancerres.aacrjournals.org
`
`on October 30, 2014. © 1989 American Association for Cancer
`Research.
`
`IMMUNOGEN 2294, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Table 5 Antibody responses to immunotoxin XomaZyme-791 in colorectal cancer patients
`against*Patient1234567891011121314151617Immunotoxin
`Peak antibody titers
`
`MoAb-RICIN A CHAIN IMMUNOTOXIN XomaZyme-791
`
`tested
`dose per kg/
`thru study
`IgGa
`IgGc.
`anticombining