Therapy of Patients with Malignant Melanoma Using a
`Monoclonal Antimelanoma Antibody-Ricin A Chain
`Immunotoxin
` 
`Lynn E. Spitler, Michael del Rio, Archie Khentigan, et al.
`Cancer Res  
`
`1987;47:1717-1723.
`
`Updated version
` 
`
`Access the most recent version of this article at:
`http://cancerres.aacrjournals.org/content/47/6/1717
` 
`
`
`
` 
` 
` 
` 
` 
` 
`
`E-mail alerts
` 
`Reprints and
`Subscriptions
` 
`Permissions
` 
`
`Sign up to receive free email-alerts
`
` related to this article or journal.
`
`To order reprints of this article or to subscribe to the journal, contact the AACR Publications
`.
`pubs@aacr.org
`Department at
` 
`To request permission to re-use all or part of this article, contact the AACR Publications
`.
`permissions@aacr.org
`Department at
` 
`
`
`
`Downloaded from Downloaded from
`
`on October 30, 2014. © 1987 American Association for Cancercancerres.aacrjournals.org on October 30, 2014. © 1987 American Association for Cancercancerres.aacrjournals.org
`
`
`
`
`
`Research. Research.
`
`IMMUNOGEN 2193, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`[CANCER RESEARCH 47, 1717-1723, March 15, 1987)
`
`Therapy of Patients with Malignant Melanoma Using a Monoclonal Antimelanoma
`Antibody-Ricin A Chain Immunotoxin1
`
`Lynn E. Spitler,2 Michael del Rio, Archie Khentigan, Nancy I. Wedel, Nathalie A. Brophy, Langdon L. Miller,
`W. Scott Harkonen, Linda L. Rosendorf, Howard M. Lee, Ronald P. Mischak, Russell T. Ka«aliata,
`John B. Stoudemire, Linda B. Fradkin, Eddie E. Bautista, and Patrick J. Scannon
`Department of Medicine, Children's Hospital [L. E. S., M. d. R., N. I. W., N. A. B., L. L. M., W. S. H.J, San Francisco, California 94118, and XOMA Corporation
`[L. E. S., M. d. R.. A. K., N. l. W., N. A. B., W. S. H., L L. R., H. M. L, R. P. M., R. T. K., J. B. S., L. B. F., E. E. B., P. J. S.], Berkeley, California 94710
`
`ABSTRACT
`
`We conducted a trial of a murine monoclonal am ¡melanomaantibody-
`ricin A chain immunotoxin (XOMAZYME-MEL)
`in 22 patients with
`metastatic malignant melanoma. The dose of immunotoxin administered
`ranged from 0.01 mg/kg daily for 5 days to l mg/kg daily for 4 days
`(total dose: 3.2 to 300 mg). Side effects observed in most patients were a
`transient fall in serum albumin with an associated fall in serum protein,
`weight gain, and fluid shifts resulting in edema. In addition, patients
`experienced mild to moderate malaise,
`fatigue, myalgia, decrease in
`appetite, and fevers. There was a transient decrease in voltage on electro
`cardiograms without clinical symptoms, change in serial echocardiograms
`or elevation of creatine phosphokinase MB isozyme levels. Symptoms
`consistent with mild allergic reactions were observed in three patients.
`The side effects were related to the dose of immunotoxin administered
`and were generally transient and reversible. Encouraging clinical results
`were observed, even after a single course of a low dose of immunotoxin.
`In addition,
`localization of antibody and A chain to sites of metastatic
`disease was demonstrated by immunoperoxidase staining of biopsy spec
`imens. Additional studies are being conducted to continue the evaluation
`of safety and efficacy of immunotoxin therapy for malignancy.
`
`INTRODUCTION
`
`of cultures of
`the development
`The technology permitting
`fused cells secreting antibody of predefined specificity was first
`described in 1975 (2). Since then, hybridoma
`technology has
`developed rapidly and has permitted the production of mono
`clonal antibodies
`for a variety of purposes,
`including the diag
`nosis and therapy of malignancy in humans.
`The first attempts
`to apply this technology in the therapy of
`malignancy involved the use of unmodified monoclonal
`anti
`bodies (3). The toxic effects encountered from the injection of
`unmodified monoclonal
`antibodies
`in human subjects have
`generally been minimal. There has also been little evidence of
`clinical benefit
`to cancer patients,
`even when the antibodies
`were administered in massive doses.
`In a single, widely cited case, a dramatic, prolonged clinical
`response was observed in a patient with B-cell lymphoma given
`an anti-idiotype antibody (4). In a subsequent
`study, this group
`reported objective incomplete
`remissions which were of rela
`tively short duration in 5 of 10 additional patients (5). In other
`reports, only a transient decrease in circulating cells was found
`following treatment with anti-idiotype
`antibody (6) or antibod
`ies reactive with T-cells (7, 8) with no apparent
`therapeutic
`benefit.
`In patients with malignant melanoma,
`injection of
`unmodified antimelanoma
`antibody 9.2.27 resulted in localiza
`tion of the antibody in tumor sites, as determined by immuno-
`
`12/17/86; accepted 12/19/86.
`revised 8/25/86,
`Received 4/24/86;
`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.
`1This project was supported by funds from XOMA Corporation. Presented in
`part at the First International Congress on Malignant Melanoma, Venice, Italy,
`1985(1).
`2To whom requests for reprints should be addressed, at Xoma Corporation,
`2910 Seventh Street, Berkeley, CA 94710.
`
`(9). In one study,
`peroxidase staining, but no clinical benefit
`encouraging results were obtained in patients with malignant
`melanoma using the anti-ganglioside
`antibody R24 (10).
`in
`We anticipated
`that unmodified monoclonal
`antibodies,
`general, would not be of significant
`therapeutic benefit
`to pa
`tients with solid tumors. Unmodified antibodies may kill target
`cells via complement-mediated
`cytotoxicity
`or antibody de
`pendent cell-mediated cytotoxicity, both of which are indirect
`and require involvement of the host immune system. Monoclo
`nal antibodies can also be used to target cytotoxic agents to
`tumor cells for direct killing. Such an approach has the advan
`tage of increasing selective tumoricidal activity and decreasing
`the systemic toxicity of these cytotoxic agents. Studies utilizing
`currently available chemotherapeutic
`agents conjugated with
`monoclonal
`antibodies
`thus far have demonstrated
`relatively
`low cytotoxic activity (11). Another approach to targeted ther
`apy involves monoclonal
`antibodies coupled to therapeutic
`ra-
`dioisotopes. An example of the use of this approach in mela
`noma has been reported by Larson et al. (12).
`Since 1981, we have directed our efforts toward the devel
`opment of immunotoxins
`consisting of monoclonal antibodies
`coupled to ribosomal
`inhibiting proteins, such as ricin A chain,
`as therapeutic modalities. These ribosomal
`inhibiting proteins
`are enzymes that
`inhibit protein synthesis by ribosomal
`inacti
`va!inn. thereby causing cell death (13). Internalization
`of the A
`chain, which is necessary for cytotoxic action, occurs following
`the binding of the immunotoxin
`to the cell surface via the
`monoclonal
`antibody. There is evidence that ribosomal
`inhib
`iting proteins
`are so potent
`that one molecule entering the
`cytosol
`is sufficient
`to cause cell death (14).
`In a radioimmunoimaging
`study, conducted under a United
`States FDA3 Investigational New Drug exemption notice, using
`our monoclonal
`antimelanoma
`antibody labeled with indium-
`111, we showed that the antibody localizes in melanomas
`(15).
`This same antibody was coupled with ricin A chain to create an
`immunotoxin
`for therapy in which the antibody would bind to
`tumor cells and the A chain would kill the cells to which the
`antibody bound. We report herein the results of our trial of this
`monoclonal
`antimelanoma
`antibody-ricin A chain immuno
`toxin (XOMAZYME-MEL).
`
`MATERIALS AND METHODS
`
`Patients. The patients eligible for this study had Stage III metastatic
`malignant melanoma (disease spread beyond regional
`lymph nodes).
`The first eight patients were required to have failed chemotherapy with
`the last dose having been administered more than 4 weeks before
`immunotoxin therapy. After we gained clinical experience in immuno
`toxin administration with the first eight patients, we eliminated the
`requirement
`that patients must have received prior chemotherapy.
`Other criteria for entry into the study included that
`the patients have
`good performance status with an expected survival time of greater than
`
`Downloaded from
`
`cancerres.aacrjournals.org
`
`3The abbreviations used are: FDA, Food and Drug Administration; EKG,
`electrocardiogram;
`id.,
`intradermal.
`1717
`on October 30, 2014. © 1987 American Association for Cancer
`Research.
`
`IMMUNOGEN 2193, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`IMMUNOTOXIN
`
`THERAPY
`
`3 months, a WBC >4,000 cells/cu mm, platelet count >100,000/mm3,
`good renal and liver function, and no severe systemic disease aside from
`melanoma. Signed,
`informed consent was obtained from all patients
`prior to entry into the study.
`the
`the generation of the hybridoma,
`Monoclonal Antibody. For
`hybridization, cloning, and recloning were performed according to the
`conventional procedure described by Kohler and Milstein (2) with
`minor modifications (16). BALB/c mice were immunized with cultured
`human melanoma cells. The spleen cells were harvested and fused with
`the 8-azaguanine-resistant murine myeloma line P3-X63-Ag8 in the
`presence of polyethylene glycol. The cells were cultured overnight,
`resuspended in medium containing hypoxanthine,
`aminopterin,
`and
`thymidine. They were then cloned. Hybridomas
`secreting antibodies
`with the appropriate
`specificity were subcloned twice by limiting dilu
`tion using BALB/c splenocytes as feeder cells.
`The monoclonal antibody used in the preparation of the ¡mimmo-
`toxin was produced from murine ascites and was purified by XOMA
`Corporation (Berkeley, CA) using a staphylococcal Protein A column
`with elution at pH 3.5.
`It
`is an IgG2a antibody and reacts with
`melanoma-associated
`antigens having molecular weights of 220,000
`and over 500,000. The hybridoma and the monoclonal antibody have
`been fully characterized in accordance with guidelines proposed by the
`FDA in the document entitled "Points
`to Consider
`in the Production
`of Monoclonal Antibody Products for Human Use." On frozen sections,
`the antibody shows minimal
`reactivity with normal
`tissues except for
`vascular endothelium,
`in which the reactivity appears to be cytoplasmic.
`The antibody also cross-reacts with nevus cells. The hybridoma contains
`xenotropic and ecotropic viruses; however,
`the purified antibody con
`tains neither these viruses nor parental hybridoma DNA or RNA (17).
`Both the hybridoma and the purified antibody are free of other murine
`viral contaminants
`as determined by the mouse antibody production
`test (a test in which contamination with 12 murine viruses are evaluated
`by injection of the test article into mice and determination of antibody
`production to the viruses of interest).
`XOMAZYME-MEL Immunotoxin. The immunotoxin, consisting of
`the murine monoclonal antimelanoma
`antibody conjugated to ricin A
`chain,
`is produced by XOMA Corporation. The conjugation technique
`has been described in detail elsewhere (18). The ricin A chain is purified
`by affinity chromatography using an anti-ricin B chain column. Briefly,
`the
`antibody
`is
`activated
`with
`/V-succinimidyl-3-(2-pyridyldi-
`thio)propionate
`followed by addition of affinity purified ricin A chain
`which has been reduced with dithiothreitol. The immunotoxin is then
`purified by gel chromatography.
`It is provided in a sterile, pyrogen-free
`formulation
`at a concentration
`of 1.0 mg/ml
`in 0.9% phosphate-
`buffered saline solution, pH 7.O. Preclinical
`testing demonstrated bind
`ing specificity of the immunotoxin
`similar to that of the unmodified
`antibody and cytotoxic antimelanoma
`activity both in vitro and in vivo.
`Associated toxicology studies indicated safety of the drug.
`Protocol. This study was conducted under a United States FDA
`Investigational New Drug exemption notice. Signed, informed consent
`was obtained from each patient. The patients' active participation
`in
`the study lasted for approximately
`2 months,
`including an initial
`hospitalization of 7 to 12 days. Immediately after admission and prior
`to beginning therapy, each patient was skin tested by i.d. injection of
`100 ng of the unmodified antibody is 0.1 ml phosphate-buffered saline,
`and the test site was observed during a 30-min interval
`for signs of
`immediate reactivity. All of the patients had negative skin tests.
`Patients
`received the monoclonal
`antimelanoma
`antibody ricin A
`chain immunotoxin in doses ranging from 0.01 mg/kg/day for 5 days
`to 1 mg/kg/day for 4 days (Table 1). Two patients received a dose of
`0.2 mg/kg/day
`for 10 days. The total dose administered during this
`study ranged from 3.2 to 300 mg. The immunotoxin was diluted to
`approximately 200 ml in phosphate-buffered
`saline and was adminis
`tered by i.v. infusion over a period of 0.5 to 1.5 h.
`examination,
`The patients were evaluated
`serially by physical
`ophthalmological
`examination,
`and laboratory tests including la-ma
`tology panel, chemistry panel, urinalysis, creatinine
`clearance, pro-
`thrombin time, partial
`thromboplastin
`time, stool guaiac, complement
`levels,
`follicle stimulating
`hormone,
`estrogen,
`testosterone,
`chest
`X-ray, computerized
`tomography of the head, chest, and abdomen,
`
`EKG, and other laboratory tests as indicated. The IgG immune response
`to murine immunoglobulin
`and ricin A chain was also determined
`serially.
`Immune Response. The antibody response to the immunotoxin com
`ponents was measured in an enzyme immunoassay. Various dilutions
`of patients'
`sera were added to microtiter plates containing absorbed
`ricin A chain or the murine monoclonal antimelanoma antibody. Goat
`antihuman IgG antibody conjugated to alkaline phosphatase
`(Zymed
`Laboratories, South San Francisco, CA) was added followed p-nitro-
`phenylphosphate (Sigma Laboratories, St. Louis, MO). Titration curves
`were generated for each patient, and the immune response was ex
`pressed as the ratio of the titration end point dilution of the serum
`sample showing maximum response to the titration end point dilution
`of the pretreatment
`serum sample.
`Immunoperoxidase Studies. Biopsy specimens of tumors were ob
`tained from study patients,
`and frozen sections were prepared for
`evaluation by two immunoperoxidase
`techniques,
`the avidin-biotin
`system and the indirect conjugate peroxidase method (19). Sections
`were stained for the presence of the immunoglobulin component of the
`immunotoxin
`by incubation with biotinylated horse anti-murine
`im
`munoglobulin antibody (Vector Laboratories, Burlingame, CA), fol
`lowed by the addition of avidin-biotin complex (Vector). Parallel sec
`tions were stained for the ricin A chain component of the immunotoxin
`using a goat anti-ricin A chain antiserum,
`followed by a biotinylated
`rabbit anti-goat
`immunoglobulin
`reagent
`(Vector) and avidin-biotin
`complex. Additional studies were performed using an indirect conjugate
`peroxidase method with similar
`results. Sites of localization of the
`immunoperoxidase were revealed by the use of 3-amino-9-ethylcarba-
`zole as the chromogen, giving a red color that
`is distinguishable
`from
`melanin pigment. Tissues were examined independently by two observ
`ers.
`
`RESULTS
`
`Twenty-two patients were evaluated in this study (Table 1).
`There were 13 males and 9 females with ages ranging from 28
`to 69 years. All had Stage III melanoma;
`13 had failed prior
`chemotherapy
`and 9 had not received chemotherapy.
`are
`Side effects associated with immunotoxin
`administration
`summarized
`in Table 2. The severity of the side effects was
`generally related to the dose of immunotoxin
`administered.
`In
`doses up to and including 0.5 mg/kg/day for 5 days, these side
`effects were transient
`and reversible in all patients evaluated.
`The major dose-limiting side effect of this immunotoxin was a
`
`Table 1 Patient population included in the trial of XOMAZYME-MEL
`monoclonal antimelanoma antibody ricin A chain immunotoxin
`
`lactic
`Daily dose
`dose
`
`
`
`Patient1234S6189IO11121314IS16171819202122Age(years)31563936285152355344333845415536544162586857PrestudySex dehydrogenase(mg/kg)FFFMMMMFFMMFFMMFMMMMFMtfNNENEEENEEEEENENNNNNN0.010.050.20.20.50.51.01.00.750.20.50.750.20.50.750.750.50.50.50.50.50.5Dosesreceived555555445105510542555555Total
`(mg)3.216686726018030020029813519019510625026580300183235190170200
`
`
`
`" N, normal; E, elevated.
`
`Downloaded from
`
`cancerres.aacrjournals.org
`
`1718
`on October 30, 2014. © 1987 American Association for Cancer
`Research.
`
`IMMUNOGEN 2193, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Table 2 Clinical and laboratory findings in patients undergoing
`immunotoxintherapyObservationsClinicalMalaise/fatigueFeverTachycardiaDecreased
`
`
`5-,
`
`IMMUNOTOXIN
`
`THERAPY
`
`of
`patients1514141266521°120201612/13*8/11876"2"2"2"2/2I"></11
`4.
`
`appetiteNauseaWeight
`
`
`
`gain(>5%)MyalgiaFlushDeathPruritusLaboratoryAlbumin
`
`decrease(>10%)Total
`
`
`serum proteindecreaseLow
`
`voltage onEKGFibrinogen
`increaseErythrocyte
`sedimentation rateincreaseLeukocytosisCalcium
`
`
`decreaseSerum
`glutamic oxaloacetate transaminaseincrease'Lactic
`
`
`dehydrogenaseincrease'Thrombocytopenia
`(<50,000 platelets/cu
`mm)PTT
`prolongationC
`
`reactive proteinincreaseEosinophiliaCreatinine
`
`increaseHemoconcentrationMetabolic
`
`acidosisNo.
`" Relationship to immunotoxin therapy not established (see text).
`* Denominator
`represents number of patients evaluated if observations were
`not made in all 22 patients.
`' Increase to two or more times the baseline value.
`''Some of the abnormal values were attributed to disease progression or other
`causes (see text).
`
`6 ,
`
`2
`
`6
`
`2 -
`
`RTA
`1
`
`RTA
`234
`
`RTA
`
`RTA
`
`RTA
`
`RTA
`56
`
`RTA
`789
`
`RTA
`
`RTA
`
`RTA
`10
`
`RTA
`11
`
`Patient Number
`
`RTA
`12
`
`RTA
`13
`
`RTA
`14
`
`RTA
`15
`
`RTA
`16
`
`RTA
`17
`
`RTA
`18
`
`RTA
`19
`
`RTA
`20
`
`RTA
`21
`
`RTA
`22
`
`PatientNumber
`
`Indi
`Fig. 1. Results of albumin levels in 22 patients receiving immunotoxin.
`vidual prestudy levels and nadir of albumin levels are shown for each patient.
`Dosages are indicated in Table 1. Lighter bars, prestudy albumin level; darker
`bars, modes of albumin level; gm %, g/100 ml.
`
`3-
`
`2.
`
`1.
`
`a
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Study Day
`
`receiving 5
`Fig. 2. Results of serial serum albumin levels in 10 patients
`infusions of immunotoxin at a dose of 0.5 mg/kg/day within an 8-day period.
`Numbers, mean ±SE (bars); gm %, g/100 ml.
`
`108
`
`106 .
`
`104 .
`
`102
`
`100
`
`98 .
`
`96
`
`051015202530
`
`Study Day
`
`in 10 patients receiving 5
`Fig. 3. Results of serial body weight determinations
`infusions of immunotoxin at a dose of 0.5 mg/kg/day within an 8-day period.
`Numbers, mean ±SE (bars).
`
`ml. There was no associated proteinuria. Serum albumin levels
`stabilized and began to return to normal within 48 h of the last
`treatment with immunotoxin.
`of
`least 0.2 mg/kg/day
`All patients
`receiving a dose of at
`immunotoxin
`gained weight during the course of hospitaliza-
`tion for immunotoxin
`treatment. The weight change for the 10
`patients who received 5 infusions of immunotoxin
`at a dose of
`0.5 mg/kg is illustrated in Fig. 3. For this group,
`the weight
`gain was 9.1 ±1.3 Ib (range, 1 to 17.4 Ib). Associated with this
`were clinical signs of edema in some patients. There were no
`signs of pulmonary edema in any patient. The patients' weights
`decreased to baseline levels as the serum albumin returned to
`pretherapy levels.
`Along with the clinical evidence of fluid retention, most
`patients
`receiving a dose of at least 0.5 mg/kg/day
`of immu
`notoxin exhibited signs of mild hypovolemia as manifested by
`tachycardia
`and,
`in some cases, a modest
`fall
`in orthostatic
`blood pressure. These signs and symptoms were not severe and
`responded to conservative management.
`In some patients, daily
`doses of immunotoxin were interrupted for 1 to 2 days to allow
`for partial
`reversal of these transient effects before continuing
`therapy.
`I.v. hydration was administered to some patients.
`Many of the patients manifested fevers several hours after
`infusion of immunotoxin,
`and they generally ranged between
`38 and 39°C.In addition, most patients experienced malaise,
`fatigue, and decrease in appetite during the course of hospital-
`1719
`
`decrease in serum
`in serum albumin with a concomitant
`fall
`total protein values. Weight gain and fluid shifts were noted
`which resulted in edema.
`The fall in serum albumin was observed in all patients receiv
`ing a dose of at least 0.2 mg/kg/day
`of immunotoxin
`(Fig. 1).
`This decrease usually occurred after the second dose of immu
`notoxin. Serum albumin levels in 10 patients given 0.5 mg/kg/
`day for five days are illustrated in Fig. 2. For those patients,
`the mean
`serum albumin
`level was
`4.2 ± 0.1
`(SE)
`g/100 ml before treatment and fell to a nadir of 2.9 ±0.1 g/100
`
`Downloaded from
`
`cancerres.aacrjournals.org
`
`on October 30, 2014. © 1987 American Association for Cancer
`Research.
`
`IMMUNOGEN 2193, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`IMMUNOTOXIN THERAPY
`
`(no. 15), who had a prior
`widespread disease. The third patient
`history of coronary artery disease with apparent unstable an
`gina, demonstrated
`hypotension,
`hemoconcentration,
`and a
`metabolic acidosis within 12 h after the fourth dose of immu
`notoxin (0.75 mg/kg/day). The patient
`initially responded to
`i.v. hydration,
`but he developed atrial
`fibrillation and had a
`cardiopulmonary
`arrest within 36 h of his last dose of immu
`notoxin. Autopsy findings confirmed his past history of coro
`nary artery disease but did not clarify the cause of death. The
`immediate cause of death was thought
`to be due to an arrhyth
`mia. The relationship
`between immunotoxin
`administration
`and this patient's death is unknown.
`and will be the
`Pharmacological
`studies were performed
`subject of a subsequent manuscript.
`lmmunoloj;ical
`analysis
`indicated that almost all patients mounted an immune response
`to both the murine immunoglobulin
`and ricin A chain compo
`nents of the immunotoxin
`following completion of the thera
`peutic course (Fig. 4). Results of these studies will be presented
`in detail elsewhere. Encouraging clinical results were observed
`in this study (Table 3). One patient had a complete response
`with disappearance of a pulmonary metastasis. The response is
`ongoing at 13 months.
`In addition,
`four patients had a mixed
`response which is defined as a 50% or greater reduction in area
`of one or more métastaseswhile concurrently
`one or more
`lesions increased in size or a new lesion appeared after treat
`ment. Although these do not meet
`the standard oncological
`definition of an objective response,
`they are noteworthy in view
`
`1000^
`
`100 _
`
`10 -
`
`1-
`
`oc
`lü
`co
`
`zoC
`
`L
`CO
`LU
`K
`
`RICIN A CHAIN
`
`MURINE
`IMMUNOGLOBULIN
`IgG antibody response to immunotoxin components. For each patient,
`Fig. 4.
`the immune response is expressed as the ratio of the titration end point dilution
`of the serum sample showing maximum response to the titration end point
`dilution of the pretreatment
`serum sample.
`
`ization. Some patients had associated myalgia. These side ef
`fects were self-limiting and well tolerated.
`A transient
`change to decreased voltage on EKG without
`other ST-T wave changes or clinical symptoms was noted. Sinus
`tachycardia was also clinically observed in most patients. Serial
`echocardiograms
`and
`creatine
`kinase
`fraction
`2
`levels
`(CK2(MB)] were obtained in four patients who showed low
`voltage on EKG. There was no evidence of significant pericar
`dia! effusions or changes
`in ventricular
`function. Levels of
`creatine phosphokinase muscle-brain isozyme did not increase.
`A review of the hematological
`evaluation showed no evidence
`of bone marrow suppression. There was a moderate
`leukocy-
`tosis with an associated decrease in the percentage of lympho
`cytes in some patients. Platelet counts generally did not fall
`below normal values.
`In 4 patients, platelet counts
`increased
`modestly during the course of therapy. Two patients had pro
`longation of partial
`thromboplastin
`time values to more than
`110s and thrombocytopenia with platelet counts of less than
`50,000 platelets/mm3. One of these patients had rapidly pro
`gressive metastatic melanoma with secondary disseminated in-
`travascular
`coagulation
`and a heparin-induced
`thrombocyto
`penia. This coagulopathy was associated with guaiac-positive
`stools. The etiology of the second patient's
`abnormal coagula
`tion parameters
`is unknown and was not associated with any
`bleeding diathesis.
`In addition,
`there was a fall in platelets from
`a pretreatment
`value of 120,000 mm3 to a nadir of 76,000 in
`one patient
`(no. 16) that was attributed to a recent course of
`chemotherapy with lomustine.
`Twelve of 13 patients
`evaluated had an increase in serum
`fibrinogen during immunotoxin
`therapy. There was also noted
`an increase in erythrocyte
`sedimentation
`rate and C reactive
`protein in some patients. Some patients also demonstrated mild
`transient elevations in lactic dehydrogenase and serum glutamic
`oxaloacetate transaminase
`levels. In only two patients was there
`an increase to five or more times the baseline value. One of
`these patients
`(no. 8) had rapidly progressive disease, and in
`the other (no. 15), the increase was a preterminal
`event. Seven
`patients demonstrated
`decreases
`in serum calcium which cor
`related with the decrease
`in serum albumin. This decrease
`probably reflects a fall in available albumin for calcium binding.
`There was no clinical evidence of hypocalcemia, and the calcium
`levels returned to normal as albumin levels returned to baseline
`values.
`(no. 17), with known preexisting gout and
`In one patient
`associated nephropathy,
`there was a transient
`increase of cre-
`atinine from 1.3 to 1.9 mg/dl associated with increased activity
`of his gout during the course of hospitalization
`for immuno
`toxin therapy. These changes were attributed to a combination
`of the preexisting
`nephropathy with mild prerenal
`azotemia
`and medication with indomethacin.
`Three patients experienced possible allergic reactions. Two
`of these had received prior murine antibody as a part of the
`previously mentioned radioimmunoimaging
`study, and one ex
`perienced the reaction after a 10-day course of infusions.
`In two
`patients,
`the reactions occurred during the infusion and con
`sisted of facial flush and slight nausea. There was associated
`pruritus in one of these patients.
`In another patient,
`the reaction
`resembled an atypical
`serum sickness with eosinophilia. All
`reactions were mild and resolved spontaneously without medi
`cation except
`that one patient was given one p.o. dose of
`diphenhydramine
`for symptomatic
`relief of pruritus.
`Three of 22 patients
`in this study expired within 2 months
`of receiving immunotoxin
`therapy. Two patients had rapidly
`progressive metastatic melanoma and died as a result of their
`
`
`
`cancerres.aacrjournals.org Downloaded from
`
`1720
`on October 30, 2014. © 1987 American Association for Cancer
`Research.
`
`IMMUNOGEN 2193, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Table 3 Response to therapy with XOMAZYME-MEL monoclonal antimelanoma antibody ricin A chain immunotoxin
`Patient's performance status (as of 12/15/85)
`is based on the Karnofsky scale.
`
`IMMUNOTOXIN
`
`THERAPY
`
`responseProgressionMixed
`
`status%0701000000010070005000010010010010080100
`
`response(13+)*Complete
`
`
`response(13+)ProgressionMixed
`
`
`
`response(2)ProgressionProgressionProgressionStability
`
`1(NPC)12(CF)13
`
`(NPC)14
`(NPC)15
`(NPC)16(CF)17
`
`s.c.
`
`(6)ProgressionProgressionProgressionProgressionProgressionUnevaluableProgressionMixed
`
`responsePulmonary',
`and description of
`PatientKCF)2(CF)3(CF)4(CF)5(CF)6(CF)7(CF)8(CF)9(CF)10(NPC)1
`
`intraabdominal,(NR)°Pulmonary,
`lymphatic
`
`s.c. (PR); central nervous system(NR)Pulmonary
`(CR)Central
`nervous system (NE); hepatic, pulmonary, adrenal, s.c.
`(NR)S.c.
`
`(PR); central nervous system, pulmonary,(NR)Central
`lymphatic
`
`nervous system, hepatic, adrenal, s.c.(NR)Central
`
`nervous system, hepatic, pulmonary, gastrointestinal,(NR)Cardiac,
`
`hepatic, pulmonary, s.c.(NR)Pulmonary
`(SD)Hepatic,
`
`pulmonary, bone, lymphatic(NR)Splenic,
`
`renal, s.c.(NR)Hepatic,
`
`pulmonary, s.c.(NR)Hepatic,
`
`bone(NR)Hepatic,
`
`pulmonary', s.c.(NR)Pulmonary,
`pancreatic, s.c.
`(NE)Central
`
`nervous system (NE); bone, s.c.(NR)Pulmonary
`response(3+)Stability
`
`
`(NR); s.c.(PR)Pulmonary
`(NPC)18
`(3+)Stability
`(SD)Pulmonary
`(NPC)19
`(3+)Stability
`(SD)Pulmonary
`(NPC)20
`(SD)Pulmonary,
`(3+)Stability
`(CF)21
`(2+)Mixed
`
`lymphatic, s.c.(SD)Pulmonary
`(CF)22
`response (2+)Performance
`(NR); s.c. (PR)Net
`(NPC)Site
`" NR, no response; CF, chemotherapy failure: NPC, no prior chemotherapy; NE, not évaluable;SD, stable disease; PR. partial reduction: CR, complete reduction.
`'' Numbers in parentheses, duration of response in months; +. ongoing response.
`
`and
`of the proposed mechanism of action of the immunotoxins
`they are presented as evidence of biological effect. The duration
`of the tumor regressions
`ranged from 2 to 13 months, and are
`ongoing in three of the four patients. Five patients have shown
`stabilization of pulmonary métastasesongoing at 2 to 6 months.
`The observed tumor
`regressions were unusual
`in that
`lesions
`continued regressing for prolonged intervals following a single
`course of immunotoxin without any additional
`therapy. Two
`additional patients had temporary regression of their cerebral
`métastasesfollowing immunotoxin
`therapy. Both of them had
`completed
`central nervous
`system radiation
`therapy 4 to 8
`weeks prior
`to the immunotoxin
`and it is not clear whether
`these regressions represent
`responses to immunotoxin or radia
`tion therapy. Therefore,
`these two patients are considered une-
`valuable. Two patients, who had failed chemotherapy with
`multiple drugs, are alive more than 13 months after a single
`course of immunotoxin
`therapy. Overall, 11 patients are alive
`and 11 have expired.
`The histories of two patients are described to illustrate the
`type of responses observed. One patient, no. 3, is a 39-year-old
`white female who had a primary melanoma of the back in 1977.
`In 1983, the patient was found to have a right axillary lymph
`node metastasis which was resected. She was subsequently
`treated with Newcastle disease viral oncolysate.
`In July 1984,
`she developed a metastasis
`to the breast
`that was excised. A
`recurrence of the breast
`lesion was excised 1 month later. She
`then underwent
`two cycles of chemotherapy with bleomycin
`sulfate, vinblastine sulfate, hydroxyurea,
`and procarbazine hy-
`drochloride. The last dose of chemotherapy was administered
`on 10/10/84. During the course of chemotherapy,
`a retrocar-
`diac metastasis
`increased in size despite the quadruple drug
`chemotherapy
`In November 1984, she received immunotoxin
`therapy at a dose of 0.2 mg/kg/daily
`for 5 days (68 mg total).
`One month after immunotoxin
`therapy,
`the patient had radio-
`graphic evidence of tumor
`regression that continued over the
`ensuing months until
`the tumor became undetectable approxi
`mately 8 months after
`immunotoxin
`administration. No new
`lesions have appeared, and she remains in complete remission
`13 months after a single course of immunotoxin
`(Fig. 5).
`The second patient, no. 2, is a 55-year-old white female who
`she was treated with melphalan heated-limb perfusions, dacar-
`had a primary melanoma of the left foot in 1976. She developed
`bazine,
`lomustine,
`radiation therapy,
`surgery, and intrarterial
`extensive métastasesto the left leg, and between 1980 and 1984
`cisplatin. Despite this, she developed a pulmonary metastasis
`1721
`on October 30, 2014. © 1987 American Association for Cancer
`Research.
`
`Downloaded from
`
`cancerres.aacrjournals.org
`
`Fig. 5. A, chest computerized tomography scan of patient 3 obtained after
`quadruple drug chemotherapy
`and immediately before immunotoxin
`therapy
`showing a retrocardiac mass (arrows) that
`increased in size in the face of chemo
`therapy. B, chest computerized tomography scan of patient 3 obtained 1 year
`after a single course of immunotoxin therapy showing complete regression of the
`retrocardiac mass. Other studies showed no evidence of new disease elsewhere.
`R. right: /.. left.
`
`IMMUNOGEN 2193, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`IMMUNOTOXIN THERAPY
`
`*
`
`B
`
`. l. section of melanoma obtained from patient 16 2 weeks after two infusions of immunotoxin showing no reaction when stained with anti-A chain antibody.
`Fig. 6.
`This is presented as a negative reaction for comparison with the positive reaction (Fig. 5B). B, section of melanoma obtained from patient 22 three and one-half h
`after the fifth infusion of immunotoxin.
`stained with anti-A chain antibody. There is strong reactivity within the cells, prominently in the areas surrounding blood
`vessels. X 400.
`
`in September 1984. The patient was subsequently involved in a
`radioimmunoimaging
`study using the same murine monoclonal
`antibody as that
`in the immunotoxin. A previously unrecog
`nized pulmonary metastasis was imaged at
`that
`time. One
`month later, she received immunotoxin
`at a dose of 0.05 mg/
`kg/daily for 5 days (16 mg total). Approximately
`2 months
`aft