Phase I Evaluation of an Anti-Breast Carcinoma Monoclonal
`Antibody 260F9-Recombinant Ricin A Chain Immunoconjugate
` 
`Louis M. Winer, Joyce O'Dwyer, Joanne Kitson, et al.
`Cancer Res  
`
`1989;49:4062-4067.
`
`Updated version
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`Access the most recent version of this article at:
` http://cancerres.aacrjournals.org/content/49/14/4062
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`cancerres.aacrjournals.org cancerres.aacrjournals.org
`
`
`on October 30, 2014. © 1989 American Association for Canceron October 30, 2014. © 1989 American Association for Cancer
`
`Research. Research.
`
`IMMUNOGEN 2162, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`(CANCER RESEARCH 49. 4062-4067. July 15. 1989]
`
`Phase I Evaluation of an Anti-Breast Carcinoma Monoclonal Antibody 260F9-
`Recombinant Ricin A Chain Immunoconjugate1
`
`Louis M. Weiner,2 Joyce O'Dwyer, Joanne Kitson, Robert L. Comis, Arthur E. Frankel, Robert J. Bauer, Michael S.
`Konrad, and Eric S. Groves
`Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 ¡L.M. H'., J. O., J. A'., R. L, C.J; Duke University Medical Center,
`Durham, \orth Carolina 27710¡A. E. F.]; and Cetus Corporation. Emeryville, California 94608 /R. J. B.. M. S. A'., E. S. G.J
`
`ABSTRACT
`Four women with metastatic breast cancer were treated with monoclo
`nal antibody 260K9-recombinant ricin A chain, a ricin A chain immuno
`conjugate (1C) which targets a M, 55,000 antigen expressed by human
`mammary carcinomas. Patients were treated by daily, 1-h i.v. injections
`for 6 to 8 consecutive days. Two patients were treated with 10 MR/kg
`daily and the two others were treated with 50 «ig/kgdaily. The trial was
`suspended after four patients had been treated because patients treated
`with a continuous infusion schedule with this 1C had developed significant
`neurological
`toxicity at doses similar to those used in this study. The
`half-life of the 1C showed a t.,a of approximately
`1.8 h, a t.,/3 of
`approximately 8.3 h, and a peak concentration of about 200 ng/ml, at the
`lower dose level, and showed a f.,or of approximately 2.5 h, t, p of about
`10.4 h, and a peak concentration of 500 and 850 ng/ml for the two
`patients at the higher dose level. All four patients developed evidence of
`a human anticonjugate antibody response within 16 days of the onset of
`therapy. The treatment was associated with significant
`toxicity, mani
`fested by a syndrome consisting of weight gain, edema, hypoalbuminemia,
`and dyspnea. Similar symptoms were observed in patients treated by
`continuous infusions of the 1C. This clinical syndrome, seen at doses of
`1C which were insufficient
`to saturate
`antigen-expressing malignant
`tumor deposits in this trial, has been seen in other 1C therapy trials and
`in clinical trials using the cytokine interleukin 2. To investigate a possible
`mechanism responsible for this toxicity, human monocytes were incu
`bated with varying concentrations of 1C. There was detectable binding of
`1C to human monocytes at 1C concentrations which were achieved clini
`cally in this trial. Furthermore,
`the binding appeared to be abrogated by
`preincubation of the monocytes with pooled human immunoglobulin, thus
`suggesting that binding occurs via Fey receptor-mediated mechanisms.
`Binding was not affected if different linkers between recombinant ricin A
`chain and the antibody were used or if a different antibody moiety was
`used in the 1C preparation. Chemically linked dimers of MOPC-21 bound
`to human monocytes at least as well as the ICs; this binding was not
`abrogated by preincubation with pooled human immunoglobulin. Since
`the 1C preparations used in this clinical trial contained small percentages
`of dimers and/or multimers,
`the clinical
`toxicity syndromes which we
`observed may be related to this series of observations. A more complete
`understanding of the relationship of this previously unreported mecha
`nism of 1C binding to human cells expressing Fc*yreceptors with the
`clinical manifestations of the capillary leak syndrome will await produc
`tion and testing of Hal)')..
`ICs or highly purified whole antibody 1C
`preparations which contain only monomers. Further
`investigations into
`the mechanisms by which 1C binding to FCT receptor-bearing cells may
`lead to disruption of endothelial cell integrity may provide important
`clues to the pathogenesis of the capillary leak syndrome seen with a
`variety of biological therapies.
`
`INTRODUCTION
`The availability of monoclonal antibody technology has made
`it possible to develop antibody-toxin conjugates that can deliver
`
`revised 4/6/89; accepted 4/14/89.
`Received 10/31/88;
`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.
`1This study was supported in part by National Cancer
`Institute Grant P30
`CA 06927.
`Investigator Award. To
`Institute Clinical
`2Recipient of a National Cancer
`should be addressed, at the Department of
`whom address requests for reprints
`Medical Oncology, Fox Chase Cancer Center. Central Avenue and Shelmire
`Street. Philadelphia, PA 19111.
`
`the toxin moiety to malignant cells in a highly specific manner.
`A variety of toxins have been tested; among the most potent of
`these is ricin. This toxin catalytically inhibits protein synthesis
`by binding to and cleaving the A'-glycosidic bond of position
`/44324in 28S RNA of 60S subunits of mammalian
`ribosomes
`(1-3). The native toxin is composed of two chains, A and B,
`linked by a disulfide bond. The B chain is responsible
`for
`binding to cell surfaces, while the A chain contains
`the enzy
`matic activity.
`ICs' containing
`purified A chains have been
`tested extensively
`in preclinical models
`and clinical
`trials
`(4-14).
`The IgGl murine monoclonal antibody 260F9 is specific for
`a A/r 55,000 antigen expressed by approximately 50% of breast
`cancer cells (15). This antibody has been conjugated to recom
`binant ricin A chain to form the immunoconjugate
`260F9 MAb-
`rRA (16). Following extensive preclinical
`testing which con
`firmed the selectivity, biological activity, and acceptable toxicity
`profiles of the 1C, Phase I evaluations of this compound were
`begun using two different administration
`schedules. This com
`munication
`describes our observations
`of a daily injection
`schedule and focuses on the capillary leak syndrome seen with
`this 1C. We show that
`this and other
`ICs bind to human cells
`that do not express the antigen identified by 260F9, via uptake
`by cellular Fc> receptors.
`
`MATERIALS
`
`AND METHODS
`
`Clinical Study Design. Four patients with metastatic breast cancer
`were treated with this immunoconjugate
`between May and August
`1987. Their clinical characteristics are summarized in Table 1. Patients
`1 and 2 were assigned to receive 10 ug/kg daily of immunoconjugate,
`administered by a 1-h infusion for 8 consecutive days. Patient 3 was
`scheduled to receive 50 Mg/kg daily by the same route for 8 consecutive
`days, while patient 4 was assigned to receive 50 /¿g/kgdaily for 6 days
`based upon pharmacokinetic
`studies in the first
`three patients. The
`patients were hospitalized to receive all treatments
`in the clinical study
`unit at Fox Chase Cancer Center.
`toxicity on a daily basis while
`The patients were evaluated for
`receiving treatment and at approximately weekly intervals thereafter.
`Unless specified otherwise,
`all
`toxicity is graded according to the
`recently developed National Cancer Institute common toxicity criteria.
`1C Preparation. 260F9 MAb-rRA 1C was prepared by the Cetus
`Corporation. Briefly, purified 260F9 MAb was linked to rRA through
`a linker containing a disulfide bond. Ricin A chain was produced in
`Escherichia coli Kl 2 from the gene sequence of ricin A chain cloned
`from the complementary DNA of the castor bean. The final 1C was
`purified free of unconjugated rRA and conjugates containing multimers
`of MAb and rRA; however, a small fraction of contaminating MAb-
`MAb-rRA persisted in the final product. Clinically used 1C preparations
`contained a small concentration
`of dimers and multimers,
`less than
`10% as assessed by nonreducing gel electrophoresis.
`ICs 260F9 MAb-Ll-rRA and 260F9-L2-rRA were prepared at Cetus
`using two thioether
`linkers, LI and L2, to aid in determining the role
`
`1The abbreviations used are: 1C, immunoconjugate: 260F9 MAb-rRA. murine
`monoclonal antibody 260F9-recombinant
`ricin A chain immunoconjugate:
`rRA.
`rccombinant
`ricin A chain; SPDP, yV-succinimidyl-3-(2-pyridylthio)propionate.
`
`4062
`
`
`
`cancerres.aacrjournals.org Downloaded from
`
`on October 30, 2014. © 1989 American Association for Cancer
`Research.
`
`IMMUNOGEN 2162, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`PHASE l TRIAL OF IMMUNOCONJUGATE 260F9-MAh-rRA
`
`Table l Patient characteristics
`
`
`(%)"909010080Priortherapy*Melph.
`Patient1234Age(yr)35713354PS
`5-FUra, CTX.MTX.DXR.
`
`
`sternalXRT5-FUra.
`
`intensity than 95-98% of the negative control cell popula
`fluorescent
`tion. Statistics were performed using paired t test analysis, as described
`sitesChest
`(19).
`andabdominalwallBoneSCNBoneLungsChest
`
`
`MTX, VLB,CTX.DXR.
`
`MMCMTX,
`5-FUra, Pred.ChlorMTX.
`
`
`MMC, TAM,CTX,VLB.
`
`5-FUra. ehestwallXRTMctastatic
`
`wall, leftSCNpleura,
`
`lungs
`
`°PS, Karnofsky performance status; Melph, melphalan: 5-FUra, 5-fluoroura-
`cil: CTX. cyclophosphamide: MTX. methotrexate: DXR. doxorubicin: XRT,
`radiation therapy; VLB. vinblastinc; MMC, mitomycin C; Pred. prednisone:
`Chlor, ehlorambucil: Tam.
`tamoxifen: SCN, supraclavicular
`lymph node.
`* Excluding surgery.
`
`RESULTS
`
`are summarized in
`Clinical Course. The courses of treatment
`Table 2. Patients 1 and 2 received their planned therapy at full
`doses for all 8 days. Patient 3 required a 1-day treatment break
`and 50% dosage reductions
`thereafter,
`so that
`she received
`therapy for 7 full days, with 62.5% of her planned dose actually
`administered. Patient 4 received full doses of a 6-day planned
`treatment course. The shorter
`treatment course was undertaken
`because of the demonstration
`of anticonjugate
`antibody re
`sponses observed in the previous patients (see below).
`One patient, #3, had a clinical response, with the disappear
`ance of her sole site of disease, a 1-cm'
`lung nodule identified
`by chest computer-assisted
`tomographic
`scan. However,
`she
`had other
`lung abnormalities,
`also seen on computer-assisted
`tomographic
`scan, of uncertain disease significance, which did
`not change with therapy. She had recurrence of her tumor at a
`chest wall/skin
`site 3.5 months
`after
`the initiation
`of her
`therapy.
`A number of toxicities were observed with this therapy, even
`at doses which were too low to yield detectable binding of the
`immunoconjugate
`to the target antigen of 260F9 (Table 3).
`Malaise and other constitutional
`symptoms
`such as fever or
`myalgias were commonly seen but were not dose limiting. While
`all patients experienced performance
`status deterioration,
`only
`patient 3 declined significantly. Peripheral
`blood monocyte
`counts did not change with therapy. Anemia was universal and
`could not be totally accounted for by multiple phlebotomies.
`Weight gain averaging 4.2 kg was observed as was edema,
`hypoalbuminemia,
`hypoproteinemia,
`and eosinophilia. Patient
`
`Table 2 Treatment summary
`Duration of ther
`apy (days)
`
`Total dose
`Planned total
`Assigned daily
`received (nig)
`Planned Actual
`dose (nig)
`dose (*ig/kg)
`Patient
`1234101050506.85.520.717.78886887"66.85.513.017.7
`
`of the disii Hide bond in the binding of the clinically tested 1C to human
`monocytes.
`MOPC-21 MAb-MAb ICs were produced at Cetus using SPDP
`linker
`technology. The final mixture was purified of free MAb and
`higher order multi-MAb conjugates by a column sizing step.
`Pharmacokinetics and Anti-Conjugate Antibody Determinations. Sam
`ples were obtained at baseline; at 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 12.0, 18.0,
`and 24.0 h; and then daily prior
`to treatment. On the last day of
`therapy, blood was sampled prior to and just following dosing, and 24
`h after
`this last
`injection. Serum levels of 260F9 MAb-rRA were
`determined using a double sandwich enzyme-linked immunosorbent
`assay. The sensitivity of this assay is less than 1.0 ng/ml. One compo
`nent of the sandwich was directed at
`the rRA and the other at
`the
`murine MAb component;
`thus the assay determined the serum concen
`tration of conjugates composed of rRA and murine MAb. Analysis was
`performed using a nonlinear,
`least squares fit of the data using a two
`compartment model, which fit the data better than a one compartment
`model. Data were weighted inversely and the fit program used was
`Simusolv (Dow Chemical Company). All serum sample times for each
`patient were incorporated into the data set used for the fitting of the
`presented parameters.
`Anti-drug and anti-drug component antibody titers were determined
`using an enzyme-linked immunosorbent
`assay. Briefly, the drug or one
`of its components
`(260F9 MAb or rRA) was coated on plates. Appro
`priate dilutions of patient serum were incubated in the plate, unbound
`material was washed free, and the plate was developed with peroxidase
`anti-human IgG or peroxidase anti-human IgM. The isotype-matched
`murine antibody 520C9 was used as a plate coat to distinguish between
`responses directed at
`the binding specificity of 260F9 (antiidiotype)
`and those directed at the constant
`regions of murine IgG I antibodies.
`Cell Phenotype Studies. A variety of antibodies and ICs were used
`for in vitro analysis of binding to monocytes. Pooled human IgG,
`MOPC-21, and UPC-10 myeloma proteins were obtained from Sigma
`Chemical Co., St. Louis, MO. Monoclonal antibody 260F9 was gen
`erously provided by Dr. David Ring (Cetus Corporation). 260F9 MAb-
`rRA, MOPC-21 MAb-rRA, 260F9 MAb-Ll-rRA,
`and 260F9 MAb-
`ToxicityMaximum
`L2-rRA were all prepared by Cetus Corporation. F(ab')2 fragments of
`
`Karnofskyperformancestatus
`goat anti-mouse immunoglobulin conjugated with fiuorescein isothio-
`
`drop(%)Highest
`cyanate were purchased from Cappel, Malvern, PA.
`
`fever(grade)Most
`Peripheral blood was obtained from normal donors and peripheral
`
`wt gain(kg)Hypotension
`blood mononuclear cells were purified on Ficoll-Isopaque gradients, as
`(grade)Tachycardia
`(grade)Edema
`described (17). In some experiments purified monocytes were obtained
`(grade)Largest
`by reversible binding to gelatin and autologous plasma-coated plastic
`
`hemoglobin drop (g/100ml)Eosinophilia
`flasks, as described (18). To evaluate binding of various proteins
`to
`(%)Serum
`
`albumin (cl ofbaseline)Scrum
`monocytes, purified monocytes or peripheral blood mononuclear cell
`
`total protein (''< ofbaseline)Rash
`suspensions were incubated with various concentrations of the binding
`(grade)Dyspnea
`proteins for 30 min at 4°C.Cells were washed and F(ab')? fragments
`(grade)Neurological
`of goat anti-mouse
`IgG labeled with fiuorescein isothiocyanate were
`(grade)Nausea,
`added. Following another 30-min incubation at 4°Cthe cells were
`
`vomiting(grade)Flu-like
`symptoms (grade)110050003.14697100000210050012.410778302a00034015.23021.686069101»2I41021.70011.73798600Ie11
`washed and fluorescence was evaluated by flow cytometry using a
`°Delayed onset of dyspnea in absence of pulmonary tumor, edema, or further
`FACScan flow cytometer
`(Becton Dickinson, Mountain View, CA).
`weight gain.
`Gates were set on monocytes by forward and side scatter criteria; greater
`* Delayed onset of fingertip parcsthesias (see text).
`than 95% of the cells in the monocyte gates expressed Leu-M3 (data
`' Left arm weakness and diminished sensation. Tumor in brachial plexus found
`not shown). Results are expressed as percentage of cells with more
`on magnetic resonance imaging scan.
`4063
`
`" This patient received full doses for 3 days. Treatment was held for 1 day and
`then resumed at 50rr doses for the remaining 4 days.
`
`Table 3 Adverse events during therapy
`Patient
`
`
`
`cancerres.aacrjournals.org Downloaded from
`
`on October 30, 2014. © 1989 American Association for Cancer
`Research.
`
`IMMUNOGEN 2162, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`PHASE I TRIAL OF IMMUNOCONJUGATE 260F9-MAb-rRA
`
`Fig. 1. The binding of the IgGl murine antibody 260F9 (•)and 260F9 MAb-
`rRA 1C (O) to human monocytes was assessed by flow cytometry as described in
`"Materials and Methods." At protein concentrations
`exceeding 1 x 10"* M, the
`1C bound significantly more than did native antibody. *, P < 0.01; **, P < 0.05;
`***,/>< 0.001.
`
`hypotension which was as
`3 experienced grade 3 orthostatic
`sociated with weight gain, edema, hypoalbuminemia,
`and hy-
`poproteinemia. Her clinical picture closely matched that of the
`capillary leak syndrome which has been observed in patients
`receiving high dose interleukin 2 therapy (20, 21). In addition,
`this patient experienced a grade 2 nonpruritic rash on her trunk
`and extremities
`following the last dose of immunoconjugate.
`Three of the four patients
`experienced
`delayed symptoms
`which were possibly attributable
`to the immunoconjugate
`ther
`apy. Patient 2 experienced grade 2 dyspnea 6 days after
`the
`conclusion
`of
`therapy
`in the absence of any demonstrated
`pulmonary tumor, edema, or weight gain. She was thought
`to
`have congestive heart
`failure by her local
`treating physicians,
`although this was never documented by noninvasive or invasive
`studies. Patient
`3 developed the delayed onset of fingertip
`paresthesias
`in the arm contralateral
`to her prior mastectomy
`incision. These symptoms resolved completely within 6 months.
`She has since developed evidence of local skin recurrence but
`has had no evidence of axillary or brachial plexus involvement.
`Finally, patient 4 experienced progressive
`left arm weakness
`and diminished sensation in that arm following therapy. How
`ever, extensive tumor was found in the left brachial plexus upon
`magnetic resonance imaging scanning.
`Pharmacokinetics
`and Anti-Drug Antibodies. Pharmacoki-
`netics are displayed in Table 4A. The two patients treated with
`10 Mg/kg/day developed peak serum concentrations
`of approx
`imately 200 ng/ml.
`tv,a was approximately
`1.8 h and tv,ßwas
`approximately 8.3 h for these two patients. For the two patients
`treated with doses of 50 ^g/kg/day,
`tv,a was approximately
`2.5
`h, Ã(cid:141)././3was was approximately
`10.4 h and the peak serum levels
`were approximately
`500 and 850 ng/ml,
`respectively. Trough
`levels of 1C were noted to decline in patient 1 during the last 2
`days of therapy (from 25 to 10 ng/ml). The role of anti-drug
`antibodies
`in mediating this decrease is unclear. Peak serum
`levels did not change in this patient
`from days 1 to 8 (264 to
`248 ng/ml).
`As shown in Table 4B, all four patients developed anticon-
`jugate antibody titers, which in all cases exceeded the titers to
`either the antibody or ricin A chain components of the conju
`gate. Significant
`IgG titers were noted against 260F9 antibody;
`
`Table 4 Pharmacokinetics: one and two compartment Jit models
`A. Pharmacokinetics
`
`Daily dose (pg/kg) (1-h in
`fusion)Peak
`
`serumconcentration(ng/ml)Half-life
`
`(h)Volume
`
`of distributionofcentral
`compartment(ml/kg)'*«
`
`Patient
`10
`
`Patient 2
`10
`
`Patient 3
`50
`
`Patient 4
`SO
`
`±1.339.1
`
`±0.21.1
`
`±1.769.1
`
`±0.52.6
`
`±1.163.7
`
`±0.13.0
`
`±1.990.7
`
`
`±0.22.0
`
`in patient 1, this appeared to be largely an antiidiotype response
`since the human anti-mouse antibody response against the IgGl
`murine
`antibody 520C9 was substantially
`less than that of
`260F9. The development
`of an antiidiotype
`response distin
`guishable from a human anti-mouse antibody response was not
`clear in the other patients tested. In general,
`the IgG peak titers
`substantially exceeded the IgM peak titers.
`In all patients,
`a
`human anticonjugate
`response was noted with 16 days of the
`onset of treatment and as early as 8 days following the onset of
`therapy in patients 1 and 3.
`Patient Tumor Studies.
`In patient 1 an abdominal wall me
`tastasis was biopsied and found to demonstrate
`the antigen
`recognized by antibody 260F9. A chest wall metastasis was
`biopsied at the conclusion of 8 days of treatment
`and analyzed
`for antigen expression and persistence of the immunoconjugate.
`While the malignant cells in this lesion expressed antigen, as
`measured by the method of
`immunoperoxidase
`staining of
`frozen lesion sections with 260F9 antibody, no persistent
`im
`munoconjugate was detectable in the lesion (data not shown).
`This finding is not unexpected since higher doses have been
`required to achieve saturation of tumor binding sites in clinical
`trials with other antibodies (22).
`Toxicity Mechanism Studies. The identification of significant
`capillary leak syndrome toxicities
`in association with immu
`noconjugate
`therapy suggested the possibility that
`the immu
`noconjugate was delivering recombinant
`ricin A chain to unin
`tended targets and that this delivery might be inducing the toxic
`syndrome. We chose to investigate
`the possibility that
`the
`addition of the ricin A chain had altered the structure of the
`IgGl murine antibody 260F9 so that
`the modified protein was
`capable of binding to human Fc7 receptors. To test this, human
`monocytes were incubated in vitro with varying concentrations
`of immunoconjugate
`or with 260F9. As shown in Fig. 1, the
`native antibody did not bind to monocytes over a broad range
`of concentrations.
`In contrast,
`the immunoconjugate
`exhibited
`a concentration-dependent
`increase in binding so that detecta
`ble binding was noted at concentrations
`as low as 10~8 M. It
`should be noted that
`the peak serum 1C concentrations
`even at
`the lower dose level (Table 4A) were associated with substantial
`binding to monocytes
`in vitro. This enhanced
`binding was
`observed not only when the monocytes were incubated with
`these proteins in small volumes for immunophenotypic
`analyses
`but also when the monocytes were incubated in tissue culture
`media at 1 x IO6cells/ml
`in the appropriate
`concentrations
`of
`immunoconjugate
`(data not shown).
`4064
`
`±0.047.9
`±0.049.9
`
`±0.38.7
`
`±0.110.9
`
`+0.10.42
`±0.10.49
`
`±1.30.32
`±0.50.15
`±0.0116206.262.71704.4
`±0.0996010.495.58505.0
`
`
`±0.01480810.491.95007.1±0.01423711.81
`
`(h)<»0
`(h)Amplitude
`fH0Area
`to
`
`under curve(ng/ml-h)Clearance
`
`(ml/kg-h)K„
`(ml/kg)2604.9
`
`B. Human anti-conjugate antibody responses
`
`IgG (peak tiler)
`
`IgM (peak titer)
`
`39.0
`
`Patient
`1
`2
`3
`4
`
`First day
`of rise
`8
`16
`10
`15
`
`Conju
`gate
`80
`20
`300
`10
`
`260F9 rRA 520C9
`40
`35
`3
`555
`40
`917
`
`30
`
`30
`
`Conju
`gate
`15
`4
`30
`2
`
`260F9 rRA 520C9
`7
`<1
`4
`2
`<1
`4
`20
`4
`20
`<1
`<1
`<1
`
`Downloaded from
`
`on October 30, 2014. © 1989 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2162, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`PHASE I TRIAL OF IMMUNOCONJUGATE 260F9-MAb-rRA
`
`50^
`
`40LUW90)coaiB-
`
`30ooE§|O).E
`
`20.Cu>"àio"oCog
`
`10£--ITTHumanigI1260F9Ii260F9-
`
`Human Ig +
`260F9-MAb-rRA
`MAb-rRA
`Fig. 2. Purified human monocytes were incubated with 10 * M 260F9 or
`260F9 MAb-rRA as described in the text. Cells were preincubated with either
`medium or 50 »<g/mlof pooled human immunoglobulin (Human Ig) for 30-45
`min. These conditions saturate human monocyte Fc-yreceptors (data not shown).
`The binding of human immunoglobulin to monocytes could not be detected in
`this assay since l-'(ali').. fragments of goat anti-mouse immunoglobulin were used
`to detect the presence of adhered immunoglobulin.
`
`by which this binding may
`the mechanisms
`To investigate
`occur, monocytes were incubated with a variety of ICs and other
`proteins as shown in Table 5. To evaluate the role of the 260F9-
`binding portion on monocyte binding, monocytes were exposed
`to an 1C identical
`to 260F9 MAb-rRA except
`that the antibody
`portion of the 1C was the MOPC-21 myeloma protein. To
`evaluate the possibility that the specific linker used to conjugate
`the antibody to ricin A chain was responsible for the observed
`results
`(through either disulfide exchange or another
`linker-
`specific mechanism),
`two ricin A chain 260F9 ICs prepared
`using different
`thioether
`linker technologies, designated 260F9
`MAb-Ll-rRA and 260F9 MAb-L2-rRA, were tested as well.
`The myeloma protein UPC-10, an IgG2a murine protein which
`interacts with the Fc7 receptor expressed by human monocytes,
`was tested as well. Finally,
`the ability of preincubation with 50
`Mg/ml pooled human IgG to inhibit 1C binding via saturation
`of monocyte Fc7 receptors was evaluated as well. As shown by
`the representative
`experiment
`depicted on Table 5, all of the
`ICs bound well to monocytes. Furthermore,
`the binding of each
`1C was inhibited to control
`levels when the monocytes were
`preincubated with human immunoglobulin.
`The ICs did not
`bind to lymphocytes. To further
`evaluate the Fc7 receptor-
`mediated uptake of the immunoconjugate,
`a number of exper
`iments were performed in which human monocytes were prein
`cubated with human immunoglobulin
`prior
`to the addition of
`the 1C. As shown in Fig. 2, preincubation with pooled human
`IgG clearly suppressed binding of the 1C to human monocytes.
`The clinically used 1C preparations
`contained less than 10%
`dimcrs and multimers by nonreducing gel electrophoresis. To
`further
`investigate the mechanisms by which this 1C may bind
`to the Fc7 receptor expressed by human monocytes,
`the ability
`of chemically constructed SPDP-linked
`dimcrs of MOPC-21
`to bind to human monocytes was investigated. Fig. 3 demon
`strates the results of a series of experiments
`in which monocytes
`were incubated with either MOPC-21,
`260F9 MAb-rRA, or
`MOPC-21 dimers at varying concentrations. As expected,
`the
`immunoconjugate
`bound to human monocytes;
`this binding
`was inhibited by preincubation with human immunoglobulin.
`The MOPC-21 dimer bound to monocytes as least as well as
`the immunoconjugate.
`Furthermore,
`this binding was only par
`tially reversed by preincubation with human immunoglobulin.
`Since the preparation
`used may have contained higher multi
`mers of MOPC-21,
`it is possible that
`these immunoglobulin
`aggregates possessed substantially higher affinity for Fc7 recep
`tors
`than did the dimers. These data suggest one possible
`explanation for the binding of this 1C to human Fc7 receptor-
`bearing cells.
`
`DISCUSSION
`
`trial of bolus 1C admin
`This dose escalation phase I clinical
`istration was suspended after
`four patients had been accrued
`
`Table 5 Ricin A chain immunoconjugaies bina to human monocytes via Fey
`Preincubation with human
`immunoglobulin
`
`infusion of the 1C in
`treated by continuous
`because patients
`another phase I trial developed severe and unexpected neuro
`logic toxicity.4 No serious neurotoxicity was observed with the
`bolus schedule, and we speculate that
`the continuous
`infusion
`schedule may have resulted in an increased area under
`the
`curve, permitting observation of the neurotoxicity at equivalent
`daily 1C doses. We found two additional problems which may
`limit
`the likelihood of reaching therapeutically
`useful dosages
`of this 1C. The first, not unexpectedly,
`is the development of
`anticonjugate
`antibodies. This response was seen as early as 8
`days following the onset of therapy in our series. This response
`does not seem to be trivial
`in that patient 3 experienced a rash
`and in patient 1, who developed anticonjugate
`antibodies by
`day 8, peak serum levels of 1C decreased late in treatment. The
`second problem was the toxic syndrome consisting of edema,
`hypoalbuminemia, weight gain, and, occasionally,
`congestive
`heart failure or pulmonary edema. This toxicity syndrome was
`not related to the schedule of administration
`of 260F9 MAb-
`positive)No8.110.910.412.012.912.011.68.2Yes—__—8.48.28.18.5
`rRA because similar toxicity was noted in patients treated with
`a continuous
`infusion schedule at doses of either 50 or 100 ^g/
`kg daily for 8 days.
`trials
`This symptom complex has been reported in clinical
`using other
`ICs incorporating
`the ricin A chain. Spitler et al.
`MAb-rRAMOPC-21
`-MAb-rRA260F9-Ll-rRA260F9-L2-rRANo9.79.33.691.448.242.329.064.4Yes_"—__—7.48.16.45.7Lymphocytes(%
`* B. J. Gould, M. J. Horowitz, P. W. Carter, J. Moore. E. Groves, D. Anthony,
`L. M. Weiner, and A. Frankel. A phase I study of a continuous
`infusion anti-
`breast cancer immunotoxin: observation of a targeted toxicity not predicted by
`animal studies, submitted for publication.
`
`positive)Tested
`
`Monocytes(%
`
`protein (25>ig/ml)Human
`
`immunoglobulin260F9MOPC-21UPC-10260F9
`
`1—¿(cid:3),not done.
`
`
`
`cancerres.aacrjournals.org Downloaded from
`
`on October 30, 2014. © 1989 American Association for Cancer
`Research.
`
`4065
`
`IMMUNOGEN 2162, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`PHASE l TRIAL OF IMMUNOCONJUGATE
`
`2601 <) MAb-rRA
`
`co7°+1Cco
`
`0)60(01"o
`
`E40C
`
`CO1
`
`30O).C1
`
`20ut"
`
`10Percent
`o0--".-__mì%W
`
`Ti
`
`rh*MOPC-21
`
`5x10'8M
`
`260F9-MAb-rRA
`10'7M
`
`MOPC-21
`DIMER
`5X10-8M
`
`MOPC-21
`DIMER
`5X10-9M
`
`MOPC-21
`DIMER
`SxlO-'OM
`
`Fig. 3. In this set of four experiments, monocytcs were incubated with MOPC-
`21, 260F9 MAb-rRA. or SPDP-linked
`dimers of MOPC-21. The cells were
`prcincubated without (D) or with (D) pooled human immunoglobulin. MOPC-21
`dimers bound to monocytes at least as well as did 260F9 MAb-rRA. Furthermore,
`the binding of the dimers was not reversible by preincubation with pooled human
`immunoglobulin (P = 0.26). Since the dimers may have contained multimers of
`immunoglobulin.
`the enhanced affinity of these complexes for Fc-yreceptors may
`be responsible for the relative inability of human immunoglobulin
`to abrogate
`binding.
`
`relevance of
`the physiological
`of normal human serum so that
`this in vitro model may be assessed more fully. The relationship
`of these in vitro observations with the capillary leak syndrome
`will be clarified when agents which do not bind to monocytes
`are evaluated clinically.
`An additional mechanism for some of the reported 1C toxicity
`in other trials is hepatic uptake due to recognition of mannose-
`containing oligosaccharide
`chains of native ricin A chain by
`specific surface receptors expressed by nonparenchymal
`hepatic
`cells. Byers et al. (26) have shown that mannose-containing
`blocking agents can prolong blood circulation of a native ricin
`A chain-containing
`immunotoxin
`and decrease liver uptake as
`well in a murine model. Dose-dependent binding of native ricin
`A chain ICs to rabbit alveolar macrophages
`following i.v. infu
`sion has been observed (27). In that study, in vitro binding could
`be inhibited by preincubation
`of alveolar macrophages with
`various mannose-containing
`compounds,
`although the role of
`possible Fc7 receptor-mediated
`uptake was not evaluated. How
`ever, the 80% inhibition of binding by mannose argues for the
`primacy of mannose receptor-mediated
`uptake of 1C by rabbit
`alveolar macrophages.
`In another
`study the ability of ricin A
`chain to act directly as a ligand for
`the mannose
`receptor
`expressed by rat bone marrow macrophages was demonstrated
`with corresponding effects on cellular protein synthesis (28). In
`this latter study, antibody-ricin A conjugates were not evalu
`ated. Since our 1C preparations
`utilized recombinant
`nongly-
`cosylated ricin A chain, we do not believe that mannose recep
`tor-mediated uptake is an important mechanism for the toxic-
`ities we observed.
`Capillary leak syndrome is commonly seen in patients treated
`with high doses of the cytokine interleukin 2, particularly when
`the treatment
`is combined with infusions of autologous
`inter
`leukin 2-activated lymphocytes
`(20, 21). The clinical observa
`tion that
`this syndrome worsens when autologous
`activated
`lymphocytes are added suggests that these cells may be involved
`in the pathogenesis
`of the syndrome. Damle et al. (29) have
`demonstrated
`that
`these lymphokine-activated
`killer cells bind
`to endothelial
`cell surfaces,
`further supporting the association
`of these cells with the syndrome. Recently it has been demon
`strated that endothelial cells from interleukin 2-treated patients
`express activation antigens, although Fc-y receptors were not
`evaluated in that study (30). The mechanisms by which such
`activated cells may induce this syndrome
`remain unknown,
`although lymphokine-activated
`killer cells mediate detachment
`and lysis of human umbilical vein endothelial
`cells (29, 31).
`Although we observed little binding of 260F9 MAb-rRA to
`human lymphocytes (Table 5), studies which evaluate the inter
`actions of ricin A chain ICs with interleukin 2-activated large
`granular
`lymphocytes are required. The recent demonstration
`that human lymphokine-activated
`killer cells lyse autologous
`monocytes (32) suggests that monocyte lysis may be a common
`mechanism for disparate etiologies and manifesta