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`1999 93: 3678-3684
`
`Selective Ablation of Acute Myeloid Leukemia Using Antibody-Targeted
`Chemotherapy: A Phase I Study of an Anti-CD33 Calicheamicin
`Immunoconjugate: Presented in part at the 1997 Annual Meeting of the
`American Society of Clinical Oncology, Denver, CO; the 1997 European
`Cancer Conference, Hamburg, Germany; and the 1997 Annual Meeting of
`the American Society of Hematology, San Diego, CA.
`
`E.L. Sievers, F.R. Appelbaum, R.T. Spielberger, S.J. Forman, D. Flowers, F.O. Smith, K. Shannon-Dorcy,
`M.S. Berger and I.D. Bernstein
`
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` (3965 articles)Clinical Trials and Observations
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`Copyright 2011 by The American Society of Hematology; all rights reserved.
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`IMMUNOGEN 2187, pg. 1
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`CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
`Selective Ablation of Acute Myeloid Leukemia Using Antibody-Targeted
`Chemotherapy: A Phase I Study of an Anti-CD33 Calicheamicin
`Immunoconjugate
`
`By E.L. Sievers, F.R. Appelbaum, R.T. Spielberger, S.J. Forman, D. Flowers, F.O. Smith, K. Shannon-Dorcy,
`M.S. Berger, and I.D. Bernstein
`
`Leukemic blast cells express the CD33 antigen in most
`patients with acute myeloid leukemia (AML), but this anti-
`gen is not expressed by hematopoietic stem cells. We
`conducted a study to determine whether normal hematopoi-
`esis could be restored in patients with AML by selective
`ablation of cells expressing the CD33 antigen. In a dose
`escalation study, 40 patients with relapsed or refractory
`CD331 AML were treated with an immunoconjugate (CMA-
`676) consisting of humanized anti-CD33 antibody linked to
`the potent antitumor antibiotic calicheamicin. The capacity
`of leukemic cells to efflux 3,3’-diethyloxacarbocyanine iodide
`(DiOC2) was used to estimate pretreatment functional drug
`
`PATIENTS WITH refractory or recurrent acute myeloid
`
`leukemia (AML) have a dismal prognosis. Toxic effects
`associated with additional conventional chemotherapy are often
`life-threatening, and few patients achieve a complete remission
`(CR). A therapy that more specifically targets leukemia is likely
`to be safer, and possibly more effective than current nonspecific
`chemotherapeutic agents.
`During hematopoietic development, stem cells capable of
`establishing long-term multilineage hematopoiesis give rise to
`progenitors with diminished self-renewal capacity and a greater
`degree of differentiation. During this process, hematopoietic
`cells express distinct cell surface antigens that are also ex-
`pressed by their malignant counterparts. Some of these antigens
`(eg, CD33) are present on maturing normal hematopoietic cells
`and on AML cells, but not on normal hematopoietic stem
`cells.1-4 These findings raise the possibility that an antibody
`can be used to deliver cytotoxic agents to selectively ablate
`
`From the Clinical Research Division, Fred Hutchinson Cancer
`Research Center, Seattle; the Departments of Pediatrics and Medicine,
`University of Washington, Seattle, WA; the Department of Hematology
`and Bone Marrow Transplantation, City of Hope National Medical
`Center, Duarte, CA; and Wyeth Ayerst Research, Radnor, PA.
`Submitted August 19, 1998; accepted January 29, 1999.
`Supported by Wyeth-Ayerst Research. E.L.S. is supported by an
`American Cancer Society Clinical Oncology Career Development Award.
`I.D.B. is an American Cancer Society Clinical Research Professor.
`Presented in part at the 1997 Annual Meeting of the American
`Society of Clinical Oncology, Denver, CO; the 1997 European Cancer
`Conference, Hamburg, Germany; and the 1997 Annual Meeting of the
`American Society of Hematology, San Diego, CA.
`Address reprint requests to E.L. Sievers, MD, Fred Hutchinson
`Cancer Research Center, 1100 Fairview Ave N, D5-280, PO Box 19024,
`Seattle, WA 98109-1024; e-mail: esievers@fhcrc.org.
`The publication costs of this article were defrayed in part by page
`charge payment. This article must therefore be hereby marked ‘‘adver-
`tisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
`this fact.
`r 1999 by The American Society of Hematology.
`0006-4971/99/9311-0018$3.00/0
`
`resistance. Leukemia was eliminated from the blood and
`marrow of 8 (20%) of the 40 patients; blood counts returned
`to normal in three (8%) patients. A high rate of clinical response
`was observed in leukemias characterized by low dye efflux in
`vitro. Infusions of CMA-676 were generally well tolerated,
`and a postinfusion syndrome of fever and chills was the
`most common toxic effect. Two patients who were treated
`at the highest dose level (9 mg/m2) were neutropenic G5
`weeks after the last dose of CMA-676. These results show
`that an immunoconjugate targeted to CD33 can selectively
`ablate malignant hematopoiesis in some patients with AML.
`r 1999byTheAmericanSocietyofHematology.
`
`malignant myeloid and immature normal cells while sparing
`normal stem cells.
`The myeloid cell surface antigen CD33 is an attractive target
`for this approach, as it is expressed on AML blast cells from
`about 90% of patients.2,3 CD33 also may be expressed by most,
`if not all, of the malignant precursors in at least some cases of
`AML, as precursor cells that lack CD33 from some patients
`give rise to normal granulocyte/monocyte precursors in a
`marrow long-term culture system.5,6 The possibility that anti-
`CD33 antibody could be used to deliver a cytotoxic agent to
`malignant cells was suggested by studies showing rapid satura-
`tion of leukemic blast cells in peripheral blood and marrow after
`intravenous administration of approximately 5 mg/m2 radioio-
`dinated anti-CD33 antibodies.7,8 Further, in vitro studies showed
`rapid internalization of the antibody by the target cell.9 To test
`the concept of selective ablation of malignant hematopoiesis
`and to evaluate the safety of this approach, we treated patients
`with relapsed or refractory AML with escalating doses of
`CMA-676, antibody-targeted chemotherapy consisting of an
`engineered human anti-CD33 antibody linked with the potent
`antitumor antibiotic calicheamicin.10
`
`MATERIALS AND METHODS
`
`Patients. Patients were required to have AML that was either
`refractory to standard therapy or that had recurred after remission.
`Patients whose leukemia relapsed after a marrow or peripheral blood
`stem cell transplant were required to have had successful engraftment
`(platelets $20 3 103/µL without transfusion and an absolute neutrophil
`count [ANC] $500/µL) before relapse. Each prospective patient
`underwent a bone marrow aspirate to confirm that morphologic
`evidence of leukemia was present and to document leukemic blast cell
`surface expression of the CD33 antigen. Patients were required to have
`a Karnofsky performance status $60%, a white blood cell count #30 3
`103/µL, serum creatinine #2.0 mg/dL, and serum bilirubin #1.5 mg/dL,
`to have recovered from toxic effects of previous antineoplastic therapy,
`and to be able to give informed consent. Patients were ineligible if they
`had previously received anti-CD33 antibody treatment, were pregnant
`or nursing, had a prior malignancy, or had active symptomatic central
`nervous system leukemia or an uncontrolled life-threatening infection.
`Recombinant humanized anti-CD33-calicheamicin drug conjugate.
`CMA-676 was produced and provided for clinical study by Wyeth-
`
`3678
`
`Blood,Vol 93, No 11 (June 1), 1999: pp 3678-3684
`
`IMMUNOGEN 2187, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
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`SELECTIVE ABLATION OF AML USING ANTIBODY-TARGETED CHEMOTHERAPY
`
`3679
`
`Ayerst Research (Radnor, PA) under an Investigational New Drug
`Application. Celltech Therapeutics, Ltd (Slough, UK) transformed the
`murine-derived IgG1 monoclonal antibody (p67.6)8 into an engineered
`human IgG4 antibody (hP67.6), which was then conjugated to the
`enediyne calicheamicin10 using a Nac-gamma linker molecule (Hamann
`et al, manuscript in preparation). Calicheamicin binds in a sequence-
`specific manner to the minor groove of DNA and induces double-strand
`DNA breaks that ultimately induce cell death.10 Specific cytotoxicity of
`CMA-676 against CD331 leukemia cells was documented in colony-
`forming assays in vitro, against leukemic cell lines in vitro, and in vivo
`in nude mice (data not shown).
`In this open, single-arm, phase I dose escalation
`Protocol design.
`study, CMA-676 was administered to patients with relapsed or refrac-
`tory CD331 AML at two study sites. Informed consent was obtained
`from all patients in accordance with the institutional review boards of
`the participating institutions. In a single-dose tolerance study with
`CMA-676 in chimpanzees, an intravenous dose of 0.5 mg/m2 (twice the
`initial clinical dose of 0.25 mg/m2) was well
`tolerated, with no
`compound-related clinical signs of clinical pathology changes evident
`throughout a 15-day postinfusion period (data not shown). In previous
`clinical studies in patients using radiolabeled mP67.6 antibody,8 dose-
`limiting toxicity was not observed at doses up to and including 17
`mg/m2 antibody trace-labeled with 131I. However, saturation of CD33
`binding sites was achieved at a dose level of approximately 5 mg/m2.
`Hence, at
`least
`three patients were treated at each dose level of
`CMA-676 with up to three infusions of 0.25, 0.5, 1, 2, 4, 5, 6, and 9 mg
`protein per square meter of body surface area. If one or two of the first
`three of these patients experienced a grade III toxic effect (see below),
`three additional patients were to be treated at the same dose. The
`maximum tolerated dose was defined as one dose level below the dose
`that was associated with unacceptable CMA-676–related toxicity.
`Dose-limiting toxicity was defined as severe marrow hypoplasia of
`more than 6 weeks duration after a single dose of CMA-676, one grade
`IV study drug-related toxic effect or two grade IV toxic conditions of
`ambiguous relationship to CMA-676.
`CMA-676 was administered as a single 2-hour intravenous infusion.
`All cytokines or chemotherapeutic agents were withdrawn before
`treatment. Patients without leukemic progression and drug-related,
`nonhematologic toxic effects judged to be grade III or less could receive
`one or two subsequent cycles of CMA-676 with at least 14 days
`between cycles. Patients were treated with acetaminophen 650 mg
`orally and diphenhydramine 25 to 50 mg intravenously 15 to 30 minutes
`before infusion of CMA-676. Patients who achieved a CR and
`subsequently had a relapse of their leukemia could receive retreatment
`at the dosage level being evaluated at the time of retreatment.
`Patient evaluation. Patients were examined for acute toxic effects
`in accordance with the World Health Organization toxic effect grades.
`Peripheral blood specimens were obtained to study the pharmacokinet-
`ics of CMA-676, to evaluate for evidence of CMA-676 binding to
`CD33-positive cells, and to measure the effects of CMA-676 on
`hematologic variables and serum chemistry parameters. A complete
`blood count was obtained before the initial dose and each day for 2
`weeks thereafter, or until recovery of granulocyte and platelet levels to
`the prestudy levels. Hepatic and renal function were measured before
`initial dose administration, three times per week thereafter, and on day
`28 after the last dose of CMA-676.
`Bone marrow aspirates were obtained before initial dose administra-
`tion with CMA-676 on days 1, 7, and 14 of each treatment cycle and on
`days 1, 7, 14, and 28 of the last treatment cycle. All marrow specimens
`were examined by light microscopy to estimate cellularity and to detect
`any residual leukemic blast cells. Patients with progressive disease were
`removed from the study. Disappearance of leukemia was defined as the
`absence of peripheral blasts and the presence of #5% blast cells in the
`bone marrow by light microscopic evaluation. CR was defined as
`
`disappearance of leukemia in addition to an ANC .1,500/µL and a
`platelet count .100 3 103/µL, without transfusions.
`Laboratory investigations. Total hP67.6 antibody concentrations in
`plasma samples were determined using an enzyme-linked immunosor-
`bent assay (ELISA). Formation of antigen-antibody CMA-676 bound to
`peripheral blood mononuclear cells was detected by flow microfluorim-
`etry. Cells were incubated with biotinylated goat monoclonal anti-
`human IgG4, followed by avidin-fluorescein isothiocyanate. Cells
`incubated with avidin-fluorescein isothiocyanate alone comprised the
`negative control. The saturation percentage was defined as 100 times the
`ratio of the fluorescence intensity of patient mononuclear cells (minus
`the negative control) over the maximum fluorescence intensity (minus
`the negative control). Maximum achievable saturation was determined
`by incubating patient mononuclear cells from the same time point with
`saturating amounts of CMA-676 in vitro before the addition of the
`anti-human IgG4 antibody. The efflux of 3,3’-diethyloxacarbo-
`cyanine iodide (DiOC2) from CD33-positive blast cells was measured
`as an indication of functional drug efflux.11 Serum samples obtained
`from each patient before CMA-676 administration, on day 7 after
`initial doses, and on days 7, 14, 21, and 28 after administration of the
`final dose of CMA-676 were analyzed for anti-hP67.6 (humanized
`mouse antibody) or anti-calicheamicin/linker immune response by
`ELISA.
`
`RESULTS
`
`Patient characteristics. A total of 40 patients with relapsed
`or refractory AML were enrolled. Patient characteristics are
`shown in Table 1. Three to eight patients were treated at each of
`eight dose levels of CMA-676: 0.25, 0.5, 1, 2, 4, 5, 6, and 9 mg
`protein per square meter of body surface area.
`Nonhematologic toxicity. The most frequently reported
`drug-related adverse events are summarized in Table 2. The
`most commonly observed nonhematologic side effect, a syn-
`drome of fever and chills, occurred in 32 of 40 (80%) patients
`beginning 2 to 4 hours after the start of the 2-hour intravenous
`infusion. The syndrome was limited to grade I to II except in
`three instances. Grade III fever and chills occurred in two
`patients after receiving CMA-676 at 6 and 9 mg/m2, respec-
`tively. The third patient (COH-008) who had asymptomatic
`
`Table 1. Patient Characteristics (N 5 40)
`
`Age: median (range)
`Sex
`Stage of disease
`First relapse
`$Second relapse
`Prior stem cell transplant
`Allogeneic
`Autologous
`None
`Cytogenetic abnormality
`t(8;21)
`inv(16)
`t(4;11)
`t(6;11)
`t(9;11)
`Complex abnormality
`None detected
`Unknown
`Prior myelodysplastic phase
`Present
`Absent
`
`54 (24-73)
`21M:19F
`
`20
`20
`
`14
`4
`22
`
`1
`2
`1
`1
`2
`22
`10
`1
`
`9
`31
`
`IMMUNOGEN 2187, pg. 3
`Phigenix v. Immunogen
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`
`3680
`
`SIEVERS ET AL
`
`Table 2. Most Frequently Reported Drug-Related Adverse Events (No. of Patients)
`
`Event*
`Grade 0-I fever/chills
`Grade II fever/chills
`Grade III† fever/chills
`Grade IV fever/chills
`Grade III hepatic enzyme
`elevation (AST or ALT)
`Grade IV hepatic enzyme
`elevation (AST or ALT)
`
`0.25
`
`(n 5 4)
`4
`—
`—
`—
`
`—
`
`—
`
`0.5
`
`(n 5 3)
`2
`1
`—
`—
`
`1
`
`—
`
`1
`
`(n 5 4)
`1
`3
`—
`—
`
`1
`
`—
`
`CMA-676 Dose (mg/m2)
`
`2
`
`(n 5 3)
`1
`2
`—
`—
`
`—
`
`—
`
`4
`
`(n 5 6)
`4
`2
`—
`—
`
`—
`
`—
`
`5
`
`(n 5 6)
`1
`5
`—
`—
`
`4
`
`—
`
`6
`
`(n 5 8)
`3
`3
`1‡
`1§
`
`1
`
`1\
`
`9
`
`(n 5 7)
`3
`3
`1
`—
`
`1
`
`—
`
`Abbreviations: AST, alanine serum transaminase; ALT, alanine aminotransferase.
`*Grades are defined according to modified World Health Organization toxicity grading scale.
`†Modified to include fever ,40°C with asymptomatic hypotension.
`‡This patient (FH-007) is represented in the table twice. He experienced grade II fever/chills after his initial treatment at 1 mg/m2. He developed
`shortness of breath in association with retreatment with CMA-676 after his leukemia relapsed, presumably caused by an immune reaction to
`CMA-676.
`§This patient (COH-008) who had asymptomatic hypotension and was receiving low-dose dopamine before CMA-676 administration, had a
`temperature .40°C and reversible symptomatic hypotension 5 hours after the initiation of the CMA-676 infusion at 6 mg/m2.
`\This patient (FH-020) with documented concurrent cholelithiasis had a pretreatment AST of 13 U/L that increased to 304 U/L 4 days after
`receiving the first dose of CMA-676, and rapidly declined to the normal range within a week. Immediately before receipt of the second dose of
`CMA-676, her AST increased to 1,804 U/L and again rapidly returned to the normal range. Elevated enzymes were thought to be primarily due to
`the patient’s cholelithiasis.
`
`hypotension and was receiving low-dose dopamine before
`CMA-676 administration, had a temperature .40°C and revers-
`ible symptomatic hypotension 5 hours after the initiation of the
`CMA-676 infusion at 6 mg/m2. Another patient (FH-007)
`developed transient shortness of breath in association with
`retreatment with CMA-676 after his leukemia relapsed, presum-
`ably caused by an immune reaction to CMA-676 (see below).
`Nausea and fatigue were among other less frequent toxic effects
`thought possibly related to CMA-676.
`Reversible, possibly drug-related hepatic transaminase eleva-
`tions from 5 to 10 times the normal range were observed in eight
`patients (Table 2). One additional patient with documented
`concurrent cholelithiasis had a pretreatment alanine serum
`transaminase (AST) of 13 U/L that increased to 304 U/L 4 days
`after receiving the first dose of CMA-676, and rapidly declined
`to the normal range within a week. Immediately before receipt
`of the second dose of CMA-676, her AST increased to 1,804
`U/L and again rapidly returned to the normal range. In this
`patient, elevated enzymes were thought to be primarily due to
`the patient’s cholelithiasis. No patient had a drug-related
`bilirubin elevation of greater than five times the normal range.
`No significant study drug-related central nervous system,
`cardiac, or renal toxic effects were observed. With regard to
`nonhematologic toxic effects, a maximum tolerated dose was not
`reached, and dose-limiting toxicity was not observed.
`Hematologic toxicity. Because most patients had concur-
`rent neutropenia and thrombocytopenia caused by active AML
`at the time of enrollment, whether hematologic toxic effects
`were directly attributable to CMA-676 was often impossible to
`ascertain. However, among nine patients with neutrophil counts
`.1,500 cells/µL before treatment, eight had severe neutropenia
`(,200 cells/µL) within 14 days of CMA-676 infusion regard-
`less of dose level. Figure 1 shows the decline and recovery of
`the neutrophil count from a representative patient during the 2
`weeks after each infusion. Although dose-limiting toxicity was
`
`not formally observed, two of seven evaluable patients had
`prolonged drug-related neutropenia after treatment at a dose
`level of 9 mg/m2. The first patient (FH-023) required 38 days to
`recover to an ANC .500 cells/µL after the third dose of
`CMA-676, and the second patient (COH-015) did not achieve
`neutrophil recovery and died of sepsis 50 days after receiving
`the second dose (Table 3).
`Elimination of blast cells from the peripheral blood and bone
`marrow. Blast cells were identified on a peripheral blood
`smear in 31 (78%) patients before infusion of CMA-676. One
`week after treatment, 22 (71%) of these patients had fewer blast
`cells and in six (19%) of these patients, blast cells had
`completely disappeared from the peripheral blood smears. The
`proportion of patients who experienced reductions in peripheral
`blast cell counts was highest among those who received higher
`
`Fig 1. Relationship between hematologic parameters and time for
`a representative patient (FH-012) who received CMA-676 at 4 mg/m2
`per dose. Arrows denote infusions of CMA-676. All counts refer to
`peripheral blood counts.
`
`IMMUNOGEN 2187, pg. 4
`Phigenix v. Immunogen
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`SELECTIVE ABLATION OF AML USING ANTIBODY-TARGETED CHEMOTHERAPY
`
`3681
`
`Table 3. Clinical Responses and Recovery of Hematologic Function in Patients Who Received Treatment With CMA-676
`
`Time in Days From Last Dose to:
`
`Clinical
`Response
`
`Patient
`No.
`
`Age
`(yr)
`
`Cytogenetic
`Abnormality
`
`Dose
`Level
`(mg/m2)
`
`Peripheral
`Blasts
`(per µL)
`
`Percentage
`of Marrow
`Blasts
`
`ANC
`.1,500/
`µL
`
`Platelet
`Count
`.100 3 103/µL
`
`50
`83
`
`60
`
`88
`67
`8
`
`40
`23
`95
`
`12
`22,377
`
`748
`
`1,912
`3,560
`0
`
`2,445
`0
`300
`
`1
`4
`
`9
`
`5 6 9 9 9 6
`
`CR*
`
`FH-007
`FH-012†
`
`43 t(6;11)
`29 t(6;9)
`
`FH-023†
`
`34 None
`
`Elimination of
`blast cells from
`peripheral
`blood and
`marrow‡
`
`FH-013
`FH-019§
`FH-022
`
`FH-024
`COH-015§
`FH-007\
`
`74
`53
`65
`
`52
`34
`43
`
`None
`t(8;21)
`None
`
`t(1;7); t(12;17)
`Trisomy 11; del(15)
`t(6;11)
`
`4
`20
`
`42
`
`7
`35
`
`160
`
`Relapse
`
`132
`188
`
`.623
`
`Not attained
`Not attained
`18
`
`Not attained
`Not attained
`Not attained
`
`70
`Not applicable
`40
`
`20
`Not attained
`8
`
`Not evaluable¶
`Not attained
`Not attained
`
`14
`Not applicable
`56
`
`Site of
`Recurrence
`
`Bone; marrow
`Testes; central
`nervous system
`None
`
`Marrow
`Not applicable
`Marrow; central
`nervous system
`Marrow
`Not applicable
`Marrow
`
`*Patient achieved transfusion independence with ANC .1,500/µL, platelet count .100 3 103/µL, and ,5% blasts in the bone marrow aspirate.
`†Patient received donor lymphocyte infusion after attaining CR (FH-012) or before achieving platelet count .100 3 103/µL (FH-023).
`‡Patient had ,5% blasts in the bone marrow aspirate with various degrees of hematopoietic recovery.
`§Patients died of infection 12 (FH-019) and 50 (COH-015) days after receiving a third dose of CMA-676.
`\Patient attained CR after receiving CMA-676 at 1 mg/m2 and then was retreated with CMA-676 at a dose of 6 mg/m2.
`¶Patient received a bone marrow transplant shortly after leukemia disappeared from the bone marrow and eventually recovered a normal
`platelet count.
`
`doses of CMA-676. Reduced numbers of peripheral blast cells
`were observed in 10 of 11 patients treated with either 6 or 9
`mg/m2 of CMA-676 compared with 12 of 20 patients treated at
`dose levels from 0.25 to 5 mg/m2.
`Morphologic evidence of leukemia within the bone marrow
`(.5% blast cells) was seen in all patients before treatment.
`After treatment with one to three doses of the drug conjugate, 8
`(20%) of 40 patients had ,5% leukemic blast cells on
`morphologic examination of bone marrow aspirate and biopsy
`specimens (Table 3). In one of these patients (FH-007), blast
`cells were also eliminated from the bone marrow on a second
`occasion after retreatment with CMA-676 (see below).
`Recovery of normal blood counts. Recovery of blood
`neutrophil counts to greater than 1,500 cells/µL was observed in
`five of eight patients who had ,5% leukemic blasts by
`morphologic examination of bone marrow aspirate and biopsy
`specimens (Table 3). In addition, three of these five patients
`achieved normal platelet counts. The first patient, FH-007,
`achieved a complete hematologic and cytogenetic remission 7
`days after receiving the third dose of CMA-676 at 1 mg/m2.
`After 140 days, local leukemia recurrences in his femurs and
`iliac wing were identified by magnetic resonance imaging and
`confirmed by surgical biopsy. He was treated with conventional
`chemotherapy and local radiation and achieved another CR. He
`experienced relapse again 293 days after completing the first
`course of CMA-676, was treated with two additional doses of
`CMA-676 at 6 mg/m2, and his leukemia disappeared again for
`56 days. His leukemic cells expressed CD33 at the time of each
`of his relapses.
`The second patient, FH-012, achieved a complete hemato-
`logic and cytogenetic remission 35 days after the third dose of 4
`mg/m2. In an attempt to consolidate his remission, he was given
`an infusion of lymphocytes from his bone marrow donor 40
`days after infusion of the third dose of CMA-676. He remained
`
`in CR for 214 days after treatment until testicular and central
`nervous system relapses occurred.
`The third patient, FH-023, received three doses of CMA-676
`at 9 mg/m2, and his ANC recovered to .1,500/µL 42 days after
`he received the third dose of CMA-676. His platelet count
`reached a peak of 36 3 103/µL 23 days after he received the
`third dose of CMA-676, but the patient subsequently became
`platelet
`transfusion-dependent for approximately 3 weeks.
`Evidence of antibody on the patient’s platelet surfaces was
`documented. Megakaryocytes were present and platelet matura-
`tion appeared normal on examination of the marrow aspirate. In
`an attempt
`to consolidate his remission, he was given an
`infusion of lymphocytes from his bone marrow donor 54 days
`after receiving the third dose, and his platelet count eventually
`rose to .100 3 103/µL 106 days later. At the time of this report,
`he remained in continuous cytogenetic and hematologic remis-
`sion .623 days after receiving the third dose of CMA-676.
`Pharmacokinetics and saturation of peripheral CD33 sites.
`Detectable plasma levels of CMA-676 were identified in all
`patients immediately after intravenous administration and the
`half-life of CMA-676 was estimated to be 38 6 21 hours for
`patients receiving the 9 mg/m2 dose. Figure 2 shows the
`relationship between the total number of cell surface CD33 sites
`and CD33 sites bound with CMA-676 in peripheral blood cells
`for a characteristic patient over time. Thirty minutes after
`infusion, CD33 sites were almost completely saturated with
`CMA-676. Twenty-four hours after administration, fewer CD33
`sites and bound drug conjugate were evident, suggesting
`internalization of the CD33-immunoconjugate complex. In the
`patients who received the maximum dose (9 mg/m2), a median
`of 92.2% (range, 79.2% to 100%) of CD33 binding sites on
`peripheral blood blast-sized cells were bound by the drug
`conjugate 30 minutes after infusion of a dose. Peak CD33
`saturation levels in relation to treatment response are shown in
`
`IMMUNOGEN 2187, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
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`3682
`
`SIEVERS ET AL
`
`relationship between measured leukemic blast cell drug efflux
`levels and saturation of CD33 sites with response. Elimination
`of leukemia appeared to be correlated with a low capacity by
`leukemic blast cells to extrude DiOC2. For example, of the 30
`patients evaluated with the assay in whom doses of CMA-676
`saturated .75% of available CD33 sites on peripheral blood
`blast cells, 8 of 17 patients with leukemic blast cells that showed
`#40 channel numbers of DiOC2 efflux had ,5% blasts in the
`bone marrow after treatment. In contrast, none of the 13 patients
`with leukemic blast cells expressing .40 channel numbers of
`DiOC2 efflux entered remission.
`Immune responses. No humoral responses to anti-hP67.6
`antibody were detected. A humoral response to the calicheamicin-
`linker complex was documented in one patient after receiving a
`third dose of CMA-676 and in a second patient during
`retreatment with CMA-676.
`
`DISCUSSION
`
`This study describes the use of an antibody-drug conjugate
`capable of targeting and safely eliminating target leukemic cells
`in vivo. Disappearance of AML cells from the bone marrow and
`peripheral blood of eight patients with complete restoration of
`normal hematopoiesis in three patients was observed after
`treatment with antibody-targeted chemotherapy. These results
`show that eradication of CD331 AML cells can allow restora-
`tion of normal hematopoiesis by remaining CD332 precursors.
`For some patients, we hypothesize that the CD332 precursors
`are predominantly or completely nonmalignant. This is based
`on findings that, in some cases of AML, the clonal abnormality
`originated in either a committed progenitor or an early multipo-
`tent cell whose proliferative expression is mainly restricted to
`the granulocyte/monocyte lineage.12 Because selection of CD332
`precursors from some of these leukemias allowed normal
`hematopoietic growth in culture,
`the malignant clone may
`involve few, if any, CD332 precursors.5,6
`In other cases of AML, particularly in older patients, the
`clonal abnormality has been found in both the erythroid and
`myeloid lineages, showing malignant involvement of multipo-
`tent precursors.12 In addition, clonal karyotypic abnormalities
`have been found in primitive precursor cells from some AML
`patients.13,14 It is conceivable that normal hematopoiesis could
`also be restored in these patients even if a substantial portion of
`CD332 precursors were malignant because normal precursors
`express at least a short-term proliferative advantage. While this
`explanation appears inconsistent with the observed leukemic
`growth in a immunodeficient mouse model after infusion of
`isolated primitive (CD341 CD382) precursors from human
`leukemia specimens,15 it is consistent with the report that a
`patient undergoing allogeneic transplantation who was inadver-
`tently given an infusion of donor AML cells initially recovered
`with normal donor hematopoiesis.16
`Each patient whose blood counts returned completely to
`normal had experienced relapse after an allogeneic bone
`marrow transplant (data not shown). Therefore, it is possible
`that a graft-versus-leukemia effect eliminated the remaining
`CD332 leukemic cells after elimination of the bulk of AML
`cells. However,
`in a recently initiated phase II study of
`CMA-676, normal hematopoiesis was restored in some patients
`
`Fig 2. Relationship between total number and CMA-676–bound
`CD33 sites on peripheral blood cells in a characteristic patient over
`time. The solid line represents the number of CD33 sites available for
`binding to CMA-676 as estimated by the maximal fluorescence
`intensity obtained by incubating an aliquot of cells in vitro with
`excess CMA-676. The dashed line represents the fluorescence inten-
`sity of bound CMA-676 to cell surfaces. Near-complete saturation is
`seen 30 minutes after the start of the infusion.
`
`Fig 3. These results show that saturation of sites is not, by itself,
`sufficient to insure treatment response.
`Response correlation with drug efflux. We evaluated the
`efflux of DiOC2 from pretreatment leukemic blast cells as a
`measure of functional drug resistance. Figure 3 shows the
`
`Fig 3. Relation of leukemic blast cell dye efflux and maximum
`CMA-676 saturation of CD33 sites on peripheral blood blast-sized
`cells with treatment response (N 5 36). (m) Denotes patients who had
`F5% leukemic blasts by morphologic examination of bone marrow
`aspirate and biopsy specimens after treatment. (d) Denotes patients
`whose leukemia did not disappear. Peripheral blood samples from
`four patients were unavailable for analysis. The efflux from the
`dominant population is represented in the five instances in which
`efflux profiles were bimodal. *Leukemic cell specimens from patients
`FH-023 and FH-024 were 2 and 3 days old, respectively, at the time of
`efflux measurement. Because blast-sized cells from each showed
`uncharacteristically low DiOC2 loading, obtained efflux values may
`underestimate true efflux.
`
`IMMUNOGEN 2187, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
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`
`SELECTIVE ABLATION OF AML USING ANTIBODY-TARGETED CHEMOTHERAPY
`
`3683
`
`who had not received a transplant.17 Molecular remissions have
`been observed in a portion of patients with acute promyelocytic
`leukemia who received unconjugated anti-CD33 antibody
`(HuM195).18 Although an AML patient with 8% blasts in his
`bone marrow achieved a CR with HuM195 given at a supersatu-
`rating dose, complete clinical responses have not been reported
`in patients with large tumor burdens.19 Hence, the observed
`elimination of leukemia after treatment with CMA-676 was not
`likely due to antibody-mediated effects alone.
`Myelosuppression was the most clinically significant adverse
`event. The severe marrow hypoplasia observed in two patients
`who received 9 mg/m2 is consistent with depletion of CD331
`hematopoietic progenitor cells and with the time required for
`CD332 cells to restore hematopoietic function.20 To prevent
`prolo