Monoclonal Antibodies to Myeloid Differentiation Antigens: In Vivo
`
`Studies of Three Patients With Acute Myelogenous Leukemia
`
`By Edward D. Ball. George M. Bernier, Gibbons G. Cornwell Ill. 0. Ross Mcintyre, Joseph F. O'Donnell, and
`Michael W. Fanger
`
`However, the induction of human antibody to murine MoAb
`Three patients with acute myelogenous leukemia IAML) in
`relapse were treated with intravenous infusions of one or
`was observed in one patient who was treated over a
`70-day period. Toxicities encountered were minimal and
`more purified murine monoclonal antibodies (MoAbsl spe­
`included fever (3 patients). back pain (1 patient). and
`
`cific for differentiation antigens on normal and malignant
`arthralgias and myalgias (1 patient}. This is the first
`myeloid cells. Three of the MoAbs used were lgM immuno­
`reported clinical trial of (1) lgM MoAbs. (21 MoAb therapy
`
`globulins that react with glycolipids, while the fourth. an
`in patients with AML. (3) combinations of MoAbs directed
`lgG2b. reacts with a protein antigen. Peripheral blood
`
`toward different myeloid differentiation antigens. and (4)
`
`leukemia cell counts decreased significantly. but transient­
`
`MoAbs directed to glycolipids. The relative lack of toxicity
`ly, during treatment. Evidence of in vivo binding of each
`
`and the positive effects of MoAb treatment in the reduc­
`MoAb to leukemia cells was obtained. although two of the
`tion of leukemia cell counts permit the continued study of
`four MoAbs could not be detected in the plasma following
`
`more innovative approaches to the treatment of AML with
`
`infusion, perhaps due to circulating blocking factors. Anti­
`MoAbs.
`genic modulation was not encountered in these studies.
`
`SEVERAL INVESTIGATORS have reported in
`vivo treatment of human leukemias and lympho­
`mas with murine monoclonal antibodies (MoAbs)
`directed to a variety of tumor-associated antigens. •-5
`Patients with acute lymphocytic leukemia (ALL),1
`chronic lymphocytic leukemia (CLL),2 T-cell lympho­
`mas,1 and nodular lymphocytic lymphoma4·5 have all
`been treated in this manner, with variable results.
`Several obstacles to such therapy have been identified,
`including temporary internalization (modulation) of
`the cell surface antigen targeted for therapy 1•6 and
`circulating blocking factor(s).2-5 While most trials of
`MoAb therapy have produced only temporary
`decreases in tumor cell counts, toxicity has been mini­
`mal. Of interest is the report of the successful induc­
`tion of complete remission in a patient with a 8-cell
`nodular lymphocytic lymphoma using a monoclonal
`antibody directed to the idiotype expressed on the
`patient's tumor cells.5
`We have recently prepared four MoAbs reactive
`with different normal myeloid differentiation antigens
`that are also expressed on leukemia cells from patients
`with acute myelogenous leukemia (AML).1-9 In this
`report, we describe the treatment of three patients with
`acute myelocytic leukemia (AML) with these MoAbs.
`These studies were conducted to determine ( I ) the
`safety of MoAb administration in patients with AML,
`(2) changes in concentrations of circulating blasts and
`polymorphonuclear cells (PMNs), (3) the presence of
`free and cell-bound MoAb after therapy, (4) rate of
`clearance of circulating MoAb, and (5) the presence of
`factor(s) in patient and normal plasma that block
`MoAb binding to cells. Three of the antibodies
`employed in this study are lgM and react with glyco­
`lipids. To our knowledge, this article describes the first
`trial of in vivo MoAb treatment with lgM MoAbs, the
`
`first report of human MoAb treatment targeted to
`glycolipids, and the first treatment of AML with
`MoAb directed to myeloid differentiation antigens.
`The lack of serious toxicity with these reagents permits
`numerous future applications of these and other
`MoAbs.
`
`MATERIALS AND METHODS
`
`Patient Selection
`Patients with AML who were refractory to standard chemother­
`apy or who were poor risks for further chemotherapy were consid­
`ered for MoAb treatment. Treatment of patients with MoAb was
`approved by the Committee for the Protection of Human Subjects.
`and all patients gave informed consent for all aspects of the
`therapeutic protocol.
`
`Case Reports
`Pa1ient I. R.B. was a 27-yr-old white female who developed
`AML in September 1981. At that time. her WBC was 8.600/ µI with
`2% blasts, and her bone marrow was hypcrcellular. with 15% blasts
`and 8% promyelocytes. Auer rods were present, and she was
`classified as FAB type M2.'0 She achieved a complete remission
`(CR) with cytosine arabinoside (Ara-C). daunorubicin (DNR). and
`6-thioguanine (6-TG). but relapsed in February 1982. A second
`
`From the Departments of Medicine and Microbiology, Dart­
`mouth-Hitchcock Medical Center. and the Norris Cotton Cancer
`Center. Hanover. NH.
`Supported in part by Grants CA 31918. CA 23108. CA 31888.
`and Al 19053 awarded by the National Cancer Institute and the
`Institute of Allergy and Infectious Diseases. DHHS. respectively,
`and by the York Cross of Honour Research Foundation. The
`cytojfuorograph was the generous gift of the Fannie E. Rippel
`Foundation and is partially supported by the core grant of the
`Norris Cotton Cancer Center (CA 23108).
`Submitted April 25, /983;accepted July 9, 1983.
`Address reprint requests to Dr. Edward D. Ball. Department of
`Microbiology. Dartmouth Medical School. Hanover. NH 03756.
`© 1983 by Grune & Stratton. Inc.
`0006-4971 /83/6206-()()()9$01.00/0
`
`Blood, Vol. 62, No. 6 (December), 1983: pp. 1203-1210
`
`1203
`
`1 of 9
`
`BI Exhibit 1009
`
`

`

`1204
`
`BALL ET Al.
`
`rem1ss1on was attained with intermediate doses of Ara·C and
`L·asparaginase.11 but the patient relapsed and MoAb therapy was
`started.
`Patient 2. P.S. is a 65-yr-old white male who presented in
`February 1981 with fatigue, sore throat, and a platelet count of
`12,000/µI. Bone marrow was diagnostic of AML (FAB MI), and his
`leukemic cells exhibited trisomy No. 8 on chromosomal analysis. He
`achieved a CR with Ara-C, DNR, and 6-TG. but relapsed in
`January 1982. Following reinduction with intermediate dose Ara-C,
`transient aplasia complicated by bacterial and fungal sepsis
`occurred, but leukemic cells reappeared. In May 1982, he was
`treated with cis-retinoic acid (provided by Hoffmann-LaRoche, Inc.,
`Nutley, NJ) in the hope of inducing differentiation. He developed
`significant cutaneous toxicity. no improvement in his peripheral
`counts, and increasing transfusion dependency. He was begun on
`MoAb therapy in September 1982.
`Patient 3. L.S. was a 65-yr-old white male who was well until
`September 1981, when he developed fever. adenopathy, and a rash.
`At that time, he was found to have a WBC of 46,000/µI, with 48%
`blasts and a bone marrow consistent with acute myelomonocytic
`(FAB M4) leukemia. A CR was attained with Ara-C and DNR, and
`he was maintained with Ara-C, DNR, 6-TG, and vincristine. In
`June 1982, he had a biopsy-proven recurrence of leukemia in the skin
`of his right buttock and posterior thigh. At that time, his peripheral
`blood and bone marrow were normal, and he responded to 1,500 rad
`of radiation therapy. In October 1982. he developed diffuse arthral­
`gias and increasing cutaneous nodules of the face and neck, which
`biopsy showed to be leukemic. His bone marrow remained normal.
`and he was treated with MoAbs.
`
`Monoclonal Antibodies
`The MoAbs used in this study included PMN 6. PMN 29. and
`PM-81. all lgM class immunoglobulins. and AML-2-23. an lgG2b
`MoAb (Table I). PMN 6 and PMN 29 react with mature granulo­
`cytes. while PM-81 and AM L-2-23 react with both granulocytes and
`monocytcs. Each of these MoAbs are cytotoxic in the presence of
`rabbit serum to normal and leukemic myeloid cells.1·• None of these
`four MoAbs arc reactive with the colony-forming unit-granulocytc/
`monocyte (CFU-GM) or burst-forming unit-erythroid (BFU-E).9
`PMN 6 and PMN 29 react with blast cells from patients with AML
`of the M4 subtype. PM-81 with all subtypes of AML. and AML-
`2-23 with the M4 and M5 subtypes.•·• An lgM and an lgG2b MoAb
`of irrelevant specificity were used as controls for in vitro studies.
`
`Antibody Purification
`Hybridoma cells producing each of the above antibodies were
`grown in the peritoneal cavity of pristane-primed BALB/c mice.
`Ascitic fluid was collected aseptically and pooled. lgM MoAbs were
`purified by gel filtration through Sepharose CL-6B (Pharmacia Fine
`Chemicals. Piscataway, NJ) using sterile pyrogen-free phosphate­
`buffered saline (PBS) (pH 7.4) as running buffer. AML-2-23 was
`purified on a protein·A-Sepharose column by elution at pH 3.5_11
`All antibody preparations were passed through a 0.2-µ filter and
`stored at 4°C. Cultures for aerobic and anaerobic bacteria and fungi
`
`Teble 1. Monoclonal Antibodies Employed in Thia Study
`
`Specificity
`
`MoAb
`PM-81
`AML-2-23
`PMN 29
`PMN6
`
`lsoiype
`
`AML
`Normal Cells Subelass Ref ere.,.,.
`8
`7.9
`7,9
`7.9
`
`Granulocyte/monocyte M1·M5
`lgM
`lgG2b Monocyte/granulocyte M4-M5
`lgM
`M4
`Granulocyte
`lgM
`G<anulocyte
`M4
`
`were negative. Endotoxin assays, performed by the Limulus amoeba­
`cyte lysate assay (Associates of Cape Cod, Inc .• Woods Hole, MA),11
`consistently revealed less than 2 ng endotoxin/mg MoAb.
`
`Cell Separations
`Leukemia cells from patients treated in this study and cells from
`normal volunteers were harvested from the peripheral blood as
`previously described." Leukemia cells were separated by Ficoll·
`Hypaque gradient centrifugation. Normal polymorphonuclear leu­
`kocytes (PMNs) were harvested by sedimentation of Ficoll­
`Hypaque dense cells in 2% dextran.
`
`Antibody Binding Assays
`Binding of MoAbs to leukemic and normal cells was determined
`by indirect immunofluorescence and flow cytometry. Target cells
`(2 x I 06} were incubated with 50 µl of purified MoAb at 20 µg/ml
`for 30 min at 4°C, followed by the addition of a fluorescein
`isothiocyanate (FITC) coupled goat F(ab')i antibody directed to
`mouse immunoglobulin (FITC-GAM) (Boehringer-Mannheim,
`Indianapolis, IN) for another 30 min at 4°C. The cells were then
`analyzed for fluorescence on the Ortho (Westwood, MA) Cytofluor­
`ograph System 50H, with multichannel distribution analyzer 2103
`and Ortho 2150 computer systems. Positive binding was defined as
`fluorescence on MoAb-reacted cells greater than on control MoAb­
`reacted cells.
`
`Blocking Studies
`Plasma from leukemic patients and normal controls was studied
`for the presence of blocking factors that might interfere with the
`binding of MoAb to cell surfaces. Plasma was incubated with equal
`volumes of varying concentrations of purified MoAb for 15 min at
`4cc prior to addition of this mixture to target cells. Following
`incubation at 4°C for 30 min. FITC-GAM was added for an
`additional 30 min at 4°C. Cells treated in this manner were then
`analyzed by flow cytometry.
`
`Determination of Patient Antibody Production to
`Mouse Antibody by Enzyme-linked lmmunosorbent
`Assay (ELISA)
`MoAbs PM-81. PMN 29. and AML-2-23 were applied to individ­
`ual wells of 96-well flat-bottom plates at 20 µg/ml in PBS and the
`plates incubated at room temperature (RT) overnight. The plates
`were washed with PBS and incubated with 5% bovine serum albumin
`(BSA) in PBS for I hr at 37°C. Control wells were treated with 5%
`BSA only. After washing, serially diluted normal and patient plasma
`(pre-, intra-. and posttreaiment) were applied in duplicate to MoAb­
`treated and control wells. The plates were incubated for 2 hr at 37°C
`and washed with PBS. Alkaline phosphatase-labeled affinity­
`purified goat anti-human antibodies specific for either lgM or lgG
`(Kirkegaard & Perry Labs. Inc., Gaithersburg. MO) were added
`and the plates incubated at RT for 16 hr. The plates were again
`washed and p-nitrophenyl phosphate disodium (Sigma Chemical
`Corp., St. Louis, MO) was applied. The plates were observed for the
`appearance of yellow color, and the optical density (OD) at 405 nm
`of each well was determined on an ELISA reader after 15 min. The
`mean OD of duplicates of each serum dilution assayed against BSA
`alone (background) was subtracted from that of the same dilution
`assayed against MoAb.
`
`Monoclonal Antibody Administration
`Purified MoAb was administered by intravenous infusion. The
`dose to be administered was diluted into a final volume of 500 ml of
`
`2 of 9
`
`BI Exhibit 1009
`
`

`

`MONOCLONAL ANTIBODY THERAPY OF AML
`
`1205
`
`Teble 2. Binding of MoAb to Leukemie Cella From AML Petienta•
`MonocloMI Antibody (% Cells Positive)
`
`Patient
`PM-81
`(FABClassl
`86 (41)
`1(M2)
`0(01
`0(0)
`O(Olt
`75 (991
`11 (7)
`2(M1)
`22 (20)
`59 (61)
`54 (110)
`70 (1101
`94 (135)
`3(M4)
`92 (1201
`•Binding was determined by indirect immunofluoreseence and flow
`cytometry. MoAb-treated cells were considered positive when fluores·
`cence exceeded the backg:ound level detected on cells treated with
`control MoAb.
`tThe number in parentheses is the mean fluorescence intensity (MF I)
`of MoAb-treated cells corrected for the MFI of control MoAb-treated
`cells.
`
`normal saline containing 5% human albumin. Following preparation
`of the final solution to be infused into the patient, culture for bacteria
`and fungi was performed. Solutions of antibody were usually admin­
`istered over 8 hr. while the patient was being monitored closely for
`changes in vital signs or symptoms of allergic reaction. The duration
`of infusion in patient 2 was 24 hr on one occasion and 7 days on
`another (sec Fig. 3). In order to minimize potential allergic reac­
`tions. patients 2 and 3 were premedicated with 50 mg diphenhy­
`dramine hydrochloride and 100 mg hydrocortisone immediately
`before each MoAb infusion.
`
`Rationale for MoAb Dose and Infusion Rate
`The dosage of MoAb was initially calculated based on the amount
`of MoAb that saturated a given number of leukemia cells in vitro
`and an estimate of the patient's leukemia cell burden. We infused
`each dose over 8 hr because other investigators had noted less
`toxicity with slow compared to rapid infusions.2 We modified this
`approach in patient 2 after the first series of infusions revealed
`favorable effects on the blast cell counts. with little toxicity. There­
`fore. we increased both the amount and the duration of the MoAb
`infusion in an effort to produce a more sustained depression in the
`blast count.
`
`Study Parameters
`
`Patients treated with MoAbs were observed for changes in the
`white blood cell and differential counts, hemoglobin. platelet count.
`prothrombin time. partial thromboplastin time. urinalysis, serum
`creatinine. alkaline phosphatase, glutamic oitaloacetic transaminase
`(GOT). glutamic-pyruvic transaminase (GPT), lactic dehydrogen­
`asc (LOH). and isocn7.yme profile and 5' nucleotidase.
`Free MoAb in the serum of patients during and after treatment
`was measured by incubating PMNs sequentially with plasma sam­
`ples and FITC-GAM before. during. and after therapy, and studied
`by now cytometry. Blast cells from patients treated with MoAb were
`harvested at various times during treatment and studied for the
`presence of cell surface mouse antibody by adding FITC-GAM.
`Aliquots of the same cell preparations were treated in parallel, with
`the addition of purified MoAb followed by the FITC-GAM and flow
`cytometric analysis to quantify free binding sites for MoAb.
`A skin nodule biopsy was performed on patient 3 (who had
`leukemia cutis) during the infusion of the AM L-2-23 antibody. Cells
`from the skin nodule were dissociated by forceps and passed through
`a steel screen to make a single-cell suspension. Aliquots of these cells
`were then incubated either with FITC-GAM or with purified
`AML·2-23 antibody followed by FITC-GAM and studied by flow
`cytometry.
`
`RESULTS
`
`Monoclonal Antibody Binding
`The results of pretreatment cytofluorographic anal­
`ysis of MoAb binding to the leukemia cells from the
`patients treated in this study are shown in Table 2. The
`leukemia cells of patient 1 reacted strongly with the
`PM-81 MoAb but not with PMN 6, PMN 29, or
`AML-2-23. The cells of patient 2 reacted strongly with
`PM-81 and, to a lesser extent, with PMN 29. At the
`time of this analysis, significant numbers of more
`differentiated cells (promyelocytes and myelocytes)
`were present, which probably accounted for the rela­
`tively strong reaction of PMN-29 with these cells.
`
`PMN6
`
`PMN 29
`
`AML·2·23
`
`Blast cells from patient 3 obtained at his first relapse
`reacted with all four of the MoAbs. Binding studies of
`PM-81 to normal PMNs and blast cells revealed that
`both cell populations (2 x 106 cells, respectively)
`showed saturable binding at approximately 3 µg/ml of
`the PM-81 MoAb.
`
`Effects of Treatment
`Patient I
`Following the first infusion (60 mg of PM-81 over 8
`hr), the peripheral blood blast cell count fell from
`54,000/µI to 22,000/µI (Fig. 1). Twelve hours later,
`however, the blast cell count returned to 48,000/ µI.
`The next administration of PM-81 (60 mg) produced a
`depression in the blast count to 19,000/ µI. Again,
`however, the blast count rose to 43,000/ µI within 12 hr
`and to 55,000/ µI 24 hr later. While more antibody was
`being prepared, the patient was treated with DNR (25
`mg/sq m), which decreased the blast count to 5,000/ µI
`before it began to rise. On day 1 O. a 30-mg dose of
`PM-81 was administered, and her blast count fell from
`
`- - DNR
`+
`
`- -
`
`$!
`• 40
`§.. ..
`
`z
`:::>
`
`0 0 20 _, _, Ill 0
`
`2
`
`I
`
`5
`
`4
`DAYS
`
`10
`
`11
`
`12
`
`Fig. 1. Circulating blast count in petient 1 following MoAb
`PM-81 infusion. The peripherel blood blaat cell count ia plotted
`epinat time in relation to infusion of PM-81 MoAb <•I. The
`emountl of PM-81 infused wMe 60. 60. 30. end 20 mg. respec­
`tively. during uch of the indicated trNtmenta. A dose of DNR (85
`mg) wH given on day 6. The blast cell count decreHed to 9,000/ µI
`by day 8 end remained et this level for the 3 deya prior to the next
`MoAb infusion.
`
`3 of 9
`
`BI Exhibit 1009
`
`

`

`1206
`
`BALL ET AL.
`
`:-coN
`
`fl C ,_..- In rlro BINDING
`t1
`i:,
`1:
`
`FLUORESCENCE
`INTENSITY
`
`Fig. 2. Binding of MoAb PM-81 to bleat cella from patient 1
`obtained during PM-81 infusion. Bleat cell• were aepareted by
`Ficoll-Hypaque gradient centrifugation end incubated either with
`FITC-GAM alone (in vivo binding) or with aeturating amounts of
`PM-81 followed by FITC-GAM (in vitro binding) end analyzed on
`the cytofluorogreph. Cella (CON) obteined before therapy were
`incubated with control lgM MoAb end FITC-GAM end compared to
`cells reacted with PM-81 MoAb. Evidence of in vivo binding is seen
`from this enelyaia. although aeturetion of ell PM-81 binding sites
`was not achieved. The degree of binding of control cells incubated
`with PM-81 (not ahownl is identical to thet of cells exposed in vivo
`to PM-81. end these in vitro binding curves ere auperimpoaeble.
`
`9,000/ µI to 5,000/ µI. Another dose of PM-8 1 (20 mg)
`produced a modest decrease in white count to 4,000/
`µI. Due to the patient's grave clinical condition. which
`included probable sepsis. the patient and her physi­
`cians elected to withhold any further therapy, and she
`expired I wk later.
`During the first infusion of the PM-8 1 antibody to
`patient I, she developed an urticaria) eruption on her
`legs. which responded promptly to a single dose of
`diphenhydramine hydrochloride (50 mg i.v.) and
`hydrocortisone (I 00 mg i. v.). Prior to the next three
`infusions, she was premcdicated with these drugs and
`experienced no further skin reactions or other adverse
`effects. with the exception of mild febrile reactions.
`
`Blast cells harvested al I hr and 5 hr during the first
`MoAb infusion and I hr after completion of the
`infusion revealed detectable amounts of the PM-8 1
`antibody bound to leukemia cells. However, addition of
`saturating amounts of PM-81 in vitro showed that only
`about 25% of available antigenic sites had been occu­
`pied in vivo (Fig. 2). Plasma obtained I hr after the
`infusion was completed revealed high levels of free
`PM-81 antibody, as determined by the ability of this
`patient's plasma to bind to normal PMNs in vitro.
`These studies suggested that antigenic modulation did
`not occur during the period of infusion of the PM-8 1
`MoAb, since the number of antigenic sites was
`unchanged on cells exposed to PM-8 1 in vivo compared
`to pretreatment cells. Similar results were obtained
`during the subsequent infusions, though the percent­
`age of cells demonstrating in vivo antibody binding
`during the third and fourth infusions was lower than
`that observed in the first and second infusions.
`
`Patient 2
`The first treatment involved a series of infusions of
`PMN 6, PMN 29, and PM-81 MoAbs; the second
`treatment consisted of PMN 29 and PM-81; while the
`final treatment involved the use of PM-81 only (Fig.
`3). Al the onset of therapy, the patient had a white
`blood cell count of 18,000/ µ),with 22% blasts and 50%
`myeloid cells at various levels of maturation. The
`treatment plan was to use two different MoAbs (PMN
`6 and PMN 29) to remove more mature myeloid cells
`before using PM-81, which reacted more strongly with
`the patient's blast cells. Infusion of PMN 6 resulted in
`a drop of the mature myeloid cell (PMNs, bands,
`myelocytes, and promyelocytes) count from 9,000/ µI
`
`Cl 1!21 -
`
`1!21•-
`
`-----·---·
`
`CJ• PllN •
`ell• PllN 29
`•. ,. .... ,
`e • IL.UT CELLS
`0 • MATURE llYELOID CELLS
`
`...
`
`20
`
`11
`
`•
`
`12
`
`� �
`
`z
`�
`
`0 � I
`
`....
`...
`u
`
`..
`
`2
`
`3
`
`.. �5 11
`
`11 20
`DAYS
`Fig. 3. Circulating blast end mature myeloid cell counts in patient 2 during MoAb treatment.
`
`17
`
`11
`
`4 of 9
`
`BI Exhibit 1009
`
`

`

`MONOCLONAL ANTIBODY THERAPY OF AML
`
`1207
`
`to 5,000/ µI. The following day, treatment with PMN
`29 produced a fall in the mature myeloid count from
`7 ,400 /µI to 1,600 /µI and a decrease in the blast count
`from 3,400/µI to 1,400/µ1. On the third day, PM-81
`produced a fall in the mature myeloid count from
`6,600/µl to 400/µl and a fall in the blast count from
`5,300/ µl to 1,400/ µI. A return to pretreatment levels
`occurred within I day. Cells from a bone marrow
`aspirate performed on day 3 revealed no measurable
`cell-bound MoAb. During this treatment course, toxic­
`ity was limited to complaints of back pain during
`infusion of the PMN 6 antibody, which diminished as
`the rate of infusion was decreased.
`On day 15, infusion of PMN 29 ( 60 mg over 8 hr)
`decreased the mature myeloid count from 12,000/ µl to
`4, I 00/ µI and the blast count from 4,300/ µl to 2,200/
`µI. PM-81 was then administered over 8 hr (60 mg)
`and 24 hr (210 mg). The blast count remained in the
`range of 1-2,000/µl, while the mature myeloid count
`dropped to 0 and remained low for nearly 24 hr. No
`toxicity, other than mild fever, was observed during
`this treatment. In view of the prolonged effect on
`reduction of the blast cell count, a larger dose ( 600 mg)
`of PM-81 was given by continuous infusion (4 mg/hr)
`over 7 days, starting on day 62 (with interruptions for
`blood transfusions) (Fig. 3). A fresh bottle of MoAb
`was prepared daily for this longer infusion. The mature
`myeloid count fell from 17,300/µl to 180/µl and the
`blast count from 4,500/µl to 1,800/µl. On day 65,
`when the infusion had been stopped for blood transfu­
`sion, major increases in both mature myeloid and blast
`counts were noted. but these counts fell again upon
`resumption of the infusion. On day 68, after therapy
`
`was stopped, the blast count peaked at 11.400/ µI
`before falling again to the pretreatment level. The
`serum LOH concentration tripled (200-600 IU/liter,
`isoenzymes 2 and 3) during each of these treatments.
`During the infusion of PM-81, MoAb could not be
`detected in the patient's plasma or bound to cells from
`the peripheral blood or bone marrow. On day 64, a
`bolus of 1251-labeled PM-81 (0.25 mg PM-81, 7 x 108
`cpm/mg) was injected and serial measurements of
`plasma and cell-bound counts were performed. Rapid
`decrease in free plasma radioactivity occurred (T1/2 -
`3.5 hr}, with plasma counts still detectable at 24 hr but
`not at 48 hr. Peripheral blood cell-bound radioactivity
`was detectable only immediately after infusion of the
`1251-labeled PM-81.
`
`Patient 3
`This patient was treated with MoAbs PMN 29 and
`AML-2-23. Since this patient had no circulating leu­
`kemia cells at the time of therapy and only a small
`percentage of blasts in the bone marrow, we monitored
`normal blood cell counts and investigated the ability of
`the AML-2-23 antibody to bind to leukemia cells in
`the patient's leukemic skin nodules. Reductions in the
`peripheral blood neutrophil count and monocyte count
`occurred during infusion of MoAb AML-2-23 (60 mg
`over 12 hr) (Fig. 4). Infusion of PMN 29 (70 mg over
`12 hr) produced a decline in the neutrophil count to
`500/µI. Several hours later, infusion of AML-2-23 (60
`mg over 12 hr) had no effect on the blood cell counts.
`Unbound PMN 29 was detectable in plasma following
`the PMN 29 infusion, but free AML-2-23 was not
`present during or after the infusion. Serum LOH
`
`800
`
`800
`
`r-0
`
`
`%
`400=
`c:
`.......
`
`200
`
`-
`
`c:::::i-
`
`D• PMN 29
`•• AML·2·23
`---• LDH
`
`5
`
`..
`0 ...
`)( 4
`
`,'
`,'
`,'
`, ,
`,
`I
`I
`I
`I
`I
`I
`I
`I
`·/
`
`Fig. 4. Circulating blood cell counts end serum LOH
`levels in patient 3 during MoAb treatment. The effect of
`treatment on PMNs (0) end monocytH l•I is plotted in
`relationship to each MoAb infusion.
`
`1
`
`2
`
`3
`
`5
`
`e
`
`7
`
`..
`DAYS
`
`5 of 9
`
`BI Exhibit 1009
`
`

`

`1208
`
`too
`
`BALL ET AL.
`
`Fig. 5. Effect of in vitro incuba­
`tion of pleama from patienta 1-3 on
`binding of MoAb PM-81 to PMNa.
`Plasma aamplH obtained before
`MoAb treatment from patient 1 (01.
`patient 2 (&). end patient 3 ( 0 I end
`from • normal donor ( 0 I were incu­
`bated with varying concentration• of
`PM-81. The binding of these mix­
`tures and purified PM-81 (/\) wH
`detected by the addition of FITC­
`GAM end the percentage of positive
`cells determined by cytoftuorogre­
`phy.
`
`Pll011 IJ.lllfmtt
`
`'
`
`to
`
`concentrations (isoenzymes 2 and 3) increased from
`300 to 800 IU/litcr after MoAb treatment (Fig. 4).
`Removal of one of the patient's leukemic nodules
`during the last infusion and isolation of these cells in
`vitro revealed that delectable levels of leukemia cell­
`bound MoAb AML-2-23 were present. However, the
`binding sites were well below saturation (approxi­
`mately 20% of saturation). During infusions of both
`antibodies, the patient experienced myalgias, arthral­
`gias. and fever. none of which necessitated discontin­
`uation of therapy. During treatment with AML-2-23,
`microscopic hematuria was noted. However, no change
`in the scrum crcatininc or BUN occurred.
`
`Evidence of Plasma Inhibitors of MoAb Binding
`
`
`to Cells
`
`Plasma samples from the patients treated in this
`study were found to have varying amounts of PM-81
`inhibitors (Fig. 5). Binding studies of purified PM-81
`in the presence and absence of pretreatment plasma
`from patient 2 indicated that 2-10 µg/ml of PM-81
`was neutrali7.cd by a plasma substance. while plasma
`from patients I and 3 and normal donors had no
`blocking activity. Plasma samples obtained during the
`treatment of patient 2 with PM-81 did not block
`PM-81 binding in vitro and did not contain detectable
`
`amounts of free PM-81 (as determined by binding to
`PMNs in vitro). These findings provided presumptive
`evidence that soluble factors and cell-bound antigen
`were capable of complexing all of the infused PM-81
`MoAb. In contrast, the plasma of patient 1, which did
`not contain inhibitors of PM-81, did have free MoAb
`following treatment with PM-8 I.
`Analysis of plasma samples obtained from patient 3
`before and after an infusion of AML-2-23 revealed
`that the quantity of AML-2-23 blocking factor
`increased after the infusion (Fig. 6). Free plasma
`AML-2-23 MoAb was not detectable immediately
`after a 60-mg infusion. These results also suggested
`that soluble and cell-bound antigens (on skin leukemia
`cells) were capable of binding all of the infused MoAb.
`Plasma from patient 2 and from normal donors did not
`contain significant levels of blocking factors for
`MoAbs PMN 29 and PMN 6 (data not shown)­
`findings that are consistent with our ability to detect
`free MoAb PMN 6 and PMN 29 in the circulation of
`this patient after infusion of these MoAbs.
`
`Patient Antibody Production to Mouse lg
`
`Plasma from patient 2 was found to contain human
`antibody of the IgG class reactive with murine MoAb,
`first noted on day 62 of therapy. The titer (defined as
`
`Fig. 6. Effect of in vitro incubation of
`plasma from patient 3 on binding of MoAb
`AML-2-23 to PMNa. Plasma samples
`obtained before (day 2. •I end during
`AML-2-23 infusion (day 4. O. and day 5. /\I
`and from • normal donor (01 were incu­
`bated with varying concentrations of
`AML-2-23. The binding of thHe mixtures
`and purified AML-2-23 alone l•I to normal
`PMNs was then detected by the addition
`of FITC-GAM. end the percentage of posi­
`tive cells was determined by cytoftuorog­
`rephy.
`
`100
`
`w 80
`> �
`iii
`0 60
`....
`I/)
`_,
`_,
`w 40
`(,)
`�
`
`0.2
`
`0.5
`
`2
`AML·2·23 ( ftglmll
`
`10
`
`6 of 9
`
`BI Exhibit 1009
`
`

`

`MONOCLONAL ANTIBODY THERAPY OF AML
`
`1209
`
`the dilution that gave 50% maximal binding) of this
`antibody increased from 1: 100 on day 62 to 1: 1,600 on
`days 73 and 77. This antibody reacted similarly with
`lgM MoAb PMN 29 and the control lgM MoAb, but
`did not react with lgG MoAb AML-2-23, suggesting
`that the patient's antibody is directed mainly towardµ
`chain.
`
`DISCUSSION
`
`These studies represent the first reported clinical
`trials of (I) lgM class MoAbs, (2) MoAb therapy in
`patients with AML. (3) combinations of MoAbs
`directed toward different myeloid differentiation anti­
`gens. and (4) MoAbs directed to glycolipid antigens.
`No significant toxicity was observed in any of the
`patients. Perhaps more importantly, the reduction in
`circulating leukemia cell count and the serum rise of
`intracellular enzymes indicated that leukemia cell kill
`was achieved in these patients.
`MoAb therapy in mice has demonstrated that lgG
`(but not lgM) antibodies lead to an enhanced ability of
`phagocytic cells to mediate leukemia cell lysis, 16•17
`probably through antibody-dependent cellular cyto­
`toxicity (ADCC) or by opsonization of target cells.
`Although it is possible that, in the present study,
`MoAb coating led to transient sequestration of leu­
`kemic cells in extravascular sites, the depression of
`peripheral blood neutrophil and blast cell counts con­
`comitant with a rise in leukocyte L D H isoenzymes
`indicate that myeloid cell lysis did occur in vivo. The
`mechanisms of this cytolysis are at present unclear.
`They probably do not include direct complement­
`mediated lysis, since the MoAbs employed in this
`study did not mediate significant cell lysis in vitro
`using human complement. Furthermore, there is no
`evidence for the existence of lgM-specific Fe receptors
`on human neutrophils or monocytes.18 On the other
`hand,
`lgM MoAb-mediated cytolysis may occur
`through fixation of C3b to complement receptors on
`phagocytic cells in the absence of activation of the
`subsequent complement components. Alternatively, or
`in addition. since these MoAbs bind to normal phago­
`cytic cells as well as leukemia cells, bridging of target
`and effector cells might have occurred without the
`participation of complement or Fe receptors.
`With the introduction of the concept that tumors
`may issue from a pool of proliferating and self­
`renewing cells representing only a fraction of the
`tumor.19 and the increasing evidence that various fac­
`tors may influence the rate of proliferation and degree
`of differentiation in cells that have emerged from this
`pool. our findings gain greater significance. Thus,
`although it would be desirable to eliminate or greatly
`reduce the size of the tumor stem cell pool (as presum­
`ably occurs in that fraction of patients achieving
`
`long-term remissions or cures with chemotherapy), it is
`possible that treatments eliminating cells at stages
`beyond the stem cell level could reduce the disease
`morbidity or could induce kinetic events in the stem
`cell pool, thereby rendering it more sensitive to chemo­
`therapy.
`Following infusion of MoAbs, in vivo binding to
`leukemia cells, although subsaturating, was demon­
`strated in each patient. Moreover, in patient I, the
`inability to saturate leukemia cells occurred despite
`the presence of 0.5- 1 µg/ml of free PM-81, suggesting
`the existence of different subpopulations of leukemia
`or normal myeloid cells with diverse affinities for the
`antibody. Differences were demonstrated between
`patients in the quantity of free plasma MoAbs subse­
`quent to