`THE JOURNAL OF lMMUNOLOGY
`Copyright 0 1982 by The American Association of Immunologists
`
`Vol. 126, No. 3. March 1982
`Pnnted In U S.A.
`
`STUDIES ON THE ABILITY OF MONOCLONAL ANTIBODIES TO SELECTIVELY MEDIATE
`COMPLEMENT-DEPENDENT CYTOTOXICITY OF HUMAN MYELOGENOUS LEUKEMIA
`BLAST CELLS’
`
`EDWARD D. BALL,’ JAMES M. KADUSHIN, BERNICE SCHACTER, AND MICHAEL W. FANGER
`From the Department of Microbiology, Dartmouth Medical School, Hanover, NH 03755 and the Department of Pathology, University Hospitals
`of Cleveland, Case Western Reserve University, Cleveland, O H 44 106
`
`A panel of monoclonal antibodies that bind to leukemic
`finding was that the antigens determined by these antibodies
`blast cells from patients with acute myelocytic leukemia
`were found to some extent on all cells tested, both normal and
`and chronic myelocytic leukemia in blast crisis was stud-
`leukemic. Even so, we have determined that some of these
`ied for their ability
`to mediate complement-dependent
`antibodies can permit complement-mediated lysis of leukemic
`lysis of a variety of cell populations from patients with
`myeloblasts but not normal cells. This paper describes in detail
`leukemia, normal blood cells, and human leukemia cell
`the patterns of cytotoxicity of 20 different monoclonal anti-
`lines. Several of these monoclonal antibodies were selec-
`bodies to cells from patients with leukemia, normal individuals,
`(AML-1-99,
`tively cytotoxic to myeloid leukemia cells
`and human leukemia cell lines. Several of these monoclonal
`AML-1-211, AML-2-30, CML-75, CML-115, and CML-150).
`antibodies appear exclusively cytotoxic to myeloid leukemia
`Although they were all capable of binding to normal cell
`cells, whereas others mediate lysis of myeloid leukemia cells
`populations, none of these hybridomas were cytotoxic to
`as well as subpopulations of normal cells. The potential clinical
`normal cells. Three of these antibodies (AML-1-211, CML-
`utility of this panel of monoclonal antibodies is considered.
`75, and CML-150) were cytotoxic to some leukemia cell
`samples only after dilution of the hybridoma supernatant,
`i.e., they showed a prozone. Binding of these three anti-
`bodies, as well as another, AML-1-201, as determined in
`a radioimmunoassay, also showed a prozone. Other mon-
`oclonal antibodies are described (AML-2-23 and AML-2-
`9) that mediate complement-dependent cytotoxicity to
`myeloid leukemia cells as well as selected normal cell
`types (monocytes and lymphocytes, respectively). The
`potential clinical utility of these monoclonal antibodies is
`considered in the context of recently encountered prob-
`lems in the use
`of monoclonal antibodies to mediate
`leukemia cell lysis in vivo.
`
`MATERIALS AND METHODS
`
`Hybridomas. Details of the production of these hybridomas and partial
`characterizations of the monoclonal antibodies are reported el~ewhere.~
`Briefly, monoclonal antibodies were developed by immunizing BALB/c mice
`with human myelogenous leukemia cells and fusing spleen lymphocytes
`with cells from the P3-XAg63 murine myeloma cell line by using polyethylene
`glycol (m.w. 1000, J. T. Baker Chemical Co., Phillipsburg, NJ) according to
`the method of Kohler and Milstein (2). A panel of 20 different monoclonal
`antibodies were included in this study. All were IgM immunoglobulins except
`AML-2-23, which is an IgG of the y, subclass. The hybridomas were grown
`in Dulbecco’s minimal essential medium (DMEM) supplemented with 20%
`fetal calf serum (FCS). 2 mM L-glutamine, 5 mM HEPES4 buffer, and
`gentamicin, 50 pg/ml. All medium components were obtained from K. C.
`Biologicals, Kansas City, MO. except gentamicin (Schering Corp., Kenil-
`worth, NJ).
`Cells. Leukemic cells were obtained from patients with AML, acute
`lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and CML
`in blast crisis, and were separated from blood by Ficoll-Hypaque gradient
`centrifugation (3). Normal blood cells were obtained from the mononuclear
`cell populations of normal laboratory personnel. Monocytes were separated
`from the mononuclear cell fraction by adherence to plastic. T and B
`lymphocyte separations were accomplished by filtering lymphocyte prepa-
`rations through nylon wool columns as described by Daynilovs et a/. (4). T
`cells were collected in the initial filtrate while the adherent B cells were
`detached by vigorous agitation and rewashing of the column with medium.
`The human leukemic cell lines CCRF-CEM. KG-1 a, HL-60. and U937 were
`also studied. CCRF-CEM, a lymphoblastoid cell line, derived from a patient
`with T-ALL (obtained from the American Type Culture Collection), was
`maintained in culture in RPMl 1640 containing 20% FCS (5). The KG-la
`cell line, a subline of the KG-1 cell line derived from a patient with AML (6).
`was grown in Alpha medium (Flow Laboratories, Inc., Rockville. MD) con-
`taining 20% FCS and gentamicin. This line was a gift from Dr. P. Koeffler.
`Division of Hematology-Oncology, UCLA. Los Angeles. CA. The HL-60 cell
`line was derived from a patient with acute promyelocytic leukemia (7). This
`line was a gift of Dr. Robert Gallo, Laboratory of Tumor Immunology,
`National Institutes of Health and was maintained in RPMl supplemented with
`10% FCS. The U937 cell line was derived from a patient with histiocytic
`lymphoma and has some functional and morphologic characteristics of
`macrophages (8). This line was a gift of Dr. Paul Guyre. Department of
`Physiology, Dartmouth Medical School, and was maintained in RPMl 1640
`supplemented with 10% FCS.
`Binding. Supernatants from hybridomas were serially diluted in DMEM
`containing 20% FCS and were added to wells coated with glutaraldehyde-
`fixed leukemic cells, normal lymphocytes, or normal monocytes and incu-
`the method of Kennetrt (9). After
`bated for 2 hr at 37°C according to
`washng off unbound antibody with phosphate-buffered saline (PBS) and
`
`Although monoclonal antibodies that recognize tumor-spe-
`cific antigens would be extremely useful in the diagnosis and
`treatment of human malignant diseases, it is as yet unclear
`whether truly tumor-specific antigens exist on human tumor
`cells (1). Nonetheless, it seems likely that quantitative differ-
`ences in antigen expression will be found on tumor
`cells
`compared to normal cells, and such differences may be useful
`in demonstrating subgroups of selected tumor types or in
`mediating lysis of tumor vs normal cells.
`In a previous paper,3 we reported the preparation and prop-
`erties of a panel of monoclonal antibodies that bind to rnyelo-
`blasts from patients with acute myelocytic leukemia (AML)4 or
`chronic myelocytic leukemia (CML) in blast crisis. An important
`
`’ This work was supported by Grants CA31918 and AI 19053 awarded by the
`National Cancer Institute and the Institute of Allergy and Infectious Diseases,
`DHHS. respectively.
`Address correspondence to: Edward D. Ball. M.D., Department of Microbi-
`ology. Dartmouth Medical School, Hanover. NH 03755.
`Ball. E. D.. Fanger. M. W.: Monoclonal antibodies reactive with human
`myeloid leukemia cells. Clin. Exp. Immunol. In press.
`Abbreviations used in this paper: AML. acute myelocytic leukemia: CML.
`chronic myelocytic leukemia; ALL, acute lymphocytic leukemia; CLL, chronic
`lymphocytic leukemia; CML-BC. chronic myelocytic leukemia in blast
`crisis;
`HEPES. N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid.
`
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`'251-anti-mouse K-antibody was added and incubated for 2 hr at 37°C. After
`washing with PBS the wells were counted in a gamma Counter (Beckman
`4000).
`cytotoxicity. Cytotoxicity was tested by dye exclusion in microtiter trays
`according to the method of Amos (10). Cells from patients with leukemia
`and mormal peripheral blood were suspended in Verona1 Buffer (Oxoid, K.
`c. Biologicals, St. Louis, MO) or calcium and magnesium free Hanks'
`balanced salt solution (M. A. Bioproducts, Walkersville. MD). Hybridoma
`supernatants were diluted in hybridoma culture medium and incubated with
`cells for 30 min at 22°C before a wash step and the addition of rabbit Serum
`(diluted 1 :4.2 in Verona1 buffer) as a source of complement (Low-Tox H,
`Cedarlane Laboratories, Ltd., Hornby. Ontario. Canada). Incubation with
`complement proceeded for 1 hr at 22°C. Trypan blue exclusion was used
`to estimate cytolysis. Controls included anti-pz microglobulin and anti-la
`antisera (Accurate Chemical and Scientific Corp.. Westbury, NY). Negative
`controls included human AB serum, the myeloma parent Supernatant, and
`heat-inactivated complement. Each experiment was done at least in dupli-
`cate and in many cases, in quadruplicate. The data reported are the means
`of replicate assays in which deviation from the mean was leSS then 10%.
`
`RESULTS
`Sixteen of the 20 monoclonal antibodies studied gave maxi-
`mal binding to both leukemic and normal cells in undiluted or
`weakly diluted supernatant. The titer, defined as the dilution
`that gave 50% of maximal binding, ranged from 1 :2 to 1 :lo24
`for this group of monoclonal antibodies. The binding of several
`representative monoclonal antibodies to AML cells is seen in
`Figure 1A. Either a plateau or linear decline in binding was
`seen with decreasing concentration of antibody.
`In contrast, four of the 20 monoclonal antibodies studied,
`AML-1-201, AML-1-211. CML-75, and CML-150, showed an
`increase in binding to AML cells as the hybridoma supernatant
`was diluted, with maximal binding observed between 1 :8 and
`1 /32 dilution (Fig. 1 B).
`Binding of the majority of these monoclonal antibodies to
`mormal lymphocytes paralleled that to AML cells (Fig. 2). A
`notable exception was AML-2-23, which showed binding to
`lymphocytes only slightly above background while binding
`significantly to AML cells over a broad range of dilutions. The
`binding of monoclonal antibodies AML-2-23, AML-1-211, AML-
`2-9, AML-1-99, and CML-75 to monocytes is shown in Fig. 3.
`With the exception of monoclonal antibody AML-2-23, the
`binding patterns of these monoclonal antibodies to monocytes
`was similar
`to their binding to lymphocytes and AML cells.
`AML-2-23 showed similar binding to both monocytes and AML
`cells.
`Several monoclonal antibodies selectively mediated comple-
`ment-dependent lysis of leukemic myeloblasts (AML-1-21 1,
`AML-1-99. CML-75, CML-115, and CML-150). Three of these
`clones, AML-1-211, CML-75. and CML-150, mediated lysis of
`some leukemia cell samples only upon dilution of the super-
`natant, whereas other samples were lysed by both diluted and
`neat supernatant. Monoclonal antibody AML-1-211 permitted
`diluted
`lysis of four different AM1 cell samples only when
`between 1 :8 and 1 : 128 (see Figs. 4 and 5 for representative
`cytotoxicity curves). Monoclonal antibody CML-75 permitted
`lysis of one of these AML cell samples at dilutions between 1 :
`16 and 1:512 (Figs. 4 and 5 ) . The dilutions of these
`two
`monoclonal antibodies that mediated cytotoxicity corre-
`sponded to those that yielded maximal binding. CML-150,
`another monoclonal antibody that had a prozone in binding to
`AML cells, was able to lyse one sample of leukemic myeloblasts
`in undiluted supernatant. However, in contrast to monoclonal
`antibodies AML-1-211 and CML-75, CML-150 was still not
`in Figures 4 and 5.
`cytotoxic to the AML samples shown
`Subsequently, other AML samples were lysed by hybridomas
`CML-75, AML-1-211, and CML-150 only at dilutions of super-
`natant between 1 : 1 0 and 1 :loo. It is notable that each of these
`
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`I:W
`1:256 1:1024
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`I: 16
`Dilutlon of Antlbody
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`1:4
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`1256
`1:16
`1 : 6 4
`Dilutlon of Antlbody
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`I:IOM
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`figure 1. Binding of serially diluted hybridoma supernatant to 1 O5 AML cells.
`A. Hybridomas represented are AML-2-17 (0). AML-2-23 (A). AML-1-22 (0). and
`(A), AML-1-211
`AML-1-104 (0). 6 , Hybridomas represented are AML-1-201
`(O), CML-75 (0). and CML-150 0. The binding of the P3-X63Ag8 parent
`myeloma supernatant (A) is also represented in both 1 A and 16. Supernatants
`were diluted with DMEM containing 20% calf serum and incubated for 2 hr at
`37°C on glutaraldehyde-fixed leukemia cells. Bound monoclonal antibody was
`detected with a rabbit-anti-mouse kappa chain antibody labeled with '251. Each
`sample was counted for 2 min in a gamma counter.
`
`three monoclonal antibodies (AML-1-211, CML-75, and CML-
`150) mediated lysis of some leukemic cells only upon dilution
`while other samples were lysed by neat Supernatant. Moreover,
`a prozone was seen with some leukemic cells with only one or
`two of these monoclonal antibodies while another antibody
`permitted lysis in neat supernatant, yet the pattern was oppo-
`site for other cell samples. A summary of the collected cytotox-
`icity data for these monoclonal antibodies is shown in Table I.
`Neither AML-1-211. AML-1-99. CML-75, CML-115, or
`CML150 mediated lysis of any normal T and B lymphocytes or
`monocytes studied at any dilution of antibody (Figs. 6 and 7).
`Monoclonal antibodies AML-2-9 and AML-2-23 were cyto-
`toxic to a number of leukemia cell samples as well as selected
`normal cell populations. AML-2-23 mediated lysis of eight of
`13 myeloblast samples as well as all normal monocytes studied
`(Fig. 61, but was not cytotoxic to any lymphocytic leukemia cell
`samples or normal lymphocytes (Fig. 7). In contrast, AML-2-9
`mediated lysis of four of 12 myeloid leukemias as well as some
`lymphocytic leukemias and normal lymphocyte samples.
`One monoclonal antibody, AML-1-201, mediated lysis of
`nearly every cell population studied, both normal and leukemic.
`The exception was the cell line Daudi, which does not express
`class I HLA antigens or P2-microglobulin on the cell surface
`(1 1, 12). This antibody appeared specific to µglobulin
`as determined by an enzyme-linked imrnunosorbent assay with
`purified human ,&-microglobulin (a gift of Dr. George Bernier,
`Department of Medicine, Dartmouth Medical School). Finally,
`a number of monoclonal antibodies did not lyse any cell pop-
`ulation studied despite the demonstration that significant bind-
`ing occurred and the fact they were IgM immunoglobulins.
`Several monoclonal antibodies (AML-1-22, AML-1-99, AML-
`1-1 16, AML-1-201, AML-2-9, and AML-2-23) mediated lysis
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`I
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`Dllution of Antlbody
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`
`
` 1:1024
`
`1:256
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`
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`1:64
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`
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`1:16
`
`
`
`1:4
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`-
`-
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`-
`-
`Dilution of Antibody
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`*
`
`12 r
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`12 -
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`z 4 -
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`V
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`1:16
`
`
`
`1:4
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`1:64 1:1024 1:256
`
`
`
`Dilution of Antlbody
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`1:16
`
`
`
`1:4
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`
`1:256
` 1:1024
`1 : 6 4
`Dilution of Antibody
`
`F;gure 2. Binding of serially dlluted hy-
`bridoma supernatant to lo5 normal lympho-
`cytes and 1 O5 AML cells. Figure 2A shows the
`binding of AML-1-211 to lymphocytes (0) and
`AML cells (0). The P3-X63Ag8 myeloma cell
`line supernatant is also represented (A) in
`Figures 2A, 6, C. D, and E. Figure 28 shows
`the binding of AML-2-23 to lymphocytes (0)
`and AML cells (0). Figure 2C shows the bind-
`ing of AML-1-201 to lymphocytes (0) and AML
`cells (0). Figure 2 0 shows the binding of CML-
`150 to lymphocytes (0) and AML cells (0).
`Figure 2E shows the binding of CML-75 to
`lymphocytes (0) and AML cells (0). Samples
`were counted for 2 min.
`
`I
`
`E
`7
`1:4
`
`-
`
`
`
`t
`1:16
`1256
`1:64
`Dilution of Antibody
`
`1:1024
`
`of one or more of the CCRF-CEM, KG-1 a, HL-60, or U937 cell
`lines (Table I). Only AML-1-99 and AML-1-201 were capable
`of lysing all cell lines while AML-2-9 and AML-1-22 permitted
`lysis of CCRF-CEM and AML-1-1 16 permitted lysis of KG-1 a
`cells. Both AML-2-9 and AML-2-23 permitted lysis of the HL-
`60 cell line.
`
`DISCUSSION
`
`We presented data previously that described the binding of
`a panel of monoclonal antibodies to leukemic and normal cell
`populations. None of these hybridomas were specific for any
`leukemia cell type, yet significant quantitative differences in
`antigen expression on leukemic cells compared to normal cells
`were shown with several of these antibodies. In spite of a lack
`of absolute binding specificity, however, several antibodies are
`capable of selective complement-dependent cytotoxicity of
`leukemic, and, in some cases, myeloid leukemia cells. The
`present report documents these observations and demon-
`strates that some monoclonal antibodies can, under appropri-
`ate conditions, express specific cytotoxic activities that were
`not evident in the initial screenings.
`Monoclonal antibodies AML-1-99. AML-1-211. AML-2-30,
`CML-75, CML-115, and CML-150 all demonstrated cytotoxic-
`ity to myeloid leukemias exclusively while sparing normal cell
`populations. AML-1-21 1 mediated lysis of seven of 13 myeloid
`leukemia samples. This antibody showed a prozone effect with
`
`Dilution of Antibody
`Figure 3. Binding of serially diluted hybridoma supernatant to lo5 normal
`monocytes. Hybridomas represented are AML-1-99 03. AML-1-211 (0). AML-2-
`9 (A-A), AML-2-23 (A). and CML-75 (0). The P3-X63Ag8 myeloma cell line
`supernatant is also represented (A). Each sample was counted for 2 mln.
`
`four of these AML samples that coincided with the binding
`behavior of the hybridoma supernatant. Lysis of normal lym-
`phocytes or monocytes was not observed with this antibody at
`any dilution. Monoclonal antibody CML-75 was able to cause
`complement-dependent lysis of five of 13 myeloid leukemia
`samples but was not cytotoxic to normal lymphocytes and
`monocytes despite the demonstration of large amounts of CML-
`75 antigen on these cells. Antibodies AML-1-99. CML-1 15,
`and CML-150 were not cytotoxic to normal cells yet AML-1-99
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`TO MYELOID LEUKEMIA CELLS
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`divalent or trivalent binding. More easily dissociable complexes
`would be formed that would result in some loss of antibody by
`washing during the assay. As less antibody is presented to the
`cell surface more cross-linking of receptors might occur, lead-
`ing to a more stable complex. Although this behavior was noted
`for four of the 20 antibodies studied, all of which were IgM
`immunoglobulins, 15 other IgM monoclonal antibodies showed
`either a plateau or a nearly linear decrease in binding upon
`dilution. Differences in antigen density or distribution and/or
`that some of the monoclonal antibodies were of a different IgM
`subclass may also contribute to these results. The monoclonal
`antibodies that showed a prozone were among those with the
`highest binding to a variety of cells under standardized condi-
`tions, whereas none of the monoclonal antibodies recognizing
`less densely expressed epitopes had a prozone in the binding
`assay. The prozone effect noted in the cytotoxicity assay may
`have the same explanation because a wash step is included in
`the assay.
`These studies illustrate that for selected monoclonal anti-
`bodies, binding and/or effector functions can either be under-
`estimated or undetectable if screenings are limited to undiluted
`hybridoma supernatants. This has implications for both the
`initial selection of hybridomas and attempts to demonstrate
`specificity of hybridomas as it is clear that cytotoxicity can be
`demonstrated for some monoclonal antibodies only at critical
`antibody concentrations. Furthermore, this behavior needs to
`be considered in trials of serotherapy of human
`leukemia.
`
`Cytotoxicity of leukemia cells and cell lines mediated by monoclonal antibodies
`
`TABLE I
`
`in the presence of complement
`
`
`Leukemia Cell
`
`Cell
`
` Lineb
`Monoclonal
`
`
`
`
`
` Type'
`
`19821
`
`
`
`
`
`MONOCLONAL ANTIBODIES CYTOTOXIC
`
`1 9
`
`1:512
`
`I
`k2048
`
`1:32
`1A28
`1:8
`Dilution of Antibody
`Figure 4. C-dependent cytotoxicity mediated by monoclonal antibodies to
`cells from a patient with AML. Equal volumes of hybridoma supernatant and cells
`at 2 X 1 06/ml were incubated for 30 min at 22°C in microtiter wells. Rabbit C
`was added and incubation continued for 60 min. Cytolysis was estimated by
`Trypan blue exclusion. Hybridomas represented are AML-1-211 (0). AML-2-23
`(A). and CML-75 (0). The P3-X63Ag8 myeloma supernatant is also represented
`(A). All assays were done in duplicate.
`
`1:512
`
`
`
`1328
`
`
`
`
`
`1:2 1:32
`
`
`
`I:8
`
`~~
`
`-
`I:2048
`
`and CML-1 15 each permitted lysis of one of the AML samples
`studied and CML-150 permitted lysis of three AML samples.
`Monoclonal antibodies AML-2-9 and AML-2-23 were cyto-
`toxic to some myeloid leukemias as well as some normal cell
`types. AML-2-9 permitted lysis of some samples of
`normal
`lymphocytes but not of normal monocytes. AML-2-23 was
`highly active in killing monocytes and six of eight AML and
`three of five CML-blast crisis cell samples but was not cytotoxic
`to lymphoid cells. The specificity of this antibody is similar to
`that of monoclonal antibodies reported by Linker-Israeli et a/.
`(1 3) and Ugolini et a/. (1 4) that also were capable of mediating
`lysis of monocytes (13) and some myelomonocytic leukemia
`cells. Todd et a/. (1 5) reported the production of a monoclonal
`antibody, Mo2, that binds to normal monocytes and some
`leukemic myeloblasts. This antibody does not bind to the HL-
`60 cell line, which suggests that AML-2-23 and Mo2 bind to
`different antigenic determinants because AML-2-23 both binds
`to and permits complement-dependent lysis of this cell line.
`The explanation for the prozone effect in the binding of some
`of these IgM monoclonal antibodies is not clear. One possibility
`is that when excess antibody is present, binding may be
`relatively more monovalent and thus inherently less stable than
`
`AML CML-BC ALL CLL
`
`
`
`AML-1-99 0 / 4 1 / 7 0 / 4 0 / 3
`
`
`
`
`AML-1-201
`
`8 / 8 3/4 4/4 3/3
`
`
`
`AML-1-211 0/3 0/4 6/8 1/5
`
`
`
`
`
`AML-2-9 113 114 2/a 2/4
`
`
`
`
`
`
`5/8 3 / 5 0 / 3 0 / 4
`AML-2-23
`Dilution of Antibody
`AML-2-30
`1 / 5
`0 / 4
`
`
`
`CML-75 0 / 4 0 / 3 4 / 8 1 / 5
`
`
`
`
`FIgure 5. C-dependent cytotoxicity mediated by monoclonal antibodies to
`CML-115
`2/8
`0 / 3
`0 / 4
`0 / 3
`cells from a patient with AML (a different patient from that shown in Fig. 6).
`Hybridomas represented are AML-1-211 (a), AML-2-23 (A), and CML-75 (0).
`0/3 013
`CML-150
`218
`114
`The P3-X63Ag8 myeloma supernatant is also represented (A). All assays were
`a Cells were obtained from patients with a variety of leukemias. The numerator
`done in duplicate.
`refers to the number of different individuals whose cells showed positive reactions
`and the denominator refers to the number of individuals whose cells were tested.
`Positive is defined as >50% lysis of leukemia cells by hybridoma supernatant,
`+ + refers to >50?'0 lysis Of the cell line mediated by monoclonal antibody.
`No score indicates that no lysis was observed.
`
`KH-la CCRF HL-60 U937
`++ ++ ++ ++
`++ ++ ++ ++
`++ ++
`++
`
`
`
`0 / 2
`
`0 / 4
`
`IO
`
`Dilution d Antibody
`normal human monocytes. Hybridomas represented are AML-1-201 (0). AML-1-
`Figure 6. C-dependent cytotoxicity mediated by monoclonal antibodies to
`21 1 (O), AML-2-23 (A). and CML-75 (0). The P3-X63Ag8 myeloma supernatant
`is also represented (A). All assays were done in duplicate.
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`these specific markers on cells
`tative changes. if any, of
`stimulated to differentiate are in progress.
`Although the monoclonal antibodies reported here did not
`detect leukemia-specific antigens, there are several reasons to
`consider their utility in leukemia treatment. Some experimental
`evidence exists that monoclonal antibodies directed toward
`normal Cell surface antigens can be used to effectively treat
`animal tumors that bear that antigen without necessarily caus-
`ing untoward effects. The studies of Bernstein et a/. (1 9) using
`a monoclonal anti-Thy-I .1 antibody to treat a Thy-1.1 -positive
`murine leukemia in vivo have shown efficacy in the elimination
`of tumor metastases while demonstrating no serious toxicity as
`a result of normal T lymphocyte binding and lysis. The use of
`monoclonal antibodies directed toward antigens that are ex-
`pressed more, quantitatively, on leukemic cells may be equally
`efficacious and yet not seriously deleterious to normal host
`cells. It has also been shown that under appropriate conditions
`subpopulations of tumor cells can develop that do not express
`certain surface antigens (20). Thus, monoclonal antibody ther-
`apy directed toward a given single antigen could result in
`selection of a subpopulation of tumor cells. Finally, modulation
`of cell surface antigens can occur as a result of monoclonal
`antibody binding (1 8).
`It seems likely that successful treatment of human tumors
`may require the use, in combination, of panels of monoclonal
`antibodies such as AML-1-99. AML-1-211, AML-2-9. AML-2-
`23, AML-2-30, CML-75, CML-115, and CML-150. In this man-
`ner, the problem already encountered in tlials of serotherapy
`in humans, that of antigen modulation (21 -231, may be circum-
`vented, as multiple determinants are used as targets for anti-
`body-mediated leukemia cell lysis. Studies utilizing the panel
`of monoclonal antibodies described in this report are being
`conducted to evaluate tumor cell escape from antibody-medi-
`ated lysis and its prevention.
`Several of these monoclonal antibodies have immediate di-
`agnostic value based on their ability to selectively lyse leukemic
`myeloblasts but not leukemic lymphoblasts. This panel of mon-
`oclonal antibodies may also be useful in defining subsets of
`myeloid leukemia cells based on variable binding to and lysis
`of cells from individual patients. Such data can be correlated
`with cellular morphology and histochemical staining. It may be
`possible to show characteristic "fingerprints" of certain sub-
`types of leukemia defined by binding to panels of monoclonal
`antibodies that could aid in both diagnosis and possibly in
`determining optimal therapy.
`
`Acknowfedgmenfs. We wish to thank Drs. Hillard Lazarus
`and Roger Herzig for providing some of the leukemia cells used
`in these studies.
`
`REFERENCES
`
`1, Old. L. J. 1981. Cancer Immunology. The search for specificity. G. H. A.
`Clowes Memorial Lecture. Cancer Res. 41 :361.
`2. Kohler, G.. and C. Milstein. 1975. Continuous cultures of fused cells se-
`creting antibody of predefined specificity. Nature 256:495.
`3. Boyum, A. 1976. Isolation of lymphocytes, granulocytes, and macrophages.
`Scand. J. Immunol. 5 9 .
`4. Daynilovs, J.. G. Ayoub. and P. I. TerasakiT 1980. B-lymphocyte isolation by
`thrombin-nylon. Histocompatibility testing report of the 8th International
`Histocompatibility Workshop, 287.
`5. Foley, G. E.. H. Lazarus. S. Farber. €3. G. Uzman. 8 . A. Boone. and R. E.
`McCarthy. 1965. Continuous culture of human lymphoblasts from peripheral
`blood of a child with acute leukemia. Cancer 18:522.
`6. Koeffler. H. P.. R. Billing. A. J. Lusis. R. Sparkes. and D. W. Golde. 1980.
`An undifferentiated variant derived from the human acute myelogenous
`leukemia cell line (KG-1 1. Blood 56:265.
`7. Collius. S. J., R. C. Gallo, and R. E. Gallagher. 1977. Continuous growth
`and differentiation of human myeloid leukaemic cells in suspension culture.
`Nature 270:347.
`
`-
`50
`-
`40
`30 -
`20 .
`
`IO
`
`1:2
`
`1:s
`Id26 1512
`I:8
`Dilution of Antibody
`C-dependent cytotoxicity mediated by monoclonal antibodies to
`normal human lymphocytes. Identical results were obtained with unfractionated
`peripheral blood lymphocytes, T lymphocytes, and B lymphocytes. Hybridomas
`represented are AML-1-201 0. AML-1-211 (01% AML-2-23 (A), and CML-75
`(0). The P3-X63Ag8 myeloma supernatant is also represented (A), All assays
`were done in duplicate.
`
`1:-
`
`Optimal conditions for leukemia cell lysis may vary with the
`monoclonal antibody used and the leukemia cell population.
`There are now several human leukemia cell lines available
`for in vitro studies of leukemia cell biology (1 6). It is of interest
`that few of the monoclonal antibodies from the panel reported
`here mediated lysis of the myeloid leukemia cell lines, KG-1 a
`or HL-60. Only one of the hybridomas that showed selective
`complement-dependent cytotoxicity for fresh leukemia sam-
`ples from patients, AML-1-99, was cytotoxic to both of these
`cell lines despite evidence that significant binding occurred
`with all hybridomas but AML-2-23. Antibody AML-1-99, how-
`ever, mediated lysis of the KG-la, HL-60, U937, and CCRF-
`CEM cell lines, suggesting that the AML-1-99 antigen is ex-
`pressed similarly on these cell lines. Because these cell lines
`are of diverse lineage, their susceptibility to lysis by AML-1-99
`may possibly be due to binding to an antigen expressed on
`rapidly proliferating cells, a "division antigen." Monoclonal
`antibody AML-2-23, which mediated lysis of 8 of 13 myeloid
`leukemia cell populations, also mediated lysis of the HL-60 cell
`line but not of another myeloid cell line, KG-1 a. It is of interest
`that AML-2-23 did not permit lysis of the U937 cell line. These
`cells seem to require activation by medium conditioned by
`mixed lymphocyte cultures to assume some of the functional
`characteristics of monocytes (1 7). The failure of AML-2-23 to
`bind to and permit complement-dependent lysis of this cell line
`in its unstimulated state also shows that at least one normal
`surface marker of normal monocytes is absent. Studies with
`stimulated U937 cells to determine if the AML-2-23 antigen is
`expressed on stimulated cells are planned.
`In contrast, the expression of the AML-2-23 antigen on cells
`of the HL-60 cell line shows that these cells express an antigen
`characteristic of mature monocytes. We have also found that
`cells of the HL-60 cell line express an antigen characteristic of
`polymorphonuclear leukocytes that is recognized by a mono-
`clonal antibody, PMN-29, developed in our laboratory (unpub-
`lished observation). The expression of two different antigens
`on these cells in the native state that correlate with specific
`states of differentiation is interesting in light of the demon-
`strated ability of these cells to differentiate into either mature
`granulocytes or monocytes with appropriate chemical inducers
`(18). The present report suggests that certain markers of
`differentiated cells are present before induction of differentia-
`tion with chemical mediators. Studies to determine the quanti-
`
`PETITIONER'S EXHIBITS
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`MONOCLONAL ANTIBODIES CYTOTOXIC
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`TO MYELOID LEUKEMIA CELLS
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`8. Sundstrom, C.. and K. Nilsson. 1976. Establishment and characterization of
`a human histiocytic lymphoma cell line (U-937). Int. J. Cancer 17:565.
`9. Kennett. R. H. 1980. Enzyme-linked antibody assay with cells attached to
`polyvinyl chloride plates, in Monoclonal Antibodies, Edited by Kennett. R.
`H., T. J. McKearn. and K. E. Bechtol. Plenum Press, New York. P. 376.
`10. Amos, D. 8. Cytotoxicity testing. In Manual of Tissue Typing Techniques,
`Department of Health, Education, and Welfare publication (National Institutes
`of Health). Government Printing Office, Washington, D.C. 80-545. P. 42.
`11. Fellous. M., F. Martchelewicz, M. Kamorn. and S. Davsset. 1975. The use
`of a lymphoid cell line to define new E-lymphocyte specificities probably
`controlled by the MHC Region. Histocompatibility Testing. Edited by Kiss-
`meyer-Nielson. Munksgaard, Copenhagen. P. 708.
`12. Evrin. P. E., and K. Nilsson. 1974. p2 microglobulin production in vitro by
`human hematopoietic, mesenchymal, and epithelial cells. J. Immunol. 1 12:
`137.
`13. Linker-Israeli, M.. R. J. Billing, K. A. Foon. J. H. Fitchen. and P. I. Terasaki.
`1981. Monoclonal antibodies reactive with acute myelogenous leukemia
`cells. Fed. Proc. 40:1118 (Abstr.)
`14. Ugolini. V., G. Nuney. G. R. Smith, P. Stasney. and J. D. Capra. 1980. Initial
`characterization of monoclonal antibodies against human monocytes. Proc.
`Natl. Acad. Sci. 77:6764.
`15. Todd, R. F. 111, L. M. Nadler. and S. F. Schlossman. 1981. Antigens on
`human monocytes identified by monocl