JOURNAL OF HEMATOTHERAPY 1:85-94 (1992)
`Mary Ann Liebert, Inc., Publishers
`
`Initial Trial of Bispecific Antibody-Mediated
`Immunotherapy of CD15-Bearing Tumors:
`Cytotoxicity of Human Tumor Cells Using a
`Bispecific Antibody Comprised of Anti-CD 15
`(MoAb PM81) and Anti-CD64/Fc7RI (MoAb 32)
`
`EDWARD D. BALL,1 PAUL M. GUYRE,3 LETHA MILLS,1 JAN FISHER,4
`NATHAN B. DINCES,4 and MICHAEL W. FÄNGER3
`
`ABSTRACT
`The high-affinity receptor for IgG, Fc-yRI, expressed on monocytes and interferon-7
`(IFN-7)-stimulated neutrophils, is a trigger molecule for cell-mediated cytotoxicity. We have
`prepared murine monoclonal antibodies (MoAb 22 and MoAb 32) that bind to Fc^RI outside
`the ligand binding site and thus bind to and trigger cytotoxicity that is not competed by other
`immunoglobulins. Because of these properties, it seemed that these MoAbs would be very
`useful for the development of bispecific antibodies (BsAb) for targeting normal cellular
`immune defense mechanisms as a new form ofimmunotherapy for treatment ofcancer. BsAbs
`incorporate into a single molecule the binding specificities of two different antibodies, and,
`thus, can be used to target myeloid cells to tumors, ensure activation of cellular cytotoxic
`mechanisms, and target cell lysis and/or phagocytosis. BsAbs were prepared using anti-FcyRI
`MoAb and an anti-myeloid cell MoAb, PM81, reactive with the CD15 antigen, for studies of
`antibody-dependent cellular cytotoxicity. Conjugates were made by cross-linking sulfhydryl
`groups of Fab fragments of MoAb 32 or 22 (both IgG,) and sulfhydryl groups added to intact
`PM81 (an IgM) using A^-succinimdyl-acetyl-S-thioacetate (SATA). The resulting product was
`purified by high-performance size-exclusion chromatography. The ability of the BsAbs to
`mediate attachment of human monocytes to tumor target cells was confirmed in a microtiter
`well assay of binding of MTT-labeled U937 cells (a human Fc7RI-bearing cell line) to SKBR-3
`(PM81-reactive breast carcinoma) target cells. The ability of the BsAbs to mediate killing of
`HL-60 promyelocytic leukemia cells was studied using a 6-hour Chromium-51 release assay.
`Effector cells were monocytes obtained by cytopheresis and cultured for 18 hours with IFN-7.
`Monocytes alone caused minimal killing (5-20%), monocytes plus BsAb caused moderate
`killing (20-50%), and monocytes plus BsAb plus human serum resulted in maximal killing
`(50-80%). Experiments were performed to test the ability of the BsAb to purge bone marrow
`
`Departments of 'Medicine, 2Microbiology, and 3Physiology, Dartmouth Medical School, Hanover, NH.
`4Medarex, Inc., West Lebanon, NH.
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`of small numbers of leukemia cells using bone marrow mononuclear phagocytes treated for 18
`hours with IFN-7 prior to adding target cells. Without the addition of human serum as a
`source of complement, a 90% depletion of clonogenic HL-60 cells could be demonstrated.
`With human complement, up to 95 % depletion was seen. Thus, this BsAb possessed the ability
`to lyse tumor cell targets by two different mechanisms, complement and cell-mediated lysis. In
`a Phase I clinical trial, 4 patients with CD15+ tumors were treated with up to 48 mg of this
`BsAb with no toxicity. Of this group, the patient with acute myelogenous leukemia experi-
`enced a transient 30-60% reduction in circulating leukemic blast cells during each of six
`infusions over a 2-week period. Although in vitro assays indicated maximal effectiveness
`between 1 and 10 |xg/ml of this BsAb, in vivo reduction in circulating cell counts was observed
`when peak serum concentrations were as low as 50 ng/ml. As such, this BsAb may be useful for
`in vivo therapy of high-risk tumors, especially after induction of remission or after bone
`marrow transplant, i.e., for treatment of minimal residual disease in patients with CD15-
`expressing tumors, including acute myeloid leukemia, small cell carcinoma of the lung, and
`colon and breast carcinoma.
`
`INTRODUCTION
`
`Although MURiNE monoclonal antibodies (MoAb) directed to tumor-associated antigens have consid-
`erable therapeutic potential, there are also several limitations to their use in vivo (Dillman, 1989). These
`include lack of host cellular or humoral effector mechanisms, immune response to the foreign protein, and
`delivery of adequate amounts of the MoAb to the tumor. For a MoAb to be an effective anticancer agent, it is
`necessary that it work in concert with an effector mechanism such as complement (Stepan et al, 1984) or a
`cellular effector, as in antibody-dependent cellular cytotoxicity (ADCC)' (Ortaldo et al, 1987). Many
`potentially useful MoAbs are not able to use these effector mechanisms because they are not of the appropriate
`subclass of immunoglobulin. It has become clear that murine Ig of the IgG2a and IgG3 subclasses are the most
`effective at mediating ADCC by virtue of their ability to bind to the high-affinity Fc receptor, FC7RI (Lübeck
`et al, 1985). Murine IgG, and IgG2b MoAb mediate ADCC by human effector cells far less efficiently, if at
`all (Van de Winkel et al, 1991). In addition, human complement is not as capable of mediating cytotoxicity
`with murine MoAb as guinea pig or rabbit complement. Thus, only MoAbs of the IgG2a and IgG3 subclasses
`have shown activity during in vivo therapy, presumably through ADCC mediated by FcyRI-bearing cells
`(Steplewski et al, 1988).
`In this paper, we describe an approach to overcoming some of the limitations of certain MoAbs for in vivo
`therapy. We have prepared murine MoAbs (MoAb 22 and MoAb 32) that bind to FC7RI outside the ligand
`binding site, and thus their ability to bind to and trigger cytotoxicity through this receptor is not competed by
`other immunoglobulins. Bispecific antibodies (BsAbs) were previously shown to mediate ADCC of red blood
`cells that was not inhibited by nonimmune human IgG (Shen et al, 1986a). We now report use of BsAbs
`linkage using Fab fragments of anti-Fc7RI MoAb 32 or 22 and a whole IgM
`prepared by chemical
`anti-myeloid cell MoAb, PM81, reactive with the CD 15 antigen, for studies of ADCC in vitro and therapy.
`This BsAb was able to mediate ADCC of HL-60 leukemia cells. Moreover, when testing for the ability of
`human plasma to block this effect, we found an enhancement of killing mediated at least partly by human
`complement. Thus, this BsAb lysed tumor cells by two different mechanisms, complement and cellular-
`mediated lysis. In a Phase I clinical trial, 4 patients with CD 15 + tumors were treated with up to 48 mg of this
`BsAb with no toxicity, and in one of these patients who had acute myelogenous leukemia and could be
`evaluated, there was a transient 30-60% reduction in circulating leukemic blast cells during each of six
`infusions over a 2-week period. Thus, this BsAb may be useful for treatment of minimal residual disease in
`patients with CD15-expressing high-risk tumors.
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`THERAPEUTIC BISPECIFIC ANTIBODY TO Fc-yRI AND CD 15
`
`MATERIALS AND METHODS
`
`Cells
`
`HL-60 and U937 cells (American Type Culture Collection (ATCC), Rockville, MD) were cultured in
`RPMI-1640 (GIBCO, Grand Island, NY) containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) as
`previously described (Ball et al, 1983). SKBR-3 cells (ATCC) were cultured in DMEM + 10% FBS.
`Monocytes were purified as previously described (Shen et al, 1986b). Briefly, mononuclear cells were
`harvested from the peripheral blood of normal donors by leukapheresis. The monocytes were further purified
`by separation over Histopaque (Sigma Chemical Co., St. Louis, MO), and incubation with rotation at 4°C
`(Shen et al, 1986b). The resulting aggregates were allowed to sediment and then sedimented again through
`FBS. The resulting preparations were >95% monocytes as assessed by morphological examination of
`Wright's-Giemsa-stained cytospins and expression of CD14.
`The hybridomas PM81 (Bell etal, 1983), 32 (Anderson et al, 1986), and 22 (Guyre era/, 1989) were tested
`for mycoplasma, sterility, and murine viruses by the MAP test and found to be free of contamination. The
`hybridomas were cultured in MF-1 medium (modified Excell-300, J.R. Scientific) containing 0.5% FBS.
`
`Production of MoAbs
`Antibodies were produced using hollow-fiber technology, with a serum-free,
`low-protein media feed
`stream. Each MoAb was purified from harvests of antibody-rich supernatant from a hollow-fiber cartridge
`using high-performance liquid chromatography (HPLC). The MoAb was purified using a semipreparative
`anion exchange (DEAE) column with a combined pH and salt gradient. The gradient was optimized for each
`MoAb to achieve a product of greater than 90% purity, with extremely low endotoxin (0.48-0.96 EU/ml or
`0.42-0.84 EU/mg antibody) and DNA contamination levels (<5 pg/mg).
`
`Flow cytometry
`Briefly, cells (106) were incubated for 1 hour with varying concentrations of MoAb or BsAb (in the
`presence of normal human IgG ( 10^5 moles/liter), Sigma), washed with phosphate-buffered saline containing
`0.1% bovine serum albumin and 0.05% sodium azide (PBA), and further incubated with fluorescein
`isothiocyanate (FITC)-coupled, affinity-purified F(ab')2 goat anti-mouse (GAM) Ig (Caltag, Inc., S. San
`Francisco, CA) for 1 hour at 4°C. Cells were then washed once in PBA and resuspended in PBA containing 1 %
`paraformaldehyde (Sigma). The cells were analyzed on the Ortho Systems 50H flow cytometer (Ortho
`Diagnostics, Westwood, MA) interfaced to a 2150 computer using a logarithmic amplification scale.
`
`Bispecific antibody preparation and purification
`BsAbs composed of PM81 conjugated to 32 Fab (or 22 Fab) were prepared by cross-linking the sulfhydryl
`of the 32 Fab fragment to sulfhydryl groups added to the PM81 MoAb. Sulfhydryl groups were added to PM81
`using A'-succinimidyl-acetyl-S-thioacetate (SATA). 32 was digested with pepsin to produce the F(ab')2
`fragment, which was then purified by high-performance size-exclusion chromatography. Reduction with
`mercaptoethylamine and reaction with 5,5'-dithio-bis(2-nitrobenzoic acid) converted the F(ab')2 fragment to
`a Fab fragment with endogenous sulfhydryl groups activated with thionitrobenzoic acid (Fab-TNB). The
`Fab-TNB was purified by gel filtration and assayed to make sure that there were enough TNB groups attached
`to the Fab to make a good coupling partner. An excess of 32 Fab-TNB was added to the PM81 SATA to drive
`the reaction to complete conversion to bispecific antibody. The bispecific antibody was then purified using
`high-performance size-exclusion chromatography. Endotoxin levels, as determined by the Limulus ameboe-
`cyte lysate assay, were =£1.74 EU/mg.
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`Cytotoxicity assays
`HL-60 cells expressing CD 15 on their surface were labeled for 1 hour at 37°C with 200 mCi of5 ' Cr sodium
`chromate in normal saline (New England Nuclear, Boston, MA) and used as target cells. Effector cells were
`normal monocytes isolated from a leukopack as described and cultured overnight with 50 units/ml of IFN-7.
`To quantify cytotoxicity of HL-60 cells, equal volumes of test reagents (control antibodies and conjugates),
`51Cr-labeled HL-60 cells, and effector cells were mixed in round-bottomed microtiter wells. Plates were
`incubated for 6 hours at 37°C, after which half of the supernatant was removed and counted for release of 51Cr.
`Maximal lysis was obtained by addition of 2% sodium dodecyl sulfate in water. Percent cytotoxicity was
`— spontaneous lysis) + (maximal
`lysis — spontaneous
`calculated as lOOx (counts released with effectors
`lysis). In all experiments, tests were conducted in triplicate and the results are expressed as the mean ± SD.
`
`MTT cell binding assay
`An assay to determine bispecificity was developed that relies on the binding of a BsAb to CD 15 on a target
`cell (SKBR-3 cells, American Type Culture Collection, Rockville, MD) and subsequently to FC7RI on model
`effector cells (U-937 cells, American Type Culture Collection, Rockville, MD)
`labeled with MTT
`(3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) (Green et al, 1984). This binding induces
`stable association between effector and target cells. SKBR-3 cells were plated at 4 x 104 cells per well into
`96-well flat-bottomed plates (Costar) in DMEM + 50 u.g/ml gentamicin + 10% FBS, and cultured for 24-48
`hours. Culture media was then removed by rapid inversion, and antibodies (PM81 X 32, PM81, 32,
`PM81 + 32 or P3, a nonspecific murine IgG[ MoAb) were added in a volume of 50 p.1 at the concentrations
`shown. The plate was rotated at 4°C for 30 minutes, and then each well was washed twice with 200 u.1 of
`PBS-BSA at 4°C. After washing, 4 X 105 MTT-labeled U-937 cells were added and the plate rotated for an
`additional 30 minutes at 4°C. MTT labeling of U-937 cells was accomplished by adjusting cells to 2 x 106
`cells/ml in RPMI-1640 + 10% FBS and incubating at 37°C, 5% C02 for 1 hour with 0.5 mg/ml MTT. After
`incubation of MTT-labeled U-937 cells with the SKBR-3 monolayer for 30 minutes, each well was washed
`five times very gently with 200 jjlI of PBS-BSA. The plate was forcefully inverted between washes, without
`centrifugation, to remove nonadherent cells. After the last wash, 100 (¿1 of 95% isopropanol + 0.04 N HC1
`was added to each well, the cells were thoroughly mixed by pipetting to dissolve the MTT reaction product,
`and the plate was read in an ELISA reader (Dynatech, MR650). Results are reported as O.D. units, and
`represent absorbance at 570 nm.
`
`Colony-forming assay
`Peripheral blood mononuclear cells from a normal donor were cultured for 18 hours in IFN-7 ( 100 units/ml)
`prior to addition of HL-60 cells (to 1% final concentration of HL-60 cells). This mixture of cells was then
`cultured for another 18 hours under a variety of conditions (with or without autologous plasma as a source of
`complement; with or without bispecific or monospecific MoAb, including the negative control MoAb, Thy-1 )
`and then seeded into methylcellulose cultures with HL-60 conditioned media (as a source ofgrowth factors for
`HL-60 cells) (Howell & Ball, 1985).
`
`In vivo administration of bispecific antibody
`With the approval of the DHMC Committee for the Protection of Human Subjects, and after informed
`consent, 4 patients with CD15+ tumors were treated in a Phase I study of BsAb infusions. The first 2 patients
`were treated with 0.065 mg/kg per dose and the second 2 patients with 0.125 mg/kg dose. The antibody dose
`was diluted in 500 ml of normal saline. Each patient was treated six times (three times per week over 2 weeks)
`with 6-hour infusions of antibody at a single dose level per patient.
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`THERAPEUTIC BISPECIFIC ANTIBODY TO Fc-yRI AND CD 15
`
`-A— 32 F(ab')2
`PM81
`PM81x32Fab
`
`30000 -
`
`25000 -
`
`20000 -
`
`15000 -
`
`10000 -
`
`5000 -
`
`01U
`
`
`oB3OM 3U0
`
`>
`"o
`
`s <
`
`0<
`.001
`
`r i ifii|
`100
`mAb or BsAb Concentration ((ig/ml)
`FIG. 1. Bindingof BsAb to normal human monocytes. The binding of BsAb PM81 x 32 Fab, orthe component MoAb,
`to freshly isolated human leukocytes was analyzed by flow cytometry (gated on light scatter to include only the monocytes)
`as described in Materials and Methods. Values are the mean for duplicate measurements of the number of second Ab
`molecules bound per cell.
`
`i
`
` r iiimq
`1000
`
`RESULTS
`
`Characteristics of BsAb
`The ability of the BsAb to bind to antigen-positive target cells was assessed by flow cytometry. As shown
`in Fig. 1, the BsAb recognized approximately the same number ofantigenic sites on normal monocytes as the
`the avidity of binding may have been slightly reduced by the chemical
`constituent MoAb. However,
`conjugation. The MTT cell-binding assay was used to demonstrate that the antibody was functioning as a true
`BsAb. This assay indicated that maximal attachment of the CD64+ U937 cells to the CD15+ SKBR-3 cells
`occurred at 5 p,g/ml of BsAb and that both the PM81 x 32andthePM81 x 22 BsAbs were similarly effective
`in mediating conjugate formation between these cells (Fig. 2).
`
`BsAbPM81X32
`BsAbPM81X22
`
`80 -i
`
`60
`
`4<H
`
`20 H
`
`QZ Os
`
`a V
`
`iJ
`-J
`
`W «oHOwb
`
`.
`i*,
`w
`t£
`
`0
`
`5
`25
`10
`15
`20
`BsAb Concentration (|ig/mL)
`FIG. 2. Demonstration of the ability of the BsAb to mediate attachment of U937 cells (Fc7RI+) to SKBR-3 cells
`(CD15+). MTT-labeled U937 cells were added to monolayers of SKBR-3 cells in the presence or absence of two different
`BsAb. Cell attachment was measured by spectrophotometry as described in Materials and Methods. Values are
`mean ± SD for triplicate measures of bound U937 cells. The broken line indicates the maximum binding in the absence
`of antibody or in the presence of the uncoupled component MoAb.
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`BsAbPM81x32
`-A-
`PM81
`
`-
`
`— BsAb PM81x22
`PM81x32 + PM81x22
`
`.05
`.5
`5
`.1
`1
`mAb or BsAb Concentration (¡ig/ml)
`FIG. 3. ADCC of HL-60 cells by BsAb. 5lCr-labelled HL-60 cells were cultured for 6 hours at 37°C in the presence of
`MoAb PM81, BsAb PM81 x 32, BsAb PM81 x 22, or a combination of the two BsAb. Specific cytotoxicity was
`calculated as described in Materials and Methods.
`
`ADCC mediated by BsAb
`Both the PM81 x 32 Fab BsAb, and a similar BsAb (PM81 x 22 Fab) which targets to a separate epitope
`on Fc-yRI, were capable of mediating ADCC of HL-60 cells (Fig. 3). Using the chromium release assay and
`correcting for background lysis, 30-35% killing was demonstrated using 0.5-1.0 |xg/ml of BsAb, with lesser
`killing at both higher and lower concentrations. Monospecific PM81 had little activity in this assay in the
`absence of serum, and combining two bispecifics made from the same tumor-specific MoAb did not
`significantly increase cytotoxicity. Thus, these BsAbs mediate ADCC of HL-60 target cells whereas PM81
`alone does not. However, in the presence of fresh human serum, collected with precautions to keep the serum
`chilled, both the BsAb (Fig. 4) and PM81 alone (not shown) lysed up to 80% of chromium-labeled HL-60
`target cells. When monocytes were labeled with chromium and subjected to incubation with BsAb plus
`
`PM81
`PM81 x32Fab
`PM81
`PM81 x32
`
`100
`
`xoo O u
`
`IX
`
`.01
`
`1 0
`
`mAb or BsAb Concentration (|j.g/ml)
`FIG. 4. Augmented ability of BsAb to mediate ADCC in the presence of human serum. The PM81 x 32Fab BsAb and
`PM81 alone were tested by the 5lCr-release assay for cytotoxicity of HL-60 cells in the absence (closed symbols) or
`presence (open symbols) of fresh human serum as a source of complement.
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`unlabeled monocytes as effectors, no chromium release over background levels was observed (data not
`shown), indicating that the BsAb does not cause lysis of the effector cells.
`Mixing experiments
`As seen in Table 1, when evaluated in a colony formation assay, both unconjugated PM81 and PM81 x 32
`Fab eliminated 92% of HL-60 cells that had been mixed with normal bone marrow cells plus autologous
`plasma as a source ofcomplement. BsAbPM81 x 32 Fab eliminated 85% of HL-60 colonies via ADCC and
`in the absence of autologous plasma.
`In vivo therapy
`Four patients were treated with BsAb infusions. Their diagnoses were acute myelogenous leukemia
`lung cancer (SCCL), breast cancer, and pancreatic islet cell carcinoma. All were in
`(AML), small cell
`advanced stages of disease and had chemotherapy-resistant disease at the times of treatment. No acute
`toxicities were observed other than a transient period (several minutes) of substernal discomfort in 1 patient.
`The infusion was interrupted until the symptoms resolved and then completed without further incident.
`During the infusion in the patient with AML, the circulating blast cells decreased by about 50% for several
`hours after each infusion, but returned to baseline or greater levels by the next morning. The reduction in blast
`cells was somewhat surprising since less than 2% of the available binding sites were occupied by BsAb in vivo
`(Fig. 5). Peak serum levels of free BsAb were achieved at the end of the infusion period in all patients, and
`ranged from 0.08 to 0.27 p,g/ml.
`In the patients with the solid tumors, it was not possible to monitor the effects of BsAb on the tumor
`population. However, the function of the antibody was indicated by the fact that the circulating neutrophils
`decreased dramatically after each infusion. Despite this phenomenon, there were no associated signs or
`symptoms of acute reaction or of infection.
`
`DISCUSSION
`
`Redirected cellular cytotoxicity mediated by BsAb is a new form of immunotherapy with potential
`advantages over unmodified antibody therapy and other immunoconjugates such as immunotoxins. These
`advantages include a probable lack of toxicity, since no inherently toxic compounds would be introduced into
`in vivo cytotoxic mechanisms, and the ability to recruit very large numbers of
`the host, the use of normal
`cytotoxic cells to the target.
`We are particularly interested in the role of mononuclear phagocytes in mediating cytotoxicity of tumor
`cells through FC7RI (CD64) (Anderson etal, 1986; Guyre et al, 1989). Using bispecific antibodies comprised
`
`Table 1. Effect of MoAb PM81 and BsAb in the Presence or Absence
`of Human Complement on the Killing of HL-60
`Colony-Forming Cells"
`
`Without human
`complement"
`
`With human
`complement
`MoAb or BsAb
`Thy-1
`540
`PM81
`405
`40
`PM81 x 32
`60
`45
`"HL-60 cells were incubated with IFN-7-activated monocytes and MoAb
`PM81, BsAb, or a control IgM MoAb (Thy-1), in the presence or absence of
`human serum as a source of complement. This mixture was then seeded into
`methylcellulose cultures as described in Materials and Methods.
`"Colonies/200,000 cells plated.
`
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`Ab Bound in vitro
`
`Ab Bound in vivo
`
`58247
`
`116
`
`731
`
`827
`
`96
`
`80000-f
`
`60000 H
`
`40000 H
`
`20000
`
`o.
`S»
`W3
`
`A<
`
`6
`2
`4
`.2
`2
`20
`.02
`PM81x32 Fab (u.g/ml)
`Treatment Time (hours)
`FIG. 5. Binding of BsAb to AML patient blast cells in vitro and in vivo. Peripheral leukocytes (>96% blast cells) were
`isolated from patient 1 and examined by indirect immunofluorescence plus flow cytometry to determine their maximum
`binding capacity for BsAb (left panel). Cells were also isolated, kept at 0°C, and rapidly assayed by the same method for
`the amount of BsAb that had bound in vivo (right panel).
`
`8
`
`0
`
`of anti-Fc7RI MoAb (32 and 22) and antitumor-associated antigens, it may be possible to lyse tumor cells in
`vivo specifically with little toxicity. Moreover, the anti-Fc7RI mAb 32 and 22 bind to the FC7RI outside the
`domain occupied by the Fc portion of human IgG, so that the presence of physiological levels of human IgG
`does not block binding of the these MoAbs nor of BsAb made from these MoAbs to this cytotoxic trigger
`molecule (Guyre et al, 1989).
`We have prepared bispecific antibodies comprised of whole IgM against the CD 15 antigen and Fab
`fragments of 32 and 22. These BsAb are capable of mediating cellular cytotoxicity by IFN-7-activated
`monocytes against CD15+ target cells. In addition, the conjugation of Fab to the IgM anti-CD 15 did not
`abrogate its ability to mediate human complement-dependent cytotoxicity. Thus, in the presence of normal
`human serum, or plasma containing high concentrations of IgG, the cxytotoxicity of this BsAb is actually
`augmented. Moreover, the dose-response curves of the two mechanisms demonstrate that the two activities
`are optimal at different concentrations. Complement-mediated killing was more efficient in the range of 10
`u-g/ml BsAb or more, whereas ADCC was optimal at about 1 p-g/ml. Since intratumoral concentrations of
`antibody are likely to be lower than plasma concentrations, the ADCC activity curve suggests that ADCC may
`be the predominant mechanism of killing in tumors.
`We envisage two therapeutic applications of this BsAb. One is in vivo therapy of patients with CD15 +
`tumors such as AML (Ball et al, 1983), SCCL (Huang et al, 1983), colorectal cancer (Brockhaus et al, 1982),
`and breast carcinoma (Vredenburgh et al, 1991). It is likely that the BsAb will be most useful to treat minimal
`disease, such as in patients with high-risk breast cancer following primary surgical therapy or patients with
`AML after remission is induced by chemotherapy. Another application is as an adjunct to chemotherapy in
`vivo, either for remission-induction or as part of a preparative regimen used for bone marrow transplantation
`(Ball etal, 1990).
`There are theoretical drawbacks to the particular combination of antibodies we have chosen because the
`tumor-associated antigen is expressed on both the target and effector cells. Thus, the possibility exists that
`monocytes can kill monocytes by engaging the CD15 antigen on one cell and FC7RI on an adjacent monocyte.
`Our experiments with 51Cr-labeled monocytes show that this does not appear to occur. Another consequence
`of the use of the PM81 x 32 F(ab')2 construct is that normal neutrophils and monocytes that express CD15
`may interfere with binding of the bispecific antibody to tumor target cells in vivo. Binding of the antibody to
`neutrophils may also produce neutropenia and/or the release of inflammatory mediators leading to toxicity.
`However, the preliminary data from a Phase I clinical trial of this antibody has not been associated with
`toxicity, though transient neutropenia has been observed. Nevertheless, it would seem most efficacious to use
`
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`THERAPEUTIC BISPECIFIC ANTIBODY TO Fc^RI AND CD 15
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`this antibody for minimal residual disease, such as after bone marrow transplant
`for AML, or after
`myelosuppressive chemotherapy, at a time when there are few circulating neutrophils to interfere with
`binding. The in vitro and in vivo studies reported here indicate that such an approach will mediate killing of
`significant numbers of tumor cells, will have little if any toxicity, and, as an adjunct to other therapies, could
`increase the proportion of patients with tumor or leukemia who achieve long-term disease-free survival. From
`the standpoint of clinical research, one complication of studies using the BsAb is that dissociating the effects
`of the monospecific PM81 (which mediates complement-dependent killing) from those of the BsAb
`PM81 x 32 (which mediates both complement dependent killing and ADCC), may require a comparison of
`therapy using PM81 alone versus therapy with the BsAb.
`Although a very recent development, BsAbs have been used in several other Phase I clinical trials with
`In one trial, 12 patients with lung, ovarian, or breast carcinoma were treated with
`encouraging results.
`intraperitoneal or intrapleural infusions of activated T lymphocytes targeted with an anti-CD3 x anti-tumor
`(MOC31 ) BsAb (deLeij et al, 1991 ). These targeted cells induced considerable local lysis of tumor cells and
`a mild inflammatory reaction, but no toxic side effects or anti-mouse antibody responses.
`In a very
`preliminary trial of an anti-CD3 x anti-CD19 BsAb in a patient with a B-cell malignancy, significant
`reduction in peripheral tumor cell counts was also achieved (Clark et al, 1991). In the most promising and
`extensive series of studies to date, IL-2-activated lymphocytes and an anti-CD3 x anti-glioma BsAb were
`injected intracranially in 20 patients with malignant glioma (Nitta et al, 1990). After 2 years, 76% of patients
`treated with BsAb and lymphocytes were tumor free, as compared with 33% of patients given IL-2-activated
`lymphocytes and no BsAb.
`In summary, our results and those cited above indicate that BsAb may be most efficacious for high-risk
`tumors after induction of remission, after bone marrow transplant, and for treatment of minimal residual
`disease.
`
`REFERENCES
`
`Anderson, C.Guyre, P., Whitin, J., Ryan, D.H., Looney, R.J., and Fanger, M.W. (1986): Monoclonal antibodies to Fe
`receptors for IgG on human mononuclear phagocytes. J. Biol. Chem. 261: 12856.
`Ball, E., Graziano, R., and Fänger, M. (1983): A unique antigen expressed on myeloid cells and acute leukemia blast cells
`defined by a monoclonal antibody. J. Immunol. 130: 2937.
`Ball, E.D., Mills, L.E., Cornwell, G.G., Davis, B.H., Coughlin, CT., Howell, A.L., Stukel, T.A., Dain, B.J.,
`McMillan, R., Spruce, W., Miller, W.E., and Thompson, L. (1990): Autologous bone marrow transplantation for
`acute myeloid leukemia using monoclonal antibody-purged bone marrow. Blood 75: 1199.
`Brockhaus, M., Magnani, J., and Herlyn, M. (1982): Monoclonal antibodies directed against the sugar sequence of
`lacto-N-fucopentaose III are obtained from mice immunized with human tumors. Arch. Biochem. Biophys. 217: 647.
`Clark, M., Bolt, S., Tunnacliffe, A., and Waldman, H. Use of bispecific monoclonal antibodies to treat hematological
`malignancies: a model system using CD3 transgenic mice. In Bispecific Antibodies and Targeted Cellular Cytotoxicity,
`eds. Romet-Lemonne, J.L., Fanger, M.W., and Segal, D.M.: 1991, pp243-247.
`deLeij, L., de Jonge, M., Ter Haar, A., Spakman, H., The, H., de Vries, L., Mulder, N., Berendsen, H., Elias, M., and
`Smit-Sibinga, C. Intrapleural and intraperitoneal application of bispecific antibody retargeted lymphocytes to cancer
`patients. In Bispecific Antibodies and Targeted Cellular Cytotoxicity, eds. Romet-Lemonne, J.L., Fänger, M. W., and
`Segal, D.M.: 1991, pp249-253.
`Dillman, R. (1989): Monoclonal antibodies for treating cancer. Ann. Int. Med. Ill: 592.
`Green, L., Reade, J., and Ware, C. (1984): Rapid colorimetric assay for cell viability: application to the quantitation of
`cytotoxic and growth inhibitory lymphokines. J. Immunol. Meth. 70: 257.
`Guyre, P., Graziano, R., Vance, B., Morganelli, P., and Fänger, M. (1989): Monoclonal antibodies that bind to distinct
`epitopes on FcyRI are able to trigger receptor function. J. Immunol. 143: 1650.
`Howell, A. and Ball, E. (1985): Monoclonal antibody mediated cytotoxicity of human myeloid leukemia cells: an in vitro
`model for estimating efficiency and optimal conditions for cytolysis. Blood 66: 649.
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`BALL ET AL.
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`Huang, L., Brockhaus, M., Magnani, J., Cuttitta, F., Rosen, S., Minna, J., and Ginsburg, V. (1983): Many monoclonal
`antibodies with an apparent specificity for certain lung cancers are directed against a sequence found in lacto-N-
`fucopentaose III. Arch. Biochem. Biophys. 220: 318.
`Lübeck, M., Steplewski, Z., Baglia, F., Klein, M., Dorrington, K., andKoprowski, H. (1985): The interaction of murine
`IgG subclass proteins with human monocyte Fc receptors. J. Immunol. 135: 1299.
`Nitta, T., Sato, K., Yagita, H., Okumura, K., and Ishii, I. (1990): Preliminary trial of specific targeting therapy against
`malignant glioma. The Lancet 335: 368.
`Ortaldo, J., Woodhouse, C, Morgan, A., Herberman, R., Cheresh, D., and Reisfeld, R. (1987): Analysis of effector
`cells in human antibody-dependent cellular cytotoxicity with murine monoclonal antibodies. J. Immunol. 138: 3566.
`Shen, L., Guyre, P.M., Anderson, C.L., and Fanger, M.W. (1986a): Heteroantibody-mediatedcytotoxicity: antibody to
`the high affinity Fc receptor for IgG mediates cytotoxicity for human monocytes that is enhanced by interferon-g and is
`not blocked by human IgG. J. Immunol. 137: 3378.
`Shen, L., Guyre, P., Ball, E., and Fänger, M. (1986b): Glucocorticoid enhances gamma interferon effects on human
`monocyte antigen expression and ADCC. Clin. Exp. Immunol. 65: 387.
`Stepan, D., Bartholomev/, R., and LeBien, T. (1984): In vitro cytodestruction of human leukemia cells using murine
`monoclonal antibodies and human complement. Blood 63: 1120.
`Steplewski, Z., Sun, L., Shearman, C, Ghrayeb, J., Daddona, P., and Koprowski, H. (1988): Biological activity of
`human-mouse IgGl, IgG2, IgG3, and IgG4 chimeric monoclonal antibodies with antitumor specificity. Proc. Nati.
`Acad. Sei. USA. 85: 4852.
`Van de Winkel, J.G.J. and Anderson, C.L. (1991): Biology of human immunoglobulin Fc receptors. J. Leukocyte Biol.
`49:511.
`Vredenburgh, J., Simpson, W., Memoli, V., and Ball, E. (1991): Reactivity of anti-CD15 monoclonal antibody PM81
`with breast cancer and elimination of breast cancer cells from human bone marrow by PM81 and immunomagnetic
`beads. Cane. Res. 51: 2451.
`
`Address reprint requests to:
`Edward D. Ball, M.D.
`Division ofHematologylBone Marrow Transplantation
`Montefiore University Hospital
`University of Pittsburgh Medical Center
`3459 Fifth Ave.
`Pittsburgh, PA 15213
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