serotherapy of a patient with a monoclonal antibody directed … · lee m. nadler,2 philip...

[CANCER RESEARCH 40, 3147-3154, September 1980]0008-5472/80/0040-OOOOS02.00

Serotherapy of a Patient with a Monoclonal Antibody Directed against aHuman Lymphoma-associated Antigen1

Lee M. Nadler,2 Philip Stashenko, Russell Hardy, William D. Kaplan, Lawrence N. Button, Donald W. Kufe,

Karen H. Antman, and Stuart F. Schlossman

Divisions of Tumor Immunology Â¡L.M. N.. P. H., S. F. S./. Nuclear Medicine [W. D. K.J. and Medical Oncology ID. W. K., K. H A.Â¡.Sidney Farber Cancer Institute.Harvard Medical School: the Department of Hematology, Children s Hospital Medical Center Â¡L.N. B.I; and the Department of Immunology. Forsyth Dental CenterÂ¡P.S.]. Boston, Massachusetts 02115

ABSTRACT

A preliminary serotherapeutic trial was undertaken with amonoclonal antibody designated antibody 89 (Ab 89) directedagainst a lymphoma-associated antigen. In vitro studies demonstrated that Ab 89 could mediate complement-dependentlysis and macrophage adherence but not antibody-dependentcell-mediated cytotoxicity. To evaluate toxicity and therapeutic

efficacy, two courses of Ab 89 were administered to a patientwith an Ab 89-reactive tumor. Transient decreases in the

number of circulating tumor cells and the appearance of circulating dead cells were noted with the infusion of Ab 89.Following administration of 150 mg or more of Ab 89, smallamounts of antibody could be demonstrated on circulatingtumor cells at a time when no free antibody was found in theserum. The inability to deliver a significant amount of Ab 89 totumor cells in vivo is thought to be secondary to a circulatingtumor antigen. Following each infusion, the amount of thisblocking antigen decreased but could not be entirely clearedfrom the serum. This study provides preliminary evidence forthe lack of clinical toxicity of a monoclonal antibody andidentifies circulating blocking antigens as a significant obstacleto serotherapy.

INTRODUCTION

Passive administration of antibody in an attempt to producetumor regression has been pursued sporadically in the treatment of human malignant disease for more than 50 years (6,10, 21, 23, 39, 43). Recently, such trials have been limitedwith accumulation of evidence that cell-mediated rather than

humoral immunity is important in tumor resistance (5, 14) aswell as findings that passive antibody may enhance rather thaninhibit tumor growth (26, 33). Nevertheless, numerous reportsin the literature have demonstrated the antitumor effects ofpassive antibody administration in both animal model systemsand humans (9, 11, 28, 34, 37, 38, 42).

In general, attempts at serotherapy have resulted in severesystemic side effects and only transient tumor response (7, 21,29, 34, 39). One major reason for this may have been thedifficulty in preparing xenogeneic antisera with sufficient specificity and titer against the putative tumor antigen. More recently, the development of antisera by the technique of somaticcell hybridization has potential for producing very-high-titered

' This work was supported in part by NIH Grants Al 12069. CA 19589. CA

06516, RR 05526, and DE 04881.2 To whom requests tor reprints should be addressed, at Division of Tumor

Immunology, Sidney Farber Cancer Institute. 44 Binney Street, Boston, Mass02115.

Received March 26, 1980; accepted May 20, 1980.

antibodies directed against tumor-associated antigens (27). In

a previous paper (35), we reported the development of such amonoclonal antibody [designated Ab 893) directed against the

tumor cells of a patient (N. B.) with DPDL. Extensive screeningon normal and malignant tissues demonstrated that reactivitywas limited to a subset of B-cell non-Hodgkin's lymphomas. Ab89 was shown to be of high titer (10~b) by indirect Â¡mmunoflu-

orescence and complement-mediated lysis on N. B. tumor cells

but not on N. B. normal cells.Patient N. B.'s lymphoma was no longer responsive to stan

dard and experimental chemotherapy. In this setting and giventhe extraordinary specificity of this reagent, serotherapy wasundertaken with Ab 89. An immunoglobulin fraction of Ab 89was administered in 2 separate courses 1 month apart toevaluate the toxicity and therapeutic efficacy of this approach.Our results to be reported below demonstrated that: (a) PatientN. B. tolerated infusion of Ab 89 without any side effects; (b)tumor cells were transiently cleared from the circulation; and(c) a more significant response was probably prevented by thepresence of a circulating blocking antigen.

MATERIALS AND METHODS

Case Report

N. B. was a 54-year-old white male who presented in August

1976 with a 1 year history of diffuse lymphadenopathy, fatigue,and night sweats. Physical examination at presentation revealed generalized adenopathy and marked hepatosplenomeg-

aly. The WBC was 110,000/cu mm with circulating lymphomacells. Biopsy of an inguinal lymph node revealed a histologicalpattern consistent with the diagnosis of DPDL. Initial therapeutic regimens (1976 to 1977) included high-dose methotrexatewith citrovorum factor rescue, bleomycin-Adriamycin-Cytoxan-vincristine-dexamethasone, and high-dose Cytoxan, each re

sulting in lowering of circulating tumor cell count transiently. Inlate 1977, N. B. underwent a splenectomy for persistent hy-

persplenism and splenic pain. He was treated with Leukeranuntil December 1978 when his WBC began to rise rapidly. Hewas then treated in 1979 with several agents, including epi-podophyllotoxin VP-16, W-(phosphonacetyl)-L-aspartate, andthis drug in combination with 5-fluorouracil without any evi

dence of objective tumor response. At that point, when he wasconsidered unresponsive to standard chemotherapy and several Phase I and II agents, serotherapy was undertaken with amonoclonal antibody directed against an antigen on N. B.'s

' The abbreviations used are: Ab 89. antibody 89: DPDL, diffuse, poorly

differentiated lymphocyte lymphoma: FACS, fluorescence-activated cell sorter:ADCC. antibody-dependent cell-mediated cytotoxicity.

SEPTEMBER 1980 3147

on May 8, 2020. © 1980 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

L. M. Nad!ereta!.

tumor cells. Prior to serotherapy, Human Protection Committeevalidation (Protocol 79-032, dated April 10, 1979) and informed consent were obtained.

Tumor Cell Preparation and Storage

Heparinized blood from N. B. was collected at specifiedtimes, and tumor cells were harvested by Ficoll-Hypaque sedimentation (3) (Pharmacia Fine Chemicals, Piscataway, N. J.).Isolated tumor cells were stored at â€”196Â°in the vapor phaseof liquid nitrogen in 10% dimethyl sulfoxide and 20% humanAB serum until the time of characterization.

added, and cells were pelleted at 400 x g for 10 mm. Onehundred ,zIof supematant were counted in a Packard y counter(Packard Instrument Company, Downer's Grove, Ill.). Specific51Crrelease was determined by the following equation:

% of specific 51Cr release =@@ :: x 100%

where Exp is the mean of the observed triplicate, SR is thespontaneous release from cells incubated with complementalone, and MR is the maximum release obtained by treatingcells with the detergent Triton x (1% solution).

Antibody-dependent Cellular Cytotoxicity

Four X 1O@fresh N. B. tumor cells were incubated with 0.4ml of 51Cr(292 @tCi/ml;New England Nuclear) at 37Â°for 60mm and washed twice in ice-cold media. In separate groups,51Cr-labeledtumor cells (5 x 106) were then incubated with a1:100 dilution of monoclonal antibodies including: (a) Ab 89;(b) control ascites; (C) anti-IgM4; (d) anti-sc5;(e) anti-la6; or ( f)anti-$2. In addition, 51Cr-Iabeled N. B. tumor cells (5 x 106)were surface labeled with a 1:40 dilution of a polyspecificrabbit anti-human lymphocyte serum. Each group was incubated with antibody for 30 mm at 4Â°,washed twice, andresuspended at 1 x 1O@cells/mi. Target cells (1 x 1O@cells!well) were placed in V-bottomed microtiter plates (Linbro Scientific, Inc., Hamden, Conn.). Fresh mononuclear cells wereprepared and added as â€œkiller'â€˜cells to each well in effector:target ratios of 40:1 , 20:1 , and 10:1 . Microtiter plates werespun at 120 x g for 5 mm and then incubated at 37Â°for 4 hr.After incubation, plates were spun at 400 x g for 10 mm, and100 @ilof supernatant were counted in a Packard y counter.

Adherence of Ab 89-coated N. B. Tumor Cells to MacrophageMonolayers

Preparation of Macrophage-enriched Monolayers. Peripheral blood mononuclear cells were suspended in Roswell ParkMemorial Institute Medium 1640 supplemented with 20% heatinactivated human AB serum at a concentration of 15 x 106/ml. Two ml of this suspension (30 x 106)were placed in 35- x10-mm plastic Petri dishes (Falcon Plastics, Oxnard, Calif.) andincubated at 37Â°for 4 hr to allow the macrophage fraction tobecome adherent to the dishes. Nonadherent lymphoid cellswere then aspirated from the culture dishes and washed 3times, leaving a monolayer of cells. Adherent cells were >85%reactive with the monocyte-specific antibody anti-Mi (4).

Absorptionof 51CrandAntibody-labeledN.B.TumorCellson Monolayers. One hundred x 106 fresh N. B. tumor cellswere labeled with 0.9 ml of 51Cr for 90 mm at 37Â°.After 2washes, 30 x 1 @6cells were coated with 5 ml of medium, a 1:100 dilution of Ab 89, or a 1:100 dilution of control antibodyfor 30 mm at 4Â°.After 2 additional washes, 10 x 1 @6antibodyor medium-coated 51Cr-labeledN. B. tumor cells were layeredin triplicate onto Petri dishes coated with macrophage monolayers and incubated at 37Â°for 3 hr. Additional control groupsincluded macrophage monolayers incubated with untreated51Cr-IabeledN. B. tumor cells and uncoated Petri dishes incu

4 L. Nadler, manuscript in preparation.

5 V. Raso, manuscript in preparation.

6 L. Nadler, P. Stashenko, A. Hardy, J. M. Pesando, E. M. Yunis, and S. F.

Schlossman. Monoclonal antibodies against HLA-D/DR related la-like antigensin man, submitted for publication.

Detailed methods have been described previously (35). inbrief, normal or tumor cells, thawed or fresh, were washedextensively. Viability exceeded 85% on all populations at thetime of study. Cells were first treated with 0. 15 ml of Ab 89 ata dilution of 1:5000, incubated at 4Â°for 30 mm, and washedtwice. Cells were subsequently reacted with 0. 15 ml of a 1:40dilution of a fluoresceinated lgG fraction of goat anti-mouseIgG pool (Meloy Laboratories Inc., Springfield, Va.) for 30 mm,centrifuged, washed 3 times, and analyzed on the FACS (Becton, Dickinson Electronics Laboratory, Mountain View, Calif.).Background staining was obtained by substituting for Ab 890.15 ml of a 1:5000 dilution of an unrelated lgG2 subclasshybridoma antibody which was unreactive with N. B. tumorcells.

N. B. Serum Blocking Antigen Experiments

The blocking antigen has been described previously in detail(35). In order to assess the amount of serum blocking antigen,100 @s1of Ab 89 or a control antibody at several dilutions (1:500, 1:5,000, or 1:25,000) were incubated either with 100 @lof N. B. serum or human serum at several dilutions (undiluted,1:5, 1:25, 1:50, 1:100, and 1:250). The resulting â€˜â€˜blockedâ€•antibodies were incubated with N. B. tumor cells, and theirreactivity was evaluated by indirect immunofluorescence onthe FACS.

Complement-mediated Lysis

Five x 106 fresh N. B. tumor cells were incubated with 0.3ml of 51Cr(292 @sCi/ml;New England Nuclear, Boston, Mass.)at 37Â°for 90 mm and then were washed extensively withEagle's minimum essential medium and 5% fetal calf serum. InCooke V-bottomed microtiter plates (Dynatech Laboratories,Inc., Alexandria, Va.), 51Cr-labeledcells (2 x 10@)in triplicatewere placed and incubated with 150 @lof dilutions of Ab 89,control antibody, and anti-$@-microgIobuImnfor 60 mm at 200.Anti-fl@,-microglobulmnmonoclonal antibody was derived fromthe same fusion as was Ab 89, and specificity was determinedby reactivity with all cell lines excluding Daudi (no surface $2@microglobulin), ability to block reactivity with purified $2-mic?o-globulin (kindly provided by Dr. C. Terhorst, Sidney FarberCancer Institute), and immunoprecipitation. After incubation,the cells were washed twice. One hundred @dof dilutions offresh N. B. complement (1:2, 1:5, 1:10, and 1:50), humancomplement (1:2, 1:5, and 1:10), and rabbit or guinea pigcomplement (1:5, 1:10, and 1:20) were incubated with N. B.tumor cells at 37Â°for 60 mm. One hundred @lof media were

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Serotherapy with a Monoc!ona! Antibody

bated with 61Cr-IabeledN. B. tumor cells (labeled with medium,Ab 89, or control antibody). At the end of the incubation, plates

were washed gently with 5 ml of medium for each wash, andthe nonadherent cells were sequentially collected. After a totalof 6 washes/plate, 5 ml of cold Roswell Park Memorial InstituteMedium i 640 containing 25 m@EDTA were added to eachdish and incubated for 15 mm on ice. Adherent lymphoidfractions from each dish were recovered by thoroughly scraping the bottom of the dishes with a rubber policeman to disruptthe monolayer. Adherent fractions and washes were collectedseparately from replicate plates and counted in a Packard ycounter.

Antibody Purification and Sterilization

Crude Ab 89 ascites were prepared as described previously(35). Antibody was heat inactivated at 57Â°for 30 mm and spunat 100,000 x g for 2 hr. The Ig fraction was prepared byammonium sulfate precipitation and dialysis under sterile conditions as described previously (24). This preparation waspassed through a 0.45-sm Swinnex filter (Nalge Co., Rochester, N. V.); culturing for bacteria and fungus was negative.Endotoxin was absent by Limu!us lysate assay (MicrobiologicalAssociates, Bethesda, Md.), and pyrogens were excluded byinjection of 100 @dof antibody into rabbits with no resultantfever.

Infusion Method and Clinical Parameters Followed

For each infusion, Ab 89 was placed in 500 ml of 0.9% NaCIsolution and 5% human albumin and infused via a centralcatheter over 6 hr. Prior to the daily infusion, 0.1 ml of a 1:100dilution of Ab 89 was given intradermally, and no dermal orsystemic side effects were noted. Clinical parameters werefollowed prior to and during therapy. In an effort to excludeimmunological, renal, cardiac, pulmonary, and hepatic toxicities, data were collected prior to and at least once a day duringtherapy. Complete blood counts, differential, prothrombin time,partial thromboplastin time, fibrin split products, and reticulocyte counts were obtained prior to therapy and at specifiedtime intervals. A bone marrow was obtained prior to the firstcourse of therapy. To evaluate renal function, parametersincluding urinalysis, blood urea nitrogen, creatinine, 24-hrurine for creatinine clearance, protein, immunoelectrophoresis,urine electrolytes, and uric acid were followed. The patient wascardiac monitored during the entire time of infusion, and creatine phosphokinases and isoenzymes were obtained. Arterialblood gases were followed, and chest X-ray was performeddaily. Serum electrolytes, magnesium, and calcium were followed daily. To exclude hepatic toxicity, a liver-spleen scanwas initially done and then serum albumin, serum glutamicoxaloacetic acid transaminase, lactate dehydrogenase, bilirubin, and alkaline phosphatase were followed daily.

Evaluation of Tumor Cell Viability

To evaluate whether cells were killed by in vivo administrationof Ab 89, 0.1 ml of whole blood from each time point wasmixed with 0.9 ml of phosphate-buffered saline (147 m@NaCl,7.7 mM K2HPO4,and 2.4 mM KH2P04). To this dilution, 5 @Iofdiacetyl fluorescein (400 @g/ml;Eastman Kodak Co., Rochester, N. V.) and 30 @lof ethidium bromide (4 mg/mI in water;Calbiochem, La Jolla, Calif.) were added, and the mixture was

incubated for 10 mm at 20Â°.A wet mount was prepared andread on a fluorescence microscope with epiillumination (CarlZeiss, Inc., Wellesley, Mass.). (Live cells stained bright greenand dead cells stained bright red.)7 Samples prior to, during,and after therapy each day were evaluated for cell death.

In Vitro Evaluation of Serotherapy with Ab 89

Amount of Ab 89 Delivered to N. B. Tumor Cells via Ab 89Infusion. At specifiedtimespriorto and duringthe administration of Ab 89, 5 ml of fresh heparinized blood were drawn fromN. B. Buffy coats were obtained, and 1 to 2 x 106 tumor cells(>95%) were incubated with 0.1 5 ml of fluorescein-conjugatedgoat anti-mouse immunoglobulin for 30 mm at 4Â°.After 2washes, cells were analyzed on the FACS.

Demonstration of Free Ab 89 in N. B. Serum. Cryopreserved N. B. tumor cells from the day prior to antibody treatment were thawed daily during therapy and incubated with N.B. serum samples obtained during therapy for 30 mm at 4Â°.These cells were then washed, stained with fluorescein-conjugated goat anti-mouse immunoglobulin for 30 mm at 4Â°,washed twice, and analyzed on the FACS.

Change in Blocking Antigen during Therapy with Ab 89.N. B. serum from specified times during the administration ofAb 89 was collected. As described earlier in this section, N. B.serum at these time points was tested for blocking activity.

Tumor Cell Survival and Distribution in Vivo

Two x 10@Â°fresh N. B. tumor cells were withdrawn into ananticoagulant citrate dextrose triple pack. Several centrifugations were undertaken to remove: (a) platelet-rich plasma; (b)platelet-poor plasma; and (c) RBC (32). The resulting buffycoat (WBC) was incubated with 750 @zCiof sodium [51Cr]chromate (specific activity, 100 @tCi/ml)for 40 mm at 25Â°.Labeledcells were washed with autologous platelet-poor plasma at4,000 x g for 5 mm, and then 100 ml of a 1:100 dilution of Ab89 in Eagle's minimum essential medium with 5% human serumalbumin were added and placed on a rocking platform for 40mm at 20Â°.Cells were washed twice and then placed in 50 mlof autologous plasma and reinfused into Patient N. B. in 10mm. Blood samples (10 ml) were obtained at 5, 30, and 60 mmand at 2, 4, 8, 24, 48, and 72 hr. One ml of packed cells and1 ml of serum were counted for each time interval. In addition,the RBC were separated by Ficoll-Hypaque sedimentation andcounted for reactivity at times of 30 mm, 2 hr, and 24 hr. Forall tumor cell determinations, the RBC were removed by ammonium chloride lysis.

RESULTS

Ab 89 was produced by somatic cell hybridization against atumor-associated antigen on the DPDL tumor cells from PatientN. B. Hybrid clones were screened for production of antibodyby indirect immunofluorescence. Producer clones were testedfor reactivity on a panel of normal and malignant lymphoidcells, and one clone, designated Ab 89, reacted only with N.B. tumor cells (35). The reactivity of a saturating concentrationof Ab 89 on cryopreserved tumor cells from the time of presentation in 1976 was compared to cryopreserved tumor cellsfrom 1979. As shown in Chart 1, the amount of Ab 89 as

7 H. Shapiro, manuscript in preparation.

SEPTEMBER1980 3149



% of specificlysisAnti-$@-microgIobu

Antibody dilutionAb89un1:1023721:10030661:1,00038481:10,0001938

L. M. Nad!er et a!.

Ab 89 to N. B. tumor cells was blocked by a 1:5 or 1:25 dilutionof N. B. serum but not a dilution of 1:250. Moreover, noconcentration of pooled human serum could produce blocking.

In Vitro Cytotoxicity for N. B. Tumor Cells. To determinewhether Ab 89 could specifically induce killing of N. B. tumorcells in vitro, 3 antibody-dependent reactions were investigated. As shown in Table 2, significant lysis of Ab 89-coatedN. B. tumor cells occurred in the presence of fresh N. B. serumas a source of complement. Although 100% of N. B. tumorcells were reactive with Ab 89 by indirect immunofluorescence,only 38% maximal lysis was achieved at an antibody dilution of1:1000. Approximately the same number of tumor cells werelysed (20 to 36%) when fresh guinea pig, rabbit, or humancomplement were similarly used. In contrast, treatment of N. B.tumor cells with anti-fl2-microglobulin antibody and fresh N. B.serum induced up to 72% lysis.

In the second in vitro cytotoxic reaction, N. B. tumor cellswere coated with a saturating concentration of Ab 89 and thenwere used as targets in an ADCC system. N. B. tumor cellslabeled with Ab 89 were not lysed when used as targets forADCC at effector:target ratios as high as 40: 1. Of interest wasthe finding that N. B. tumor cells coated with other monoclonalantibodies including anti-igM, ic-, Ia-, or $2-microglobulin (including mouse immunoglobulin subtypes lgG,, IgG25, andIgG2b)were also not killed in ADCC. In contrast, when N. B.

Chart 2. Blocking Ab 89 reactivity with N. B. serum (schematic). Varyingdilutions of N. B. serum were incubated with a 1:5000 dilution of Ab 89, and thereactivity with N. B. tumor cells was evaluated. Background fluorescence(â€”â€”â€”â€”)was obtained by incubating N. B. tumor cells with a 1 :5000 dilution of acontrol monoclonal antibody. Normal human serum at a dilution of 1:5 wasincubated with an equal volume of a 1:5000 dilution of Ab 89 (â€”). Whenincreasing amounts of N. B. serum (â€”- â€”â€”,1:250; â€”â€”,1:25; and , 1:5)were incubated with Ab 89, progressively greater blocking of the reactivity of N.B. tumor cells was noted.

Table 2Complement-mediatedIysis of N. B. tumor cells

N. B. tumor cells were labeled with â€œCrand then incubated with severaldilutions of control antibody, Ab 89, and anti-fl,-microglobulln. After 1 hr. severaldilutions of fresh N. B. serum were added as a source of complement andincubated for 1 hr. Cells were pelleted, and an aliquot of supernatant was countedto calculate specific lysis.

V@iiTiNN@FluorescenceIntensify

Chart 1. Schematic immunofluorescence profile of N. B. tumor cells with Ab89. Cryopreserved N. B. tumor cells from 1976 (â€”) and 1979 ( ) werevirtually all reactive with Ab 89. Background fluorescence staining (â€”â€”â€”â€”)wasobtained by incubating N. B. tumor cells from 1976 or 1979 with a 1:5000dilution of ascitic fluid from a mouse given injections of an lgG2 monoclonal

antibody unreactive (control) with N. B. tumor cells.

Table 1

Summary of evidence that Ab 89 is a tumor-associated antigen

1. unreactive with normal lymphoid tissues (0/60): separated peripheral bloodlymphocytes, monocytes, granulocytes, bone marrow, tonsil, lymph node,spleen, thymus, and fetal tissues.

2. Unreactive with normal and tumor cell lines (0/ 13): Epstein-Barr virustransformed B-cell lines, T-lymphoblastoid cell lines, chronicmyelogenous and acute lymphoblastic leukemic lines, and Burkitt'slymphoma lines.

3. Unreactive with mitogen-stimulated peripheral cells (0/8):phytohernagglutinin, concanavalin A, tetanus, pokeweed mitogen, andIipopolysaccharide.

4. Unreactive with cryopreserved leukemic cells (0/20): null and T-cell acutelymphoblastic leukemia, chronic myelogenous leukemia, hairy cellleukemia, and T-cell chronic lymphocytic leukemia.

5. Non-Hodgkin's lymphomas (4/69): unreactive with all T-celI lymphomas (0-PDL and Diffuse Hystiocytic)(0/4), nodular lymphomas, B- or NullDiffuse Hystiocytic, Waldenstrom's, undifferentiated, and Burkitt'slymphoma (0/20); reactIve with chronic lymphocytic Ieukemias (2/17)and B-cell DPDL's (2/18).

6. Unreactive with N. B. separated T-lymphocytes, B-lymphocytes, monocytes.erythrocytes, granulocytes, and platelets.

7. Unreactive with panel of lymphocytes of identical HLA Subtype as N. B.8. Unreactive with nonlymphoid tumors (0/7).

determined by indirect immunofluorescence which bound to N.B. tumor cells decreased between 1976 and 1979. This decrease in fluorescence intensity suggested that the amount ofantigen on the surface of N. B. tumor cells decreased over this3-year interval. In contrast, the other cell surface antigens (s-,DC-, Ia-, fl2-microglobulmn) were unchanged over the identical

time period (data not shown).Table 1 presents a summary of the evidence that Ab 89

defines a tumor-associated antigen. As shown, Ab 89 does notreact with allogeneic or syngeneic normal tissues, mitogenstimulated lymphocytes, fetal tissues, or nonlymphoid tumorcells. In contrast, Ab 89 was reactive with 10% of B-cell chroniclymphocytic leukemias and DPDL's.

It was previously demonstrated that serum from Patient N. B.contained an antigen which specifically blocked the binding ofAb 89 to N. B. and other Ab 89-reactive tumor cells (35). Inaddition, it was shown that this circulating antigen was specifically retained by a Sepharose Ab 89 column but not by othercolumns to which irrelevant monoclonal antibodies were coupled. An estimation of the amount of blocking antigen in N. B.serum was then undertaken. As seen in Chart 2, the binding of

FluorescenceInfensify

3150 CANCER RESEARCH VOL. 40



At#perenceof 5'Cr-Iabeled N. B. tumor cells to macrophagemonolayersN.B. tumor cells were labeled with â€œCrand then incubated with medium,Ab89,

or control antibody. These â€œCr-labeledcells were then layered ontoPetndishesor onto dishes coated with macrophage monolayers (in triplicate)andIncubated

for 3 hr. Afterincubation, nonadherentcells were removed bywashing(6tImes), and then adherent fractions were removed and counted. Therewasnegligibie

sticking of 5'Cr-labeled cells to uncoated Petri dishes(7000 Â±5000).No.

of cpm bound toadherentN.B. 51Cr-Iabeledtumor cells coated with mononuclearcellsMedium

88,000 Â±15,000k1:100 dilutionofAb 89 264,000 Â±36,0001:100 dIlution of control antibody 97,000 Â±12,500a

M@ Â±S.D.

Serotherapy with a Monoc!ona! Antibody

tumor cells were coated with a polyspecific rabbit anti-humanlymphocyte sera, 90% of the cells were Iysed in an ADCCassay. This finding demonstrated that N. B. tumor cells weresensitive to lysis but that Ab 89 and the other monoclonalantibodies tested could not facilitate ADCC.

Lastly, we determined whether Ab 89-coated tumor cellscould adhere to a macrophage monolayer. As shown in Table3, N. B. tumor cells were approximately 3 times more adherentto macrophage monolayers than were N. B. tumor cells treatedwith medium or an unreactive monoclonal antibody.

Therapeutic Trials with Ab 89. The administration of Ab 89to Patient N. B. was undertaken to evaluate possible clinicaltoxicity and/or potential therapeutic efficacy of this hybridomaantibody. Detailed clinical and laboratory data (see â€˜â€˜Materialsand Methodsâ€•)were monitored. During the antibody administration, it was determined whether: (a) tumor cells were killed;(b) Ab 89 could be demonstrated on the surface of circulatingN. B. tumor cells following infusion; (C) following infusion, free

Ab 89 was present in the circulation; and (d) a decrease in

circulating antigen occurred.Patient N. B. was infused with Ab 89 daily for 3 days.

Following the infusion of 25 mg of Ab 89 on Day 1, there wasno evidence of clinical toxicity or decrease in the WBC count(Chart 3). In addition, Ab 89 could not be detected on thecirculating tumor cells, nor was free antibody detected in N. B.serum. Furthermore, no change in the amount of circulatingantigen was demonstrated when the capacity of N. B. serum toinhibit the binding of Ab 89 to N. B. tumor cells was tested.

Table 3

FluorescenceIntensify

Chart 4. Amount of Ab 89 delivered to N. B. tumor cells in vivo. Seventy-fivemg of Ab 89 were administered to Patient N. B. The amount of Ab 89 deliveredto the tumor cell surface as measured by staining N. B. tumor cells withfluorescein-conjugated goat anti-mouse immunoglobulin (â€”â€”) was slightlygreater than the fluorescence intensity of a 1:100,000 dilution of Ab 89 (on N. B. tumor cells. Background staining on N. B. tumor cells was accomplishedwith a 1:5000 dilution of a control antisera (â€”â€”â€”â€”),and maximal fluorescencestaining was achieved with a 1:5000 dilution of Ab 89 (â€”).

On the second day, 75 mg of Ab 89 were infused andproduced a transient fall in the circulating tumor cell count(from 388,000 to 240,000/cu mm). The number of nonviablecirculating tumor cells was estimated by both ethidium bromideand fluorescein diacetate uptake. Both methods showed thatthere were only 4% circulating nonviable cells prior to infusioncompared to 30% dead cells at the end of the infusion. Following the infusion of Ab 89, Patient N. B. complained for the firsttime of lymph node and liver tenderness. At this time, there wasa drop in the creatinine clearance from 86 to 38 mI/mm, andsimultaneously there was a decrease in the circulating antigen(complete blocking at a 1:10 dilution of N. B. serum). Ab 89was now detectable on the surface of N. B. tumor cells, but stillno free antibody was present in the serum. The amount of Ab89 on the circulating tumor cells was equivalent to a 1:50,000dilution of Ab 89 (Chart 4). Thus, most of Ab 89 infused wasnot present on the circulating tumor cell surface. To determinewhether or not Ab 89 was lost from the tumor cell surface andwhether these tumor cells could bind additional Ab 89, N. B.tumor cells obtained prior to, during, and after infusion werereacted with a saturating concentration of Ab 89. These studiesdemonstrated that N. B. tumor cells obtained during the infusion could bind additional antibody. Moreover, when thesecells were maximally saturated with antibody, the amount ofsurface antigen 89 was similar whether the cells were obtainedprior to, during, or after infusion.

Given the fact that maximal surface staining with Ab 89 wasnot achieved, the patient was next given 150 mg of Ab 89 onDay 3. Once again, as seen in Chart 3, there was a transientdrop in the number of circulating tumor cells accompanied bythe identification of 19% dead cells circulating (compared to3% prior to therapy). Circulating N. B. tumor cells were againfaintly stained with Ab 89, and no free antibody was detectedin the serum. In contrast to the previous studies, a very significant decrease in circulating antigen was demonstrated (complete blocking at a 1:5 dilution of N. B. serum), as shown inChart 5. The creatinine clearance dropped to 56 mI/mm.Lymph node and liver tenderness was noted, but no other

Hours

3151

25mg 75mg 50mgAb89 Ab89 Ab89â€” â€” â€”

Chart 3. WBC during serotherapy of Patient N. B. with Ab 89. Ab 89 wasgiven daily for 3 days. Each infusion was administered over 6 hr with frequentmonitoring of the WBC. The WBC fell transiently during the infusion on thesecond and third days.

SEPTEMBER1980



L. M. Nad!er et a!.

clinical toxicity was noted. On the following day, the WBC hadreturned to 392,000/cu mm, and the creatinine clearance roseto 82 mI/mm. The above studies suggested that the majorlimitation to the delivery of Ab 89 to circulating N. B. tumorcells was the presence of the circulating blocking antigen.

One month later, the patient's WBC was 320,000/cu mm. Afinal course of Ab 89 was given to determine whether a veryhigh dose of antibody could: (a) remove circulating antigen;and (b) be delivered to the tumor cell surface. After an infusionof 1.5 g of antibody over a 6-hr period, there was only atransient fall in the WBC to 200,000/cu mm, and only faintstaining of the tumor cell surface was demonstrable. The quantity of antibody bound to the tumor cell was equivalent to a 1:50,000 dilution of Ab 89 and was not significantly differentfrom that seen with 150 mg of Ab 89. No free antibody wasfound, but now there was a marked reduction of circulatingantigen so that complete blocking was detected at only a 1:2dilution of N. B. serum (complete blocking prior to this infusionwas at a 1:50 dilution of N. B. serum). The patient toleratedthis infusion without side effects except for a transient decreasein creatinine clearance to 54 mI/mm. These studies suggestedthat the quantity of circulating antigen was too great to effectively deliver Ab 89 to the patient's tumor cells.

To determine whether optimally antibody-coated tumor cellswould be cleared from the circulation, 51Cr-labeledtumor cellswere coated with a saturating dilution of Ab 89 in vitro andreinfused into the patient. The distribution and survival of thesecells was then examined. Chart 6 illustrates the number ofcirculating 51Cr-labeled cells in the leukocyte fraction of 1 mlof whole blood at varying time intervals. All the 51Crwas foundin the WBC fraction, and there was no evidence of free 51Crinthe serum. After an initial equilibration, there was a slow andgradual decrease in the number of circulating labeled cellsover 72 hr. Scintigraphic images were performed daily todetermine the sites of accumulation of the 51Cr-and Ab 89-labeled tumor cells. Fig. 1 demonstrates that initially radiochromium was primarily localized in the blood pool (Day 1).

Over the ensuing 72 hr, the tracer within the cardiac blood pooldecreased and localized in the liver, suggesting hepatic clearance. No detectable collection of radionuclide was seen in thepatient's massively enlarged paraaortic lymph nodes. Previous

in

splenectomy precluded radionuclide accumulation within thisorgan. At the end of this initial 72-hr period, most of theradiochromium was localized in the liver, suggesting hepatictrapping without lysis. Because of the rapid progression of thepatient's disease despite continuing chemotherapy, no controlsurvival studies using 51Cr-Iabeledtumor cells without surfaceAb 89 were undertaken. One can only present several historicalcontrols of the survival of 51Cr-labeledtumor cells (acute lymphoblastic leukemia, chronic lymphocytic leukemia, andchronic myelogenous leukemia) from studies in the literature(8, 31).

DISCUSSION

In this study, we present a case report of a serotherapeutictrial in which a monoclonal antibody against a human tumorassociated antigen was used. Previous studies indicated thatAb 89 was reactive with the tumor cells from Patient N. B. anda small subgroup of patients with B-cell non-Hodgkin's lymphoma but not with other tumors, normal lymphoid cells, ornormal cells from N. B. !n vitro characterization of Ab 89demonstrated that this antibody mediated complement-dependent lysis and macrophage adherence but failed to mediateADCC. A total dose of 2 g of Ab 89 was then administered tothe patient in 2 courses separated by a 3-week interval. Administration of Ab 89 led to transient decreases in circulatingtumor cells and increases in circulating dead tumor cells. It wasimportant to note that no significant clinical toxicity was observed. Following infusions, Ab 89 could be found on the tumor

Hours Post Infusion

Chart 6.@ â€˜Survival@ of Ab 89-coated N. B. tumor cells. N. B. tumor cells were5Cr-iabeled in vitro and then coated with a 1:100 dilution of Ab 89 and infusedinto Patient N. B. At several time intervals, whole blood was drawn, and theamount of 5Cr was measured in the serum and in the WBC and RBC. After aninitial distribution phase, the percentage of 51Cr-labeledtumor cells graduallydecreased over 72 hr (â€”). Historical control curves for the â€˜â€˜survivalâ€•of â€œCrlabeled acute lymphoblastic leukemia (â€”â€”â€”â€”),chronic lymphocytic leukemia( ), and chronic myelogenoma leukemia (â€”â€”)cells are presented forcomparison.

Preâ€”Ab89Infusion

FluorescenceIntensity

Chart 5. Decrease in blocking activity during serotherapy (schematic). Varying dilutions of N. B. serum obtained prior to and after infusion of 150 mg of Ab89 were incubated with a 1:5000 dilution of Ab 89, and reactivity with N. B.tumor cells was evaluated. Background fluorescence (â€”â€”â€”â€”)was obtained byincubating N. B. tumor cells with a 1:5000 dilution of control ascites. Normalhuman serum at a dilution of 1:5 was incubated with an equal volume of a 1:5000 dilution of Ab 89 (â€”). After Ab 89 infusion, the amount of blockingantigen decreased in N. B. serum at dilutions of 1:5 ( ) and 1:25 (â€”â€”).

CANCERRESEARCHVOL. 403152

Post- Ab89 Infusion



Serotherapy with a Monoclonal Antibody

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Fig. 1. Tumor cells were labeled with 5'Cr and Ab 89 in vitro and reinfused into Patient N. B. on Day 1. On each day, scintophotographic images of the thorax andabdomen were collected on a Searle large-field-of-view â€˜ycamera fitted with a medium-energy parallel-hole collimator. Images were simultaneously collected on aPDP 11/40 computer in a 64 x 64 matrix mode. Digital images, normalized for counts and time, show that initial radioactivity within the cardiac blood pool slowlydissipated to localize within the liver over the 4 days of the study.

have been due to the purity and restricted specificity of Ab 89.One of the major obstacles to serotherapy in Patient N. B.

appears to be the presence of a circulating blocking antigen.In animal tumor models, several blocking factors have beenextensively studied (15, 16, 20, 40) and, in humans, preliminary descriptionsof blockingfactors havebeen reported(13,1 7â€”19). These reports suggest that blocking factors are a

combination of circulating tumor antigens and circulating antigen-antibody complexes, and it has been suggested that thesefactors provided one mechanism for tumor cell escape fromantibody-mediated tumor cell rejection (2, 12, 41). The presentstudy demonstrated that binding of Ab 89 to N. B. tumor cellscan be blocked in vitro by a circulating antigen. More importantly, following the infusion of Ab 89 in N. B., there was onlyminimal tumor cell binding, and there was no free circulatingantibody. With the infusion of 1.5 g of Ab 89, there was amarked decrease in circulating blocking antigen but there wasalso a significant quantity of blocking antigen still present.Given the immense tumor burden in this patient, it is possiblethat the circulating antigen represented tumor breakdown products rather than shed tumor antigen. In any case, one couldnot totally eliminate circulating antigen by the infusion of antibody. The patient's low platelet count and debilitated statusprecluded the removal of the circulating antigen by leukapheresis or exchange transfusion prior to antibody infusion. Moreover, there is no evidence in animal model systems that blocking antigen can be removed from the blood or lymph node bythese methods (25, 36).

Serotherapy has been previously attempted in several humantumors, including some of the non-Hodgkin's lymphomas (9,28, 34, 42). The administration of allogeneic antilymphocyte

cell surface, although its density was very low. Circulatingtumor antigen decreased following large doses of Ab 89;however, no circulating free antibody was seen at any time inthe patient's serum. These studies suggested that the circulating antigen and the large tumor mass may have prevented thebinding of a significant amount of Ab 89 to N. B. tumor cells.

The precise mechanisms by which Ab 89 caused transientdecreases in circulating N. B. tumor cells are not known. Therapid appearance of circulating dead cells suggested thatcomplement-mediated lysis may have contributed to tumor cellkilling. The appearance of lymph node and hepatic tendernessand the evidence that 51Cr-labeled Ab 89-coated tumor cellswere removed slowly by the liver would alternatively suggestthat the reticuloendothelial clearance of antibody-coated tumorcells was primarily responsible. However, the extraordinarytumor burden as well as a compromised reticuloendothelialsystem (secondary to splenectomy and tumor infiltration) coupled with the large quantity of circulating antigen obviouslyobscured the precise mechanism by which the antibody wasâ€â€˜partially effective.â€•

Ab 89 was administered without evidence of significant toxicity. Following each antibody infusion, transient decreases inthe creatinine clearance were noted. These decreases werepresumably due to antigen-antibody complexes deposited inthe kidney, and it is important to note that the renal functionreturned to normal within 24 hr. The total amount of Ab 89 (2g) infused was greater than the range where serum sickness isfrequently reported (30); however, no symptoms developed inN. B. after these infusions. The absence of either acute allergicreactions or serum sickness may have been due to the â€˜â€˜compromised' â€˜immune status of this patient or alternatively may

SEPTEMBER 1980 3153

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sera has led to reports of decrease in circulating leukemic cellsand shrinkage of parenchymal organs (1, 22, 34). In most ofthese series, tumor responses have been reported only withminimal tumor burden. Similar observations have been reportedin animal systems (11, 37, 38). A number of factors probablycontributed to the lack of response to Ab 89 including: (a) sizeof tumor burden; (b) quantity of surface tumor antigen; and (C)quantity of circulating antigen. While this patient did not respond significantly, better effects might be achieved in patientswith a smaller tumor burden and less circulating antigen. Nofurther serotherapeutic trials with Ab 89 have been attemptedbecause of the rarity of expression of this antigen on B-cellIymphomas. it is hoped that other monoclonal antibodies defining more common lymphoma-associated antigens will soon bedescribed. In any case, it is possible that monoclonal antibodiesmay eventually be used successfully for immune therapy considering their high titer, specificity, and unlimited availability.Moreover, these antibodies may be used as agents to specifically deliver cytotoxic agents to the tumor cell surface.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Charles Dinarello for his help in demonstrating that Ab 89 was endotoxin and pyrogen free. We would also like to thankJoan McDowell and Carla Golden for technical assistance, and L. M. Grappi forher secretarial assistance during the preparation of this manuscript.

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1980;40:3147-3154. Cancer Res Lee M. Nadler, Philip Stashenko, Russell Hardy, et al. against a Human Lymphoma-associated AntigenSerotherapy of a Patient with a Monoclonal Antibody Directed

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