Monoclonal Antibodies Reactive With Small Cell
`Carcinomaof the Lung*”’
`
`Edward D. Ball,*°.° Robert F. Graziano, Olive S. Pettengill,’
`George D. Sorenson,’ and Michael W. Fanger*5
`
`ABSTRACT—Murine monoclonal antibodies (MoAb) reactive with
`antigens associated with small cell carcinoma of the lung (SCCL)
`were prepared and partially characterized. Four were selected for
`further study on the basis of their lack of reactivity with normal
`leukocytes and erythrocytes. These MoAb, designated SCCL-41,
`SCCL-114, SCCL-124, and SCCL-175, are all IgM immunoglobu-
`lins. The binding of these MoAb to patient-derived SCCL tumor
`cells, SCCL cell
`lines, and non-SCCL cell lines was studied by
`indirect
`immunofluorescence and flow cytometry. Considerable
`heterogeneity in the expression of these cell surface antigens was
`noted among both the patient-derived tumor cells and the SCCL
`ceil
`lines. One of the MoAb, SCCL-175, reacted with 7 of 7
`patient-derived tumor cell samples and 9 of 10 SCCL cell lines.
`None of the antigens defined by these MoAb were expressed on
`non-SCCL lung tumor cell
`lines. SCCL-175 reacted with cells
`from both a choriocarcinoma and a colon carcinoma cell tine,
`whereasthe other 3 MoAb were unreactive with these and several
`
`in the
`lines. These MoAb may be useful
`tumor cell
`other
`diagnosis and subclassification of SCCL tumors.—JNCI! 1984;
`72:593-598.
`
`SCCL is a heterogeneous disease in which several
`morphologic subclasses of tumor cells have been identi-
`fied (7, 2). In addition, SCCL tumorsoften contain foci
`of non-SCCL lung tumorcells (3, 4). To study these
`and other parameters of heterogeneity in SCCL with
`specific probes, as well as to develop improved abilities
`to diagnose and treat
`this disease, we have prepared
`MoAb reactive with cells from SCCL tumors. Cells
`from patients with SCCL and cell
`lines derived from
`patients with SCCL were found to be reactive with
`these MoAb. In this paper we describe the preparation
`and specificity of these MoAb.
`
`MATERIALS AND METHODS
`
`Cell lines. ——Cell lines studied included DMS 44, 47,
`53, 79, 153, 187, 235, 406, 431, and 483, all of which
`were derived from patients with SCCL and have pheno-
`typic characteristics of SCCL (5-7). These cell
`lines
`were cultured in either Waymouth’s MB 752/1 medium
`containing 20% FBS (DMS 44, 53, 153, 187, 235, 406,
`431, and 483) or RPMI-1640 medium with 20% FBS
`(DMS79).
`Non-SCCL tumorcell lines studied included IMR 32,
`a neuroblastoma line (obtained from the ATCC, Rock-
`ville, Md.); DMS 351, a malignant melanomacell line
`(isolated from lymph node biopsy specimen); Squ Ca, a
`squamouscell lung carcinoma cell line (provided by K.
`Havemann and C. Gropp, Marburg, Federal Republic
`of Germany); DMS 485, a large cell undifferentiated
`
`lung tumorcell line (isolated from pleural fluid); A549,
`an adenocarcinoma lung tumorcell line (obtained from
`ATCC) (8); Ca Lu-1, a squamouscell lung carcinoma
`line (ATCC); SK-Lu-1, a lung adenocarcinomacell line
`(obtained from Dr. J. Fogh, Sloan Kettering Cancer
`Institute)
`(9); BeWo,
`a choriocarcinoma cell
`line
`(ATCC); DLD-1, a colon carcinomacell line (provided
`by D. Dexter, Providence, R.I.)
`(J0); and HE-lung,
`embryonic lung fibroblasts (obtained from M.A. Bio-
`products, Walkersville, Md.). Ca Lu-] was cultured in
`McCoy’s 5-A medium (GIBCO, Grand Island, N.Y.)
`with 10% FBS. A549, SK-LU-1, IMR 32, and HE-lung
`were cultured in Dulbecco’s modified Eagle minimum
`essential medium with 10% FBS (HE-lung; 16% FBS);
`BeWo, DLD-1, DMS 485, and DMS 351 were all
`cultured in RPMI-1640 with 20% FBS.
`The following leukemia cell lines were also studied;
`HL-60, a promyelocytic leukemia cell
`line (obtained
`from R. C. Gallo, National
`Institutes of Health,
`Bethesda, Md.) (71); K562, an undifferentiated myeloid
`leukemia cell line (12); Daudi, and Epstein-Barr virus-
`transformed B-cell line (13); and CCRF-CEM, a T-cell
`leukemia line (/4). These lines were all cultured in
`RPMI-1640 with 20% FBS.
`from patients with
`Patient cells——Tumor cells
`SCCL were obtained from either
`surgical biopsy
`specimens (3 patients) or at autopsy performed within 4
`hours of death (3 patients). Cells freshly isolated from
`
`ABBREVIATIONS USED: ATCC=American Type Culture Collection;
`AZ=sodium azide; BSA=bovine serum albumin; FBS=fetal bovine
`serum; MoAb=monoclonal antibody (antibodies); PBS=phosphate-
`buffered saline; RIA=radioimmunoassay; SCCL=small cell carcinoma
`of the lung.
`
`' Received June 16, 1983; accepted November 8, 1983.
`? Supported in part by Public Health Service (PHS) grants
`CA-31918, CA-31888, and CA-25845 from the National Cancer
`Institute and by PHS grant AI-19053 from the National Institute of
`Allergy and Infectious Diseases. The Cytofluorograph was
`the
`generous gift of the Fannie E. Rippel Foundation and is partially
`supported by the Norris Cotton Cancer Center core grant CA-23108.
`*This study was presented in part at
`the annual meeting of the
`American Federation for Clinical Research, Washington, D.C.,
`May 2, 1983, and appeared in abstract form in Clinical Research, Vol.
`31, No. 2, p. 402A, 1983.
`“Department of Medicine, Dartmouth Medical School, Hanover,
`N.H. 03756.
`* Department of Microbiology, Dartmouth Medical School.
`® Address reprint requests to Dr. Ball, Department of Microbiology,
`Dartmouth Medical School.
`"Department of Pathology, Dartmouth Medical School.
`
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`594 Ball, Graziano, Pettengill, et al.
`
`to
`lacto-N-fucopentaose HI,
`terminal pentasaccharide,
`which several reported anti-SCCL MoAbreact (17-19),
`we performed inhibition studies. Purified LNF II
`(provided by V. Ginsburg, National
`Institutes of
`Health, Bethesda, Md.) was incubated with each of the
`MoAb and a known positive control MoAb reactive
`with LNF III, PM-81 (20), at a concentration of 5
`mg/ml for 1 hour before addition of these mixtures to
`SCCL tumorcells from patient B (text-fig. 2). Quanti-
`tative comparisons of the binding of each MoAbeither
`in the presence or absence of LNF III was determined
`by indirect immunofluorescence and flow cytometry.
`
`RESULTS
`
`primary or metastatic tumors, which in all cases were
`densely involved with SCCL, were gently teased apart
`into single-cell
`suspensions and passed through a
`stainless-steel filter. Cell viability was assessed by stain-
`ing with acridine orange and ethidium bromide. Only
`samples with more than 50% viable cells were included
`in this study.
`leukocytes were obtained
`Normal cells—Normal
`from volunteers and separated into granulocyte, mono-
`cyte, and lymphocyte fractions as previously described
`(15). Erythrocytes typed with antisera to Lewis A and B
`blood group antigens were obtained from the Blood
`Bank of Mary Hitchcock Memorial Hospital, Hanover,
`N.H.
`Preparation of hybridomas.—BALB/c mice were
`immunized ip three times over a 3-month period with
`Specificity of hybridomas.—Thereactivity of the 4
`2x10" cells, which had been dissociated from the
`MoAb with the immunogen is shown in text-figure |}.
`Intense but variable fluorescence was observed with
`primary lung tumor of a patient with SCCL. This
`tumor was classified as “intermediate’’ in the modified
`each MoAb. None of
`the MoAb reacted at all by
`World Health Organization classification (2). Fusion of
`indirect immunofluorescence with lymphocytes, mono-
`spleen cells from an immunized mouse with murine
`cytes, or granulocytes from the patient from whom the
`myelomacells of the NS-1 cell line was performed with
`primary tumor was obtained or from 6 normal donors
`(data not shown), thus indicating that they are probably
`the use of polyethlene glycol as the fusing agent as
`previously described (16). Hybridomas making MoAb
`not reactive with histocompatibility or other common
`reactive with the immunogen were selected by solid-
`cell surface antigens. In addition, none of the MoAb
`reacted with erythrocytes from 4 normal donors (2
`phase RIA with the use of glutaraldehyde-fixed cells as
`Lewis B and 1 Lewis A-positive, and | Lewis antigen-
`previously described (15). Of these, 4 were selected for
`more extensive study on the basis of
`their relative
`negative). The reactivities of
`these MoAb with cells
`specificity for the immunogen and lack of reactivity
`from the primary lung tumor of a second patient with
`with normal leukocytes. These hybridomas were desig-
`SCCL are shown in text-figure 2.
`In addition,
`liver
`nated SCCL-41, SCCL-114, SCCL-124, and SCCL-175.
`metastases from the same patient were examined and
`found to have a similar antigenic profile, although
`All 4 of the MoAb produced were IgM antibodies.
`staining was less intense in each case. A summary of
`Indirect
`immunofluorescence and cytofluorographic
`the reactivities of the MoAb with tumorcells obtained
`analysis.—The reactivity of these MoAb with SCCL
`cells was determined by indirect
`immunofluorescence
`fromatotal of 7 sites from 6 different patients is shown
`in table 1.
`and flow cytometry. Cells freshly isolated from primary
`or metastatic tumors were prepared as described above.
`Reactivity with SCCL cell lines.—-The expression of
`Adherent cell
`lines were dissociated from the culture
`these antigens on several SCCL cell lines developed by
`flask after 10-minute incubation with 0.01% EDTA
`Pettengill et al.
`(5) was evaluated. Consistent with
`disodium in calcium and magnesium-free Hank’s
`other evidence of phenotypic heterogeneity in these
`balanced salt
`solution followed by washing with
`cell lines, including differential peptide hormonesecre-
`RPMI-1640. Cells were incubated for 30 minutes at 4°C
`tion (6, 21, 22) and morphology (5), we have found
`with 0.5 ml hybridoma supernatant, unbound MoAb
`considerable heterogeneity in antigen expression. As
`were removed by washing with 7 volumes of PBS (pH
`examples,
`the reactivities of MoAb SCCL-124 and
`SCCL-175 with 4 different SCCL cell lines are shown
`7.4) containing 0.1% BSA and 0.05% AZ followed by the
`addition of
`fluorescein isothiocyanate-labeled F(ab’)2
`in text-figures 3 and 4. Marked heterogeneity in the
`goat anti-mouse antibody (Boehringer-Mannheim,
`expression of antigens defined by each MoAb was
`noted.
`Indianapolis, Ind.) for an additional 30 minutes. After
`another wash in PBS-BSA-AZ, the cells were analyzed
`these MoAb with 10
`The pattern of reactivity of
`for fluorescence on the Cytofluorograph 50H (Ortho
`different SCCL cell lines is shown in table 2. Interest-
`Instruments, Westwood, Mass.) with the 2150 computer
`ingly,
`this pattern of
`reactivity is
`similar
`to that
`system. Simultaneous gating on both viable andsingle-
`obtained with fresh tumor cells from patients. For
`cell populations was performed, and the data reported
`example, SCCL-175 reacted significantly with all of the
`are those obtained from these populations. Positive
`samples of fresh tumor tissue as well as the cell lines.
`control antibodies included an MoAbto beta-2-micro-
`In contrast, SCCL-41 reacted with only 4 of the 7 fresh
`globulin, AML-1-201, and a mouse antiserum obtained
`tumor samples and with 3 of the 10 SCCL cell lines.
`by immunization with fresh SCCL cells. The negative
`Reactivity with non-SCCL tumor cell
`lines.—To
`control antibody was an IgM MoAb, SCC1,
`reactive
`further study the specificity of these MoAb, we exam-
`with an irrelevant antigen (sheep erythrocytes).
`ined a number of lung tumorcell lines of non-SCCL
`Hapten inhibition studies.—To determine if any of
`histology, other solid tumor cell
`lines, and leukemia
`cell
`lines. None reacted with cells from several non-
`these MoAb react with molecules that possess
`the
`
`5
`
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`PATIENT A: PRIMARY LUNG TUMOR
`
`Antibodies to Small Cell Lung Carcinoma 595
`
`SCCL 41
`62% +
`MFI 69
`
`SCCL 114
`60% +
`MFI 35
`
`FLUORESCENCE INTENSITY
`
` >
`
`
`
`CELLNUMBER
`
`CELL
`
`TEXT-FIGURE 1.—Reactivity of 4 MoAb
`with patient A-derived SCCL cells.
`Cells from the primary lung tumor
`that were used as the immunogen for
`these hybridomas were studied bycyto-
`fluorography. The specific staining of
`tumorcells (black area) for each MoAb
`is
`shown in individual panels
`(A,
`SCCL-41; B, SCCL-114; C, SCCL-124;
`D, SCCL-175). The percentage of
`positive cells, as defined by fluores-
`cence greater than on control MoAb-
`treated cells (dotted line), is shown as
`well as the meanfluorescence intensity
`(MFI). The vertical line at the right of
`each panel represents highly fluores-
`cent cells.
`
`4 MoAb
`EXT-FIGURE 2.—Reactivity of
`with patient B-derived SCCL cells.
`Cells obtained from the primary lung
`tumor of a patient with SCCL were
`studied by cytofluorography. See legend
`for text-figure 1 for details.
`
`SCCL 124
`64% +
`MFI 75
`
`SCCL 175
`77% +
`MFI 127
`
`SCCL 124
`21% +
`MFI 8
`
`SCCL 175
`64% +
`MFI 35
`
`
`
`
`PATIENT B: PRIMARY LUNG TUMOR
`c
`
`NUMBER
`
`
`SCCL 41
`43% +
`MFI 21
`
`scci 114
`22% +
`MFI9
`
`
`
`FLUORESCENCE INTENSITY
`
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`596 Ball, Graziano, Pettengill, et al.
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`TABLE 1.—Reactivity of MoAb with SCCL tumorcells
`freshly isolated from patients*?
`
`MoAb
`
`Percentpositive cells
`
`Patientfor patient Patienta E
`Bl
`B2
`A
`C
`OD
`
`
`
`SCCLAL
`SCCL-114
`SCCL-124
`SCCL-175
`
`62
`60
`64
`77
`
`43
`22
`21
`64
`
`42
`12
`8
`36
`
`16
`5
`22
`386
`
`15
`22
`23
`53
`
`-
`=
`+
`+
`
`+
`+
`+
`+
`
`“Reactivity of MoAb with SCCL cells was determined by
`indirect
`immunofluorescence and flow cytometry for patients
`A-D. The numbers reported for these patients’ cells are the
`percentage of cells stained with specific MoAb fluorescing
`greater than cells stained with control IgM MoAb. Cells from
`patient E were examined by indirect
`immunofluorescence of
`tissue sections and patient F, by RIA. In the latter case, positive
`(+)
`indicates a significant reaction over background fluores-
`cence. RIA counts were greater than three times background
`and similar to those obtained with an anti-SCCL antiserum.
`Samples B1 and B2 were primary lung tumorcells (B1) and
`liver metastases (B2) from the same patient. Cells obtained from
`patients A, B, and E were obtained at autopsy. Cells from
`patients C, D, and F were obtained by surgical biopsy. Samples,
`A, Bl, C, and F were obtained from the primary lung tumor,
`samples B2 and E were liver metastases, and sample D was
`from a supraclavicular lymph node metastasis.
`
`lines of epidermoid, adeno-
`SCCL lung tumor cell
`carcinoma, and large cell phenotype (table 3). Only
`SCCL-175 reacted with cells from other tumorcell lines
`(choriocarcinoma and colon carcinoma, table 3). None
`of the MoAbreacted with cells from the leukemia cell
`lines K562, HL-60, Daudi, and CCRF-CEM (table 3).
`Finally, none of
`the MoAb reacted with HE-lung
`fibroblasts.
`Hapten inhibition studies—The binding of each
`MoAb to SCCL tumor cells was not
`inhibited by
`concentrations of purified LNF III
`that have been
`shown to inhibit completely binding of LNF III-
`reactive MoAb (17) and to abolish completely the
`binding of
`the PM-81 MoAb. The percentages of
`positive cells and their mean fluorescence intensities
`were nearly identical
`to those shown in text-figure 2
`both in the presence and absence of LNFIII.
`
`DISCUSSION
`
`Using the approach of immunizing with fresh SCCL
`tumor tissue, we have prepared and partially character-
`ized 4 hybridoma-derived MoAb that appear
`to be
`relatively specific for SCCL. Our reason for selecting
`
`\|\||
`
`c
`
`! |\\
`
`|\||\\\||
`
`TEXT-FIGURE 3,—Reactivity of MoAb
`SCCL-124 with 4 DMS SCCLcell lines
`as determined by cytofluorography.
`
`SCCL 124
`
`
`
`
`
`CELLNUMBER
`
`FLUORESCENCE INTENSITY
`
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`SCCL 175
`
`597
`
`TEXT-FIGURE 4.—Reactivity of MoAb
`SCCL-175 with 4 DMS SCCLcell lines
`as studied by cytofluorography.
`
` Antibodies to Small Cell Lung Carcinoma
`
`
`
`CELLNUMBER
`
`DMS 153
`54% +
`
`FLUORESCENCE INTENSITY
`
`fresh tumortissue rather than an SCCL cell line as an
`immunogen was because of the possibility that a cell
`line(s) might not mirror the antigen profile of an in
`vivo tumor. Our approach was successful
`in that we
`were able to produce MoAbreactive with the cells used
`as immunogen as well as other patient-derived SCCL
`tumor cells and SCCL cell lines. We also showed that
`the MoAb are not
`reactive with common or histo-
`compatibility antigens on leukocytes from either the
`patient from whom the tumor immunogen wasderived
`or from normal donors. Moreoever, these MoAb do not
`react with glycolipids and,
`in particular, with the
`glycolipid molecules with which manyreported anu-
`tumor cell, including anu-SCCL, MoAb react (/7-/9).
`This conclusion is based on the lack of reactivity of our
`MoAbwith lipid extracts of SCCL tumorcells (Gins-
`
`TABLE 2.—Reactivity of MoAb withcells of the
`DMS SCCLcell lines®
`
`DMS SCCL cell lines”
`MoAb eee
`44
`47
`53 79 158
`187 285
`406
`431 483
`
`~~
`-
`SCCL41
`—-
`-
`SCCL114.
`SCCL 124 +++ +4+4+
`SCCL175
`+
`—
`
`FOF Rp
`+
`-
`=
`=
`=
`=~
`$+
`+
`4
`-
`=
`+
`+
`-
`-
`+
`+
`Ft
`th +
`fF
`tet
`$F
`+
`
`burg V: Personal communication) and with human
`granulocytes, cells known to express molecules with the
`terminal pentasaccharide lacto-N-fucopentaose III
`to
`which manyother anti-tumorcell and SCCL MoAbare
`directed (17-19). The inability of purified LNF III to
`block the binding of each MoAb supports this con-
`clusion.
`This
`
`report demonstrates another aspect of
`
`the
`
`TABLE 3.—Reactivity of MoAb with non-SCCL tumor cell lines*”
`
`Cell lines
`
`MoAb
`
`Tissue origin
`
`Designation
`
`SCCL SCCL SCCL SCCL
`41
`114
`(124)
`«175
`
`Squ Ca
`Lung, squamous
`Ca Lu-l
`Lung, squamous
`DMS485
`Lung,large cell
`Lung, adenocarcinoma A549
`Lung, adenocarcinoma SK-LU-1
`Neuroblastoma
`1MR 32
`Melanoma
`DMS 351
`Choriocarcinoma
`BeWo
`Colon carcinoma
`DLD-1
`Promyelocytic leu-
`HL-60
`kemia
`Myeloid leukemia
`B-cell leukemia
`T-cell leukemia
`
`-
`K562
`-
`Daudi
`CCRF-CEM -
`
`Prttdbrpadi
`
`iisttbyout
`
`-
`-
`-
`
`
`
`+Pepitrrretl
`
`-
`-
`-
`
`-
`~
`-
`-
`
`-
`-
`-
`
`“Reactivity of MoAb with SCCL cell lines was determined by
`flow cytometry.
`Reactions were scored as follows: —, <20% of cells positive; +,
`20-40% positive; ++, 40-60% positive; +++, >60% of cells positive.
`
`* Reactivity of MoAb with cell
`cytometry.
`” See footnote b, table 2.
`
`lines was determined by flow
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`598 Ball, Graziano, Pettengill, et al.
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`heterogeneity within SCCL tumors. Of considerable
`interest
`is
`the similar pattern of
`reactivity of cells
`freshly isolated from patients and those obtained from
`cell
`lines. That each of the 4 MoAb reacted with a
`similar percentage of both the cell lines and the freshly
`isolated tumor cell samples indicates that the cultured
`cell lines maintain an antigenic profile similar to cells
`in vivo. Weare presently studying tumorcells immedi-
`ately after their removal
`to directly follow changes in
`antigen expression with time.
`An attempt was madeto relate the expression of the
`antigens defined by these MoAbto other heterogeneous
`phenotypic characteristics of the SCCL cell lines. These
`included morphology (5), ultrastructure (5), hormone
`secretion (6, 21, 22),
`tissue site of origin (5, 7),
`treatment status of the patient (5), growth rate (, 7),
`and karyotype (Wurster-Hill D.: Personal communica-
`tion.) No meaningful correlations could be made
`between qualitative or quantitative antigen expression
`and any of these features of the cell lines.
`As noted by ourselves and other investigators, most
`reported tumor cell-reactive MoAb are also reactive
`with other selected tumor types (19, 23, 24) and with
`normal cells of the same or different lineage (15, 76, 19,
`23, 24). SCCL-175 reacted with cells from a colon
`carcinoma line and a choriocarcinoma line. However,
`none of the MoAbreported here reacted with any of the
`non-SCCL lung tumorcell lines studied. Although it is
`premature to conclude that any of
`these MoAb are
`relatively or absolutely specific for SCCL, further study
`of specificity will reveal whether they will be useful in
`distinguishing SCCL from non-SCCL lung tumors. It
`seems likely that combinations of MoAbreported to be
`reactive with non-SCCL lung tumors but not SCCL
`(25, 26) and MoAbspecific for SCCL such as those
`reported here might make a useful panel of reagents for
`the immunodiagnosis of lung tumors.
`In contrast
`to the leukemias,
`the study of solid
`tumors is hampered by the difficulties of both obtain-
`ing and utilizing freshly isolated cells from patients.
`Therefore, the use of well-characterized cell lines is of
`great importance to the continued study of cancers such
`as SCCL. Ourfinding that SCCLcell lines express cell
`surface antigens characteristic of freshly isolated tumor
`tissue from patients with SCCL further validates the
`use of
`these lines as models of SCCL cell biology.
`Moreover,
`the potential utility of MoAb in the diag-
`nosis and treatment of
`this disease may be studied
`utilizing cell
`lines that mimic the in vivo antigenic
`properties of SCCL tumorcells.
`
`REFERENCES
`
`the lower respiratory
`(1) CARTER D, EGGLESTON JC. Tumors of
`tract. In: Atlas of tumor pathology, second series, fascicle 17.
`Washington, D.C.: Armed Forces Inst Pathol, 1980.
`(2) World Health Organization, Geneva, Switzerland. Histological
`typing of lung tumors. Tumori 1981; 67:253-272.
`(3) BRERETON HD, MATHEWS MM, Costa J, Kenr CH, JOHNSON
`RE. Mixed anaplastic small-cell and squamous-cell carcinoma
`of the lung. Ann Intern Med 1978; 88:805-806.
`(4) ABELOFF MD, EGGLESTON JC, MENDELSOHN G, ETTINGER DS,
`BayLin SB. Changes in morphologic and biochemical charac-
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`teristics of small cell carcinoma of the lung. Am J Med 1979;
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`(5) PETTENGILL OS, SORENSON GD, WwuRSTER-HILL DH, et al.
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`from small-cell carcinoma of the lung. Cancer 1980; 45:906-
`918.
`(6) SORENSON GD, PETTENGILL OS, CATE CC, DELPRET SA. Bio-
`markers in small cell carcinoma of the lung. In: Aisner J, ed.
`Lung cancer. New York: Churchill-Livingstone. In press.
`(7) PETTENGILL OS, SORENSON GD. Tissue culture and in vitro
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