Monoclonal Antibodies Reactive With Small Cell
`
`Carcinoma of the Lung‘“
`
`Edward D. Ball,“-5-6 Robert F. Grazlano,5 Olive 8. Pettenglll,’
`George D. Sorenson,’ and Michael W. Hanger“-5
`
`
`
`ABSTRACT—M urine 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 oi their lack oi reactivity with normal
`leukocytes and erythrocytes. These MoAb. designated SCCL-41,
`SCCL-114. SCCL—124, and SCCL—175. are all lgM 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 surlace antigens was
`noted among both the patient-derived tumor cells and the SCCL
`cell
`lines. One of the MoAb, SCCL—175, reacted with i’ 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
`line.
`whereas the other 3 MoAb were unreactive with these and several
`
`in the
`lines. These MoAb may be uselul
`other tumor cell
`diagnosis and subclaseification 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 (I, 2). In addition, SCCL tumors often contain loci
`of non-SCCL lung tumor cells {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
`
`lines derived front
`from patients with SCCL and cell
`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
`
`Ceil lines—Cell lines studied included DMS 44. 47.
`53. 79, 153, 187, 235, 406, 43], and 183, 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/] medium
`containing 20% FBS (DMS 44, 53.
`I53,
`[87, 235, 405,
`431. and 483} or RPMI-lfi40 medium with 20% FBS
`(DMS 79).
`Non—SCCL tumor cell lines studied included [MR 32.
`a neuroblastoma line (obtained from the ATCC, Rock—
`ville, MIL); DMS 35], a malignant melanoma cell line
`{isolated from lymph node biopsy specimen); Squ Ca. a
`squamous cell lung carcinoma cell line (provided by K.
`Havemann and C. Gropp. Marburg, Federal Republic
`of Germany); DMS 485, a large cell undifferentiated
`
`lung tumor cell line (isolated from pleural fluid}; A549.
`an adenoearcinoma lung tumor cell line (obtained from
`ATCC) (8); Ca Lu-l, a squamous cell lung carcinoma
`line (ATCC): SK-Lu-l, a lung adenocarcinoma cell line
`(obtained from Dr. J. Fogh, Sloan Kettering Cancer
`Institute)
`(9): BeWo,
`a choriocarcinoma cell
`line
`(A'FCC); DLD-l. a colon carcinoma cell line (provided
`by D. Dexter, Providence, R.I.)
`(10); and [IE-lung.
`embryonic lung fibroblasts {obtained from MA. Bio-
`products, Walkersville, Md.). Ca Lu—l was cultured in
`McCoy's 5-A medium (GIBCO, Grand Island, N.Y.)
`with 10% FBS. A549, SK-I.U-l, [MR 32, and HE-lung
`were cultured in Dulhecco's modified Eagle minimum
`essential medium with 10% FBS (HE-lung;
`lfi% FBS):
`BeWo, DLD-l, DMS 485, and DMS 35] were all
`cultured in RPMI-lfi40 with 20% F38.
`
`The following leukemia cell lines were also studied;
`111.60, a promyelocytic leukemia cell
`line (obtained
`from R. C. Gallo, National
`Institutes of Health,
`Bethesda, Md.) (II); K562, an undifferentiated myeloid
`leukemia cell line (I2); Daudi, and Epstein—Barr virus—
`transformed B-cell line (13); and CCRF-CEM, a T-cell
`leukemia line (14). These lines were all cultured in
`RPMl-lfi'lO 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: A'I‘CC=Amcrican Type Culture Collection;
`AZZsodiurn azide; RSAZhovirte serum albumin; FBS=fetal bovine
`serum: MoAb=monoclonal antibody (antibodies); PBS=phospliate—
`buffered saline; RIA=radioimrnunoasesay: SCCL=sruall cell carcinoma
`of the lung.
`
`' Received june [6, 1983: accepted November 8. 1983.
`2Supported in part by Public Health Service {PHS) grants
`CA-3l9l8. CA—SIBSB. and (IA—25845
`from the National Cancer
`Institute and by PHS grant Al-ll-Nlfig limit the National Institute of
`Allergy and Infectious Diseases. The Cytofluorograph was
`the
`generous gift oi the Fannie E. Rippel Foundation and is partially
`supported by the Norris Cotton Cancer Center core grant (TA-23l08.
`IThis study was presented in part at the annual meeting ol the
`American Federation for Clinical Research. Washington. D.C..
`May 2. l983. and appeared in abstract form in Clinical Research. Vol.
`3], No. 2. [1. 402A. 1983.
`4Department of Medicine. Dartmouth Medical School, Hanover.
`N.H. 03756.
`
`5 Department of Microbiology. Dartmouth Medical School.
`6z‘l'ridress reprint requests to Dr. Ball, Department ol Microbiology,
`Dartmouth Medical School.
`
`I Department of Pathology, Dartmouth Medical School.
`
`593
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`594 Ball, Grazlano, Peltenglll, el at.
`
`primary or metastatic tumors. which in all cases were
`densely involved with SCCL, were gently Leased 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). Erythrcvcytes 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.—BALBXC mice were
`immunized ip three times over a 3-month period with
`2XIOT cells, which had been dissociated from the
`primary lung tumor of a patient with SCCI... This
`tumor was classified as "intermediate" in the modified
`
`disodium in calcium and magnesium-free Hank's
`balanced salt
`solution followed by washing with
`RPMI-1640. Cells were incubated for 30 minutes at 40C
`
`with 0.5 ml hybridon‘ta supernatant, unbound MoAl)
`were removed by washing with 7 volumes of PBS (pH
`7.4} containing 0.1% BSA and 0.05% AZ followed by the
`addition of
`fluorescein isothioeyanate-labeled F(ab')2
`goat
`anti—mouse antibody (Boehringer—Mannheim,
`Indianapolis, Ind.) for an additional 30 minutes. After
`another wash in PBS—BSA—AZ, the cells were analyzed
`for fluorescence on the Cytofluorograph 50H {Ortho
`Instruments. Westwood. Mass.) with the 2l50 computer
`system. Simultaneous gating on both viable and single-
`cell populations was performed, and the data reported
`are thOse obtained from these pOpulations. Positive
`control antibodies included an MoAb to beta-2-micro-
`
`globulin, AML-l-20], and a mouse antiserum obtained
`by immuniration with fresh SCCI. cells. The negative
`control antibody was an IgM MoAb, SCCI.
`reactive
`with an irrelevant antigen (sheep erythrocytes).
`Hapten inhibition studies.—-To determine if any of
`these MoAb react with molecules that possess
`the
`
`JNCI. VOL, 72, N0. 3. MARCH |984
`
`World Health Organiration classification (2). Fusion of
`spleen cells from an immunized mouse with murine
`myeloma cells of the NS-l cell line was performed with
`the use of polyethlene glycol as the fusing agent as
`previously described (16). Hybridomas making MoAb
`reactive with the immunogen were selected by solid—
`phase RIA with the use of glutaraldehyde—fixed cells as
`previously described (15). Of these, 4 were selected for
`more extensive study on the basis of
`their relative
`specificity for the immunogen and lack of reactivity
`with normal leukocytes. These hybridomas were desig-
`nated SCCL-41, SCCL-ll4, SCCL-124, and SCCL-l75.
`All 4 of the MoAb produced were IgM antibodies.
`Indirect
`immunofluorescence and cytofltiorogmphic
`anaiysis.—The reactivity of these MoAb with SCCI.
`cells was determined by indirect
`immunofluorescence
`and flow cytometry. Cells freshly isolated from primary
`or metastatic tumors were prepared as described above.
`Adherent cell
`lines were dissociated from the culture
`flask after 10—minute incubation with 0.01% EDTA
`
`
`
`to
`lacto-N-fucopentaose III,
`terminal pentasaccharide.
`which several reported anti~SCCL MoAb react (I7—19).
`we performed inhibition studies. Purified LNF III
`(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.
`I’M-81 (20), at a concentration of 5
`mg/ml for 1 hour before addition of these mixtures to
`SCCL tumor cells from patient B (text-fig. 2). Quanti-
`tative comparisons of the binding of each MoAb either
`in the presence or absence of LNF III was determined
`by indirect immunofluorescence and flow cytornetry.
`
`RESU LTS
`
`Specificity of hybridomas.——The reactivity of the 4
`MoAb with the immunogen is shown in text-figure l.
`Intense but variable fluorescence was observed with
`
`the MoAb reacted at all by
`each MoAh. None of
`indirect immunofluorescence with lymphocytes. mono-
`cytes, or granulocytes from the patient from whom the
`primary tumor was obtained or from 6 normal donors
`(data not shown). thus indicating that they are probably
`not reactive with histocompatibility or other common
`cell surface antigens. In addition, none of the MOM)
`reacted with erythrocytes from 4 normal donors (2
`Lewis B and I Lewis A—positive, and l Lewis antigen-
`negative). The reactivities of these MoAb with cells
`from the primary lung tumor of a second patient with
`SCCL are shown in text-figure 2.
`In addition,
`liver
`metastaseas from the same patient were examined and
`found to have a similar antigenic profile, although
`staining was less intense in each case. A summary of
`the reactivities of the MoAb with tumor cells obtained
`
`from a total of 7 sites from 6 different patients is shown
`in table 1.
`
`Reactivity with SCCL cell lines—'I‘he expression of
`these antigens on several SCCL cell lines developed by
`Pettengill et al.
`(5) was evaluated. Consistent with
`other evidence of phenotypic heterogeneity in these
`cell lines, including differential peptide hormone secre-
`tion (6, 21, 22} and morphology (5). we have found
`considerable heterogeneity in antigen expression. As
`examples,
`the reactivities of MoAb SCCL—l24 and
`SCCL-175 with 4 different SCCL cell lines are shown
`
`in text-figures 3 and 4. Marked heterogeneity in the
`expression of antigens defined by each MoAb was
`noted.
`
`these MoAb with [0
`reactivity of
`The pattern of
`different SCCL cell lines is shown in table 2. Interest—
`
`to that
`similar
`reactivity is
`this pattern of
`ingly,
`obtained with fresh tttmor cells from patients. For
`example, SCCL-l?5 reacted significantly with all of the
`samples of fresh tumor tissue as well as the cell
`lines.
`In contrast, SCCL-41 reacted with only 4 of the 7 fresh
`tumor samples and with 3 of the 10 SCCL cell lines.
`Reactivity with non—SCCL tumor calf
`lines—To
`further study the specificity of these MoAb, we exam-
`ined a number of lung tumor cell
`lines of non-SCCL
`histology, other solid tumor cell
`lines, and leukemia
`cell
`lines. None reacted with cells from several non-
`
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`Antibodies to Small Cell Lung Carcinoma
`
`PATIENT A: PRIMARY LUNG TUMOR
`
`SCCL 41
`62% +
`MFI 69
`
`SCCL 124
`64% +
`MFl 75
`
`SCCL11A
`60% +
`MFI 35
`
`SCCL 175
`77% +
`MFI 127
`
`FLUORESCENCE INTENSITY
`
`PATIENT B: PRIMARY LUNG TUMOR
`
`SCCL 41
`43% +
`MFI 21
`
`SCCL124
`21% +
`MFI 8
`
`4 MoAh
`Ext-FIGURE 2.—Reat'tivity of
`with patient B-dcrived SCCL cells.
`(‘rlls obtained from the primary lung
`tumor of a patient with SCCL were
`studied by (‘ylolluorogt'aplnu See legend
`for text-figure l for details.
`
`I.—Reactivity of 4 MoAb
`Tun-Iona:
`with patient A-dcrived SCCL cells.
`Cells from the primary lung tumor
`that were used as the immunogen [or
`these hybridomas were studied by cyto-
`lluorography, The specific staining of
`tumor cells (blark area) {or each MoAb
`is
`shown in individual panels
`(A.
`SCCL-4|; B, SCCL-1M; C, SCCL-124:
`D. SCCL-I75). 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 mean fluorescence intensity
`(MFI). The vertical line at the right 0]
`each panel represents higth fluores-
`cent cells.
`
`
`
`
`
`CELLNUMBER
`
`
`
`CELLNUMBER
`
`SCCL 175
`64% +
`MFI 35
`
`FLUORESCENCE INTENSITY
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`596 Ball, Graziano, Pettengill, el al.
`
`TABLE l.—-Reactim'ty of MoAb with SCCL tumor cells
`freshly isolated from patients”
`
`MoAb
`
`Percent positive cells
`for patient
`ABIB2CD
`
`Patient
`E
`
`Patient
`F
`
`SCCL-41
`SCCL-114
`SCCL~124
`SCCL-175
`
`62
`60
`64
`77
`
`43
`22
`21
`64
`
`42
`12
`8
`36
`
`16
`5
`22
`36
`
`15
`22
`23
`53
`
`lines of epidermoid. adeno-
`SCCL lung tumor cell
`carcinoma, and large cell phenotype (table 3). Only
`SCCL-I75 reacted with cells from other tumor cell lines
`(choriocarcinoma and colon carcinoma, table 3). None
`of the MoAb reacted with cells from the leukemia cell
`lines K562, HL-GO, 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 (l7) and I0 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 LNF III.
`
`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
`
`“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 BI and B2 were primary lung tumor cells (BI) 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.
`
`
`
`TEX'LI‘IGIIRE 3l-Rl’adlvily of MoAb
`SCCL-I24 wiIh 4 DMS SCCL cell lines
`as determined by cylolluorogiaphy.
`
`SCCL 124
`
`C
`
`I IIIIIIIIIIIIIIIII
`
`
`
`CELLNUMBER
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`FLUORESCENCE INTENSITY
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`SCCL 175
`
`Antibodies to Small Cell Lung Carcinoma
`
`597
`
`TEXT-HOUR}: 4.~Reactivity ol MoAb
`SCCL~l75 with 4 DMS SCCL cell lines
`as studied by cytolluorography.
`
`
`
`CELLNUMBER
`
`DMS 153
`54% +
`
`burg V: Personal communication) and with human
`granulocytes, cells known to express molecules with the
`terminal pentasaccharide lacto-N-fucopentaose Ill
`to
`which many other anti-tumor cell and SCCL MoAb are
`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
`
`.
`.
`Desmmm"
`
`SCCL SCCL SCCL SCCL
`41
`114
`124
`175
`
`Squ Ca
`Lung. squamous
`Ca Lu-l
`Lung. squamous
`DMS 485
`Lung. large cell
`Lung. adenocarcinoma A549
`Lung. adenocarcinoma SK—LU-l
`Neuroblastoma
`lMR 32
`Melanoma
`DMS 351
`Choriocarcinoma
`BeWo
`Colon carcinoma
`DLD-l
`Promyelocytic leu-
`HL-60
`kemia
`Myeloid leukemia
`B-cell leukemia
`T-cell leukemia
`
`K562
`Daudi
`CCRF-CEM
`
`I|lll|Ill|
`
`|
`
`|Ill1|llll
`
`FLUORESCENCE INTENSITY
`
`fresh tumor tissue 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 MoAb reactive 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 was derived
`or from normal donors. Moreoever, these MoAb do not
`react with glycolipids and,
`in particular, with the
`glycolipid molecules with which many reported anti-
`tutnor cell. including anti.SCCL, MoAb react (17-19).
`This conclusion is based on the lack of reactivity of our
`MoAb with lipid extracts of SCCL tumor cells (Gins-
`
`TABLE 2.—Reaetit'ity of MoAb with cells of the
`DMS SCCL cell lines"
`
`MoAb
`
`DMS SCCL cell linesb
`
`44
`
`47
`
`53 79 153
`
`187 235
`
`406
`
`431 483
`
`++ ++
`
`—
`—
`+
`+
`
`+
`—
`—
`+++
`
`—
`+
`—
`+
`
`+
`v
`—
`SCCL 41
`+
`—
`—
`SCCL 114
`SCCL 124 +++ +++ +
`SCCL 175
`+
`—
`+
`
`—
`—
`—
`—
`—
`—
`+ ++
`
`
`
`“Reactivity of MoAb with SCCL cell lines was determined by
`flow cytometry.
`hReactions 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, Grazlano, Pettenglll, et al.
`
`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 botlt 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. We are presently studying tumor cells immedi-
`ately after their removal to directly follow changes in
`antigen expression with time.
`An attempt was made to relate the expression of the
`antigens defined by these MoAb to other heterogeneous
`phenotypic characteristics of the SCCI. cell lines. These
`included morphology (5}, ultrastt‘ucture (5). hormone
`secretion (6, 21. 22},
`tissue site of origin (5, 7),
`treatment status of the patient (5). growth rate {5, 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, ntost
`reported tumor cell-reactive MoAb are also reactive
`with other selected tumor types {$9. 23, 24) and with
`normal cells of the same or different lineage (15, I6, 19,
`23, 24). SCCL-l'l'ft
`reacted with cells from a colon
`carcinoma line and a choriocarcinorna line. However,
`none of the MoAb reported here reacted with any of the
`non-SCCL lung tumor cell 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 ttott-SCCL lung tumors. It
`seems likely that combinations of MoAb reported to be
`reactive with non—SCCL lung tumors but not SCCL
`(25, 26) and MoAb specific for SCCL such as those
`reported here might make a ttseful 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 Our finding that SCCL cell lines express cell
`surface antigens characteristic of freshly isolated tumor
`tissue from patients with SCCI. 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 tumor cells.
`
`
`
`teristics of small cell carcinoma of the lung. Am ] Med 1979:
`66:757—164.
`(5) PFTTENGIIL OS. SORENSON GD. WURSTER-lllll. DH. el al.
`Isolation and growth characteristics of continuous cell
`lines
`front small—cell carcinoma of the lung. Cancer [98f]; 45:906—
`918.
`{6) SORENSON GD. PETTENGILL 05. Can: CC. DELPRE'I‘ SA. Bio-
`markers in small cell carcinoma of the lung. In: Aisner j. ed.
`Lung cancer. New York: Churchill-l.ivingstone. In press.
`(7) PE’I‘I'ENGILL OS. SORENSUN CD. Tissue culture and in vitro
`characteristics.
`In: Greco FA. Buttn PA. Oldham RK. eds.
`Small cell lung cancer. New York: Grurte. [931:5l-77.
`{8) GIARI) I)_|. Aattoatsotst SA, Tonalto G]. et al. In vitro cultivation
`of human tumors: Establishment of cell lines derived from a
`series of solid tumors] Natl (lancer Inst 1973: 5I:I4I7—|423.
`{9} FOGH J. WRIGH'I‘ WC. LIN-'ELESS JD. Absence of HeLa contami-
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`Celltrion, |nc., Exhibit 1006
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`6 of 6
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`Celltrion, Inc., Exhibit 1006
`
`