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[CANCER RESEARCH 53,5244-5250. November 1, 1993]
Molecular Cloning of a Human Monoclonal Antibody Reactive to Ganglioside GM3
Antigen on Human Cancers1
Dave S. B. Hoon,2 Yuan Wang, Lan Sze, Hidetoshi Kanda, Takeshi Watanabe, Sherie L. Morrison, Donald L. Morton
and Reiko F. Irie
John Wayne Institute for Cancer Treatment and Research, Santa Monica, California 90404 [D. S. B. H., Y. W., L. S., D. L. M., R. E /./, Department of Molecular Immunology,
Medical Institute of Bioregulalion, Kyushu University, Fukuoka 812, Japan ¡H.K.. T. W.¡:and Department of Microbiology and Molecular Genetics, The Molecular Biology
InstÃ-lale, University of California, Los Angeles, California 90024 /S. L. M.¡
ABSTRACT
In this study we report the characterization of a human monoclonal
antibody (HuMab), L612, that reacts with ganglioside GM3and has thera
peutic application for the treatment of human neoplasms, particularly
melanoma. A permanent IgM-secreting Epstein-Barr virus-transformed
B-cell line L612 was established. L612 HuMab bound specifically to neo-
plastic cell lines in culture and in tissue biopsy specimens such as mela
noma, colon, breast, and lung cancer. The antibody did not bind to normal
cells or biopsy tissue. HuMab L612 showed the highest reactivity to mela
noma cells, particularly to those with high concentrations of GM3. Im-
munostaining on high-performance thin-layer chromatography plates
demonstrated that L612 HuMab bound to (.\,, purified from melanoma
cells. Removal of the sialic acid from GM.<abolished antibody binding.
HuMab L612 also reacted to (.,,, purified from egg yolk, indicating that
it recognizes an NeuAca2-3 galactose antigen determinant. HuMab L612
heavy and light chains were sequenced and determined to belong to the /.i
heavy chain variable subgroup III and K chain variable subgroup IV
families, respectively. The studies indicate that the L612 HuMab has
significant therapeutic potential for a wide variety of human cancers.
INTRODUCTION
In previous studies we and others have observed an aberrant ex
pression of gangliosides (sialic acid-containing glycolipids) on the cell
surface of various human neoplasms (2-6). Several of these ganglio
sides have been shown to induce antibody responses in humans (7—
10). We have shown that the humoral immune response to specific
gangliosides on tumor cells plays an important role in host protective
immunity and correlates with prolonged survival (7). There are a
number of murine Mabs3 to carbohydrate determinants on glycolipid
molecules that are associated with human cancer. These antibodies
have provided an important tool in identifying specific immunogenic
carbohydrate determinants and sequences (11-16) and several of these
murine Mabs have been used in clinical trials in the treatment of
cancer (16-18), but only in a very few cases have they produced
significant therapeutic benefit. This benefit was transient because the
formation of human antibodies to murine antibodies precludes re
peated and prolonged treatment periods. In addition the murine im
mune system can recognize different antigen determinants from the
human immune system and often overlooks the polymorphic antigen
determinant(s) on the cell surface of human cancer cells, instead
producing antibodies that also react with normal human cells.
Received 6/14/93: accepted 8/23/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1This study was supported by USPHS Grant ÇAI2582 and the California Research
Institute Fund (D. S. B. H.).
2To whom requests for reprints should be addressed, at John Wayne Institute for
Cancer Treatment and Research, Division of Molecular and Cellular Immunology, 2200
Santa Monica Blvd.. Santa Monica. C'A 90404.
3 Mah, monoclonal antibody; IA, immunoadherence assay; GMi. ganglioside.
H'NcuAc-LacCer [Svennerholm classification (1)]; HPTLC, high-performance thin-layer
chromatographv; HuMab. human monoclonal antibody; PCR. polymerase chain reaction;
V. variable region of antibody gene; EBV, Epstein-Barr virus; cDNA. complementary
DNA; dNTP. deoxynucleotide triphosphntc.
Considerable effort has been expended to develop HuMabs for
therapeutic purposes and as a tool to identify immunogenic determi
nants on human tumor cells. Successful production of HuMabs to
tumor-associated antigens has lagged far behind mouse Mab. The
main problem is the development of long-term stable lines that secrete
antibodies continuously. Our laboratory has succeeded in establishing
several HuMabs to tumor-associated ganglioside antigens by trans
forming patients' B-cells with EBV (19). This approach has been
successfully used to establish monoclonal antibodies to tumor-asso
ciated ganglioside antigens GM2 and GD2 (19, 20). We have demon
strated that intralesional injections of these HuMabs can induce mela
noma tumor regression (20, 21). HuMabs with specificity to dominant
tumor-associated ganglioside antigenic determinants may have better
therapeutic potential than HuMabs with specificity to less dominant
antigenic determinants.
One of the antigens that have been biochemically identified as
dominating the cell surface of several human tumors, particularly
melanoma, is GM3 (2). GM3 is found on human normal cells but at a
much lower density than human melanoma (2). A murine Mab M2590
was reported to react with GM3 antigen on B16 melanoma but not on
normal cells because of the greater density and tertiary structure of
GM3 on the cell surface of melanoma cells (14). We also have pro
duced a murine Mab 202 which recognizes the terminal disaccharide
(sialyla2-3Gal) on GM3and/or GM2; Mab202 reacts to GM3 of human
melanoma and not normal tissue (13). The specificity of this Mab for
melanoma cells also may be due to the density of the antigen. HuMab
recognizing carbohydrate epitopes may have a restricted usage of VH
or V, regions (22). Establishing and cloning HuMabs specific to
carbohydrate epitopes will allow us to answer this question.
We were interested in developing a therapeutic HuMab to GM3. In
this study we characterized the binding specificity and variable gene
sequence of a HuMab L612 that binds strongly to GM3and reacts with
human cancers but not normal tissue.
MATERIALS AND METHODS
Establishment of L612 Cell Line. HuMab L612 was established by trans
formation of a patients' B-cells with EBV as described previously (19). The
B-cclls were from regional lymph nodes of a breast cancer patient who had
undergone elective lymphadenectomy. Briefly, lymphocytes were obtained by
passage of dissected lymph nodes through a steel mesh wire and by separation
on Ficoll-Hypaque gradient (Pharmacia, Piscataway, NJ). The T-cells were
removed by E-rosetting (19). The enriched B-cell fraction was then washed 3
times with Hanks' buffered saline solution and incubated with EBV for 20 h in
RPM1 1640 (GIBCO, Grand Island, NY) containing 10% heat-inactivated fetal
calf serum (Gemini Bioproducts, Calabasas, ÇA).B-cells were then cloned by
limiting dilution and assessed for antibody secretion by the IA assay as de
scribed previously (23).
In screening for anti-GM1 antibody-producing B-cell clones the M12 mela
noma cell line was used as the target cell in the IA assay. Previously we had
determined that M12 expresses a high density of GM, on its cell surface (24).
Of 43ft B-cell clones that secreted antibodies, those that produced a consistent
amount of antibodies and reacted to M12 in the IA assay were recloned. Clones
were adapted to grow in serum-free medium containing growth factors
(SGF-6S in CEM 2000; Scott Lab, Carson, CA) and subsequently recloned for
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HUMAN MONOCLONAL ANTIBODY TO GANGLIOSIDE GM<
Table 1 HuMab I.(yl2 binding specificity lo neoplasie and normal cells
An IA assay was performed with 0.116 jig HuMab L612/ml to evalúale antibody
binding. The scale used to quantitate binding was 4+ (strongest) to 0 {no binding) as
described in "Materials and Methods."
Human celllinesMelanomaLung
adenocarcinomaBreast
carcinomaGastrointestinal
adenocarcinomaErythroleukemiaT-cell
leukemiaMyelomaNeuroblastomaEBV
B-lymphoblastsNo.
positive/
no.tested12/164/45/72/7(I/II/I11,10/10/11Av.
cell
reactivity+
+++
++
++II+000
high antibody secretion. From these clones we selected one B-cell clone line
(L612) that produced antibodies highly reactive to M12. The doubling time of
the cell line, which is grown in suspension culture, is about 24 h. The cell line
has now been secreting antibodies for over five years.
HuMab L612 was purified for analysis and therapy as described previously
(25). Briefly, the L612 line was grown in serum-free medium. IgM in the
concentrated spent medium was purified by salt and hypotonie precipitation
followed by Sephacryl S-300 column chromatography (Pharmacia Biotech,
Inc., Alameda, CA). Each fraction was tested for protein concentration by
spectrophotometric analysis and anti-GM, antibody tiler by a GM.i-enzyme-
linked immunoabsorbent assay (24). Each fraction containing an equal con
centration of protein and human IgM with high anti-melanoma cell activity
was pooled and concentrated to 5 mg/ml. Purified HuMab was filtered
through a 0.22 (¿mfilter and cryopreserved until needed. Each lot was tested
for the presence of bacteria, Mycopiasma, endotoxins, hepatitis virus, and
other viruses.
Antibody Specificity Analysis: Immunoadherent Assay. An IA assay
was performed on cultured cell lines as described previously (23). The IA is a
very sensitive method to detect complement binding antibody to tumor cells.
Briefly, the assay detects IgM bound to tumor cells by specific rosette forma
tion of human erythrocytes in the presence of complement (23). The adherence
of one or more pre-screened human erythrocytes to the cell surface was graded
using the following scale: >9()% of cells adhered, 4+; 75%, 3+; 50%, 2+;
and 0 if no adherence was observed. Immune absorption was performed in
several experiments prior to the IA assay. The immunoabsorption assay in
volved incubating HuMab with purified antigen, such as gangliosides or cells,
before performing the IA assay (23). This assay allows analysis of antibody
specificity by antigen-competitive blocking. Melanoma cell lines MIO, M12,
M14, M15, and M24 were established in our laboratory. From several of the
patients both melanoma lines and EBV-transformed B-lymphoblast lines were
established.
Antibody Staining of Human Tissues. Tissue sections 4 p.m thick were
cut from human biopsy tissue frozen in tissue-freezing compound. The sections
were immediately fixed in cold formaldehyde buffer and air-dried. Slides were
incubated with Tris buffer for 5 min and treated with 3% hydrogen peroxide for
10 min to quench endogenous peroxidase activity (26). Slides were then
washed in running water for 5 min and treated with human serum for 20 min.
A three-step immunoperoxidase staining technique was used to detect L612
HuMab binding to tissue sections as described previously (26). Briefly, tissue
sections were first incubated with L612 HuMab and then incubated with a
specific a-anti-idiotypc mouse Mab to L6I2 (18C10) (24). Biotinylated goat
anti-mouse IgG (Vector Laboratories. Burlingame, CA) and peroxidasc-con-
jugated Strcptavidin (Zymed Laboratories, San Francisco, CA) were used as
indicator antibodies. Sections were countcrstained with hematoxylin and cov-
erslips were applied using glycerol-gelatin. Photographs of slides were taken
using a Nikon light microscope equipped with a Nikon camera (Nikon, Mel
ville, NY).
Ganglioside Analysis. The ganglioside fraction of cultured melanoma cells
was isolated and purified as described previously (2). Purified gangliosides
were applied to HPTLC and identified by resorcinol-HCl method (27). Gan
glioside standards were purified from human and bovine sources.
Immunostaining with L612 HuMab against purified gangliosides was per
formed on HPTLC plates (Merck, Darmstadt, Germany). Briefly, after chro
matography, gangliosides on HPTLC plates were soaked in 0.01% polyisobu-
tylmethacrylate in n-hexane. air-dried, and incubated with 1% gelatin-
phosphate-buffered saline solution at 37°Cfor 1 h. HPTLC plates were then
incubated with L612 HuMab (20 fig/ml) at 37°Cfor 90 min. Plates were then
incubated with horseradish peroxidase-conjugated goat anti-human IgM
(Boehringer Mannheim Biochemicals, Indianapolis, IN) at 37°Cfor 30 min
followed by five washings and the addition of substrate solution of 2 mg/ml
o-phenylenediamine (Sigma, St. Louis, MO) in 0.1 Mcitrate-phosphate buffer
(pH 5.0) containing 0.006% H2O2. When the color of the bands developed, the
reaction was stopped and the plates were washed in phosphate-buffered saline
five times.
PCR Cloning of the Variable Regions of L612 HuMab. The guanidium
thiocyanate-cesium chloride procedure was used to prepare total RNA from the
L612 B-lymphoblast line (28). Ten /¿gof RNA were mixed with 60 pmol of
either ¡iheavy or K light chain 3' primers and heated at 7()°Cfor 10 min. The
mixture was then added to 50 /j.1 reverse transcriptase reaction solution con
taining 10 fil 5X reverse transcriptase buffer (Promega, Madison, WI). 4 fj.1 10
HIMdNTP mix (200 fjLMdATP, dCTP, dGTP, and dTTP final concentration), and
3 ¿¿I(600 units) reverse transcriptase (Promega). The mixture was incubated at
37°Cfor 1 h.
The PCR reaction was performed as follows. Ninety-seven fil of PCR
mixture were added to 3 /¿Iof RNA-cDNA mixture. The PCR mixture con
tained 10X PCR buffer, 10 mw dNTP mix at 60 ¡J.Mfinal concentration of each
dNTP, 5 units of Taq polymerase (Promega), and 60 /IM appropriate 5' and 3'
heavy and light chain primers. The mixtures were subjected to 35 cycles of
amplification at 91°Cfor 1 min, 52°Cfor 2 min, and 72°Cfor 1.5 min followed
by a final incubation at 72°Cfor 10 min in a Perkin Elmer/Cetus thermal
cycler. An aliquot of the PCR product was run on a 2% agarose gel to verify
the correct size band product. The n heavy chain and K light chain primers
produced a 495- and 603-base pair cDNA product, respectively. When the gel
showed a correct size of single product, the remainder of the RNA-cDNA
product was subjected to PCR to obtain more product.
The gel products were isolated, pooled, and subjected to phenol-chloroform
extraction and ethanol precipitation. The DNA was then digested with appro
priate restriction enzymes, extracted, precipitated, purified with GeneClean
(BiolOl; San Diego, CA), and ligated into Bluescript (Stratagene, La Jolla.
CA) (29). Ten independent clones of the variable region /j. chain and four
independent clones of the variable region K chain were isolated and sequenced
(29). A JH heavy chain probe was used for screening and verifying the heavy
chain clones. Sequencing was done by the dideoxynuclcotide method with T7
DNA polymerase (Sequenase; United States Biochemicals, Cleveland, OH)
according to the manufacturer's protocol. Three heavy /Achain leader primers
were used (30): GGGAATTCATGGACTGGACCTGGAGG(AG)TC(CT)-
TCr(GT)C;GGGjMLT£ATGGAG(CT)TTGGGCTGA(CG)CTGG(CG)TTT-
(CT)T; and GGGAALTÇATG(AG)A(AC)(AC)(AT)ACT(GT)TG(GT)(AT)-
(CG)C(AT)(CT)(CG)CT(CT)CTG. These contain an £coRI (underlined)
restriction site to facilitate cloning. The heavy fj. chain ./ region primer was
CCAAGCTTAGACGAGGGGGAAAAGGGTT. This contains an Hindm site
(underlined). Two light «chain leader primers were used (31): GACATC-
GAGCTCACCCAGTCTCCA: and GAAATTGAGCTCACCCAGTCTCCA.
These primers contain an Sad site (underlined). The light K chain J region
primer was GCGCCGTCTAGAACTAACACTCTCCCCTGTTGAAGCTCT-
TTGTGACGGGCAAG. This primer contains an A7>al site (underlined). Ini
tially we used the light K chain leader primers of Larrick t-i a!. (30); however,
we obtained poor PCR-cDNA product yield and switched to another set of
primers (31).
Transfection. The pAG4202 mammalian expression vector containing spe
cific promotors engineered for human immunoglobulin expression and a neo-
Table 2 ¿.672binding correlation to melanoma cell ganglioside expression
Gangliosides were purified from cell lines as described in "Materials and Methods" and
fun on HPTLC for quantitation (2). Gangliosides were expressed as dry wcight/g of cell.
Gangliosides described according to the classification of Svcnnerholm and Friedman ( 1)
and 1UPAC-IUBC (47). IA was performed on cell lines with HuMab L612 as described in
Table 1. L612 reactivity to cells was measured on a scale of 4+ to 0.
CelllinesM15
M12
M14
Mid
M24Gangliosides
(nmol/gcell)GM.I20.6
18.1
7.3
7.1
0.3GM20.5
0
4.0
8.2
8.2GD32.0
2.8
4.2
2.6
0.4ÖD200
2.4
4.5
0.7IA
binding
(L612
reactivity)0
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HUMAN MONOCLONAL ANTIBODY TO OANOLIOSIDE GM3
GM3-
GDIb-
GTIb-
STD M12
Fig. 1. Purified gangliosides from melanoma cell line M12 run on HPTLC. Ganglio
sides were isolated and purified as described in "Materials and Methods" and visualized
with resorcinol-HCl reagent. Ordinate, ganglioside standards (STD).
mycin drug marker (developed by Dr. S. Morrison) was linearized at a unique
Pvu\ site. The human IgM constant region gene (a kind gift from Dr. M. J.
Shulman) was isolated and inserted into the pAG4202 vector. The pCN4203
mammalian expression vector containing the human K constant region gene
with a xanthine-guanine phosphoribosyl drug marker (developed by Dr. S.
Morrison) was linearized al a unique Pvwl site. The genomic cloned L612 VL
and VH genes were inserted into their respective vectors. Vectors with their
respective inserts were transfected into Sp2/0-Agl4 nonproducing mouse my
eloma cells as described previously (29). The transfected cell lines were
selected for both drug markers and cloned. Supernatants from the clones were
collected, concentrated, and tested in the IA assay with M12 cells for reactivity.
RESULTS
Characterization of L612 HuMab. The L612 cell line is stable,
secreting approximately 20 fig/ml IgM antibody/48 h. This cell line
also expresses cell surface membrane IgM which could be detected by
flow cytometric analysis using a ß-anti-idiotype murine Mab (4C10)
specific for L612 (data not shown). The L612 IgM light chain was
determined to be K using specific anti-human light chain Mabs (Bec-
ton-Dickinson, Mountain View, CA) by flow cytometry and Western
blotting.
The binding of HuMab L612 to different tumor cell lines was
assessed (Table 1). The IA assay showed antibody binding on several
neoplastic cell lines of different histogenèses.Although the percentage
of melanoma lines showing binding of L612 was lower than that of
lung lines, melanoma cell lines demonstrated the strongest reactivity.
No binding of HuMab L612 was observed to peripheral blood lym
phocytes (T- and B-cells, monocytes, and granulocytes) and erythro-
cytes tested from 32 and 44 individual volunteers tested, respectively.
For comparison purposes, HuMab L612 binding to cultured mela
noma cell lines and the level of GM3 expression of the individual lines
were assessed (Table 2). Melanoma lines with a high GM3 content,
such as M12 and M15, had strong antibody binding. The cell lines
MIO, M14, and M24, which express low levels of GM3, showed
poorer binding of L612. As a control, anti-GM2 HuMab IgM (L55
HuMab) (21) binding was assessed using the same melanoma lines.
Cell lines with high GM2 had strong antibody binding, whereas those
with no GM2, such as M12, had no binding (data not shown). This
suggested that the IgM HuMabs binding to the melanoma cells was
specific and not general, nonspecific binding. The ganglioside profile
run on HPTLC of M12 is shown in Fig. 1. Treatment of M12 and M15
Table 3 HuMab L612 binding specificity to gangliosides
Gangliosides described according to the classification of Svennerholm and Friedman
(1) and IUPAC-IUBC (47). Specificity binding analysis of HuMab L612 to gangliosides
was assessed by an IA immunoabsorption inhibition assay. L612 HuMab was incubated
with individual antigens (5 nmol) before being assessed for binding in the IA assay with
M12 cells (see "Materials and Methods").
Glycolipid
nameNeutral
glycolipidCTH"GlobosideAsialo-GviiGangliosidesGM4GMÕGM.IGM2GM2,
GMUSPGSPGGDJGD2GDI»GDihGUI,IA
adsorptioninhibitionGlycolipid
structureGbOsejCerGbOse4CerGbOse4CerI'NeuAc-GalCerII'NeuAc-LacCerII'NeuGc-LacCerII'NeuAc-GgOseiCerIFNeuAc-GgOse4CerlV'NeuAc-nLcOse4CerIV'NeuGc-nLcOse4CerII^NeuAc^-LacCerIF(NcuAc)2-GgOse.,CerIV'NeuAc,II3NeuAc-GgOse4CerlI3(NeuAc)2-GgOse4CerIV'NeuAc.IPfNeuAcb-GgO^CerAntigen
liter0(1(I6464000160(1(1(1(1Q
" CTH, ceramide trihexoside; SPG, sphingosine.
(strongest L612 binding) with Vibrio choleras neuraminidase abol
ished HuMab L612 binding (IA assay), while treatment with trypsin
did not (several experiments; data not shown). These experiments
suggested that the HuMab L612 binding to cell surface was related to
sialic acid recognition.
L612 Specificity to Gangliosides. To verify the binding of L612
HuMab to purified gangliosides, an IA immune absorption inhibition
GM3
Fig. 2. Immunostaining with HuMab L612 of gangliosides purified from melanoma
cell lines run on HPTLC; see "Materials and Methods" for procedures. Lane l, M12
melanoma gangliosides treated with Vïhriochoierai" neuraminidase. No staining with
HuMab L612 was observed. Lane 2, total M12 gangliosides run without neuraminidase
treatment. GM.i band stains with L612 HuMab only. Lane 3, lower band of GM.i from M12
run only on HPTLC. Lane 4, total M12 gangliosides run. GMj band stained only with
HuMab L612.
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Fig. 3. Photomicrograph of HuMah L612
staining. Three-step immunoperoxidase staining
of hiopsied tumor tissue. Primary eutaneous
melanoma (A), eolon adcnocarcinoma (/i), and
lung adenocarcinoma (C'( were first incubated
with MuMah 1.M2, then with murine anti-idio-
typc <«18Cd antibody, followed by biotinylated
anti-murinc IgG. For controls, tissues were
stained as described above without HuMab L612.
No staining occurred in controls (data not
shown). X250.
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HUMAN MONOCLONAL ANTIBODY TO GANGLIOSIDE GM,
HuMab L612 heavy chain variable region sequence'
-19 -15 -10 -5
Met Glu Phe Gly Leu Thr Trp Leu Phe Leu Val Ala Asn Leu Lys
ATG GAG TTT GGG CTG ACC TGG CTT TTT CTT GTG GCT AAT TTA AAA
15 10
Gly Val Gin Cys Glu Val Gin Leu Leu Asp Ser Gly Gly Gly Leu
GGT GTC GAG TGT GAG GTG GAG CTG TTG GAT TCT GGG GGA GGC TTG
15 20 25
Val Gin Pro Gly Gly Cys Leu Arg Leu Ser Cys Ala Ala Ser Gly
GTA CAG CCT GGG GGG TGC CTG AGA CTC TCC TGT GCA GCC TCT GGA
30 35 40
Phe Thr Phe Ser Ser Cvs Ala Met Ser Trp Val Arg Gin Ala Pro
TTC ACC TTT AGC AGC TGT GCC ATG AGC TGG GTC CGC CAG GCT CCA
45 50 52 52a 55
Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Glv Ser Glv Gly
GGG AAG GGG CTG GAG TGG GTC TCA GCT ATT AGT GGT AGT GGT GGT
60 65 70
Ser Thr Tvr Tvr Ala Asp Ser Val Lys Glv Arg Phe Thr Ile Ser
AGC ACÕ TAC TAC GCA GAC TCC GTG AAG GGC CGC TTC ACC ATC TCC
75 80 82 82a 82b 82c
Arg Asp Lys Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu
AGA GAC AAA TCC AAG AAC ACG TTG TAT CTG CAÕ ATG AAC AGC CTG
85 90 95
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Glv Glv Asn
AGA GCC GAG GAC ACG GCC GTA TAT TAC TGT GCG AAA GGT GGC AAC
100 A B C 105
Asp Ile Leu Thr Glv Tvr Tvr Ala Trp Gly Gin GLy Thr Leu Val
GAT ATT TTG ACT GGT TAT TAT GCT TGG GGC CAG GGA ACC CTG GTC
110
Fig. 4. Nuclcotide and deduced amino acid sequence of (he heavy chain of HuMab
L612. Underlined amino acids represent the complementary determining regions CDRI.
CDR2, and CDR3 (95-102) in order. * The sequence data are under accession no. LI 1699
from GenBank.
test was performed (Table 3). Purified gangliosides (5 nmol) isolated
from melanoma cells and other sources were assessed for their ability
to competitively block HuMab L612 binding to M12 in the IA assay.
Both GM4 and GM1 showed the strongest blocking. Sphingosine
showed some blocking but was much weaker. None of the neutral
glycolipids or other gangliosides blocked L612 HuMab binding.
To confirm that HuMab L612 reacts to ganglioside GM3, we de
veloped HPTLC plates with purified gangliosides from melanoma cell
lines M12 and M14. Fig. 2 shows a representative immunostaining
experiment indicating the specificity of HuMab L612 binding to GM3.
In Lanes 2 and 4, L612 bound only to GM3. In Lane 1, gangliosides
were treated with Vibrio cholerae neuraminidase to remove sialic acid.
No antibody binding was observed, indicating that sialic acid on GM1
was part of the recognition site of HuMab L612. These studies con
firmed the above studies on the ability to abolish L612 binding to cells
by treatment with neuraminidase. GM3 from melanoma cells has two
bands which represent different lengths of fatty acid chains (2). The
HuMab L612 bound to the lower band as well as the upper band.
Immunostaining Tissue Sections. A three-step immunoperoxi-
dase staining technique was used to evaluate the specificity of the
HuMab L612 to various types of tumor tissue. One of the difficulties
of assessing HuMab staining of tumor tissue sections is that the
indicator antibody will also bind to natural antibodies in the tissue
sections. We circumvented this problem by using an a-anti-idiotype
murine IgG Mab (18C10) specific to HuMab L612 as an indicator
antibody. Fig. 3, A, B, and C show HuMab L612 immunostaining of
frozen sections of an invading cutaneous melanoma, colon adenocar-
cinoma, and lung adenocarcinoma, respectively. HuMab L612
strongly stained invading melanoma cells in the skin; however, it did
not stain adjacent tissue cells. Tissue sections of melanoma biopsies
consistently showed very strong binding with the HuMab. Strong
HuMab staining was also demonstrated with colon and lung adeno-
carcinomas. No binding to normal adjacent tissues in the colon or lung
was observed.
Cloning and Sequencing of HuMab L612 Variable Regions.
The variable regions of the heavy and light chain genes were cloned
and sequenced using PCR. Specific oligonucleotide primers were
constructed that corresponded to the 5' signal peptide and conserved
3' constant region of both chains. PCR was performed on RNA
extracted from L612 cells and the PCR cDNA products were cloned
into Bluescript vector. Multiple clones containing the heavy and light
chain V regions were selected for sequencing. Analysis of the gene
sequence verified that the PCR cDNA products encoded functional
domains. All the clones had identical sequences demonstrating no
error in cloning. RNA from different passages of the L612 cell line
was run separately in the PCR, and the PCR products were cloned and
sequenced to verify gene sequences.
Analysis of the sequence of the heavy chain indicated that it be
longed to the heavy chain subgroup III (Fig. 4) (32). The leader
sequence is similar to many of the other antibodies that belong to this
group (32). L612 HuMab is unusual in that it has two additional
cysteine residues in the V,, region at position 17 in the framework
segment of the antibody, FRI, and at position 32 in CDRI; no other
V//III sequences have these additional cysteine residues. CDR3 is 11
amino acids (GGNDILTGYYA) long and constitutes a region of con
siderable variability among the members of this subgroup.
The sequence analysis of the light chain indicated it belonged to K
light chain variable subgroup IV (Fig. 5). The L612 light chain is
100% identical to the reported V K IV germline sequence from posi
tion 1 (FRI region) to position 95 (CDR3 region) (31), suggesting that
no somatic mutation has occurred. The CDR3 is 9 amino acids
(QQYYSTPPT) long and constitutes a region with many similarities
among this subgroup. The light chain uses JK\ with a proline residue
located at the V-J junction.
We also performed genomic DNA sequencing as described previ
ously (33) of the variable antibody region genes of L612 cells. The
genomic DNA sequence of L612 V light chain and heavy chain
showed that the nucleic acid sequences were identical to the PCR
analysis (data not shown). Analysis of genomic antibody sequences by
digestion with restrictive enzymes and Southern blot analysis with a
Ju probe indicated the cell line was monoclonal (data not shown).
HuMab L612 light chain variable region sequence*
15 10 15
Asp Ile Val Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu
GAC ATC GTG ATG ACC CAG TCT CCA GAC TCC CTG GCT GTG TCT CTG
20 25 27 27a 27b 27c
Gly Glu Arg Ala Thr ILe Asn Cys Lvs Ser Ser Gin Ser Val Leu
GGC GAG AGG GCC ACC ATC AAC TGC AAG TCC AGC CAG AGT GTT TTA
27d 27e 27f 30 35
Tyr Ser Ser Asn Asn Lvs Asn Tvr Leu Ala Trp Tyr Gin Gin Lys
TAC AGC TCC AAC AAT AAG AAC TAC TTA GCT TGG TAC CAG CAG AAA
40 45 50
Pro Gly Gin Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg
CCA GGA CAG CCT CCT AAG CTG CTC ATT TAC TGG GCA TCT ACC CGG
55 60 65
Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
GAA TCC GGG GTC CCT GAC CGA TTC AGT GGC AGC GGG TCT GGG ACA
70 75 80
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Ala Glu Asp Val Ala
GAT TTC ACT CTC ACC ATC AGC AGC CTG CAG GCT GAA GAT GTG GCA
85 90 95
Val Tyr Tyr Cys Gin Gin Tvr Tvr Ser Thr Pro Pro Thr Phe Gly
GTT TAT TAC TGTCAG CAÕ TAT TAT AGT ACT CCT CCG ACG TTC GGC
100 105
Gin GLy Thr Lys Val Glu Ile Lys Arg
CAÕ GGG ACC AAG GTG GAA ATC AAA CGA
Fig. 5. Nucleotide and deduced amino acid sequence of the light chain of HuMab L612.
Underlined amino acids represent the complementary determining regions CDRI, CDR2.
and CDR3 (90-97) in order. * The sequence data are under accession no. LI 1700 from
GenBank.
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HUMAN MONOCLONAL ANTIBODY TO GANGLIOSIDE GMJ
Transfection and Expression of Human-Human Chimeric IgM-
L612. HuMab L612 variable genes were genetically engineered to be
expressed in a myeloma cell line. The objective was to determine the
functional activity of the cloned variable genes. The isolated L612
genomic V, and V,/ genes were cloned into specific expression vectors
and were simultaneously transfected into nonproducing myeloma cell
lines. Stably transfected cells (IgM-L612) were selected for both drug
markers. Transfectomas producing both chimeric heavy and light
chains were isolated and cultured, and the supernatant was collected.
Chimeric antibodies were concentrated and assessed for binding to
M12 cells using an IA assay. Two separate selected transfectoma lines
of chimeric HuMab bound and reacted strongly to M12. These studies
indicated that the cloned V/, and V, genes were functional.
DISCUSSION
This study characterizes the binding specificity and sequence of a
HuMab that binds to GM3 on the cell surface of human neoplastic
cells. HuMab L612 binds not only to GM3 but also to GM4. The
removal of the sialic acid component of GM1 abolished the binding of
L612. The specificity of L612 to the sialic acid on GM3 was verified
by the lack of blocking of asialo-GM3 in the IA inhibition assay. These
studies suggest that the HuMab L612 detects the terminal sugar of the
gangliosides that have a NeuAc a2-3-galactose residue. There is a
possibility that L612 may react with other glycoconjugates with N-
and O-linked structures. Future studies will examine other potential
glycoconjugates as antigens for L612.
Immunostaining of frozen sections with HuMab L612 indicated a
strong specificity for neoplastic tissue, including melanoma, colon
adenocarcinomas, and lung adenocarcinomas. Recently, we have
shown that HuMab L612 binds to renal cell carcinomas (35). GM3 is
an excellent target antigen on melanoma cells in that it is expressed in
>95% of melanoma biopsies and often at high density. There are
several hypotheses why HuMab L612 recognizes GM3 antigen on
tumor cells and not on normal cells. One reason why L612 may bind
to neoplastic cells is the high density of GM1 compared to normal
cells. Another possible explanation is that the ganglioside is more
assessable on the tumor cell surface (14). Further studies are needed
to determine the mechanism of the recognition of cell surface GM, by
L612 HuMab. A similar differential binding specificity between mela
noma and normal cells has been reported with a murine Mab recog
nizing GM3 (14, 34).
The expression of GMs on the neoplastic cell surface has been
shown to influence several functional properties including cell adhe
sion, spreading, and motility (36). These are essential processes during
invasion and metastasis of tumors (37). It is interesting to note that
sialidase treatment can neutralize the effect of GM1 in these processes
(36). The sialic acid in GM1 plays an important role in the functional
activity of the molecules as well as a role as an antigenic determinant.
The HuMab L612 may have potential application in blocking mela
noma cell invasion and metastasis.
The development of PCR-based cloning techniques allows one to
rapidly clone and sequence antibody variable regions. The sequencing
of the light chain of HuMab L612 showed it to be identical to the
germline VKIVgene and to use 7^1. Except for the proline residue at
the V-J junction, the light chain of HuMab L612 was identical to the
light chain of FK-001; a IgM antibody produced by an EBV trans
formed B-cell line specific to Pseudomonas (¡eruginosa exotoxin A
(40). The heavy chain of the FK-001 belongs to the variable subgroup
(V,/) (31). The Vf, region of L612 had two additional cysteine resi
dues. These two additional cysteine residues have the potential of
producing disulfide bonds, thus making the IgM molecule more rigid,
possibly leading to changes in functional activity (39). Several of the
light chains in the V K IV subgroup are from cold agglutinins with
anticarbohydrate specificities (41). It is interesting to note that most
cold agglutinins bind to carbohydrate structures consisting of lactos-
amine disaccharides on RBC (41). GM1 and sphingosine, which L612
binds to, are normal components of human erythrocytes; however,
L612 does not bind to erythrocytes.
There are only a few HuMabs to tumor-associated antigens (19, 21,
42, 43) produced by stable secreting EBV-transformed cloned B-cell
lines that have been reported. Only a subset of these antibodies rec
ognize carbohydrate epitopes. None have been sequenced to assess
restriction of heavy and light chain variable family usage (44). The V,,
of L612 HuMab belongs to a large family and shows considerable
homology to the sequences of 18/2, 18/17, 18/9, and 1/17, which are
anti-DNA antibodies (45, 46), and the anti-tumor-associated antigen
antibody 4G12 (46). Interestingly, the latter represents a Mab (mouse-
human hybridoma) to a tumor-associated glycoprotein antigen on
human malignant tumor cells. Thus, the L612 HuMab may belong to
a family of autoantibodies. Immune responses to melanoma have been
thought to represent autoimmune responses in the host, and anti-
ganglioside antibody responses in humans are considered as possible
autoimmune responses (42). Preliminary studies have shown the pres
ence of anti-GM1 antibodies in melanoma patients (48). Currently, we
are evaluating the presence of anti-GM, immune responses against
human melanoma. The presence of IgM anti-GM1 antibodies may be
a natural autoantibody defense mechanism against cells, such as neo
plastic cells, that overproduce this antigen.
Cloning and sequencing of these human variable genes will allow
us to genetically engineer immunoglobulin class switches (38) and
"rescue" antibodies from unstable EBV-transformed B-cell lines, as
well as allow the engineering of recombinant chimeric molecules and
production of functional recombinant immunoglobulin-like molecules
such as single chain Fv (39). The availability of human antibody genes
now permits us to engineer antibodies to make them better therapeutic
agents, stabilize their structure, use site-directed mutagenesis to ex
plore and control specificity, etc. Sequencing of the amino acid struc
ture of HuMabs specific to ganglioside antigens may provide insights
into the mechanism of interaction between the antibody and the car
bohydrate epitope they recognize.
Currently, clinical pilot studies are being planned to test the efficacy
of the HuMab L612 to treat patients with malignant melanoma. Pre
liminary studies on one melanoma patient have shown that intra-
lesional injection of HuMab L612 with control HuMab IgM signifi
cantly induced tumor regression.
ACKNOWLEDGMENTS
We wish to thank Dr. A. Walsh for her editorial assistance.
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1993;53:5244-5250. Cancer Res
Dave S. B. Hoon, Yuan Wang, Lan Sze, et al.
Antigen on Human Cancers
M3
Ganglioside G
Molecular Cloning of a Human Monoclonal Antibody Reactive to
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