Note: Descriptions are shown in the official language in which they were submitted.
-- 1 3398 1 6
- TUMOR IMMUNOTHERAPY US-NG
- ANTI-IDIOTYPIC ANTIBOD-ES
TABLE OF CONT~TS
Page
1. Field of the Invention............................... 7
2. Background of the Invention.......................... 7
~ 2.1. Anti-Idiotypic Antibodies...................... 7
2.2. Tumor-Associated Antigens...................... 9
2.3. Idiotypic Manipulations of Tumor Immunity.. 10
2.4. Suppressor Cells and Suppressor Factors.... 12
3. Summary of the Invention............................. 13
3.1. Definitions.................................... 16
4. Description of the Invention......................... 17
5. Detailed Description of the Figures.................. 23
5.1. Idiotypic Interactions Initiated by
Antigenic Stimuli............................. 27
20 5.2. Manipulation of Idiotope Expression by
Anti-Idiotypic Antibodies............................ 30
5.2.1. Selection and Amplification of
Pre-existing Antitumor Idiotopes... 31
5.2.1.1. Genetic Restriction................. 33
5.2.1.2. Route of Immunization............... 35
- 5.2.1.3. Idiotope Specificity................ 37
5.2.2. Internal Image Antibodies..................... 39
5.3. Production of Anti-Idiotypic Monoclonal
Antibodies Specific to an Idiotype which
Recognizes a Defined Tumor Antigen................... 42
5.3.1. Production of Anti-Idiotypic Mono-
clonal Antibodies by Immunization
with an Antibody (ABl) that Recog-
nizes a Defined Tumor Antigen................ 42
~ - ~33~B1~
-2-
5.3.2. Production of Anti-Idiotypic Mono-
clonal Antibodies which are Direc-
ted against an Idiotope on T Cells
5that Recognizes a Defined Tumor
Antigen............................... 44
5.4. Evaluation and Demonstration of
Immunopotency by Induction of
Tumor-Specific CMI............................ 45
105.5. Further Characterization of Anti-Idiotypic
Antibody...................................... 46
S.6. Uses in Immunoprophylaxis, Immunotherapy,
and Immunoassay............................... 46
-~ 5.6.1. Immunization Against Tumors............ 46
5.6.2. Adoptive Immunotherapy................. 48
5.6.3. Inhibition of Immune Suppression of
Anti-Tumor Reactivity................. 51
5.6.4. Immunoaffinity Applications............ 51
5.6.5. Immunoassays........................... 51
6. Immunotherapy of Murine Sarcomas with Auto-Anti-
Idiotypic Monoclonal Antibodies which Bind to
~ Tumor-Specific T Cells.............................. 52
6.1. Materials and Methods.......................... 53
6.1.1. Mice................................... 53
6.1.2. Tumors................................. 53
6.1.3. Assay of Delayed-Type Hyper-
- - sensitivity............................ 53
6.1.4. Generation of Auto-Anti-Idiotypic
Monoclonal Antibodies.................. 54
6.1.5. T Cell Hybridomas....................... 56
6.1.6. Isolation of Suppressor Factors by
Affinity Chromatography with
Monoclonal Antibody..................... 56
6.1.7. Binding of Suppressor Factor K54SF
to MCA-1490 Cells..................... 57
1 33981 6
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.~
Page
6.1.8. Isolation and Culture of T Cell
Lines................................ 58
6.1.9. In Vivo Manipulation of Tumor
Growth............................... 59
6.2. Subcutaneous Administration of mAb 4.72 or
mAb 5.96 Induces Tumor-Specific DTH to
MCA-1490 and MCA-1511........................ 59
6.3. Intravenous Administration of mAb 4.72
; Suppresses DTH to MCA-1490................... 61
6.4. Expression of an Idiotope Defined by mAb
4.72 on T Cells Mediating DTH to MCA-1490.... 63
6.5. Expression of an Idiotope Recognized by
mAb 4.72 on Factors Derived from Suppressor
T Cells...................................... 65
6.6. Serotherapy of Mice with MCA-1490 or
MCA-1511 Tumors.............................. 77
6.7. Discussion........... ,................... ~...................... .79
20 7. Anti-Idiotypic Antibody Specific to an Idiotype
which Recognizes a Human Melanoma-Associated
GD3 Ganglioside Antigen....................... ..... .................. .83
7.1 Materials and Methods.......................... 84
7.1.1. Mice.................................. 84
- 25 7.1.2. Target Cells.......................... 84
7.1.3. Glycolipid............................ 84
7.1.4. Monoclonal Antibodies................. 84
7.1.5. Coupling of Antibody with Keyhole
Limpet Hemocyanin.................... 86
7.1.6. Production of Monoclonal
Anti-Idiotypic Antibodies (Ab2)
Specific for MG-21 (Abl)............. 86
- 7.1.7. mAb (Abl) Binding Inhibition Assay. 87
- _4_ 1 ~ 3 ~ 6
,, .
Page
7.1.8. Determination of Immunoglobulin
Isotype.............................. .................. 88
7.1.9. Anti-Idiotypic Antibody (Ab2)
Binding Assay........................ .................. 88
7.1.10. Radioiodination of Antibody and
Direct 125I-Ab2 Binding Assay......... 89
7.1.11. Antibody-FITC Conjugation............. 89
7.1.12. Fluorescence Activated Cell Sorter
Analysis.............................. 90
7.1.13. Complement-Dependent Cytotoxicity
Assay................................. 90
7.1.14. Antibody-Dependent Cellular
Cytotoxicity Assay.................... 91
7.1.15. Competition Assay for Detecting
Anti-MG-21 Antibodies in Patient
' Sera.................................. 92
7.2. Results....................................... 93
7.2.1. Selection of Hybridomas................ 93
7.2.2. mAb 2Cl (Ab2) is Specific
for MG-21............................. 94
7.2.3. mAb 2C1 (Ab2) Inhibits the Binding
of MG-21 (Abl) to M-2669 Cells and
GD3 Antigen in a Dose-Dependent
Manner................................ 94
25 7.2.4. Antibody 2C1 Inhibits the CDC and
~-- ADCC Activity of MG-21 Against
M-2669 Cells......................... 97
7.2.5. Detection of Anti-MG-21 Antibodies
in Patient Sera Using mAb 2C1 as a
Probe................................ 101
8. Monoclonal Anti-Id~otypic Antibodies Related to
the p97 Melanoma Antigen........................... 103
8.1. Materials and Methods......... -................... 104
8.1.1. Animals.............................. 104
~339816
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Page
8.1.2. Human Melanoma Cells............. .......104
8.1.3. Mouse Melanoma Cells............. .......104
8.1.4. Antibodies....................... .......104
8.1.5. Screening of Hybridomas.......... .......105
8.-1.6. Studies on Purified Anti-Idiotypic
Antibodies...................... .......106
8.1.7. Competition of Radiolabelled p97
for Binding to Fab Fragments of
mAb 96.5........................ .......107
8.1.8. Searches for Anti-Idiotypic
Antibodies (Ab3) In Vivo........ .......107
8.2. Results.................................. .......108
8.2.1. Generation of Ab2 Binding to
Idiotypic Determinants on mAb 96.5. 108
8.2.2. Tests on Ab2 Specificity for the
Antigen-Binding Site of mAb 96.5... 109
8.2.3. Analysis of the Binding of Ab2 to
a Series of mAb which Specify p97
- Epitopes other than p97a......... .......110
8.2.4. Induct-ion of an Ab3 Response.... .......113
8.3. Discussion............................... .......116
9. Anti-Idiotypic Antibodies Specific for
Anti-Carcinoma Antibody L6.................... .......118
~' 9.1. Materials and Methods.................... .......119
9.1.1. Animals.......................... .......119
9.1.2. Cell Lines....................... .......119
9.1.3. Antitumor Antibodies (nAbln)..... .......119
9.1.4. Generation of Anti-Idiotypic
Antibodies (nAb2n)............... .......120
9.1.5. Ab2 Purification................. .......120
9.1.6. Inhibition Assay to Detect Ab2
Binding to the Paratope Region
of L6........................... .......121
9.1.7. Blocking Assay to Detect
1 33981 6
Competition between Ab2 and Antigen
for L6 Binding Sites................. 121
9.1.8. Induction of an Ab3 Response......... 122
59.1.9. Purification of Polyclonal Ab3....... 123
9.1.10. Induction of an Ab3 Response......... 124
9.1.11. Assay for Binding of Ab3 to Cells.... 124
9.1.12. Measurement of the Ability of Ab3
to Inhibit the Binding of Ab2 to
L6 Fab............................... 125
9.1.13. Assay for Binding of Ab3 to Cells..... 125
9.2. Results....................................... 126
9.2.1. Generation of Anti-Idiotypic
mAb (Ab2) to L6 (Abl)................. 128
9.2.2. Characterization of Ab2 Binding to
Idiotopes on L6...................... 129
9.2.3. Specificity of the L6-Generated Ab2 129
9.2.4. Ab2 Epitope Specificity............... 129
9.2.5. Ability of Ab3 to Bind to the
Antigen Defined by L6................. 130
9.2.6. Ability of Ab3 to Bind to the
Antigen Defined by L6................. 131
9.3. Discussion.................................... 131
10. Deposit of Microorganisms.......................... 132
; 25
~7~ 1 33981 6
l. FIELD OF THE INVENTION
The present invention is directed to methods which
utilize anti-idiotypic antibodies for tumor immunotherapy
and immunoprophylaxis. The invention relates to the
- manipulation of the idiotypic network of the immune system
for therapeutic advantage, e.g. by use of anti-idiotypic
antibody for immunization against tumor, for inhibition of
immune suppression mediated by suppressor T cells or
suppressor factors expressing an idiotope directed against a
tumor antigen, for activation of lymphocytes used in
adoptive immunotherapy, etc. In a specific embodiment,
monoclonal anti-idiotypic antibodies which were raised
against the idiotype of an antibody that defines a self-
differentiation antigen, such as an oncofetal, ordifferentiation antigen, can be used in vivo to induce an
immune response against tumors bearing the oncofetal
antigen.
~ The anti-idiotypic monoclonal antibodies of the present
invention are valuable in tumor immunotherapy and
immunoprophylaxis, and of general importance in human
medicine. The molecules of the present invention may also
be used as reagents in immunoassays such as ELISA tests and
radioimmunoassays which are useful as diagnostic tools for
25 the detection of antitumor antibodies or tumor antigens, and
in immunoabsorption assays which are useful for the
isolation and identification of anti-tumor antibodies. In
addition, these reagents will be valuable tools in
understanding the development and growth of neoplasia.
2. BACKGROUND OF THE INVENTION
2.l. ANTI-IDIOTYPIC ANTIBODIES
Anti-idiotypic antibodies or anti-idiotypes are
35 antibodies directed against the antigen-combining region or
1 33q8 1 6
variable region (called the idiotype) of another antibody
molecule. In theory, based on Jerne's network model of
idiotypic relationships (Jerne, N.K., 1974, Ann. Immunol.
(Paris) 125c:373; Jerne, N.K., et al., 1982, EMB0 1:234),
immunization with an antibody molecule expressing a paratope
(antigen-combining site) for a given antigen should produce
a group of anti-antibodies, some of which share with the
antigen a complementary structure to the paratope.
Immunization with a subpopulation of the anti-idiotypic
antibodies should in turn produce a subpopulation of
antibodies or immune cell subsets that are reactive to the
initial antigen.
A network of idiotopes and anti-idiotopes has been
invoked to explain immune regulation, with common or related
idiotopes of antibodies, B lymphocytes, and various subsets
of T lymphocytes and their soluble products interacting with
anti-idiotopes (Jerne, N.K., 1974, supra; Urbain, J., et
al., lg77, Proc. Natl. Acad. Sci. U.S.A. 74:5126; Rajewski,
R. and Takemori, T., 1983, Ann. Rev. Immunol. 1:569).
20 Studies which have been done on immunity to both haptens and
viral antigens indicate that B cell derived anti-idiotypic
antibodies can induce T cell responses (Rajewski, R. and
Takemori, T., supra; Urbain, J., et al., supra; Binz, H. and
Wigzell, H., 1978, J. Exp. Med. 147:63). For example, Ertl
25 et al. immunized mice with a Sendai virus-specific T cell
clone and produced an anti-idiotypic mAb which regulated the
DTH response to Sendai virus (Ertl, H.C.J., et al., 1982,
Proc. Natl. Acad. Sci. U.S.A. 79:7479). As evidence for a B
cell antibody arising in response to a T cell idiotope,
30 Kennedy et al. observed tumor rejection, but no antitumor
antibodies, in mice treated with anti-idiotopic antibodies
- relating to the SV40 T antigen (Kennedy, R.C., et al., 1985,
J. Exp. Med. 161:1432). The administration of exogenous
anti-idiotypic antibody can exert enhancing or suppressive
35 influences, dependent on, among other variables, the dose of
1 33~81 6
the antibody (Reth, M., et al., 1981, Nature (London)
290:257).
2.2. TUMOR-ASSOCIATED ANTIGENS
A variety of tumor-associated antigens (TAA) have been
described. One class of TAA is the tumor-specific
transplantation type of cell-surface antigen (TSTA), which
has been recognized by induction of immune responses in
tumor transplant experiments.
Another class of TAA is the oncofetal or differenti-
ation antigen. Oncofetal antigens are mainly embryonic or
fetal cell products which are expressed by malignant cells
due to derepression of the embryonic genes. One example of
human oncofetal antigens are the carcinoembryonic antigens
(CEA) of the colon. This set of antigens is found on
tissues derived from the fetal gastrointestinal tract, and-
on tumors of the gastrointestinal tract. Alpha-fetoprotein
is another known oncofetal antigen, which is secreted by
hepatocarcinoma cells, as well as malignant yolk sac and
20 fetal liver cells, and the proliferating fraction of adult
liver cells.
A third class of TAA includes virally-induced tumor
antigens. These include the T antigen induced by DNA tumor
viruses, and the envelope antigens of RNA tumor viruses.
A variety of human cell-surface TAA has been detected
- in human neoplasms by mouse monoclonal antibodies
(Hellstrom, K.E., et al., 1982, Human Tumor-Associated
Antigens Identified by Monoclonal Antibodies, in, Springer
Seminars in Immunopathology: Mechanism of Host Resistance
30 in Cancer. Springer, New York, pp. 127-146; Herlyn, M., et
al., 1984, in, Contributions to Oncology, Karger, Basel,
Switzerland, Vol. 19 pp. 160-170; Reisfeld, R.A. and Sell,
S., eds., 1985, Monoclonal Antibodies and Cancer Therapy,
UCLA Symposia on Molecular and Cellular Biology, New Series,
35 Vol. 27, Alan R. Liss, Inc., New York). Many of these
1 33981 6
--10--
antigens are termed oncofetal, since they are expressed
strongly by tumors and certain embryonic cells and much more
weakly by normal cells from the adult host. Other tumor-
associated antigens have been detected by their ability to
stimulate host cell-mediated immunity (CMI) in human cancer
(Hellstrom, K.E., and Hellstrom, I., 1969, Adv. Cancer Res.
12:167-223; Halliday, W.J., and Maluish, A.E., 1982 in,
Assessment of Immune Status by the Leukocyte Adherence
Inhibition Test, Academic, New York pp. 1-26; Herberman,
R.B., 1974, Adv. Cancer Res. 19: 207-263; Thomson, D.M.P.,
1980, Cancer Res. 29: 627-629; Halliday, W.J., et al; 1975,
Int. J. Cancer 16:645-654) and in tumor-bearing animals
(Taranger, L.A., et al., 1972, Science 176: 1337-1340;
Halliday, W.J., et al., 1974, Cell Immunol. 10:467-475;
Steele, G., et al., 1975, J. Natl. Cancer Inst. 54: 959-
967). Some of these antigens are also oncofetal, but their
molecular nature and relationship to the antigens defined by
mouse monoclonal antibodies are unclear.
Rat monoclonal antibodies to antigens shared by mouse
20 bladder carcinomas have been obtained recently (Hellstrom,
I., et al., 1982, Int. J. Cancer 29: 175-180; Hellstrom, I.,
et al., 1985, Cancer Res. 45: 2210-2218). One of the
antibodies, 6.10, was shown to be specific for a bladder
tumor oncofetal antigen (Hellstrom, I., et al., 1985, Cancer
25 Res- 45:2210-2188).
Unmodified tumor antigen (Hoover, H.C., et al., 1985,
Cancer 55:1236-1243; McIllmurray, M.B., et al., 1978, Br.
Med. J. 1:579-580) and live recombinant viruses (Earl, P.L.,
et al., 1986, Science 23:728-731; Lathe, R., et al., 1987,
30 Nature 236:878-880) have been used in attempts to induce
therapeutically beneficial anti-tumor immune responses.
2.3. IDIOTYPIC MANIPULATIONS OF TUMOR IMMUNITY
- Several idiotypic manipulations of tumor immunity have
35 been reported. Nepom et al. (1984, Proc. Natl. Acad. Sci.
-11- 1339816
U.S.A. 81:2864-2867) described induction of tumor immunity
where an oncofetal antigen was introduced into a xenogeneic
host. Polyclonal anti-idiotypic antibodies were prepared
which recognized idiotypic determinants on a mouse antibody
to the pg7c epitope of human melanoma antigen p97. The
polyclonal antisera could induce, in mice, both CMI and an
- Ab3 response to p97. Forstrom et al. (1983, Nature (London)
303:627-629) used an anti-idiotypic antibody to induce CMI
in mice to a syngeneic chemically induced sarcoma. In this
study, the anti-idiotypic antibody was an auto-antibody
produced by hyperimmunization to the tumor, and the tumor
antigen was not a defined molecule. The studies of Flood et
al. (1980, Proc. Natl. Acad. Sci. U.S.A. 77:2209-2213) and
Binz et al. (1982, Int. J. Cancer 29:417-423) demonstrated
idiotypic manipulations of tumor immunity in syngeneic
systems, with undefined antigen molecules. Flood et al.
showed evidence that murine anti-idiotypic T lymphocytes
could participate in an autoimmune reaction to
fibrosarcoma-specific T lymphocytes, and thus adversely
20 affect an individual's immune response to a tumor. Binz et
al. used anti-idiotypic antibodies to induce in vitro
proliferation of T lymphocytes specifically cytotoxic to rat
sarcoma cells.
Additional studies have looked at the effect of anti-
25 idiotypic antibodies on tumor growth. Tilkin et al. (1981,
Proc. Natl. Acad. Sci., U.S.A. 78:1809-1812) showed that
immunization of mice with lymph node cells sensitized to an
unidentified sarcoma antigen resulted in tumor rejection and
growth inhibition. Kennedy et al. (1985, J. Exp. Med.
30 161:1432-1449) described the suppression of tumor formation
in mice challenged with SV40-transformed cells, after
injection with polyclonal anti-idiotypic antibodies related
to the SV40 antigen.
Koprowski et al. (1984, Proc. Natl. Acad. Sci. U.S.A.
35 81:216-219) showed the presence of anti-idiotypic antibodies
-12- 1339816
in patients who had a remission of carcinoma after
administration of a monoclonal antibody directed against
human gastrointestinal cancer.
2.4. SUPPRESSOR CELLS AND SUPPRESSOR FACTORS
The suppressor cell/factor cascade has been recognized
in tumor and model systems (Nepom, G.T., et al, 1983,
Experientia 39:235; Asherson, G.L., et al., 1984, Immunology
53:491; Dorf, M.E. and Benacerraf, B., 1984, Ann. Rev.
Immunol. 2:127). Suppressor cells play an important role in
regulating tumor immunity (Greene, M.I., et al., 1977, Proc.
Natl. Acad. Sci. U.S.A. 74:5118; Hellstrom, K.E., et al.,
1978, J. Exp. Med. 148:799; Nepom, G.T., et al., 1983
Experientia 39:235; North, R.J., 1982, J. Exp. Med.
155:1063; Yamauchi, K., et al., 1979, J. Immunol. 123:1653).
Some of these cells produce suppressor factors (SF) (Nelson,
K., et al., 1975, Int. J. Cancer 16:539; Greene, M.I., et
al., 1977, J. Immunol. 119:759; Koppi, T.A. and Halliday,
W.J., 1983, Cell. Immunol. 76:29) which can be detected in
20 sera from tumor-bearing animals and human patients through
the inhibition (Nblocking~) of in vitro manifestations of
cell-mediated immunity (CMI) (Hellstrom, K.E., et al., 1978,
J. Exp. Med. 148:799; Hellstrom, I., et al., 1969, Proc.
Natl. Acad. Sci. U.S.A. 62:362; Baldwin, R.W., 1973, Adv.
25 Cancer Res. 18:1; Halliday, W.J., et al., 1974, Cell.
Immunol. 10:467; Steele, G., et al., 1975, J. Natl. Cancer
Inst. 54:959; Hellstrom, K.E., et al., 1977, Biochim.
Biophys. Acta 473:121; Halliday, W.J., et al., 1980, J.
Natl. Cancer Inst. 65:327; Koppi, T.A. and Halliday, W.J.,
30 1981, J. Natl. Cancer Inst. 66:1089; Kuchroo, V.K., et al.,
1983, Cancer Res. 43:1325; Koppi, T.A., et al., 1981, J.
Natl. Cancer Inst. 66:1097). Some SF have tumor specificity
and can be removed from serum by absorption with the
respective tumor or tumor-related antigen but not with
35 tumors expressing different antigens (Kuchroo, V.K., et al.,
-13- 1 33q81 6
1983, Cancer Res. 43:1325; Baldwin, R.W., 1973, Adv. Cancer
Res. 18:1; Hellstrom, K.E., et al., 1977, Biochim. Biophys.
Acta 473:121; Koppi-Reynolds, T.A. and Halliday, W.J., 1984,
Immunol. Lett. 8:219). This suggests that there is a
binding site or idiotope on SF molecules, complementary to
the tumor antigen determinants (Nepom, G.T., et al., 1983,
Experientia 39:235; Hellstrom, K.E., et al., 1977, Biochim.
Biophys. Acta 473:121). It has been reported that
circulating SF bind to antibodies from mice hyperimmunized
with tumor cells, suggesting that the antibodies are
complementary to idiotypic determinants on the SF
(Hellstrom, K.E., et al., 1977, Biochim. Biophys. Acta
473:121; Nepom, G.T.j et al., 1977, Proc. Natl. Acad. Sci.
U.S.A. 74:4605); in these studies, both the antibody and
suppressor cell responses were assumed to be polyclonal.
Certain immune sera, obtained after tumor removal or
regression, abrogate (~unblock~) the antigen-specific
suppressive ("blocking~) activity of tumor-bearer sera as
measured in vitro (Halliday, W.J., et al., 1974, Cell.
20 Immunol. 10:467; Hellstrom, I. and Hellstrom, K.E., 1970,
Int. J. Cancer 5:195). It has been theorized that this
Nunblockingn effect was mediated by anti-idiotypic
antibodies (Hellstrom, K.E., et al., 1977, Biochim. Biophys.
Acta 473:121). It was reported that Nunblocking" antibodies
25 had a therapeutic effect in rats with primary or
transplanted polyoma virus-induced tumors (Bansal, S.C. and
Sjogren, H.O., 1972, Int. J. Cancer 9:490; Sjogren, H.O. and
Bansal, S.C., 1971, in Progress in Immunology, Amos, B.,
ed., Academic Press, New York, p. 921; Bansal, S.C. and
30 Sjogren, H.O., 1971, Nature (New Biol.) 233:76).
3. SUMMARY OF THE INVENTION
The present invention is directed to methods which
utilize anti-idiotypic antibodies, or fragments thereof, for
35 tumor immunotherapy and immunoprophylaxis. The invention
-1.- 133981~
relates to the manipulation of the idiotypic network of the
immune system for therapeutic advantage. Particular
embodiments include the use of anti-idiotypic antibodies for
immunization against tumor, for activation of lymphocytes to
be used in adoptive immunotherapy, and for inhibition of
immune suppression mediated by suppressor T cells or
suppressor factors expressing an idiotope directed against a
tumor antigen. In specific embodiments, monoclonal anti-
idiotypic antibodies, or fragments thereof, (a) which were
raised against the idiotype of an antibody that defines a
tumor antigen, such as an oncofetal or differentiation
antigen, and (b) which exhibit tumor-specific properties
such as induction of tumor-specific cell mediated immunity
(as measured by various assays, e.g. the leukocyte adherence
inhibition assay or the delayed-type hypersensitivity
assay), inhibition of anti-tumor antibody binding, etc. are
identified. The monoclonal anti-idiotypic antibodies, or
fragments thereof, which demonstrate immunopotency can be
used ln vivo in a patient to induce an immune response
20 directed against tumor cells that bear the tumor antigen.
The anti-idiotypic antibodies, or fragments thereof, can
also be used to monitor anti-antibody induction in patients
undergoing passive immunization to a tumor antigen by
administration of anti-tumor antibody.
In another embodiment, the induction of anti-idiotypic
antibodies in vivo by administration of anti-tumor antibody
or immune cells or factors exhibiting the anti-tumor
idiotopes, can be of therapeutic value.
The invention is also directed to the monoclonal anti-
30 idiotypic antibody molecules, antibody fragments, or
chemically modified antibodies or fragments, which recognize
an idiotype directed against a defined tumor antigen. The
molecules of the invention may be produced by any technique
known in the art, including the hybridoma technique
35 originally described by Kohler and Milstein (1975, Nature
-15- l 33981 6
256:495-497), the human B-cell hybridoma technique (Kozbor
et al., 1983, Immunology Today 4:72), and the EBV-
transformation technique (Cole et al., 1985 Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-
96).
The invention is illustrated by way of example in which
serotherapy of mice by injection with anti-idiotypic
antibodies related to a murine fibrosarcoma delayed the
appearance and caused regression of transplanted sarcoma. A
T cell line was established which expressed an idiotope
directed against a fibrosarcoma, and which could transfer
delayed-type hypersensitivity to the fibrosarcoma.
In another example of the invention, a mouse monoclonal
anti-idiotypic antibody which is specific to an idiotype
which recognizes a human melanoma-associated GD3 ganglioside
antigen is described. The anti-idiotypic antibody was
demonstrated to prevent binding of the anti-GD3 antibody to
its antigen, and to inhibit both complement- and antibody-
dependent cytotoxicity of the anti-GD3 antibody. By using
20 the anti-idiotypic antibody as a probe, an assay was
developed to monitor human antibodies to the anti-GD3
antibody in patients receiving the anti-GD3 antibody for
therapeutic or diagnostic purposes.
In a third example of the invention, mouse monoclonal
25 anti-idiotypic antibodies which recognize an idiotype
directed against a human melanoma-associated p97 antigen are
described. Monoclonal anti-idiotypic antibodies were
obtained which could competitively inhibit the binding of
p97 to anti-p97 antibody, and which could induce antibodies
30 to p97 ln vivo.
In another example, we describe murine monoclonal
anti-idiotypic antibodies which recognize an idiotype on
monoclonal antibody L6 which defines a carbohydrate antigen
of human carcinomas. Anti-idiotypic antibodies were
-16- l 33981 6
obtained which were capable of inducing antibodies in vivo
reactive with the carcinoma antigen defined by antibody L6.
The antibody molecules of the invention, fragments of
the antibody molecules containing the idiotype of the
molecules, or chemical modifications of these molecules can
be used to assay for the presence of anti-tumor antibodies,
tumor antigen by competition assays, and the induction of
cell-mediated tumor immunity in immunoprophylactic and
immunotherapeutic applications.
3.1. DEFINITIONS
As used herein, the following abbreviations will have
the meanings indicated:
Abl = antibody 1; the initial antibody of an
anti-idiotypic antibody cascade
Ab2 = antibody 2; anti-idiotypic antibody directed
against an idiotype of Abl
Ab3 = antibody 3; anti-anti-idiotypic antibody,
directed against an idiotype of Ab2
ADCC = antibody-dependent cellular cytotoxicity
anti-Id = anti-idiotypic antibody(ies)
BTCC = bladder transitional cell carcinoma
CDC = complement-dependent cytotoxicity
CMI = cell-mediated immunity
DTH = delayed-type hypersensitivity
FACS = fluorescence-activated cell sorter
FCS = fetal calf serum
FITC = fluorescein isothiocyanate
HRP = horseradish peroxidase
Id = idiotope
Ig = immunoglobulin
i.p. = intraperitoneal
i.v. = intravenous
kDa = kiloDalton
KLH = keyhole limpet hemocyanin
-17- l 3398 1 6
LAI = leukocyte adherence inhibition
mAb = monoclonal antibody(ies)
MCA = 3-methylcholanthrene
OPD = O-phenylene diamine
par = parental (p97-negative) C3H/HeN mouse
melanoma line K-1735-M2
PBS = phosphate buffered saline
PC = peritoneal cells
SC = spleen cell
4. DESCRIPTION OF THE-FIGURES
Figure 1. A schematic diagram of four types of
idiotypic and anti-idiotypic responses initiated by
antigenic stimuli. TH (Fig. lB, lD) or TS (Fig. lC)
substituting for Abl in the stimulation of anti-idiotypic
- antibody production is illustrated.
Figure 2. A schematic diagram of three pathways by
which Ab2 can induce antitumor immunity. Figure 2A shows
the induction of antigen specific TH upon immunization with
20 Ab2. The elicitation of normally ~silent~ Abl specificities
is illustrated (Fig. 2B), as is the triggering of Id TS
(Fig. 2C).
Figure 3. A schematic diagram of immunization with
internal-image anti-idiotypic antibody to elicit anti-
25 antigen response.
Figure 4. T cells of line 90.3 (closed circles)inhibit the growth of MCA-1490 (Panel A) but not MCA-1510 or
MCA-1511 (Panels B and C). The growth of tumor mixed with
control T cells (open circles) was used for comparison.
Figure 5. Treatment of tumor-bearing mice with auto-
anti-idiotypic mAb inhibits tumor growth. mAb were given
intraperitoneally, on days 8, 13, 17, and 21. Tumor size is
presented as the average area of the tumors for the 10 mice
in each treatment group. Mice were treated with mAb 4.72
(closed circles), mAb 5.96 (open squares3, mAb 8.2 (open
-18- l3 3 98l6
circles) or PBS (dashed line). Difference significant at *p
less than 0.05,**p less than 0.01, ***p less than 0.001.
Figure 6. Treatment of tumor-bearing mice with auto-
anti-idiotopic monoclonal antibodies induces regression of
growing sarcomas. The number of mice with tumors greater
than 0.2 cm2 after treatment with mAb 4.72 (closed circles)
- or mAb 5.96 (open circles) is presented for tumor MCA-1490
(top panel) or MCA-1511 (bottom panel).
Figure 7. Inhibition of mAb MG-21 binding to M-2669
clone 13 cells by culture supernatant of anti-idiotypic
antibody-producing hybridoma 2C1.
Figure 8. Direct binding of monoclonal anti-idiotypic
antibody 2C1 to mAb MG-21. Various concentrations of mAb
2C1 (closed circles) or control immunoglobulin Pl.17 (closed
triangles) were added to polyvinyl chloride wells precoated
with mAb MG-21.
Figure 9. Specificity of anti-idiotypic mAb 2Cl for
MG-21 as compared to various mAbs.
Figure 10. Cell-sorting profiles of FITC-conjugated
20 MG-21 against M-2669 cells in the presence or absence of
mAb 2Cl. Tumor cells were stained with FITC-conjugated MG-
21 alone (40 ug/ml, panel a) or remained unstained to obtain
the background level (panel b). Panels c through f show
inhibition of staining with FITC-conjugated MG-21 in the
25 presence of mAb 2C1 at different concentrations (panel c,
160 ug/ml; panel d, 80 ug/ml; panel e, 40 ug/ml; panel f,
20 ug/ml). Panels g and h show staining of FITC-conjugated
MG-21 in the presence of mAb 26.8 (160 ug/ml) and Pl.17
(160 ug/ml).
Figure 11. Binding of purified anti-idiotypic antibody
(Ab2) to Fab fragments of anti-p97 mAb 96.5. Fab antibody
fragments of mAb 96.5 were plated and blocked, various
dilutions of Ab2 added, and the binding of Ab2 detected by
addition of HRP-labelled goat anti-mouse IgG, as described
35 in Sectlon 8.1.5.
-19- 1 3398 1 6
Figure 12. Ab2 can inhibit the binding of mAb 96.5 to
SK MEL-28 cells. Anti-p97 mAb 96.5 (0.33 ug/ml) was mixed
with purified anti-idiotypic antibody (Ab2) (10 ug/ml) and
added to SK MEL-28 cells, which express approximately
400,000 molecules of p97 per cell and which had been plated
onto the wells of Immunolon plates. The binding of mAb 96.5
to the celIs was detected by an ELISA and the inhibition of
this binding by Ab2 was calculated.
Figure 13. Competitive inhibition by anti-idiotypic
antibody (Ab2) of the binding of 125I-labelled p97 antigen
to Fab fragments of-anti-p97 mAb 96.5. Various dilutions of
each Ab2 were mixed with radioiodinated p97, and the binding
of p97 to Fab fragments of mAb 96.5 was determined in a
solid phase assay.
Figure 14. Anti-idiotypic antibody (Ab2) can inhibit
the binding of Fab fragments of anti-p97 mAb 96.5 to
radiolabelled p97. Fab fragments of mAb 96.5 were plated
onto the wells of Immunolon plates, and Ab2 (10 ug/ml) was
added. The Ab2 were tested for their ability to decrease
20 the binding of radiolabelled p97 to the Fab-coated wells, by
blocking of the p97-binding sites on the Fab fragments.
Figure 15. Assays of the ability of mAb which
recognize different epitopes of the p97 antigen to inhibit
the binding of Ab2 to Fab fragments of anti-p97 mAb 96.5.
25 Each anti-p97 mAb was mixed with the Ab2 to be tested, and
added to plates coated with Fab fragments of mAb 96.5,
followed by addition of goat anti-mouse IgG1-HRP to detect
the binding of the respective Ab2 to the Fab fragments. The
binding of a given Ab2 was tested alone (closed circles), or
30 in the presence of P1.17 control immunoglobulin (X--X), and
compared with the binding seen after various mAb specific
for p97 had been added (open circles).
Figure 16. Binding of plated anti-idiotypic antibody
(Ab2) to different anti-p97 mAb, and the effect thereof on
35 the abiIity of the anti-p97 mAb to subsequently bind p97.
-20- 1339816
Each Ab2 was plated, various concentrations of anti-p97 mAb
8.2, 4.1, or 96.5 were added, and the binding of any bound
anti-p97 mAb to radioiodinated p97 was measured.
Figure 17. Binding of plated anti-idiotypic antibody
(Ab2) to different anti-p97 mAb, and the effect thereof on
the ability of the anti-p97 mAb to subsequently bind p97.
Each Ab2 was plated, various concentrations of anti-p97 mAb
96.5 (Panel A, solid line), mAb 133.1 (Panel A, dashed
line), or mAb 133.3 (Panel B) were added, and the binding of
any bound anti-p97 mAb to radioiodinated p97 was measured.
Figure 18. Panel A: Binding, to plated p97, of mouse
- sera containing Ab3. The sera were derived from BALB/c mice
which had been immunized with anti-idiotypic antibodies
conjugated to KLH. Panel B: Binding, to plated p97, of mAb
96.5 and of sera pooled from mice immunized to p97.
Figure 19. Inhibition of the binding of p97 to Ab3, by
p97-positive cells but not by p97-negative cells. p97-
positive 2A (p97 gene-transfected mouse melanoma K-1735-M2
line) cells, but not p97-negative (parental K-1735-M2)
20 cells, were able to inhibit the binding, to plated p97, of
Ab3 from immunized BALB/c mice. Panel A shows data from
mice immunized with Ab2 #3, and Panels B, C, and D show data
from mice immunized with Ab2 #7, #4, and #5, respectively.
Figure 20. Antibody binding to plated p97 was tested
25 after absorption of the antibody with p97-positive 2A cells
(2a), p97-negative parental K-1735-M2 cells (par), or
- antibody alone. Panel A: Absorption of mAb 96.5 with 2A
cells inhibits the binding of mAb 96.5 to plated p97. Panel
B: Absorption, with 2A cells, of serum antibodies (alpha-
30 p97) from mice immunized with p97, inhibits the binding of-
the serum antibodies to plated p97. Panel C: Serum
antibodies (alpha pl.17) from mice immunized with pl.17 do
not bind p97. Panel D: Normal mouse serum (NMS) does not
bind p97.
-21- 1 33981 6
.
Figure 21. Binding to plated p97 of Ab3 in serum of
immunized mice. C3H/HeN mice were immunized with anti-
idiotypic antibodies (Ab2) conjugated to KLH. Binding of
Ab3 in serum from the immunized mice, to p97 antigen, was
detected by a solid phase ELISA.
Figure 22. Binding to plated p97 of mAb 96.5. Binding
of anti-p97 mAb 96.5 to plated p97 antigen was detected by a
solid-phase ELISA.
Figure 23. Inhibition of the binding of mAb L6 to
H-3347 carcinoma cells by purified Ab2. Ab2 concentrations
were as follows: E~3 50 ~g/ml; ~e~ 5.0 ~g/ml; E~ 0.5
~g/ml; _ 0.05 ~g/ml. mAb L6 was used at 1.0 ~g/ml.
Figure 24. Binding of FITC-labelled Ab2 to Fab L6
saturated H-3347 cells. Part A: Results are shown for the
following Ab2: ~ 3; 7; -~S- 9; A 11;
O 13; -15. Part B: Results are shown for the
following Ab2: -~ t- 2;~ f\ 4; 0 6; ~ 8; C~3 10;
0 12; - ~ 14. LFE (linear fluorescence equivalent)
Ratio is equal to the ratio of sample fluorescence to
20 background fluorescence.
Figure 25. Binding of FITC-labelled Ab2 to PE-labelled
L6 saturated H-3347 cells. The tested Ab2 is indicated
below each graph. FITC-labeled Ab2;
---- Phycoerythrin-labelled mAb L6 at 1:25 dilution of
25 approximately 1 mg/ml solution. RFL LFE Ratio: red
fluorescence linear fluorescence equivalent (ratio of sample
fluorescence to background fluorescence of phycoerythrin);
GRFL LFE Ratio: green fluorescence linear fluorescence
equivalent (ratio of sample fluorescence to background
30 fluorescence of FITC)
Figure 26. Inhibition of binding of mAb F26 to H-3347
cells by L6 Ab2. Ab2 concentrations were as follows: ~3
200 ~g/ml; E2~ 20 ~g/ml; E~ 2.0 ~g/ml. mAb F26 was used
at 1.0 ~g/ml.
-22-
~ 1339816
Figure 27. Ab2 epitope specificity. Competition
assays for L6 anti-idiotype variable region specificity were
performed as described in Section 9.1.9, infra. Competitors
were added at a concentration of 6 ~g/ml for 0.5 hour prior
to the addition of biotinylated chimeric L6 antibody,
resulting in a final concentration of 4 ~g/ml for the
latter. Competitors were as follows: _ chimeric L6
mAb; e8a the variable heavy chain of chimeric L6 mAb plus
J558L lambda I light chain; I I variable light chain of
1o chimeric L6 mAb; ~3 irrelevant human IgGl (PW P328lB8).
The irrelevant human IgG1 showed no inhibition for any Ab2.
Direct binding studies showed no recognition of any of the
V-kappa Ab2 for the chimeric L6 heavy chain and vice versa.
Figure 28. Inhibition of binding of Ab2 to Abl in the
presence of BALB/c (Part A) or C3H/HeN (Part B) sera
containing Ab3. Dilutions of Ab3-containing sera were as
follows: ~ 1:20; ~Ea 1:200, ~ 1:2000; _ 1:20,000.
Ab2 was used at 1.0 ~g/ml.
- Figure 29. Binding to H-3347 carcinoma cells of
affinity-purified sera from C3H/HeN mice immunized with Ab2.
Sera was taken 6 to 20 weeks after the first immunization.
Antisera were as follows: anti-P1-17; anti-Ab2
#3; 0 anti-Ab2 #11; ~ anti-Ab2 #12; 0 anti-Ab2 #13;
~ anti-Ab2 #14; -~- anti-Ab2 #15. The binding of mAb L6
'~ 25 at a concentration of 0.016 ~g/ml (----) is also shown. LFE
(linear fluorescence equivalent) Ratio is equal to the ratio
of sample fluorescence to background fluorescence.
Figure 30. Binding to H-3347 carcinoma cells of
affinity-purified sera from BALB/c mice immunized with Ab2.
30 Sera was taken 6 to 24 weeks after the first immunization.
Antisera were as follows: anti-Pl-17; ~ anti-Ab2
#3; anti-Ab2 #11; 0 anti-Ab2 #12; ~ anti-Ab2
#13; _ anti-Ab2 #14; 1 1 anti-Ab2 #15. The binding of
mAb L6 at a concentration of 0.08 ~g/ml (----) is also
35 shown. LFE (linear fluorescence equivalent) Ratio is equal
-23- 1 3 3q 81 6
to the ratio of sample fluorescence to background
fluorescence.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to methods which
utilize anti-idiotypic antibodies for tumor immunotherapy
and immunoprophylaxis. The invention relates to the
manipulation of the idiotypic network of the immune system
for therapeutic advantage. Immunization with anti-idiotypic
antibodies (Ab2) can induce the formation of anti-anti-
idiotypic immunoglobulins, some of which have the same
antigen specificity as the antibody (Abl) used to derive the
anti-idiotype. This creates a powerful paradigm for
manipulation of immune responses by offering a mechanism for
15 generating and amplifying antigen-specific recognition in
the immune system. An immune response to tumors appears to
involve idiotype-specific recognition of tumor antigen; the
present invention relates to strategies for manipulating
this recognition towards achieving therapeutic benefit.
20 Particular embodiments of the invention include the use of
anti-idiotypic antibody for immunization against tumor, for
activation of lymphocytes used in adoptive immunotherapy,
and for inhibition of immune suppression mediated by
suppressor T cells or suppressor factors expressing an
25 idiotope directed against a tumor antigen. The anti-
idiotypic antibodies, or fragments thereof, can also be used
to monitor anti-antibody induction in patients undergoing
passive immunization to a tumor antigen by administration of
anti-tumor antibody.
In a specific embodiment, the induction of anti-
idiotypic antibodies in vivo, by administration of anti-
tumor antibody or immune cells or factors exhibiting the
anti-tumor idiotope, can be of therapeutic value.
In another embodiment of the present invention,
- 35 monoclonal anti-idiotypic antibodies, or fragments thereof,
-24- 133~815
raised against the idiotype of an antibody that defines a
self-differentiation antigen, such as an oncofetal, or
differentiation antigen can be administered in vivo to
induce a specific immune response directed against tumor
cells that bear the oncofetal antigen. Patients having
tumors may be immunotherapeutically treated with the
monoclonal anti-idiotypic antibodies of the present
invention whereas patients identified as having a predis-
position may be so treated immunoprophylactically.
The present invention is also directed to anti-
idiotypic mAb molecules, or fragments of the anti-idiotypic
mAb molecules, or modifications thereof, that recognize an
idiotype that is directed against a defined antigen specific
for a tumor. Such tumor antigens include antigens of
fibrosarcoma, self-differentiation antigens such as
-~ oncofetal, or differentiation, antigens which are expressed
by malignant cells, including but not limited to oncofetal
antigens such as carcinoembryonic antigens (CEA) of the
colon, alpha-fetoprotein, the human antigenic counterpart or
functional equivalent of the 175 kDa murine antigen of
transitional cell bladder carcinomas, the melanoma
associated antigen p97 or GD3, and differentiation antigens
of human lung carcinomas such as L6 and L20, described in
more detail infra.
~ 25 The mAb molecules of the present invention include
- whole monoclonal antibody molecules and fragments or any
chemical modifications of these molecules, which contain the
antigen combining site that binds to the idiotype of another
antibody molecule(s) with specificity to a defined tumor
30 antigen. Monoclonal antibody fragments containing the
idiotype of the mAb molecule could be generated by various
techniques. These include, but are not limited to: the
F(ab')2 fragment which can be generated by treating the
antibody molecule with pepsin, the Fab' fragments which can
35 be generated by reducing the disulfide bridges of the
-25- 1 33~81 6
F(ab')2 fragment, and the 2Fab or Fab fragments which can be
generated by treating the antibody molecule with papain and
a reducing agent to reduce the disulfide bridges.
Depending upon its intended use, the molecules of the
invention may be chemically modified by the attachment of
any of a variety of compounds using coupling techniques
known in the art. This includes but is not limited to
enzymatic means, oxidative substitution, chelation, etc., as
used, for example, in the attachment of a radioisotope for
immunoassay purposes.
The chemical linkage or coupling of a compound to the
molecule could be directed to a site that does not
participate in idiotype binding, for example, the Fc domain
of the molecule. This could be accomplished by protecting
the binding site of the molecule prior to performing the
coupling reaction. For example, the molecule can be bound
to the idiotype it recognizes, prior to the coupling
reaction. After completion of coupling, the complex can be
disrupted in order to generate a modified molecule with
20 minimal effect on the binding site of the molecule.
The antibodies, or fragments of antibody molecules of
the invention, can be used as immunogens to induce, modify,
or regulate specific cell-mediated tumor immunity. This
includes, but is not limited to, the use of these molecules
in immunization against syngeneic tumors.
The method of the invention may be divided into the
following stages solely for the purpose of description:
(a) production of anti-idiotypic mAb(s) (which may be auto-
anti-idiotypic) directed against an idiotype which binds a
30 defined antigen of a tumor; (b) evaluation and demonstration
of tumor idiotype specificity of the anti-idiotypic mAb
molecules or their derivative fragments, for example, by
demonstration of immunopotency by induction of specific CMI,
of binding to specific suppressor T cells or suppressor
35 factors, of binding to specific helper T cells, of
-26-
~339816
inhibition of the binding of antibody directed against the
tumor antigen, of inhibition of the cytotoxicity properties
of the antibody directed against the tumor antigen, etc.;
and (c) formulation of immunoprophylactic, immunotherapeu-
tic, and immunodiagnostic regimens.
In a model system described in a specific example ofthe present invention, treatment of mice with auto-anti-
idiotypic antibodies related to a fibrosarcoma antigen was
shown to specifically reduce the growth of established
sarcomas. In another example of the invention, a murine
monoclonal anti-idiotypic antibody which recognizes an
idiotype directed against a GD3 ganglioside antigen of human
melanoma is described. This antibody was able to block the
binding and cytotoxicity properties of the antibody
containing the idiotype which it recognizes. In a third
- example of the invention, murine monoclonal anti-idiotypic
antibodies related to the human melanoma-associated p97
antigen were described, which antibodies can competitively
inhibit the binding of p97 to anti-p97 antibody, and which
20 can induce antibodies to p97 in vivo. In yet another
example of the invention, we describe the generation and
characterization of murine monoclonal anti-idiotypic
antibodies which recognize an idiotype on monoclonal
antibody L6 which defines a carbohydrate antigen of human
25 carcinomas. Several of the antibodies are shown to be
capable of inducing antibodies in vivo to the carcinoma
antigen defined by antibody L6. However, the methods
described are not limited to melanoma or fibrosarcoma or
carcinoma antigens, but can be applied to the production and
30 use of anti-idiotypic mAbs related to any specific tumor
antigens.
The use of anti-idiotypic antibodies to induce immune
responses to tumors can be viewed as two separate issues.
First, such antibodies may be used to select or amplify a
35 pre-existing antitumor repertoire, that is, to recruit, via
-27- 1 3398 1 6
idiotypic selection, T and/or B cells with specificity for
tumor antigen. Second, anti-idiotypic antibodies can be
employed as ~internal imagesn of antigens to induce a
primary immune response which is anti-anti-idiotypic, and a
portion of which is directed against the nominal tumor
antigen. In the latter case, the-immune specificity will be
against anti-idiotope, rather than antigen.
By using anti-idiotypic antibodies to induce immunity,
T and B cells may be selected which are different from those
- 10 which participate in a naturally occurring antitumor
response, either as a result of upregulation of an immune
response that is normally suppressed or by the de novo
induction of a response. Of significant therapeutic
importance is the potential to induce effective antitumor
reactivity in hosts which are otherwise incapable of
mounting such reactivity.
5.l. IDIOTYPIC INTERACTIONS INITIATED
BY ANTIGENIC STIMULI
In order to effect therapeutic manipulations of the
idiotypic recognition of tumor antigens, one must consider
the nature of the naturally occurring idiotypic response to
a growing syngeneic tumor. We shall thus discuss the nature
of the idiotypic repertoire in the response to stimulation
with any antigen.
Four possible idiotypic and anti-idiotypic responses
which can be induced by antigens are illustrated in Figure
l. Figure lA shows a sequential progression of induced
complementary specificities. In this type of idiotypic
cascade, immunization with antigen leads to a population of
30 immunoglobulins which carry distinct idiotypes, the sum of
which is known as the idiotype or Abl response. The
presence of the Abl then induces an anti-idiotypic response,
characterized by a heterogeneous population of antibodies,
known as Ab2, which have specificity for the various
1 33~81 6
-28-
idiotopes in the Abl population. Directionality of the
antigen-elicited response is implied by the arrows in Figure
1. The induction of Ab2 by Abl is independent of antigen
(Urbain, J., et al., 1982, Ann. Immunol. 133D:179-189;
Rodkey, L.S., 1974, J. Exp. Med. 139:712; Kelsoe, G. and
Cerny, J., 1979, Nature 279:333). There is support for this
model (Fig. lA) in tumor immunity (Lee, V.K., et al., 1986,
Biochim. Biophys. Acta 865:127-139; see examples sections
infra).
; 10An idiotypic cascade may also lead to the generation of
Ab2 according to the pathway shown in Figure lB. Tumor
: antigen recognition induces antigen-specific TH cells, which
bear particular idiotopes, presumably on their antigen
receptor molecules. These T cells then stimulate the
generation of an anti-idiotypic immunoglobulin response in
the form of a population of Ab2.
What are the implications of a model in which the
idiotypic cascade proceeds through alternate antibody and T
cell components? In an analysis of idiotopes associated
20 with the response to MCA-induced mouse sarcomas or
carcinomas (Lee, V.K., et al., 1986, Biochim. Biophys. Acta
865:127-139; see examples sections 6,7,8, infra), immuniza-
tion with monoclonal Ab2 failed to generate Ab3 with Abl-
- like specificity, whereas antitumor TH were easily induced.
25 This observation, coupled with the finding of idiotype-
positive TH in the naturally occurring antitumor response,
supports a model in which there can be a direct regulatory
'~- interaction between Id+ T cells and anti-idiotypic B cells
~ (and Ab2) (Nelson, R. and Nepom, G.T., 1986, in Paradoxes in
- 30 Immunology, Hoffman, G., et al., eds., CRC Press, Boca
Raton, Florida, pp. 177-185; Bismuth, G., et al., 1984, Eur.
J. Immunol. 14:503; Thomas, W.R., et al., 1983, J. Immunol.
130:2079). In vivo, the Id+ T cells can provide the
stimulus for production of anti-idiotype.
-29- 1 33q8 1 6
The lack of Id+ B cell recognition in certain cases,
may reflect a defect in the genetic capacity to generate
antitumor idiotopes on immunoglobulin molecules; alternativ-
ely, the regulatory state in the tumor-bearing host may
effectively suppress Id+ Abl. As discussed infra (see
Section 5.2.1),-this apparent defect may be circumvented
therapeutically in a host which does not normally develop
Id+ antibody responses.
In addition to idiotypic interactions which result in a
TH response leading to tumor rejection, exposure to tumor
antigen (or antigen-antibody complexes) can lead to the
generation of antigen-specific suppressor T cells (Ts)
(Fig. lC). In several tumor systems, suppressor T cells
have been shown to function as inducer cells, triggering and
amplifying tumor antigen-specific suppression (Nepom, G.T.,
et al., 1983, Experientia 39:235-242). If Id+ TS are
directly induced by antigen stimulation, as in the model
shown in Figure lC, it is possible that these cells, like
- the TH in Figure lB, can serve as stimuli for the generation
20 of anti-idiotypic Ab2.
In a model where idiotypic TS are generated subsequent
to stimulation by antigen, an alternate pathway can also be
considered. As diagrammed in Figure lD, Id+ TS may arise as
a consequence of anti-idiotypic stimulation. In a recent
25 analysis of IgH-restricted T cell responses to the hapten
azobenzenearsonate, the nature of immunoglobulin idiotopes
was found to determine the development of TS idiotopes
(Hayglass, K., et al., 1986, J. Exp. Med. 164:36-49;
Hayglass, K., et al., 1986, Immunol. Today 7:179-183). T
30 cells developing in IgH congenic mice acquired the idiotypic
repertoire of the host, and treatment of neonatal animals
with antibodies to the ~ immunoglobulin chain abolished the
establishment of a normal repertoire of functional T cell
idiotypes. Thus, the immunoglobulin compartment could
1 339~ 1 6
determine the development of complementary T cell recogni-
tion elements.
As illustrated by the examples sections infra, the
idiotypic pathway depicted in Figure lD appears to exist
also in the context of tumor antigen recognition. In our
studies on mice which had either sarcomas or bladder
carcinomas induced with MCA, a single dominant T cell
idiotope was found to be prevalent in the suppressor
response mediated by both TS and soluble factors (see
Kuchroo, V.K., et al., 1987, Cellular Immunol. 104:105-114,
and examples section 6, infra). That is, instead of a
heterogeneous mixture of idiotypes, some shared "public"
idiotopes predominate. This was documented by removing
suppressor factors using affinity absorbents made from
either monoclonal or polyclonal anti-idiotypic antibodies.
Our findings indicated that most of the apparent idiotypic
specificities in the suppressor response to any particular
tumor antigen are shared.
In model systems, the presence of "public" idiotopes
20 has been attributed to regulatory idiotopes important for
network interactions (Bona, C., et al., 1982, J. Exp. Med.
156:986). The presence of such putative regulatory
idiotopes in antitumor immunity may reflect either a genetic
selection in the generation of idiotypic Ts, or an influence
25 of immunoglobulin selection in the generation of the TS
repertoire. If the latter is the case, the induction of
idiotypic TS can include the pathway we have shown in Figure
lD, in which the Ab2 response influences the nature of Ts.
5.2. MANIPULATION OF IDIOTOPE EXPRESSION
BY ANTI-IDIOTYPIC ANTIBODIES
There are essentially two approaches for using anti-
idiotypic antibodies to manipulate the immune response to
tumor antigens for therapeutic benefit. One is based on
selecting and amplifying pre-existing antitumor idiotopes
-31- 1 3 3 q 81 6
within the T and B cell repertoires, and the other entails
the priming of a de novo response using an anti-idiotype
which acts as an internal image of tumor antigen.
5.2.1. SELECTION AND AMPLIFICATION OF-
PRE-EXISTING ANTITUMOR IDIOTOPES
In the therapeutic production of anti-idiotypic
antibodies based on an alteration of preexisting antitumor
idiotope representation, therapeutic efforts are based on a
reversal of the pathways illustrated in Figure 1, in which
the Ab2 compartment is used as the initiator of idiotypic
interactions.
Three pathways by which Ab2 can induce tumor antigen-
specific immunity are illustrated in Figure 2. Figure 2A
shows that immunization with Ab2 can lead to the development
of antigen specific TH. This result has been accomplished
against infectious agents and found to give protection from
disease (Sacks, D., et al., 1982, J. Exp. Med. 155:1108;
Sharp, A., et al., 1984, J. Exp. Med. 160:1195-1205; Fons,
G., et al., 1985, J. Immunol. 134:1225-1229). Analogous
findings have been made for several chemically and virally-
induced tumors (Kennedy, R.C., et al., 1985 , J. Exp. Med.
161:1432; Binz, H., et al., 1982, Int. J. Cancer 19:417-423;
Tilken, A.F., et al., 1981, Proc. Natl. Acad. Sci. U.S.A.
78:1809; Flood, P., et al., 1980, Proc. Natl. Acad. Sci.
25 U.S.A. 77:2209-2213). The Ab2 which is utilized can arise
as a result of an antigen-induced idiotypic cascade, or can
be induced by immunization with antigen-specific T cells
(see Section 5.3, infra).
Anti-idiotypic antibodies can also stimulate "silent"
30 clones, i.e., clones which are normally suppressed even in
genetically competent individuals (Bona, C.A., et al., 1981,
J. Exp. Med. 153:951). Thus, the anti-idiotypic antibodies
appear to be able to nreprogram~ the immune system to
generate antibodies which would not otherwise be made (Fig.
-32- 13398~6
2B). Thus, immunization with anti-idiotypic antibodies
related to capsular polysaccharides of E. coli produces
protective immunity in neonatal mice that do not normally
develop antibodies against the capsular polysaccharides
(Stein, K., et al., 1984, J. Exp. Med. 160:1001).
For Ab2 to select and stimulate both id+ B and T cell
-~ clones, it is probable that shared idiotopes between B and T
cell receptors must exist (Rajewski, K. and Takemori, T.,
1983, Ann. Rev. Immunol. 1:569-607; Ertl, H.C.J., et al.,
1982, Proc. Natl. Acad. Sci. U.S.A. 79:7479; Nadler, P.I.,
et al., 1982, Eur. J. Immunol. 12:113), and correspond to
antigen-specific receptors which confer the same antigen
specificity for both B and T cells. In a preferred
embodiment of the invention, one can select for use in
immunization those Ab2 which induce T cell immunity, in
which a T cell subset is stimulated which mediates the
desired response. One technique to select appropriate Ab2
for use is to raise Ab2 experimentally by immunization with
antigen-specific T cells (Infante, A.J., et al., lg82, J.
- 20 Exp. Med. 155:1100). In this particular embodiment of the
invention, the T cell receptor is used as the stimulatory
idiotype so that the Ab2 population will be targeted
specifically to the T cells of choice. Immunization with
such an Ab2, then, will select and stimulate T cell clones
25 which share receptor idiotopes with the immunizing cells.
Several investigators have reported successful cloning
of tumor-infiltrating T lymphocytes recovered from tumor
biopsies or surgical specimens. These lymphocytes, after
culture in vitro with IL-2, are effective in eliciting
30 antitumor responses when reinfused to the host (Rosenberg,
S.A., et al., 1986, Science 233:1318). In a preferred
embodiment of the invention, such cells should be ideal
immunogens for raising Ab2 directed against the particular
idiotopes associated with such antitumor clones. These Ab2,
35 then, would serve as anti-idiotypes which potentially will
133q816
select and amplify T cells with similar antitumor specific-
ity.
As discussed supra, anti-idiotypic specificities in
tumor-immunized mice appear to be complementary not only to
TH but also to TS and soluble suppressor factors (Hellstrom,
K.E., et al., 1977, Biophys. Biochim. Acta Reviews on Cancer
473:121-148; see Section 8, infra; Binz, H., et al., 1982,
Int. J. Cancer 19:417-423; Tilken, A.F., et al., 1981, Proc.
Natl. Acad. Sci. U.S.A. 78:1809; Flood, P., et al., 1980,
Proc. Natl. Acad. Sci. U.S.A. 77:2209-2213). The evidence
that anti-idiotypic antibodies recognize a dominant public
' idiotope on Ts-derived factors suggests that there is a
regulatory relationship between the id+ cellular compartment
and the Ab2 compartment (see Section 6, infra; Kuchroo,
V.K., et al., 1987, Cellular Immunol. 104:105-114; Nelson,
K. and Nepom, G.T., 1986, in Paradoxes in Immunology,
Hoffman, G., et al., eds., CRC Press, Boca Raton, Florida,
pp. 177-185). Thus, in order to avoid the preferential
activation of the TS compartment upon administration of
anti-idiotypic antibodies (as illustrated in Fig. 2C), and
in order to achieve a therapeutically effective result,
anti-idiotypic therapy for cancer should employ proper
manipulation of the idiotypic networks. Proper manipulation
involves the consideration of three issues: the genetic
25 restriction between anti-idiotype and the T cell
compartment; the route of anti-idiotype administration; and
the choice of idiotope specificity.
5.2.1.1. GENETIC RESTRICTION
Two types of genetic restriction can potentially
present barriers to cellular recruitment by injected anti-
idiotypes: MHC restriction and IgH restriction. MHC
restriction confers an element of genetic control of T cell
activation in an antigen-specific response. To the extent
35 that an anti-idiotype mimics antigen in the recognition
_34_ 133~816
process, it is logical to consider possible requirements for
histocompatibility. Indeed, examples have been reported in
which antigen-primed T cells are MHC restricted not only for
their specific antigen recognition, but also for their
ability to recognize cell-bound anti-idiotype in vitro
(Ertl, H., et al., 1986, Int. Rev. Immunol. 1:61-66).
What are the implications of these findings for the in
vivo administration of anti-idiotype? In a study using
anti-idiotype priming to generate anti-viral immunity, a
genetically non-restricted response was observed (id.).
This may have been due to the direct recruitment of TH which
subsequently activated other T cell compartments. Thus,
even if the MHC restricting element for anti-Id presentation
is different from that of nominal antigen presentation, T
cell recruitment can still occur. With an appropriate route
of administration (see below), anti-idiotypic antibodies can
be introduced to the immune system by antigen-presenting
cells in such a way that they may be "seen" in the context
of the right restriction elements. In fact, a host which is
a genetic non-responder to the nominal antigen can
conceivably respond to challenge with anti-idiotype,
depending on the mechanism maintaining the non-responsive-
ness. Thus, MHC restriction appears to be of minor
importance except where cell-bound anti-idiotype is used.
In a preferred embodiment of the invention, anti-idiotype
administration should seek to optimize the presentation of
the Ab2 immunogen to the host immune system, even where
MHC-incompatible.
In a preferred embodiment of the invention, the issue
30 of IgH restriction, i.e., the necessity for genetic matching
of allotypic markers associated with immunoglobulin genes,
should be considered. Since variable region antibody genes
are linked to constant region genes, the genetic potential
for specific idiotypic determinants is linked to Ig
35 allotypic markers. IgH restriction appears to govern many
-35- 133~5
steps in the idiotypic cascade (Bach, B.A., et al., 1979, J.
Exp. Med. 149:1084; Nadler, P.I., et al., 1982, Eur. J.
Immunol. 12:113; Yamamoto, H., et al., 1983, J. Exp. Med.
158:635-640; Forstrom, J.W., et al., 1983, Nature 303:627-
629). In essence, it acts as a permissive barrier which
requires the presence of appropriate V genes, and linked
allotypic markers, for idiotype-anti-idiotype recognition.
A strict IgH restriction of the immune response to Ab2
likely reflects requirements for direct recognition of
network V genes; this has been referred to as a ntrue
idiotypic~ interaction (Nisonoff, A. and Lamoyi, E., 1981,
Clin. Immunol. Immunopathol. 21:397). This requirement
limits the type of antibody which can be used as an anti-
idiotype immunogen, to one which can elicit complementary V
genes in the host. Thus, in a particular preferred
embodiment of the invention, experimentally derived anti-
idiotypic antibodies should be IgH-matched with the host.
When is IgH-matching for anti-idiotypic administration
not necessary? Anti-idiotypic antibodies which act as
internal images of the nominal antigen can substitute for
this immunization with antigen. Since such de novo
immunization is not based on specific selection of network V
genes, internal image immunogens are generally not IgH
restricted (id.), and thus do not need to be host IgH-
25 matched.
- 5.2.1.2. ROUTE OF IMMUNIZATION
The route of immunization with anti-idiotype can also
influence the nature of the immune response. Depending upon
30 the route of administration, anti-idiotypic antibodies have
been found to either enhance or suppress immune responses
(Rajewski, K. and Takemori, T., 1983, Ann. Rev. Immunol.
1:569; Urbain, J., et al., 1982, Ann. Immunol. 133D:179).
For example, in a viral system, reovirus-specific immunity
35 could be established following immunization against
-- 1 3398 1 6
idiotypic determinants, and DTH, cytolytic T cells, and
antigen-binding antibodies were observed. If soluble Ab2
was used as the immunogen, only the DTH response was seen,
- while immunization with cell-associated anti-idiotype, in
the form of a hybridoma-producing Ab2, also induced
cytolytic T cells (Ertl, H., et al., 1986, Int. Rev.
Immunol. 1:61-66). When Ab2 is used as an internal-image
immunogen (as in the reovirus system), it essentially
substitutes for antigen in the initial priming; many of the
immune manipulations which facilitate antigen responsiveness
also augment immunity to Ab2 and can be used in various
embodiments of the invention (see Section 5.3.2, infra).
Many methods may be used to introduce the immunizing
formulations; these include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, and intranasal routes. In particular
embodiments, induction of immunity with anti-idiotypic
antibodies can utilize subcutaneous (s.c.) or intramuscular
~ injection of the antibodies in the presence of various
adiuvants.
In the case of nominal antigen immunization, adminis-
tration of a haptenated protein s.c. with adjuvant has been
shown to yield a vigorous TH response, while the same
antigen intravenously (i.v.) preferentially induced TS
(Greene, M., et al., 1982, Adv. Immunol 32:253). However,
when monoclonal antitumor antibodies were used therapeutic-
ally, the opposite situation was reported by Koprowski et
al. (Koprowski, H., et al., 1984, Proc. Natl. Acad. Sci.
U.S.A. 82:216-219), who described an anti-idiotypic response
30 to systemically injected mouse antibodies to human
gastrointestinal cancer, which was apparently accompanied by
a therapeutic effect in the patient.
There is also evidence that Ab2 can effectively inhibit
the ability of an Abl to mediate antibody-dependent cellular
~ -37~ 1339816
cytotoxicity or kill tumor cells in the presence of
complement (see Section 7.2.4, infra).
Since route of Ab2 administration, as well as dosages
used, are likely to have an important impact on what kind of
response is induced, in a preferred aspect of the invention,
preliminary studies on dosage and administration effects
should be carried out. These studies can be done in an
animal model such as mouse, rat, primate, etc. For example,
in a particular embodiment, various routes of administration
of anti-idiotypic antibody related to the melanoma antigen
p97 can be tested for the in vivo and in vitro responses
which they induce upon challenge with a mouse melanoma line
expressing the p97 antigen (obtained following transfection
with the cloned p97 gene; Brown et al., 1981, J. Immunol.
127:539-546).
5.2.1.3. IDIOTOPE SPECIFICITY
Complex antigens, including tumor antigens, contain
multiple epitopes. The immune recognition which they evoke,
therefore, contains many idiotopes which are determined not
only by the heterogeneity of the epitopes, but also by the
heterogeneity of the V genes selected among Ig and T cell
receptor genes in the host. These idiotopes are largely
defined by the anti-idiotopes which they induce. Thus
25 private idiotopes elicit Ab2 responses which are unique for
a particular Abl, public idiotopes elicit Ab2 to a
specificity shared by many Abl, and Ab2 of the internal
image type are induced against antigen-binding structures on
Abl which are complementary to the antigen (Urbain, J., et
30 al., 1982, Ann. Immunol. 125C:373-389; Augustin, A., et al.,
1983, Surv. Immunol. Res. 2:78; Mosier, D. and Feeney, A.,
1984, in The Biology of Idiotypes, Greene, M. and Nisonoff,
A., eds., Plenum Press, New York, pp. 403-416).
In particular embodiments of the invéntion, likely
35 shifts in the idiotypic repertoire can be manipulated in an
.
-38- - 1339816
attempt to achieve the desired idiotypic response. For
example, idiotope selection depends in large part on the Ab2
used as immunogen. Thus, in a preferred embodiment, an Ab2
used as immunogen is one determined to be likely to cause
the expression of therapeutically desirable idiotopes. For
example, in a system with a dominant public idiotope on both
TH and TS cells, anti-idiotypic antibodies may prime for TH,
if administered s.c. with adjuvant, or they may interact
with soluble TS factors. As an illustration, in studies in
mice which were immune to haptens and in which public
idiotopes were recognized, a dominant Abl response with a
shared public idiotope elicited a strong anti-Id response in
mice of IgH-compatible strains. If anti-Id was given i.v.
in neonates, or if anti-Id TS were transferred, the public
Abl idiotope was suppressed, and this was shown to cause the
expression of alternate idiotopes on antigen-specific Abl
(Kekoe, G., et al., 1980, Immunol. Rev. 52:75). When
immunization with anti-Id is applied towards tumor therapy,
a similar shift in the idiotypic repertoire can occur.
In a system lacking a dominant public idiotope, or when
no Abl is identified, the adaptability of the idiotope
selection process offers encouraging alternatives. In a
particular embodiment of the invention, since administration
of Ab2 can select for alternate idiotypic responses,
antibodies to idiotopes which do not occur naturally in the
tumor-bearing host can be chosen to direct an antitumor
response. In other words, one can immunize with an Ab2
which selects a TH repertoire which cannot be selected by
exposure to tumor antigen. For example, one can immunize
30 with a monoclonal, internal-image Ab2 raised against a
xenogeneic antitumor antibody. Many of the existing
monoclonal antitumor antibodies are, indeed, specific for
antigens which may not even be immunogenic in the tumor-
bearing host (Hellstrom, K.E. and Hellstrom, I., 1985, ln
35 Monoclonal Antibodies for Tumour Detection and Drug
39 1 3398 1 6
Targeting, Baldwin, R.W. and Byers, V.S., eds., Academic
Press, London, pp. 17-51), and can be used in this
embodiment of the invention.
5.2.2. INTERNAL IMAGE ANTIBODIES
Ab2 immunization can lead to Ab3 elicitation not only
through specific V gene network interactions but also by
virtue of internal-image mimicry (Urbain, J., et al., 1982,
Ann. Immunol. 133D:179-189; Augustin, A., et al., 1983,
Surv. Immunol. Res. 2:78). That is, when the anti-idiotype
represents the conformational mirror-image of the antigen,
it can substitute for nominal antigen and elicit an Abl-like
response (Nisonoff, A. and Lamoyi, E., 1981, Clin. Immunol.
Immunopathol. 21:397) (Fig. 3). In a preferred embodiment,
therefore, such anti-idiotypic antibodies can be used as
immunogens for tumor therapy in IgH-mismatched hosts.
In an embodiment of the invention employing monoclonal
anti-idiotypes, the appropriate Ab2 should be carefully
selected. The experimental verification that any particular
20 Ab2 is an internal image type of anti-idiotype hinges on its
ability to mimic the conformational characteristics which
define recognition of the antigen. Internal image anti-
idiotypes compete in vitro with antigen for binding to
idiotype-positive Abl, and prime in vivo for Ab3 which mimic
25 Abl, and this priming occurs in a IgH unrestricted fashion
(Nisonoff and Lamoyi, supra).
In addition to the inhibition of id+ binding to
antigen, internal image Ab2 can substitute for antigen in
terms of immune recognition. For example, Ab2 may stimulate
30 antigen-specific clones in vitro in the absence of antigen,
or Ab2+ cells may serve as a target for antigen-specific CTL
(Ertl, H.C.J., et al., 1982, Proc. Natl. Acad. Sci. U.S.A.
79:7479). Since the Ab2 is substituting for antigen
conformation, the Ab2 may be ~presented" to T cells in these
35 assays in the context of MHC molecules, and therefore the
1339816
response can appear to be MHC restricted, just as for
antigen-specific response (id.).
In an embodiment of the invention in which internal
image immunogens are desired, xenogeneic anti-idiotypic
antibodies may be used. It is possible that internal image
immunogens fail to stimulate suppressor cells, while at the
same time inducing TH. This supposition is based on the
notion, outlined in Figures lD and 2C, that regulatory
idiotopes which are represented by the expression of
dominant V genes determine the communication between Ab2 and
Ts, and that some anti-idiotypic antibodies may be selected
which lack this particular idiotope yet retain the
internal-image characteristics priming for TH.
In a specific embodiment of the invention, internal
image antibodies with tumor antigen activity can be used as
tumor nvaccines~ towards induction of specific tumor
immunity. For example, such vaccines can be therapeutically
valuable for patients whose primary neoplasms have been
removed but who are at risk for development of metastases.
The ability of internal image Ab2 to compete with
antigen for binding to Abl (and vice versa? is an integral
part of their behavior. However, Ab2 which do not function
as internal images may still compete, due to steric
hindrance (and perhaps other mechanisms as well). In a
25 preferred aspect of the invention, one should investigate
the ability of an Ab2 to induce an immune response over IgH
(and MHC) barriers in experimental animals as part of their
characterization as potential internal images.
Since, on occasion, the distinction between internal-
30 image anti-Id and ntrue~ anti-Id becomes blurred, anti-
idiotypic antibodies which do not have internal image
specificity may still display internal image-associated
properties, which can be therapeutically valuable. For
example, in our study of monoclonal anti-idiotypes raised
35 against a murine bladder carcinoma antigen-associated
-41- l 3398 1 6
monoclonal antibody, immunization with the anti-idiotypes
elicited a vigorous Ab3 response which lacked any detectable
antigen-binding (Lee, V.K., et al., Biochim. Biophys. Acta
865:127-139). The Ab3 raised against a particular anti-Id
were apparently directed against "private" specificities
associated with that anti-idiotype. Although the anti-
idiotypic antibodies were shown to inhibit antigen binding
by the Abl, this was presumed due to steric inhibition, and
we tentatively interpreted these data to mean that the Ab2
were not internal image antibodies. Surprisingly, however,
these same monoclonal Ab2 were able to prime mice for
antitumor responses and also to bind to tumor-specific T
cell suppressor factors (Lee, V.K., et al., 1985, Proc.
Natl. Acad. Sci. U.S.A. 82:6286-6290; Kuchroo, V.K., et al.,
1987 Cellular Immunol. 104:105-114). Since the anti-
idiotypic monoclonal antibodies in these experiments were
raised in mice, and were directed against xenogeneic
antitumor monoclonal antibodies raised in rats, the
antitumor response elicited in mice by Ab2 immunization
20 would not be çxpected to be based on specific V gene
selection, and could instead be attributed to some internal
image Ab2.
A second example comes from experiments analyzing
idiotypic responses to TMV-associated antigens, in which
25 mice were immunized with rabbit anti-idiotypic antibodies
which were specific for "private~ rabbit idiotopes
(Francotte, M. and Urbain, J. 1984, J. Exp. Med. 160:1485).
Surprisingly, these mice made anti-TMV antibodies which were
idiotypically cross-reactive with the rabbit idiotopes.
30 Thus, while the stimulatory anti-idiotype was not an
~internal image~ specificity, it nevertheless elicited
antigen-specific information in the face of apparent V gene
incompatibility.
~ -42- 1339816
5.3. PRODUCTION OF ANTI-IDIOTYPIC MONOCLONAL
ANTIBODIES SPECIFIC TO AN IDIOTYPE
WHICH RECOGNIZES A DEFINED TUMOR ANTIGEN
5.3.1. PRODUCTION OF ANTI-IDIOTYPIC MONOCLONAL ANTI-
BODIES BY IMMUNIZATION WITH AN ANTIBODY (ABl)
THAT RECOGNIZES A DEFINED TUMOR ANTIGEN
In a specific embodiment of the invention, production
of anti-idiotypic monoclonal antibodies specific to an
idiotype which recognizes a defined tumor antigen requires
immunization of a host with antibodies that recognize the
defined tumor antigen. As previously explained, such tumor
antigens include but are not limited to oncofetal, or
differentiation, antigens such as CEA, alpha-fetoprotein,
the human antigenic functional equivalent of the 175 kDa
murine antigen of transitional cell bladder carcinoma,
melanoma associated antigen p97 (see Brown et al., 1981, J.
Immunol. 127:539-546), differentiation antigens of human
lung carcinoma such as L6 and L20 (see Hellstrom et al.,
1986, Cancer Res. 46:3917-3923), and the differentiation
antigen associated with human melanoma, GD3 ganglioside
antigen, antigens of fibrosarcoma, and the l-ike.
The possible host species include but are not limited
to experimentaI animals such as mice, rabbits, and
chimpanzees; and humans. Various adjuvants can be used to
enhance the immunological response to the antibodies,
depending on the host species and including but not limited
to, mineral gels, e.g., aluminum hydroxide; surface active
substances such as lysolecithin; pluronic polyols;
polyanions; peptides; oil emulsions; and potentially useful
human adjuvants such as BCG (bacille Calmette-Guerin) and
corynebacterium parvum. The Ab2 can also be coupled to an
immunogenic carrier, including but not limited to LPS, or
cross-linked with glutaraldehyde (Primi, C.D., et al., 1982,
J. Immunol. 129:1124-1129). The immunogen may also be
incorporated into liposomes, or conjugated to polysaccha-
~43~ 13398~6
- rides and/or other polymers, or otherwise chemically
modified for use. Allotypic determinants on the Ab2
molecule itself can also be used to enhance immunogenicity.
In the reovirus system, when the immunization with Ab2
crossed allotypic barriers, that is, when the host was IgH
mismatched, a vigorous Ab3 response was seen (Ertl, H., et
al., 1986, supra). This result suggests that Ig allotypic
determinants on the anti-idiotype acted as helper determin-
- ants towards augmenting the immune responses. Thus, in a
particular embodiment, IgH mismatching may be used to
enhance immunogenicity.
The anti-idiotypic antibodies, or fragments of the
anti-idiotypic antibodies, or chemically modified fragments
or antibodies may be used for immunization. In addition, a
mAb fragment containing the idiotype of the mAb molecule
could be used, including but not limited to the Fv, Fab,
Fab', or F(ab')2 fragments, which can be generated by known
techniques.
The monoclonal antibody can be prepared by using any
technique which provides for the production of antibody
molecules by continuous cell lines in culture. These
include but are not limited to the hybridoma technique
originally described by Kohler and Milstein (1975, Nature
256:495-497), and the more recent human B-cell hybridoma
technique (Kozbor et al., 1983, Immunology Today 4:72) and
EBV-transformation technique (Cole et al., 1985, Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-
96). Selection of the hybridoma can be carried out by any
; of numerous assays, ~ for binding to Abl, or for
inhibition of Abl binding to tumor cells (Nepom, G.T., et
-~' al., 1984, Proc. Natl. Ac~d. Sci. U.S.A. 81:2864-2867;
Holbeck, S.L. and Nepom, G.T. 1983, J. Immunol. Methods
-' 60:47-52; see Section 7.1.7, infra).
1339816
5.3.2. PRODUCTION OF ANTI-IDIOTYPIC MONOCLONAL
ANTIBODIES WHICH ARE DIRECTED AGAINST AN
IDIOTOPE ON T CELLS THAT RECOGNIZES
- A DEFINED TUMOR ANTIGEN
In another specific embodiment of the invention, an
anti-idiotypic antibody is produced, which is related to a
tumor antigen, and which is directed against an idiotope
associated with T cells that recognizes the tumor antigen.
The idiotope may also be associated with suppressor factors
that exhibit the idiotope directed against the tumor
antigen. Such tumor antigens include but are not limited to
those listed in Section 5.3.l, supra. In one embodiment
(see Section 6, infra), the anti-idiotypic antibody can be
produced by immunization with tumor antigen, formation of
hybridomas, and screening for auto-anti-idiotypic monoclonal
antibodies by methods including but not limited to assay of
tumor-specific DTH, tumor-specific LAI, binding to
monoclonal antibody directed against the tumor antigen, etc.
In particular embodiments, the tumor antigen used for
production of auto-anti-idiotypic antibody can be specific
to fibrosarcoma, transitional cell bladder carcinoma; it can
be melanoma antigen p97 or GD3, or human lung carcinoma
antigen L6 or L20. Alternatively, T cells (TH or Ts) or
suppressor factors which bind tumor antigen can be used to
immunize a host for the production of the anti-idiotypic
antibody. In particular embodiments, T cells which express
an idiotope defined by an antibody specific to fibrosarcoma,
transitional cell bladder carcinoma, p97 melanoma antigen,
GD3 melanoma antigen, L6 or L20 lung carcinoma antigen, may
be used for immunization. Since injection of TS cells may
30 be therapeutically detrimental, immunization with TH cells
is preferred. The T cells used for immunization may be
obtained, for example, from the tumor-carrying patient
himself or from an appropriate (preferably histocompatible)
donor whose immune system has been exposed to the tumor
35 antigen. The T cells can then be isolated for injection by
.
1 3398 1 6
various techniques known in the art, e.g. fluorescence-
tagged monoclonal anti-tumor antibody binding and FACS.
(See Section 6.4, infra). Suppressor factors for immuniza-
tion can be isolated by many techniques known in the art,
including but not limited to immunoaffinity chromatography
(to a tumor-antigen coated column), generation of T-T
hybridomas by fusing T cells from tumor-bearing hosts
(Nelson, K.A., et al., 1980, Proc. Natl. Acad. Sci. U.S.A.
77:2866) followed by screening e.g. for suppression of DTH
to tumor antigen, specific binding to tumor antigen, etc.
(see Section 6.5, infra).
5.4. EVALUATION AND DEMONSTRATION OF IMMUNOPOTENCY
BY INDUCTION OF TUMOR-SPECIFIC CMI
Where the anti-idiotypic antibody of the invention is
envisioned for use in immunization against tumors (see
Section 5.6.1, infra), in a preferred embodiment, the
immunopotency of the antibody should be tested. Any method
which can demonstrate immunopotency of the anti-idiotypic
molecule by showing induction of tumor-specific CMI upon
immunization with the anti-idiotypic antibody or its
derivative fragment(s) is within the scope of the invention
for evaluation of the immunopotency of the anti-idiotypic
mAb; such assays include, but are not limited to DTH (for a
description of the DTH assay procedure see Forstrom et al.,
1983, Nature (London) 303:627-629) and/or LAI (Halliday,
W.J. ~ Maluish, A.E., 1982, in Assessment of Immune Status
by the Leukocyte Adherence Inhibition Test, Academic, New
York pp. 1-26; Koppi, T.A., and Halliday, W.J., 1982, Cell.
Immunol. 66:394-406; Koppi, T.A., and Halliday, W.J., 1981,
- 30 J. Natl. Cancer Inst. 66:1089-1096). Further specificity
testing can include but is not limited to immunoabsorption
assays and treatment of peritoneal cells with antibodies and
complement.
-46- 1 3398 1 6
5 . 5 . ~'UK'l'~:~ CHARACTERIZATION
OF ANTI-IDIOTYPIC ANTIBODY --
In order to further determine anti-idiotypic antibody
specificity, Abl binding assays and/or assays of inhibition
of Abl-tumor binding may be carried out. Such assays may be
accomplished by any methods known in the art, ~ those
described in Section 7, infra. Two additional assays that
may be done include tests of inhibition of complement-
dependent cytotoxicity or antibody dependent cellular
cytotoxicity properties of Abl (see Sections 7.1.1.3 and
7.1.1.4 infra).
5. 6. USES IN IMMUNOPROPHYLAXIS,
IMMUNOTHERAPY, AND IMMUNOASSAY
The purpose of this embodiment of the invention is to
describe uses of the anti-idiotypic antibody molecules of
this invention, or fragments of the antibody molecules,
which may or may not be chemically modified, in the field of
medicine.
, .
5 . 6 .1. IMMUNIZATION AGAINST TUMORS
Patients having tumors may be treated therapeutically
by immunization with the anti-idiotype monoclonal antibodies
of the present invention whereas patients with a disposition
for the tumor may be treated immunoprophylactically by such
immunization. An advantage of using anti-idiotypic mAb over
antigen in a tumor vaccine formulation is that large
quantities of identical material can be obtained for use as
immunogen. This is especially valuable when the antigen is
a glycolipid or a carbohydrate, which itself may be
30 difficult to obtain in pure form and in sufficient amounts.
In addition, if the antigen is a protein, the availability
of an anti-idiotypic antibody avoids the necessity of having
the cloned gene for the antigen, in order to obtain
sufficient quantities of the antigen for use in vaccines.
-47- 1 3 3 9 8~ 6
The anti-idiotypic antibodies, or fragments of the anti-
idiotypic antibodies, or chemically modified fragments or
antibodies may be used to immunize against tumors. Any mAb
fragment containing the idiotype of the mAb molecule could
be used, including but not limited to the Fv, Fab, Fab', or
F(ab')2 fragments, which can be generated by known
t~chn;ques. The anti-idiotypic antibody molecule or its
derivative fragments may be formulated with a suitable
adjuvant in order to enhance the immunological response.
These adjuvants may include, but are not limited to, mineral
gels, e.g. aluminum hydroxide; surface active substances
such as lysolecithin, pluronic polyols; polyanions;
peptides; oil emulsions; and potentially useful human
adjuvants such as BCG (bacille Calmette-Guerin) and
corynebacterium parvum. The immunogen may also be
incorporated into liposomes, or conjugated to polysaccha-
rides and/or other polymers, or otherwise chemically
modified for use.
Many methods may be used to introduce the immunizing
formulations; these include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, and intranasal routes.
In particular embodiments of the invention, hosts with
- fibrosarcoma can be injected intraperitoneally with an
anti-idiotypic monoclonal antibody specific to an idiotope
which recognizes a fibrosarcoma antigen.
Immunization in test animals may be assayed by
injection of the anti-idiotypic antibody or related
derivative molecule, followed by challenge with a tumori-
30 genic agent(s) such as syngeneic tumor cells or chemicalcarcinogens, and observing tumor development and progres-
sion.
-48- 1 3398 1 6
5.6.2. ADOPTIVE IMMUNOTHERAPY
In another embodiment of the invention, T cells
(preferably TH) which express an idiotope which recognizes a
tumor antigen which can be, but is not limited to, an
antigen described in Section 5.3.1, supra, can be introduced
into a host, for immunoprophylaxis or immunotherapy. Such
cells can be obtained from the tumor-growing host himself,
expanded in vitro, selected for the proper idiotypic
specificity (either before or after expansion), and
0 reintroduced into the host (see Section 6.4, infra).
Alternatively, the T cells can be obtained from an
appropriate (preferably histocompatible) donor.
In an alternative approach, it is possible to activate
tumor-specific lymphocytes in vitro and to treat the patient
with the activated tumor-specific leukocytes. Recently,
regression of cancer was observed in response to an adoptive
immunotherapeutic treatment involving the administration of
lymphokine activated killer cells (LAK) to tumor-bearing
hosts (Rosenberg et al., 1985, New Engl. J. Medicine
313:1485-1492). However, this therapy resulted in a number
of undesirable side effects, including severe toxicity,
pulmonary edema, and respiratory distress. By contrast, the
method of this embodiment of the present invention involves
administering to a patient stimulated lymphocytes which are
specific for the tumor associated antigen (which can be, but
is not limited to, an antigen described in Section 5.3.1,
supra) which may result in a more specific therapy and
reduced side effects. For example, peripheral lymphocytes
can be withdrawn from the patient, or a histocompatible
30 donor who was exposed either to the tumor antigen or to a
monoclonal anti-idiotypic antibody raised to the idiotype of
an antibody that defines the tumor antigen as described
above. The lymphocytes can then be stimulated in vitro in
the presence of the tumor-specific anti-idiotypic antibodies
35 of the present invention. Such specific stimulation can be
- _49_ l 33981 6
accomplished using the monoclonal anti-idiotypic antibodies
of the present invention in a method such as the one
described by Binz et al. (1982, Int. J. Cancer 29:417-423).
The activated T cells can then be expanded in cell culture.
This expansion can be accomplished by repeated stimulation
of the T cells with the anti-idiotypic antibodies of the
invention, with or without IL-2, or by growth in medium
containing IL-2 alone. Other methods of T cell cultivation
(for example, with other lymphokines, growth factors, or
other bioactive molecules) can also be used. The activated
lymphocytes may then be tested for cell-mediated antitumor
immune reactivity. If desired, confirmation of identity of
the activated lymphocytes as T cells can be accomplished by
e~rination of the cells with regard to cell-surface
expression of T and B cell markers. This can be carried
out, for example, by immunofluorescence analysis using
fluorescein-conjugated monoclonal antibodies to T and B cell
antigens. Expression of known T cell markers, such as the
CD4 and CD8 antigens, confirms the identity of the activated
lymphocytes as T cells.
The activate~ T cells are then tested for antitumor
reactivity. This can be accomplished by any of several
techniques known in the art for assaying specific cell-
mediated immunity. For example, a cytotoxicity assay, which
measures the ability of the stimulated T cells to kill the
tumor cells in vitro, may be accomplished by incubating the
lymphocytes with 51Cr-labelled tumor cells, and uninfected
labelled cells, and measuring 51Cr release upon lysis. Such
assays have been described (see, for example, Zarling, J.M.,
et al., 1986, J. Immunol. 136:4669). The activated
lymphocytes can also be tested for T helper cell activity by
measuring their ability to proliferate, as shown by 3H-
thymidine incorporation, following stimulation, and/or by
measuring their ability to produce lymphokines such as IL-2
or interferon upon stimulation, in the absence of exogenous
50 ' - 1 33~8 1 6
IL-2. Other assays of specific cell-mediated immunity known
in the art, such as leukocyte-adherence inhibition assays
(Thomson, D.M.P. (ed.), 1982, Assessment of Immune Status by
the Leukocyte Adherence Inhibition Test, Academic Press, New
York), may also be used. The selected lymphocytes can then
be inoculated into the patient. Inoculation of the
activated T cells is preferably through systemic
administration although other methods of administration (for
example, direct infusion into an artery) can be used. The T
cells can be administered intravenously through a central
venous catheter or into a large peripheral vein. In a
preferred embodiment, approximately 1 x 108 cells are
infused initially and the remainder are infused over the
following several hours. In some patients, recombinant
human IL-2 may be used and can be infused intravenously
every 8 hours beginning at the time of T cell infusion.
Injections of IL-2 will preferably be at doses of 10,000 to
100,000 units/kg bodyweight, as previously used in cancer
patients (Rosenberg, S.A., et al., 1985, N. Engl. J. Med.
313:1485). The IL-2 infusion can be continued for several
days after infusion of the activated T cells if tolerated by
the patient.
5.6.3. INHIBITION OF IMMUNE SUPPRESSION
OF ANTI-TUMOR REA~llVllY
In another embodiment of the invention, anti-idiotypic
antibodies which specifically-recognize an idiotope which is
directed against a tumor antigen and which idiotope is
present on suppressor T cells and/or suppressor factors, can
be administered in vivo in order to inhibit suppression of
30 anti-tumor reactivity. In particular embodiments, such a
tumor antigen can include but is not limited to those listed
in Section 5.3.1, supra.
~ -51- 1 33981 6
5.6.4. IMMUNOAFFINITY APPLICATIONS
The anti-idiotypic antibodies or related molecules of
this invention may be used to isolate antibodies directed
against a defined tumor antigen. Techniques known in the
art by which this could be accomplished include but are not
limited to immunoaffinity columns and immunoabsorption
reactions. Anti-tumor antibody isolated through use of the
anti-idiotypic antibody or related molecules could be a
valuable tool in tumor immunotherapy.
5.6.5. IMMUNOASSAYS
In an alternate-embodiment of the present invention,
the anti-idiotypic antibodies or related molecules of the
present invention may be used as antigens in immunoassays.
These immunoassays would allow the detection of anti-tumor
antibody in animals or patients. The molecules of the
present invention would also be used in competition
immunoassays to test for the presence of tumor antigens.
The molecules of the present invention may be used in
any immunoassay system known in the art, including but not
- limited to radioimmunoassays, ELISAs, nsandwich" assays,
precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-
fixation assays, protein A immunoassays, fluorescent
immunoassays, and immunoradiometric assays.
In a specific embodiment, the anti-idiotypic antibodies
of the invention, directed against an idiotype of nAntibody
1~ which defines a tumor antigen, can be used in competitive
immunoassays to monitor the presence of antibodies in
30 patients being administered Antibody 1, for therapeutic or
diagnostic purposes (see Sections 7.1.1.S, 7.2.5., infra).
In another embodiment, anti-idiotypic antibodies can be used
to identify anti-tumor antibodies during purification
procedures.
-52- l 3 3 q 8l 6
6. IMMUNOTHERAPY OF MURINE SARCOMAS WITH
- AUTO-ANTI-IDIOTYPIC MONOCLONAL ANTIBODIES
WHICH BIND TO TUMOR-SPECIFIC T CELLS
According to the network theory (Jerne, N.K., 1974,
Ann. Immunol. 125C:373; Rajewski, K. and Takemori, T., 1983,
Ann. Rev. Immunol. 1:569; Urbain, J., et al., 1982, Ann.
Immunol. 133D:179), mice forming an immune response to
syngeneic sarcomas should have antibodies to the idiotypes
of the responding lymphocytes. As described in the examples
infra, based on this prediction, we immunized BALB/c mice to
either of two syngeneic, transplanted fibrosarcomas, and
formed hybridomas which produced monoclonal antibodies which
primed syngeneic mice for tumor-specific DTH. One of these
auto-anti-idiotypic monoclonal antibodies is shown to define
an idiotope present both on T cells which have antitumor
activity and on products of T suppressor cells, which
inhibit this activity. We also show that treatment of mice
with either of the two monoclonal anti-idiotypic antibodies
significantly reduced growth of established sarcomas, an
effect that was specific for the appropriate mAb-tumor
combination.
6.1. MATERIALS AND METHODS
6.1.1. MICE
BALB/c mice were bred in the Division of Animal Health
Resources, Fred Hutchinson Cancer Research Center (FHCRC),
Seattle, Wash., and were matched for age and sex in each
experiment. Females older than ten weeks were chosen since
they gave optimal responses in pilot tests. CB-20 mice were
30 raised at FHCRC from breeding pairs obtained from Dr.
Michael Potter, National Institute of Health.
_53_ l 339~1 6
6.1.2. TUMORS
Fibrosarcomas MCA-1490, MCA-1510, and MCA-1511 were
induced in BALB/c mice by intramuscular injection of 3-
methylcholanthrene in trioctanoin and maintained by serial
syngeneic transplantation of tissue frozen in the second
generation. They were shown to be free of LDH, Sendai and
ectromelia viruses, and from mycoplasma, and, like other
chemically induced mouse sarcomas, to express individually
unique tumor-specific transplantation antigens.
BW5147.G.1.4.ouar.1, is a drug-marked AKR thymoma which was
obtained from the Cell Distribution Center of the Salk
Institute. NS1 cells came from Dr. Ingegerd Hellstrom's
laboratory. Both BW5147 and NS1 cells were shown to be free
of mycoplasma.
6.1.3. ASSAY OF DELAYED-TYPE HYPERSENSlllVllY
An assay was used which measured delayed-type hyper-
sensitivity (DTH) as mediated by Thy 1 ,Lyt 1 lymphocytes,
and was characterized by the typical morphological manifes-
tations of a DTH reaction (Forstrom, J.W., et al., 1983,Nature 303:627; Cory, J., et al., 1981, in Monoclonal
Antibodies and T Cell Hybridomas, Hammerling, G.J., et al.,
eds., Elsevier/North-Holland Biomedical Press, p. 503).
Mice were immunized by subcutaneous injection of either a
total of 1 x 106 irradiated (15,000 rad) tumor cells into
two sites, one on each flank, or by injection of 3-10 ug of
mAb (diluted in phosphate-buffered saline, PBS) at four
subcutaneous sites. When material was assayed for
suppression of DTH, it was always assayed for suppression of
30 its inductive phase. In these experiments, the putative
suppressive material was diluted to 100 ul in PBS and in-
jected into a tail vein immediately following immunization.
In each test for DTH, treatment or control groups
consisted of 5 mice, and they were always coded. Five days
35 after immunization, DTH was elicited by injection of 5 x 10
~54~ l 33981 6
tumor cells into one of the two hind footpads. After 24
hours, the thickness of both the injected and the uninjected
contralateral footpads was measured, using a dial micro-
meter. For each treatment group, the data are presented as
the mean increase in thickness (i.e., the swelling) of the
injected footpads. The significance of the differences
between treatment groups and corresponding controls was
determined using a two-tailed Student's t test.
6.1.4. GENERATION OF AUTO-ANTI-IDIOTYPIC
MONOCLONAL ANTIBODIES
- BALB/c mice were immunized by subcutaneous injection of
1 x 107 trypan blue unstained, cultured MCA-1490 or MCA-1511
cells, followed three weeks later by excision of the
resulting tumor nodules. After two more weeks, the mice
- 5 were injected with 2 x 106 irradiated (15,000 rads) cells
from the respective tumors, and two weeks later they were
injected once more with the same dose. Splenic cells were
obtained 7 days after the last injection and fused to NS-l
myeloma cells, using techniques which have been described
(Yeh, M.Y., et al., 1979, Proc. Natl. Acad. Sci. U.S.A.
76:2927).
Hybridomas were first screened for production of IgG
antibodies using a radioimmunocompetition assay (Brown,
J.P., et al., 1980, J. Biol. Chem. 255:4980), and those
; 25 producing greater than 4 ~g IgG per ml were expanded.
Culture supernatants were pooled, with each pool consisting
of supernatants from five hybridomas. Antibodies were
purified from each pool by affinity chromatography on S.
aureus protein A covalently coupled to Sepharose CL-4B
(Sigma Chemical Co., St. Louis, MO) (Brown, J.P., et al.,
1980, J. Biol. Chem. 255:4980). The p~ of the antibody
solutions was adjusted to 8.5 prior to chromatography to
facilitate the isolation of IgGl. BALB/c mice were
immunized by injection with the pooled antibodies and five
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133~8~6
days later were challenged with the appropriate tumor cells
(MCA-1490 or MCA-1511 cells) for elicitation of DTH. The
individual supernatants of a pool testing positive were
assayed for the priming of DTH to MCA-1490 and MCA-1511,
with one of the two tumors serving as the control. Two to
four percent of the wells from each fusion were found to
contain antibodies with the desired activity. Hybridomas
making antibodies that primed for tumor-specific DTH were
cloned twice by limiting dilution, after which positive
clones were expanded and adapted to grow as ascites tumors
in pristane-primed-BALB/c mice (Yeh, M.Y., et al., 1979,
Proc. Natl. Acad. Sci. U.S.A. 76:2927). Monoclonal
antibodies were purified from ascites fluid by affinity
chromatography on protein A Sepharose. mAb 4.72 (relating
to MCA-1490) and mAb 5.96 (relating to MCA-1511) were used
for the present study. They were both of the IgGl isotype,
as was mAb 8.2, which was used as a control in some
experiments.
6.1.5. T CELL HYBRIDOMAS
Several hybridoma lines were obtained by fusion of
BW5147 cells with thymocytes of mice carrying MCA-1490
tumors, as previously described (Nelson, K.A., et al., 1980,
Proc. Natl. Acad. Sci. U.S.A. 77:2866; Nelson, K., et al.,
1985, in T-Cell Hybridomas, Taussig, M.J., ed., CRC Press,
p. 129). The four hybridomas used in this study produced
factors which suppressed the lysis of MCA-1490 cells by
specifically immune T cells as tested in a 51Cr-release
assay (Nelson, K., et al., 1985, supra). Cultures of these
30 hybridomas and of the BW5147 fusion partner were grown in
Dulbecco's modified Eagles' medium (Grand Island Biological
Co., Grand Island, N.Y.), which was supplemented with fetal
bovine serum (150 ~l/ml), penicillin (100 units/ml),
streptomycin (100 ~g/ml) and L-glutamine (290 ~g/ml), and
35 was buffered with sodium bicarbonate. Spent medium was
c
-56- 1339816
taken from cells in log phase growth when there were
approximately 2 x 106 cells per ml. The medium was filtered
(0.4 microns, Millipore) and stored at -70~C until assayed.
6.1.6. ISOLATION OF SUPPRESSOR FACTORS BY
AFFINITY CHROMATOGRAPHY WITH mAb
mAb 4.72 or 5.96 was used together with a control mAb,
8.2, which is also of the IgGl isotype and is specific for a
human melanoma antigen, p97 (Brown, J.P., et al., 1981, J.
Immunol. 127:539). The mAb were diluted in PBS to 2 mg per
ml and then coupled to an equal volume of Affi-Gel 10 (Bio-
Rad Laboratories, Richmond, CA) by overnight incubation at
4~C. The gels were washed, incubated with 0.1 M ethanola-
mine and used to prepare columns. The columns were washed
: extensively with PBS and pre-eluted with 3 M NaSCN prior to
use. Spent culture media of T cell hybridomas (as a source
of suppressor factor) or BW5147 cells (as a control) were
diluted 1:10 in PBS, after which 0.2 ml of the diluted media
were passed through columns of 0.2 ml of gel. The columns
were washed with PBS until 2 ml of effluent had been
collected, after which the effluents were diluted 1:10 in
PBS and assayed for suppression of the inductive phase of
DTH.
Experiments were also performed with sera from BALB/c
mice which had been transplanted with either MCA-1490 or
25 MCA-1511 tumors or which were untreated littermates. The
- sera were diluted 1:10 in PBS, after which 0.5 ml of the
diluted sera was passed through columns of 0.5 ml gel. The
columns were washed with PBS~ The first 1.5 ml was
collected as effluent and an additional 5 ml was discarded.
30 Bound proteins were then eluted from the columns by addition
of 0.5 ml of 3 M NaSCN, and the eluates were desalted by
passage through columns of Sephadex G-25 equilibrated with
PBS. Eluates and effluents were diluted in PBS to yield a
1:100 dilution relative to the original sera. They were
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, "~,s -
1 33~81 6
- -57-
subsequently tested for suppression of the inductive phase
of DTH.
6.1.7. BINDING OF SUPPRESSOR FACTOR
K54SF to MCA-1490 CELLS
Adherent cultured MCA-1490 cells were incubated in
ethylene-diamine tetraacetic acid (5 mM), washed with PBS,
and incubated in a 0.1% solution of glutaraldehyde for 10
minutes at 4~C. The cells were subsequently incubated in
PBS with 0.5% bovine serum albumin, followed by washing with
PBS. Spent culture media of T hybridoma I-K54 or control
BW5147 cells were diluted 1:100 in PBS containing 10 ug of
either mAb 4.72 or mAb 8.2 (as a control), and incubated on
ice for 30 minutes. Subsequently, they were added to 100 ul
of packed tumor cells, which were suspended and incubated at
room temperature for 10 minutes and on ice for an additional
30 minutes. In one experiment, the tumor cells were
pretreated by incubation with antibody and washed three
times with PBS before the addition of diluted spent media
from I-K54 or BW5147 cells. After incubation on ice, the
tumor cells were washed 5 times with 15 ml of PBS and
suspended in 0.5 ml of glycine-HCl buffer (pH 3.0). After
further incubation on ice for 10 minutes, the cells were
pelleted by centrifugation at 200 X g. The pH of the
supernatant was immediately adjusted by passage through
Sephadex G-25 equilibrated with PBS. The eluates were
diluted 1:10 in PBS and assayed for suppression of the
inductive phase of DTH.
~ _'h
6.1.8. ISOLATION AND CULTURE OF T CELL LINES
3 T cells reacting with mAb 4.72 were obtained from lymph
nodes draining the site of a progressively growing,
transplanted MCA-1490 sarcoma. The cells were isolated by
centrifugation on a Percoll gradient (Pharmacia) and mixed
with mAb 4.72 or mAb 8.2 coupled with biotin. Subsequently,
Trade Mark
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~.,,
-58-
133~816
they were washed, treated with avidin-fluorescein
isothiocyanate (FITC), washed again and analyzed on a
fluorescence-activated cell sorter (FACS-II, Becton-
Dickinson). Brightly stained cells, which were seen only in
the sample treated with mAb 4.72, and which represented less
than 1% of the sample, were collected. These cells, termed
90.3, were cultured in Click's medium supplemented with 25%
medium from a culture of rat spleen cells which had been
stimulated with concanavalin A for 24 hours. Vigorous
growth was observed after 25 days and was maintained by
expanding the cultures into fresh medium every 72 hours.
Another cell line, 11.2, was established from Lyt-1 positive
lymphocytes which had been obtained from nodes draining the
site of a MCA-1511 tumor; it was maintained in a way similar
to line 90.3. The cells were assayed between 6 and 18 weeks
after establishment of the 90.3 or 11.2 lines. Normal T
cells from lymph nodes of tumor-free mice were cultured for
48 hours before they were used as controls for some of the
assays.
6.1.9. IN VIVO MANIPULATION OF TUMOR GROWTH
Tumor tissue was obtained from mice 14-21 days after
injection of tumor cells from serial passage. A suspension
was prepared by mechanical disruption and brief treatment
with trypsin. Viability was assessed by exclusion of trypan
blue. In the experiment presented in Fig. 4, tumor cells
were mixed with cultured T cells at a ratio of 1:10 and the
mixture injected in one subcutaneous site. Ten mice were
used per treatment group. Tumor growth was monitored at 2-4
day intervals by two perpendicular measurements of the
growing tumor nodule. Tumors with an area greater than 0.2
cm2 rarely regressed in untreated controls and were
considered established tumors.
In the experiments measuring the effect of mAb
treatment, tumor cells were injected subcutaneously in one
_59_ 1 3 3 9 8 1 6
site per mouse. mAb was diluted in PBS and injected
intraperitoneally (i.p.), 10 ug per injection. Tumor growth
- was monitored as described above. A two-tailed Student's t
test was used to determine the significance of the
difference between the various treatment groups.
6.2. SUBCUTANEOUS ADMINISTRATION OF mAb 4.72
OR mAb 5.96 INDUCES TUMOR-SPECIFIC DTH
TO MCA-1490 and MCA-1511
We have previously reported that mAb 4.72 can induce
- 10 DTH to MCA-1490, when injected subcutaneously, and that it
reacts with an idiotope on tumor-specific cells, i.e., that
it is auto-anti-idiotopic (Forstrom, J.W., et al., 1983,
Nature 303:627). We have now developed a similar mAb, 5.96,
using lymphocytes from mice sensitized to an antigenically
different sarcoma, MCA-1511 (id.) This allowed us to assess
the immunological specificity of the DTH effect by testing
mAb 4.72 and 5.96 in parallel (Table I).
- 25
-60- 1 33q8 1 6
TABLE I
mAbs 4.72 AND 5.96 PRIME BALB/C MICE FOR DTH THAT
5IS TUMOR-SPECIFIC AND ALLOTYPE RESTRICTED*
Mean Footpad Swelling
Mice (x 10 3 in + SE)
Mouse Injected in Mice Challenged with
Strain with MCA-14g0 MCA-1511
BALB/c mAb 4.72 15.3 + 1.2t 5.0 + 1.6
(H-2d~ MCA-1490 cells18.4 + 1.6tNot done
Igh-la) mAb 5.96 8.0 + 1.2 13.7 + 0.3t
MCA-1511 cellsNot done 18.7 + 0.9
Diluent 8.3 + 1.2 5.0 + 1.8
CB-20 mAb 4.72 7.7 + 0.4 6.7 + 0.3
(H-2 , MCA-1490 cells18.3 + o.gtNot done
Igh-lb) mAb 5.96 8.2 + 0.6 6.7 + 1.2
MCA-1511 cellsNot done 20.0 + 1.8t
Diluent 8.0 + 1.2 5.3 + 0.6
* Five mice in each group were injected subcutaneously
with 5 ug of mAb or 10 tumor cells in 100 ul. Five days
later, all mice were given 5 x 105 tumor cells in 20 ul into
- one hind footpad. The increase in the thickness (swelling)
of the injected pad over the contralateral pad was
determined 24 hours later and is presented as the mean (+
SE) for each group.
t The response of these mice was significantly greater
than that of mice injected with diluent at P less than 0.01
by Student's t test.
-61- l 3398 1 6
As shown in Table I, BALB/c mice primed with mAb 4.72
responded with DTH to a subsequent challenge with MCA-1490
cells but not to challenge with MCA-1511 cells. mAb 5.96
behaved in an analogous way in that it primed BALB/c mice
for DTH to MCA-1511 but not MCA-1490. The specificity of
the DTH indicates that different idiotopes were involved in
the immune responses to MCA-1490 and MCA-1511.
- In agreement with previous data for mAb 4.72 (Forstrom,
J.W., et al., 1983, Nature 303:627), the priming required
identity at genes linked to the Igh-1 allotype locus (Table
I), since neither of the two mAb primed CB-20 mice.
Immunization with tumor cells did induce DTH in the CB-20
mice. When tested with antibody binding assays (Cory, J.,
et al., 1981, in Monoclonal Antibodies and T Cell Hybrido-
mas, Hammerling, G.J., et al., eds., Elsevier/North-Holland
Biomedical Press, p. 503), mAb 5.96 was similar to mAb 4.72
(Forstrom, J.W., et al., 1983, Nature 303:627) in that it
did not bind to sarcoma cells.
We conclude that mAb 4.72 and mAb 5.96 are functionally
anti-idiotopic to the immune responses to MCA-1490 and MCA-
1511, respectively.
6.3. INTRAVENOUS ADMINISTRATION OF
mAb 4.72 SUPPRESSES DTH TO MCA-1490
We tested the effect of intravenous (i.v.) administra-
tion of mAb 4.72 on the DTH reactivity of mice which had
been immunized by subcutaneous injection of either mAb 4.72
or MCA-1490 cells or with MCA-1511 cells, the latter being
used as control. Immediately following the subcutaneous
immunization, mice were injected via a tail vein with
approximately 5 ug of mAb 4.72 or mAb 8.2 (as a control).
The DTH response was measured 5 days later (Table II).
1339816
-62-
TABLE II
mAb 4.72 SUPPRESSES DTH TO MCA-1490 WHEN INJECTED
INTRAVENOUSLY (I.V.) INTO MICE IMMEDIATELY AFTER
IMMUNIZATION BY SUBCUTANEOUS INJECTION OF EITHER
mAb 4.72 OR MCA-1490 TUMOR CELLS*
~' .
Mean Footpad
0 Mice Mice Antibody (orSwelling
Immunized Challenged Fab fragments)(x 10 3
with with Injected i.v. in. + SE)
mAb 4.72 MCA-1490 mAb 4.72 4.8 + 0.8
mAb 8.2 12.2 + 0.7
None (diluent) 13.6 + 0.5
MCA-1490 MCA-1490 mAb 4.72 6.8 + 1.6t
Fab 4.72 7.2 + 1.3t
~ mAb 8.2 17.0 + 1.3
None (diluent) 19.8 + 0.7
Nothing MCA-1490 None 4.0 + 0.9
MCA-1511 MCA-1511 mAb 4.72 15.0 + 2.6
Fab 4.72 15.6 + 1.2
mAb 8.2 14.8 + 2.1
None (diluent) 16.4 + 2.6
Nothing MCA-1511 None 4.6 + 1.3
* Mice were immunized by subcutaneous injection, and DTH
was measured as described for Table I. Material assayed for
suppression of DTH was diluted, and 100 ul injected i.v.
immediately following immunization. Each mouse received 5
ug of whole mAb or 3.5 ug of Fab fragments.
t The response of these mice was significantly lower than
that of mice receiving diluent at P less than 0.001 accord-
ing to Student's t test.
63- 1 3 3 ~ 8 1 5
-
As shown in Table II, i.v. injection of mAb 4.72 suppressed
the ability of subcutaneously injected mAb 4.72 or MCA-1490
cells to prime mice for DTH to MCA-1490. Furthermore, i.v.
injection of Fab fragments prepared from mAb 4.72 suppressed
immunization with MCA-1490 cells. Immmunization with MCA-
; 1511 cells was not suppressed, and i.v. injection of a
control mAb, 8.2, had no effect.
We conclude that the route of administration influencedthe DTH reactivity of mice receiving mAb 4.72, with
subcutaneous injection inducing DTH (see Section 6.2.,
supra) and i.v. injection suppressing this effect.
6.4. EXPRESSION OF AN IDIOTOPE DEFINED
BY mAb 4.72 ON T CELLS MEDIATING
DTH TO MCA-1490
We studied whether the mAb 4.72 defined idiotope was
present on T cells mediating DTH to MCA-1490. Mononuclear
cells from lymph nodes draining a growing MCA-1490 tumor
were analyzed on a fluorescence-activated cell sorter (FACS)
using mAb 4.72 coupled to biotin and avidin-FITC. Brightly
stained cells were observed. Although the brightly stained
cells represented less than one percent of the total
population of mononuclear cells, they were not seen in
samples analyzed with a biotinylated control monoclonal
antibody, mAb 8.2, used at the same dose. The stained cells
- 25 were isolated and cultured in the presence of interleukin-2
(IL-2), from which a cell line was established, which was
called 90.3. When the 90.3 cells were analyzed for surface
phenotype after six weeks of culture, they bound mAb 4.72,
although the fluorescence intensity was lower than that of
- 30 the original cells. These cells were Thy-l positive and
expressed Lyt-1 but not Lyt-2 antigens.
We then investigated the effect of the 90.3 cells on
the DTH response to MCA-1490 (Table III).
- - 1 33q8 1 5
-64-
-TABLE III
TUMOR-SPECIFIC DTH IS TRANSFERRED BY
T CELLS FROM LINES 90.3 AND 11.2*
Mice Injected with Mixture of Mean Footpad Swelling
Tumor Effector (x 10 3 in. + SE)
Cells Cells
- Experiment 1 Experiment 2
MCA-1490 90.3 19.2 + 1.6t 15.0 + 2.2t
MCA-1490 Normal T 9.6 + 1.1 4.8 + 0.6
MCA-1490 None (diluent) 7.0 + 0.7 4.0 + 0.4
MCA-1511 90.3 9.4 + 1.0 4.0 + 0.4
MCA-1511 11.2 17.3 + 2.6 l0.7 + 1.2
MCA-1511 Normal T10.2 + 2.3 3.0 + 0.3
MCA-1511 None (diluent) 8.0 + 0.7 3.0 + 0.4
None (diluent) 90.3 0.4 + 1.2 0.2 + 0.4
20 None (diluent) 11.2 2.6 + 0.6 1.2 + 0.6
None (diluent) Normal T 4.8 + 1.0 3.6 + 1.2
* Effector T cells (2 x 105) derived from lines 90.3 or
11.2 or from naive BALB/c mice were mixed with MCA-1490 or
MCA-1511 cells (5 x 105) and injected into one footpad of a
naive BALB/c mouse. Five mice were used per group. After
24 hours, DTH was measured and calculated as described in
Table I.
t The response of these mice was significantly greater
than that of mice receiving diluent and MCA-1490 cells at P
less than 0.02 by Student's t test.
- 13398~6
-65-
As shown in Table III, there was DTH to MCA-1490 when
mixtures of 90.3 cells and MCA-1490 cells were injected into
the footpads of naive BALB/c mice. There was no DTH to
MCA-1511, and neither was there any footpad swelling in mice
which received only the 90.3 cells but no MCA-1490 cells. A
second line of T cells, 11.2, was established from lymph
nodes of mice draining the antigenically different sarcoma
MCA-1511. The 11.2 line gave DTH to MCA-1511 but not to
MCA-1490. Lymph node cells from naive mice were cultured in
medium containing IL-2 for 48 hours prior to the assays and
was used as another control; they did not transfer DTH to
MCA-1490.
90.3 cells, which had been cultured for nine weeks,
were tested for their effect on the in vivo growth of the
MCA-1490 tumor. Cultured T cells from naive mice were used
as controls. The T cells were mixed with MCA-1490, MCA-1510
or MCA-1511 tumor cells; each mixture was injected
subcutaneously into ten naive BALB/c mice. Time to
appearance of tumors was monitored, as well as the growth
rate of the tumors. As shown in Figure 4, one of ten mice
which had received a mixture of MCA-1490 together with 90.3
cells developed palpable tumors, as compared to nine of ten
mice in which control T cells rather than 90.3 cells were
admixed. The 90.3 cells had no effect on two antigenically
different sarcomas, MCA-1510 or MCA-1511.
We conclude that mAb 4.72 reacted with a small
population of T cells from mice bearing sarcoma MCA-1490,
and that cell line 90.3, which was derived from such cells,
was specifically reactive to MCA-1490.
6.5. EXPRESSION OF AN IDIOTOPE RECOGNIZED
BY mAb 4.72 ON FACTORS DERIVED FROM
SUPPRESSOR T CELLS
We have previously described the generation of T-T
hybridomas by fusing T cells from mice bearing sarcoma MCA-
-66- 1 33~81 6
1490 (Nelson, K.A., et al., 1980, Proc. Natl. Acad. Sci.
U.S.A. 77:2866). The monoclonal products (suppressor
factors~ made by four of these hybridomas, I-K54, II-15,
II-32, and II-122, have been demonstrated to suppress DTH to
MCA-1490, but not to MCA-1511, and to bind specifically to
MCA-1490 cells, while they differed as to the kinetics and
genetic restriction of their suppression (Nelson, K., et
al., 1985, in T-Cell Hybridomas, Taussig, M.J., ed., CRC
Press, p. 129).
; 1O We selected one of the four suppressor factors, K54SF,
to test whether the idiotope defined by mAb 4.72 was
associated with the portion of that factor which could bind
to MCA-1490. K54SF (obtained from supernatant of cultured
I-K54 cells) and MCA-1490 cels were inoculated together,
after which the tumor cells were washed and any K54SF that
had bound to them was eluted. Suppression was assayed by
DTH tests after injecting the eluates i.v. into mice
immediately after these had been immunized subcutaneously
with MCA-1490 or MCA-1511 cells. Tumor-specific suppressive
activity was recovered in the eluates.
The ability of mAb 4.72 to inhibit the binding of K54SF
to MCA-1490 was investigated in more detail. Medium
containing K54SF was mixed with either mAb 4.72 or mAb 8.2
(control) before it was added to MCA-1490 cells, and eluates
~f the cells were tested for suppression (Table IV).
- 35
133~816
-67-
TABLE IV
INCUBATION OF K54SF (A SUPPRESSOR FACTOR
MADE BY T-T HYBRIDOMA I-K54) WITH mAb 4.72
INHIBITS ITS BINDING TO MCA-1490 CELLS*
Anti-MCA-1490 Immune Mice Mean Footpad Swelling
Injected with (x 10 in. + SE)
Suppressor Subsequently in Mice Challenged
Source of Factor Incubated with MCA-1490
Suppressor First with and - Experiment Experiment
Factor Mixed with Eluted from 1 2
(medium)
I-K54 mAb 8.2 MCA-1490 6.4 + 0.6t 4.0 + 0.7t
I-K54 mAb 4.72 MCA-1490 15.6 + 1.1 15.2 + 0.9
BW5147
(control) mAb 8.2 MCA-1490 17.4 + 0.9 Not done
BW5147
(control) mAb 4.72 MCA-1490 17.2 + 1.6 Not done
I-K54 Diluent MCA-1490 Not done 6.6 + 1.5t
pre-incu-
bated with
mAb 8.2
I-K54 Diluent MCA-1490 Not done 17.6 + 1.2t
pre-incu-
bated with
mAb 4.72
* Media of I-K54 or BW5147 (control) cells were first mixed
30 with mAb 4.72 or mAb 8.2, after which they were incubated
with MCA-1490 cells. In Experiment 2, there were also groups
(lines 5 and 6 in the Table) in which MCA-1490 cells were
incubated with mAb 8.2 or mAb 4.72, before they were
incubated with I-K54 medium. After incubation, the tumor
1 339ûl 6
-68-
cells were washed, and material bound to them was eluted and
assayed for suppression of DTH to MCA-1490. Mean footpad
- swelling in unimmunized (control) mice was 5.0 + 0.3 in
Experiment 1 and 5.2 + 0.6 in Experiment 2, and mean footpad
swelling in anti-MCA-1490 immune mice challenged with MCA-
1490 was 15.8 + 1.4 in Experiment 1 and 17.6 + 1.0 in
Experiment 2.
t The response of these mice was significantly lower than
that of mice receiving diluent at P less than 0.001 by
Student's t test.
As shown in Table IV, eluates of MCA-1490 cells which had
been incubated with a mixture of mAb 8.2 and K54SF,
suppressed DTH to MCA-1490. In contrast, eluates of MCA-
1490 cells incubated with a mixture of K54SF and mAb 4.72
were not suppressive. This result indicates that the
ability of K54SF to bind to MCA-1490 was inhibited by its
binding to mAb 4.72. Eluates of MCA-1490 cells which had
been incubated with BW5147 (control) medium and mAb 8.2 were
not suppressive; thus suppression was not due to eluted
tumor antigen. Pretreatment of MCA-1490 cells with mAb 4.72
did not prevent them from binding K54SF (see line 6 of Table
XV), and thus the inhibition seen when K54SF was incubated
with mAb 4.72 was not due to competition between mAb 4.72
and K54SF for binding to antigen on MCA-1490 cells. The
results suggest that the idiotope recognized by mAb 4.72 is
expressed at the site of K54SF which binds to MCA-1490 or,
at least, in close proximity to that site. Similar results
were obtained for the suppressor factor produced by a T cell
hybridoma, II-32.
Since K54SF suppresses DTH to MCA-1490, we assayed the
effect of K54SF on the function of 90.3 cells, since these
cells could transfer DTH specific to MCA-1490 (see Section
6.4, supra). Mice were injected, in one footpad, with a
~339816
-69-
mixture of cells from the 90.3 line and cells from the MCA-
1490 tumor. One hour later, K54SF (as supernatant of the
I-K54 hybridoma) was injected into the same footpad; BW5147
supernatant was used as control. As shown in Table V,
injection of K54SF suppressed the ability of the g0.3 cells
to mediate DTH to MCA-1490.
-~ 20
- 1 3398 1 6
-70-
- TABLE V
DTH TO MCA-1490, AS TRANSFERRED BY T CELLS
FROM LINE 90.3, IS SUPPRESSED BY A FACTOR
PRODUCED BY T-T HYBRIDOMA I-K54*
Source of
Suppressor Mean Footpad Swelling
10 Cells Trans- Factor (cul- (x 10 in. + SE)
ferring DTH ture medium)
Experiment 1 Experiment 2
15 90.3 None (diluent) 19.2 + 1.6 15.0 + 2.2
I-K54 1.0 ul8.2 + 0. 8 t Not done
I-K54 0.1 ul9.4 + o.gt 3.0 + 0.4t
BW5147 1.0 ul 20.3 + 1.4 Not done
BW5147 0.1 ul 19.6 + 1.6 13.3 + 1.2
20 Normal T None (diluent) 9.6 + 1.1 4. 8 + o . 6
Diluent None (diluent) 7.0 + 0.7 4.0 + 0.6
* Line 90.3 T cells were mixed with MCA-1490 cells and
injected into the footpads of naive BALB/c mice to assay for
25 DTH, as described in Table III. Medium of I-K54 or BW5147
(control) cells was diluted to 5 ul and injected into the
same footpad as the T cell-tumor cell mixture. Five mice
were used per group.
t The response of these mice was significantly lower than
30 that of mice receiving 90.3 cells, tumor cells and diluent,
at P less than 0.01 by Student's t test.
-71- l 3 3 q 8l 5
Since both 90.3 effector cells and K54 suppressor cells
appeared to carry a mAb 4.72-defined idiotope, we evaluated
whether this idiotope was present also on suppressor factors
made by T cell hybridomas other than K54. Suppressor
factors derived from four different T cell hybridomas were
assayed for suppressive activity after incubation with
-~ immobilized mAb 4.72. Spent medium from the four hybridomas
and from BW5147 (control) cells, was passed through columns
of either mAb 4.72 or mAb 8.2 (control), which had been
10 covalently coupled to agarose. The effluents were assayed
; for suppression of DTH to MCA-1490, by injecting them i.v.
into BALB/c mice immediately after they had been subcutane-
ously immunized to MCA-1490 (Table VI).
- ~3398~6
-72-
TABLE VI
5mAb 4.72 BINDS TO FACTORS WHICH ARE PRODUCED
BY T CELL HYBRIDOMAS AND WHICH SUPPRESS DTH TO MCA-1490*
Mean Footpad Swelling
Source of Suppressor(x 10 3 in. + SE) in Mice Injected
Factors (culturewith Medium Adsorbed with
medium from) mAb 8.2 mAb 4.72
BW5147 (control) 24.2 + 1.8 23.4 + 0.8
Hybridoma I-K54 7.8 + 1.0 t 22.9 _ 1.2
Hybridoma II-15 9.6 + 2.8** 18.4 + 2.2
Hybridoma II-32 10.8 + 2.0** 24.8 + 2.4
Hybridoma II-122 11.0 + 1.2 t 23.6 + 2.0
* Spent culture medium of T cell hybridomas I-K54, II-15,
20 II-32 and II-122 or of BW5147 (control) cells were passed
through columns of mAb 4.72 or 8.2 covalently coupled to
agarose. The effluents were assayed for suppression of the
inductive phase of DTH to MCA-1490 as described in Table II.
Statistical significance of differences in footpad swelling,
25 as compared to control mice receiving diluent was estimated
by Student's t test.
t p less than 0.001
** P less than 0.01
As shown in Table VI, media derived from each of the four T
hybridomas and passaged through a control column (of mAb
8.2) suppressed the induction of DTH to MCA-1490, while this
suppression was removed by passage through a mAb 4.72
1 33~8 1 6
-73-
column. All four suppressor factors thus appeared to
express the idiotope defined by mAb 4.72.
This result suggested that the idiotope defined by
mAb 4.72 was dominant in regulating the suppressor response
to MCA-1490. However, as factors made by only four
hybridomas were studied, we next analyzed the presumably
polyclonal suppressor response in mice bearing a growing
MCA-1490 sarcoma. This experiment was done on the basis of
the demonstration that sera from mice carying MCA-1490 or
MCA-1511 sarcomas can suppress the induction of DTH to the
respective tumors (Table VII).
13398 16
-74-
- TABLE VII
5SERA FROM MICE BEARING SARCOMA MCA-1490 OR MCA-1511
SUPPRESSES DTH TO THE RESPECTIVE TUMOR*
Mean Footpad Swelling
(x 10 in. + SE) in Mice
Immunized and Challenged with-
Serum Donor MCA-1490 MCA-1511
MCA-1490 tumor-bearer mice 6.7 + 1.3 t 20.0 + 0.4
MCA-1511 tumor-bearer mice 15.9 + 2.6 10.4 + 2.3 t
Naive mice 17.5 + 4.5 22.2 + 2.6
None (diluent) 15.0 + 1.4 21.0 + 1.4
* Sera from mice bearing progressively growing MCA-1490 or
MCA-1511 tumors or from control mice were diluted and 100 ul
injected i.v. into mice immediately before immunization to
MCA-1490 or MCA-1511. DTH was elicited and measured as
described in Table I. Mean footpad swelling in unimmunized
mice was 5.3 + 1.2 after challenge with MCA-1490 and 6.3 +
0.3 after challenge with MCA-1511.
t The response of these mice was significantly lower than
that of mice receiving diluent at P less than 0.01 by
Student's t test.
30 Sera from tumor-bearing mice were chromatographed on columns
of mAb 4.72 or mAb 8.2 (control), the material bound to the
columns was eluted, and effluents and eluates were assayed
for suppression of DTH (Table VIII).
1 33981 6
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~77~ ' 1 3398 1 6
As shown in Table VIII, sera from mice bearing MCA-1490
suppressed DTH to MCA-1490 after passage through a control
column (of mAb 8.2) but not-after passage through a mAb 4.72
column, and material which suppressed DTH to MCA-1490 was
recovered in an eluate of the latter column. Immunoadsorp-
tion with mAb 4.72 did not remove the ability of sera from
mice bearing a different tumor, MCA-1511, to suppress DTH to
that tumor.
Our results indicate that suppression of DTH to MCA-
1490 was associated with an idiotope recognized by mAb 4.72
and that suppression of DTH to MCA-1511 did not involve that
idiotope.
6.6. SEROTHERAPY OF MICE WITH
MCA-1490 OR MCA-1511 TUMORS
We investigated whether injection of mAb 4.72 and 5.96
protected mice against challenge with the appropriate tumor
cells (MCA-1490 or MCA-1511, respectively) and whether it
had any therapeutic effect on established tumors. The mAb
20- were injected intraperitoneally; the i.v. route was not
chosen in view of the evidence (see Section 6.3, supra) that
mice so injected showed decreased DTH reactivity to tumor
antigens.
In the first set of experiments, mice (20 per group)
25 were injected with either mAb 4.72 or (control) mAb 8.2.
They were challenged, 5 days later, with MCA-1490 cells at a
dose which, according to pilot tests, caused progressive
tumor growth in approximately 90% of the recipients.
Priming with mAb 4.72 delayed the appearance of transplanted
30 MCA-1490 sarcoma by 5 to 9 days compared to controls, but
there was no significant difference between the percentage
of mice dying from progressively growing tumors. The growth
of the antigenically unrelated MCA-1511 sarcoma was not
affected. Varying the amount of mAb 4.72 injected did not
-78- 13398~6
improve the results. Rather, in one experiment in which
mice received 100 times the amount of mAb 4.72 which could
prime for DTH, the growth of MCA-1490 was accelerated as
compared to both control mice and control tumors. A dual
effect has also been reported in the manipulation of
immunity to viral antigens, using anti-idiotypic antibodies
(Kennedy, R.C. and Dreesman, G.R., 1984, J. Exp. Med.
159:655; Kennedy, R.C., et al., 1984, J. Virol. 50:951).
In a second series of experiments, mice were first
injected with MCA-1490 cells subcutaneously, and this was
followed 7 or 8 days later by 100 ug mAb i.p., at this point
in time, tumors had become barely palpable in about 50% of
the mice. The antibody injection was repeated at 4 to 5 day
intervals for a total of four injections. In all of the 10
mice given mAb 4.72, the original small tumor nodules had
regressed when the experiment was terminated after 6 weeks.
At that time, 6 of 10 mice given the control mAb 8.2 had
progressively growing tumors with a surface area greater
than or equal to 0.20 cm2. This difference between the ~wo
20 groups was significant at P less than or equal to 0.05.
To test the limits of this form of therapy, the dose of
tumor cells was increased by giving tumor cells at a dosage
twice that required to grow out in 100% of the recipients.
The two antigenically distinct sarcomas, MCA-1490 and MCA-
1511, were treated in parallel with the appropriate anti-
idiotypic mAb (4.72 and 5.96, respectively). Groups of ten
mice were injected with mAb 4.72 (related to MCA-1490), mAb
5.96 (related to MCA-1511), mAb 8.2 (as a control), or
diluent (as another control), starting nine days after
30 transplantation when all the mice had barely palpable tumors
(greater than 0.2 cm ). As shown in Figure 5, treatment
with mAb 4.72 limited the growth of MCA-1490, but not of
MCA-1511, and treatment with mAb 5.96 similarly inhibited
MCA-1511, but not MCA-1490. The differences between mice
35 receiving the appropriate anti-idiotypic mAb and any of the
~79~ l 33 q 8l 6
three controls (the inappropriate anti-idiotopic mAb, mAb
8.2 or diluent) was statistically significant at P less than
or equal to 0.001. Figure 6 presents these data in more
detail by showing the number of mice with tumors in each
group treated with mAb 4.72 or mAb 5.96. In each of the two
groups receiving appropriate anti-idiotypic antibody, tumors
regressed in 5 or 7 of the 10 treated mice, and three of
these mice (in each of the two groups) remained free of
tumor for two weeks after the last injection of antibody.
0 At that time, all the mice in the control groups had tumors
with a surface area greater than 1.5 cm2, and some of the
mice had already died with tumor.
We conclude that treatment of mice with established
sarcomas MCA-1490 and MCA-1511, using intraperitoneal
injection of the appropriate anti-idiotypic mAb, had
significant antitumor activity.
6.7. DISCUSSION
We herein describe evidence that T cells and anti-
idiotypic B cells were generated in mice which either bore agrowing sarcoma or had been immunized to such a tumor. T
cells bearing the same idiotope included cells which
mediated tumor-specific DTH and cells which made a soluble,
tumor-specific suppressor factor. By employing anti-
idiotopic mAb, we could manipulate the antitumor response soas to limit the growth of established syngeneic sarcomas
expressing the relevant tumor antigens.
Two mAb, 4.72 and 5.96, were used. They were isolated
from BALB/c mice which had been immunized to sarcomas MCA-
30 1490 or MCA-1511, respectively. We regard the two mAb as
anti-idiotopic in view of three findings which relate to
their functions. First, each mAb induced tumor-specific DTH
in syngeneic mice in the absence of tumor antigen. Second,
this induction was allotype restricted. Third, neither mAb
35 bound to the immunizing tumor.
-80- ~ 1339816
The idiotope defined by mAb 4.72 was identified on T
cells which mediated DTH to MCA-1490. This was done in an
experiment in which mAb 4.72 was used to isolate from lymph
nodes of mice responding to MCA-1490, a small fraction of
lymphocytes which expressed the 4.72-defined idiotope and
from which a T cell line, 90.3, could be established. Naive
mice receiving the 90.3 cells displayed DTH to MCA-1490 but
not to MCA-1511, and the 90.3 cells prevented the outgrowth
of sarcoma MCA-1490 but not sarcoma MCA-1510 or MCA-1511.
Cheever et al. (1986, J. Exp. Med. 163:1100) have demonstra-
ted the ability of Lyt 1 2 T cells to limit the progression
of murine leukemia. However, their protocol includes
restimulation of the cultured cells with antigen as well as
IL-2. The tumor specificity of the 90.3 cells argues
against their being lymphokine-activated killer cells.
Products of T cells which suppressed the DTH response
to MCA-1490 were found to express the idiotope defined by
mAb 4.72. This was shown in studies on four T cell hybrido-
mas which had been obtained by fusing thymocytes from mice
20 carrying growing MCA-1490 tumors with BW5147 cells (Nelson,
K., et al., 1985, in T-Cell Hybridomas, Taussig, M.J., ed.,
CRC Press, p.129). The products of these hybridomas had
previously been found to suppress the lysis of 51Cr labelled
MCA-1490 cells by specifically immune cytolytic T cells
(Nelson, K.A., et al., 1980, Proc. Natl. Acad. Sci. U.S.A.
77:2866) and to suppress the induction of DTH to MCA-1490
(Cory, J., et al., 1981 in Monoclonal Antibodies and T Cell
Hybridomas, Hammerling, G.J., et al, eds., Elsevier/North-
Holland Biomedical Press, p. 503); this suppression was
30 speciflc for the response to MCA-1490 and was allotype
restricted. Furthermore, the suppressor factors had been
found to bind to MCA-1490 cells from which they could be
recovered by elution (Nelson, K.A., et al., 1980, supra).
In the examples described herein, we demonstrate that mAb
35 4.72 inhibits the binding of all four suppressor factors to
1 33981 6
-81-
MCA-1490 cells. This suggests that the idiotope defined by
mAb 4.72 is associated with the site on the suppressor
factor which binds to a tumor antigen on MCA-1490 cells. T
suppressor factors bearing the idiotope defined by mAb 4.72
could both suppress the priming for DTH by mAb 4.72 and the
transfer of DTH to MCA-1490 by T cells from line 90.3
(which, as discussed supra, express the mAb 4.72-defined
idiotope).
Based on these data, we suggest the following model:
0 mAb 4.72 defines an idiotope which occurs on tumor-specific
regulatory T cells and is dominant in the immune response of
BALB/c mice to MCA-1490. During this response, the same or
a cross-reacting idiotope activates a B cell clone producing
anti-idiotypic antibody. The latter antibody is presumed to
regulate both suppressor T cells and DTH-reactive T cells.
Our model assumes, as supported by various studies
(Rajewski, K. and Takemori, T., 1983, Ann. Rev. Immunol.
1:569; Urbain, J., et al., Ann. Immunol. 133D:179; Binz, H.
and Wigzell, H., 1978, J. Exp. Med. 147:63; Ertl, H.C.J., et
20 al., 1982, Proc. Natl. Acad Sci. U.S.A. 79:7479), that T and
B cells interact in idiotypic nètworks. Our model also
assumes that a B cell derived anti-idiotypic antibody (mAb
4.72 in our example) arose in response to a T cell idiotope
and not to a parallel set of idiotope-positive B cells. In
25 support of this, we have no evidence for tumor cell binding
antibody in the response of mice to MCA-1490, nor have we
been able to induce such antibody by immunizing with mAb
4.72. This is in contrast with the ease in demonstrating
idiotope-positive, antigen-reactive T cells. The data
30 support the hypothesis that a T cell idiotope can induce an
anti-idiotypic response in the B cell compartment, which in
its turn can regulate T cell responses in both upwards and
downwards directions.
- An alternative model assumes that the mAb 4.72-defined
35 idiotope displays an internal image of the MCA-1490 tumor
1 33~81 6
-82
antigen. However, it is difficult to reconcile that model
with the allotype (Igh-1) restriction of the induction of
DTH by mAb 4.72.
In the present study, we used the anti-idiotypic mAb
4.72 in a successful attempt to remove the suppressive
activity of sera from mice bearing MCA-1490. All the
circulating serum factors which could be detected by
measuring suppression of DTH to MCA-1490 appeared to express
the 4.72-defined idiotope.
As described supra, we next examined the feasibillty of
manipulating the immune response to tumor by using anti-
idiotypic mAb. mAb 4.72 and 5.96 were chosen, as they could
both induce DTH reactive cells in naive mice, and as the
idiotope defined by one of the mAb, 4.72, was expressed on
DTH reactive cells in immune mice. Our first set of
experiments addressed the question of tumor prevention, by
giving mAb before tumor transplantation, while a second
series of experiments dealt with therapy of already
established tumors. When mice received mAb 4.72 before they
20 were challenged with MCA-1490 cells, tumor outgrowth
commonly was delayed by a few days. These antitumor effects
did not improve by increasing mAb dose; in fact, an
experiment with a 100-fold increase in mAb dose showed
accelerated tumor growth.
In a second set of experiments, we investigated the
therapeutic value of injecting mice with established
sarcomas intraperitoneally with the appropriate anti-
idiotypic mAb. Injection of mAb 4.72 (but not of mAb 5.96)
limited the growth of sarcoma MCA-1490 and, more impor-
30 tantly, it induced the regression of 3 out of 10 such
sarcomas and prolonged the survival of the treated mice.
Injection of mAb 5.96 (but not of mAb 4.72) had a similar
effect in mice with sarcoma MCA-1511, also inducing
regression of 3 out of 10 sarcomas.
35 -
-83- 1339815
7. ANTI-IDIOTYPIC ANTIBODY SPECIFIC TO AN
IDIOTYPE WHICH RECOGNIZES A HUMAN
MELANOMA-ASSOCIATED GD3 GANGLIOSIDE ANTIGEN
Mouse monoclonal antibody (mAb) MG-21 (Hellstrom
et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:1499-1502)
recognizes a GD3 ganglioside antigen expressed on the
surface of cells from most human melanomas and in trace
amounts on normal cells (Dippold, W.G., et al., 1980, Proc.
Natl. Acad. Sci. U.S.A. 77:6114-6118; Nudelman, E., et al.,
1982, J. Biol. Chem. 257:12752-12756; Yeh, M.Y., et al.,
1982, Int. J. Cancer 29:269-275). mAb MG-21 demonstrates
complement-dependent cytotoxicity (CDC) to GD3-positive
cells with human serum as the source complement, and
antibody-dependent cellular cytotoxicity (ADCC) to GD3-
positive cells with human lymphocytes (Hellstrom et al.,
1985, supra). The GD3 antigen to which MG-21 binds has been
used with some success as the target for a passively
administered mouse mAb, R24 (Houghton, A.N., et al., 1985,
Proc. Natl. Acad. Sci. U.S.A. 82:1242-1246), whose
specificity and biological actlvity is similar to that of
20 MG-21. As described in the examples infra, we have used
MG-21 (Abl) as an immunogen to generate a mouse monoclonal
anti-idiotypic antibody (Ab2), which was designated 2C1.
This mAb, which is an IgG2a, was selected by its capacity to
inhibit the binding of MG-21 to a GD3-positive melanoma cell
25 line. mAb 2Cl was found to bind to MG-21 with high avidity,
but not to any of six other mouse mAb. It could completely
abrogate the binding of MG-21 to GD3-positive melanoma cells
as well as to purified GD3, and it inhibited the CDC and
ADCC properties of MG-21 in a dose-dependent fashion. By
30 using mAb 2Cl as a probe, an assay was developed to monitor
human antibodies to mAb MG-21 in patients receiving mAb MG-
21 for therapeutic or diagnostic purposes. A detailed
description of each step in this embodiment of the invention
is presented in the subsections below.
1339816
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7.1. MATERIALS AND METHODS
7.1.1. MICE
Eight to twelve week-old BALB/c female mice were
purchased from the Animal Facilities of the Fred Hutchinson
- Cancer Research Center (Seattle, WA).
7.1.2. TARGET CELLS
Human melanoma cell line M-2669 clone 13 was used; for
0 simplicity, it is referred to here as M-2669. It had been
established from a metastatic melanoma and cloned (Beaumier,
P.L., et al., 1986, J. Nucl. Med. 27:824-828). Cells from
this clone strongly express the GD3 antigen defined by mAb
MG-21 as determined by a binding assay (id.). The melanoma
cells were grown in 6% CO2 in air in RPMI 1640 culture
medium (Gibco, Grand Island, New York) containing 15% heat-
inactivated fetal calf serum (Hyclone Laboratories, Inc.,
Logan, Utah) buffered with NaHCO3 and supplemented with
penicillin (100 U/ml), streptomycin (100 mg/ml) and L-
20 glutamine (290 mg/liter).
7.1.3. GLYCOLIPID
GD3 ganglioside antigen was purified from M-2669 clone
13 melanoma cells as described (Nudelman, E., et al., 1982,
25 J. Biol. Chem. 257:12752-12756), and was provided by Dr.
Sen-itiroh Hakomori at Fred Hutchinson Cancer Research
Center (Seattle, WA).
7.1.4. MONOCLONAL ANTIBODIES
MG-21 (Hellstrom, I., et al., 1985, Proc. Natl. Acad.
Sci. U.S.A. 82:1499-1502) is an IgG3 antibody which binds to
a GD3 ganglioside antigen expressed strongly on about 80-90%
of human melanomas (Nudelman, E., et al., 1982, J. Biol.
Chem. 257:12752-12756; Yeh, M.-Y., i982, Int. J. Cancer
35 29:269-275). MG-21 can mediate CDC in the presence of human
1339816
-85-
serum and ADCC in the presence of human peripheral blood
lymphocytes (PBL), and it can inhibit the outgrowth of human
melanoma xenografts in nude mice (Hellstrom, I., et al.,
1985, Proc. Natl. Acad. Sci. U.S.A. 82:1499-1502).
mAb 2A-14 is an IgG3 which also binds to the GD3
ganglioside antigen, but to an epitope which by cross-
- blocking experiments appears to be different from that
recognized by MG-21.
mAb 96.5 is an IgG2a which binds to p97, a melanoma-
associated cell surface glycoprotein (Brown, J.P., et al.,
- 1981, J. Immunol. 127:539-546).
mAb L6 is an IgG2a which binds to a carbohydrate
antigen expressed strongly on cells from most human
carcinomas but not melanomas (Hellstrom, I., et al., 1986,
Cancer Res. 46:3917-3923).
- mAb L20 is in IgG1 which identifies a 110 kd protein
expressed on the surface of cells from most human carcinomas
but not melanomas (id.).
mAb 7T1.1 is an IgG3 immunoglobulin specific for blood
group A antigen strongly expressed on many human carcinomas.
mAb lG3.10 is an IgG3 antibody specific for blood group
A-like antigen also expressed on many human carcinomas.
mAb 26.8, which was used as an Ab2 control, is an IgG1
which binds to an idiotope on mAb 96.5 specific for the p97
25 melanoma antigen. mAb 26.8 can inhibit the binding of
mAb 96.5 to p97.
- P1.17 is an IgG2a mouse myeloma protein which was
obtained from the American Type Culture Collection (ATCC
Accession No. TIB 10). -~ ~
Antibodies were purified either from spent culture
medium or ascitic fluid by affinity chromatography on
protein A-Sepharose CL-4B as described by Ey et al. (1978,
Immunochemistry 15:429-436).
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7.1.5. COUPLING OF ANTIBODY WITH
KEYHOLE LIMPET HEMOCYANIN --
Antibody MG-21 was coupled to keyhole limpet hemocyanin
(KLH) by chemical cross-linking in the presence of
glutaraldehyde according to the procedures of Bona et al.
(1979, J. Exp. Med. 149:815-823). In brief, 1 ml of
mAb MG-21 solution (3.6 mg/ml) was mixed with 1 ml of KLH
solution (3 mg/ml) in 0.1 M phosphate buffer, pH 7.5.
Coupling was initiated by the addition of 1 ml of a 0.25%
solution of glutaraldehyde (Sigma Chemical, St. Louis, MO).
The mixture was shaken at room temperature for 1 hour. The
reaction was stopped by adding 250 ~l of 1 M glycine.
Antibody-KLH conjugate was stored frozen at -20~C before
use.
7.1.6. PRODUCTION OF MONOCLONAL ANTI-IDIOTYPIC
ANTIBODIES (Ab2) SPECIFIC FOR MG-21 (Abl)
Two 8-week-old BALB/c female mice were immunized
intraperitoneally (i.p.) with 100 ~g of MG-21-KLH conjugate
in complete Freund's adjuvant. Two weeks later, the same
amount of conjugate was given i.p. in incomplete Freund's
adjuvant. Another 8 weeks later, the mice were boosted
again with conjugate in saline. Four days after the last
immunization, spleens were removed and the harvested cells
were fused with NS-l mouse myeloma cells by use of
25 polyethylene glycol. Hybridomas secreting anti-idiotypic
antibodies specific for MG-21 were selected, grown in HAT
medium, and cloned using established procedures (Yeh, M.-Y.,
- et al., 1979, Proc. Natl. Acad. Sci. U.S.A. 76:2927-2931).
7.1.7. mAb (Abl) BINDING INHIBITION ASSAY
Enzyme-linked immunosorbent assays (ELISAs) of antibody
binding were used for the initial screening of hybridomas
secreting anti-idiotypic antibodies. In this assay,
hybridoma culture supernatants were tested for inhibition of
- ~339816
-87-
binding of MG-21 (~Abl") to M-2669 cells, as evidence for
the presence of anti-idiotypic antibodies ("Ab2"). M-2669
melanoma cells, seeded into polyvinyl chloride plates (105
cells/well) and fixed with 0.5% glutaraldehyde, were used as
targets. One hundred ul of mAb MG-21 (5 ~g/ml) were mixed
with equal volumes of hybridoma supernatants, incubated at
4~C for 2-4 hours, and then added to the target cells.
After another incubation for 1 hour at 37~C, the plates were
washed three times with 0.05% Tween-20 in PBS (PBS-Tween
buffer). The cells were then incubated with 100 ul of goat
- anti-mouse IgG antibody-peroxidase conjugate (Boehringer
Mannheim Biochemicals, Indianapolis, IN) diluted 1/10,000 in
PBS-Tween buffer at 37~C for 30 minutes, and washed again.
One hundred ul of o-phenylenediamine (OPD) containing 0.015
H2O2 in citrate phosphate, pH 5.0, were dispensed in each
well as substrate for peroxidase. About 3-5 minutes later,
100 ul of 1.3 N H2SO4 were added for blocking the enzyme-
substrate reaction. Absorbance was measured at 492 nm/630
nm dual wavelength for each well in a GSC microplate reader
(Genetic System Corp., Seattle, WA).
To investigate the dose-dependency of the inhibitory
effect of anti-idiotypic antibody (Ab2) on the binding of
MG-21 (Abl) to cells and GD3 antigen, 100 ul of a fixed
concentration (5 ~g/ml) of MG-21 were mixed with 100 ul of
25 various concentrations of appropriate anti-idiotypic or
control antibodies, incubated at 37~C for 1 hour, and then
added to wells precoated with M-26~9 cells (105/well) or
purified GD3 antigen (200 ng/50 ~l/well). The remaining
procedures were the same as those above. ~ -~
7.1.8. DETERMINATION OF IMMUNOGLOBULIN ISOTYPE
Goat antisera to the specific class of mouse immuno-
globulin were used (Southern Biotechnology Assoc.,
Birmingham, AL). Fifty ~1 of each such anti-serum was
35 diluted in PBS (1 ~l/ml), plated into 96-well plates
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(Dynatech, Alexandria, VA), and incubated overnight at 4~C.
The plates were washed once with PBS-Tween buffer, and then
incubated for 1 hour at room temperature with 100 ~1 per
well of RPMI 1640 medium containing 15% fetal calf serum
(FCS). After washing, 50 yl of hybridoma spent culture
medium were added, followed by incubation of the plates at
room temperature for 1 hour and one washing with PBS-Tween
buffer. Subsequently, 50 ~1 of goat anti-mouse IgG antibody
conjugate diluted 1/10,000 in PBS-Tween buffer were added to
0 each well. The plates were incubated for 30 minutes at 37~C
and washed five times with PBS-Tween buffer, after which
100 ~1 of OPD substrate were added to each well. Five
minutes later, the reaction was stopped by adding 100 ~1 of
1.3 N H2SO4, and the absorbance was measured in a GSC
15 microplate reader.
7.1.9. ANTI-IDIOTYPIC ANTIBODY (Ab2) BINDING ASSAY
A binding assay was used to determine whether the
ability of Ab2 to inhibit the binding of MG-21 (Abl) was
20 specific. Purified MG-21 at 0.5 ~g/100 ~1 was added to coat
each well of 96-well polyvinyl chloride plates (Costar,
Cambridge, MA), after which 200 ~1 per well of RPMI 1640
medium containing 15% FCS was added as a "blocker" to
prevent antibody binding to plastic. After washing, 100 ~1
25 of purified Ab2 or control immunoglobulin Pl.17 were added
at various concentrations. The plates were incubated at
37~C for 30 minutes, and then washed three times with PBS-
Tween buffer. One hundred ~1 of peroxidase-conjugated
rabbit anti-mouse IgG which had the same isotype as the Ab2
30 were then added after dilution in PBS-Tween buffer. After
incubation for 30 minutes at 37~C, followed by extensive
washing, 100 ~1 of OPD substrate were added and incubated
for 3-5 minutes in the dark. Finally, 100 ~1 of 1.3 N H2SO4
were added. The plates were read by a GSC microplate
35 reader.
- 1339816
-89-
7.1.10. RADIOIOlD~ATION OF ANTIBODY AND
DIRECT I-Ab2 BINDING ASSAY
One hundred ~l of purified mAb were incubated with 500
uCi Na125I (Amersham Corporation, Arlington Heights, IL) and
40 ug of chloramine-T in 500 ~l of PBS at 4~C for three
minutes. Labeled mAb was separated from free 125I by gel
filtration on a column of Sephadex G-25; the specific
activity was approximately 4 x 106 cpm/~g. The labeled mAb
was diluted in 15% FCS in PBS before use.
For a direct 125I-Ab2 binding assay, 100 ~l of various
purified mAb (50 ~g/ml) in 15 mM NaHCO3 at pH 9 were plated
into 96-well plates (Dynatech, Alexandria, VA) at 4~C
overnight. After washing, the wells were blocked by
incubation overnight at 4~C with 200 ~l of RPMI 1640 medium
containing 15% FCS. The plates were washed three times with
PBS-Tween buffer, and 2.5 x 105 cpm of labeled Ab2 in 100 ~l
of 30% FCS were added to each well and incubated at room
temperature for 1 hour. After thorough washing, the bound
radioactivity was dissolved in 100 ~l of 2 M NaOH,
transferred to test tubes, and counted-in a Gamma counter
(Beckman, Irvine, CA).
7.1.11. ANTIBODY-FITC CONJUGATION
Fluorescein isothiocyanate (FITC) was conjugated to mAb
MG-21 as described by Goding (Goding, J.W., 1976,
25 J. Immunol. Methods 13:215-226). Briefly, 2 mg of purified
MG-21 was dialyzed overnight in 0.2 M carbonate/bicarbonate
buffer, pH 9.5. FITC (Molecular Probes Inc., Junction City,
OR) dissolved in dimethyl sulfoxide (1 mg/ml) was added at a
ratio of 40 ~g FITC/mg of antibody. The mixture was
30 incubated at 37~C for 45 minutes, after which conjugated mAb
was separated from free FITC by passage through a G-25
Sephadex column equilibrated with PBS containing 0.1% NaN3.
The fluorescein/antibody conjugation ratio was about 3.5 to
~; 133~816
--so--
4Ø The conjugated mAb was stored at -20~C in PBS
containing 1% bovine serum albumin (BSA).
7.1.12. FLUORESCENCE ACTIVATED CELL SORTER ANALYSIS
For binding inhibition analysis using a fluorescence
activated cell sorter (FACS), 100 ul of FITC-conjugated MG-
21 were incubated for 30 minutes at 37~C with 100 ~1 of
various concentrations of purified Ab2 or control antibodies
in 10% normal mouse serum. The antibody mixtures were then
added to test tubes containing 1 x 106 paraformaldehyde-
fixed M-2669 cells in 100 ~1 of PBS. After 30 minutes of
- incubation, the cells were washed two times with PBS, and
then analyzed with a Coulter Epics C fluorescence activated
cell sorter (Coulter Corporation, Hialeah, FL). Data were
expressed as linear fluorescent equivalence (LFE), which
represents relative fluorescent intensity.
7.1.13. COMPLEMENT-DEPENDENT CYTOTOXICITY ASSAY
To test whether Ab2 specific for MG-21 can inhibit
20 complement-dependent cytotoxicity (CDC) of mAb MG-21, a 4-
hour Cr-release assay (Hellstrom, I., et al., 1985, Proc.
Natl. Acad. Sci. U.S.A. 82:1499-1502) was used. Briefly,
106 target cells were labeled with 100 ~Ci of 51Cr for 2
- hours at 37~C. After labelling, the cells were washed three
25 times, resuspended in RPMI 1640 medium containing 15~ FCS,
and 20,000 labeled cells, suspended in 45 ~1 of RPMI 1640
medium, and seeded into each well of Microtiter V-bottom
plates (Dynatech Laboratories, Alexandria, VA). Various
concentrations of purified M6~21 were combined with ' -
30 different concentrations of purified Ab2 (or control mAb~ to
test for inhibition of CDC. They were added in 90 ~1 per
well, followed by 65 ~1 of undiluted unheated human serum
per well. After incubation for 4 hours at 37~C, the plates
were centrifuged at 400 x g, 100 ~1 of supernatants from
35 each well were removed, and the level of radioactivity was
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determined by a Gamma counter (Beckman, Irvine, CA).
Spontaneous release was defined as the cpm released into the
medium from target cells which had not been exposed to
antibody or complement, and total release was estimated as
the cpm released from target cells that were osmotically
lysed. Percent cytotoxicity was calculated by the following
formula:
Experimental - Spontaneous
release release
Percent cytotoxicity = x 100
Total - Spontaneous
release release
7.1.14. ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY ASSAY
To test whether Ab2 can inhibit the antibody-dependent
cellular cytotoxicity (ADCC) of mAb MG-21, a 4-hour release
assay was employed. PBL from healthy human subjects were
used as effector cells. They were separated on Ficoll-
Hypaque and prescreened for low natural killer (NX) cell
activity. Only lymphocytes with low NK activity (less than
10% Cr-release over four hours) were used. After labeling
of the target cells, they were plated (2 x 104 cells/50 ~l)
into Microtiter plates as for the CDC assay. Fifty ~l of
purified MG-21 and 50 ~l of purified Ab2 (or control) mAb
were added at various concentrations, followed by 2 x 106
lymphocytes per well in 50 ~l of medium; the ratio of
lymphocytes to target cells was 100:1. The mixtures were
incubated for 4 hours at 37~C in a 6% CO2 in air atmosphere.
Subsequently, the plates were centrifuged, and 100 ~l of
supernatants were transferred from each well for radioactiv-
ity measurement. Percent cytotoxicity was calculated as for
30 the CDC assay-
- - 133~8~5
-92-
7.1.15. COMPETITION ASSAY FOR DETECTING ANTI-MG-21
ANTIBODIES IN PATIENT SERA
A competition assay using monoclonal anti-idiotypic
antibody (Ab2) as a probe was developed for detecting anti-
MG-21 antibodies in the sera of patients treated with mAb
MG-21. Briefly, 100 ~1 of Ab2 (5 ~g/ml) in 15 mM NaHCO3
buffer, pH 9, were added to each well of Falcon pro-bind
assay plates (Becton Dickinson, Oxnard, CA), incubated at
room temperature for 1 hour, and subsequently washed with
PBS-Tween buffe,r. Seventy-five ~1 of mAb MG-21'(1 ~g/ml)
were preincubated for 45 minutes with equal volumes of sera
from a patient or with pooled normal human serum; the sera
were diluted 1:2.5, 1:5 and 1:10 in PBS. Subsequently,
- 100 ~1 of the mixtures were added to the Ab2-precoated
plates. After an additional incubation for 30 minutes at
room temperature, the plates were washed twice with PBS-
Tween buffer. One hundred ~1 of rabbit anti-mouse IgG3
antibody-peroxidase conjugate (Zymed Laboratories rnc.,
South San Francisco, CA), diluted 1:1,000 in PBS-Tween
buffer, were added and incubated for 30 minutes at room
2 temperature. After three washes with PBS-Tween buffer, the
wells were filled with 100 ~1 of OPD substrate containing
0.015% H2O2 in citrate phosphate, pH 5Ø Five minutes
later, 100 ~1 of 1.3 N H2SO4 were added, and the plates were
read by a GSC microplate reader. Percentage of inhibition
of MG-21 binding to mAb 2Cl was calculated by the following
formula:
OD in the presence
of patient serum
Percent inhibition = (1- ) x 100
OD in the presence
of normal human serum
Serum samples from three of eight patients with
advanced melanoma, which were enrolled in a phase I trial of
mAb MG-21, were tested by this assay. Briefly, MG-21 in 5%
1339816
--93--
human serum albumin had been infused to these patients over
a 4-6 hour time period with the infusions being repeated
daily for 7 days. The serum samples were taken prior to
treatment and at various intervals after, and were stored at
5 -80~C until analysis.
- 7.2. RESULTS
7.2.1. SELECTION OF HYBRIDOMAS
Spleen cells from mice immunized with MG-21 were fused
with NS-1 cells to generate hybridomas which could produce
mAb to idiotypic determinants on MG-21; the latter mAb are
referred to as Ab2. Two weeks after fusion, hybridoma
supernatants were tested for antibodies inhibiting the
15 binding of MG-21 to M-2669 cells. One hybridoma, 2Cl, which
had such activity, was cloned and expanded. As shown in
Figure 7, supernatant of the 2Cl hybridoma strongly
inhibited the binding of MG-21 to M-2669 cells but did not
itself bind to M-2669 cells. Supernatant from the NS-l
20 myeloma was used as a control and did not inhibit the
binding of MG-21.
Hybridoma 2Cl grew as an ascites tumor, when inoculated
intraperitoneally into pristane-primed BALB/c mice. mAb 2C1
was found to be an IgG2a by solid phase enzyme immunoassay
25 with goat anti-mouse Ig specific class antisera.
As shown in Figure 8, mAb 2Cl gave strong binding to
MG-21 when tested in an ELISA at concentrations between
0.08 ~,g/ml and 2 ~g/ml with significant binding still being
observed at 6.4 ng/ml. No binding was seen to Pl.17, an
30 IgG2a myeloma protein (Fig. 8).
7.2.2. mAb 2Cl IS SPECIFIC FOR MG-21
To determine the degree of specificity of 2Cl for MG-
21, a binding assay was performed using 12 I-labeled 2C1.
35 Six mouse mAbs, which had been generated against a variety
~33~816
--94--
of human tumors, were included as controls. As shown in
Figure 9, mAb 2C1 bound strongly to MG-21 but not to any of
the six control mAbs, two of which (2A-14 and 96.5) were
specific for melanoma-associated antigens that were
5 different from the epitope recognized by MG-21. Of these
two mAb, 2A-14 reacts with an epitope of the GD3 antigen
which is different from that recognized by MG-21.
7.2.3. mAb 2C1 (Ab2) INHIBITS THE BINDING OF
MG-21 (Abl) TO M-2669 CELLS AND GD3
- ANTIGEN IN A DOSE-DEPENDENT MANNER
A binding assay was used to titrate how much mAb 2Cl
was required as Ab2 to inhibit the binding of MG-21 (Abl) to
M-2669 cells. As shown in Table IX, mAb 2Cl completely
inhibited the binding of MG-21 to M-2669 cells when present
15 at a concentration equal to or greater than that of MG-21;
two control immunoglobulins, mAb 26.8 and P1.17, gave no
significant inhibition.
1339816
TABLE IX
DOSE-DEPENDENT INHIBITORY EFFECT OF mAb 2Cl
ON THE BINDING OF mAb MG-21 TO M-2669 CELLS
Antibody 1 Antibody 2 Absorbance
concentration concentration at 492/630 nm
MG-21 (2.5 ~g/ml) None 0.324 + 0.0152
10 MG-21 (2-5 ~g/ml) mAb 2Cl (10 ~g/ml) 0.071 + 0.029*
MG-21 (2.5 ~g/ml) mAb 2Cl (5 ~g/ml) 0.043 + 0.026*
MG-21 (2.5 ~g/ml) mAb 2Cl (2.5 ~g/ml) 0.053 + 0.002*
MG-21 (2.5 ~g/ml) mAb 2Cl (1.25 ~g/ml) 0.129 + 0.019**
MG-21 (2.5 ~g/ml) mAb 2Cl (0.625 ~g/ml) 0.156 + 0.023**
15 MG-21 (2.5 ~g/ml) mAb 26.8 (10 ~g/ml) 0.305 + 0.007
MG-21 (2.5 ~g/ml) mAb 26.8 (5 ~g/ml) 0.329 + 0.035
MG-21 (2.5 ~g/ml) mAb 26.8 (2.5 ~g/ml) 0.315 + 0.024
- MG-21 (2.5 ~g/ml) mAb 26.8 (1.25 ~g/ml) 0.337 + 0.035
MG-21 (2.5 ~g/ml) mAb 26.8 (0.625 ~g/ml) 0.330 + 0.036
20 MG-21 (2.5 ~g/ml) Pl.17 (10 ~g/ml) 0.322 + 0.049
MG-21 (2.5 ~g/ml) Pl.17 (5 ~g/ml) 0.368 + 0.019
MG-21 (2.5 ~g/ml) Pl.17 (2.5 ~g/ml) 0.331 + 0.019
MG-21 (2.5 ~g/ml) Pl.17 (1.25 ~g/ml) 0.318 + 0.035
MG-21 (2.5 ~g/ml) Pl.17 (0.625 ~g/ml 0.318 + 0.031
lData are presented as mean + SE.
2Control
Differences statistically significant from control without Ab2,
30 *, P less than 0.01; **, P less than 0.025.
133~8~6
In a parallel study, purified GD3, which is the
antigen recognized by MG-21, was used instead of M-2669
cells. mAb 2Cl inhibited the binding of MG-21 to the
purified GD3 ganglioside in a dose-dependent manner
(Table X).
TABLE X
DOSE-DEPENDENT INHIBITORY EFFECT OF mAb 2C1 ON THE
BINDING OF mAb MG-21 TO GD3 GANGLIOSIDE ANTIGEN
Antibody 1 Antibody 2 Absorbance
concentration concentration at 490 nm/630 nm
MG-21 (2.5 ~g/ml) None 0.486 + 0.092
MG-21 (2.5 ~g/ml) mAb 2C1 (25 ~g/ml) 0.085 + 0.006*
MG-21 (2.5 ~g/ml) mAb 2C1 (12.5 ~g/ml) 0.073 + 0.010*
MG-21 (2.5 ~g/ml) mAb 2C1 (6.25 ~g/ml) 0.075 + 0.008*
MG-21 (2.5 ~g/ml) mAb 2C1 (3.125 ~g/ml) 0.379 + 0.009
20 MG-21 (2.5 ~g/ml) mAb 2C1 (1.563 ~g/ml) 0.502 + 0.075
MG-21 (2.5 ~g/ml) mAb 2C1 (0.782 ~g/ml) 0.482 + 0.033
MG-21 (2.5 ~g/ml) mAb 2C1 (0.391 ~g/ml) 0.533 + 0.078
lData are presented as mean + SE.
Control
Differences statistically significant from control without Ab2,
*, P less than 0.01.
The inhibitory effect of mAb 2Cl on the binding of MG-
21 to M-2669 cells was confirmed by FACS analysis using
FITC-conjugated MG-21. Starting with 40 ~g/ml of FITC-
conjugated MG-21, which approximately corresponded to the
35 saturation concentration, excess amounts of mAb 2Cl or
_97_ ~3398~6
control antibodies were added to M-2669 cells. Figure 10
shows that 2C1 completely inhibited the binding of FITC-
conjugated MG-21 to the tumor cells, while control
antibodies had no effect.
7.2.4. ANTIBODY 2C1 INHIBITS THE CDC AND ADCC
A~llVl~lY OF MG-21 AGAINST M-2669 CELLS
Previous experiments have shown that MG-21 gives strong
CDC and ADCC with GD3-positive melanoma cells (Hellstrom,
I., et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:1499-
1502). We now tested whether 2C1 had any effect on these
activities. As shown in Table XI, CDC of MG-21 was
completely abrogated by adding mAb 2C1 at a concentration
greater than that of MG-21, but not by adding either of two
control antibodies, mAb 26.8 or P1.17.
-98- l 33~8 1 6
TABLE XI
INHIBITION OF CDC OF mAb MG-21
AGAINST M-2669 CELLS BY mAb 2C1
Antibody 1 Antibody 2 Cytotoxicity
Final Concentration Final Concentration %
MG-21 (5 ~g/ml) None 100 (Control)
MG-21 (5 ~g/ml) mAb 2C1 (10 ~g/ml) 2*
MG-21 (5 ~g/ml) mAb 2C1 (1 ~g/ml) 84
MG-21 (5 ~g/ml) mAb 2C1 (0.1 ~g/ml) 100
MG-21 (5 ~g/ml) mAb 26.8 (10 ~g/ml) 100
MG-21 (5 ~g/ml) mAb 26.8 (1 ~g/ml) 100
MG-21 (5 ~g/ml) P1.17 (10 ~g/ml) 100
MG-21 (5 ~g/ml) Pl.17 (1 ~g/ml) 100
MG-21 (1 ~g/ml) None 64 (Control)
MG-21 (1 ~g/ml) mAb 2C1 (10 ~g/ml) 1*
MG-21 (1 ~g/ml) mAb 2C1 (1 ~g/ml) 0*
MG-21 (1 ~g/ml) mAb 2Cl (0.1 ~g/ml) 56
MG-21 (1 ~g/ml) mAb 26.8 (10 ~g/ml) 69
MG-21 (1 ~g/ml) mAb 26.8 (1 ~g/ml) 60
MG-21 (1 ~g/ml) Pl.17 (10 ~g/ml) 67
MG-21 (1 ~g/ml) P1.17 (1 ~g/ml) 69
1Cytotoxicity was determined in a 4-hour 51Cr-release assay
using normal human serum as a source of complement. No
cytotoxicity was seen with human serum alone. Antibodies
30 alone did not give any cytotoxicity. Significance was
determined by student's t-test and is indicated by *, P less
than 0.01.
133q816
Antibody 2C1 also completely inhibited the ADCC activity of
MG-21 against M-2669 cells when its concentration was
greater than that of MG-21, while no significant inhibition
was seen with the two control antibodies (Table XII).
-loo- 1 3398 1 6
TABLE XII
INHIBITION OF ADCC OF mAb MG-21
-- AGAINST M-2669 CELLS BY mAb 2C1
Antibody 1 Antibody 2 Cytotoxicity
Final Concentration Final Concentration
MG-21 (5 ~g/ml) None 40 (Control)
MG-21 (5 ~g/ml) mAb 2C1 (10 ~g/ml) 4*
MG-21 (5 ~g/ml) mAb 2C1 (1 ~g/ml) 16*
MG-21 (5 ~g/ml) mAb 2Cl (0.1 ~g/ml) 33-
MG-21 (5 ~g/ml) mAb 26.8 (10 ~g/ml) 44
MG-21 (5 ~g/ml) mAb 26.8 (1 ~g/ml) 40
MG-21 (5 ~g/ml) P1.17 (10 ~g/ml) 34
MG-21 (5 ~g/ml) P1.17 (1 ~g/ml) 39
MG-21 (1 ~g/ml) None 32 (Control)
MG-21 (l ~g/ml) mAb 2C1 (10 ~g/ml) 7*
MG-21 (1 ~g/ml) mAb 2C1 (1 ~g/ml) 3*
MG-21 (1 ~g/ml) mAb 2C1 (0.1 ~g/ml) 16*
MG-21 (1 ~g/ml) mAb 26.8 (10 ~g/ml) 26
MG-21 (1 ~g/ml) mAb 26.8 (1 ~g/ml) 23
MG-21 (1 ~g/ml) P1.17 (10 ~g/ml) 28
MG-21 (1 ~g/ml) P1.17 (1 ~g/ml) 23
1Cytotoxicity was determined in a 4-hour 51Cr-release assay
using normal human peripheral blood lymphocytes as
effectors. The ratio of effector cells to target cells was
100:1. Antibodies alone gave no cytotoxicity and lympho-
30 cytes alone gave 6.9~ cytotoxicity. Significant differencescompared to control without Ab2 were calculated by student's
t-test, *, P less than 0.01.
-lol- 1339816
7.2.5. DETECTION OF ANTI-MG-21 ANTIBODIES IN
PATIENT SERA USING mAb 2Cl AS A PROBE
Since mAb 2Cl is specific for MG-21, it can be used as
a reagent for detecting human anti-MG-21 antibodies in the
sera of patients treated with MG-21. A competition assay
was developed (see Section 7.1.15., supra), by which we
tested whether sera from any of 3 patients injected with
MG-21 inhibited the binding of MG-21 to mAb 2Cl. As shown
in Table XIII, sera obtained from all these three patients
17, 18 or 21 days, respectively, (or later) after adminis-
tration of MG-21, strongly inhibited the binding of MG-21 to
mAb 2Cl with 20-83%. The pretreatment sera gave less than
13% inhibition as compared to pooled normal human serum.
-102- l 33~8 1 6
TABLE XIII
INHIBITION OF BINDING OF mAb MG-21 TO mAb 2C1
BY SERA FROM PATIENTS RECEIVING mAb MG-21
Days After % Inhibition
Patient Dose of MG-21/day Start of mAbof Binding
No. for 7 Days*Treatment
1:2.5** 1:5 1:10
1 5 mg/M /day 0 0 0 0
14 58 53 33
18 60 58 43
24 65 62 47
28 83 87 85
2 5 mg/M2/day 0 10 12 0
21 55 46 31
28 53 32 25
49 62 46 20
61 59 32 27
3 50 mg/M /day 0 6 13 2
18 28 21 20
28 30 30 26
31 15
66 77 60 45
127 73 50 43
Patient received 4-6 hour infusion of MG-21 daily for 7
30 days in dose noted. Serum samples were drawn at various
times after the start of treatment.
**Serum samples were diluted 1:2.5, 1:5 and 1:10 in PBS.
1339~16
-103-
The anti-idiotypic mAb antibody 2Cl, which we describe
here, recognizes an idiotype specific to a human melanoma-
associated GD3 ganglioside antigen. mAb 2C1 was shown to
bind to mAb MG-21 even at a low concentration (0.08 ~g/ml),
but not to other mAb of the same or different isotypes. It
inhibited, in a dose-dependent manner, the binding of MG-21
to the GD3 ganglioside antigen as well as to GD3-positive
M-2669 melanoma cells. Furthermore, mAb 2C1 completely
abrogated the CDC and ADCC activities of mAb MG-21, as long
as its concentration was greater than that of MG-21.
Using mAb 2C1 as a probe, we have developed an assay
for human anti-MG-21 antibodies in the sera of patients
treated with MG-21. Analogous assays may be developed for
other types of anti-tumor antibodies. Since human
antibodies binding to MG-21 were present at short times
(14-21 days) after treatment of patients with MG-21, unless
such antibodies are highly effective in inducing an immune
response leading to tumor rejection, procedures minimizing
such antibody deveiopment can be envisioned for use when
prolonged treatment of patients by administered anti-tumor
antibodies is desired.
8. MONOCLONAL ANTI-IDIOTYPIC ANTIBODIES
RELATED TO THE P97 MELANOMA ANTIGEN
We have made monoclonal anti-idiotypic antibodies (Ab2)
related to the p97 antigen of human melanoma. This was
accomplished by immunizing BALB/c mice with 96.5, a
monoclonal antibody (mAb) specific for epitope p97a,
hybridizing the mouse spleen cells with NS-l myeloma cells,
and selecting for hybridomas which made antibody that bound
to Fab fragments prepared from mAb 96.5 (Fab 96.5). The Ab2
were tested for binding to mAb 96.5 and to mAb defining
other epitopes of the pg7 antigen, as well as for their
ability to inhibit the binding between mAb 96.5 and p97.
Three monoclonal Ab2 were identified which competitively
- -104- 1 3 3 q 81 6
inhibited the binding between p97 and mAb 96.5. When
injected into either BALB/c or C3H/HeN mice, two of them
induced Ab3 which expressed the same idiotype as mAb 96.5
and which were specific for p97. These two Ab2 thus behaved
as ~internal images" of p97.
8.1. MATERIALS AND METHODS
8.1.1. ANIMALS
0 Approximately 6-8 week-old female BALB/c and C3H/HeN
mice were used throughout this study.
8.1.2. HUMAN MELANOMA CELLS
Line SK MEL-28 was used as a source of p97 antigen-
positive target cells. Each SK-MEL 28 cell expresses, at
its surface, approximately 400,000 molecules of p97.
8.1.3. MOUSE MELANOMA CELLS
Cells from the B16 (C57BL) mouse melanoma, which had
been transfected with the p97 gene (Plowman, G.D., 1986,
Characterization and expression of the melanotransferrin
(p97) gene, Ph.D. dissertation, University of Washington)
were used to produce soluble p97 antigen. We also employed
a line of cells (2A) from the C3H/HeN mouse melanoma line
K-1735-M2 (Fidler, I.J. and Hart, I.R., 1981, Cancer Res.
41:3266-3267) which, after transfection with the gene for
p97, express approximately 1o6 p97 molecules per cell. The
K-1735-M2 cells, which entirely lack p97, are referred to as
parental (par) cells, since the 2A line was derived from
30 them.
8.1.4. ANTIBODIES
Seven mAb to the human melanoma-associated antigen p97
(Brown, J.P., et al., 1981, J. Immunol. 127:539-546) were
35 used in the study. Three epitopes of p97 have been defined
3 3 9 8
-105-
by using these mAb in competitive binding inhibition assays,
-namely p97a (by mAb 96.5 and 4.1), pg7b (by mAb 118.1,
133.1, and 133.3), and pg7c (by mAb 8.2 and 133.2).
Hybridoma 96.5, which produces a mAb to epitope p97a, was
- 5 obtained by fusing spleen cells from an immunized BALB/c
mouse with NS-l myeloma cells (id.). Fab fragments were
made from mAb 96.5 by papain digestion and are referred to
as Fab 96.5 (id.). mAb F6 is an IgG2a specific for a
proteoglycan antigen on human melanoma cells. It was used
to prepare Fab fragments (referred to as Fab F6) which were
employed as controls.
In order to raise anti-Id, BALB/c mice were injected
subcutaneously with 100 ~g of purified mAb 96.5 which had
- been conjugated with keyhole limpet hemocyanin (KLH), and
subsequently mixed with Freund's complete adjuvant (Bacto
H37Ra, Difco Labs, Detroit, MI). One month later, they were
injected intraperitoneally with the same amount of KLH-
conjugated mAb 96.5 in Freund's incomplete adjuvant (Difco).
The mice were subsequently injected with mAb 96.5 in saline
-20 at 2-week intervals for 1 or 2 more times. Three days after
the last injection, they were killed and a spleen cell
suspension was prepared and fused with NS-l mouse myeloma
cells, using standard techniques (Kohler, G. and Milstein,
C., 1975, Nature 256:495-497).
8.1.5. SCREENING OF HYBRIDOMAS
Primary screening was performed by an ELISA (Kohler, G.
- and Milstein, C., 1975, Nature 256:495-497). Fab 96.5, at a
concentration of 4 ~g/ml in ~osphate buffered saline (PBS),
30 were plated onto Immunolon plates (Dynatech Laboratories,
Chantilly VA). The next day, the plates were washed with
PBS containing 0.05% Tween 20 and "blockedn by incubation
for 1 hour with PBS containing 0.05~ Tween and 1~ fetal calf
serum (FCS). Supernatants (50 ~1) from each well with
growing hybridoma cells were added. One hour later, a
Trade Mark
r ~
1339816
- -106-
mixture was added, containing goat anti-mouse IgGl (Zymed,
San Francisco, CA) which had been coupled with horseradish
peroxidase (HRP), 0.05% Tween 20, and 1~ FCS in PBS. After
a 1 hour incubation, antibody binding to the plated Fab 96.5
was detected by adding O-phenylene diamine (OPD) according
to the directions of the manufacturer (Zymed). The plates
were read in an automatic microplate reader (Genetic Systems
Corporation, Seattle, WA) at an absorbance of 492 nm/630 nm.
Fab fragments from mAb F6 were employed as controls, and
only those hybridomas which made mAb that bound to Fab 96.5,
but not to Fab F6, were retained for further testing. The
employed screening procedures detected only hybridomas
making IgG1 antibodies.
To further test supernatants for activity, an assay was
used in which the supernatants were diluted two-fold,
combined with one part of mAb 96.5, and added to Immunolon
wells onto which Fab 96.5 had been plated. After addition
of goat anti-mouse IgG and OPD, the ability of the added mAb
96.5 to prevent binding of the supernatants to the plated
Fab was assessed. This assay was also employed to test
anti-Id for binding to mAb defining p97 epitopes other than
p97a .
Hybridomas which made antibodies binding to Fab 96.5
but not to Fab F6 were cloned twice by limiting dilution,
after which they were expanded and injected into pristane-
primed BALB/c mice for ascites production.
8.1.6. STUDIES ON PURIFIED ANTI-IDIOTYPIC ANTIBODIES
Ab2 were purified by precipitation with saturated
30 ammonium sulfate (Mishell, B. and Shiigi, S., 1979, ln
Select Methods in Cellular Immunology, W.H. Freemen & Co.,
pp. 278-281). To identify anti-Id which could interfere
with the binding of mAb 96.5 to p97, SK MEL-28 melanoma
cells were plated at 104 cells/well. Purified Ab2 were
35 mixed with mAb 96.5 (1 ~g/ml). Inhibition of binding of mAb
- 1 33981 6
-107-
96.5 to the melanoma cells was detected by adding a goat
anti-mouse HRP conjugate as above; a few tests of this type
were also performed on hybridoma supernatants.
To study whether the purified Ab2 could block the
antigen binding site of Fab 96.5, various concentrations of
Ab2 were added to wells of Immunolon plates onto which Fab
96.5 had been plated. p97 antigen isolated from transfected
B16 mouse melanoma cells was radioiodinated (Rose, T.M., et
al , 1986, Proc. Natl. Acad. Sci. U.S.A. 83:1261-1265), 2 x
cpm of labelled p97 were added, and the number of counts
bound per well was determined.
8.1.7. COMPETITION OF RADIOLABELLED P97 FOR
BINDING TO Fab FRAGMENTS OF mAb 96.5
Various concentrations of purified Ab2 were mixed with
a constant amount of 125I-labelled p97 antigen, and the
mixtures were added to plates which had been coated with Fab -
96.5. After one hour, the plates were washed, 2 N NaOH was
added, and the contents of the wells were counted in a gamma
counter.
8.1.8. SEARCHES FOR AB3 IN VIVO
BALB/c and C3H/HeN female mice, 6-8 weeks old, were
injected intraperitoneally (i.p.) with 50 ~g of Ab2
conjugated to KLH and mixed with complete adjuvant. Five
days later, they were boosted by an injection of Ab2 in
incomplete adjuvant and subsequently injected with Ab2 in
saline at 5-day intervals. After a total of 4 and 6
immunizations, respectively, the mice were bled. They
continued to be boosted at 2-week intervals for several
weeks. In some cases, the immunization protocol was
initiated at 2-week intervals with 4-5 boosts.
Sera from the immunized mice were titered for the
presence of antibodies binding to Ab2 and referred to as
Ab3. This was done by mixing diluted sera with Ab2, and
-108- 1339816
adding the mixture onto Immunolon plates which had been
coated with Fab 96.5 (as Abl), after which goat anti-mouse
IgG1-HRP and OPD were added. Data were expressed as percent
inhibition of the binding of Ab2 to Abl. They were
calculated by determining the O.D. (optical density) value
for (Ab3 + Ab2), dividing it by the O.D. value for Ab2
alone, and subtracting the quotient from 100.
The sera were also tested for Ab3 binding to the p97
antigen. Purified p97 was plated onto Immunolon plates at 5
0 ug/ml in PBS and left overnight. After blocking, diluted
sera were added, followed by a goat antiserum to mouse
immunoglobulin which reacted with IgG, IgM, and IgA, and was
coupled to HRP.
A solid phase inhibition assay was also employed, in
which the mouse sera were mixed with 2A mouse melanoma cells
which express p97 at the cell surface, or with par mouse
melanoma cells, which do not. The mixture was first
incubated for 1 hour and then added to Immunolon plates
coated with the p97 antigen. As above, binding was detected
by adding OPD in the presences of anti-mouse-HRP conjugates.
8.2. RESULTS
8.2.1. GENERATION OF Ab2 BINDING TO
IDIOTYPIC DETERMINANTS ON mAb 96.5
BALB/c mice were immunized with mAb 96.5, their spleen
cells fused, and hybridoma supernatants screened for IgGl
antibodies to mAb 96.5, as described supra. Approximately
3,000 hybridomas were obtained from 8 different fusions.
Supernatants from 70 of these hybridomas were found to bind
to Fab 96.5 and not to the control Fab F6; most of the
hybridomas from which they were derived, therefore, were
presumed to make Ab2. Seven of these hybridomas were
cloned, and the mAb which they made were purified and tested
for binding to Fab 96.5. As shown in Figure 11, mAb made by
- 1 3398 1 6
- --109--
all the seven hybridomas bound to Fab 96.5, although there
was variation between Ab2 in the binding values observed at
high antibody concentration. None of the mAb bound to Fab
F6.
8.2.2. TESTS ON Ab2 SPECIFICITY FOR THE
ANTIGEN-BINDING SITE OF mAb 96.5
We investigated whether any of the seven Ab2 on which
data are presented in Figure 11 identified the antigen-
binding site of mAb 96.5. First, we measured the ability of
the mAb to inhibit the binding of mAb 96.5 to the p97
antigen expressed by SK MEL-28 cells; this was carried out
as described in Section 8.1.6. Three of the seven Ab2, #3,
#5 and #7, strongly inhibited this binding, while two Ab2
(#4 and #6) gave a weak inhibition, and two Ab2 (#1 and #2)
gave no inhibition (Fig. 12).
Second, we studied the ability of the seven Ab2 to
compete with soluble p97 for binding to mAb 96.5. Various
dilutions of each Ab2 were mixed with radioiodinated p97,
and the binding of p97 to Fab 96.5 was determined in a solid
phase assay (Fig. 13). Anti-Id #3, #5, and #7 competed with
radioiodinated p97 while the four other anti-Id (#1, #2, #4,
#6) did not compete. The results were thus similar to those
presented in Figure 12.
Third, we demonstrated that the same three Ab2 with the
ability to compete with p97, namely #3, #5, and #7, can
block the antigen-binding sites of Fab 96.5 so as to
decrease 50-60% of the binding of radioiodinated p97 to Fab
96.5 (Fig. 14). One Ab2, #2, gave 25% inhibition of this
binding, and the remaining three Ab2 (#1, #4, #6) gave 0-10%
inhibition.
The data thus suggested that three Ab2, #3, #5, and #7,
were capable of mimicking the p97a epitope as an ninternal
imagen. However, the possibility still existed that steric
hindrance-was responsible for the observed effects. We
-llo- 1 33~81 6
therefore tested the ability of the Ab2 to induce an Ab3
response in vivo, as described in Section 8.2.4, infra.
8.2.3. ANALYSIS OF THE BINDING OF Ab2 TO A SERIES OF
mAb WHICH SPECIFY p97 EPITOPES OTHER THAN p97a
Seven mAb to p97 were selected for study. According to
assays measuring competitive inhibition of mAb binding to
p97-positive cells, the seven mAb identify three different
epitopes on the p97 antigen (Brown, J.P., et al., 1981, J.
Immunol. 127:53-546), namely p97a (mAb 4.1 and mAb 96.5),
pg7b (mAb 118.1, mAb 133.1, mAb 133.3, and mAb 8.2) and pg7c
(mAb 133.2). mAb 133.3 is an IgG2b, mAb 4.1 and 8.2 are
IgGl, and the other mAb are IgG2a, except 4.1 and 8.2 which
are IgG21.
In the experiment presented in Figure 15, each of the 5
IgG2a mAb were mixed with the Ab2 to be tested, and added to
plates coated with Fab 96.5, followed by addition of goat
anti-mouse IgG1-HRP to detect the binding of the respective
Ab2 to Fab 96.5. As shown in Figure 15, mAb 133.3 inhibited
the binding of six of the tested Ab2 (#1, #3, #4, #5, #6,
and #7) to Fab 96.5 (Fig. 13); the inhibition was of
approximately the same degree as that seen with mAb 96.5.
The binding of #2, which like the other Ab2 had been
selected for binding to mAb 96.5, was not inhibited. mAb
118.1, 133.1, and 133.2 did not inhibit the binding of any
of the tested Ab2-
A different assay was used to analyze the degree of
binding between the Ab2 and various anti-p97 mAb of the IgG1
isotype, and the effect of any such binding upon the
subsequent binding of radiolabelled p97 by the anti-p97 mAb.
The two IgG1 anti-p97 mAb, 4.1 and 8.2, were tested, as was
mAb 96.5. Each Ab2 was plated, various concentrations of
mAb 8.2, mAb 4.1, or mAb 96.5 were added, and the binding of
the anti-p97 mAb to radioiodinated p97 was measured (Fig.
16). Three Ab2, #3, #5, and #7, interfered with the binding
1 339~1 6
- --111--
of mAb 96.5 to p97 (in agreement with the results described
in Section 8.2.2, supra), while four other Ab2, #1, #2, #4,
and #6, did not interfere. None of the Ab2 bound to mAb 8.2
or mAb 4.1. The experiment was repeated, testing mAb 133.1
and mAb 133.3 in parallel with mAb 96.5, since the data
presented in Figure 15 showed that mAb 133.3 (but not 133.1)
- could bind to Ab2. Figure 17 shows that all of the seven
Ab2 tested bound to both mAb 96.5 and 133.3. In contrast,
none of the Ab2 bound to mAb 133.1.
An assay was performed which demonstrated that six of
the Ab2 (with the exception of #2) prevented mAb 133.3 from
binding to SK MEL-28 melanoma cells. In addition, none of
the Ab2 could inhibit mAb 4.1 or 8.2 from binding to SK MEL-
28 cells. None of the seven Ab2 bound to Pl.17, an IgG2a
myeloma protein, or to the two mAb used as controls, L6 (an
IgG2a anti-carcinoma antibody) or MPG24 (an IgG2a antibody to
a melanoma-associated proteoglycan), or to a goat antiserum
to mouse IgG2b (Table XIV).
1 3398 1 6
-112-
TABLE XIV
mAb 96.5 INHIBITS THE BINDING OF SOME Ab2 TO Fab 96.5*
Inhibitor
Ab2 - Goat Anti-
Number PBS 96.5 -pl.17 MPG24 L6 Mouse IgG2b
#1 0.961 0.125 0.941 0.993 0.876 1.026
#2 1.074 0.514 0.942 1.064 0.953 1.049
#3 0.655 0.084 0.586 0.618 0.567 0.623
#4 0.649 0.066 0.629 0.674 0.611 0.743
#5 0.555 0.061 0.521 0.557 0.506 0.541
#6 0.447 0.091 0.436 0.443 0.418 0.551
#7 0.554 0.074 0.515 0.577 0.504 0.627
*As detected by an ELISA, in which various inhibitors at 80
~g/ml were mixed with Ab2 (0.4 ~g/ml) and added to plates coated
with Fab 96.5. HRP-conjugated goat anti-mouse IgGl was added to
the plates and incubated for 1 hour, followed by addition of
25 OPD. OD was measured at 492 nm.
-113- l 3398 1 6
Taken together, our findings indicate that the seven Ab2
tested could bind to mAb 133.3, perhaps at its antigen-
binding site, with the exception of #2 which does not bind to
mAb 133.3 at its antigen-binding site. (#2 does however bind
to mAb 133.3, apparently at other than its antigen-binding
site; see Figure 17.)
8.2.4. INDUCTION OF AN Ab3 RESPONSE
0 The seven Ab2 were tested for their ability to induce an
Ab3 antibody response in mice. In the first sets of
experiments, syngeneic (BALB/c) mice were immunized with Ab2.
For most of these experiments, the Ab2 were conjugated with
KLH.
After the immunization with Ab2, sera of the mice were
tested for the presence of Ab3 detectable by its ability to
bind to Ab2. Several dilutions of the mouse sera were mixed
with the respective Ab2, and added to Immunolon plates coated
with Fab 96.5 (as Abl). Goat anti-mouse IgGl-HRP was then
used to detect the binding of the Ab2 to the Fab 96.5 (Table
XV) .
- 1 33q~1 6
-114-
TABLE XV
INHIBITION OF THE BINDING OF Ab2 TO Fab 96.S
(AS Abl) BY SERA FROM BALB/c MICE WHICH HAD
BEEN INJECTED WITH THE RESPECTIVE Ab2*
,,
% Inhibition
Ab21st Bleeding 2nd Bleeding
Number
.
- #1 90 96
100 99
#2 96 97
98
#3 85 100
83 98
. #4 82 96
86 87
#5 74 92
94
#6 0 89
#7 - 73 91
77 92
*Sera were diluted 1:10. The respective Ab2 was
conjugated to KLH prior to injection into mice. Data are
given for each of 2 mice/group and for 2 subsequent bleedings
of the mice.
1339816
-115-
As shown in Table XV, serum derived solely from the
first bleeding inhibited the binding of Ab2 to Fab 96.5. The
amount of inhibitory activity increased after a second boost.
We next tested whether sera from the immunized mice
could bind to p97 (like an Abl). Soluble p97 antigen was
plated, and diluted mouse sera added (after blocking),
followed by goat anti-mouse IgG-HRP. Sera from mice
immunized with either of two Ab2, #3 or #7, bound to p97,
while sera from mice immunized with any of the other five
Ab2, including #5! did not bind to p97 (Fig. 18). As a
positive control, we titrated the binding to p97 of either
mAb 96.5 or a mouse anti-p97 serum, and compared the binding
observed with that seen using sera from mice immunized with
either Ab2 #3 or #5. The data showed that the latter sera
contained p97-specific Ab3 in a concentration of
approximately 1-5 ~g/ml. The ability of Ab2 #3 or #7 to
induce an Abl-like Ab3 response thus indicated that these Ab2
were internal image antibodies.
To further test the specificity of the Ab3 for p97, sera
- 20 from Ab2-immunized mice were absorbed with 1 x 1o6 cells from
either the p97-positive mouse melanoma line 2A or from its
p97-negative parent (as a control), before the sera were
added to p97 which had been coated onto plates. The binding
of the Ab3 to p97 was then detected by using a goat anti-
25 mouse IgG-HRP conjugate. As shown in Figure 19 (Panels A and
B), sera from mice immunized with either Ab2 #3 or #7
contained Ab3 which bound to p97, and this Ab3 activity was
removed by absorption with 2A cells but not by absorption
with par cells. There was a lower amount of binding to p97
30 with sera from mice immunized with either Ab2 #4 or #5, and
this binding was also inhibited by absorption with 2A cells
(Fig. 19, Panels C and D). Neither normal mouse serum nor
serum from mice immunized with P1.17 (as a control) contained
antibodies which bound to p97. While the binding of purified
35 mAb 96.5 to p97 could be completely abrogated by absorption
,
1 33q8 1 6
-116-
with 2A cells (Fig. 20), the binding of serum antibodies from
mice immunized with p97 was only partially inhibited, as was
the Ab3 activity of sera from mice immunized with either Ab2
#3 or #7.
An Ab3 response was also detected in C3H/HeN mice after
immunization with Ab2 conjugated to KLH. Serum from the
immunized C3H/HeN mice bound to soluble p97 antigen in a
solid phase ELISA (Figs. 21, 22). Absorption of the mouse
sera with either 2A or par cells before they were tested for
0 binding to soluble p97 verified that the binding was to p97.
Experiments were also done in which BALB/c and C3H/HeN
mice were immunized with an Ab2 which had not been conjugated
to KLH. Sera of these mice were found to contain antibodies
which bound to Fab 96.5. To determine whether these
antibodies were Ab2 which still remained in the circulation,
or whether any Ab4 had been induced, an ELISA was performed
with HRP conjugates which could identify not only IgGl (the
isotype of the Ab2), but also IgG2b and IgG3. The findings
suggested that the mouse sera contained Ab4 which could bind
to Abl and belonged to the IgG2b and IgG3 classes.
8.3. DISCUSSION
We have made a series of mouse mAb to idiotypic
determinants on mAb 96.5, which defines ps7a~ an epitope of
the p97 melanoma antigen, and have analyzed them with
respect to specificity and ability to induce an Ab3 response
in mice. Seven Ab2 were studied in some detail. Four of
these Ab2 did not appear to identify the region of mAb 96.5
involved in binding p97, since they neither appreciably
inhibited the binding between mAb 96.5 and p97, nor was their
binding to mAb 96.5 inhibited by soluble p97. However, three
of the Ab2, referred to as #3, #5, and #7, respectively,
prevented the binding between soluble p97 antigen and mAb
96.5, and soluble p97 prevented the binding between Fab 96.5
35 and these Ab2.
t 3398 1 6
-117-
mAb 133.3 behaved similarly to mAb 96.5 when tested in
binding assays with the seven Ab2, while mAb 4.1 did not.
This was unexpected, since mAb 133.3 has been reported to
identify a different epitope (pg7b~ than mAb 96.5, while mAb
4.1 has been reported to be specific for the same epitope
(p97 ) (Brown, J.P., et al., 1981, J. Immunol. 127:539-546)
as mAb 96.5. The reasons for the discrepancy between results
obtained using an assay for competitive inhibition of mAb
binding to target cells (id.), and our findings, obtained
with an assay measuring the binding between Abl and Ab2, are
unknown.
The antigen binding inhibition data were consistent with
the view that three of our Ab2, #3, #5, and #7, were of the
~internal image~ type. The data did not, however, exclude
alternative explanations such as steric hindrance. We
therefore investigated whether any of these three Ab2 could
induce an immune response in mice. Since humoral antibody
responses can generally be studied more easily and precisely
than cell-mediated responses, we searched for Ab3 which could
20 bind to p97.
Two Ab2, #3 and #7, induced an Abl-like Ab3 response in
both BALB/c and C3H/HeN mice, thus indicating that they are
internal image antibodies. A third Ab2, #5, whose behavior
was similar to #3 and #7 when tested in vitro, did not induce
an Abl-like Ab3 response. The binding of the Ab3 to p97 was
competitively inhibited by absorption with 2A cells, which
express ps7, but not with cells from the p97-negative par
line. The fact that a response was also observed in the
allogeneic C3H/HeN strain indicates that the response was not
30 controlled by Th genes, but more likely induced by the Ab2
acting as an "internal image" of the p97 antigen (Lee, V.K.,
et al., 1986, Biochim. Biophys. Acta 865:127-139).
To obtain solely an Abl-like response ln vivo, the
conjugation of an Ab2 to KLH was necessary. When KLH was not
35 used, the sera of the immunized mice contained both
1 3398 1 6
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antibodies of the IgGl isotype of the Ab2 and antibodies
which were of other isotypes (IgG2b, IgG3), and which may
have been Ab4. If Ab4 were, indeed, generated, an Ab3
response may have occurred at some point.
9. ANTI-IDIOTYPIC ANTIBODIES SPECIFIC
FOR ANTI-CARCINOMA ANTIBODY L6
Murine monoclonal antibody (mAb) L6 (Abl) is an IgG2a
specific for a tumor-associated carbohydrate antigen which is
found at the surface of cells from many different human
carcinomas (Hellstrom, I., et al., 1986, Cancer Res. 3917-
3923). Anti-idiotypic mAb (Ab2) against L6 have been made.
Twenty-four Ab2 were obtained which bound to Fab fragments
prepared from L6 but not to Fab prepared from a control
IgG2a. Eight of these 24 mAb could bind at high
concentrations to one of two mAb which were independently
derived but have the same specificity as L6. Fourteen of the
24 Ab2 could inhibit the binding of L6 to antigen on cells.
Six of these 14 Ab2 were unable to bind to Fab fragments
prepared from L6 and already bound to L6 antigen-positive
20 cells. Using cloned variable region gene segments, it was
found that two of these 6 Ab2 could specifically-recognize
the L6 heavy chain variable region associated with an
irrelevant light chain, while the remaining 4 recognize a
combinatorial determinant, requiring L6 heavy and light chain
25 variable regions to be associated. In contrast, all of 8
tested Ab2 which bound to L6 Fab attached to cells, could
also bind to the isolated light chain variable region. The 6
Ab2 which did not bind to L6 Fab fragments attached to cells
were injected into BALB/c and C3H/HeN mice. Two of them
(possibly more) induced polyclonal antibodies (Ab3) which
expressed the same idiotype as L6, bound to L6-positive tumor
cells and competed with L6 for its antigen-binding site.
- - -119- t 3398 1 6
9.1. MATERIALS AND METHODS
9.1.1. ANIMALS
Six-to-eight week old BALB/c and C3H/HeN female mice
were obtained from the Animal Facilities at the Fred
Hutchinson Cancer Research Center. They were used throughout
the study unless otherwise indicated.
., .
9.1.2. CELL LINES
0 Human colon carcinoma line H-3347 (Hellstrom, I., et
al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:7059-7063)
expresses high levels of the antigen defined by L6. The CEM
human T cell line, which does not bind L6, was included as a
negative control.
9.1.3. ANTITUMOR ANTIBODIES ("ABl~)
Monoclonal antibody L6 (IgG2a) was employed as the
antitumor mAb (Abl) in this study. Its development and
characterization is described elsewhere (Hellstrom, I., et
al., 1986, Cancer Res. 46:3917-3923; Hellstrom, I., et al.,
1986, Proc. Natl. Acad. Sci. U.S.A. 83:7059-7063). mAb F26
(IgGl) and 012/28-24 (IgG2a) are made by hybridomas which
resulted from the fusion of spleen cells from BALB/c mice
immunized with carcinoma tissue; hybridization and selection
25 was similar to that resulting in L6. The latter two mAb
compete with L6 for binding to carcinoma cells. mAb 96.5
(Brown, J.P., et al., 1981, J. Immunol. 127:539-546) and
myeloma protein Pl.17 (American Type Culture Collection
Accession No. TIB 10), which are both IgG2a, were used as
30 controls. Fab fragments were made from L6 and 96.5 by papain
- digestion (id.), and are termed Fab L6 and Fab 96.5,
respectively.
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9.1.4. GENERATION OF ANTI-IDIOTYPIC ANTIBODIES (AB2)
A protocol was used which had been successfully employed
to generate Ab2 relating to a different tumor antigen, p97
(see Section 8, supra). BALB/c mice were immunized with 100
5 ~g of L6 which had been coupled to keyhole limpet hemocyanin
(KLH) as described (Streicher, H.Z., et al., 1986, J.
Immunol. 136:1007-1014). The first immunization was given
subcutaneously in complete H37Ra adjuvant (Difco, Detroit,
MI), and a second dose was given in incomplete Freund's
0 adjuvant intraperitoneally (i.p.) 4 weeks later. Two to four
subsequent immunizations were done i.p. in saline at two-week
intervals.
Spleens were removed 3 days after the last boost, and
the spleen cells were fused with NS-l myeloma cells by
centrifugation with polyethylene glycol. After 10 days, the
hybridomas were screened by an ELISA against Fab fragments
which had been prepared from L6 (referred to as L6 Fab) and
were coated onto 96-well Immunolon II plates (Dynatech,
Chantilly, VA). The binding to L6 Fab was detected by
20 separately using each of three different reagents: a rabbit
antiserum to mouse IgG1 which had been coupled to horseradish
peroxidase (HRP) (referred to as IgG1-HRP), a HRP-conjugated
rabbit antiserum to mouse IgG3 (referred to as anti-mouse
IgG3-HRP), or protein A coupled to HRP ("protein A-HRPN);
25 these reagents were obtinaed from Zymed (South San Francisco,
CA). Antibodies binding to Fab L6 were tested for binding to
Fab 96.5 to exclude nonspecific binders. Hybridomas
appearing to produce mAb to the idiotype of L6 were cloned
twice by limited dilution, followed by testing of all
30 subclones.
9.1.5. AB2 PURIFICATION
Four-to-six week old male BALB/c mice were primed with
pristane. Ten days later, they were injected with 5 x 106
35 Ab2-producing hybridoma cells and ascites was collected 3 to
.
1 33981 6
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8 weeks subsequently. Ascites containing IgG2a and IgG2b
antibodies was purified over protein A columns (Brown, J.P.,
et al., 1981, J.-Immunol. 127:539-546), and IgGl-containing
ascites was purified by ammonium sulfate precipitation,
followed by DEAE-Sephacel (Pharmacia, Uppsale, Sweden)
chromatography. Purified Ab2 were tested for binding to
Fab L6 as well as for inhibition of the binding of L6 to
H-3347 cells (as described below).
0 9.1.6. INHIBITION ASSAY TO DETECT AB2 BINDING
TO THE PARATOPE REGION OF L6
An assay was used similar to one previously employed in
the p97 system (see Section 8, supra). Supernatants
containing Ab2, or purified Ab2 were mixed with mAb L6 for a
final concentration of 0.1-0.4 ~g/L6 per ml. The mixture was
incubated in a 96-well tissue culture plate (Falcon, Becton
Dickinson, Oxnard, CA) for 30 minutes at room temperature,
after which it was added to glutaraldehyde-fixed H-3347
carcinoma cells which had been attached to the wells of a
96-well plate. Binding of L6 to the H-3347 cells was
20 detected by an ELISA using a goat antiserum to mouse IgG
coupled to HRP, or protein A coupled to HRP. Data were
expressed as percent inhibition of the binding of L6 to the
cells in the presence of the Ab2.
9.1.7. BLOCXING ASSAY TO DETECT COMPETITION
BETWEEN AB2 AND ANTIGEN FOR L6 BINDING SITES
Thirty ~g/ml of Fab L6 were added to 5 x 105 H-3347
carcinoma cells in a propylene tube. After a 30 minute
incubation at 4~C, the cells~ere washed to remove any
30 unbound Fab. Purified Ab2 were labelled with flourescein
isothiocyanate (FITC). Various concentrations of the
labelled Ab2 were added to the propylene tube, followed by
incubation at 4~C for 30 minutes and washing. Ab2 binding
was detected using an EPIC-C model flourescein-activated cell
*
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~ 13398'6
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sorter (FACS), as previously described (Hellstrom, I., et
al., 1986, Cancer Res. 46:3917-3923). This experiment was
also performed with phycoerythrin (PE) labelled L6 instead of
unlabelled L6 Fab fragments.
9.1.8. CLONING OF L6 HEAVY AND LIGHT
CHAIN VARIABLE REGION GENE SEGMENTS
DNA was isolated as described in principle by Blin and
Stafford (1976, Nucl. Acids Res. 3:2303-2308) and in more
detail by Ledbetter et al. (1987, Mol. Immunol. 24:1255-
1261). It was digested with either EcoRI or HindIII and size
fractionated on sucrose gradients (Maniatis, T., et al.,
1982, Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor Laboratory, New York). Gradient fractions containing
specific variable (V) regions were identified by Southern
blot analysis (Southern, E.M., 1975, J. Mol. Biol. 98:503-
507). The expressed alleles were identified by virtue of
their lack of detection in Southern Blot analysis of non-
producing L6 sublines. EcoRI-digested DNA containing the
expressed L6 heavy chain V region gene segment was cloned
20 into the EMBL3 lambda phage vector (Promega Biotech, Madison,
WI) with Gigapack packaging extracts (Stratagene, La Jolla,
CA), plated, and screened using an XbaI/EcoRI fragment
containing the murine heavy chain enhancer.
A 2.3 kb HindIII fragment spanning the murine heavy
25 chain enhancer was cloned into a pSV2-gpt vector (Mulligan,
R.C. and Berg, P., -1980, Science 209:1422-1427) containing
the human C exons as a 10.5 kb BamHI fragment derived from
the HG3A phage clone (Ellison, J.W., et al., 1982, Nucl.
Acids Res. 10:4071-4079). The expressed variable heavy chain
30 gene segment was then transferred into the vector as a 10.5
kb EcoRI fragment.
Gradient fractions containing HindIII-digested DNA with
the expressed L6 light chain were cloned into the XbaI site
of Lambda Zap (Stratagene) by filling in the HindIII
133~816
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overhangs with the dinucleotide AG and the XbaI overhangs
with CT thus leaving compatible overhangs for cloning. The
resulting library was screened with a 1 kb PstI/HindIII
fragment from the intron separating JK and CK in the pBR322
clone containing the 9.5 kb BamHI fragment with the expressed
K gene from MPCll (Kelley, D.E., et al., 1982, Cell 29:681-
689). For the chimeric light chain expression construct, a 1
kb HindIII/XmnI fragment containing the murine K enhancer
(from MPCll) was cloned upstream of a 2.7 kb EcoRI fragment
0 encoding the human CK exon (Heiter, P.A., et al., 1982, J.
Biol. Chem. 257:1516-1522) in a pUC-gpt vector. The
expressed 6 VK gene segment was then transferred into this
vector as a EcoRI-NotRI fragment linkered into the unique
PstI site.
The chimeric light chain and heavy chain gene constructs
were transfected into the mouse myeloma cell line Ag 8.653
using a BioRad electroporator per the manufacturer's
instructions, with subsequent selection on 0.5 ~g/ml
mycophenolic acid. Similar transfections were performed
using only the chimeric light chain gene construct and the
SP2/0 mouse myeloma cell line, or with the chimeric heavy
chain construct and the J588L murine myeloma which expresses
a murine lambda I light chain. Cell lines expressing the
chimeric proteins were identified by ELISA. Chimeric L6
antibody was purified over protein A-Sepharose and
biotinylated as described previously (Pohlit, H.M., et al.,
1979, in Immunological Methods, I. Lefkovits and B. Pernis,
eds., Academic Press, New York, pp. 181-194).
9.1.9. ASSAYS FOR L6 ANTI-IDIOTYPE
VARIABLE REGION SPECIFICITY
Competition assays were performed by coating Immulon II
plates with 100 ~l of mAb 187.1 (provided by Dr. Dale
Yelton), followed by three washes, and then supernatant from
cultures of the L6 anti-idiotypic mAb. The plates were then
~ 3398 1 6
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washed again, and 100 ~1 of unlabelled competitor was added
at a concentration of 6 ~g/ml. The unlabelled competitors
were either culture supernatants from transfectants
expressing the L6 chimeric variable heavy chain associated
with the J558L light chains, or the L6 chimeric variable
light chain only, or media spiked with purified chimeric L6
mAb or PW P3281B8 (a control, irrelevant human IgGl
antibody). After 30 minutes at 30~C, 50 ~1 of a 1 ~g/ml
solution of purified biotinylated chimeric L6 antibody was
0 added to the 100 ~1 volume already present in the well. This
was allowed to incubate for an additional hour at 37~C, then
the plate was washed, incubated with Avidin-HRP (TAGO,
Burlingame, CA) (1:1000 dilution for 30 minutes at room
temperature), washed again, and developed with TMB chromogen
in buffered substrate (Genetics Systems, Seattle, WA). The
data was expressed as % inhibition, with no competitor (media
only) taken to be 100% with the background value (no Ab2)
subtracted.
9.1.10. INDUCTION OF AN AB3 RESPONSE
Six-to-eight week old BALB/c and C3H/HeN females were
immunized with 50 ~g Ab2 which had been conjugated with KLH
(Streicher, H.Z., et al., 1986, J. Immunol. 136:1007-1014).
A similar second immunization was done one week after the
first one, followed by a third and fourth immunization
performed at l-week intervals with the Ab2 given in phosphate
buffered saline (PBS), and subsequent immunizations were done
at 2-week intervals with the Ab2 in PBS. All immunizations
were done i.p. After 4 weeks~ the mice were bled
30 periodically and their sera were tested for the presence of
(polyclonal) Ab3.
9.1.11. PURIFICATION OF POLYCLONAL AB3
Sera from mice immunized with Ab2 were collected between
6 and 20 weeks after the first immunization and pooled. Ten
Trade Mark
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mg of the respective Ab2 were conjugated to 1 ml of cyanogen
bromide-activated Sepharose 4B (Pharmacia, Piscataway, NJ),
and chromatography was done accor*ing to the manufacturer's
instructions, so as to enrich for Ab3 which could bind to the
respective Ab2.
9.1.12. MEASUREMENT OF THE ABILITY OF AB3 TO
INHIBIT THE BINDING OF AB2 TO L6 Fab
A previously described assay was used (see Section 8,
supra). Sera or purified Ab3 to be tested for Ab3 were mixed
with a fixed concentration (2.0 ~g/ml) of purified Ab2 and
added to an Immunolon II plate whose wells had been coated
with Fab L6; mAb L6 and PBS were used as positive and
negative controls, respectively. Following 30 minutes of
incubation, the binding of Ab2 to Fab L6 was detected by an
ELISA using a rabbit anti-mouse IgGl HRP/protein A-HRP
cocktail. Data were expressed as percent inhibition of the
binding of Ab2 to Fab L6 by the Ab3 serum.
9.1.13. ASSAY FOR BINDING OF AB3 TO CELLS
Various dilutions of mouse serum to be tested for Ab3 or
purified polyclonal Ab3 (in 50 ~1 amounts) were incubated at
4~C with 5 x 105 H-3347 cells for 30 minutes in polypropylene
- tubes. Culture medium, serum from mice immunized with mAb
96.5 or with P1.17, and normal mouse serum were used as
negative controls. Cells were washed twice in media.
Binding of Ab3 to target cells was detected by a goat anti-
mouse IgG labelled with FITC (Tago, Burlingame, CA); 50 ~1
was added to the mixture of mouse serum and tumor cells and
incubated for 30 minutes. After washing twice in media, the
cells were analyzed using FACS. CEM cells, which do not
express the antigen defined by L6, were used as a negative
target cell control.
* Trade Mark
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9.2. RESULTS
9.2.1. GENERATION OF ANTI-IDIOTYPIC mAb (AB2) TO L6 (ABl)
Hybridomas producing anti-idiotypic mAb were obtained by
immunizing BALB/c mice with L6 and fusing their spleen cells
with myeloma NS-l, followed by screening of the hybridoma
supernatants for binding to Fab L6. Cells from wells with
supernatants binding to Fab L6 were cloned twice and the
clones tested for their ability to make mAb binding to Fab L6
and not to Fab 96.5 (used as a negative control). From 8
fusions, 24 hybridomas which made mAb to Fab L6 were
isolated, stabilized and used for ascites production; they
represented several different isotypes (Table XVI).
- 1 33981 6
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TABLE XVI
ANTI-IDIOTYPIC mAb TO mAb L6
Anti-idiotypic Anti-idiotypic Binding to-p~ratope
hybridoma mAb (Ab2) region of L6 Isotype
4/1-6-1 L6 anti-Id 1 Yes IgG2b
4/2-4-1 L6 anti-Id 2 Yes IgG2b
4/3-2-1 L6 anti-Id 3 Yes IgG2a
4/5-1-1 L6 anti-Id 4 Yes IgG2b
5/4-2-2 L6 anti-Id 5 No IgG3
6/14-2-2 L6 anti-Id 6 Yes IgGl
6/13-3-1 L6 anti-Id 7 Yes IgGl
6/20-4-5 L6 anti-Id 8 Yes IgGl
7/10-2-3 L6 anti-Id 9 Yes IgGl
5/14-2-6 L6 anti-Id 10 Yes IgGl
5/15-3-1 L6 anti-Id 11 Yes IgGl
10/24-1-5 L6 anti-Id 12 Yes IgGl
10/20-2-3-1 L6 anti-Id 13 Yes IgGI
11/48-3-14 L6 anti-Id 14 Yes IgGl
11/75-1-3 L6 anti-Id 15 Yes IgG2a
11/81-1-2 L6 anti-Id 16 No IgM
11/80-3-8 L6 anti-Id 17 No IgM
10/30-1-6-28 L6 anti-Id 18 No IgG3
10/36-1-1 L6 anti-Id 19 No IgG3
11/5-31-2-4 L6 anti-Id 21 No ~ IgG3
11/26/2-4 L6 anti-Id 22 No IgG3
11/27-5-5-4 L6 anti-Id 23 No IgG3
30 9/66-11-2 L6 anti-Id 24 No IgG3
10/46-6-4 L6 anti-Id 25 No IgG3
*Defined as the ability to inhibit mAb L6 from binding to its
target antigen on H-3347 carcinoma cells.
1 3398 1 6
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Ab2 purified from the ascites was used for further testing
(see below).
9.2.2. CHARACTERIZATION OF AB2 BINDING TO IDIOTOPES ON L6
An initial screening identified 14 of 24 hybridomas
tested which made Ab2 inhibiting 90-100% of the binding of L6
(tested at 0.2-0.4 ~g/ml) to carcinoma cells and were further
tested for the ability to inhibit the binding of L6 to its
target antigen on cells. Such inhibition is expected to
0 occur if the Ab2 reacts with the idiotope region of L6. An
ELISA was used for this purpose, utilizing glutaraldehyde-
fixed H-3347 cells as a source of antigen. As shown in
Figure 23, the binding of L6 (0.2 ~g/ml) to carcinoma cells
was inhibited when Ab2 was added at a concentration of 0.5
15 ~g/ml and 5 ~.g/ml. Two of the inhibiting Ab2 were IgG2a, 3
were IgG2b, and the remaining 9 were IgG1, while none of 7
IgG3 or 2 IgM antibodies was inhibitory.
The 14 Ab2 which could inhibit the binding of L6 in the
above-described solid-phase assay were labelled with FITC for
20 analysis. Each conjugate was analyzed by an ELISA for the
ability to bind to Fab L6 as well as the ability to prevent
L6 from binding to antigen-positive carcinoma cells. This
was done to ascertain that the labelled antibodies behaved
exactly like unlabelled antibodies. When the FITC-labelled
25 Ab2 were added to the Fab L6, which had been added in a
saturation dose, to H-3347 carcinomas cells, it was found
that 8 of the 14 Ab2 could still bind to the Fab L6, while
the binding of 6 Ab2 was completely inhibited (Fig. 24).
When PE-labelled L6 was used instead of Fab L6 in this assay,
30 it was found that the L6 remained bound to the cells and was
not displaced in the presence of the 6 non-binding Ab2 (Fig.
25).
The 6 Ab2 which inhibited the binding of L6 to its
target antigen, and which were unable to bind to previously
35 bound Fab L6 or cells, were selected for further testing.
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The studies reported in the following sections were performed
on samples of these 6 Ab2 and primarily aimed to identify Ab2
capable of acting as Ninternal images~ of the L6-defined
antigen.
9.2.3. SPECIFICITY OF THE L6-GENERATED AB2
There are two mAb, F2-6 and 012/28-24, which have the
same specificity as L6 when tested on the FACS in competitive
binding inhibition assays. Samples of these two mAb were
10 mixed with various concentrations of Ab2 to test whether the
latter could inhibit the binding of the two ("L6-look-alike")
mAb to carcinoma cells. None of the 14 Ab2 tested inhibited
the binding of 012/28-24 to the L6-defined antigen, when
assayed similarly as shown for L6 in Fig. 26. Eight of the
fourteen Ab2 tested could inhibit binding of F26 at high
concentrations, 20 to 200 ~g/ml inhibiting 1 ~g/ml of F26.
This suggests that 012/28-24 has a different idiotype from
that of L6, and that F26 and L6 may have overlapping
idiotopes.
- 20
9.2.4. AB2 EPITOPE SPECIFICITY
The epitope specificity of the L6 anti-idiotypic
monoclonal antibodies was demonstrated in competition
experiments in which either L6, the chimeric L6 heavy chain
25 associated with the J558L murine lambda 1 light chain, the
free chimeric L6 light chain, or an irrelevant human IgGl,
was tested for their ability to compete for each anti-
idiotypic antibody's binding to biotinylated chimeric L6
(Fig. 27). Each of the 14 Ab2 were shown to specifically
30 recognize the cloned L6 V regions by virtue of their ability
to bind the biotinylated chimeric L6 molecule. The binding
was shown to be inhibitable by unlabelled chimeric L6, but
not an irrelevant human IgGl. Eight Abs could be inhibited
to an appreciable degree by the free chimeric L6 light chain
(#1, #2, #4, #6, #7, #8, #9, and #15) but not by the chimeric
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heavy chain associated with mouse lambda 1, and therefore the
eight Abs recognize an L6 variable heavy chain associated
-determinant. These eight are the Ab2 which are able to bind
the L6 Fab bound to cells. Two of the Ab2 (#12 and #13) were
inhibitable by the L6 chimeric heavy chain associated with
the J558L light chain, but not by the free chimeric light
chain, and they thus recognize an L6 V-associated
determinant. The remainder of the Ab2 (#3, #10, #11, and
#14) were not inhibitable by either of the cloned V regions
separately, and must therefore, be specific for a
combinatorial determinant formed only by the assembly of the
appropriate V regions. These results were also confirmed
through direct binding studies.
9.2.5. INDUCTION OF AB3
Five of-the 6 Ab2 which appeared to be binding-site-
related were conjugated to KLH and used to immunize BALB/c
and C3H/HeN mice, as described in Section 9.1.4. The mice
were periodically bled and boosted, and their sera were
20 analyzed.
A binding inhibition assay was first performed to
determine whether the immunized mice appeared to make any Ab3
which could be identified by its ability to bind specifically
to Ab2. Various dilutions of sera from the mice were mixed
25 with Ab2 (1.0 ~g/ml), the mixtures were applied to plates
coated with Fab L6, and the binding of Ab2 to the Fab L6 was
determined by an ELISA. Results were expressed as percent
inhibition of the binding of Ab2 to Fab L6 in the presence of
the given mouse serum. As shown in Fig. 28, the binding of
30 Ab2 (1.0 ~g/ml) to Fab L6 was inhibited over 90% at a 1:200
dilution of several of the immune mouse sera. The affinity
purified antisera were found to inhibit binding of Ab2 in the
same assay. This was seen for all the Ab2 tested and
indicates that Ab3 were produced to the Ab2 paratope region.
-131- l 3398 t 5
The induction of Ab3 anti-idiotypic to Ab2 was similar in
BALB/c and C3H/HeN mice.
9.2.6. ABILITY OF AB3 TO BIND TO THE ANTIGEN DEFINED BY L6
If an Ab2 acts as an ~internal image~ of the L6 antigen,
it should be able to invoke an Ab3 that binds specifically to
the antigen defined by L6 (Abl). To test the antigen
specificity of the Ab3 generated in mice immunized with Ab2,
various dilutions of the affinity purified immune mouse sera
were tested for binding to cells from the L6-positive
carcinoma lines H-3347; CEM cells to which L6 does not bind
were used as the negative control. Sera from C3H/HeN mice
(Fig. 29) which had been immunized with Ab2 #11 and #12 and
sera from BALB/c mice (Fig. 30) immunized with Ab2 #14 bound
to the carcinoma cells.
9.3. DISCUSSION
We have generated monoclonal anti-idiotypic antibodies
(Ab2) to a mouse mAb, L6, which binds to a carbohydrate
20 antigen expressed on many human carcinomas. The Ab2 were
first screened for binding to Fab L6 fragments, followed by
screening for their ability to inhibit the binding of L6 to
its target antigen on carcinoma cells. Out of a total of 24
Ab2 initially selected for binding to Fab L6, 14 Ab2
inhibited the binding of L6 to antigen-positive carcinoma
cells. Six of the latter Ab2 were unable to bind to L6 Fab
fragments previously bound to antigen on cells, suggesting
that they identify a region of L6 which is associated with
its antigen-binding site. When these 6 Ab2 were tested for
30 binding to cloned L6 light and heavy chain variable regions,
4 Ab2 bound only to a combination of the appropriate V
regions, while 2 Ab2 recognized the variable heavy chain
region associated with an irrelevant variable light chain,
and none could bind light chains. In contrast, all of 8
1339815
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tested Ab2 which bound to free chimeric Fab L6 attached to
cells could bind to chimeric L6 light chains.
An Abl-like response _ vivo was obtained only when
using those Ab2 which did not bind to Fab L6 attached to
5 cells and which bound to either a combinatorial or a variable
heavy chain-associated determinant. In contrast, of those
Ab2 which bound to Fab L6 attached to carcinoma cells and to
isolated chimeric L6 light chains, all failed to induce an
Abl-like Ab3 response in mice.
0 Ab2 #11, #12, and #14 behaved as ~internal image" Ab2 in
several different types of assays.
Two Ab2 (#11, #12) could induce an Abl-like Ab3 response
in mice. Sera from mice immunized with any of these Ab2 were
able to bind with the same specificity as L6 to carcinoma
cells. Since an Ab3 response was induced in allogeneic
C3H/HeN mice, it was not allotype restricted, as would have
been expected if the Ab2 had served as an immune regulator
(Lee, V.K., et al., 1986, Biochim. Biophys. Acta 865:127-
139). Rather, the A-b2 behaved as an ~internal image"
(Urbain, J., et al., 1982, Ann. Immunol. 133D:179-189; Lee,
V.K., et al., 1986, Biochim. Biophys. Acta 865:127-139) of
the L6-defined antigen. It was also found that one Ab2 (#14)
could induce an Abl-like Ab3 response in the syngeneic
system.
10. DEPOSIT OF MICROORGANISMS
The following hybridoma cell lines, producing the
indicated monoclonal antibody, have been deposited with the
American Type Culture Collection, Rockville, MD, and have
30 been assigned the listed accession number.
. ' -133- 1 3398 1 6
Monoclonal Date
Antibody Related of Accession
Hybridoma (Ab2) Antigen DepositNumber
Cell line 2C1 2Cl human melanoma- 7-17-87 HB 9484
associated GD3
ganglioside
antigen
Cell line #3 #3 human melanoma- 8-13-87 HB 9498
(24.89/1.3 c1.5) associated p97
anti.gen
Cell line ~7- #7 human melanoma- 8-13-87 HB 9497
(24.6/28.2 cl.l) associated p97
antigen
5/15-3-1 ~11 human carcinoma- 9-18-87HB 9544
associated L6
antigen
10/24-1-5 #12 human carcinoma- 4-1-88HB 9681
associated L6
antigen
11/48-3-14 ~14 human carcinoma- 4-1-88HB 9680
associated L6
antigen
4/1-6-1 #1 human carcinoma- 9-18-87HB 9546
associated L6
antigen
6/14-2-2 #6 human carcinoma- 9-18-87HB 9545
associated L6
antigen
Cell line L6-20-4 L6 Human carcinoma 12-6-84 HB 8677
associated L6
antigen
The present invention is not to be limited in scope by
the cell line deposited since the deposited embodiment is
intended as a single illustration of one aspect of the
invention and any cell lines which are functionally
equivalent are within the scope of this invention. Indeed,
various modifications of the invention in addition to those
shown and described herein will become apparent to those
skilled in the art from the foregoing description and
,~,
1 33q81 6
-134-
accompanying drawings. Such modifications are intended to
fall within the scope of the appended claims.
It is also to be understood that all base pair sizes
given for nucleotides are approximate and are used for the
5 purpose of description.
.
