Note: Descriptions are shown in the official language in which they were submitted.
",..
_2013966
1
This application relates to active specific tumor
immunotherapy.
Active specific tumor immunotherapy is an attempt
antigenically to stimulate the endogenous antitumor
immunity of a host. This is typically done by immunizing
the host with whole cells or extracts obtained from the
host's own tumors, from other patient's tumors, or from
established tumor cell lines. The immunizing agent often
includes a microbial or chemical adjuvant for non-
specifically stimulating the reticuloendothelial system.
Rather than using whole cells or their lysates, one
may resort to purified tumor-associated antigens. While
such agents are more specific than antigenically complex
cells or lysates, they may induce a more equivocal immune
response in that fewer antigenic determinants are
presented. Since tumor cells are heterogeneous, and
undergo immunological changes with time, it is uncertain
whether, at the time of intervention, all tumor cells will
express the immunizing antigen.
2~1~.~~6~
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2
Vertebrates have two basic immune responses:
humoral or cellular. Humoral immunity is provided by
the special class of cells referred to as B
lymphocytes. These cells produce antibodies which
circulate in the blood and lymphatic fluid. On the
other hand, cell mediated immunity is provided by the T
cells of the immune system.
The cellular immune response is particularly
effective against fungi, parasites, intracellular viral
infections, cancer cells and foreign matter, whereas
the humoral response primarily defends against the
extracellular phases of bacterial and viral infections.
Containment of antigen at its point of entry is
accomplished by walling off the area by local
inflammation. Acute inflammation is characterized by
the influx of plasma proteins and polymorphonuclear
leukocytes. Chronic inflammation is characterized by
the infiltration of T-lymphocytes and macrophages.
When acute (antibody induced) and chronic (T-cell
induced) inflammations occur in the skin, they are
called immediate and delayed type hypersensitivity
reactions respectively. ITH peaks at 24 hours, and
subsides in 48 hours: p~Ti appears in 24-48 hours and
peaks at 48-72 hours. The subset of T cells involved
in DTH reactions are called here DTH-Effector cells and
have the CD4+ phenotype.
T-lymphocytes can also differentiate into a subset
of T cells which have cytotoxic activity. These T
cells can destroy target cells either directly or
through the secretion of cytotoxic factors. It is
believed by some that another subset of T-cells have a
suppressive or regulatory role. (They may, of course,
/'""", ,.... , a _.
3
be the same subset of T cells, but differently
activated). Most cytotoxic T cells and suppressor T
cells have the CD8+ phenotype. Suppression may be
antigen-specific, and it may affect either or both
limbs of the immune system.
Mucins are molecules which originate in the mucous
membranes of mammals and are characterized by a
molecular weight in excess of 150,000 daltons, a
carbohydrate content in excess of 70%, and the capacity
to form chemical bonds with water to provide a
mucilaginous or lubricating fluid. Several mucins,
such as epiglycanin, have been associated with
adenocarcinomas.
While mucins may be purified for use in active
specific tumor immunotherapy, an alternative is the
preparation of a synthetic antigen: a conjugate of
numerous tumor-associated carbohydrate hapten molecules
with a suitable carrier molecule.
Epiglycanin (epi) is the major cell surface
glycoprotein (500,000 daltons) produced by the mammary
adenocarcinoma transplantable cell line TA3Ha.
Friberg, Jr., J.N.C.I., 48:1463 (1972); Codington, et
al., Canc. Res., 43:4373 (1983). It should be noted
that TA3Ha is very deadly. The normal post
transplantation life expectancy of a mouse is only 15
20 days. Moreover, it has been reported that TA3Ha is
immunoresistant.
The TA3Ha carcinoma cells are covered by a mucin-
like glycocalix composed mainly of epiglycanin.
Codington, et al., J.N.C.I., 60:811 (1978); Miller, et
al., J.N.C.I., 68:981 (1982). Epiglycanin resembles
4
many human tumor-associated mucins. Rittenhouse, et
al., Lab. Med., 16: 556 (1985). Antigens which cross
react with epiglycanin have been found in human breast,
lung, colon, and ovarian cancers. Codington, JNCI, 73:
1029 (1984).
Epi is mainly carbohydrate (75-80%) in
composition, and expresses multiple T and Tn
determinants. T and Tn are general carcinoma
autoantigens. Springer, Science, 224:1198 (1984). T-
alpha antigen, also known as the TF (Thomsen-
Friedenreich) antigen, is the immediate precursor of
the human blood group MN antigens. Tn, in turn, is the
immediate precursor of the T-alpha antigens. Normally,
T-alpha antigens are not accessible to the human immune
system because they are masked by sialic acid.
Friedenreich exposed the T-alpha antigen by treatment
of red blood cells with neuraminidase, whereupon they
were bound by anti-T antibodies of human sera.
Kim and Ohlenbruck determined that the
immunodominant portion of the T antigen was the
disaccharide beta-D-Gal-(1-3)-alpha-D-GalNac. Z.
Immun-Forsch. 130:88-89 (1966). It was later
established that in contrast to healthy tissues,
certain adenocarcinomas presented T-alpha and Tn
determinants in reactive, unmasked form. Springer, et
al., Cancer, 45: 2949-54 (1980). Both TF and Tn
determinants are found in approximately 90% of human
adenocarcinomas. Springer, Science, 224: 1198 (1984).
This T-alpha determinant has been prepared
synthetically. Ratcliffe, et al., Carbohydrate Res.,
93: 35-41 (1981); Lemieux, EP Patent 44,188. Example
11 in the latter reference describes the use of T-alpha
2013966
hapten on an HSA carrier (at incorporations of 7, 12, 14
and 22 haptens per HSA molecule) to detect a delayed type
hypersensitivity response. The use of such haptens in the
5 production of anti-T-alpha monoclonal antibodies was not
mentioned. A synthetic T-alpha hapten is also described by
Kolar, U.S. 4,442,284.
Rahman and Longenecker, J. Immunol. 129: 2021-2024
(1982) used the natural form of the T-alpha antigen
(neuraminidase-treated erythrocytes) for the production of
monoclonal antibodies whose binding to these cells was
competitively inhibited by synthetic T-alpha hapten. Thus,
their use of synthetic T-alpha hapten was as a character-
izing agent.
Asialo-GMl, a gangliotetraosyl ceramide with the
structure Gal(beta 1-3)GalNac et 1-4)Gal(beta 1-4)Glc(beta 1-1)ceramide, is
found in brain tissue as part of the ganglioside GM1. The
immunodominant portion of this molecule (the terminal
disaccharide), differs from that of TF by the substitution
of a beta linkage (underlined) for an alpha linkage, and
hence is referred to herein as T-beta (as distinct from T-
alpha). Lemieux, U.S. 4,137,401 discloses reaction
conditions for linking a bridging arm to an aldose by a
beta-D-anomeric glycosidic linkage. Synthetic T-beta
haptens have been used in a number of immunological
studies. Hoppner et al, Vox-Sang., 48: 246-53 (1985);
Rahman and Longenecker, supra; Longenecker et al, Int. J.
Cancer, 33: 123-129 (1984).
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5a
Synthetic T-alpha, T-beta and Tn antigens have been
E
used to stimulate anticancer T cell immunity. Henningsson
~a~~~~~
6
Cyclophosphamide (N, N-bis[2-cholorethyl]-
tetrahydro-2H-1,3,2-oxazaphosphorine-2-amine-2-oxide),
a nitrogen mustard derivative, is a cytotoxic agent
which causes cross-linking of DNA. It is most
effective against rapidly dividing cells, hence its use
in cancer chemotherapy. Since it also destroys
lymphocyte cells, it is also useful as a
immunosuppressive agent, indeed, it is one of the most
potent immunodepressants known.
Although most chemotherapeutic agents suppress
host immunity, it has been demonstrated that certain
chemotherapeutic agents, under specific conditions, are
able to augment host anti-tumor immunity. Berd and
Mastrangelo, Cancer Res., 48: 1671-75 (1988);
Mastrangelo, et al., Seminars in Oncology, 13: 186-94
(1986). Campbell, et al., J. Immunol., 141: 3227
(November 1, 1988) reported that cyclophosphamide
reduced the tumor burden in mice implanted with a
murine B cell lymphoma, rendering the tumor more
amenable to active specific immunotherapy with an anti-
idiotype antibody vaccine. Nothing in the article
suggests that the idiotype resembled any carbohydrate
epitope of the lymphoma. No immunosuppressive mucins
are known to be associated with lymphomas. See also
Reissman, et .al., Cancer Immunol. Immunotherap., 28:
179-84 (1989) (leukemias).
Mitchell, et al., Cancer Res., 48: 5883 (October
15, 1988) treated melanoma patients with
cyclophosphamide and, several days later, immunized
them with a melanoma cell lysate. The value of
cyclophosphamide pretreatment was unclear. While
cyclophosphamide seemed to favor increases in
20139fi6
circulating cytolytic lymphocyte precursors, it had no
effect on concanavalin A-inducible suppressor T-cell
levels, and "the patients who received cyclophosphamide
here had no better frequency of clinical response than
those given the lysate mixture alone." In any event, no
immunosuppressive mucins are known to be associated with
melanomas.
In some cancers, the tumors themselves seem to release
immunosuppressive factors. The most striking example of
this phenomenon is Hodgkin's disease, in which a small
tumor in a single lymph node releases or induces the
release of immunosuppressive factors that have a powerful
effect on the entire cell-mediated immune system. Patients
with Hodgkin's disease have a poor delayed hypersensitivity
response and are abnormally sensitive to intracellular
parasitic infections, e.g., tuberculosis and herpes virus
infections. Jessup, et al, Cancer Res., 48: 1689 (1988)
mentions that when lymphocytes from patients with
colorectal carcinoma are incubated with carcinoembryonic
antigen (CEA), a factor is secreted that inhibits immune
responses. It has not been recognized previously, however,
that tumor immunosuppressive activity can be mediated by
mucins. It has been reported that TA3Ha cells are
immunoresistant; this is not the same as immunosuppressive.
20139fi6
8
It has been discovered that mucins, including the
epiglycanin of adenocarcinomas and bovine submaxillary
mucins, have an immunosuppressive effect on subsequent
immune response to cross-reactive antigens. The
present invention relates to the enhancement of the immune
response to active specific adenocarcinoma tumor immuno-
therapy with antigens cross-reactive with adenocarcinoma
tumor-associated mucins by pretreatment with an agent which
inhibits the immunosuppressive effect of the tumor-asso-
ciated mucin. The preferred agent is cyclophosphamide.
Thus, by one broad aspect of the present invention, a
synergistic composition is provided for inhibiting the
growth of an adenocarcinoma tumour, such tumour being
associated with a mucin having immunosuppressive activity,
the composition comprising an immune-response-potentiating
amount of cyclophosphamide, and an immunogenically-effec-
tive amount of a carbohydrate epitope-bearing antigen, that
antigen being immunologically cross-reactive with a mucin
having immunosuppressive activity associated with the
tumour.
By variants of such composition, the antigen may be a
synthetic glyconconjugate, or epiglycanin.
By another variant thereof both the tumour-associated
mucin and the antigen are characterized by a T or a Tn
determinant.
..
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9
By yet another variant thereof, the synthetic glycon-
conjugate is a conjugate of a T-alpha hapten and a pharma-
ceutically-acceptable, immunogenic protein carrier.
By still another variant thereof, the tumour is a
mammary adenocarcinoma.
By another aspect of this invention, mucin-depleted
blood fraction is provided for imparting enhanced respon-
siveness to active specific tumour immunotherapy, such
tumour being associated with a circulating mucin having
immunosuppressive activity, the mucin-depleted blood
fraction having been incubated with a specific adsorbent
for the mucin.
By one variant thereof, the adsorbent is an
immobilized antibody which preferentially binds the mucin.
By yet another aspect of this invention, a combined
therapeutic agent is provided for treating an adenocarci-
noma tumour, the tumour being associated with a circulating
mucin having immunosuppressive activity, the combined
therapeutic agent comprising a mucin-depleted blood
fraction, the blood having been incubated with a specific
adsorbent for the mucin, and a vaccine comprising an
antigen which immunologically cross-reacts with the tumour.
By still another aspect of this invention, a
synergistic composition is provided for inhibiting the
growth of an adenocarcinoma tumour, the tumour being
associated with a mucin having immunosuppressive activity,
.-..
io ''20 1 396 6
the synergistic composition comprising an immune-response-
potentiating amount of an agent which antagonizes the
immunosuppressive activity, and an immunogenically-
effective amount of a carbohydrate epitope-bearing antigen
which is immunologically-cross-reactive with the mucin.
By one variant thereof, the antigen is a blood group
antigen or precursor thereof, or is a synthetic glycon-
conjugate bearing the immunodominant carbohydrate epitope of
a blood group antigen or precursor thereof.
By a still further aspect of this invention, lymph node
cells adapted for inhibiting the growth of an adenocarcinoma
tumour, the lymph node cells having been derived from a donor
treated with a synergistic composition for inhibiting the
growth of an adenocarcinoma tumour, the tumour being
associated with a mucin having immunosuppressive activity, the
synergistic composition comprising an immune-response-
potentiating amount of cyclophosphamide, and an
immunogenically-effective amount of a carbohydrate epitope-
bearing antigen which is immunologically cross-reactive with
a mucin having immunosuppressive activity associated with the
tumour.
~c
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11
By still another aspect of this invention, the use is
provided of a carbohydrate epitope-bearing antigen which is
immunologically cross-reactive with a mucin associated with an
adenocarcinoma tumour and having an immunosuppressive
activity, in the manufacture of a composition for the
treatment of an adenocarcinoma tumour on a patent previously
treated with an immune response-potentiating amount of a
cyclophosphamide.
By a still further aspect of this invention, the use is
provided of cyclophosphamide and a carbohydrate epitope
bearing antigen which is immunologically cross-reactive with
a mucin associated with an adenocarcinoma tumour and having an
immunosuppressive activity, in the manufacture of a
composition for the treatment of an adenocarcinoma tumour.
In the accompanying drawings,
Figure 1 shows the results of a second TA3Ha tumor
challenge of four groups of mice which survived a previous
TA3Ha implantation as a result of combined therapy with
cyclophosphamide and a TFa-bearing natural or synthetic
antigen in Ribi adjuvant. The ordinate is the percent
survival; the abscissa, the number of days after challenge.
The groups are as follows: (3) cyclophosphamide+Epi-Rbi (4
immunizations, subcutaneously); (4) TFa/KLH-Ribi (4 s.c.); (5)
cyclophosphamide+TFa/KLH-Ribi (4 s.c.); (c) control mice.
u.
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lla
Figure 2 shows the comparison of an expanded set of
treatments. The groups are as follows: (1) no CY, no
immunization (i.e. , control) ; (2) CY only (day 1) ; (3) CY
(day 1)+TFa/KLH-Ribi (day 2)+CY (day 5)+TFa/KLH-Ribi (days
6, 10, 17) ; (7) CY only (day 5) ; (8) CY (day 5) , TFa/KLH-
Ribi (days 6, 10, 14, 21); (9) CY (day 1)+CY (day 5).
Figure 3 shows the results of a local Winn assay of
the ability of lymph node cells from mice surviving TA3Ha
implantations thanks to therapy with cyclophosphamide and
a TFa-bearing antigen to transfer adoptively the ability to
inhibit TA3Ha tumor growth to other mice. Tumor growth is
shown by trend lines, marked as follows:
Marking Donor Recipient
filled circles CY+TFa/KLH-Ribi saline
open circles same CY
filled triangles CY on day 5 saline
open triangles same CY
filled squares normal saline
open squares same CY
It has been found that a synthetic conjugate of the T
alpha hapten and a conventional carrier protein, keyhole
limpet hemocyanin (KLH) emulsified in a conventional
adjuvant, trehalose dimycloate and monophosphoryl lipid A
(MPL) (available in combination form Ribi Immunochem.
Research, Inc. , Hamilton, Montana and referred to herein as
"Ribi"), when administered subcutaneously into hosts
bearing tumors which express the T-alpha epitope provided
25% long-term survival. When administration of this
conjugate was preceded by treatment with cyclophosphamide,
50% survival was seen for hosts in which the tumor had been
4
~r .
a 203966
llb
established for five days and 90% survival when the tumor
had been established for only two days.
Moreover, it has been found that lymph node cells
obtained from mice that had been treated with
cyclophosphamide and T-alpha-KLH-Ribi and had survived this
active specific tumor immunotherapy were able to inhibit
tumor growth completely in a Winn-type assay.
The present invention, in its various aspects, is not
limited to the use of any particular adjuvant. Other
chemical and microbial adjuvants, e.g., CFA, SAF-1, MDP,
BCG, liposomes, and Bordetella pertussis toxin may be used
in place of Ribi. The tumor-associated hapten may be
conjugated to other carrier proteins, e.g., tetanus or
diphtheria toxoid, or retrovirus peptides (e. g., VP6 viral
peptide), rather than KLH, and the hapten/molecule-to-
carrier molecule substitution ratio may be varied. Either
natural or synthetic antigens which cross-react with the
immunosuppressive mucin may be employed.
While the experimental example relates to therapy of
a mammary adenocarcinoma in a mouse model, it is believed
that the treatment method of the present invention is
adaptable to other mammals including human subjects, and to
treatment of other adenocarcinomas. Synthetic tumor-
associated glycoproteins (S-TAGS) and other carbohydrate
antigens are known in the art and may be preferred. For
synthetic methods, see Kaifu and Osawa, Carbohydr. Res.,
58:235 (1977); Ratcliffe et al, Id., 93:35 (1981); Paulsen
,,r.,
2013966
llc
et al, Id., 104:195 (1982); Bencomo and Sinay, Id., 116:69
(1983) .
While the preferred antigen presents a T-alpha
disaccharide epitope, a T-beta or Tn epitope might be
provided instead. Also, the immunodominant carbohydrate
epitopes of other blood group antigens and precursors might
be presented. Moreover, anti-idiotype antibodies may be
used in place of synthetic or natural antigens.
The time interval between administration of the cyclo-
phosphamide and administration of the synthetic tumor-
associated glycoconjugate is not fixed, but is dependent on
the time of onset and duration of action of the cyclophos-
phamide's inhibitory effect on suppressor T cell activity
or on the induction of such activity by tumor-expressed
mucins. The dosage of cyclophosphamide may be selected to
increase the antigenic specificity of the anti-suppressor
T cell activity effect.
In place of cyclophosphamide, another antagonist of
immunosuppression may be employed, e.g., other oxazaphos
phorines, cimetidine or an anti-(suppressor cell) or anti
(suppressor factor) monoclonal antibody. Numerous
antibodies of these two types are offered for sale (see
Linscott's Directory of Immunological and Biological
Reagents, p. 10, 5th ed., 1988-89).
4
v 2013966
lld
The present invention, in its various aspects, is not
to be restricted on the basis of the present interpretation
of the mechanism whereby cyclophosphamide or a similar
agent exercises an immunopotentiating effect. An agent
antagonizes the immunosuppressive effect of a tumor-
associated mucin if it interacts with the mucin or the T
cell so that the mucin no longer activates suppressor T
cell activity, or if it interacts with a T cell so
activated or its suppressor factors so as to diminish the
suppressor activity induced by that mucin, or if it
interacts with other components of the cellular immune
system so as to render them less vulnerable to suppressor
T cells activated by that mucin or to suppressor factors
released by such cells.
.s
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12
In another embodiment, a monoclonal antibody
specific for an epitope of a tumor-associated,
immunosuppressive mucin is attached to a suitable
support to form an immunosorbent. Circulating tumor-
s associable immunosuppressive mucins recognized by the
immunosorbent are removed from the patient's
bloodstream by plasmapheresis. The immune response to
the tumor, with or, without further stimulating the
immune system by active specific tumor immunotherapy,
is thereby enhanced. (Lectins or other binding
substances might be used in place of anti.bodies).
In a third embodiment, such a monoclonal antibody
is administered to the patient, so that it complexes
the circulating mucin and thereby hinders its adverse
interaction with the cellular immune system.
MATERIALS AND METHODS
Animal: l0 week old pathogen free female CAF1/J
mice purchased from Jackson Laboratory were used
throughout the investigation.
Tumor cell line: TA3-Ha tumor cell line was
originally provided by Dr. J. F. Codington (Mass.
General Hospital, Boston, Mass). The tumor cells were
grown in vivo by weekly passage (i.p.) in CAF1/J mice.
Synthetic tumor-associated glycoconiuQate fS-TAG)
and control antigens: S-TAGs (~QGall->3GalNAca-Ser-Gly-
carrier) of Ta-KLH and Ta-HSA synthesized by Biomira,
Inc., Edmonton, Alberta. KLH was purchased from Cal
Biochem and HSA was purchased from Sigma, St. Louis,
MO. Hapten substitution ratios were 10-35:1 for HSA
and 800-3,000:1 for KLH.
.
,~-.~
13
CLrclophosphamic~e ICYI treatments:
Cyclophosphamide purchased from Sigma was dissolved in
sterile saline. Mice were injected intravenously with
CY at a concentration of 100 mg/Kg per mouse.
Tumor vaccine formulation and protocol for active
specific immunotherapy: Mice were first injected
intraperitoneally with approximately 700 TA3-Ha tumor
cells on day 0. The animals were divided into various
groups of 8 mice and the following tumor vaccine
formulation was administered. Group 1: control mice
received CY treatment and no immunization: Group 2 mice
received CY on day 1 only: Group 3: mice received CY
treatment on day 1, followed by subsequent subcutaneous
immunization of Ta-KLI-i-Ribi emulsion on day 2, 6, 10
and 17; Group 4: mice received subcutaneous
immunization of Ta-KLH-Ribi emulsion on day 2, 6, 10
and 17. In a separate experiment, the above
experimental set up was repeated with two additional
groups of animal added. Group 5: CY treatment was
given on day 5, followed by subsequent subcutaneous
immunizations of TA-KLH-Ribi emulsion on day 6, 10, 14
and 21. Group 6: CY treatment given on day 5 only and
received no further immunization.
Separate groups of tumor injected mice (also 8 per
group) were set up for experimental controls. Group l:
mice received CY treatment on day 1 followed by
subsequent subcutaneous immunizations of KLH-Ribi
emulsion on day 2, 6, 10 and 17: Group 2: mice received
CY on day 1 followed by subsequent subcutaneous
immunizations of only Ribi compound on day 2, 6, 10 and
17. All animals were monitored for survival daily for
60 or more days. CY was administered intravenously at
~ . ~...i --
14
a volume of 0.2 ml. Ta-KLH was emulsified in 2.0 ml of
Ribi compound and 0.2 ml of the emulsion was
distributed equally among 2 subcutaneous sites in the
upper belly and 1 site at the base of the tail (for day
2 immunization only).
ELISA: Levels of anti-TFa antibodies (IgG and
IgM) in sera of surviving mice were determined about 7
weeks after initial tumor transplantation in an ELISA.
l0 Briefly, test and control sera were serially diluted in
wells of microtiter plate coated with TFa-HSA at a
concentration of 0.25 ug/well. Bound anti-TFa IgG and
IgM antibodies was detected with horseradish
peroxidase-conjugate goat anti-mouse IgG and IgM
antibodies, respectively.
Footpad testing for DTH responses: DTH was
evaluated by testing mice in the footpad with Ta-HSA
(50~,g) glycoconjugate on day 54 after initial tumor
transplantation. Mice were injected in the right
(test) or left (control) hind footpads with 30-50~C1 of
antigen in sterile saline or, for a control, sterile
saline alone. Just before and 24-48 hours after
injection, footpad thickness was measured with a
vernier caliper. The results were calculated as the
increase in footpad thickness of glycoconjugate-in
sterile saline-injected pads at 24 or 48 hours after
footpad challenge minus the increase in footpad
thickness of sterile saline only-injected pads over the
same time period.
Second challenge of surviving mice with TA3-Ha-
tumor: Four days after footpad testing, surviving
animals were further challenged with 1 x 104 TA3-Ha
~4~~.~~~
tumor cells intraperitoneally. Mice were monitored
daily for survival over a period of at least 60 days.
Immunoassays for the Measurement of
5 ImmunocomgetenceLImmunosuQpressive in Cancer Patients
The DTH response is an immunological specific
cell-mediated response which develops after the
intradermal injection or topical application of a test
10 antigen. Cancer patients can be tested for DTH
reactivities toward (i) an autologous tumor antigen in
the form of synthetic tumor associated glycoconjugate,
e.g., TaHSA, and (ii) a neoantigen such as 2,4-dinitro-
chlorobenzene (DNCB). If the patient has been
15 previously sensitized to the antigen, an inflammatory
reaction characterized by induration will develop 24 to
48 hours later. Failure to respond to these antigens
is indicative of immunosuppression in the patient.
Lymphocyte transformation is an extremely popular
in vitro technique used to measure cellular
immunocompetence. Small resting lymphocytes are
exposed to a mitogen (such as phytohemagglutin and
concanavalin A) and are transformed into large
lymphoblastic cells. The simplest method for assaying
lymphoproliferation upon exposure to the mitogens is
tritiated thymidine ([3H] thymidine) incorporation.
This measures the counts per minute (cpm) of tritiated
thymidine incorporated into DNA for a standard number
of cells. In addition to the mitogens mentioned,
synthetic tumor-associated glycoconjunate of TaHSA can
be used as an immunostimulant in the assay. A
significantly lower Stimulation Index (net
cpm/unstimulated cpm) is indicative of active
immunosuppression.
16
The identification of surface markers for
lymphocyte subpopulations and the development of
specific monoclonal antibodies to these markers enable
one to detect as well as quantify specific lymphocyte
subpopulation (such as T-helper (OKT4) and T-
suppressor (OKT8)) in cancer patients. Active
immunosuppression induced by a suppressor T lymphocyte
subpopulation can be revealed by measuring the T4 and
T8 lymphocyte subpopulation in the patient.
Natural Killer (NK) cells are of lymphoid
appearance. Their cytotoxic capabilities are not
dependent on prior sensitization. Measurement of NK
cell activity is usually done using a chromium release
assay, in which cells that are to be tested for NK
activity are incubated with chromium labeled K562
cells. After 3 to 4 hours, the supernatant from each
test well is collected, and the amount of chromium
released into the supernatant is measured. Cytotoxic T
lymphocyte activities against TF antigen-carrying
carcinomas also can be tested by a chromium release
assay as described above.
,,..,.,. ,
17
Examgle 1- Observation of Immunosuppression of Mucins
At least two mucins, epiglycanin and bovine
submaxillary mucins, are able to suppress DTH effector
cells (CD4+).
For the experiment whose results are shown in
Table 1 below, mice were first injected with various
l0 amounts of Epiglycanin or just saline as control. Six
to seven days later all mice were immunized with 50 ~,g
epiglycanin emulsified in complete Freund's adjuvant.
Foot-pad testings were preformed 7 days after
immunization and net foot-pad swellings were measured
at 24 and 48 hours. It will be seen that pretreatment
with epiglycanin reduced the degree of foot-pad
swelling (a classic measure of DTH response) by 70-95%.
We have also shown that this immunosuppressive
effect is adoptively transferred (see Table [lAJ
below). Mice were immunized subcutaneously with 50 ~g
Epiglycanin-CFA immediately after cell transfer and
were footpad-tested 7 days later with 30 ~tg of the
immunizing antigen. An 83% reduction in footpad
swelling was observed.
. ~ ~.~ _
18
Table 1- Immunosunpressive Effect of Epialvcanin on DTH
Response in Mice
Net Foot-pad swelling**
(MM)
Expt. Treat- Immuni- 24 hr 48 hr % Reduc-
ment zation@ tion
to
1 0.4 mL 50 ~.g Epi- 0.35 0.33
2 saline CFA 6-7 0.29 0.18 -
3 i.v. days later 0.26 0.21 - .
1 100 ~Cg 50 ~g Epi- 0.06 0.00 81.1
2 Epi CFA 6-7 ND ND _
3 i.v. days later 0.14 0.01 70.6
1 200 ~g 50 /.1g Epi- 0.01 0.05 90.5
2 Epi CFA 6-7 0.03 0.00 94.3
3 i.v. days later 0.15 0.00 71.2
** Average of 3-5 mice, Subcutaneously, multiple
sites of
injections
Table lA' Immunosuppressive Effect of Epicrlvcanin on
DTH Response in Mice
Net Foot-pad swelling***
Cells 24 hr 48 hr % Reduction
Treatments Transferred at 24 hrs.
Injected 6.4 x 107 0.37 0.35 -
0.4 mL spleen cells
saline
i.v.
Injected 6.4 x 107 0.05 0.07 83.0
200 ~cg Epi spleen cells
in 0.4 mL
saline i.v.
** Average of 5 mice
_ ~0~.~~~
,...
19
For the determination of the suppressor activity
of bovine submaxillary mucins (BSM), mice were first
injected intravenously with 200 ~g of sterile saline as
control. Six days later, animals were divided into
various groups and were immunized with 50 ~.g BSM
emulsified either in complete Freund's adjuvant or Ribi
compound. All animals were footpad tested for DTH
responsiveness 7 days after immunization with BSM. As
shown in Table 2 below, net swelling was depressed by
85-95%.
~i ..
Table 2
5 T r a a t m a n t
immunized with
Immuni- 50~g BSM-Ribi or 0 rs 24 Net
hou hours
zation CFA (S. C.) R L R L Swelling
10
1. BSM-CFA Treated with 0.2m1 2.10 2.10 2.10 2.10 0.00
2. BSM-CFA saline footpad 2.05 2.10 2.10 2.10 0.05
3. BSM-CFA with BSM(50~g) 2.00 2.05 2.05 2.05 0.05
-85% .033+.029
15
1. BSM-Ribi Treated with 20O~Cg 2.05 2.10 2.15 2.10 0.10
2. BSM-Ribi BSM i.v. footpad 2.05 2:05 2.10 2.10 0.00
3. BSM-Ribi with BSM (50~tg 2.10 2.10 2.10 2.10 0.00
-95% .033+.066
20
1. BSM-CFA Treated with 2~Cg 2.05 2.05 2.35 2.00 0.30
2. BSM-CFA BSM i.v. footpad 2.05 2.05 2.35 2.10 0.25
3. BSM-CFA with BSM (50~.g) 2.15 2.15 2.30 2.10 0.25
.23+.076
1. BSM-Ribi Treated with 0.2m1 2.05 2.05 2.55 2.00 0.50
2. BSM-Ribi saline footpad 2.05 2.05 2.60 2.05 0.55
3. BSM-Ribi with BSM (50~Cg) 2.05 2.00 3.00 2.00 0.95
.67+.25
21
Example 2: Cyclophosphamide Inhibition of
Immunosup~ressive Effect of Mucin
Mice were injected either with 0.4m1 (containing
either 200 ~,g or 100 ~g Epiglycanin) Epiglycanin or
sterile saline solution intravenously. Six days after
initial injection, mice were treated intravenously with
CY (100m1/kg) or sterile saline solution as control,
twenty four hours prior to immunization with 50 ~Cg
Epiglycanin emulsified in equal. volume of complete
Freund's adjuvant. Seven days after immunization, mice
were foot-pad tested with 50y~g Epiglycanin. As shown
in Table 3, pretreatment with cyclophosphamide enhanced
the immune response to epiglycanin in mice previously
given an immunosuppressive dose.
202~~~
,~
22
0 hours 24 hours 48
hours
treatment L R L R Net L R det
1. 100 ~.tg Epi 2.00 2.00 2.05 2.00 0.05 2.00 2.00 0.00
iv 1/ 10
2. saline 1/16 2.10 2.00 2.10 2.00 0.00 2.05 2.00 0.00
50 ~Cg Epi
3. CFA 2.00 2.00 2.05 2.00 0.05 2.05 2.00 0.05
4. 2.00 2.00 2.05 1.95 0.05 2.00 2.00 0.00
(-82.14%)* 03.025 .0125.025
.
1. 100 ~Cg Epi 2.00 2.00 2.50 2.00 0.50 2.20 2.00 0.20
2. CY 2.00 2.00 2.05 2.00 0.05 2.05 2.00 1.05
3. 50 ~,t,g Epi 2.00 2.00 2.30 2.00 0.30 2.20 2.00 0.20
4. CFA 2.00 2.00 2.15 2.00 0.15 2.25 2.00 0.25
(+19%)** 25.195 .175.087
.
1. Saline (0.4m1) 2.00 2.05 2.10 2.00 0.10 2.05 2.05 0.05
2. 50 /~,g Epi 2.00 2.00 2.30 2.00 0.30 2.25 2.00 0.25
3. CFA 2.00 2.00 2.25 2.00 0.25 2.15 2.00 0.15
4. 2.00 2.05 2.20 2.05 0.20 2.15 2.00 0.15
.2 1.085 .1 5.08
Clone
1. 200 ~g Epi 2.00 2.00 2.05 2.00 0.05 2.00 2.00 0.00
2. saline 2.00 2.00 2.00 2.00 0.00 2.00 2.00 0.00
3. 50 ~.tg Epi 2.00 2.00 2.05 2.00 0.05 2.05 2.00 0.05
4. CFA 2.10 2.05 2.15 2.00 0.05 2.10 2.00 0.00
(-82.14%)* 5
.037.02
1. 200 ~g Epi 2.05 2.00 2.35 2.00 0.35 2.25 2.00 0.20
2. saline 2.15 2.10 2.20 2.10 0.05 2.10 2.10 0.00
3. 50 ~.tg Epi 2.10 2.05 2.55 2.00 0.45 2.40 2.00 0.30
4. CFA 2.05 2.05 2.50 2.00 0.40 2.30 2.00 0.25
(+47. 6%)** .31.1 79
23
1. 200 ~,g Epi 2.00 2.00 2.20 2.000.20 2.102.00 0.10
2. saline 2.00 2.05 2.25 2.000.25 2.152.00 0.15
3. 50 ~g Epi 2.05 2.00 2.30 2.000.25 2.202.00 0.15
4. CFA 2.05 2.05 2.20 2.050.15 2.202.05 0.15
.21+.048
Example 3- Thera peutic Effect of Treatment
Combined
with Cvclop hosphamide T-Algha Gl~ cocon-iuaate
and
The sequential administration of cyclophosphamide
and a T-alpha epitope/bearing synthetic glycoconjugate
improved survival (Table 3) of mice challenged with
Ta3Ha mouse mammary adenocarcinoma tumor cells, which
express epiglycanin.
Table 3. Effect of combined treatment and S-TAG
of CY
on the development of TA3-Ha tumor in CAF1/J
Mice
No . of
tumor-
M a d i a f r a
n a
Group survival mice/-
No. Treatments time (days) total
1 none 17-19 0/16
2 CY(day 1) only 18-21 0/16
3 CY(day 5) only 23 2/9
4 CY(day 1&5) only 26 1/9
5 CY(day 1) + KLH-Ribi 17 0/8
6 CY(day 1) + HSA-Ribi 20 0/8
7 CY(day 1) + Ribi 20 0/8
8 CY(day 1) + TaKLH-Ribi >100 14/17
9 CY(day 5) + TaKLH-Ribi 35 4/8
10 CY(day 1&5) + TaKLH-Ribi 46 4/8
11 TaKLH-Ribi only 20-22 4/16
12 TaKLH-Ribi + CY(day 5) 23 1/8
Four days after f ootpad testing, surviving
animals from active specific immunotherapy were further
challenged with 1 x 104 TA3-Ha tumor cells intraperit-
oneally. (This dose is greatly in excess of the LD50).
~J ',.i
24
Mice were monitored for survival daily for 60 or more
days.
The results are shown in Figure 1. It will be
seen that the best surviving group was the one given
both cyclophosphamide and T-alpha/KLH in Ribi adjuvant.
A more complex experimental comparison is shown in
Figure 2. It will be seen here that administration of
cyclosphamide alone had only an ephemeral effect on
survival. Best results (group 5) were obtained with
repeated courses of T-alpha/KLH in Ribi adjuvant after
the initial treatment with cyclophosphamide.
Example 4' Adoptive Transfer of Tumor Resistance From
Lonct-Term Survivors of Active Specific Immunotherapy
Long term survivors from the active specific
immunotherapy experiment were used in a local Winn
assay to test whether their immune splenic and lymph
node cells were able to inhibit tumor growth in vivo.
Splenic and lymph node cells obtained from various
treatment groups of mice were mixed with live TA3-Ha
tumor cells at a effector:target cell ratio of 100:1
and were injected subcutaneously into the footpads of
recipient mice pretreated either in cyclophosphamide
(100mg.kg i.v.) or saline in the same manner. The
footpad swelling was measured at 24-48 hours and every
2 days thereafter. Tumor size in the footpad was
expressed in terms of net swelling of footpad thickness
(mm).
Lymph node cells obtained from mice (treated with
CY and TA-KLH-Ribi immunizations) which survived
,.~ ..~ 2 0 1 3 9 6 6
inhibit tumor growth completely in a Winn type assay.
Spleen cells, on the other hand, did not transfer immunity.
The present invention, in the various aspects, extends
5 to the adoptive transfer of cell-mediated immunity to
adenocarcinomas expressing immunosuppressive mucins by
means of lymph node cells obtained from subjects who
responded favourably to the combined anti-immunosuppres-
sion, active specific immunotherapy taught herein. For a
10 general protocol for adoptive immunotherapy, see Rosenberg,
U.S. 4,690,915.
