U.S. patent application number 09/445903 was filed with the patent office on 2002-10-31 for adjuvant compositions for vaccines.
Invention is credited to BOON, THIERRY, SILLA, SILVIA, UYTTENHOVE, CATHERINE.
Application Number | 20020160011 09/445903 |
Document ID | / |
Family ID | 10814118 |
Filed Date | 2002-10-31 |
United States Patent
Application |
20020160011 |
Kind Code |
A1 |
BOON, THIERRY ; et
al. |
October 31, 2002 |
ADJUVANT COMPOSITIONS FOR VACCINES
Abstract
The present invention provides improved adjuvant compositions
comprising QS21/3DMPL and Interleukin 12. These find utility in a
range of prophylatic and therapeutic vaccines, including cancer
vaccines. 09/445903
Inventors: |
BOON, THIERRY; (BRUSSELS,
BE) ; SILLA, SILVIA; (BRUSSELS, BE) ;
UYTTENHOVE, CATHERINE; (CHAUMONT GISTOUX, BE) |
Correspondence
Address: |
SMITHKLINE BEECHAM CORPORATION
CORPORATE INTELLECTUAL PROPERTY-US, UW2220
P. O. BOX 1539
KING OF PRUSSIA
PA
19406-0939
US
|
Family ID: |
10814118 |
Appl. No.: |
09/445903 |
Filed: |
February 8, 2000 |
PCT Filed: |
June 9, 1998 |
PCT NO: |
PCT/EP98/03685 |
Current U.S.
Class: |
424/184.1 ;
424/198.1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 31/00 20180101; A61K 2039/55572 20130101; A61K 2039/55577
20130101; A61K 2039/55538 20130101; A61K 39/39 20130101 |
Class at
Publication: |
424/184.1 ;
424/198.1 |
International
Class: |
A61K 039/00; A61K
045/00; A61K 039/38 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 14, 1997 |
GB |
9712347.5 |
Claims
1. A vaccine composition comprising a saponin adjuvant,
monophosphoryl lipid A or derivative thereof, Interleukin 12, and
an antigen.
2. A vaccine composition as claimed in claim 1 wherein the
monophosphoryl lipid A is 3-O-deacylated monophosphoryl lipid
A.
3. A vaccine composition as claimed in claim 1 or claim 2 wherein
the saponin adjuvant is QS21.
4. A vaccine composition as claimed in claim 3 additionally
comprising cholesterol.
5. A vaccine composition as claimed in claim 1 to 4, wherein the
antigen is a tumour rejection antigen.
6. A vaccine composition as claimed herein for use in medicine.
7. Use of a saponin adjuvant, monophosphoryl lipid A or a
derivative thereof and Interleukin 12 and an antigen in the
manufacture of a vaccine for the treatment of prophylaxis of
pathogenic infections or cancer.
8. A method of enhancing an immune response to an antigen
comprising administering the antigen with an adjuvant composition
as described in cairns 1 to 4:
9. A method of treating or preventing a pathogenic infection in a
patient comprising administering a vaccine as claimed herein.
10. A method of producing a vaccine composition as claimed in claim
1, comprises admixing a saponin adjuvant, monophosphoryl lipid A or
derivative thereof, Interleukin 12 and an antigen.
Description
[0001] The present invention relates to improved adjuvant
compositions, for the stimulation of an immune response suitable
for immunotherapy applications. In particular the present invention
relates to compositions comprising mixture of a saponin adjuvant
with monophosphoryl lipid A or derivative thereof and interleukin
12. In particular, the invention relates to compositions comprising
3 de-o-acylated monosphoryl lipid A, QS21, and IL12. Such
compositions are particularly useful in the immunotherapy of
tumours.
[0002] Cancer is a disease developing from a single cell due to
genetic changes. Clinical detection of these tumours occurs mostly
in a relatively late stage of disease, when the primary tumour can
be removed by surgery, and the existence of micro metastases
settled in different organs has often already occurred.
Chemotherapy does often not completely eliminate these cells, which
then remain as a source for recurrent disease.
[0003] Immune cells are able to control all different tissues (with
the exception of the brain) and, due to their memory function, can
also eliminate hidden cells reentering the circulation
(metastasis). Therefore, an activated immune response to tumour
cells is expected to be of clinical benefit. Despite their
undifferentiated growth, tumour cells are in many aspects
indistinguishable from normal cells, and over-expression of certain
proteins or expression of mutated proteins is in most cases not
sufficient to activate the immune response. This situation results
in failure of immune surveillance. Thus, strategies for therapy of
disseminated tumours need to specifically activate the immune
response to tumour cells and to trigger migratory activity of
cytotoxic T cells for example leading to elimination of most and
possibly every single tumour cell. Genetic mutation in tumour cells
is intense, and strong immune responses are therefore required to
prevent further genetic changes of the tumour cells (escape
variants) under the pressure of the immune system.
[0004] It is now well established that cellular antigens which are
not cell surface proteins per se can be the targets of immune
rejection through their recognition by immune regulatory and
cytotoxic T cells. New potential target antigens for
immune-mediated tumour rejection are being identified, based on
their recognition by immune T cells, rather than by antibodies.
Such antigens may or may not induce antibody formation. It is now
recognized that the expression of tumour antigens by a cell is in
itself not sufficient for induction of an immune response to these
antigens. Initiation of a tumour rejection response requires a
series of immune amplification phenomena dependent on the
intervention of antigen presenting cells, which are responsible for
delivery of a series of activation signals which ultimately leads
to the rejection of the tumour.
[0005] Tumour rejection antigens which are presented on tumour
cells and which are recognised by cytotoxic T cells can lead to
lysis of the cell. To achieve this, in a clinical setting a vaccine
composition comprising a tumour rejection antigen needs to be
presented in a suitable adjuvant system to enable a suitable immune
response to be mounted. However, activation of the immune systems
requires activation signals which are initiated by antigen
presenting cells and are not activated by the tumour cells
themselves.
[0006] Vaccination with isolated tumour rejection antigens has been
envisaged either by recombinant proteins, by the use of live
recombinant vectors or by DNA vectors. Preferably subunit antigens
will be used. However, to ensure these are effective, powerful
adjuvant systems are required.
[0007] Accordingly, the present invention provides an adjuvant
composition comprising a combination of a saponin adjuvant in
combination with monophosphoryl lipid A or derivative thereof
together with the cytokine Interleukin 12.
[0008] Immunologically active saponin fractions having adjuvant
activity derived from the bark of the South American tree Quillaja
Saponaria Molina are known in the art. For example QS21, also known
as QA21, is an Hplc purified fraction from the Quillaja Saponaria
Molina tree and it's method of its production is disclosed (as
QA21) in U.S. Pat. No. 5,057,540. Quillaja saponin has also been
disclosed as an adjuvant by Scott et al, Int. Archs. Allergy Appl.
Immun., 1985, 77, 409.
[0009] Monosphoryl lipid A and derivatives thereof are known in the
art. A preferred derivative is 3 de-o-acylated monophosphoryl lipid
A, and is known from British Patent No. 2220211.
[0010] Interleukin 12 (IL-12) is known. For a review see Trinchieri
G. Interleukin-12-A proinflammatory cytokine with immunoregulatory
functions that bridge innate resistance and antigen-specific
adaptive immunity. Immunology 13: 251.276, 1995. It is a
heterodimeric cytokine produced mostly by phagocytic cells in
response to bacteria, bacterial products, and intracellular
parasites, and to some degree by B lymphocytes. In particular,
IL-12 is produced by antigen presenting cells and instrumental in
induction of TH-1 cell responses. IL-12 induces IFN-gamma from NK
and T cells, acts as a growth factor for activated NK and T cells,
enhances the cytotoxic activity of NK cells, and induces cytotoxic
T lymphocyte generation.
[0011] IL-12 and IL-12-induced IFN-gamma favor Th1 cell
differentiation by priming CD4 (+) T cells for high IFN-gamma
production. However, we surprisingly found that other cytokines,
such as IFN-.gamma., IL-2, IL-6, IL-7, GM-CSF or MCP were unable to
enhance the effect of the QS21/MPL adjuvant.
[0012] Preferably the compositions of the invention contain the
immunologically active saponin fraction in substantially pure form.
Preferably the compositions of the invention contain QS21 in
substantially pure form, that is to say, the QS21 is at least 90%
pure, preferably at least 95% pure and most preferably at least 98%
pure. Other immunologically active saponin fractions useful in
compositions of the invention include QA17/QS17.
[0013] In a preferred embodiment the composition also comprises a
sterol such as cholesterol wherein the sterol is present in an
excess ratio to that of the saponin. These show decreased
reactogenicity when compared to compositions in which the
cholesterol is absent, while the adjuvant effect is maintained. In
addition it is known that QS21 degrades under basic conditions
where the pH is about 7 or greater. Thus a further advantage is
that the stability of QS21 to basemediated hydrolysis is enhanced
in formulations containing cholesterol.
[0014] Although the adjuvant compositions can be utilised for the
treatment or prophylaxis of a range of disease, they find
particular utility in the field of cancer immunotherapy.
[0015] In particular, the adjuvant formulation finds utility
particularly with tumour rejection antigens such as those for
prostrate, breast, colorectal, pancreatic, renal or melanoma
cancers. Exemplary antigens include MAGE 1 and MAGE 3 or other MAGE
antigens for the treatment of melanoma, BAGE or GAGE LAGE (NY-eso1)
1)PRAME or Her-2/neu; Robbins and Kawakami (1996), Current Opinions
in Immunology 8, pps 628-636; Van den Eynde et al., International
Journal of Clinical & Laboratory Research (submitted 1997);
Correale et al. (1997), Journal of the National Cancer Institute
89, p293. Indeed these antigens are expressed in a wide range of
tumour types such as melanoma, lung carcinoma, sarcoma and bladder
carcinoma. Other classes of antigens useful in the context of the
present invention include tissue specific antigens such as Prostate
Specific antigen (PSA); Prostate Specific Membrane antigen (PMSA),
Melan A/Mart 1, gp100, tyrosinase TRP1 or TRP2.
[0016] Accordingly in one aspect of the present invention there is
provided a vaccine comprising an adjuvant composition according to
the invention and a tumour rejection antigen, or tissue specific
antigen.
[0017] Other antigens or antigenic compositions include for
example, polysaccharide antigens, protein antigens or DNA encoding
antigens or antigenic compositions derived from HIV-1, (such as gp
120 or gp 160), any of Feline Immunodeficiency virus, human or
animal herpes viruses, such as gD or derivatives thereof or
Immediate Early protein such as ICP27 from HSV1 of HSV2,
cytomegalovirus (especially human) (such as gB or derivatives
thereof), Varicella Zoster Virus (such as gpl, II or III), or from
a hepatitis virus such as hepatitis B virus for example Hepatitis B
Surface antigen or a derivative thereof, hepatitis A virus,
hepatitis C virus and hepatitis E virus, of from other viral
pathogens, such as Respiratory Syncytial virus (for example RSV F
and G proteins or immunogenic fragments thereof disclosed in U.S.
Pat. No. 5,149,650 or chimeric polypeptides containing immunogenic
fragments from HSRV proteins F and G, eg GF glycoprotein disclosed
in U.S. Pat. No. 5,194,595), antigens derived from meningitis
strains such as meningitis A, B and C, Streptococcus Pneumonia,
human papilloma virus, in particular from strains HPV6, 11, 16 and
18, Influenza virus, Haemophilus Influenza B (Hib), Epstein Barr
Virus (EBV), or derived from bacterial pathogens such as
Salmonella, Neisseria, Borrelia (for example OspA or OspB or
derivatives thereof), or Chlamydia, or Bordetella for example P.69,
PT and FHA, or derived from parasites such as plasmodium or
toxoplasma.
[0018] The P815 tumour is a mastocytoma, induced in a D BA/2 mouse
with methylcholanthrene and cultured as both an in vitro tumour and
cell line. This represents an excellent model system for the
human.
[0019] The model system described in this application is a murine
system whereby a a murine tumour antigen, P1A, expressed in the
mouse mastocytoma P815, is being tested for its ability to
stimulate CTL in the mouse with and without adjuvant. The
significance of this system is that P1A is a true tumour rejection
antigen in that its gene is the same in both normal and tumour
cells but the gene is silent in normal cells and only expressed in
tumour cells. This is in comparison to other P815 antigens that
were previously found and which are created by mutation of normal
alleles. These are called tum- variants or tum- antigens. Mutations
in the tumantigens create new antigenic peptides which can then be
recognised by CTL.
[0020] Tum- antigens are likely to be tumour-specific whereas true
tumour antigens will be shared between different tumours and
patients and therefore the latter will be better candidates for
vaccine formulations. Human tumour rejection antigens analogous to
P1A include the MAGE, BAGE, GAGE etc. families as described
earlier. These genes are found in both normal and tumour tissues
but the corresponding proteins are expressed only in tumours and in
normal testis. As the testis is an immune privileged site it is
unlikely to be affected by any vaccine.
[0021] P1A is a true murine TRAs. Therefore, one can test a large
number of adjuvants with a variety of different assays in mice
giving a good indication as to which formulations should be used
with human tumour rejection antigens in human clinical trials.
[0022] Preferred compositions of the invention are those forming a
liposome structure. Compositions where the sterol/immunologically
active saponin fraction forms an ISCOM structure also form an
aspect of the invention.
[0023] The ratio of QS21 : sterol will typically be in the order of
1:100 to 1:1 weight to weight. Preferably excess sterol is present,
the ratio of QS21: sterol being at least 1:2 w/w. Typically for
human administration QS21 and sterol will be present in a vaccine
in the range of about 1 ng to about 100.mu.g, preferably about 10
.mu.g to about 50 .mu.g per dose.
[0024] The liposomes preferably contain a neutral lipid, for
example phosphatidylcholine, which is preferably non-crystalline at
room temperature, for example eggyolk phosphatidylcholine, dioleoyl
phosphatidylcholine or dilauryl phosphatidylcholine. The liposomes
may also contain a charged lipid which increases the stability of
the lipsome-QS21 structure for liposomes composed of saturated
lipids. In these cases the amount of charged lipid is preferably
1-20% w/w, most preferably 5-10%. The ratio of sterol to
phospholipid is 1-50% (mol/mol), most preferably 20-25%.
[0025] The compositions of the invention also contain a 3
deacylated monophosphoryl lipid A derivative (3-de-0-acylated
monophosphoryl lipid A, also known as 3D-MPL) and is manufactured
by Ribi Immunochem, Montana. A preferred form is disclosed in
International Patent Application 92/116556.
[0026] Suitable compositions of the invention are those wherein
liposomes are initially prepared without 3D-MPL, and 3D-MPL is then
added, preferably as 100nm particles. The 3D-MPL is therefore not
contained within the vesicle membrane (known as 3D-MPL out).
Compositions where the 3D-MPL is contained within the vesicle
membrane (known as 3D-MPL in) also form an aspect of the invention.
The antigen can be contained within the vesicle membrane or
contained outside the vesicle membrane or encapsulated. Preferably
soluble antigens are outside and hydrophobic or lipidated antigens
are either contained inside or outside the membrane. The 3D-MPL
will be present in the range of about 1Ug to 100Ug and preferably
about 10 to 50 .mu.g per dose of human vaccine.
[0027] Often the vaccines of the invention will not require any
specific carrier and formulated in an aqueous or other
pharmaceutically acceptable buffer. In some cases it may be
advantageous that the vaccines of the present invention will
further contain alum.
Example 1:
[0028] a)--Immunisation of DBA/2 mice with peptide+3
D-MPL+QS21+Lipids .+-.IL12
[0029] Human tumours express antigens that can be recognized by
autologous CTL. These antigens constitute useful targets for cancer
immunotherapy. We decided to evaluate in the P815 murine
mastocytoma model the efficacy of an immunization method that could
be applied to human patients. Syngeneic DBA/2 mice were an-injected
with antigenic peptides mixed with adjuvant and murine IL12.
[0030] An adjuvant composition comprising QS21, lipids (DQ) and 3
de-o-acylated monophosphoryl lipid A (3D-MPL) was prepared.
[0031] Briefly a mixture of lipid (such as phosphatidyicholine
either from egg-yolk or synthetic) and cholesterol in organic
solvent, is dried down under vacuum (or alternatively under a
stream of inert gas). An aqueous solution (such as phosphate
buffered saline) is then added, and the vessel agitated until all
the lipid is in suspension. This suspension is then microfluidised
until the liposome size is reduced to 100 nm, and then sterile
filtered through a 0.2 Um filter. Extrusion or sonication could
replace this step.
[0032] The cholesterol phosphatidylcholine ratio is 1:4 (w/w), and
the aqueous solution is added to give a final cholesterol
concentration of 5 to 50 ng/ml. The liposomes have a defined size
of 100 nm and are referred to as SUV (for small unilamellar
vesicles). If this solution is repeatedly frozen and thawed the
vesicles fuse to form large multilamellar structures (MLV) of size
ranging from 500nm to 15.mu.m.
[0033] QS21 in aqueous solution is added to the liposomes. This
mixture is then added to 50 .mu.g of P198 peptide (KYQAVTTTL) and
3D-MPL.
[0034] FIG. 1 Immunisation of DBA/2 mice with peptide
p198.+-.DQS21/3D-MPL +IL12
[0035] DBA/2 mice were injected s.c. in the two footpads with
50.mu.g of P198 peptide (KYQAVTTTL), corresponding to the antigen
expressed by the P198 TUM--clone (Sibille et al., J. Exp. Med.,
1990: 172, 3545), mixed with the DQS21/3DMPL adjuvant (adjuvant)
100.mu.l final. For a second group of animals we added to the
peptide and adjuvant solution 50.mu.g (500U) murine IL12. This
murine IL12 was purified from the supernatant of transfected P1HTR
cells as described in Gajewski et al. (J. Immunol. 1995, 154:
5637-5648). On d1 and 2, we injected locally an additional dose of
IL12 100ng (1000U) or PBS. On day 16, the mice were bled and
stimulation of blood lymphocytes was performed by mixing
3.times.10.sup.6 Ficoll purified lymphocytes with 10.sup.5
irradiated stimulating cells (100 Gy) and 2.times.10.sup.6
irradiated normal syngeneic spleen cells (30 Gy) as feeder cells.
The stimulating cells were P1983 cells, an azaguanine-resistant
variant derived from the P198 TUM-clone. The cells were incubated
in 48-well plates in a final volume of 0,8ml MLTC medium described
in Warnier et al. (Int. J. Cancer, 1996, 67, 303-310). Seven days
later, CTL activity was measured in a standard chromium-release
assay using 1,000 5.sup.1Cr-labelled targets. Two targets were
used: the P1983 cells or the P511 cells (azaguanine-resistant
variant derived from the P815 TUM+cells) not expressing the P198
antigen. To eliminate non-specific lysis, 10.sup.5 cold P511 cells
were added as competitors. On day 26, the mice received a second
injection of peptide, adjuvant and IL12 or PBS, followed by two
local injections of 100ng (1000U) IL12 or PBS. A second bleeding of
the mice was performed on day 41 to estimate CTL activity after two
injections. Data are expressed in lytic units (LU)/10.sup.6
lymphocytes as described in Brichard et al. (Eur. J. Inununol.,
1995, 25: 664-671). Specific lytic units were calculated by
subtracting the values obtained with the negative targets (usually
less than 0,3 LU) from those obtained with the positive target.
Mice were scored as.+-.when the LU detected were comprised between
0,1 and 1;+when LU were comprised between 1 and 10; and++above 10
LU.
[0036] FIG. 2 CTL activity in mice injected with peptide
P198.+-.DQS21/3D-MPL.+-.IL12
[0037] After the first injection, no CTL activity was detected in
the mice injected with the peptide and the adjuvant. When IL12 was
added significant CTL activity was detected in all the animals. For
the majority of the mice (13/15) the response was moderate since we
measured less than 1 LU/10.sup.6PBL. After the second immunization,
two mice out of 15, injected with the peptide and adjuvant alone
were positive. In the group injected with IL12, CTL activity had
increased and half of the mice showed a very high response. The
addition of IL12 to the peptide and the adjuvant increased strongly
the number of responding mice and the level of CTL activity
observed after only a few injections.
[0038] FIG. 3 Immunisation of DBA/2 mice with peptide
P198.+-.DQS21/3D-MPL.+-.IL12 in footpads or flanks.
[0039] In this second experiment, we applied the immunisation
protocol described before (FIG. 1) with some modifications. To
determine the relative contribution of IL12 and adjuvant in CTL
induction, we injected one group of mice in the footpads with P198
peptide and IL12 without adjuvant. To test a s.c. injection site
that is applicable to humans, we also injected 2 groups of mice
s.c. in the flank instead of the footpads; the first one receiving
the peptide, the adjuvant and the IL12 and the second receiving
only the peptide and the adjuvant. Four injections were performed
and mice were bled after the first, the second and the fourth
injection for CTL activity determinations.
[0040] FIG. 4 CTL activity in mice injected with peptide
P198.+-.DQS21/3D-MPL.+-.IL12 in the footpads or the flanks.
[0041] After the first immunisation, we observed that 4 mice out of
10 injected with peptide, the adjuvant and IL12 in the footpads
showed a significant CTL activity. In the group injected without
adjuvant 3 mice also showed CTL activity but the response was
lower. Nearly no response was obtained after injection into the
flanks since only 2 mice receiving the peptide, the adjuvant and
IL12 showed a weak CTL activity.
[0042] After the second immunisation, all the mice receiving the
peptide, the adjuvant and IL12 combination in the footpads
exhibited high CTL activity. All the mice injected with the peptide
and the IL12 without adjuvant also showed a specific CTL activity,
but much weaker. The same situation is observed for the mice
injected in the flank with the peptide, the adjuvant and the IL12
while in the absence of IL12 no response is observed after
injection in the flank. After the fourth injection, all the mice
that received peptide, adjuvant and IL12 in the footpads had a CTL
activity located in the high values. We also observed an increase
in the average of CTL activity for the mice injected without
adjuvant or receiving the peptide, the adjuvant and the IL12 in the
flank. Even after 4 injections we did not observe any response in
the mice injected in the flank without IL12.
[0043] We confirm in this experiment the potent effect of IL12 on
the generation of CTL activity after immunisation with the P198
peptide. This effect is enhanced by the combination with the
DQS21/3D-MPL adjuvant since the response is obtained earlier in all
the mice and since the average level of response is higher. The
effect of IL12 is also required to obtain CTL activity when the
antigen is injected in the flank instead of in the footpads.
[0044] FIG. 5 IL12 dose curve
[0045] Mice were injected with the P198 peptide mixed with the
DQS21/3D-MPL adjuvant. Different doses of murine IL12 3ng (30U),
10ng (100U), 30ng (300U), 100ng (1000U) were mixed with the peptide
and the adjuvant and also repeated locally the two following days.
The control group received the peptide and the adjuvant but no
IL12. Mice were bled after each of the two immunisations to monitor
the appearance and level of CTL activity.
[0046] FIG. 6 CTL activity in mice receiving peptide
P198.+-.DQS21/3D-MPL.+-.various doses of IL12
[0047] In the two preceding experiments we used a high dose of IL12
(1.mu.g/mouse/day). Even if the IL12 was injected locally we saw a
systemic toxicity with symptoms similar to those observed in a LPS
shock. We decided to try decreasing doses of IL12. The effect of
IL12 was nearly fully maintained when the dose/mouse/day was
decreased to 10ng (100U). It disappeared when the mice were
injected with only 30 ng IL12. At that dose, the systemic toxicity
of IL12 was largely reduced but not totally absent.
[0048] FIG. 7 Immunisation of DBA/2 mice with peptide
P1A.+-.DQS21/3D-MPL.+-.IL12
[0049] After several experiments with the peptide P198 showing that
high CTL activity were induced by injections of a combination of
peptide, adjuvant and IL12, we decided to apply this protocol to
the P1A peptide. This peptide presented by the Ld molecule
constitutes the P815A antigen that is a major target for the immune
rejection in vivo (Uyttenhove et al. J. Exp. Med., 1983, 157:
1040-1052). Gene P1A, which code for the P815A antigen is expressed
in several mastocytoma tumour lines (Van den Eynde et al. J. Exp.
Med., 1991, 173: 1373-1384). Like the MAGE, BAGE and GAGE genes, it
is not expressed in adult normal tissues, with the exception of
spermatogonia in the testis (Van den Eynde et al, 1991 and
Uyttenhove et al, Int. J. Cancer, 1997, 70: 349-356). Accordingly,
the P815A antigen represents a good mouse model for the human MAGE,
BAGE and GAGE antigens.
[0050] Mice were injected s.c. in the two footpads with 50.mu.g of
P1A peptide (LPYLGWLVF described in Leth et al. Eur. J. Immunol.
1992, 22: 2283-2288) mixed with the adjuvant DQS21/3D-MPL. For one
group, 100ng (1000U) of IL-12 was added to the peptide and the
adjuvant. These mice received additional doses of 100ng (1000U)
IL-12 injected locally the two following days. This injection
scheme was repeated four times and the mice were bled after the
second and the fourth injection. The lymphocytes were restimulated
in vitro for 7 days and the CTL activity was measured in a
conventional.sup.51Cr assay. We used L1210. P1A cells as
stimulating cells. The syngeneic L1210 P1A transfectant cells
expressing the antigen P815AB were generated as described in
Uyttenhove et al. Int. J. Cancer (1997) 70: 349-356. As target
cells we used P511 cells expressing all the P815 antigens and
P1-204 cells, an antigen-loss variant not expressing the P815 AB
antigen described in Uyttenhove et al (J. Exp. Med., 157,
1040-1052, 1983). To avoid problems of non specific lysis, cold
P1-204 were added as competitors.
[0051] FIG. 8 CTL activity in mice injected with the P1A
peptide.+-.DQS21/13D-MPL .+-.IL12
[0052] After two injections, 9 mice out of ten showed significant
CTL activity specific for the P815A antigen when IL12 was added to
the peptide and adjuvant. Half of those mice exhibited high CTL
activity levels. In the group injected without IL12, a positive
response was detected only in one mouse and this activity was
rather low.
[0053] After the fourth injection, all the mice receiving IL12 were
positive and the average of lytic units had increased. Without IL12
we detected a good CTL activity in four mice, one additional mouse
showed a very low response at the limit of the significance
threshold. In this system again, the addition of IL12 increased the
number of responding mice and diminished the number of injections
needed to obtain high and specific CTL activity. In this experiment
we injected a high dose of IL12 (100ng (1000U)/mouse/day). Like in
the previous experiments using the P198 peptide we observed
systemic toxic effects of the IL12.
[0054] Conclusion:
[0055] The addition of IL12 to peptide and adjuvant combination is
very effective at increasing the number of mice displaying high CTL
responses after immunisation. In addition, CTL responses appear
earlier in the presence of IL12.
* * * * *