U.S. patent application number 12/505022 was filed with the patent office on 2009-11-19 for cd 40 binding molecules and ctl pepetides for treating tumors.
This patent application is currently assigned to University Hospital Leiden. Invention is credited to Cornelis J. M. Melief, Rienk Offringa, Stephen P. Schoenberger, Rene Toes.
Application Number | 20090285814 12/505022 |
Document ID | / |
Family ID | 22199819 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090285814 |
Kind Code |
A1 |
Melief; Cornelis J. M. ; et
al. |
November 19, 2009 |
CD 40 Binding Molecules and CTL Pepetides for Treating Tumors
Abstract
Disclosed is a method and composition for treating tumors or
infectious diseases, wherein the composition includes CD40 binding
molecules together with CTL-activating peptides, e.g., tumor
antigens. Such composition is useful for enhancing the anti-tumor
effect of a peptide tumor vaccine, or for otherwise activating CTLs
so that the activated CTLs can act against tumorous or infected
cells. The CD40 binding molecules can include antibody molecules,
as well as homologues, analogues and modified or derived forms
thereof, including immunoglobulin fragments like Fab, F(ab').sub.2
and Fv, as well as other molecules including peptides,
oligonucleotides, peptidomimetics and organic compounds which bind
to CD40 and activate the CTL response.
Inventors: |
Melief; Cornelis J. M.;
(Haarlem, NL) ; Schoenberger; Stephen P.;
(Encinitas, CA) ; Offringa; Rienk; (San Mateo,
CA) ; Toes; Rene; (Leiden, NL) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
University Hospital Leiden
Leiden
NL
|
Family ID: |
22199819 |
Appl. No.: |
12/505022 |
Filed: |
July 17, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10227789 |
Aug 26, 2002 |
7563445 |
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12505022 |
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09316935 |
May 22, 1999 |
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10227789 |
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60086625 |
May 23, 1998 |
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Current U.S.
Class: |
424/133.1 ;
424/139.1 |
Current CPC
Class: |
A61K 39/0011 20130101;
A61P 31/12 20180101; A61K 48/00 20130101; A61K 2039/55516 20130101;
A61K 39/39 20130101; C07K 2317/75 20130101; C12N 2710/10322
20130101; A61K 2039/505 20130101; A61K 38/08 20130101; A61P 35/00
20180101; A61K 39/39541 20130101; C07K 2317/73 20130101; C07K
16/2875 20130101; C07K 2317/76 20130101; C07K 14/005 20130101; A61K
2039/60 20130101; C07K 16/2878 20130101; C12N 2710/20022 20130101;
A61K 39/39541 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/133.1 ;
424/139.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; A61P 31/12 20060101
A61P031/12 |
Claims
1. A pharmaceutical composition comprising (a) an activating
anti-CD40 antibody, (b) a CTL-activating peptide, and (c) a
pharmaceutically acceptable excipient or diluent.
2. The pharmaceutical composition of claim 1 wherein the
CTL-activating peptide is a tumor peptide or a viral peptide.
3. The pharmaceutical composition of claim 2 wherein the
CTL-activating peptide is a tumor peptide.
4. The pharmaceutical composition of claim 2 wherein the
CTL-activating peptide is a viral peptide.
5. The pharmaceutical composition of claim 1 wherein the
CTL-activating peptide is an HPV16 CTL-activating peptide.
6. The pharmaceutical composition of claim 1, wherein the
activating anti-CD40 antibody has reduced immunogenicity to avoid
an immune response against itself in a subject to which it is
administered.
7. The pharmaceutical composition of claim 2, wherein the
activating anti-CD40 antibody has reduced immunogenicity to avoid
an immune response against itself in a subject to which it is
administered.
8. The pharmaceutical composition of claim 3, wherein the
activating anti-CD40 antibody has reduced immunogenicity to avoid
an immune response against itself in a subject to which it is
administered.
9. The pharmaceutical composition of claim 4, wherein the
activating anti-CD40 antibody has reduced immunogenicity to avoid
an immune response against itself in a subject to which it is
administered.
11. The pharmaceutical composition of claim 6 wherein the
activating anti-CD40 antibody is human, humanized, chimeric or
Deimmunised.TM..
12. The pharmaceutical composition of claim 7 wherein the
activating anti-CD40 antibody is human, humanized, chimeric or
Deimmunised.TM..
13. The pharmaceutical composition of claim 8 wherein the
activating anti-CD40 antibody is human, humanized, chimeric or
Deimmunised.TM..
14. The pharmaceutical composition of claim 9 wherein the
activating anti-CD40 antibody is human, humanized, chimeric or
Deimmunised.TM..
15. A method of treating a tumor in a subject in need thereof,
comprising administering to the subject the pharmaceutical
composition of claim 8 in an amount effective to treat said tumor,
wherein the tumor expresses an antigen (a) that comprises or
corresponds to the tumor peptide, or (b) from which the tumor
peptide was derived.
16. The method of claim 15 wherein the subject is a human and the
activating anti-CD40 antibody is human, humanized, chimeric or
Deimmunised.TM..
17. The method of claim 15 wherein the pharmaceutical composition
is administered directly into the tumor.
18. A method of treating a viral infection in a subject in need
thereof, comprising administering to the subject the pharmaceutical
composition of claim 9 in an amount effective to treat said
infection, wherein virus-infected cells in the subject express an
antigen (a) that comprises or corresponds to the viral peptide, or
(b) from which the viral peptide was derived
19. The method of claim 18 wherein the subject is a human and the
activating anti-CD40 antibody is human, humanized, chimeric or
Deimmunised.TM..
Description
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] The invention includes CD40 binding molecules together with
CTL-activating peptides, including tumor antigens, in a
pharmaceutical composition.
Background of the Invention
[0002] Many tumors escape surveillance by our immune system. In
cancer patients there is clearly a quantitative and/or qualitative
defect in the immune system's specific mechanisms to delete tumor
cells. One of these mechanisms is provided by the cytotoxic T cells
(CTL) that can recognise and kill cells infected by virus or
transformed into cancer cells. Previously it was postulated that
dendritic cells (DC) stimulate T-helper cells which, in turn,
provide help for the activation of CTL. The present inventors have
demonstrated that the T-helper cell is not providing helper signals
directly to the CTL (by secretion of IL2), but rather, the T-helper
cell is providing a signal to the DC that induces yet
uncharacterised cell surface and/or soluble molecules that can
activate CTL in the absence of T-helper cells. The signal provided
by the T-helper cell to the DC is mediated by CD40L-CD40
interaction. This novel finding has provided an unique opportunity
for cancer immunotherapy.
[0003] The immune system is capable of killing autologous cells
when they become infected by virus or when they transform into
cancer cells. Such a potentially dangerous mechanism must, clearly,
be under tight control. The immune system's CTL circulate as
inactive precursors. To be activated, the precursor T-killer cell
must recognise its specific antigen peptide, which is presented as
MHC class I molecules on professional APC. However, in order to
prime these T cells, the APC also need to present the antigen in a
proper costimulatory context as provided by, amongst others, the
costimulatory surface molecules B7.1 and B7.2 and by the lymphokine
IL-12.
[0004] Until recently it was believed that a T-helper cell that
recognises the same antigen on the same APC is needed to fully
activate the CTL. The specific T-helper cell would supply cytokines
such as IL-2 needed for the activation of the CTL. Guerder and
Matzinger (J. Exp. Med. 176:553 (1992)), however, proposed the
"licensing" model for CTL activation. In this model it was
suggested that the T-helper cell, when recognising its antigen on a
professional APC, would deliver an activation signal to the APC
that as a result would be able to subsequently activate a CTL
without the need for the T-helper cell to be present. Only very
recently, the molecular mechanism of the licensing model has been
elucidated. Schoenberger et al. (Nature 393:480 (1998)), described
the role of the CD40L-CD40 pathway in the licensing model.
Interaction between T-helper cell and DC through the CD40-CD40L
binding results in activation of the DC, thereby enabling the DC to
efficiently prime naive CTL.
[0005] DC circulate through the tissues of our body and in this
manner can collect, process and present antigens. After collection
of antigens, they migrate to the draining lymph nodes where they
present antigen to the T cells. It is well known that a DC needs to
be activated to perform optimally. Resting DC express only modest
levels of MHC and costimulatory molecules and are poor stimulators
of T cells. DC can be activated by inflammatory cytokines and
bacterial products, which results in upregulation of MHC and
costimulatory molecules. Activation of DC into fully mature DC,
expressing optimal levels of MHC molecules, costimulatory molecules
and lymphokines such as IL-12, requires additional triggering of
these cells through the CD40 receptor. Consequently, the
combination of inflammatory cytokines at the site of antigen uptake
and the CD40L-CD40 interaction during the T-helper cell interaction
result in an optimal capacity to license the DC for CTL
activation.
[0006] Many tumors escape immune surveillance by specific CTL
mechanisms. If DC gather tumor antigens under non-inflammatory
conditions, the number of T-helper cells that are activated may be
to low to induce enough CTL to be activated to induce an
appropriate anti-tumor response. This concept has prompted
investigators to help the immune system by administration of
cytokines such as IL-2 and IL-12 that directly stimulate CTL
activity or by boosting antigen presentation by administration of
tumor cells transfected with GM-CSF. These strategies have met
variable but encouraging results.
[0007] It is clear that there is still a great need to find ways to
generate and/or enhance protective anti-tumor responses involving
cellular and humoral immunity. The CD40 activation pathway was
found to be a major immunoregulatory pathway for the generation of
primary humoral and cellular immune responses. As described above,
the CD40 pathway on DC is responsible for the induction of
anti-tumor CTL responses. In addition, activation of the CD40
pathway on macrophages stimulates a strong tumoricidal
activity.
SUMMARY OF THE INVENTION
[0008] The invention includes CD40 binding molecules together with
CTL-activating peptides, including tumor antigens in a
pharmaceutical composition. Such composition is useful for
enhancing the anti-tumor effect of a peptide tumor vaccine, or for
otherwise activating CTLs so that the activated CTLs can act
against tumorous or infected cells. The CD40 binding molecules can
include antibody molecules, as well as homologues, analogues and
modified or derived forms thereof, including immunoglobulin
fragments like Fab, F(ab').sub.2 and Fv, as well as small molecules
including peptides, oligonucleotides, peptidomimetics and organic
compounds which bind to CD40 and activate the CTL response.
CTL-activating peptides include the adenovirus-derived E1A peptide,
having the sequence SGPSNTPPEI (SEQ ID NO:2), and the HPVI6 E7
peptide derived from human papillomavirus type 16, having the
sequence RAHYNIVTF (SEQ ID NO:3).
[0009] The CD40 binding molecule and the CTL activating peptide can
be administered to a patient by suitable means, including
injection, or gene constructs encoding such a molecule and a
peptide can be administered, and the molecule and peptide thereby
produced in vivo or ex vivo. Such a gene therapy is conducted
according to methods well known in the art. If the transfection or
infection of the gene constructs is done ex vivo, the infected or
transfected cells can be administered to the patient, or these
steps can be done in vivo whereby the molecule and the peptide are
produced endogenously. The transfection or infection, if done ex
vivo, can be by conventional methods, including electroporation,
calcium phosphate transfection, micro-injection or by incorporating
the gene constructs into suitable liposomes. Vectors, including a
retrovirus, adenovirus or a parvovirus vector, or plasmids, can be
used to incorporate the gene constructs, which are then expressed
in vivo or ex vivo.
[0010] It is demonstrated herein that T-cell help for CTL priming
is mediated through CD40-CD40Ligand (CD40L) interactions, and that
lack of CTL priming in the absence of CD4.sup.+ T cells can be
restored by monoclonal antibody (mAb)-mediated CD40 activation of
bone marrow-derived APC in the presence of CTL-activating peptides
including tumor antigens.
[0011] Furthermore, blockade of CD40L, expressed by CD4.sup.+ T
cells, results in the failure to raise CTL immunity. This defect
can be overcome by in vivo CD40-triggering. In vivo triggering of
CD40 can markedly enhance the efficacy of peptide-based anti-tumor
vaccines, or otherwise activate CTLs to result in an anti-tumor or
anti-infected cell reaction.
[0012] It is also noted that a CTL-activating peptide can become
tolerogenic, meaning that the host reaction against cells
expressing such peptide is inhibited, in the absence of anti-CD40.
However, such a peptide combined with an activating anti-CD40
antibody converts tolerization into strong CTL activation.
Moreover, as noted above, CD40-ligation can provide an already
protective tumor-specific peptide-vaccine with the capacity to
induce therapeutic CTL immunity in tumor-bearing mice.
[0013] These findings together demonstrate that the CD40-CD40L pair
acts as a switch determining whether naive peripheral CTL are
primed or tolerized, Therefore CD40-binding agents such as
monoclonal antibodies and other stimulatory ligands can be
effectively used in combination with a CTL-activating peptide.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1: Cross-priming of E1B-specific CTLs requires
CD4.sup.+ T helper cells
[0015] Splenocytes from normal (a) or CD4-depleted B6 (b) mice
immunized with Ad5EI-BALB/c MECs were tested at various
effector/target ratios for lysis of syngeneic MEC target cells
loaded with the E1B.sub.192-200 peptide (solid lines), which is
derived from human adenovirus and has the sequence VNIRNCCYI (SEQ
ID NO:1) or a D.sup.d-restricted control peptide HPV-16
E7.sub.49-57 (dashed lines). Each line represents one mouse. Data
shown represent one experiment of three performed with similar
results.
[0016] FIG. 2: CD40 activation replaces CD4.sup.+ T helper
cells
[0017] Splenocytes from CD4-depleted (a, b) or class II-deficient
I-Ab-knockout (KO) (c, d) B6 mice were immunized with Ad5E1-BALB/c
MECs and treated with either the CD40-activating antibody (Ab)
FGK45 (a, c) or an isotype control antibody (b, d). These
splenocytes were tested for E1B-specific CTL activity on syngeneic
MEC target cells loaded with either the E1B.sub.192-200 peptide
(solid lines) or the HPV-16 E7.sub.49-57 control peptide (dashed
lines). Each line represents a single mouse. Data depicted are from
two independent experiments. E/T ratio, effector/target ratio.
[0018] FIG. 3: B cells are not essential as cross-priming APCs or
for anti-CD40-mediated restoration of cross-priming
[0019] Splenocytes were taken from untreated (a), CD4-depleted
B-cell-deficient B6 MT mice (b, c), 1 which were immunized with
Ad5EI-BALB/c MECs and which received either an isotype control
antibody (b) or the CD40-activating antibody FGK45 (c). These
splenocytes were tested for E1B-specific CTL activity on syngeneic
MEC target cells loaded with either the E1B.sub.192-200 peptide
(solid lines) or the HPV E7.sub.49-57 control peptide (dashed
lines). Each line represents one mouse. Data shown represent one
experiment of two performed with similar results.
[0020] FIG. 4: CD40L blockade-prevents cross-priming of
E1B-specific CTLs
[0021] Splenocytes were taken from B6 mice immunized with
Ad5E1-BALB/c MECs and treated with the CD40L-blocking antibody MR-1
(a), or control antibody (b), or from mice treated with the
CD40L-blocking antibody MR-1 in combination with the
CD40-activating antibody FGK45 (c) 24 h after immunization. These
splenocytes were tested for E1B-specific CTL activity on syngeneic
MEC target cells loaded with the E1B.sub.192-200 peptide (solid
lines) or the HPV-16 E7.sub.49-57 control peptide (dashed lines).
Each line represents one mouse. Data shown represent one experiment
of three performed with similar results. E/T ratio, effector/target
ratio.
[0022] FIG. 5: Mice injected s.c. with the E1B-peptide are no
longer able to mount E1B-specific CTL
[0023] C57BL/6 mice were injected twice s.c. (1 week interval) with
20 .mu.g E1A-peptide (a, b) or control-peptide (c, d) in IFA, and
challenged i.p. 1 day later with SAMB7 (b, d), a cell line
expressing high amounts of E1A-peptide. Bulk cultures derived from
these mice were tested for E1B-specific cytotoxicity on target
cells pulsed with the E1B-peptide (-.box-solid.-) or the HPV16
E7-peptide (-.largecircle.-). Specific lysis of representative bulk
cultures at different effector to target (E/T) ratios is shown.
This experiment has been repeated 4 times with comparable
results.
[0024] FIG. 6: Tolerizing E1B-peptide is rapidly distributed
systemically after s.c. injection in IFA
[0025] Spleen cells derived from untreated C57BL/6 mice (-), or
from mice injected s.c. 16 h earlier with 100 .mu.g of E1A- or
HPVI6 E7-peptide in IFA were used as stimulator cells for an
E1A-specific CTL clone. [.sup.3H]-thymidine incorporation
(cpm).+-.SEM is shown for different effector to stimulator
concentrations, without subtraction of background counts. Results
are representative of 5 independent experiments.
[0026] FIG. 7: CTL-tolerance induction is reverted into CTL-priming
after CD40-triggering in vivo
[0027] Wild type C57BL/6 mice (a, b) and H-2.sup.b CD40.sup.-/-
mice (c, d) were injected s.c. with 20 .mu.g E1B-peptide in IFA
alone (c), in combination with a rat IgG2a control antibody (a), or
in combination with the anti-CD40 mAb FGK-45 (b, d). Bulk cultures
from these mice were tested for E1B-specific cytotoxicity on target
cells pulsed with the E1B-peptide (-.box-solid.-), the HPV16
E7-peptide (-.largecircle.-) or Ad5E1 transformed tumor cells
(-.diamond-solid.-). Specific lysis of representative bulk cultures
at different E/T ratios is shown. This experiment has been repeated
18 (B6 mice) and 2 (CD40.sup.-/- mice) times, respectively, with
comparable results. In (e) the % specific lysis of 23 respectively
37 bulk CTL cultures derived from B6 mice injected with E1B-peptide
in IFA alone (left) or in combination with the anti-CD40 mAb
(right) at an E/T of 60 is shown. Mean plus standard deviation of
each group are shown (E1B versus E1A+anti-CD40: p<0.01, student
t-test).
[0028] FIG. 8: Therapy of HPV16 E6 and E7 transformed cells by
combination treatment of peptide together with in vivo CD40
triggering
[0029] Mice were injected s.c with 25.000 HPVI6 transformed
syngeneic cells (TC-1). C57BL/6 mice were left untreated
(-.largecircle.-) or after 6 days received 100 .mu.g HPV16
E7-peptide i.p. in IFA (-.quadrature.-), 100 .mu.g HPV16 E7-peptide
i.p. in IFA in combination with the anti-CD40 mAb FGK-45
(-.box-solid.-) or a control peptide i.p. in IFA in combination
with the anti-CD40 mAb FGK-45 (- -) The percentage of tumor bearing
mice is depicted for different treatment groups (n=10) in (a). The
differences between the group treated with the HPV16-peptide plus
the anti-CD40 mAb and the other three groups were statistically
significant (p<0.01) (Log-Rank test). In (b) the percentage of
surviving animals is shown (E7-peptide-treated group vs E7-peptide
plus anti-CD40-treated group: p=0.002, Log-Rank test).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0030] The CD40 binding molecules of the invention can be made by
conventional production and screening techniques. A rat and a
hamster anti-mouse CD40 monoclonal antibody ("Mabs") are each
described in Nature 393: 474-77 (1998) and are available
commercially (Pharmingen, Inc., Calif.). The anti-mouse CD40 MAb,
designated FGK45, which is used in the experiments described below,
is described by Rolink. A. et al., Immunity 5:319-330 (1996).
Anti-human CD40 MAbs can be made following techniques well-known in
the art, and described by G. Kohler and C. Milstein (Nature, 1975:
256:495-497). MAbs can be raised by immunizing rodents (e.g., mice,
rats, hamsters and guinea pigs) with either native CD40 as
expressed on cells or purified from human plasma or urine, or
recombinant CD40 or its fragments, expressed in a eukaryotic or
prokaryotic system. Other animals can be used for immunization,
e.g., non-human primates, transgenic mice expressing human
immunoglobulins and severe combined immunodeficient (SCID) mice
transplanted with human B lymphocytes. Hybridomas can be generated
by conventional procedures by fusing B lymphocytes from the
immunized animals with myeloma cells (e.g., Sp2/0 and NSO), as
described by G. Kohler and C. Milstein, id. In addition, anti-CD40
MAbs can be generated by screening of recombinant single-chain Fv
or Fab libraries from human B lymphocytes in phage-display systems.
The specificity of the MAbs to CD40 can be tested by enzyme linked
immunosorbent assay (ELISA), Western immunoblotting, or other
immunochemical techniques. The activating activity of the
antibodies on CTLs, in combination with a CTL-activating peptide,
can be assessed using the assays described in the Examples
below.
[0031] For treating humans, the anti-CD40 MAbs would preferably be
used as chimeric, Deimmunised.TM., humanized or human antibodies.
Such antibodies can reduce immunogenicity and thus avoid human
anti-mouse antibody (HAMA) response. It is preferable that the
antibody be IgG4, IgG2, or other genetically mutated IgG or IgM
which does not augment antibody-dependent cellular cytotoxicity (S.
M. Canfield and S. L. Morrison, J. Exp. Med., 1991:173:1483-91) and
complement mediated cytolysis (Y. Xu et al., J Biol. Chem., 1994:
269: 3468-74; V. L. Pulito et al., J Immunol., 1996;
156:2840-50).
[0032] Chimeric antibodies are produced by recombinant processes
well known in the art, and have an animal variable region and a
human constant region. Humanized antibodies have a greater degree
of human peptide sequences than do chimeric antibodies. In a
humanized antibody, only the complementarity determining regions
(CDRs) which are responsible for antigen binding and specificity
are animal derived and have an amino acid sequence corresponding to
the animal antibody, and substantially all of the remaining
portions of the molecule (except, in some cases, small portions of
the framework regions within the variable region) are human derived
and correspond in amino acid sequence to a human antibody. See: L.
Riechmann et al., Nature, 1988; 332:323-327; G. Winter, U.S. Pat.
No. 5,225,539; C. Queen et al., U.S. Pat. No. 5,530,101.
[0033] Deimmunised.TM. antibodies are antibodies in which the T and
B cell epitopes have been eliminated, as described in International
Patent Application PCT/GB98/01473. They have reduced immunogenicity
when applied in vivo.
[0034] Human antibodies can be made by several different ways,
including by use of human immunoglobulin expression libraries
(Stratagene Corp., La Jolla, Calif.) to produce fragments of human
antibodies (V.sub.H, V.sub.L, Fv, Fd, Fab, or F(ab').sub.2, and
using these fragments to construct whole human antibodies using
techniques similar to those for producing chimeric antibodies.
Human antibodies can also be produced in transgenic mice with a
human immunoglobulin genome. Such mice are available from Abgenix,
Inc., Fremont, Calif., and Medarex, Inc., Annandale, N.J.
[0035] One can also create single peptide chain binding molecules
in which the heavy and light chain Fv regions are connected. Single
chain antibodies ("ScFv") and the method of their construction are
described in U.S. Pat. No. 4,946,778. Alternatively, Fab can be
constructed and expressed by similar means (M. J. Evans et al., J
Immunol. Meth., 1995; 184:123-138). All of the wholly and partially
human antibodies are less immunogenic than wholly murine MAbs, and
the fragments and single chain antibodies are also less
immunogenic. All these types of antibodies are therefore less
likely to evoke an immune or allergic response. Consequently, they
are better suited for in vivo administration in humans than wholly
animal antibodies, especially when repeated or long-term
administration is necessary. In addition, the smaller size of the
antibody fragment may help improve tissue bioavailability, which
may be critical for better dose accumulation in acute disease
indications, such as tumor treatment.
[0036] Based on the molecular structures of the variable regions of
the anti-CD40 mAbs or the known CTL-activating peptides, one could
use molecular modeling and rational molecular design to generate
and screen molecules which mimic the molecular structures of the
binding region of the antibodies or the peptides, respectively, and
activate CTLs. These small molecules can be peptides,
peptidomimetics, oligonucleotides, or other organic compounds. The
mimicking molecules can be used for treatment of cancers and
infections. Alternatively, one could use large-scale screening
procedures commonly used in the field to isolate suitable molecules
from libraries of compounds.
[0037] The dosage for the molecules of the invention can be readily
determined by extrapolation from the in vitro tests and assays
described below, or from animal experiments or from human clinical
trials. The molecules of the invention would be preferentially
administered by injection, in the case of antibodies or proteins,
although certain small molecules may be suited for oral
administration. The assays and tests demonstrating the efficacy of
the invention are described below.
Example 1
Signaling Through CD40 can Replace CD4.sup.+ Helper T Cells in CTL
Priming
[0038] A well characterized model system to probe the mechanism of
T-cell help for the, primary activation of CD8+ CTL responses in
vivo was used. C57BL/6 (with the major histocompatibility complex
(MHC) H-2.sup.b) mice immunized with allogenic BALB/c (H-2.sup.d)
mouse embryo cells (MECs) expressing the human adenovirus type 5
early region 1 (Ad5EI-BALB/c MECs) generated strong CTL responses
against an H-2D.sup.d-restricted epitope of the adenovirus E1B
protein (E1B.sub.192-200) (FIG. 1a). As the allogeneic H-2.sup.d
MHC molecules expressed by the Ad5E1-BALC/c MECs cannot prime
H-2.sup.b-restricted host CTLs, generation of E1B-specific CTLs
must require cross-priming, that is, the uptake and
H-2.sup.b-restricted re-presentation of antigen by host APCs.
Cross-priming of E1B-specific CTLs is strictly helper-dependent
(FIG. 1b), as mice depleted of CD4.sup.+ T-helper (Th) cells before
immunization no longer mounted an E1B-specific CTL response.
[0039] To investigate whether signalling through CD40 can replace
CD4.sup.+ helper T cells in CTL priming, mice were depleted of
CD4.sup.+ T cells in vivo before immunization with Ad5E1BALB/c
MECs. One day after immunization, the mice received a single
injection of the activating antibody anti-mouse CD40 mAb FGK45, or
of an isotype-matched control antibody. Administration of FGK45 to
CD4-depleted, immunized mice resulted in the efficient restoration
of E1B-specific CTL responses (FIG. 2a) whereas treatment with the
control antibody did not (FIG. 2b). Priming of E LIB-specific CTLs
was not detected in naive mice treated with FGK45 alone (not
shown). To address the possibility that the effect of FGK45 was
mediated through remaining D4.sup.+ cells that were not depleted by
treatment with the anti-CD4 antibody, B6 I-A.sup.b knockout mice,
which lack mature functional CD4.sup.+ peripheral T cells, were
immunized with the Ad5EI-BALB/c MECs. The response to immunization
in these mice mirrors that seen in the CD4-depleted mice, in that
E1B-specific CTLs were detectable only in mice receiving the
CD40-activating antibody (FIG. 2c), and not in those receiving the
control antibody (FIG. 2d).
[0040] It was also studied whether the requirement for anti-CD40
antibodies in priming of CTLs in CD4-depleted mice could be
replaced by bacterial lipopolysaccharide (LPS) (50 .mu.g
intravenous), a potent inducer of proinflammatory cytokines, or by
administration of IL-2 (1.times.10.sup.5 units in incomplete Freund
adjuvant, subcutaneous) following immunization with Ad5EI-BALB/c
MECs. Whereas CD4-depleted mice treated with FGK45 exhibited strong
E1B-specific CTL activity, neither LPS or IL-2 treatment resulted
in detectable CTL priming (not shown).
[0041] Ligation of CD40 on B cells upregulates their costimulatory
activity, suggesting a role for these cells in the restoration of
CTL priming by treatment with CD40 activating antibodies. To
address this question, B6 MT mice, which lack mature B cells, were
immunized with the allogeneic Ad5EI-BALB/c MECs. Cross-priming of
E1B-specific CTLs did not require mature B cells (FIG. 3a).
However, when depleted of CD4.sup.+ cells, the B-cell deficient
mice did not generate an E1B-specific CTL response (FIG. 3b).
Activation through CD40 with the FGK45 monoclonal antibody
completely restored the capacity of CD4-depleted MT mice to prime
E1B-specific CTLs (FIG. 3c). Thus B cells are not required as APCs
or accessory cells for cross-priming in this model system, nor are
they required for CD40-mediated restoration of cross priming of
CTLs in the absence of CD4.sup.+ helper T cells. These results
demonstrate that activation of bone marrow derived APC through CD40
can bypass the requirement for CD4.sup.+ T-helper cells in the
cross-priming of E1B-specific CTLs.
Example 2
Blocking the Ability of CD4.sup.+ Helper T Cells to Interact with
APC Through the CD40L-CD40 Pathway Prevents Antigen-Specific CTL
Responses in Normal Mice
[0042] If the CD40L-CD40 interaction represents the physiological
pathway used by CD4.sup.+ helper T cells to help CTLs, blocking the
ability of the CD4.sup.+ T cells to interact with APC through
CD40L-CD40 interaction would be expected to diminish priming of
E1B-specific CTL responses in normal mice. B6 mice were immunized
with Ad5E1-BALB/c MECs and then treated with either the
CD40L-blocking antibody MR1, or control antibody. Blockade of CD40L
results in drastically reduced E1B-specific CTL responses (FIG. 4a)
compared to the efficient CTL priming seen in mice receiving the
control antibodies (FIG. 4b). The priming defect induced by CD40L
blockade was fully restored following CD40 signalling by FGK45
(FIG. 4c). Thus the defect in CTL-priming induced by CD40L blockade
lies in the failure of TH cells to transmit, rather than to
receive, CD40L-mediated signals.
Example 3
E1B-specific CTL Unresponsiveness after Peptide Administration
[0043] A previously described model system has been used (Toes et
al., J. Immunol. 156:3911 (1996)). It has been shown that s.c.
vaccination with the Ad5E1A-derived CTL epitope SGPSNTPPEI (SEQ ID
NO: 2) in IFA prevents mice from controlling the outgrowth of
Ad5E1A-expressing tumors. This indicates that the E1A/IFA vaccine
induced suppression rather than induction of E1A-specific CTL
immunity. Moreover, administration of the E1A/IFA vaccine to T cell
receptor (TCR)-transgenic mice, which express the TCR-.alpha. and
.beta. chains of an E1A-specific CTL clone, strongly suppressed
tumor-specific CTL-mediated immunity. These experiments examined
the effects of peptide administration on a monoclonal CTL
population. To establish whether s.c. E1A-peptide vaccination also
induces E1A-specific CTL tolerance at the polyclonal CTL level,
wild type (wt) C57BL/6 mice were injected with either E1A-peptide
(FIGS. 5a and 5b) or a control peptide FIGS. 5c and 5d). One day
later the mice were boosted with a syngeneic cell line expressing
high levels of the E1A-peptide at its surface (FIGS. 5b and 5d).
Injection of this cell line into mice primed with the control
peptide readily induces E1A-specific immunity (FIG. 5d). However,
the ability of mice to mount E1A-specific CTL responses was
abrogated after injection of the E1A/IFA vaccine (FIG. 5b). These
data indicate that injection of the E1A-peptide not only leads to
E1A-specific tolerance in TCR-transgenic mice but also in mice
expressing a polyclonal E1A-specific T cell repertoire.
[0044] Since s.c. injection of the E1A/IFA vaccine leads to
systemic CTL tolerance, it was investigated whether the E1A-peptide
is dispersed systemically and presented to precursor CTL in the
periphery. Therefore, mice were injected s.c. with the E1A-peptide
or Human Papilloma Virus (HPV) 16 E7-derived control peptide
emulsified in IFA. Spleen cells from these mice were isolated 16 h
later and used as stimulator cells for an E1A-specific CTL clone in
vitro. Splenocytes from mice injected with the E1A-peptide s.c.
induced specific proliferation, whereas splenocytes from mice
injected with the E7-peptide s.c. failed to do so (FIG. 6).
Moreover, a control CTL clone did not proliferate on spleen cells
derived from E1A-injected mice (data not shown). Thus, these data
indicate that the E1A-peptide injected s.c. in IFA is systemically
presented in the periphery by, amongst others, splenocytes.
[0045] In view of the tolerizing effects described above of the
E1A-peptide vaccine, there was a question whether CD40-triggering
in vivo is sufficient to prevent peripheral tolerization of CTL and
to restore CTL priming. Therefore, it was investigated whether
injection of tolerizing peptides combined with in vivo CD40
triggering could prevent the induction of peripheral CTL tolerance
leading to tumor-specific CTL immunity.
[0046] In Examples 1 and 2 it has been shown that CD40-triggering
in vivo can replace the requirement for CD4.sup.+ T helper cells in
priming of helper-dependent CTL responses. Since CD4.sup.+ T
cell-mediated helper activity has been implicated in the prevention
of peripheral CTL tolerance induction, the inventors addressed the
question whether CD40-triggering in vivo is sufficient to prevent
peripheral E1A-specific CTL tolerization. To this end, mice were
injected with the E1A/IFA vaccine in combination with the
activating anti-CD40 mAb FGK-45. Mice that received this
combination mounted strong E1A-specific CTL responses (FIGS. 7b and
7e), whereas mice that received the E1A/IFA vaccine (FIG. 7e) or
mAb alone did not (not shown). The combination of E1A/IFA vaccine
and anti-CD40 mAb failed to elicit CTL in CD40-deficient mice
(FIGS. 7c and 7d). Furthermore, co-injection of the E1A/IFA vaccine
with an isotype-matched control mAb (FIG. 6a) or IL-2 failed to
convert CTL tolerance induced by the E1A/IFA vaccine into CTL
priming (not shown). The range and variation of responses to the
E1A-epitope in E1A-peptide only, or E1A-peptide plus
anti-CD40-vaccinated animals, is shown in FIG. 7e. Thus, systemic
CD40 activation can reverse peptide-induced peripheral CTL
tolerance into peptide and tumor-specific CTL mediated
immunity.
[0047] The induction of E1A-specific immunity strongly correlated
with the presence of CD8.sup.+ T cells in the spleen of vaccinated
mice that stained with PE-conjugated H-2-D.sup.b-tetramers
containing the E1A-peptide (D.sup.b/E1A). Within 10 days after
vaccination, CD8.sup.+ T cells staining with D.sup.b/E1A tetramers
could be detected by flow cytometry in mice injected with
E1A-peptide and the anti-CD40 mAb, but not in mice injected with
E1A-peptide alone (not shown). In the mice injected with
E1A-peptide, the percentage of CD8.sup.+ cells that stained with
the D.sup.b/E1A tetramers was approximately 3%. In mice vaccinated
with whole adenovirus, which induces potent E1B-specific immunity,
comparable amounts of D.sup.b/E1A tetramer-reactive CD8.sup.+
spleen cells were detected. These results indicate that the
expansion of E1B-specific CD8.sup.+ T cells in mice that received
the E1A/IFA vaccine in combination with the anti-CD40 mAb was
substantial and equivalent to that found in virus vaccinated
animals.
Example 4
CD40-Triggering Strongly Enhances the Efficacy of Peptide-Based
Anti-Cancer Vaccines
[0048] Although the findings described above show that provision of
help through CD40-triggering is sufficient to prevent
CTL-tolerization after administration of a tolerogenic
peptide-vaccine, they do not address the question whether the
efficacy of anti-cancer vaccines that normally induce protective
immunity, instead of tolerance, can be enhanced by activation
through CD40. It was examined whether CD40-triggering in vivo is
beneficial to the outcome of vaccination with an HPV16 E7-derived
peptide. Vaccination with this peptide induces protective
CTL-mediated immunity against a challenge with HPV16-transformed
tumor cells. Moreover, this peptide can be used in a therapeutic
setting when loaded on in vitro activated DC suggesting that the
strength of the anti-tumor response is enhanced when presented by
activated DC.
[0049] Mice receiving the E7-peptide in combination with
CD40-triggering mounted a more potent CTL-response compared to mice
treated with E7-peptide only (data not shown), indicating that
CD40-triggering also enhances the efficacy of the HPV16 E7-peptide
vaccine and confirming the findings with the E1A peptide described
above. Moreover, mice treated 6 days after s.c. injection of
CD40-negative HPV16 E6/E7 transformed tumor cells with the HPV16
E7-peptide alone (open squares) are able to slow down tumor growth,
but eventually most animals succumb to the tumor (FIG. 8). When,
however, HPV16 E7-peptide vaccination was combined with injection
of the anti-CD40 mAb, tumor growth was markedly reduced and 7 out
of 10 mice rejected the tumor, whereas animals injected with a
control peptide and the anti-CD40 mAb were unable to control
outgrowth of the tumor. These results show that the effect of
vaccination regiments can be markedly enhanced when immunization is
combined with in vivo CD40-triggering. These data provide the basis
for the development of extremely potent and novel anti-tumor
vaccines for cancer patients.
[0050] The foregoing description, terms, expressions and examples
are exemplary only and not limiting. The invention includes all
equivalents of the foregoing embodiments, both known and unknown.
The invention is limited only by the claims which follow and not by
any statement in any other portion of this document or in any other
source.
Sequence CWU 1
1
319PRTHuman adenovirus E1B derived peptide 1Val Asn Ile Arg Asn Cys
Cys Tyr Ile1 5210PRTHuman Adenovirus E1A derived peptide 2Ser Gly
Pro Ser Asn Thr Pro Pro Glu Ile1 5 1039PRTHuman Papilloma Virus
Type 16 E7 derived peptide 3Arg Ala His Tyr Asn Ile Val Thr Phe1
5
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