U.S. patent application number 10/005341 was filed with the patent office on 2002-10-31 for immunotherapeutic combination for the treatment of tumors that over-express receptors with tyrosine kinase activity.
Invention is credited to Del Gado, Irene Beausoleil, Marinello, Gisela Maria Gonzalez, Ramos, Tania Crobet, Rodriguez, Rolando Perex.
Application Number | 20020160014 10/005341 |
Document ID | / |
Family ID | 40261022 |
Filed Date | 2002-10-31 |
United States Patent
Application |
20020160014 |
Kind Code |
A1 |
Rodriguez, Rolando Perex ;
et al. |
October 31, 2002 |
Immunotherapeutic combination for the treatment of tumors that
over-express receptors with tyrosine kinase activity
Abstract
The present invention is related to the field of immunology and
more specifically to cancer immunotherapy, particularly with
immunotherapeutic combinations and treatment methods to prevent
tumor cell growth and/or to eliminate those cells. The methods
described in the present invention are based on the blockade of
receptors with protein kinase activity in tyrosine residues
(Receptor Tyrosine Kinases, RTK) and of ligands for those
receptors. Immunotherapeutic combinations are described that cause
the blockade of RTK receptors and/or their ligands, by means of a
combination of passive and active immunotherapy. The referenced
procedures can be applied to patients in different clinical stages
with tumors of epithelial origin that over-express those receptors.
The combination of active and passive immunotherapy can be
simultaneous or sequential independent of whether the therapeutic
procedure will be used in patients with advanced disease or as
adjuvant therapy.
Inventors: |
Rodriguez, Rolando Perex;
(Habana, CU) ; Marinello, Gisela Maria Gonzalez;
(Habana, CU) ; Ramos, Tania Crobet; (Habana,
CU) ; Del Gado, Irene Beausoleil; (Habana,
CU) |
Correspondence
Address: |
ARENT FOX KINTNER PLOTKIN & KAHN
1050 CONNECTICUT AVENUE, N.W.
SUITE 400
WASHINGTON
DC
20036
US
|
Family ID: |
40261022 |
Appl. No.: |
10/005341 |
Filed: |
December 7, 2001 |
Current U.S.
Class: |
424/185.1 ;
435/194; 530/388.26 |
Current CPC
Class: |
A61K 39/001104 20180801;
C07K 16/22 20130101; A61K 39/001131 20180801; A61K 2039/6068
20130101; A61K 39/001102 20180801; C07K 16/2863 20130101; A61K
2039/55505 20130101; A61K 2039/55566 20130101; A61K 39/39541
20130101; A61K 2039/505 20130101; A61P 35/00 20180101; A61P 37/04
20180101; A61K 38/00 20130101; A61K 39/001134 20180801; A61K
39/39558 20130101; C07K 14/71 20130101; A61K 39/39541 20130101;
A61K 2300/00 20130101; A61K 39/39558 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/185.1 ;
435/194; 530/388.26 |
International
Class: |
A61K 039/00; C12N
009/12; C07K 016/40 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 8, 2000 |
CU |
CU 287/2000 |
Claims
1. A combination useful for immunotherapy, where this combination
has an effect on growth and/or proliferation of cells, whose growth
is dependent on the interaction between a receptor and its ligand,
in the receptor tyrosine kinase system (RTK), this combination
includes: a. --An antibody against a RTK receptor. b. --A vaccine
in which the active principle is the RTK receptor, and that induces
antibodies against this receptor.
2. Immunotherapy combination according to claim 1 where the RTK is
the EGF receptor.
3. Immunotherapy combination according to claim 2 where the vaccine
is directed against the EGF receptor.
4. Immunotherapy combination according to claim 2 where the
antibody against the RTK receptor is an antibody against the EGF
receptor.
5. Immunotherapy combination according to claim 4 where the
antibody against the RTK receptor is a humanized antibody against
the EGF receptor.
6. Immunotherapy combination according to claim 5 where the
humanized antibody against the EGF receptor is IOR R3.
7. A treatment combination useful for immunotherapy, where this
combination has an effect on growth and/or proliferation of cells,
whose growth is dependent on the interaction between a receptor and
its ligand, in the system of receptor tyrosine kinases (RTK), this
combination including: a. --An antibody against the ligands of the
RTK receptor and b. --A vaccine whose active principle is (are) the
RTK receptor's ligand(s) and that induces antibodies against this
(those) ligand(s).
8. A therapeutic combination according to claim 7 where the RTK
receptor's ligand is EGF.
9. A therapeutic combination according to claim 8 where the vaccine
is composed of conjugated proteins P64K and EGF.
10. A therapeutic combination according to claim 7 where the RTK
receptor ligand is TGF-alpha.
11. A therapeutic combination according to claim 10 where the
vaccine is composed of conjugated proteins P64K and TGF alpha.
12. A combination useful for immunotherapy, where this combination
has an effect on growth and/or proliferation of cells, whose growth
is dependent on the interaction between a receptor and its ligand,
in the system of receptor protein tyrosine kinases (RTK), this
combination includes: a. --A first agent selected from one of the
antibodies against the RTK receptor and of a vaccine where the
active principle is the RTK receptor that induces antibodies
against this receptor, and b. --A second agent selected from of one
of the antibodies against the ligands of the RTK receptor and of a
vaccine where the active principle is this ligand, which induces
antibodies against said ligand.
13. An immunotherapy combination according to claim 12, where the
first agent is an antibody against the RTK receptor.
14. An immunotherapy combination according to claim 13 where the
antibody against the RTK receptor is an antibody against the EGF
receptor.
15. An immunotherapy combination according to claim 14 where the
antibody against the EGF receptor is a monoclonal antibody.
16. An immunotherapy combination according to claim 15 where the
antibody against the EGF receptor is a humanized antibody.
17. An immunotherapy combination according to claim 16 where the
antibody against the EGF receptor is IOR R3.
18. An immunotherapy combination according to claim 12, where the
first agent is a vaccine whose active principle is an RTK
receptor.
19. An immunotherapy combination according to claim 18, where the
first agent is a vaccine whose active principle is the EGF
receptor.
20. An immunotherapy combination according to claim 12 where the
second agent is an antibody against an RTK receptor ligand.
21. An immunotherapy combination according to claim 20 where the
antibody against the RTK receptor's ligand is an antibody against
EGF.
22. An immunotherapy combination according to claim 20 where the
antibody against the RTK receptor is an antibody against
TGF-alpha.
23. An immunotherapy combination according to claim 12, where the
second agent is a vaccine whose active principle is an RTK
receptor's ligand.
24. An immunotherapy combination according to claim 23 where the
vaccine contains EGF as active principle.
25. An immunotherapy combination according claim to 24 where the
vaccine contains conjugated proteins p64K and EGF as active
principle.
26. An immunotherapy combination according to claim 23 where the
vaccine contains TGF-alpha as active principle.
27. An immunotherapy combination according to claims 1 to 26
inclusive, whose combination consists of a mixture of reagents
containing independent doses of effective formulations, either of
Mab or vaccines, where the combination of those independent
formulations induces decreased growth of tumors that over-express
EGF-R.
28. An immunotherapy combination according to claim 27, whose
combination consists of a mixture of reagents containing
independent doses of effective formulations, either of Mab against
the EGF receptor and its ligands or of vaccines with EGF-R and its
ligands (EGF, TGF alpha) as active principle, and where the
combination of those independent formulations induces decreased
growth of tumors that over-express EGF-R.
29. A method to control growth and/or proliferation of cells whose
growth is dependent on the interaction between a receptor and its
ligand, in the receptor tyrosine kinase (RTK) system, this method
including the treatment with one of the therapeutic combinations
defined in any one of the preceding claims.
30. A method according to claim 29 that includes the simultaneous
treatment with agents against RTK receptors and their ligands.
31. A method according to claim 29 that includes the simultaneous
treatment with vaccines and antibodies.
32. A method according to claim 29 that includes the treatment at
the first stage with this antibody and at a second stage with this
vaccine.
33. A method according to claim 29 that includes the treatment at a
first stage with this vaccine and at a second stage with this
antibody.
Description
[0001] The system of the EGF receptor (EGF-R) and its ligands
constitutes a molecular complex whose interaction regulates in a
specific way cellular growth and its impact has been demonstrated
in the uncontrolled growth of tumors of epithelial cell origin.
During tumorigenesis the paracrine and autocrine control of EGF-R
activation is deregulated, due to growth factor over-production,
because of the high rate of synthesis and/or receptor
mutations.
[0002] The EGF-R is a transmembrane glycoprotein with 1186 amino
acids and 170 kD molecular weight that it is broadly expressed in
normal tissues. It has been implicated in several stages of
embryogenic development.
[0003] The binding of its specific ligands, EGF or TGF-alpha,
induces receptor dimerization, as well as heterodimerization with
other members of the ErbB family, like HER-2 (Cohen B D et al.
(1996) J Biol Chem 271:7620-7629). The binding of ligand to
receptors releases a cascade of intracellular signals (Ullrich T O
and Schlessinger J (1990) Cell 61:203-212) that drives cellular
growth and differentiation. Overexpression of the receptor occurs
in some types of cancers, mainly of epithelial origin, which has
been a target for cancer immunotherapy. Such is the case for
breast, bladder, ovary, uterine, colon, lung, brain, prostate and
head and neck tumors. EGF-R expression has proven to be an
indication of bad prognosis in breast cancer (Prez R et al. (1984)
Breast Cancer and Treatment 4:189-193). While the role of EGFR and
its ligands in tumor growth is not yet known, there are suggestions
that EGF-R expression in tumor cells induces a mechanism for
autocrine stimulation that leads to uncontrolled proliferation of
those cells (Schlessinger J et al. (1983) Crit Rev Biochem 14
(2):93-111).
[0004] The main ligands of this system are the Epidermal Growth
Factor (EGF) and the Transforming Growth Factor alpha type
(TGFalpha). There are other ligands belonging to the EGF
superfamily, like: amphireguline (AR), cryto-1 (CR1), Heparin
Growth Factor, betacellulin, epiregulin, and others. EGF is a 53
amino acid polypeptide with a molecular weight of 6045 Da, which is
mitogenic for cells of epithelial origin. Its action is mainly
paracrine through its binding to EGF-R.
[0005] TGF alpha is a 50 amino acid polypeptide able to compete
with EGF for binding to EGF-R. Anti-EGF antibodies are not able to
recognize TGF alpha (Todaro G J et al. (1976), Nature 264:26-31),
meaning that both growth factors are two immunologically different
entities.
[0006] The EGFR--ligand system has been the target of passive
immunotherapy (PI) using monoclonal antibodies(Mab) against EGF-R,
in native form, associated with drugs, toxins, or radioactive
isotopes (Vollmar A M et al. (1987) J Cell Physiol 131:418-425) in
tumors with high expression of this receptor. These antibodies have
been selected by their capacity to inhibit the binding of EGF to it
receptor (neutralizing antibodies). Several clinical trials with
Mabs are being carried out and some have shown promising results as
it is the case of Phase II clinical trials with the Mab C225 in
breast, pancreatic and renal cancer, in addition to Phase III
trials in head and neck cancer (Mendelsohn, J et al. (1999)
American Society of Clinical Oncology Meeting). Other Phase II
clinical trials showing good results have been carried out with the
Mab IOR-R3 in lung tumors (Crombet T et al. (2000) Cancer
Biotherapy and Biopharmaceutical, manuscript accepted for
publication).
[0007] Passive immunotherapy with the IOR-R3 Mab (EP586002B1),
specific against the EGF-R, has demonstrated that the specific
binding of the IOR-R3 to the receptor inhibits EGF/EGF-R binding,
with subsequent inhibition of EGFR autophosphorylation. In turn,
passive immunotherapy with IOR-R3 inhibited the growth of human
tumor cells in nude mice, and it has reduced the rate of tumor
growth in some patients in clinical trials. This system has also
been target of specific active cancer immunotherapy. One example is
the use of a vaccine composed one of the main ligands of EGF-R,
EGF, coupled to a carrier protein (U.S. Pat. No. 5,894,018). This
vaccine is able to induce a specific antibody response against
autologus EGF, to inhibit EGF/EGF-R binding, thus blocking
proliferation mechanisms induced by this binding. Pre-clinical
studies have shown that mice immunized with autologus EGF coupled
to a carrier protein and administered with a useful adjuvant,
increases survival of mice transplanted with Ehrlich Ascitic Tumor
(EAT) cells (Gonzlez G et al. (1996) Vaccine Research 5(4):233-243;
Gonzalez G et al (1997) Vaccine Research 6(2):9 1-100).
[0008] Results from a Phase I clinical trial have been reported for
a vaccine containing human recombinant EGF, demonstrating the
immunogenicity and safety of vaccination (Gonzalez G et al (1998),
Annals of Oncology 9:1-5).
[0009] Another example of active specific immunotherapy of cancer
in this system is a vaccine composition containing EGF-R,
proteoliposomes derived from an external membrane protein complex
of Neisseria meningitidis and a ganglioside that associate
specifically with this receptor forming membrane molecular
complexes (Patent deposited in Cuba, priority date Jun. 12,
2000).
[0010] Likewise, vaccines containing other EGF-R ligands, such as
TGF alpha alone or combined with EGF and coupled to a carrier
protein, have been developed ( Patents Requested in Cuba, priority
date Jun. 12, 2000).
[0011] In the present invention the use of combined immunotherapies
is proposed, directed either against receptors with tyrosine kinase
activity (RTK) or against their ligands,. This combination has the
object of potentiating the observed effect when applying, in an
independent way, different forms of immunotherapy described in the
state of the art, directed alone against some of the
receptor/ligand systems. This potentiation is justified for the
combined blockade of both, ligands and receptor, in a treatment
method that includes both principles.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention is related to immunotherapeutic
combinations and treatment methods to inhibit growth of tumor cells
to eliminate those cells, based on the blockade of RTK receptors
and its ligands. This blockade can be achieved, among other
approaches, using combination, simultaneous or sequential, of
active immunotherapies (therapeutic vaccines) and passive
immunotherapies (Mab) directed to growth factors (i.e. EGF, TGFa)
and its receptors (i.e. EGF-R).
[0013] The blockade of growth factors or of their receptors causes
inhibition of cellular proliferation. In this invention we show
that simultaneous blockade of ligands and/or receptors potentiate
the inhibition effect on cellular proliferation. This therapeutic
concept is of great importance for treatment of malignant tumors,
which are fundamentally caused by an increase in the rate of
cellular proliferation.
[0014] Immunotherapeutic combinations are described that cause the
blockade of RTK receptors and/or their ligands, by means of active
and passive immunotherapeutical combinations. The referred
procedures can be applied to patients with tumors of epithelial
origin that over-express EGF-R, in different clinical stages.
[0015] The combination of active and passive immunotherapy can be
simultaneous or sequential, independent of the therapeutic
procedure used in patients with advanced disease, or as adjuvant
therapy.
[0016] In cases of advanced disease, the proposed therapeutic
combination is passive immunotherapy with Mab that recognizes the
RTK receptor and/or Mab that recognizes ligands of this receptor,
in combination with an onco-specific therapy of choice, as first
line therapy, followed by active immunotherapy using vaccines
directed against the ligands of the receptor and/or to the
receptor, to maintain the theraputic effect. In cases of adjuvant
therapy the proposed therapeutical combinations are:
[0017] 1. Passive immunotherapy with Mab that recognize either, the
RTK receptor and/or its ligands with active immunotherapy using
vaccines directed to the receptor's ligands or to the receptor
itself.
[0018] 2. Passive immunotherapy with Mab that recognize either, the
RTK receptor or its ligands as attack therapy, followed by active
immunotherapy with vaccines directed to the receptor's ligands or
to the receptor itself, as maintenance treatment.
[0019] PROCEDURE 1: Therapeutic combination including passive
immunotherapy with Mab that recognize the RTK receptor (i.e.EGF-R)
and/or the receptor's ligands (i.e.EGF, TGF alpha), followed by
active therapy with vaccines directed to the receptor and/or its
ligands, to be applied in patients with advanced stage epithelial
tumors.
[0020] This will be administered to patients with advanced cancer
who are not eligible for any other onco-specific therapy.
[0021] The first treatment step will be passive immunotherapy with
Mab that recognizes the RTK receptor (i.e.EGF-R), with the property
of inhibiting this receptor and/or Mab that recognize the receptors
ligands (i.e. EGF, TGF alpha). This will be an acute therapy aimed
at the goal of tumor remission, and can be used together with the
established onco-specific treatment for this stage of disease.
[0022] This will be followed by active immunotherapy using vaccines
that induce receptor blocking antibodies (i.e.anti-EGF-R) and/or
ligand blocking antibodies (i.e.anti-EGF anti-TGF alpha), with the
objective of maintaining disease stabilization for longer periods,
to avoid new metastates.
[0023] The procedure consists of administration to patients in
advanced stages of tumors of epithelial origin, of between 4 and 20
doses , ranging between 100 and 400 mg of a Mab that recognizes and
inhibits EGF-R, and/or MAb that recognizes the receptor's ligands.
The time between doses will be between 6 to 10 days. The complete
treatment can last between 1 to 24 months, concomitant with the
established onco-especific therapy. The treatment will continue up
to partial or complete tumor regression or up to the point where an
adverse reaction occurs that requires treatment cessation.
[0024] Between 1 and 4 weeks after this treatment, immunization
schedules will be initiated with vaccines directed against EGF-R or
its ligands (i.e.EGF , TGFalpha) coupled to a carrier protein
(i.e.P64K Neisseria meningitides recombinant protein) and
administered in an adequate adjuvant i.e alum (between 1 and 2
mg/dose) or Montanide ISA 51 (between 0.6 and 1.2 ml/dose). Each
dose contains between 50 and 800 ug of active ingredient (receptor
or ligand) coupled to the carrier protein, in a final volume of
between 0.6 and 5 mL. The immunization schedule is 5 to 8 initial
immunizations for response induction, given every 7 to 14 days.
Immunizations can be preceded by administration of cyclophosphamide
, between 100 and 500 mg/m.sup.2 of body weight, administered 2 to
4 days before the 1.sup.rst immunization. Vaccines can be
formulated in any other vaccine vehicle (i.e.liposomes, DNA
vaccines, viral vectors).
[0025] Vaccines can be formulated as independent products or as a
unique vaccine formulation. In this period, blood will be extracted
from patients in order to measure biochemical blood markers and
specific antibody titers against the ligand or receptor to which
the vaccine is directed. Extractions will be done weekly or
monthly.
[0026] Subsequently, re-immunizations will be done if antibody
titers decrease, every 1 to 4 months for a period of 1 to 2
years.
[0027] PROCEDURE 2: Immunotherapeutic combination including passive
immunotherapy with Mab that recognizes a RTK receptor (i.e.EGF-R)
and/or it ligands (i.e. EGF, TGF alpha) together with active
immunotherapy with vaccines directed against the receptor and/or
its ligands, as adjuvant treatment.
[0028] Passive treatment with Mab recognizing a RTK receptor
(i.e.EGF-R) inhibiting its activity and/or Mab recognizing
receptor's ligands (i.e.EGF, TGF alpha), together with an active
treatment with vaccines that induces an antibody response that
blocks the receptor and/or its ligands, will be administered to
patients immediately after diagnosis and/or surgical treatment.
[0029] Those treatments, administered together, will have a
synergistic effect, enabling a higher percentage of regression
and/or clinical disease stabilization.
[0030] Patients with tumors of epithelial origin are amenable to
this treatment, that consists of between 4 to 20 doses, ranging
between 100 and 400 mg, of Mab recognizing and inhibiting RTK
receptors and/or it ligands. The time between doses will be between
6 to 10 days and the treatment can last between 1 to 24 months. The
treatment will continue until partial or complete tumor regression
or up to the point where an adverse reaction occurs that requires
treatment cessation.
[0031] Concomitant immunizations will be administered with vaccines
according to the schedule described in procedure #1.
[0032] PROCEDURE 3:
[0033] Immunotherapeutic combination including passive
immunotherapy with Mab recognizing RTK receptors (i.e.EGF-R) and/or
its ligands (i.e. EGF, TGF alpha), followed by active immunotherapy
with vaccines directed against the receptor and/or it ligands, to
be applied as adjuvant therapy.
[0034] This will be applied to patients immediately after diagnosis
and/or surgical treatment. The goal of this treatment is to use
acute therapy to obtain tumor remission, via initial passive
immunotherary with Mab recognizing and inhibiting RTK receptors
(i.e.EGF-R) and/or Mab recognizing its ligands (i.e. EGF, TGF
alpha).Subsequently, active immunotherapy will be initiated using
vaccines inducing blocking antibodies against the receptor
(i.e.EGF-R) or it ligands (i.e.EGF, TGF alpha). The aim of the
2.sup.nd treatment is to obtain a longer period of freedom from
disease, to avoid the appearance of new metastates.
[0035] The procedure consists of administration to patients at
advanced stages of cancer of epithelial origin, from 4 to 20 doses
of between 100 and 400 mg of Mab that recognizes and inhibits the
EGF-R and/or its ligands. The time between doses will be between 6
to 10 days and the treatment duration can be between 1 to 24
months. The treatment will continue until partial or complete tumor
regression, or until any adverse reaction occurs that requires
treatment cessation.
[0036] Between 1 to 4 weeks after the end of treatment,
immunization schedules will begin with vaccines directed against
the EGF-R or some EGF-R ligand (i.e. EGF, TGF alpha), according to
the schedule described in procedure #1.
EXAMPLES
Example 1: Immunization Schedule with EGF Vaccine in Cancer
Patients, Using Alum as Adjuvant
[0037] With the main goal of demonstrating immunogenicity and
safety of EGF, a clinical trial was performed in which 10 patients
were immunized with an EGF Vaccine (U.S. Pat. No. 5,894,018), using
P64K as carrier protein and alum as adjuvant,.
[0038] Patient 1.1 (MMG) was included in the trial with a diagnosis
of metastasic epidermoid carcinoma of the lung, with progressive
disease, and not eligible for any other onco-specific
treatment.
[0039] The patient was immunized following a schedule of 5 initial
dose of the vaccine, containing 50ug of EGF and 2 mg alum,
administered on days 1, 7, 14, 21 and 51.
[0040] Blood extraction was performed on days 0, 15, 30, 45, 60 and
monthly thereafter for blood biochemical measurements and for
EGF-specific antibodies. Antibody titers were measured by means of
an ELISA test, antibody titers being determined as the maximal sera
dilution that gives a positive result in the ELISA test. (O.D
values equal or higher 2 times the blank).
[0041] Re-immunization was performed using the same vaccine dose
when a decrease in antibody titers was detected.
[0042] Patient MMG developed an anti-EGF antibody response with
maximum titers up to 1:8000. The kinetics of the antibody response
is shown in FIG. 1.
[0043] After the beginning of the vaccination schedule the patient
showed clinical and radiological stabilization of disease for 15
months. The patient died 23.2 months after the first
vaccination.
Example 2
Immunization Schedule with EGF Vaccine in Cancer Patients, Using
Montanide ISA 51 as Adjuvant
[0044] With the main goal of demonstrating immunogenicity and
safety of EGF using P64K as a carrier protein and Montanide ISA 51
as an adjuvant, a clinical trial was performed in which 10 patients
were immunized.
[0045] Patient 2.1 (AMG) was included in the trial with a diagnosis
of epidermoid carcinoma of the lung, with progressive disease,
being ineligible for any other onco-specific treatment. The patient
was immunized according to a schedule of 5 initial doses of the
vaccine containing 50 ug of EGF in 0.6 mL total volume, emulsified
with 0.6 mL of Montanide ISA 51 immediately before use, and
administered on days 1, 7, 14, 21 and 51.
[0046] Blood extractions were performed on days 0, 15, 30, 45, 60
and monthly thereafter for blood biochemical measurements and
measurement of specific anti-EGF antibodies.
[0047] The antibody titers were measured by means of an ELISA test,
antibody titers being determined as the maximal sera dilution that
gives a positive result in the ELISA test. (O.D values equal or
higher 2 times the blank).
[0048] Re-immunization was performed using the same vaccine dose
when a decrease in antibody titers was detected.
[0049] Patient AMG developed an anti-EGF antibody response with
maximum titers of up to 1: 32000, with a kinetics of response shown
in FIG. 2.
[0050] After the beginning of the vaccination schedule, the patient
showed stabilization of disease for 12 months, at which point
clinical and radiological tumor regression was diagnosed.
[0051] On the 14.sup.th month after the beginning of vaccination, a
2.sup.nd primary tumor appeared. The patient died 18 months after
inclusion from a surgical complication of this 2.sup.nd tumor.
Example 3
Immunization Schedule in Cancer Patients, with EGF Vaccine, Using
Alum as Adjuvant and Low Dose Cyclophosphamide Pre-treatment
[0052] A clinical trial was carried out in which 10 patients were
immunized with the main goal of demonstrating immunogenicity and
safety of the EGF Vaccine using P64K as carrier protein and alum as
adjuvant after cyclophosphamide pre-treatment.
[0053] Patient 3.1, FNR, was included in the trial with a diagnosis
of epidermoid carcinoma of the lung, with progressive disease,
being ineligible for any other onco-specific treatment. The patient
was treated with cyclophosphamide (100 mg/m.sup.2 of body surface),
3 days before the first immunization of the EGF Vaccine. The
vaccination schedule was 5 doses of the vaccine composition,
containing 50 ug of EGF and 2 mg of alum, administered on days 1,
7, 14, 21 and 51.
[0054] Blood extractions were performed on days 0, 15, 30, 45, 60
and then monthly for blood chemistry and specific anti-EGF antibody
determinations.
[0055] Antibody titers were measured by means of an ELISA test,
antibody titers being determined as the maximal sera dilution that
gives a positive result in the ELISA test. (O.D values equal or
higher 2 times the blank).
[0056] Re-immunization was performed using the same vaccine dose,
when a decrease in antibody titers were detected.
[0057] The patient developed an anti-EGF antibody response with
maximum titers up to 1:8000, as shown in FIG. 4.
[0058] After the beginning of the vaccination schedule, the patient
showed disease stabilization for 19 months.
Example 4
Immunization Schedule with EGF Vaccine in Cancer Patients, Using
Montanide ISA 51 as Adjuvant and Cyclophosphamide Pre-treatment
[0059] A clinical trial was carried out in which 10 patients were
immunized with the main goal of demonstrating immunogenicity and
safety of the EGF Vaccine, using P64K as carrier protein and and
Montanide ISA 51 as adjuvant after cyclophosphamide pre-treatment.
Patient 4.1, JPG, was included in the trial with a diagnosis of non
small cell lung adenocarcinoma, with progressive disease, being
ineligible for any other onco-specific treatment.
[0060] The patient was treated with cyclophosphamide (100
mg/m.sup.2 of body surface), 3 days before the first immunization
of the EGF Vaccine. The vaccination schedule was 5 doses of the
vaccine composition, containing 50 ug of EGF in 0.6 mL total
volume, emulsified with 0.6 mL of Montanide ISA 51 immediately
before use, administered on days 1, 7, 14, 21 and 51.
[0061] Blood extractions were performed on days 0, 15, 30, 45, 60
and then monthly for blood chemistry and specific anti-EGF antibody
determinations. Antibody titers were measured by means of an ELISA
test, antibody titers being determined as the maximal sera dilution
that gives a positive result in the ELISA test. (O.D values equal
or higher 2 times the blank).
[0062] Re-immunization was performed , using the same vaccine dose,
when a decrease in antibody titers was detected.
[0063] Patient JPG developed an anti-EGF antibody response with
maximum titers up to 1:400000, as shown in FIG. 5.
[0064] After the beginning of the vaccination schedule the patient
showed disease stabilization for 6 months.
Example 5
Immunogenicity of EGF Vaccination and its Relationship to Disease
Stabilization in Patients with Cancer
[0065] A Phase I trial in 20 patients was performed in which
patients were randomized to one of two groups using different
adjuvants..
[0066] Ten patients at stages III or IV of Non Small Cell Lung
cancer (NSCLC), were treated with 5 initial doses of vaccine
composition containing 50ug of EGF and 2 mg of alum, administered
on days 1, 7, 14, 21 and 51.
[0067] The other 10 patients (NSCLC, stages III or IV), were
immunized with 5 doses of the vaccine composition containing 50 ug
of EGF, in a total volume of 0.6 mls, emulsified with the same
volume (0.6 mL) of Montanide ISA 51.
[0068] Antibody titers were measured by means of an ELISA test,
with antibody titers determined as the maximal sera dilution that
gives a positive result in the ELISA test. (O.D values equal or
higher 2 times the blank).
[0069] In this trial, 50% of patients developed an anti-EGF
antibody response with antibody titers of 1:4000 or higher (, Good
Antibody Responders, GAR group) and 50% antibody titers below
1:4000 (Bad Antibody Responders, BAR group).
[0070] In the GAR group, 87.5% of patients showed clinical and
radiological disease stabilization for at least3 months after the
beginning of treatment.
[0071] In the BAR group, only 11,1% of patients showed this
stabilization profile (Table 1).
[0072] These data demonstrate the relationship between anti-EGF
antibody levels and tumor stabilization.
1CHART 1 Relationship of antibody responses and clinical and
radiological disease stabilization. Disease stabilization for at
least 3 months after % of patients beginning treatment. GAR 50%
87.5% BAR 50% 11.1%
Example 6
Immunogenicity of EGF Vaccination and Relationship to Survival of
Cancer Patient Subjected to This Treatment
[0073] Forty stage III /IV NSCLC patients were treated, in groups
of 10, with the schedules detailed in examples 1,2,3 and 4.
[0074] They were characterized as GAR and BAR according to criteria
exposed in example 6. Of the total of patients treated with the
previously described schedules, 50% turned out to be GAR and 50%
BAR.
[0075] When survival patterns were compared between GAR and BAR
patients, a statistically significant difference was observed, with
a mean survival of 9.1 months for GAR and a mean survival of 4.5
months for BAR (p <0.02). This result is showed in FIG. 6.
Example 7
Therapeutic Effect of the Combination of Radiotherapy and Mab
IOR-R3
[0076] Patient RML, diagnosed with stage IV language base
epidermoid carcinoma, was included in the clinical trial using the
combination of radiotherapy (RTP) and IOR-R3.
[0077] The patient received 200 mg of Mab once a week for 6 weeks.
The accumulated dose of Mab was 1200mg and the total radiation dose
was 60 Gy.
[0078] When the combination therapy was complete the patient showed
complete remission of the primary tumor and its metastases (FIG.
7). This response was maintained for more than 13 months.
Example 8
Therapeutic Effect of the Combination of Radiotherapy and Mab
IOR-R3
[0079] Patient EPG, diagnosed with stage III tonsil epidermoid
carcinoma with cervical adenopathies, was included in the clinical
trial using the combination of radiotherapy (RTP) and IOR-R3. The
patient received 200mg of Mab once a week for 6 weeks and a total
radiation dose of 64 Gy.
[0080] After treatment, this patient showed complete remission of
the tumor lesion (FIG. 8).
[0081] The response was maintained for more than 13 months.
Example 9
Therapeutic Effect of the Combination of Radiotherapy and Mab
IOR-R3
[0082] Patient CHA, diagnosed with a stage IV tonsil tumor, with
bilateral cervical adenopathies, was included in the clinical trial
using a combination of radiotherapy (RTP) and IOR-R3. The patient
received 400 mg of Mab once a week for 6 weeks, for an accumulated
dose of 2400 mg. Concomitantly, the patient received a total
radiation dose of 64 Gy.
[0083] When concluding the treatment this patient was in complete
remission of the primary tumor and the loco-regional metastasis
(FIG. 9). The response was maintained for 12 months.
Example 10
Evaluation, in Nude Mice, of Passive Therapy Using a Combination of
Anti-EGF-R Antibody (IOR-R3) and an Anti-EGF-R Ligand Monoclonal
(EGF-1)
[0084] Evaluation of the anti-tumor effect in relation to the
administered doses.
[0085] This experiment also simulates the possible effect of
combined administration of the anti-EGF-R Mab and an EGF vaccine.
The vaccine causes an anti-EGF antibody response with the same
effect of passive administration of Mab with that specificity, with
the additional advantage that, the achieved antibody response can
be maintained over time, as shown in examples 1, 2, 3 and 4
(kinetics of anti-EGF antibody titers in immunized patients)
[0086] Seven different groups of athymic mice, with NMRI genetic
origin (outbred population), were immunized with:
[0087] Group 1:10 doses of 0.5 mg of the EGF-1 Mab, intraperitoneal
route, daily frequency.
[0088] Group 2: 10 doses of 1 mg of the EGF-1 Mab, intraperitoneal
route, daily frequency.
[0089] Group 3: 10 doses of 0.5 mg of the IOR-R3 Mab,
intraperitoneal route, daily frequency.
[0090] Group 4: 10 doses of 1 mg of the IOR-R3 Mab, intraperitoneal
route, daily frequency.
[0091] Group 5: 10 doses of Phosphate Buffered Saline (PBS),
intraperitoneal route, daily frequency (negative control).
[0092] Group 6: 10 doses of 0.5 m g EGF-1 Mab combined with 0.5 mg
IOR-R3 Mab, intraperitoneal route, daily frequency.
[0093] Group 7: 10 doses of 1 m g EGF-1 Mab combined with 1 mg
IOR-R3 Mab, intraperitoneal route, daily frequency.
[0094] On the day of initiation of treatment with Mabs mice were
transplanted with 1 .times.10.sup.6 H125 human tumor cells. This
cell line over-expresses the EGF-R.
[0095] Results are shown in FIGS. 10 and 11. The anti-tumor effect
was potentiated when both treatments were combined, increasing with
increased doses.
Example 11
Schedule of Combined Mab IOR-R3/EGF Vaccine Treatments in Patients
With Advanced Stage Tumors
[0096] Patient ARP, diagnosed with epidermoid carcinoma of the head
and neck, received sequential treatment of Mab IOR-R3 and EGF
Vaccine.
[0097] The patient received 200 mg of Mab once a week for 6 weeks,
in combination with a total of 30 doses of radiotherapy , 5 doses
per week for 6 weeks, for an accumulated radiation dose of 60
Gy.
[0098] When concluding the treatment, the patient was in complete
remission of the primary tumor.
[0099] An immunization schedule with the EGF Vaccine began one
month after conclusion of the treatment with the Mab. The patient
received 5 doses of 50 ug of EGF conjugated to protein P64k, in a
total volume of 0.6 mls, emulsified with 0.6 ml of Montanide ISA 51
immediately before use. The immunizations were carried out on days
1,7,14,21 and 51.
[0100] The patient remains in the follow-up period.
Example 12
Schedule of Combined Mab IOR-R3/EGF Vaccine Treatments in Patients
With Advanced Stages Tumors
[0101] Patient MRM, diagnosed with epidermoid carcinoma of the
lung, was subjected to surgical intervention. One month after the
surgery the patient began a combined treatment of passive
immunotherapy with Mab IOR-R3 concomitantly with the EGF
vaccine.
[0102] FIG. 12 details the schedule of dose intervals. The patient
is in follow up.
BRIEF DESCRIPTION OF FIGURES
[0103] FIG. 1: Kinetics of anti-EGF antibody response in patient
MMG, immunized as detailed in example 1. Arrows indicate times of
re-immunizations.
[0104] FIG. 2: Kinetics of anti-EGF antibody response in patient
AMC, immunized as detailed in example 2. Arrows indicate times of
re-immunizations.
[0105] FIG. 3: Tumor regression observed in patient AMC. The tumor
mass is seen on the left at the start of treatment. On the right of
the figure it can be seen that 12 months after the start of
treatment the tumor mass disappeared.
[0106] FIG. 4: Kinetics of anti-EGF antibody response in patient
FNR, immunized as detailed in example 3. Arrows indicate times of
re-immunizations.
[0107] FIG. 5: Kinetics of anti-EGF antibody response in patient
JPG, immunized as detailed in example 4. Arrows indicate times of
re-immunizations.
[0108] FIG. 6: Kaplan-Maier survival curves of groups of patients
with high anti-EGF antibody response (GAR) and with low anti-EGF
antibody responses (BAR), as well as that of a historical control
group. As can beseen, GAR is associated with a significant increase
in survival compared with either BAR or with historical
controls.
[0109] FIG. 7: Graphic demonstration of tumor remission in patient
RML, treated as detailed in example 7.
[0110] FIG. 8: Graphic demonstration of tumor regression in patient
EPG, treated as detailed in example 8.
[0111] FIG. 9: Graphic demonstration of the tumor in patient CHA,
treated as detailed in example 9.
[0112] FIG. 10: Groups of mice immunized with 0.5 mg of both Mabs
IOR-R3 and EGF-1, and with the combination of 0.5 mg of IOR-R3+0.5
mg of EGF1, as detailed in example 10. A synergistic effect on
decreased tumor growth was observed in the group treated with the
combination of both Mabs.
[0113] FIG. 11: Groups of mice immunized with 1 mg of both Mabs
IOR-R3 and EGF-1, and with the combination of 1 mg of IOR-R3+1 mg
of EGF1, as detailed in example 10. A synergistic effect on
decreased tumor growth was observed in the group treated with the
combination of both Mabs.
[0114] FIG. 12: Combined treatment of Mab IOR-R3 and the EGF
Vaccine: Arrows above the time axis indicate the day of Mab
administration (days 1,7,14,21,28 and 35) and arrows below the time
axis indicate the day of immunization with the EGF Vaccine (days
2,8,15,22, and 52).
* * * * *