U.S. patent application number 16/855263 was filed with the patent office on 2020-08-06 for amyloid conjugate and uses and methods thereof.
The applicant listed for this patent is ARACLON BIOTECH, S.L.. Invention is credited to Manuel SARASA BARRIO.
Application Number | 20200246430 16/855263 |
Document ID | 20200246430 / US20200246430 |
Family ID | 1000004777903 |
Filed Date | 2020-08-06 |
Patent Application | download [pdf] |
United States Patent
Application |
20200246430 |
Kind Code |
A1 |
SARASA BARRIO; Manuel |
August 6, 2020 |
AMYLOID CONJUGATE AND USES AND METHODS THEREOF
Abstract
A composition includes aluminum hydroxide gel and a conjugate of
at least one CysA13(33-40) peptide linked to the keyhole limpet
hemocyanin (KLH). Maleimidobutyric acid Nhydroxysuccinimide ester
(SM) serves as cross-linking agent. The composition can produce an
effective and specific immune response against A.beta.40. The
antibodies produced are specific for A.beta.40 without
significantly binding to A.beta.42. The composition can increase
the response against A.beta.40 compared with the response produced
by other conjugates that include CysA.beta.(33-40) peptide and KLH,
and are bound or conjugated by other crosslinking agents.
Inventors: |
SARASA BARRIO; Manuel;
(Zaragoza, ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARACLON BIOTECH, S.L. |
Zaragoza |
|
ES |
|
|
Family ID: |
1000004777903 |
Appl. No.: |
16/855263 |
Filed: |
April 22, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16103810 |
Aug 14, 2018 |
|
|
|
16855263 |
|
|
|
|
PCT/EP2017/053242 |
Feb 14, 2017 |
|
|
|
16103810 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/03 20130101;
C07K 14/4711 20130101; A61K 39/39 20130101; C07K 2317/33 20130101;
A61P 25/28 20180101; C01F 7/02 20130101; C07K 2319/00 20130101;
A61K 38/1767 20130101; A61K 2039/55505 20130101; C07K 7/06
20130101; A61K 47/42 20130101; C07K 16/18 20130101; A61K 9/06
20130101; C07K 2319/31 20130101; A61K 47/643 20170801; C07K
14/43504 20130101; A61K 47/646 20170801 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C01F 7/02 20060101 C01F007/02; A61K 47/42 20060101
A61K047/42; A61K 39/39 20060101 A61K039/39; A61K 38/03 20060101
A61K038/03; A61K 9/06 20060101 A61K009/06; A61P 25/28 20060101
A61P025/28; A61K 47/64 20060101 A61K047/64; C07K 14/435 20060101
C07K014/435; C07K 7/06 20060101 C07K007/06; C07K 14/47 20060101
C07K014/47; C07K 16/18 20060101 C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 15, 2016 |
ES |
201630173 |
Claims
1. A composition comprising aluminum hydroxide gel and a conjugate
of at least one CysA.beta.(33-40) peptide (SEQ ID NO: 1) linked to
the keyhole limpet hemocyanin (KLH), wherein the crosslinking agent
connecting each CysA.beta.(33-40) peptide to the keyhole limpet
hemocyanin (KLH) of the conjugate is the maleimidobutyric acid
Nhydroxysuccinimide ester (SM), wherein at least 45
CysA.beta.(33-40) peptides (SEQ ID NO: 1) is linked to each keyhole
limpet hemocyanin (KLH).
2. A pharmaceutical composition comprising i) a conjugate
comprising at least one CysA.beta.(33-40) peptide (SEQ ID NO: 1)
linked to keyhole limpet hemocyanin (KLH) via a crosslinking agent,
and ii) an aluminum hydroxide gel, wherein the crosslinking agent
connecting each CysA.beta.(33-40) peptide to the keyhole limpet
hemocyanin (KLH) of the conjugate is maleimidobutyric acid
N-hydroxysuccinimide ester (SM), and wherein a pH of the
composition ranges from 5.8 to 7.0.
3. The pharmaceutical composition according to claim 2, wherein the
pH of the composition ranges from 6.2 to 7.0.
4. The pharmaceutical composition according to claim 2, wherein the
pH of the composition ranges from 5.8 to 6.2.
5. The pharmaceutical composition according to claim 2, wherein the
concentration of the at least one CysA.beta.(33-40) peptide (SEQ ID
NO: 1) is at least 100 .mu.g.
6. The pharmaceutical composition according to claim 2, wherein the
concentration of the at least one CysA.beta.(33-40) peptide (SEQ ID
NO: 1) is at least 150 .mu.g.
7. The pharmaceutical composition according to claim 2, wherein the
concentration of the at least one CysA.beta.(33-40) peptide (SEQ ID
NO: 1) is from 150 .mu.g to 400 .mu.g.
8. The pharmaceutical composition according to claim 2, wherein the
concentration of the at least one CysA.beta.(33-40) peptide (SEQ ID
NO: 1) is from 160 .mu.g to 240 .mu.g.
9. A glass ampule comprising the pharmaceutical composition of
claim 2.
Description
PRIORITY AND CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation Application of U.S.
application Ser. No. 16/103,810, filed Aug. 14, 2018, which is a
Continuation Application of International Application No.
PCT/EP2017/053242, filed Feb. 14, 2017, designating the U.S. and
published in English as WO 2017/140656 A1 on Aug. 24, 2017, which
claims the benefit of Spanish Patent Application No. ES201630173,
filed Feb. 15, 2016. All applications for which a foreign or a
domestic priority is claimed are identified in the Application Data
Sheet filed herewith and are hereby incorporated by reference in
their entirety under 37 C.F.R. .sctn. 1.57.
REFERENCE TO ELECTRONIC SEQUENCE LISTING
[0002] The present application includes an Electronic Sequence
Listing. The Electronic Sequence Listing is provided as a file
entitled DURC059001C2SEQLIST.txt, which is 1,265 bytes in size,
created and last modified on Apr. 19, 2020. The information in the
Electronic Sequence Listing is incorporated herein by reference in
its entirety.
FIELD
[0003] The present invention relates to the field of biochemistry,
more specifically to the field of sector of protein conjugation.
Additionally, the present invention has an application in the field
of medicine and veterinary science in the treatment of amyloid
diseases.
BACKGROUND
[0004] Multiple conjugates useful for the active or passive
immunization of patients with amyloid diseases, fundamentally for
Alzheimer's disease, have been described in the prior art. It
should be pointed out that most of said prior art focuses on
selecting peptides or transport proteins that allow generating a
suitable immune response in patients, the crosslinking agent used
not being given much importance or relevance. Said crosslinking
agent is often expressed in the form of a list of all those that
are available or known up until now indicating that any of them can
be used in an equivalent manner, or it is simply not even
indicated.
[0005] For example, Spanish patent application with publication
number ES2246105 discloses the prevention or treatment of amyloid
diseases, inter alia Alzheimer's disease, by means of the active
immunization of patients with the conjugate formed by the
A.beta.33-40 peptide and the transport protein keyhole limpet
hemocyanin (hereinafter, KLH), with accession number 4BED in the
Protein Data Bank. The use of said conjugate for generating
antibodies (for example, by means of immunizing mammals or birds
with said conjugate) which are subsequently used in a passive
immunization method for the prevention or treatment of amyloid
diseases, inter alia Alzheimer's disease, is also contemplated.
Said patent document does not specify the crosslinking agent
used.
[0006] The only prior art document of which the inventors are aware
and in which the crosslinking agent is considered important in the
immune response produced in the conjugate is PCT patent application
with publication number WO2005/072777. Said document discloses
conjugates for the active or passive immunization of patients based
on the crosslinking agent LPA with respect to which the capacity
thereof for binding with or having two peptides at the same time is
highlighted, making it appropriate for generating an immune
response suitable in patients. Within the general explanation of an
LPA-based conjugate it is contemplated that the peptide used may be
an A.beta.42 or A.beta.40 C-terminal fragment, and fragments 33-42,
35-42, 36-42, 37-42, 38-42 and 39-42 are specifically mentioned and
exemplified. Additionally, said document mentions that the
transport protein may be KLH. This same document describes
generating other conjugates, using a different crosslinking agent
(N-succinimidyl 3-(2-pyridyldithio) propionate, commonly known as
SPDP).
SUMMARY
[0007] After extensive and thorough experimentation, the inventors
have surprisingly discovered that maleimidobutyric acid
N-hydroxysuccinimide ester (hereinafter, SM), a heterobifunctional
crosslinking agent, in which each molecule thereof binds a peptide
to the transport protein, used as a crosslinking agent for
preparing CysA.beta.(33-40) peptide (SEQ ID NO: 1) and KLH
conjugates, produces conjugates that allow generating an improved
immune response in comparison to the immune response produced by
conjugates generated with other homo- or heterobifunctional
crosslinking agents of the prior art such as SPDP which also allow
the binding of a peptide to the transport protein. Such improvement
is clearly shown in examples 1 to 4 of the present specification.
In addition, said examples show that said new conjugates or the
pharmaceutical compositions comprising said conjugates produce:
[0008] an immune response that is as specific as possible, for the
purpose of minimizing the side effects associated with the
(preventive or therapeutic) vaccine treatment; [0009] the highest
possible immune response for the purpose of assuring an effective
immunization of patients and reducing the required doses of
immunogenic conjugate.
[0010] Thus in a first aspect, the present invention relates to a
conjugate characterized in that the crosslinking agent is SM.
[0011] In another aspect, the present invention relates to a
composition comprising the conjugate of the present invention.
[0012] In additional aspects of the present invention, the use of
said conjugate for preparing a medicinal product, more
specifically, a medicinal product intended for the treatment or
prevention of amyloid diseases, is contemplated.
[0013] Another aspect to which the present invention relates is to
compositions comprising the conjugate of the present invention for
use as a medicinal product, more specifically for use in the
treatment or prevention of amyloid diseases.
[0014] Additionally, the present invention also relates to a method
of treatment or prevention of an amyloid disease by means of the
delivery of a composition comprising the conjugate of the present
invention.
[0015] In yet another aspect, the present invention relates to a
method of manufacturing antibodies based on the use of the
conjugate of the present invention.
DETAILED DESCRIPTION
[0016] Definitions
[0017] As used herein, "amyloid disease" and the plural thereof
refer to diseases associated with the .beta.-amyloid accumulation.
Said accumulation can fundamentally occur in the brain, producing
diseases among which are found Alzheimer's disease, Parkinson's
disease, cerebral amyloid angiopathy, vascular dementia of an
amyloid origin and dementia with Lewy bodies. .beta.-amyloid
accumulation can also fundamentally occur in skeletal muscle,
producing inclusion-body myositis.
[0018] As used herein, "passive immunization" and the plural
thereof refer to the delivery of antibodies or fragments thereof to
a patient with the intention of conferring immunity to said
patient.
[0019] As used herein, "active immunization" and the plural thereof
refer to the delivery to a patient of peptides (in the form of
conjugates) acting as immunogens, i.e., they allow antibody
generation with the intention of conferring immunity to said
patient.
[0020] As used herein, "adjuvant" and the plural thereof refer to
immunomodulatory substances capable of being combined with the
conjugate of the present invention for increasing, improving or
otherwise modulating an immune response in a patient.
[0021] As used herein, "patient" and the plural thereof refer to
any mammal, preferably human, in which the conjugate of the present
invention or a composition comprising same can be administered for
the purpose of treating, preventing or delaying onset of an amyloid
disease.
[0022] As used herein, "CysA.beta.(33-40)" refers to the sequence
of positions 33 to 40 of A.beta.40 (SEQ ID NO: 2) to which a
cysteine has been added at the N-terminus. Said sequence is
reflected in SEQ ID NO: 1 and is: CGLMVGGVV.
[0023] Description
[0024] A first aspect the present invention relates to a conjugate
comprising at least one CysA.beta.(33-40) peptide (SEQ ID NO: 1)
and keyhole limpet hemocyanin (KLH), characterized in that the
crosslinking agent connecting each of the components of the
conjugate (each of said at least one peptide with KLH) is
maleimidobutyric acid N-hydroxysuccinimide ester (SM).
[0025] Said conjugate, in addition to allowing or producing an
effective and specific immune response against A.beta.40 (the
antibodies produced are specific for A.beta.40 without
significantly binding to A.beta.42), increases said response
compared with the response produced by other conjugates also
comprising CysA.beta.(33-40) peptide (SEQ ID NO: 1) and KLH, and in
which said elements have been bound or conjugated by means of
another crosslinking agent that also allow the binding of a peptide
to the transport protein.
[0026] In a second aspect, the present invention relates to a
composition comprising the conjugate of the present invention.
[0027] In a preferred embodiment, the composition additionally
comprises one or more adjuvants, which are preferably selected from
mineral salts (such as aluminum hydroxide, aluminum phosphate or
calcium phosphate), microparticles and active surface agents [such
as nonionic block polymer surfactants, virosomes, saponins,
meningococcal outer membrane proteins (proteosomes), immune
stimulating complexes, cochleates, dimethyl dioctadecyl ammonium
bromide, avridine, vitamin A or vitamin E], bacterial products
[such as the cell wall skeleton of Mycobacterium phlei, muramyl
dipeptides and tripeptides (threonyl-MDP, MDP-butyl ester,
dipalmitoyl phosphatidylethalonamine MTP), monophosphoryl lipid A,
Klebsiella pneumoniae glycoprotein, Bordetella pertussis, Bacillus
Calmette-Guerin, V. Cholerae and E. coli heat-labile enterotoxin,
trehalose dimycolate, CpG oligodeoxynucleotides], hormones and
cytokines (for example, interleukin-2, interferon .alpha.,
interferon-.beta., granulocyte-macrophage colony-stimulating
factor, dehydroepiandrosterone, Flt3 ligand, 1,25 dihydroxyvitamin
D.sub.3, interleukin-1, interleukin-6, interleukin-12, human growth
hormone, .beta.-microglobulin and lymphotactin), single antigen
constructs (such as multiple peptide antigens bound to a lysine
nucleus or cytotoxic T-cell epitopes bound to helper T-cell
epitopes and palmitoylated at the N-terminus), polyanions (such as
dextrans or double-stranded polynucleotides), polyacrylics (such as
polymethylmethacrylate or acrylic acid cross linked with allyl
sucrose), carriers [such as tetanus toxoid, diphtheria toxoid,
group B meningococcal outer membrane proteins (proteosomes),
Pseudomonas exotoxins A, a cholera toxin B subunit, a heat-labile
mutating enterotoxigenic E. coli enterotoxin, the hepatitis B virus
nucleus, cholera toxin A fusion proteins, CpG dinucleotides,
thermal shock proteins or fatty acids], live vectors (such as
vaccinia virus, canarypox virus, adenovirus, attenuated Salmonella
typhi, Bacillus Calmette-Guerin, Streptococcus gordonni, herpes
simplex virus, vaccine-derived poliovirus, rhinovirus, Venezuelan
equine encephalitis virus, Yersinia enterocolitica, Listeria
monocytogenes, Shigella, Bordetella pertussis or Saccharomyces
cerevisiae), vehicles [such as water-in-oil emulsions (for example
mineral oils such as complete Freund's adjuvant or incomplete
Freund's adjuvant; vegetable oils; squalene or squalane);
oil-in-water emulsions, such as a mixture of squalene, Tween-80 and
Span 85; liposomes; or biodegradable polymer microspheres of, for
example, lactides and glycolides, polyphosphazones, betal-glucans
or proteinoids], others (such as
N-acetyl-glucosamine-3yl-acetyl-L-alanyl-D-isoglutamine, gamma
insulin and aluminum hydroxide, transgenic plants, human dendritic
cells, lysophosphatidylglycerol, stearyl-tyrosine or tripalmitoyl
pentapeptide), or combinations thereof.
[0028] In a preferred embodiment of the second aspect of the
invention, said adjuvant is aluminum hydroxide, and more preferably
aluminum hydroxide gel. Therefore, a preferred embodiment of the
second aspect of the invention refers to a composition, preferably
a pharmaceutical composition, comprising i) a conjugate comprising
at least one CysA.beta.(33-40) peptide (SEQ ID NO: 1) linked to the
keyhole limpet hemocyanin (KLH) and ii) aluminum hydroxide gel,
wherein the crosslinking agent connecting each CysA.beta.(33-40)
peptide to the keyhole limpet hemocyanin (KLH) of the conjugate is
the maleimidobutyric acid N-hydroxysuccinimide ester (SM).
[0029] It is noted that, as shown in example 5, the adsorption
rates of the conjugate onto the aluminum hydroxide gel of said
preferred composition depends on the pH. In fact, the adsorption
rate increases at lower pH-values. A reduction from pH 7.4 to pH to
7.2 or even 7.0 does not result in significant higher adsorption
rates. An adsorption at pH 6.8 shows an adsorption rate around 90%.
At pH 6.0 the adsorption ratio is the highest. At a conjugate
concentration based on 200 .mu.g/mL net peptide and pH 6.7% of
conjugate is still free.
[0030] Based on these analyses, in order to increase the
immunogenic capacity of the pharmaceutical composition, a pH value
of between 5.8 to 7.0 should be used in order to increase the
adsorption rate of the conjugate onto the adjuvant and
significantly increase its immunogenic capacity.
[0031] Therefore, another preferred embodiment of the second aspect
of the invention refers to a composition, preferably a
pharmaceutical composition, comprising i) a conjugate comprising at
least one CysA.beta.(33-40) peptide (SEQ ID NO: 1) linked to the
keyhole limpet hemocyanin (KLH) and ii) aluminum hydroxide gel,
wherein the crosslinking agent connecting each CysA.beta.(33-40)
peptide to the keyhole limpet hemocyanin (KLH) of the conjugate is
the maleimidobutyric acid N-hydroxysuccinimide ester (SM), and
wherein the pH of the composition ranges from 5.8 to 7.0,
preferably from 6.2 to 7.0, more preferably from 5.8 to 6.2.
[0032] In further preferred embodiments of the second aspect of the
invention or of any of its preferred embodiments, the concentration
of CysA.beta.(33-40) peptides (SEQ ID NO: 1) in the pharmaceutical
composition is of at least 100 .mu.g, preferably at least 150
.mu.g, more preferably from 150 .mu.g to 400 .mu.g, still more
preferably from 160 .mu.g to 240 .mu.g, still more preferably about
200 .mu.g.
[0033] In yet further preferred embodiments of the second aspect of
the invention or of any of its preferred embodiments, the
KLH-SM-CysA.beta.(33-40) conjugates present in the pharmaceutical
composition comprise a ratio of at least 45 CysA.beta.(33-40)
peptides (SEQ ID NO: 1) per keyhole limpet hemocyanin (KLH)
protein.
[0034] In still another preferred embodiment of the second aspect
of the invention or of any of its preferred embodiments, the
pharmaceutical composition in contained in glass ampoules,
preferably of 1 or 1.2 ml.
[0035] It is further noted that, as reflected above, the adsorption
rates of the conjugate onto the aluminum hydroxide gel of in the
composition of the second aspect of the invention (when aluminum
hydroxide gel is used) depends on the pH. Moreover, surprisingly
the pH increases during the storage of the pharmaceutical
composition once it has been manufactured thus reducing the
adsorption rates if such increases reached pH values above 7.0, or
preferably above 6.8. In order to diminish such drawback that
clearly affects the immunogenic capacity of the pharmaceutical
composition, it is important to adjust the pH of the pharmaceutical
composition to a range of between 5.5 to 6.5, preferably 5.8 to
6.2, more preferably 5.9 to 6.1, at the time of manufacturing the
pharmaceutical composition so that storage does not or affects
minimally the immunogenic capacity of the composition.
[0036] Therefore, yet another preferred embodiment of the second
aspect of the invention refers to a method to manufacture a
pharmaceutical composition, which comprises the following steps:
[0037] a. Adding maleimidobutyric acid N-hydroxysuccinimide ester
(SM) to a composition comprising keyhole limpet hemocyanin (KLH) in
a buffer at a pH between 7.0 to 9; [0038] b. Eliminating the excess
of maleimidobutyric acid N-hydroxysuccinimide ester from the
solution of step a), preferably by using 0.02 M Na-Phosphate-buffer
at a pH of about 6.6 to 7.0; [0039] c. Adding CysA.beta.(33-40)
peptides (SEQ ID NO: 1) in DMSO to the solution of step b) at a pH
of between 6.6 to 7.0 to produce the conjugates; [0040] d.
Eliminating the free peptide from step c), preferably by using 0.01
M PBS-buffer at a pH of 6.6 to 7.0; [0041] e. Optionally filtrating
the solution of step d), preferably by using a filter of about 0.2
.mu.m.; [0042] f. Adjusting the pH of the solution of step d) or e)
to a arrange between 5.5 to 6.5, preferably 5.8 to 6.2, more
preferably 5.9 to 6.1, still more preferably about 6.0; and [0043]
g. Adding aluminum hydroxide gel to the solution of step f) once
the pH has been adjusted.
[0044] In a third aspect, the present invention discloses the use
of a composition comprising the conjugate of the present invention
for preparing a medicinal product. Preferably said composition is
the composition identified in the second aspect of the invention or
in any of its preferred embodiments. Also in a preferred
embodiment, said medicinal product is for use in the treatment or
prevention of an amyloid disease, more preferably of a amyloid
disease selected from Alzheimer's disease, Parkinson's disease,
cerebral amyloid angiopathy, vascular dementia of an amyloid
origin, inclusion-body myositis and dementia with Lewy bodies. In
the most preferred embodiment, said medicinal product is used for
the prevention or treatment of Alzheimer's disease.
[0045] In a fourth aspect, the present invention relates to a
method of treatment or of prevention of an amyloid disease in a
patient in need of same comprising the delivery of a
therapeutically effective amount of a composition comprising the
conjugate of the present invention. Preferably said composition is
the composition identified in the second aspect of the invention or
in any of its preferred embodiments. Also in a preferred
embodiment, said amyloid disease is a amyloid disease selected from
Alzheimer's disease, Parkinson's disease, cerebral amyloid
angiopathy, vascular dementia of an amyloid origin, inclusion-body
myositis and dementia with Lewy bodies, even more preferably
Alzheimer's disease.
[0046] In a final aspect, the present invention discloses a method
of manufacturing antibodies characterized in that it comprises an
immunization step for immunizing mammals or birds with a
composition comprising the conjugate of the present invention.
Preferably said composition is the composition identified in the
second aspect of the invention or in any of its preferred
embodiments.
[0047] It is contemplated that the mammals used in such method can
be ruminants, equidae, lagomorphs, primates (preferably humans) or
any other mammal that allows obtaining the suitable amounts of
serum for extracting or obtaining sufficient amounts of antibodies.
It is contemplated that the birds used in the method of the present
invention are any fowl-like birds, waterfowl, pigeons and doves or
any other bird that allows obtaining suitable amounts of serum for
extracting or obtaining sufficient amounts of antibodies. It is
further contemplated the protection of the antibodies obtained or
obtainable by the method of the final aspect of the invention as
well as their use to manufacture a pharmaceutical composition for
use in the treatment of an amyloid disease selected from
Alzheimer's disease, Parkinson's disease, cerebral amyloid
angiopathy, vascular dementia of an amyloid origin, inclusion-body
myositis and dementia with Lewy bodies, even more preferably
Alzheimer's disease.
[0048] Therefore, the present invention provides a conjugate
generated using the crosslinking agent SM, and compositions
comprising same that allow generating an greater immune response
compared with conjugates generated with other crosslinking agents
of the prior art.
[0049] Additionally, the immune response generated by said
conjugates of the present invention or the compositions comprising
said conjugates is specific for A.beta.40, i.e., it allows
generating anti-A.beta.40 specific antibodies without generating
anti-A.beta.42 antibodies.
[0050] For better understanding, the present invention is described
in further detail below in reference to the attached drawings
presented by way of example and in reference to illustrative
non-limiting examples.
EXAMPLE 1
Preparation of KLH-SM-CysA.beta.(33-40) Conjugates
[0051] For preparing these conjugates, KLH was used as a transport
protein, SM as a crosslinking agent and CysA.beta.(33-40) (SEQ ID
NO: 1) as an immunogenic peptide (peptide with residues 33-40 of
the amyloid peptide to which a cysteine has been added at the
N-terminus).
[0052] Binding took place between the available lysine residues of
the KLH and the cysteine added at the N-terminal end of the
peptide. In this case, the binding of the crosslinking agent to KLH
was done first (KLH activation step), and, in a second step, the
immunogenic peptide was added to the activated KLH so that
conjugation could take place.
[0053] The protocol followed for the foregoing is as follows:
[0054] A 250 mM stock solution of SM was prepared by dissolving 100
mg of SM in 680 .mu.L of dry DMSO. Aliquots of said stock solution
were made and stored at -20.degree. C. [0055] KLH was dissolved at
a concentration of 5 mg/mL in PBS/5 mM EDTA pH 7.4. [0056] 16 .mu.L
of stock solution of SM were added for each milliliter of KLH that
was conjugated. [0057] Said solution was left to react for 2 hours
and 30 minutes at room temperature under gentle stirring. [0058]
Then, the reaction buffer was changed to remove reaction byproducts
and excess unreacted SM. For this purpose, a PD10 column (GE
Healthcare; reference 17-0851-01) was used as follows: [0059] Step
a): The column was equilibrated with 25 mL (5 mL, 5 times) PBS (80
mM sodium hydrogenophosphate dihydrate, 20 mM sodium
dihydrogenophosphate monohydrate, 100 mM sodium chloride)/5 mM EDTA
pH 7.4. [0060] Step b): 2.5 mL of the already reacted solution were
added in the column and said solution was left to penetrate,
discarding the eluate. [0061] Step c): 3.5 mL of PBS were added in
the column and the eluate was collected. [0062] Step d): The column
was re-equilibrated with 25 mL (5 mL, 5 times) of 5 mM EDTA pH 7.4,
as explained above in step a). [0063] Steps b) to d) were repeated
all the times needed depending on the volume of solution that
reacted. After that, the column was rendered equilibrated. [0064]
Step e): The column was stored at 4.degree. C. in order to use it
on other occasions with the same peptide conjugate in the same
manner. Preceding protocol. [0065] Then the peptide that was to be
mixed with activated KLH was calculated. To that end, was taken
into account both the molecular weight of the peptide and the
molecular weight of KLH, in addition to the active sites therein
(measured according to the methods known in the prior art based on
measuring absorbance at 343 nm before and after treating the
activated KLH solution with dithiothreitol and performing the
necessary conversion). For example, for 10 mg of KLH with 1724
active sites:
[0066] 10/6725000 (mean molecular weight of
KLH)=1.48.times.10.sup.-6 mmol of KLH 1.48.times.10.sup.-6 mmol of
KLH.times.1724 active sites=2.55.times.10.sup.-3 mmol of peptide
required for covering all the active sites.
[0067] A 3-fold peptide excess was placed to favor the conjugation
reaction:
[0068] 2.55.times.10.sup.-3.times.3=7.65.times.10.sup.-3 mmol of
peptide required.
[0069] 7.65.times.10.sup.-3.times.weight molecular of the peptide
(834.4 Da)=mg of peptide required (6.38 mg of peptide) (applying
the required conversion factors). [0070] Once the peptide/activated
KLH ratio (i.e., KLH with bound SM) was determined, the following
reaction was prepared: mixture of the peptide with the activated
KLH with SM in a suitable proportion. To that end, the peptide was
prepared at 6 mg/mL in DMSO and was slowly added to activated KLH.
The proportion of DMSO in the final reaction must not exceed 30%.
If it were higher than that in any case, PBS/5 mM EDTA pH 7.4 is
added until the amount thereof was reduced to values of less than
30%. [0071] The solution was left to react at room temperature and
under stirring between 18 and 24 hours. [0072] Finally, the
obtained solutions were stored at 4.degree. C. The peptide that has
not conjugated with KLH and is therefore free may or may not be
removed. If said free peptide is not removed, the peptide
concentration in the final product is determined by the total
peptide used in the reaction, not only that which is bound to
native KLH, and the final reaction volume.
EXAMPLE 2
Comparison of the Immune Response of KLH-Crosslinking
Agent-CisA.beta.(33-40) Conjugates Generated using Different
Crosslinking Agents
[0073] In this case, the immune response strength generated in mice
(4 per group) with the following conjugates was compared: [0074]
KLH-SM-CysA.beta.(33-40) (produced according to Example 1). [0075]
KLH-SPDP-CysA.beta.(33-40): this conjugate uses a crosslinking
agent commonly used in the prior art, succinimidyl
3-(2-pyridyldithio)propionate (SPDP) and is produced by means of a
protocol very similar to the one described in Example 1
(introducing the necessary adaptations) and known in the prior
art.
[0076] The immune response strength test was conducted in BALB/c
strain mice. The protocol that was followed was:
[0077] 1. A week before the first inoculation, blood was drawn from
all the mice participating in the study to obtain pre-immune
serum.
[0078] 2. Depending on the group to which each mouse was assigned
(see Table 1 for the different analyzed groups), each mouse was
inoculated the corresponding vaccine once a week for three weeks
straight.
[0079] 3. A week after the third shot, blood was drawn again from
each of the mice participating in the study to measure the response
obtained in the serum.
TABLE-US-00001 TABLE 1 Groups used in the study and description of
the conjugate administered to each of them. Cross- Group Transport
protein linking Adjuvant 1 KLH, Manufacturer 1 SM Alhydrogel .RTM.
(aluminum hydroxide gel) 2 KLH, Manufacturer 1 SPDP Alhydrogel
.RTM. (aluminum hydroxide gel) 3 KLH, Manufacturer 2 SM Alhydrogel
.RTM. (aluminum hydroxide gel) 4 KLH, Manufacturer 2 SM None 5 KLH,
Manufacturer 2 SPDP Alhydrogel .RTM. (aluminum hydroxide gel) 6
KLH, Manufacturer 3 SM Alhydrogel .RTM. (aluminum (GMP) hydroxide
gel)
[0080] The peptide dose administered to the mice in each of the
groups shown in Table 1 is reflected in Table 2 included below.
TABLE-US-00002 TABLE 2 Peptide dose, including both total peptide
and peptide conjugated to KLH administered to each of the mice in
each of the groups of the study. Total peptide dose KLH-bound (sum
of free peptide and peptide dose Group KLH-bound peptide) (in
.mu.g) (in .mu.g) 1 120 40 2 120 40 3 120 40 4 120 40 5 120 40 6 60
60
[0081] Table 3 shows a tabulated summary of the immune response
strength results obtained for the different groups (analyzing serum
from the mice obtained the week after finishing the therapeutic or
vaccination regimen explained in the present example), together
with the increase observed in the immune response (fold number the
immune response increased a week after finishing the therapeutic
regimen with respect to the pre-immune response). The measurement
of the immune response was taken on the serum obtained from each
mouse by means of indirect ELISA, according to the protocol known
in the prior art, with respect to which it should be pointed out
that the ELISA plates were sieved with A.beta.40 peptide. Once the
corresponding steps of washing, blocking, subsequent washing,
incubation with the samples to be analyzed of plasma/serum (1:3
serial dilutions starting with a 1:30 dilution) and additional
washing were performed, each well was incubated with the Anti-mouse
IgG (H+L) antibody HRP (the secondary antibody dilution was 1:2000
in vehicle solution at pH 8). After incubating with said antibody
and washing the wells, the plate was developed by adding 100 .mu.L
per well of an ABTS solution (diammonium
2,2'-azinobis-[3-ethylbenzothiazolinesulfonate]; Roche; reference:
102 946 001) with 0.375 mg/mL in ABTS buffer (Roche; reference: 11
112 597 001). This substrate turned green upon reacting with the
peroxidase bound to the secondary antibody. The color intensity
depended on the amount of antibodies bound to the plate. The
reaction was incubated for 55 minutes at room temperature and in
the dark, and then the absorbance was read in an ELISA plates
reader at 405 nm. The obtained absorbance results were analyzed
with the GraphPad Prism 3.02 program. The "One Site Competition"
equation was used for the analysis:
Y = Minimum + ( Maximum - Minimum ) 1 + 10 x - LogEC 50
##EQU00001##
[0082] The EC.sub.50 data, which is the inflection point of the
curve, i.e., the point at which 50% of the maximum effect observed
was produced, was obtained from the aforementioned analysis. In the
present case, it was interpreted as the serum dilution at which 50%
of the peptide present in the well bound to the antibody present in
the serum.
TABLE-US-00003 TABLE 3 Mean immune response results obtained for
each of the groups of the study. It includes results for the serum
after the vaccine treatment (1 week after the three injections
according to the protocol described in the present example) and the
increase observed between said point and the pre-immune response
(before starting the therapeutic regimen). The mean pre-immune
EC.sub.50 was 18.11. Post-3 Increase in EC.sub.50 Group inoculation
EC.sub.50 (fold number) 1 12974.95 716.33 2 580.35 32.04 3 6325.50
349.22 4 924.97 51.07 5 1.87 0.10 6 5056.00 279.14
[0083] In view of the results shown in Table 3 and the conjugate
used in each of the study groups, the following can be concluded:
[0084] The different KLHs used as transport proteins (from
different manufacturers), despite having a certain effect on the
immune response, have not been shown to be relevant in determining
the magnitude of the immune response (high immune response in
groups 1, 3, 4 and 6, i.e., groups with an increase in EC.sub.50 of
50 or more, versus a weak or inexistent immune response in groups 2
and 5, i.e., groups with an increase in EC.sub.50 of less than 50).
[0085] The differences between obtaining a high immune response and
a weak or inexistent immune response lie in the crosslinking agent
used. Based on the experimental results obtained, it is deduced
that the conjugates in which SM were used produce an immune
response that is significantly greater that the one observed for
the conjugates in which SPDP was used. In fact, the groups treated
with conjugates with the crosslinking agent SM allow generating a
high immune response whereas the groups treated with conjugates in
which SPDP was used showed an immune response that was much weaker
or inexistent. [0086] In addition to the foregoing, the results
obtained for group 4 must be highlighted, as they clearly show that
the response of the conjugates generated using SM is still greater
than the one observed for the conjugates generated using SPDP, even
without the use of an adjuvant.
[0087] The foregoing shows that the use of SM as a crosslinking
agent for preparing the KLH-crosslinking agent-CysA.beta.(33-40)
conjugates provides vaccines with an immune response that is
surprisingly greater whether said conjugate is used with an
adjuvant or without an adjuvant.
EXAMPLE 3
Comparison of the Degree of Conjugation (Peptide Binding to KLH) of
KLH-Crosslinking Agent-CysA.beta.(33-40) Conjugates Generated using
Different Crosslinking Agents
[0088] As in the case of Example 2, the conjugates for which the
degree of conjugation or number of peptides bound per molecule of
transport protein (KLH) was compared are: [0089]
KLH-SM-CysA.beta.(33-40) (produced according to Example 1). [0090]
KLH-SPDP-CysA.beta.(33-40) (produced as indicated in Example
2).
[0091] Table 4 shows a tabulated summary of the experimental
results obtained for the experiment for binding the peptide to the
transport protein that was carried out.
TABLE-US-00004 TABLE 4 Description of the analyzed conjugates and
of the number of peptide molecules bound to each KLH molecule
observed by mass spectrometry. With respect to said bonds, the
table indicates one or two values depending on if one or two
batches of the corresponding conjugate were analyzed. Crosslinking
Number of peptide molecules Transport protein agent bound to each
KLH molecule KLH, Manufacturer 1 SM 81 KLH, Manufacturer 1 SPDP
35/33 KLH, Manufacturer 2 SM 70/68 KLH, Manufacturer 2 SPDP
44/31
[0092] As seen in Table 4, those KLHs from different manufacturers
had no effect on in the bonds result obtained. In contrast, the
crosslinking agent did indeed have an enormous effect on the
results obtained given that SM allowed obtaining twice the number
of bonds or more, i.e., by using SM as a crosslinking agent twice
the number of peptide molecules binds for each KLH molecule. This
result is surprising and unexpected given that the peptide used
incorporates a cysteine at the N-terminus for reacting with the
crosslinking agents. According to the prior art, the incorporation
of said cysteine should allow the peptide conjugation to be
efficient and equivalent to any of the crosslinking agents known in
the prior art. However, it was observed in this case that SM allows
a more efficient conjugation reaction than SPDP does.
[0093] These binding results allowed explaining part of the results
shown in Example 2 (i.e., part of the improvement observed in
immune response induction and subsequent antibody generation).
Nevertheless, said immune response results are not completely
assimilable to the obtained binding results, which is also
surprising in view of said obtained results and suggesting that the
crosslinking agent contributes to increasing the immune response
not only by mediating greater binding of the peptide to the
transport protein.
EXAMPLE 4
Immune Response Assays in Rabbits and Specificity of the Antibodies
Generated
[0094] A potency assay of vaccines comprising the
KLH-SM-CysA.beta.(33-40) conjugates was conducted in rabbits. In
this case, the rabbits were vaccinated with 200 .mu.g of total
bound peptide, said 200 .mu.g being bound to the transport protein
(chosen dose depending on preliminary studies). Each rabbit was
inoculated with 1 mL of the vaccine with the previously specified
dose, using 2% Alhydrogel.RTM. (aluminum hydroxide gel) as an
adjuvant.
[0095] The animals were treated with the previously indicated dose
by means of subcutaneous injection of the vaccine once a week for 3
straight weeks drawing blood a week before commencing the
vaccination protocol and a week after finishing it.
[0096] The titration of the generated antibodies and the analysis
of their specificity was done by means of ELISA, according to the
protocol known in the prior art and briefly indicated in Example 2,
with the following differences: [0097] The ELISA plates were sieved
with A.beta.40 or A.beta.42 peptide (included as SEQ ID NO: 3),
depending on if specific antibodies are to be detected or those
that bind to A.beta.40 or to A.beta.42, respectively. [0098] The
antibody used to detect the presence of antibodies in the analyzed
sera was Anti-Rabbit IgG (H+L) HRP (Invitrogen; reference: 65-6120)
(the secondary antibody dilution was 1:2000 in vehicle solution at
pH 8).
[0099] The development reagents were those indicated in Example 2,
and therefore the plates were also read at 405 nm and the same
equation was applied to the results. The EC.sub.50 data, which is
the inflection point of the curve, i.e., the point at which 50% of
the maximum effect observed was produced, was obtained from the
analysis. As indicated in Example 2, said result was interpreted as
the serum dilution at which 50% of the peptide present in the well
bound to the antibody present in the serum.
[0100] According to the aforementioned titration protocol, all the
samples obtained were titrated to detect A.beta.40 and A.beta.42
peptide antibodies. The obtained results are shown in tabulated
form in Tables 5 and 6.
TABLE-US-00005 TABLE 5 Amount of anti-A.beta.40 specific antibodies
in rabbits before and after the vaccination protocol. A column
relating to the increase observed as a consequence of treatment is
also included. Identification Post-3 Increase in EC.sub.50 of
Rabbit Pre-immune EC.sub.50 inoculation EC.sub.50 (fold number) 71
36.17 47406 1310.64 72 7.483 4021 537.35 73 14.07 73421 5218.27 74
4.482 12467 2781.57 75 45.48 5173 113.74 76 20.5 98381 4799.07
TABLE-US-00006 TABLE 6 Amount of A.beta.42 specific antibodies in
rabbits before and after the vaccination protocol. A column
relating to the increase observed as a consequence of treatment is
also included. Identification Post-3 Increase in EC.sub.50 of
Rabbit Pre-immune EC.sub.50 inoculation EC.sub.50 (fold number) 71
53.88 0.049 0.00 72 29.24 0.069 0.00 73 22.49 2.761 0.12 74 24.05
15.49 0.64 75 73.18 0.063 0.00 76 23.37 34.04 1.46
[0101] Based on what is shown in Tables 5 and 6, it is deduced that
the conjugate of the present invention (KLH-SM-CysA.beta.(33-40))
allows not only obtaining a high immune response in rabbits but
also said response is specific for A.beta.40 (without a significant
humoral response to A.beta.42), i.e., anti-A.beta.40 specific
antibodies are generated in the immune response that do not bind to
A.beta.42.
[0102] The results included in Examples 1 to 4 prove and confirm
the technical advantages and effects explained above in the
description, proving that the use of SM as a crosslinking agent
allows generating KLH-crosslinking agent-CysA.beta.(33-40)
conjugates that generate a greater immune response with respect to
when another crosslinking agent of the prior art is used.
Additionally, KLH-SM-CisA.beta.(33-40) conjugates allow generating
high immune responses in mice and rabbits, specific for A.beta.40
(without a significant humoral response to A.beta.42), i.e.,
anti-A.beta.40 specific antibodies that do not bind to A.beta.42
are generated in said immune responses. Said examples validate the
usefulness of the conjugate of the present invention in the
treatment of amyloid diseases, preferably Alzheimer's disease, in
mammals, preferably in humans.
EXAMPLE 5
Adsorption Studies for KLH-SM-CysA.beta.(33-40) Conjugates onto the
Aluminum Hydroxide Gel
[0103] 1.35 mg/ml, peptide is equivalent to 14.4 mg/ml conjugate
KLH-SM-CysA.beta.(33-40).
TABLE-US-00007 TABLE 7 equivalent to .mu.g/mL .mu.g/mL
Abeta-X40-KLH % conjugate based on peptide net supernatant free in
% peptide net supernatant [mg/mL] solution adsorbed 100 12 0.128 12
88 125 15 0.160 12 88 150 19 0.203 13 87 175 25 0.267 14 86 200 35
0.373 18 83
[0104] Study 2: Adsorption-Dependency of pH at Defined
Concentrations
TABLE-US-00008 Testing 1: 220 .mu.g peptide equivalent to .mu.g/mL
Abeta-X40-KLH % conjugate peptide net supernatant free in % pH
supernatant [mg/mL] solution adsorbed 7.4 38 0.41 17 83 7.2 37 0.39
17 83 7.0 33 0.35 15 85 6.8 25 0.27 11 89 6.5 21 0.22 10 90 6.0 15
0.16 7 93
TABLE-US-00009 Testing 2: 150 .mu.g peptide equivalent to .mu.g/mL
Abeta-X40-KLH % conjugate peptide net supernatant free in % pH
supernatant [mg/mL] solution adsorbed 7.4 18 0.19 12 88 7.2 18 0.19
12 88 7.0 15 0.16 10 90 6.8 10 0.11 7 93 6.5 11 0.12 7 93 6.0 0
0.00 0 100
TABLE-US-00010 Testing 3: 200 .mu.g peptide equivalent to .mu.g/mL
Abeta-X40-KLH % conjugate peptide net supernatant free in % pH
supernatant [mg/mL] solution adsorbed 7.4 13 0.14 13 87 7.2 13 0.14
13 87 7.0 12 0.13 12 88 6.8 9 0.10 9 91 6.5 0 0.00 0 100 6.0 0 0.00
0 100
[0105] In these studies the influence of the concentration of
conjugate on the adsorption rate as well as the influence of the pH
on the adsorption rate were determined. In the first experiment the
adsorption rate of different concentrations of conjugate in a
defined amount of aluminium hydroxide (0.35% corresponds to the
permitted dose of 1.25 mg Al per single dose) and the defined pH
7.4 (physiological pH) was tested. At the physiological testing
conditions (pH 7.4) 12 to 18% free conjugate was detected in the
supernatant. A reduction of the amount of peptide to 100 .mu.g
peptide does not yield higher adsorption rates.
[0106] In a second study the influence of the pH on the adsorption
rate was determined. The pH was altered (pH 6.0 to pH 7.4) at
defined conjugate concentrations (based on 100 .mu.g, 150 .mu.g and
220 .mu.g peptide net/mL). As illustrated in the results shown
above, the adsorption rate depends on the pH. In fact, the
adsorption rate increases at lower pH-values (see tables): A
reduction from pH 7.4 to pH to 7.2 or even 7.0 does not result in
significant higher adsorption rates. An adsorption at pH 6.8 shows
an adsorption rate around 90%. At pH 6.0 the adsorption ratio is
the highest. At a conjugate concentration based on 200 .mu.g/mL net
peptide and pH 6, 7% of conjugate is still free.
[0107] Based on these analyses, in order to increase the
immunogenic capacity of the pharmaceutical composition of the
invention, a pH value of between 5.8 to 7.0 should preferably be
used in order to increase the adsorption rate of the conjugate onto
the adjuvant.
[0108] Although the invention has been described with respect to
preferred embodiments, the latter must not be considered to be
limiting of the invention, which will be defined by the broadest
interpretation of the following claims.
Sequence CWU 1
1
319PRTArtificial sequenceAbeta fragment with cysteine added at the
N- terminus 1Cys Gly Leu Met Val Gly Gly Val Val1 5240PRTHomo
sapiens 2Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His
Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly
Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val 35 40342PRTHomo
sapiens 3Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His
Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly
Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val Ile Ala 35
40
* * * * *