U.S. patent application number 17/369177 was filed with the patent office on 2021-10-28 for enhancing immunogenicity of streptococcus pneumoniae polysaccharide-protein conjugates.
This patent application is currently assigned to Merck Sharp & Dohme Corp.. The applicant listed for this patent is Merck Sharp & Dohme Corp.. Invention is credited to Chitrananda Abeygunawardana, Jian He, Joseph G. Joyce, John E. MacNair, Patrick McHugh, Luwy Musey, Hari Pujar, Julie M. Skinner, William J. Smith, Michael A. Winters.
Application Number | 20210330777 17/369177 |
Document ID | / |
Family ID | 1000005705396 |
Filed Date | 2021-10-28 |
United States Patent
Application |
20210330777 |
Kind Code |
A1 |
He; Jian ; et al. |
October 28, 2021 |
ENHANCING IMMUNOGENICITY OF STREPTOCOCCUS PNEUMONIAE
POLYSACCHARIDE-PROTEIN CONJUGATES
Abstract
The present invention provides immunogenic compositions having
one or more polysaccharide-protein conjugates in which one or more
polysaccharides from Streptococcus pneumoniae bacterial capsules
are conjugated to a carrier protein in an aprotic solvent such as
dimethylsulfoxide (DMSO). The present invention also provides
methods for providing an enhanced immune response to a pneumococcal
polysaccharide protein conjugate vaccine comprising administering
to a human subject an immunogenic composition comprising
polysaccharide-protein conjugates prepared in DMSO conditions.
Inventors: |
He; Jian; (Blue Bell,
PA) ; MacNair; John E.; (Lansdale, PA) ;
Smith; William J.; (Harleysville, PA) ; Winters;
Michael A.; (Doylestown, PA) ; Joyce; Joseph G.;
(Lansdale, PA) ; Abeygunawardana; Chitrananda;
(Ambler, PA) ; Musey; Luwy; (Blue Bell, PA)
; Pujar; Hari; (Berwyn, PA) ; Skinner; Julie
M.; (Phoenixville, PA) ; McHugh; Patrick;
(Pipersville, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merck Sharp & Dohme Corp. |
Rahway |
NJ |
US |
|
|
Assignee: |
Merck Sharp & Dohme
Corp.
Rahway
NJ
|
Family ID: |
1000005705396 |
Appl. No.: |
17/369177 |
Filed: |
July 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16487550 |
Aug 21, 2019 |
11090374 |
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PCT/US2018/018729 |
Feb 20, 2018 |
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17369177 |
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62555444 |
Sep 7, 2017 |
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62463216 |
Feb 24, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/116 20130101;
A61P 31/04 20180101; A61K 39/092 20130101; A61K 47/646 20170801;
A61K 2039/6037 20130101 |
International
Class: |
A61K 39/09 20060101
A61K039/09; A61K 47/64 20060101 A61K047/64; A61P 31/04 20060101
A61P031/04; A61K 39/116 20060101 A61K039/116 |
Claims
1. An immunogenic composition comprising polysaccharide from S.
pneumoniae serotype conjugated to a carrier protein, wherein the
conjugation reaction whereby the polysaccharide is conjugated to
the carrier protein is in an aprotic solvent.
2. (canceled)
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4. (canceled)
5. (canceled)
6. (canceled)
7. The immunogenic composition of claim 1, wherein the conjugation
reaction is reductive amination.
8. The immunogenic composition of claim 1, wherein the aprotic
solvent is dimethylsulfoxide (DMSO).
9. The immunogenic composition of claim 1, wherein the carrier
protein is CRM.sub.197.
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54. The immunogenic composition of claim 1, wherein the carrier
protein is selected from the group consisting of Outer Membrane
Protein Complex (OMPC), tetanus toxoid, diphtheria toxoid, protein
D and CRM.sub.197.
55. The immunogenic composition of claim 1, wherein the carrier
protein is selected from the group consisting of tetanus toxoid,
diphtheria toxoid, and CRM.sub.197.
Description
FIELD OF INVENTION
[0001] The present invention provides immunogenic compositions
comprising at least one Streptococcus pneumoniae polysaccharide
conjugated to CRM.sub.197 using reductive amination in an aprotic
solvent such as dimethylsulfoxide (DMSO). The invention also
provides methods for enhancing immunogenicity of immunogenic
compositions having one or more polysaccharide-protein conjugates
in which one or more polysaccharides from S. pneumoniae bacterial
capsules are conjugated to a carrier protein using reductive
amination performed in an aprotic solvent such as DMSO.
BACKGROUND OF THE INVENTION
[0002] Streptococcus pneumoniae is a Gram-positive bacterium and
the most common cause of invasive bacterial disease (such as
pneumonia, bacteraemia and meningitis and Otitis media) in infants
and young children. Pneumococcus is encapsulated with a chemically
linked polysaccharide which confers serotype specificity. There are
over 90 known serotypes of pneumococci, and the capsule is the
principle virulence determinant for pneumococci, as the capsule not
only protects the inner surface of the bacteria from complement,
but is itself poorly immunogenic. Polysaccharides are T-cell
independent antigens, and can not be processed or presented on MHC
molecules to interact with T-cells. They can however, stimulate the
immune system through an alternate mechanism which involves
cross-linking of surface receptors on B cells.
[0003] Children less than 2 years of age do not mount an immune
response to most polysaccharide vaccines, so it has been necessary
to render the polysaccharides immunogenic by chemical conjugation
to a protein carrier. Coupling the polysaccharide, a T-cell
independent antigen, to a protein, a T-cell dependent antigen,
confers upon the polysaccharide the properties of T cell dependency
including isotype switching, affinity maturation, and memory
induction.
[0004] There are many conjugation reactions that have been employed
for covalently linking polysaccharides to proteins. Three of the
more commonly employed methods include: 1) reductive amination,
wherein the aldehyde or ketone group on one component of the
reaction reacts with the amino or hydrazide group on the other
component, and the C.dbd.N double bond formed is subsequently
reduced to C--N single bond by a reducing agent; 2) cyanylation
conjugation, wherein the polysaccharide is activated either by
cyanogen bromide (CNBr) or by 1-cyano-4-dimethylamrnoniumpyridinium
tetrafluoroborate (CDAP) to introduce a cyanate group to the
hydroxyl group, which forms a covalent bond to the amino or
hydrazide group upon addition of the protein component; and 3) a
carbodiimide reaction, wherein carbodiimide activates the carboxyl
group on one component of the conjugation reaction, and the
activated carbonyl group reacts with the amino or hydrazide group
on the other component. These reactions are also frequently
employed to activate the components of the conjugate prior to the
conjugation reaction.
[0005] Reductive amination has been utilized to conjugate S.
pneumoniae polysaccharides. See, for example, U.S. Pat. No.
8,192,746, U.S. Patent Application Publication No. 20170021006 and
International Patent Application Publication Nos. WO2011/110381 and
WO2015/110941. Reductive amination involves two steps: (1)
oxidation of the antigen, and (2) reduction of the antigen and a
carrier protein to form a conjugate. The reduction step can take
place in an aqueous solvent or an aprotic solvent such as DMSO.
See, e.g., International Patent Application Publication No.
WO2016/113644.
SUMMARY OF THE INVENTION
[0006] The present invention provides immunogenic compositions
comprising polysaccharides from one or more of S. pneumoniae
serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14,
15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31,
34, 35A, 35B, 35F, and 38 conjugated to a carrier protein, wherein
the conjugation reaction whereby the polysaccharide is conjugated
to the carrier protein is in an aprotic solvent. In one embodiment,
for compositions having identical serotypes, one or more of the
serotypes prepared in an aprotic solvent have increased
immunogenicity when compared to the same one or more serotypes
prepared under aqueous conditions.
[0007] The present invention provides immunogenic compositions
comprising polysaccharide protein conjugates prepared from one or
more of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8,
9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A,
23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, and 38 conjugated to a
carrier protein, wherein the polysaccharide protein conjugates are
made by a process comprising the step of conjugating the
polysaccharide to the carrier protein in an aprotic solvent.
[0008] The invention also provides methods of conjugating a
polysaccharide from S. pneumoniae serotype 1, 2, 3, 4, 5, 6C, 6D,
7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21,
22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, or 38 to a
carrier protein, comprising the step of conjugating the
polysaccharide to the carrier protein in an aprotic solvent.
[0009] The invention also provides methods of treating a subject
with an immunogenic composition comprising one or more
polysaccharides from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6C, 6D,
7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21,
22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, or 38
conjugated to a carrier protein, wherein the polysaccharide is
conjugated to the carrier protein in an aprotic solvent.
[0010] In certain embodiments, polysaccharides from one or more of
S. pneumoniae serotypes 1, 3, 4, 5, 9V, 11A, 12F, and 14 are
conjugated to a carrier protein in an aprotic solvent. In certain
embodiments, polysaccharides from one or more of S. pneumoniae
serotypes 2, 6C, 6D, 7B, 7C, 8, 9N, 15A, 15C, 16F, 17F, 20, 21,
22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, and 38 are
conjugated to a carrier protein in an aprotic solvent.
[0011] In certain embodiments, polysaccharides from one or more of
S. pneumoniae serotypes 3 and 18C are conjugated to a carrier
protein in an aprotic solvent. In one aspect of this embodiment,
polysaccharides from S. pneumoniae serotype 3 are conjugated to a
carrier protein in an aprotic solvent. In one aspect of this
embodiment, polysaccharides from S. pneumoniae serotype 18C are
conjugated to a carrier protein in an aprotic solvent.
[0012] In certain embodiments, the conjugation reaction used to
conjugate polysaccharide to the carrier protein is reductive
amination.
[0013] In certain embodiments, the aprotic solvent is DMSO.
[0014] In certain embodiments, the carrier protein is
CRM.sub.197.
[0015] In certain embodiments, conjugates prepared in DMSO have a
lysine loss value greater than 5.0. In one aspect, conjugates
prepared in DMSO have a lysine loss value between 7.0 and 18.0
inclusive.
[0016] In certain embodiments, the immunogenic composition further
comprises polysaccharides from one or more of S. pneumoniae
serotypes 6A, 6B, 7F, 10A, 15B, 19A, 19F, 22F, 23F, and 33F
conjugated to a carrier protein, wherein the conjugation reaction
whereby the polysaccharide is conjugated to the carrier protein is
in an aprotic solvent. In certain aspects of this embodiment, the
conjugation reaction is reductive amination. In certain aspects,
the aprotic solvent is DMSO. In certain aspects, the carrier
protein is CRM.sub.197. In one aspect, the immunogenic compostion
comprises polysaccharide from S. pneumoniae serotypes 6A, 6B, 7F,
18C, 19A, 19F, and 23F conjugated to a carrier protein, wherein the
conjugation reaction whereby the polysaccharide from S. pneumoniae
serotypes 6A, 6B, 7F, 18C, 19A, 19F, or 23F is conjugated to the
carrier protein is in an aprotic solvent. In certain aspects, the
polysaccharide is from S. pneumoniae serotypes 18C, 19A, 19F or
23F.
[0017] In certain embodiments, the immunogenic compositions of the
invention further comprises polysaccharides from one or more of S.
pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8,
9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F,
20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A,
35B, 35F, and 38 conjugated to a carrier protein, wherein the
conjugation reaction whereby the polysaccharide from S. pneumoniae
serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V,
10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21,
22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F,
or 38 is conjugated to the carrier protein is in an aqueous
solvent. In one aspect, between 35-100% of the serotypes in the
immunogenic composition are prepared using reductive amination
under DMSO conditions and the remaining polysaccharide protein
conjugates are prepared under aqueous conditions.
[0018] In one specific embodiment, the invention provides an
immunogenic composition consisting essentially of polysaccharides
from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C,
19A, 19F, 22F, 23F and 33F conjugated to CRM.sub.197
polysaccharide, wherein the conjugation reaction for S. pneumoniae
serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F is in DMSO conditions
and the conjugation reaction for S. pneumoniae serotypes 1, 3, 4,
5, 9V, 14, 22F and 33F is in an aqueous solvent, and optionally
further comprising about 0.2% w/v PS-20.
[0019] The present invention also provides methods for inducing a
protective immune response in a human subject comprising
administering any of the immunogenic compositions of the invention.
In certain embodiments, the subject is 50 years or older and/or
immunocompromised. In certain embodiments, the subject is 2 years
old or younger. In certain embodiments, the subject is
immunocompromised.
[0020] The present invention also provides methods for providing an
enhanced immune response to a pneumococcal polysaccharide (PnPs)
protein conjugate vaccine comprising administering to a animal
subject an immunogenic composition comprising
polysaccharide-protein conjugates comprising S. pneumoniae capsular
polysaccharides from a first set of two or more pneumococcal
serotypes conjugated to one or more carrier proteins, wherein the
two or more of the polysaccharide-protein conjugates from the first
set are prepared using reductive amination under DMSO conditions.
In one embodiment, said enhanced immune response is relative to a
control animal receiving an immunogenic composition wherein one or
more of the two or more polysaccharide-protein conjugates from the
first set are prepared using reductive amination in aqueous
conditions. In one embodiment, the control animal is a mouse. In
another embodiment, the control animal is a human. In certain
embodiments, the methods employ pneumococcal polysaccharide protein
conjugate vaccine which comprises additional polysaccharide-protein
conjugates comprising S. pneumoniae capsular polysaccharides from a
second set of pneumococcal serotypes conjugated to one or more
carrier proteins are prepared using reductive amination under
aqueous conditions, wherein serotypes from the second set are
different from serotypes in the first set.
[0021] In certain embodiments, the methods employ pneumococcal
polysaccharide protein conjugate vaccine where the pneumococcal
serotypes are selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C,
6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F,
17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A,
33F, 34, 35A, 35B, 35F, and 38.
[0022] In certain embodiments, the methods employ pneumococcal
polysaccharide protein conjugate vaccine where
polysaccharide-protein conjugates from serotype 3 or 18C are
prepared using reductive amination under DMSO conditions.
[0023] In certain embodiments, the methods employ pneumococcal
polysaccharide protein conjugate vaccine where the
polysaccharide-protein conjugates from the first set of
pneumococcal serotypes are selected from serotypes 6A, 6B, 7F, 18C,
19A, 19F, and 23F.
[0024] In certain embodiments, the methods employ pneumococcal
polysaccharide protein conjugate vaccine where
polysaccharide-protein conjugates from the first set of
pneumococcal serotypes comprise serotypes 6A, 6B, 7F, 18C, 19A,
19F, and 23F, which are prepared using reductive amination under
DMSO conditions, and polysaccharide-protein conjugates from a
second set of serotypes are prepared under aqueous conditions.
[0025] In one specific embodiment, the methods employ pneumococcal
polysaccharide protein conjugate vaccine where
polysaccharide-protein conjugates from serotypes 6A, 6B, 7F, 18C,
19A, 19F, and 23F are prepared using reductive amination under DMSO
conditions and polysaccharide protein conjugates from serotypes 1,
3, 4, 5, 9V, 14, 22F and 33F are prepared under aqueous
conditions.
[0026] In certain embodiments, the methods employ pneumococcal
polysaccharide protein conjugate vaccine where polysaccharide
protein conjugates from between 35-100% of the serotypes are
prepared using reductive amination under DMSO conditions and the
remaining polysaccharide protein conjugates are prepared under
aqueous conditions. In one aspect, polysaccharide protein
conjugates from between 45-80% of the serotypes are prepared using
reductive amination under DMSO conditions and the remaining
polysaccharide protein conjugates are prepared under aqueous
conditions. In another aspect, polysaccharide protein conjugates
from between 75-100% of the serotypes are prepared using reductive
amination under DMSO conditions and the remaining polysaccharide
protein conjugates are prepared under aqueous conditions.
[0027] In certain embodiments, the methods employ pneumococcal
polysaccharide protein conjugate vaccine where the carrier protein
is selected from the group consisting of Neisseria meningitides
Outer Membrane Protein Complex (OMPC), tetanus toxoid, diphtheria
toxoid, protein D and CRM.sub.197. In one aspect, the carrier
protein is CRM.sub.197.
[0028] In certain embodiments, the methods employ pneumococcal
polysaccharide protein conjugate vaccine where the conjugates
prepared using reductive amination under DMSO conditions have a
higher proportion of glycopeptide bonds formed as measured by
protein lysine loss value greater than 5.0. In one aspect of this
embodiment, the conjugates prepared using reductive amination under
DMSO conditions have a lysine loss value between 7.0 to 18
inclusive. In another aspect, the conjugates prepared using
reductive amination under DMSO conditions have a lysine loss value
greater than 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, or 10.0.
[0029] In another specific embodiment, the invention provides a
method for providing an enhanced immune response to a pneumococcal
polysaccharide (PnPs) protein conjugate vaccine consisting
essentially of polysaccharides from S. pneumonia serotypes 1, 3, 4,
5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated
to CRM.sub.197 polysaccharide, wherein the method comprises
administering to a human subject an immunogenic composition
comprising polysaccharide-protein conjugates from a first set and a
second set of pneumococcal serotypes, wherein the first set of
serotypes consists of 6A, 6B, 7F, 18C, 19A, 19F, and 23F and are
prepared using reductive amination under DMSO conditions, and the
second set of serotypes consists of 1, 3, 4, 5, 9V, 14, 22F and 33F
and are prepared under aqueous conditions.
[0030] In certain embodiments, the enhanced immune response in
animals vaccinated with immunogenic compositions produced by the
methods of the invention is measured by serum IgG or
opsonophagocytic antibody Geometric Mean Titers. In one aspect, the
enhanced immune response to a pneumococcal serotype is 10% or
greater compared to polysaccharide-protein conjugate from the same
pneumococcal serotype prepared under aqueous conditions. In one
embodiment, the animal is a mouse. In another embodiment, the
animal is a human.
[0031] In certain embodiments, the methods are employed with a
human subject which is 50 years old or older. In certain
embodiments, the methods are employed with a human subject which is
2 years old or younger. In certain embodiments, the methods are
employed with a human subject which is immunocompromised.
[0032] The invention also provides methods of preparing a
pneumococcal polysaccharide-protein conjugate by reductive
amination, the method comprising:
[0033] a) reacting a Streptococcus pneumoniae polysaccharide
selected from serotypes 3, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F,
23F, and 33F with an amount of an oxidant (e.g., a periodate) to
form an activated polysaccharide having an activation level from
0.05 to 0.22;
[0034] b) reacting the activated polysaccharide with a carrier
protein in an aprotic solvent, optionally in the presence of a
reducing agent, to form a polysaccharide-protein conjugate;
[0035] wherein the conjugate has a lysine loss value between 7.0 to
18.0 inclusive.
[0036] In certain embodiments, the activation level is from 0.09 to
0.22.
[0037] In certain embodiments, the oxidant is periodate.
[0038] In certain embodiments, the activation level is measured by
derivitizing aldehydes on the polysaccharide with
thiosemicarbazide.
[0039] In certain embodiments, the reducing agent is a
cyanoborohydride salt such as sodium cyanoborohydride.
[0040] In certain embodiments, the carrier protein is selected from
the group consisting of tetanus toxoid, diphtheria toxoid, and
CRM.sub.197. In one embodiment, the carrier protein is
CRM.sub.197.
[0041] The present invention also provides a quantitative method
for determining the aldehyde level (i.e., the level of periodate
activation) in an activated polysaccharide comprising the steps
of:
[0042] a) derivatizing the activated polysaccharide to form a
derivatized polysaccharide by reacting with a derivatizing agent
until completion (i.e., the reaction plateaus);
[0043] b) isolating the derivatized polysaccharide by high
performance size exclusion chromatography (to remove unreacted
derivatizing agent and matrix components);
[0044] c) quantifying the UV absorbance of the derivatized
polysaccharide.
[0045] The derivatizing agent may be selected from the group
consisting of thiosemicarbazide, thiosemicarbazide structural
analogs, hydrazides, hydrazine, semicarbazide, semicarbazide
structural analogs, aminooxy compounds or aromatic amines.
[0046] In one embodiment, the quantifying in step c) is by
comparison to a derivative standard. In one embodiment, the
quantifying in step c) is by measurement against predetermined
extinction coefficient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1. Extent of conjugation at different lysine sites on
CRM.sub.197 as determined by tryptic peptide mapping. Serotype 19A
Ps-CRM.sub.197 conjugates, prepared by reductive amination in
either aqueous solution or DMSO, were digested by trypsin and
analyzed by LC-UV-MS. The loss of peptide signal compared to
CRM.sub.197 control samples was plotted against the sites of
conjugation.
[0048] FIG. 2. Electrochemiluminescent (ECL) immunogenicity results
from mouse study arms comparing serotype 3 Ps-CRM.sub.197
conjugates prepared by reductive amination in either aqueous
solution or DMSO. Conjugates formulated with aluminum phosphate
adjuvant (APA). Pre-vaccination (Pre) and post-dose 3 (PD3) results
are shown. Results for APA only control are also shown.
[0049] FIG. 3. Post-dose 3 opsophagocytic activity (OPA) results
from mouse study arms comparing serotype 3 Ps-CRM.sub.197
conjugates prepared by reductive amination in either aqueous
solution or DMSO. OPA results pre-vaccination (Pre-immune) and for
APA only control are also shown.
DETAILED DESCRIPTION OF THE INVENTION
[0050] The present invention provides immunogenic compositions
comprising pneumococcal polysaccharide-protein conjugates, in which
conjugates from at least one pneumococcal serotype are prepared
using reductive amination in an aprotic solvent such as DMSO. The
present invention is based, in part, on the discovery that the use
of DMSO as a solvent during reductive amination of
polysaccharide-protein conjugates results in the unexpectedly
superior stability and enhanced immunogenicity for those serotypes
relative to the same conjugates prepared under aqueous conditions.
The present invention relates to the advantages of DMSO solvent in
enhancing the covalent associations of polysaccharide to protein
through direct consumption of lysine residues on the surface of the
carrier protein. For most serotypes tested, a "lysine loss" that
provided good immunogenicity (.gtoreq.7.0) could be achieved at a
lower polysaccharide activation level (0.05 to 0.22) through
conjugation in an aprotic solvent than in an aqueous buffer. The
increased covalent association has a direct benefit to increasing
the stability of the polysaccharide protein conjugate and in
enhancing the immune response to those particular polysaccharide
antigens conjugated in DMSO.
[0051] Without being bound by any theory, one possible mechanism
for the enhanced immunogenicity observed with glycoconjugates
prepared in DMSO include an increased number of linkages between
the carbohydrate (capsular polysaccharide) and lysine residues on
the surface of the carrier protein which would result in additional
attachment points between the protein and polysaccharide to impart
stability and counter chemical depolymerization or breakdown of the
peptide carbohydrate bond. See, e.g., Hsieh, Characterization of
Saccharide-CRM.sub.197 Conjugate Vaccines in Brown F, Corbel M,
Griffiths E (eds): Physico-Chemical Procedures for the
Characterization of Vaccines. Dev. Biol. Basel, Karger, 2000, vol
103, pp. 93-104. An additional benefit of the increased
polysaccharide-protein linkages that are created during conjugation
in the DMSO solvent could be additional opportunities for
successful presentation of peptide-carbohydrate to T-cells. It can
be appreciated that due to the genetic varability in the human
population resulting in varying abilities and sensitivity of
loading or associating with specific peptide sequences conjugated
to carbohydrate antigens, that additional points of attachment on
the carrier protein would allow for increased chances for
successful antigen presentation at the surface of an APC to allow
for a T-cell dependent response to an otherwise T-cell independent
antigen. Another possible mecahnsim of enhanced immunogenicity
observed by conjugation in the DMSO solvent could be due to the
denaturation of CRM.sub.197 in organic solvent, which exposes
additional lysines for polysaccharide linkages giving increased
chances for glycopeptide presentation at the surface of an APC for
T-cell dependent response to different peptide epitopes. See Avci
et al., 2011, Nature Medicine 17: 1602-1610.
[0052] Yet another benefit of conjugation in an organic solvent
generating denatured CRM.sub.197 in the conjugates could be reduced
immunological interference of antibodies against native CRM.sub.197
epitopes. A further benefit of the increased polysaccharide-protein
linkages that are created during conjugation in the DMSO solvent
could be the formation of larger sized polysaccharide protein
conjugates resulting in enhanced immunogenicity. The compositions
of the invention are believed to provide significant advantages in
eliciting a human response.
[0053] As shown in the Example 5, a polysaccharide protein
conjugate prepared from S. pneumoniae serotype 3 using reductive
amination in DMSO showed increased immunogenicity (compared to the
same conjugate prepared using reductive amination in water) in a
mouse model as measured by opsophagocytic activity (OPA). Moreover,
as shown in the Example 6, a 15-valent pneumococcal conjugate
vaccine having seven serotypes prepared using reductive amination
in DMSO (and the other eight prepared in an aqueous solvent) tended
to show superior immunogenicity in humans (with 4 serotypes
superior with statistical significance) for all seven serotypes
prepared in DMSO compared to the corresponding 15-valent PCV where
all 15 seroytpes were prepared in an aqueous solvent.
[0054] As shown in Example 4, plotting the peptide signal decrease
for lysine locations on the CRM.sub.197 protein in serotype 19A
conjugates against possible sites of conjugation (compared to a
CRM197 control) uncovered additional conjugation sites located in
previously identified common human T-cell peptide epitopes (See
Raju et al., 1995, Eur. J. Immunol. 25:3207-3214, located in
peptide 411-430 and peptide 431-450 of CRM.sub.197 sequence).
Accordingly, in certain embodiments, the present invention is also
directed to immunogenic compositions comprising one or more
polysaccharide-CRM.sub.197 conjugates, wherein at least one of the
polysaccharide-CRM.sub.197 conjugates is prepared in an aprotic
solvent and wherein a conjugate prepared in an aprotic solvent
demonstrates greater accessibility of lysines residues within amino
acids 411-430 or 431-450 of CRM.sub.197 compared to the same
conjugate prepared in an aqeuous solvent. In certain embodiments,
the present invention is also directed to immunogenic compositions
comprising one or more polysaccharide-CRM.sub.197 conjugates,
wherein at least one of the polysaccharide-CRM.sub.197 conjugates
is prepared in an aprotic solvent and wherein one or more lysine
residues within amino acids 411-430 or 431-450 of CRM.sub.197 in a
conjugate prepared in an aprotic solvent are conjugated more than
10%. In certain embodiments, the present invention is directed to
methods for increasing the accessibility of lysine residues within
CRM.sub.197, particularly within amino acids 411-430 or 431-450 of
CRM.sub.197, comprising conjugating a polysaccharide to CRM.sub.197
in an aprotic solvent. In certain aspects of this embodiment, one
or more lysine residues within amino acids 411-430 or 431-450 of
CRM.sub.197 in a conjugate prepared in an aprotic solvent are
conjugated more than 10%. In these embodiments, the polysaccharide
can be from any organism suitable for preparing an immunogenic
composition. In certain aspects, the polysaccharide is from N.
meningitides or S. pneumoniae. The polysaccharide may be from any
serotype of these organisms.
[0055] As used herein, the terms "aqueous solvent" or "aqueous
conditions" when used with conjugation, such as reductive
amination, refers use of water as the solvent for the conjugation
reaction. The water may contain buffers and other components except
that no organic solvent is present.
[0056] As used herein, the terms "aprotic solvent", when used with
conjugation, such as reductive amination, refers use of a polar
aprotic solvent, or a combination of polar aprotic solvents, as the
solvent for the conjugation reaction. Examples of polar aprotic
solvents include, but are not limited to, dimethylsulfoxide (DMSO),
dimethylformamide (DMF), and hexamethylphosphoramide (HMPA). The
aprotic solvent may have some water present, for example, up to 1%,
2%, 5%, 10% or 20%.
[0057] As used herein, "DMSO solvent" and "DMSO conditions" are
used interchangeably.
[0058] As used herein, the term "comprises" when used with the
immunogenic composition of the invention refers to the inclusion of
any other components (subject to limitations of "consisting of"
language for the antigen mixture), such as adjuvants and
excipients. The term "consisting of" when used with the multivalent
polysaccharide-protein conjugate mixture refers to a mixture having
those particular S. pneumoniae polysaccharide protein conjugates
and no other S. pneumoniae polysaccharide protein conjugates from a
different serotype.
[0059] As used herein, "lysine loss" refers to the lysine
consumption during conjugation and is determined by the difference
between the average measured amount of lysine in the conjugate and
the expected amount of lysine in the starting protein. Example 4
describes one method for determining "lysine loss".
[0060] As used herein, the term "polysaccharide" is meant to
include any antigenic saccharide element (or antigenic unit)
commonly used in the immunologic and bacterial vaccine arts,
including, but not limited to, a "saccharide", an
"oligosaccharide", a "polysaccharide", a "liposaccharide", a
"lipo-oligosaccharide (LOS)", a "lipopolysaccharide (LPS)", a
"glycosylate", a "glycoconjugate" and the like.
[0061] When referring to percentages of serotypes in the
immunogenic composition being prepared under in an aprotic solvent
(e.g., DMSO) and the remaining polysaccharide protein conjugates
being prepared under aqueous conditions, it is meant to simply
refer to the number of serotypes prepared in an aprotic solvent
divided by the total number of serotypes in the composition.
[0062] As used herein, all ranges, for example, pH, temperature,
and concentrations, are meant to be inclusive. For example, a pH
range from 5.0 to 9.0 is meant to include a pH of 5.0 and a pH of
9.0. Similarly, a temperature range from 4 to 25.degree. C. is
meant to include the outer limits of the range, i.e., 4.degree. C.
and 25.degree. C.
Polysaccharide
[0063] S. pneumonia capsular polysaccharides that can be prepared
according to the methods of the invention, i.e., reductive
amination in an aprotic solvent, include, but are not limited to,
serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V,
10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21,
22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F,
and 38. The polysaccharides may be used in the form of
oligosaccharides. These are conveniently formed by fragmentation of
purified polysaccharide (e.g. by hydrolysis), which will usually be
followed by purification of the fragments of the desired size.
[0064] In certain embodiments, one or more of serotypes 1, 2, 3, 4,
5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F,
18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F,
and 38 are prepared using reductive amination in an aprotic
solvent. In certain aspects, pneumococcal polysaccharides from one
or more of serotypes 1, 3, 4, 5, 9V, 11A, 12F, and 14 are prepared
using reductive amination in an aprotic solvent. In certain
aspects, pneumococcal polysaccharides from one or more of serotypes
2, 6C, 6D, 7B, 7C, 8, 9N, 15A, 15C, 16F, 17F, 19F, 20, 21, 22A,
23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, and 38 are prepared
using reductive amination in an aprotic solvent. In certain
aspects, pneumococcal polysaccharides from one or both of serotypes
3 or 18C are conjugated to a carrier protein using reductive
amination in an aprotic solvent. Polysaccharides from the other
serotypes in a multivalent composition may be conjugated using
reductive amination in an aprotic solvent or in an aqueous solvent.
Polysaccharides from the other serotypes in a multivalent
composition may also be conjugated using other chemistries which
may be in an aprotic solvent or in an aqueous solvent.
[0065] Capsular polysaccharides from Streptococcus pneumoniae can
be prepared by standard techniques known to those skilled in the
art. For example, polysaccharides can be isolated from bacteria and
may be sized to some degree by known methods (see, e.g., European
Patent Nos. EP497524 and EP497525); and preferably by
microfluidisation accomplished using a homogenizer or by chemical
hydrolysis. In one embodiment, each pneumococcal polysaccharide
serotype is grown in a soy-based medium. The individual
polysaccharides are then purified through standard steps including
centrifugation, precipitation, and ultra-filtration. See, e.g.,
U.S. Patent Application Publication No. 2008/0286838 and U.S. Pat.
No. 5,847,112. Polysaccharides can be sized in order to reduce
viscosity in polysaccharide samples and/or to improve filterability
for conjugated products using techniques such as mechanical or
chemical sizing. Chemical hydrolysis maybe conducted using acetic
acid. Mechanical sizing maybe conducted using High Pressure
Homogenization Shearing.
[0066] In some embodiments, the purified polysaccharides before
conjugation have a molecular weight of between 5 kDa and 4,000 kDa.
Molecular weight can be calculated by size exclusion chromatography
(SEC) combined with multiangle light scattering detector (MALS) and
refractive index detector (RI). In other such embodiments, the
polysaccharide has a molecular weight of between 10 kDa and 4,000
kDa; between 50 kDa and 4,000 kDa; between 50 kDa and 3,000 kDa;
between 50 kDa and 2,000 kDa; between 50 kDa and 1,500 kDa; between
50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDa
and 500 kDa; between 100 kDa and 4,000 kDa; between 100 kDa and
3,000 kDa; 100 kDa and 2,000 kDa; between 100 kDa and 1,500 kDa;
between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between
100 kDa and 500 kDa; between 100 and 400 kDa; between 200 kDa and
4,000 kDa; between 200 kDa and 3,000 kDa; between 200 kDa and 2,000
kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,000 kDa;
or between 200 kDa and 500 kDa.
[0067] The purified polysaccharides can be chemically activated to
make the saccharides capable of reacting with the carrier protein.
The purified polysaccharides can be connected to a linker. Once
activated or connected to a linker, each capsular polysaccharide is
separately conjugated to a carrier protein to form a
glycoconjugate. The polysaccharide conjugates may be prepared by
known coupling techniques.
[0068] The polysaccharide can be coupled to a linker to form a
polysaccharide-linker intermediate in which the free terminus of
the linker is an ester group. The linker is therefore one in which
at least one terminus is an ester group. The other terminus is
selected so that it can react with the polysaccharide to form the
polysaccharide-linker intermediate.
[0069] The polysaccharide can be coupled to a linker using a
primary amine group in the polysaccharide. In this case, the linker
typically has an ester group at both termini. This allows the
coupling to take place by reacting one of the ester groups with the
primary amine group in the polysaccharide by nucleophilic acyl
substitution. The reaction results in a polysaccharide-linker
intermediate in which the polysaccharide is coupled to the linker
via an amide linkage. The linker is therefore a bifunctional linker
that provides a first ester group for reacting with the primary
amine group in the polysaccharide and a second ester group for
reacting with the primary amine group in the carrier molecule. A
typical linker is adipic acid N-hydroxysuccinimide diester
(SIDEA).
[0070] The coupling can also take place indirectly, i.e. with an
additional linker that is used to derivatise the polysaccharide
prior to coupling to the linker.
[0071] The polysaccharide is coupled to the additional linker using
a carbonyl group at the reducing terminus of the polysaccharide.
This coupling comprises two steps: (a1) reacting the carbonyl group
with the additional linker; and (a2) reacting the free terminus of
the additional linker with the linker. In these embodiments, the
additional linker typically has a primary amine group at both
termini, thereby allowing step (a1) to take place by reacting one
of the primary amine groups with the carbonyl group in the
polysaccharide by reductive amination. A primary amine group is
used that is reactive with the carbonyl group in the
polysaccharide. Hydrazide or hydroxylamino groups are suitable. The
same primary amine group is typically present at both termini of
the additional linker. The reaction results in a
polysaccharide-additional linker intermediate in which the
polysaccharide is coupled to the additional linker via a C--N
linkage.
[0072] The polysaccharide can be coupled to the additional linker
using a different group in the polysaccharide, particularly a
carboxyl group. This coupling comprises two steps: (a1) reacting
the group with the additional linker; and (a2) reacting the free
terminus of the additional linker with the linker. In this case,
the additional linker typically has a primary amine group at both
termini, thereby allowing step (a1) to take place by reacting one
of the primary amine groups with the carboxyl group in the
polysaccharide by EDAC activation. A primary amine group is used
that is reactive with the EDAC-activated carboxyl group in the
polysaccharide. A hydrazide group is suitable. The same primary
amine group is typically present at both termini of the additional
linker. The reaction results in a polysaccharide-additional linker
intermediate in which the polysaccharide is coupled to the
additional linker via an amide linkage.
[0073] In one embodiment, the chemical activation of the
polysaccharides and subsequent conjugation to the carrier protein
by reductive amination can be achieved by means described in U.S.
Pat. Nos. 4,365,170, 4,673,574 and 4,902,506, U.S. Patent
Application Publication Nos. 2006/0228380, 2007/184072,
2007/0231340 and 2007/0184071, and International Patent Application
Publication Nos. WO2006/110381, WO2008/079653, and WO2008/143709).
The chemistry may entail the activation of pneumococcal
polysaccharide by reaction with any oxidizing agent which oxidizes
a terminal hydroxyl group to an aldehyde, such as periodate
(including sodium periodate, potassium periodate, or periodic
acid). The reaction leads to a random oxidative cleavage of vicinal
hydroxyl groups of the carbohydrates with the formation of reactive
aldehyde groups.
[0074] In one embodiment, the polysaccharide is reacted with 0.01
to 10.0, 0.05 to 5.0, 0.1 to 1.0, 0.5 to 1.0, 0.7 to 0.8, 0.05 to
0.5, 0.1 to 0.3 molar equivalents of oxidizing agent. In a
embodiment, the polysaccharide is reacted with about 0.1, 0.15,
0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75,
0.8, 0.85, 0.9, 0.95 molar equivalents of oxidizing agent. It is
generally preferable to use lower amounts of periodate for
activation, for example, 0.1 to 0.3 Meq, in order to achieve
limited polysaccharide activation (e.g., 0.05 to 0.22 or 0.09 to
0.22 moles of aldehyde/mole of polysaccharide repeat unit). As used
herein, "activation level" refers to moles of aldehyde/mole of
polysaccharide repeat unit. Less polysaccharide activation results
in a more native like polysaccharide, i.e., fewer hydroxyl groups
are converted to aldehydes.
[0075] In another embodiment, the duration of the oxidation
reaction is between 1 hour and 50 hours, between 10 hours and 30
hours, between 15 hours and 20 hours, between 15 hours and 17 hours
or about 16 hours.
[0076] In another embodiment, the temperature of the oxidation
reaction is maintained between 15.degree. C. and 45.degree. C.,
between 15.degree. C. and 30.degree. C., between 20.degree. C. and
25.degree. C. In another embodiment, the temperature of the
reaction is maintained at about 23.degree. C.
[0077] Coupling to the carrier protein is by reductive amination
via direct amination to the lysyl groups of the protein. For
example, conjugation is carried out by reacting a mixture of the
activated polysaccharide and carrier protein with a reducing agent
such as sodium cyanoborohydride in the presence of nickel. The
conjugation reaction may take place under aqueous solution or in
the presence of dimethylsulfoxide (DMSO). See, e.g., U.S. Patent
Application Publication Nos. US2015/0231270 and US2011/0195086 and
European Patent No. EP 0471 177 B1. Unreacted aldehydes are then
capped with the addition of a strong reducing agent, such as sodium
borohydride.
[0078] Reductive amination involves two steps, (1) oxidation of the
polysaccharide to form reactive aldehydes, and (2) reduction of the
imine (Schiff base) formed between activated polysaccharide and a
carrier protein to form a stable amine conjugate bond. Before
oxidation, the polysaccharide is optionally size reduced.
Mechanical methods (e.g. homogenization) or chemical hydrolysis may
be employed. Chemical hydrolysis maybe conducted using acetic acid.
The oxidation step may involve reaction with periodate. For the
purpose of the present invention, the term "periodate" includes
both periodate and periodic acid; the term also includes both
metaperiodate (IO.sub.4.sup.-) and orthoperiodate (IO.sub.6.sup.-)
and includes the various salts of periodate (e.g. , sodium
periodate and potassium periodate). In an embodiment, the capsular
polysaccharide is oxidized in the presence of metaperiodate,
preferably in the presence of sodium periodate (NaIO.sub.4). In
another embodiment the capsular polysaccharide is oxidized in the
presence of orthoperiodate, preferably in the presence of periodic
acid.
[0079] In an embodiment, the oxidizing agent is a stable nitroxyl
or nitroxide radical compound, such as piperidine-N-oxy or
pyrrolidine-N-oxy compounds, in the presence of an oxidant to
selectively oxidize primary hydroxyls (as described in WO
2014/097099). In said reaction, the actual oxidant is the
N-oxoammonium salt, in a catalytic cycle. In an aspect, said stable
nitroxyl or nitroxide radical compound are piperidine-N-oxy or
pyrrolidine-N-oxy compounds. In an aspect, said stable nitroxyl or
nitroxide radical compound bears a TEMPO
(2,2,6,6-tetramethyl-1-piperidinyloxy) or a PROXYL
(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In an aspect, said
stable nitroxyl radical compound is TEMPO or a derivative thereof.
In an aspect, said oxidant is a molecule bearing a N-halo moiety.
In an aspect, said oxidant is selected from the group consisting of
N-ChloroSuccinimide, N-Bromosuccinimide, N-lodosuccinimide,
Dichloroisocyanuric acid,
1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, Dibromoisocyanuric
acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione,
Diiodoisocyanuric acid and
1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione.
[0080] In certain aspects, the oxidizing agent is
2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) free radical and
N-Chlorosuccinimide (NCS) as the cooxidant (as described in
International Patent Application Publication No. WO2014/097099).
Therefore in one aspect, the glycoconjugates from S. pneumoniae are
obtained by a method comprising the steps of: a) reacting a
saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and
N-chlorosuccinimide (NCS) in an aqueous solvent to produce an
activated saccharide; and b) reacting the activated saccharide with
a carrier protein comprising one or more amine groups (said method
is designated "TEMPO/NCS-reductive amination" thereafter).
[0081] Optionally the oxidation reaction is quenched by addition of
a quenching agent. The quenching agent maybe selected from vicinal
diols, 1,2-aminoalcohols, amino acids, glutathione, sulfite,
bisulfate, dithionite, metabisulfite, thiosulfate, phosphites,
hypophosphites or phosphorous acid (such as glycerol, ethylene
glycol, propan-1,2-diol, butan-1,2-diol or butan-2,3-diol, ascorbic
acid).
[0082] The second step of the conjugation process is the reduction
of the imine (Schiff base) bond between activated polysaccharide
and a carrier protein to form a stable conjugate bond (so-called
reductive amination), using a reducing agent. Reducing agents which
are suitable include the cyanoborohydrides (such as sodium
cyanoborohydride or sodium borohydride). In one embodiment the
reducing agent is sodium cyanoborohydride.
[0083] In certain embodiments of the methods of the invention, the
reductive amination reaction is carried out in aprotic solvent (or
a mixture of aprotic solvents). In an embodiment, the reduction
reaction is carried out in DMSO or in DMF (dimethylformamide)
solvent. The DMSO or DMF solvent may be used to reconstitute the
activated polysaccharide and carrier protein, if lyophilized. In
one embodiment, the aprotic solvent is DMSO.
[0084] At the end of the reduction reaction, there may be unreacted
aldehyde groups remaining in the conjugates, which may be capped
using a suitable capping agent. In one embodiment this capping
agent is sodium borohydride (NaBH.sub.4). Suitable alternatives
include sodium triacetoxyborohydride or sodium or zinc borohydride
in the presence of Bronsted or Lewis acids, amine boranes such as
pyridine borane, 2-Picoline Borane, 2,6-diborane-methanol,
dimethylamine-borane, t-BuMe'PrN-BH.sub.3, benzylamine-BH.sub.3 or
5-ethyl-2-methylpyridine borane (PEMB) or borohydride exchange
resin. Following the conjugation (the reduction reaction and
optionally the capping), the glycoconjugates may be purified
(enriched with respect to the amount of polysaccharide-protein
conjugate) by a variety of techniques known to the skilled person.
These techniques include dialysis, concentration/diafiltration
operations, tangential flow filtration,
precipitation/elution,column chromatography (ion exchange
chromatography, multimodal ion exchange chromatography, DEAE, or
hydrophobic interaction chromatography), and depth filtration. In
an embodiment, the glycoconjugates are purified by diafilitration
or ion exchange chromatography or size exclusion
chromatography.
[0085] Glycoconjugates prepared using reductive amination in an
aprotic solvent are generally used in multivalent pneumococcal
conjugate vaccines. Thus, in certain embodiments for multivalent
compositions where not all the serotypes are prepared in an aprotic
solvent, the reduction reaction for the remaining seroytpes is
carried out in aqueous solvent (e.g., selected from PBS (phosphate
buffered saline), MES (2-(N-morpholino)ethanesulfonic acid), HEPES,
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Bis-tris, ADA
(N-(2-Acetamido)iminodiacetic acid), PIPES
(piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO
(3-Morpholino-2-hydroxypropanesulfonic acid), BES
(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS
(3-(N-morpholino)propanesulfonic acid), DIPSO
(3-Bis(2-hydroxyethyl) amino-2-hydroxypropane-1-sulfonic acid),
MOBS (4-(N-morpholino)butanesulfonic acid), HEPPSO
(N-(2-Hydroxyethyl)piperazine-N-(2-hydroxypropanesulfonic acid)),
POPSO (Piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA
(triethanolamine), EPPS
(4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid), Bicine or
HEPB, at a pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0 and
7.5).
[0086] In some embodiments, the glycoconjugates of the present
invention comprise a polysaccharide having a molecular weight of
between 10 kDa and 10,000 kDa. In other such embodiments, the
polysaccharide has a molecular weight of between 25 kDa and 5,000
kDa. In other such embodiments, the polysaccharide has a molecular
weight of between 50 kDa and 1,000 kDa. In other such embodiments,
the polysaccharide has a molecular weight of between 70 kDa and 900
kDa. In other such embodiments, the polysaccharide has a molecular
weight of between 100 kDa and 800 kDa. In other such embodiments,
the polysaccharide has a molecular weight of between 200 kDa and
600 kDa. In further such embodiments, the polysaccharide has a
molecular weight of 100 kDa to 1000 kDa; 100 kDa to 900 kDa; 100
kDa to 800 kDa; 100 kDa to 700 kDa; 100 kDa to 600 kDa; 100 kDa to
500 kDa; 100 kDa to 400 kDa; 100 kDa to 300 kDa; 150 kDa to 1,000
kDa; 150 kDa to 900 kDa; 150 kDa to 800 kDa; 150 kDa to 700 kDa;
150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 150 kDa
to 300 kDa; 200 kDa to 1,000 kDa; 200 kDa to 900 kDa; 200 kDa to
800 kDa; 200 kDa to 700 kDa; 200 kDa to 600 kDa; 200 kDa to 500
kDa; 200 kDa to 400 kDa; 200 kDa to 300; 250 kDa to 1 ,000 kDa; 250
kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700 kDa; 250 kDa to
600 kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350
kDa; 300 kDa to 1 ,000 kDa; 300 kDa to 900 kDa; 300 kDa to 800 kDa;
300 kDa to 700 kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa
to 400 kDa; 400 kDa to 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to
800 kDa; 400 kDa to 700 kDa; 400 kDa to 600 kDa; 500 kDa to 600
kDa.
[0087] In some embodiments, the glycoconjugates of the present
invention have a molecular weight of between 1,000 kDa and 10,000
kDa. In other such embodiments, the polysaccharide has a molecular
weight of between 1,000 kDa and 7,000 kDa. In other such
embodiments, the polysaccharide has a molecular weight of between
1,000 kDa and 6,000 kDa.
[0088] In certain embodiments, the conjugation reaction is
performed by reductive amination wherein nickel is used for greater
conjugation reaction efficiency and to aid in free cyanide removal.
Transition metals are known form stable complexes with cyanide and
are known to improve reductive methylation of protein amino groups
and formaldehyde with sodium cyanoborohydride (S Gidley et al.,
Biochem J. 1982, 203: 331-334; Jentoft et al. Anal Biochem. 1980,
106: 186-190). By complexing residual, inhibitory cyanide, the
addition of nickel increases the consumption of protein during the
conjugation of and leads to formation of larger, potentially more
immungenic conjugates.
[0089] Differences in starting cyanide levels in sodium
cyanoborohydride reagent lots also lead to inconsistent conjugation
performance, resulting in variable product attributes, such as
conjugate size and conjugate Ps-to-CRM.sub.197 ratio. The addition
of nickel reduced conjugation inconsistency by complexing cyanide,
eliminating differences in sodium cyanoborohydride lots.
[0090] Suitable alternative chemistries include the activation of
the saccharide with 1-cyano-4-dimethylamino pyridinium
tetrafluoroborate (CDAP) to form a cyanate ester. The activated
saccharide may thus be coupled directly or via a spacer (linker)
group to an amino group on the carrier protein. For example, the
spacer could be cystamine or cysteamine to give a thiolated
polysaccharide which could be coupled to the carrier via a
thioether linkage obtained after reaction with a
maleimide-activated carrier protein (for example using GMBS) or a
haloacetylated carrier protein (for example using iodoacetimide
[e.g. ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or
SIAB, or SIA, or SBAP). For example, the cyanate ester (optionally
made by CDAP chemistry) is coupled with hexane diamine or adipic
acid dihydrazide (ADH) and the amino-derivatised saccharide is
conjugated to the carrier protein using carbodiimide (e.g. EDAC or
EDC) chemistry via a carboxyl group on the protein carrier. Such
conjugates are described in International Patent Application
Publication Nos. WO 93/15760, WO 95/08348 and WO 96/29094; and Chu
et al., 1983, Infect. Immunity 40:245-256.
[0091] Other suitable techniques use carbodiimides, hydrazides,
active esters, norborane, p-nitrobenzoic acid,
N-hydroxysuccinimide, S--NHS, EDC, TSTU. Many are described in
International Patent Application Publication No. WO 98/42721.
Conjugation may involve a carbonyl linker which may be formed by
reaction of a free hydroxyl group of the saccharide with CDI (See
Bethell et al., 1979, J. Biol. Chem. 254:2572-4; Hearn et al.,
1981, J. Chromatogr. 218:509-18) followed by reaction of with a
protein to form a carbamate linkage. This may involve reduction of
the anomeric terminus to a primary hydroxyl group, optional
protection/deprotection of the primary hydroxyl group, reaction of
the primary hydroxyl group with CDI to form a CDI carbamate
intermediate and coupling the CDI carbamate intermediate with an
amino group on a protein.
[0092] After conjugation of the capsular polysaccharide to the
carrier protein, the polysaccharide-protein conjugates are purified
(enriched with respect to the amount of polysaccharide-protein
conjugate) by one or more of a variety of techniques. Examples of
these techniques are well known to the skilled artisan and include
concentration/diafiltration operations, ultrafiltration,
precipitation/elution, column chromatography, and depth filtration.
See, e.g., U.S. Pat. No. 6,146,902.
[0093] Another way to characterize the glycoconjugates of the
invention is by the number of lysine residues in the carrier
protein (e.g., CRM.sub.197) that become conjugated to the
saccharide which can be characterized as a range of conjugated
lysines (degree of conjugation). The evidence for lysine
modification of the carrier protein, due to covalent linkages to
the polysaccharides, can be obtained by amino acid analysis using
routine methods known to those of skill in the art. Conjugation
results in a reduction in the number of lysine residues recovered,
compared to the carrier protein starting material used to generate
the conjugate materials. In an embodiment, the degree of
conjugation of the glycoconjugate of the invention is between 2 and
18, between 2 and 13, between 2 and 10, between 2 and 8, between 2
and 6, between 2 and 5, between 2 and 4, between 3 and 15, between
3 and 13, between 3 and 10, between 3 and 8, between 3 and 6,
between 3 and 5, between 3 and 4, between 5 and 18, between 5 and
13, between 7 and 18, between 7, and 13, between 8 and 18, between
8 and 13, between 10 and 18 or between 10 and 13. In an embodiment,
the degree of conjugation of the glycoconjugate of the invention is
about 2, about 3, about 4, about 5, about 6, about 7, about 8,
about 9, about 10, about 11 , about 12, about 13, about 14 or about
15. In an embodiment, the degree of conjugation of the
glycoconjugate of the invention is between 7 and 18. In some such
embodiments, the carrier protein is CRM.sub.197.
[0094] The glycoconjugates of the invention may also be
characterized by the ratio (weight/weight) of saccharide to carrier
protein. In some embodiments, the ratio of polysaccharide to
carrier protein in the glycoconjugate (w/w) is between 0.5 and 3.0
(e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about
1.0, about 1.1 , about 1.2, about 1.3, about 1.4, about 1.5, about
1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1 , about
2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about
2.8, about 2.9, or about 3.0). In other embodiments, the saccharide
to carrier protein ratio (w/w) is between 0.5 and 2.0, between 0.5
and 1.5, between 0.8 and 1.2, between 0.5 and 1.0, between 1.0 and
1.5 or between 1.0 and 2.0. In further embodiments, the saccharide
to carrier protein ratio (w/w) is between 0.8 and 1.2. In an
embodiment, the ratio of capsular polysaccharide to carrier protein
in the conjugate is between 0.9 and 1.1. In some such embodiments,
the carrier protein is CRM197. The glycoconjugates and immunogenic
compositions of the invention may contain free saccharide that is
not covalently conjugated to the carrier protein, but is
nevertheless present in the glycoconjugate composition. The free
saccharide may be non-covalently associated with (i.e.,
non-covalently bound to, adsorbed to, or entrapped in or with) the
glycoconjugate.
[0095] In an embodiment, the glycoconjugate comprises less than
about 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% of free
polysaccharide compared to the total amount of polysaccharide. In
an embodiment the glycoconjugate comprises less than about 25% of
free polysaccharide compared to the total amount of polysaccharide.
In an embodiment the glycoconjugate comprises less than about 20%
of free polysaccharide compared to the total amount of
polysaccharide. In an embodiment the glycoconjugate comprises less
than about 15% of free polysaccharide compared to the total amount
of polysaccharide.
Multivalent Polysaccharide-Protein Conjugate Vaccines
[0096] Multivalent pneumococcal immunogenic compositions can
comprise capsular polysaccharides from S. pneumoniae serotype
selected from at least one of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B,
7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C,
19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F,
34, 35A, 35B, 35F, and 38 conjugated to one or more carrier
proteins, wherein a polysaccharide from at least one serotype is
prepared using reductive amination in an aprotic solvent such as
DMSO. The present invention contemplates multivalent pneumococcal
immunogenic compositions having polysaccharides from at least 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 serotypes. Preferably, saccharides from a particular
serotype are not conjugated to more than one carrier protein.
[0097] In certain embodiments, polysaccharides from at least 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 serotypes are prepared using reductive amination in an
aprotic solvent such as DMSO.
[0098] In certain embodiments, one or more of serotypes 3, 6A, 6B,
7F, 18C, 19A, 19F, or 23F are prepared using reductive amination in
an aprotic solvent. In certain aspects of this embodiment, one or
both of serotypes 3 or 18C are prepared using reductive amination
in an aprotic solvent.
[0099] In certain embodiments, at least 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, or 100% of the serotypes in a
multivalent composition are prepared in an aprotic solvent. The
remainder of the serotypes are prepared using an alternative
chemistry and/or in an aqueous solvent.
[0100] In certain embodiments, one or more of serotypes 1, 2, 3, 4,
5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F,
18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F,
and 38 are prepared using reductive amination in an aprotic
solvent. In certain aspects, one or more of serotypes 1, 3, 4, 5,
9V, 11A, 12F, and 14 are prepared using reductive amination in an
aprotic solvent. In certain aspects, one or more of serotypes 2,
6C, 6D, 7B, 7C, 8, 9N, 15A, 15C, 16F, 17F, 19F, 20, 21, 22A, 23A,
23B, 24F, 27, 28A, 31, 34, 35B, 35F, and 38 are prepared using
reductive amination in an aprotic solvent.
[0101] In one embodiment, a multivalent composition consists of
polysaccharides from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F
prepared using reductive amination in an aprotic solvent such as
DMSO and polysaccharides from serotypes 1, 3, 4, 5, 9V, 14, 22F and
33F prepared using reductive amination in an aqueous solvent.
[0102] After the individual glycoconjugates are purified, they are
compounded to formulate the immunogenic composition of the present
invention. These pneumococcal conjugates are prepared by separate
processes and bulk formulated into a single dosage formulation.
Carrier Protein
[0103] In a particular embodiment of the present invention,
CRM.sub.197 is used as the carrier protein. CRM.sub.197 is a
non-toxic variant (i.e., toxoid) of diphtheria toxin. In one
embodiment, it is isolated from cultures of Corynebacterium
diphtheria strain C7 (.beta.197) grown in casamino acids and yeast
extract-based medium. In another embodiment, CRM.sub.197 is
prepared recombinantly in accordance with the methods described in
U.S. Pat. No. 5,614,382. Typically, CRM.sub.197 is purified through
a combination of ultra-filtration, ammonium sulfate precipitation,
and ion-exchange chromatography. In some embodiments, CRM.sub.197
is prepared in Pseudomonas fluorescens using Pfenex Expression
Technology.TM. (Pfenex Inc., San Diego, Calif.).
[0104] Other suitable carrier proteins include additional
inactivated bacterial toxins such as DT (Diphtheria toxoid) or
fragment B of DT (DTFB), TT (tetanus toxid) or fragment C of TT,
pertussis toxoid, cholera toxoid (e.g., as described in
International Patent Application Publication No. WO 2004/083251),
E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa.
Bacterial outer membrane proteins such as outer membrane complex c
(OMPC), porins, transferrin binding proteins, pneumococcal surface
protein A (PspA; See International Application Patent Publication
No. WO 02/091998), pneumococcal adhesin protein (PsaA), C5a
peptidase from Group A or Group B streptococcus, or Haemophilus
influenzae protein D, pneumococcal pneumolysin (Kuo et al., 1995,
Infect Immun 63; 2706-13) including ply detoxified in some fashion
for example dPLY-GMBS (See International Patent Application
Publication No. WO 04/081515) or dPLY-formol, PhtX, including PhtA,
PhtB, PhtD, PhtE and fusions of Pht proteins for example PhtDE
fusions, PhtBE fusions (See International Patent Application
Publication Nos. WO 01/98334 and WO 03/54007), can also be used.
Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH),
bovine serum albumin (BSA) or purified protein derivative of
tuberculin (PPD), PorB (from N. meningitidis), PD (Haemophilus
influenzae protein D; see, e.g., European Patent No. EP 0 594 610
B), or immunologically functional equivalents thereof, synthetic
peptides (See European Patent Nos. EP0378881 and EP0427347), heat
shock proteins (See International Patent Application Publication
Nos. WO 93/17712 and WO 94/03208), pertussis proteins (See
International Patent Application Publication No. WO 98/58668 and
European Patent No. EP0471177), cytokines, lymphokines, growth
factors or hormones (See International Patent Application
Publication No. WO 91/01146), artificial proteins comprising
multiple human CD4+ T cell epitopes from various pathogen derived
antigens (See Falugi et al., 2001, Eur J Immunol 31:3816-3824) such
as N19 protein (See Baraldoi et al., 2004, Infect Immun 72:4884-7),
iron uptake proteins (See International Patent Application
Publication No. WO 01/72337), toxin A or B of C. difficile (See
International Patent Publication No. WO 00/61761), and flagellin
(See Ben-Yedidia et al., 1998, Immunol Lett 64:9) can also be used
as carrier proteins.
[0105] Other DT mutants can be used as the second carrier protein,
such as CRM.sub.176, CRM.sub.228, CRM.sub.45 (Uchida et al., 1973,
J Biol Chem 218:3838-3844); CRM.sub.9, CRM.sub.45, CRM.sub.102,
CRM.sub.103 and CRM.sub.107 and other mutations described by
Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel,
Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 to Asp,
Gln or Ser and/or Ala 158 to Gly and other mutations disclosed in
U.S. Pat. No. 4,709,017 or U.S. Pat. No. 4,950,740; mutation of at
least one or more residues Lys 516, Lys 526, Phe 530 and/or Lys 534
and other mutations disclosed in U.S. Pat. No. 5,917,017 or U.S.
Pat. No. 6,455,673; or fragment disclosed in U.S. Pat. No.
5,843,711. Such DT mutants can also be used to make DTFB variants
where the variants comprise the B fragment contain the epitiope
regions.
[0106] Where multivalent vaccines are used, a second carrier can be
used for one or more of the antigens in a multivalent vaccine. The
second carrier protein is preferably a protein that is non-toxic
and non-reactogenic and obtainable in sufficient amount and purity.
The second carrier protein is also conjugated or joined with an
antigen, e.g., a S. pneumoniae polysaccharide to enhance
immunogenicity of the antigen. Carrier proteins should be amenable
to standard conjugation procedures. In one embodiment, each
capsular polysaccharide not conjugated to the first carrier protein
is conjugated to the same second carrier protein (e.g., each
capsular polysaccharide molecule being conjugated to a single
carrier protein). In another embodiment, the capsular
polysaccharides not conjugated to the first carrier protein are
conjugated to two or more carrier proteins (each capsular
polysaccharide molecule being conjugated to a single carrier
protein). In such embodiments, each capsular polysaccharide of the
same serotype is typically conjugated to the same carrier
protein.
Pharmaceutical/Vaccine Compositions
[0107] The present invention further provides compositions,
including pharmaceutical, immunogenic and vaccine compositions,
comprising, consisting essentially of, or alternatively, consisting
of any of the polysaccharide serotype combinations described above
together with a pharmaceutically acceptable carrier and an
adjuvant.
[0108] Formulation of the polysaccharide-protein conjugates of the
present invention can be accomplished using art-recognized methods.
For instance, individual pneumococcal conjugates can be formulated
with a physiologically acceptable vehicle to prepare the
composition. Examples of such vehicles include, but are not limited
to, water, buffered saline, polyols (e.g., glycerol, propylene
glycol, liquid polyethylene glycol) and dextrose solutions.
[0109] In an embodiment, the vaccine composition is formulated in
L-histidine buffer with sodium chloride.
[0110] As defined herein, an "adjuvant" is a substance that serves
to enhance the immunogenicity of an immunogenic composition of the
invention. An immune adjuvant may enhance an immune response to an
antigen that is weakly immunogenic when administered alone, e.g.,
inducing no or weak antibody titers or cell-mediated immune
response, increase antibody titers to the antigen, and/or lowers
the dose of the antigen effective to achieve an immune response in
the individual. Thus, adjuvants are often given to boost the immune
response and are well known to the skilled artisan. Suitable
adjuvants to enhance effectiveness of the composition include, but
are not limited to:
[0111] (1) aluminum salts (alum), such as aluminum hydroxide,
aluminum phosphate, aluminum sulfate, etc.;
[0112] (2) oil-in-water emulsion formulations (with or without
other specific immunostimulating agents such as muramyl peptides
(defined below) or bacterial cell wall components), such as, for
example, (a) MF59 (International Patent Application Publication No.
WO 90/14837), containing 5% Squalene, 0.5% TWEEN.RTM. 80, and 0.5%
Span 85 (optionally containing various amounts of MTP-PE)
formulated into submicron particles using a microfluidizer such as
Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF,
containing 10% Squalene, 0.4% TWEEN.RTM. 80, 5% pluronic-blocked
polymer L121, and thr-MDP either microfluidized into a submicron
emulsion or vortexed to generate a larger particle size emulsion,
(c) Ribi.TM. adjuvant system (RAS), (Corixa, Hamilton, Mont.)
containing 2% Squalene, 0.2% TWEEN.RTM. 80, and one or more
bacterial cell wall components from the group consisting of
3-O-deaylated monophosphorylipid A (MPL.TM.) described in U.S. Pat.
No. 4,912,094, trehalose dimycolate (TDM), and cell wall skeleton
(CWS), preferably MPL+CWS (Detox.TM.); and (d) a Montanide ISA;
[0113] (3) saponin adjuvants, such as Quil A or STIMULON.TM. QS-21
(Antigenics, Framingham, Mass.) (see, e.g., U.S. Pat. No.
5,057,540) may be used or particles generated therefrom such as
ISCOM (immunostimulating complexes formed by the combination of
cholesterol, saponin, phospholipid, and amphipathic proteins) and
Iscomatrix.RTM. (having essentially the same structure as an ISCOM
but without the protein);
[0114] (4) bacterial lipopolysaccharides, synthetic lipid A analogs
such as aminoalkyl glucosamine phosphate compounds (AGP), or
derivatives or analogs thereof, which are available from Corixa,
and which are described in U.S. Pat. No. 6,113,918; one such AGP is
2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl
2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3--
tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside, which is
also known as 529 (formerly known as RC529), which is formulated as
an aqueous form or as a stable emulsion;
[0115] (5) synthetic polynucleotides such as oligonucleotides
containing CpG motif(s) (U.S. Pat. No. 6,207,646);
[0116] (6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4,
IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g.,
gamma interferon), granulocyte macrophage colony stimulating factor
(GM-CSF), macrophage colony stimulating factor (M-CSF), tumor
necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, etc;
and
[0117] (7) complement, such as a trimer of complement component
C3d.
[0118] In another embodiment, the adjuvant is a mixture of 2, 3, or
more of the above adjuvants, e.g., SBAS2 (an oil-in-water emulsion
also containing 3-deacylated monophosphoryl lipid A and QS21).
[0119] Muramyl peptides include, but are not limited to,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-normuramyl-L-alanine-2-(1'-2'
dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE),
etc.
[0120] In certain embodiments, the adjuvant is an aluminum salt.
The aluminum salt adjuvant may be an alum-precipitated vaccine or
an alum-adsorbed vaccine. Aluminum-salt adjuvants are well known in
the art and are described, for example, in Harlow, E. and D. Lane
(1988; Antibodies: A Laboratory Manual Cold Spring Harbor
Laboratory) and Nicklas, W. (1992; Aluminum salts. Research in
Immunology 143:489-493). The aluminum salt includes, but is not
limited to, hydrated alumina, alumina hydrate, alumina trihydrate
(ATH), aluminum hydrate, aluminum trihydrate, alhydrogel,
SUPERFOS.RTM., Amphogel, aluminum (III) hydroxide, aluminum
hydroxyphosphate sulfate, Aluminum Phosphate Adjuvant (APA),
amorphous alumina, trihydrated alumina, or trihydroxyaluminum.
[0121] APA is an aqueous suspension of aluminum hydroxyphosphate.
APA is manufactured by blending aluminum chloride and sodium
phosphate in a 1:1 volumetric ratio to precipitate aluminum
hydroxyphosphate. After the blending process, the material is
size-reduced with a high-shear mixer to achieve a monodisperse
particle size distribution. The product is then diafiltered against
physiological saline and steam sterilized.
[0122] In certain embodiments, a commercially available
Al(OH).sub.3 (e.g. Alhydrogel or SUPERFOS.RTM. of Denmark/Accurate
Chemical and Scientific Co., Westbury, N.Y.) is used to adsorb
proteins in a ratio of 50-200 g protein/mg aluminum hydroxide.
Adsorption of protein is dependent, in another embodiment, on the
pI (Isoelectric pH) of the protein and the pH of the medium. A
protein with a lower pI adsorbs to the positively charged aluminum
ion more strongly than a protein with a higher pI. Aluminum salts
may establish a depot of antigen that is released slowly over a
period of 2-3 weeks, be involved in nonspecific activation of
macrophages and complement activation, and/or stimulate innate
immune mechanism (possibly through stimulation of uric acid). See,
e.g., Lambrecht et al., 2009, Curr Opin Immunol 21:23.
[0123] Monovalent bulk aqueous conjugates are typically blended
together and diluted to target 8 .mu.g/mL for all serotypes except
6B, which will be diluted to target 16 .mu.g/mL. Once diluted, the
batch will be filter sterilized, and an equal volume of aluminum
phosphate adjuvant added aseptically to target a final aluminum
concentration of 250 .mu.g/mL. The adjuvanted, formulated batch
will be filled into single-use, 0.5 mL/dose vials.
[0124] In certain embodiments, the adjuvant is a CpG-containing
nucleotide sequence, for example, a CpG-containing oligonucleotide,
in particular, a CpG-containing oligodeoxynucleotide (CpG ODN). In
another embodiment, the adjuvant is ODN 1826, which may be acquired
from Coley Pharmaceutical Group.
[0125] "CpG-containing nucleotide," "CpG-containing
oligonucleotide," "CpG oligonucleotide," and similar terms refer to
a nucleotide molecule of 6-50 nucleotides in length that contains
an unmethylated CpG moiety. See, e.g., Wang et al., 2003, Vaccine
21:4297. In another embodiment, any other art-accepted definition
of the terms is intended. CpG-containing oligonucleotides include
modified oligonucleotides using any synthetic internucleoside
linkages, modified base and/or modified sugar.
[0126] Methods for use of CpG oligonucleotides are well known in
the art and are described, for example, in Sur et al., 1999, J
Immunol. 162:6284-93; Verthelyi, 2006, Methods Mol Med. 127:139-58;
and Yasuda et al., 2006, Crit Rev Ther Drug Carrier Syst.
23:89-110.
Administration/Dosage
[0127] The compositions and formulations of the present invention
can be used to protect or treat a human susceptible to infection,
e.g., a pneumococcal infection, by means of administering the
vaccine via a systemic or mucosal route. In one embodiment, the
present invention provides a method of inducing an immune response
to a S. pneumoniae capsular polysaccharide conjugate, comprising
administering to a human an immunologically effective amount of an
immunogenic composition of the present invention. In another
embodiment, the present invention provides a method of vaccinating
a human against a pneumococcal infection, comprising the step of
administering to the human an immunogically effective amount of a
immunogenic composition of the present invention.
[0128] Optimal amounts of components for a particular vaccine can
be ascertained by standard studies involving observation of
appropriate immune responses in subjects. For example, in another
embodiment, the dosage for human vaccination is determined by
extrapolation from animal studies to human data. In another
embodiment, the dosage is determined empirically. We have
demonstrated that the vaccine is immunogenic in Infant Rhesus
Monkey animal data.
[0129] "Effective amount" of a composition of the invention refers
to a dose required to elicit antibodies that significantly reduce
the likelihood or severity of infectivitiy of a microbe, e.g., S.
pneumonia, during a subsequent challenge.
[0130] The methods of the invention can be used for the prevention
and/or reduction of primary clinical syndromes caused by microbes,
e.g., S. pneumonia, including both invasive infections (meningitis,
pneumonia, and bacteremia), and noninvasive infections (acute
otitis media, and sinusitis).
[0131] Administration of the compositions of the invention can
include one or more of: injection via the intramuscular,
intraperitoneal, intradermal or subcutaneous routes; or via mucosal
administration to the oral/alimentary, respiratory or genitourinary
tracts. In one embodiment, intranasal administration is used for
the treatment of pneumonia or otitis media (as nasopharyngeal
carriage of pneumococci can be more effectively prevented, thus
attenuating infection at its earliest stage).
[0132] The amount of conjugate in each vaccine dose is selected as
an amount that induces an immunoprotective response without
significant, adverse effects. Such amount can vary depending upon
the pneumococcal serotype. Generally, for polysaccharide-based
conjugates, each dose will comprise 0.1 to 100 .mu.g of each
polysaccharide, particularly 0.1 to 10 .mu.g, and more particularly
1 to 5 .mu.g. For example, each dose can comprise 100, 150, 200,
250, 300, 400, 500, or 750 ng or 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8,
9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60, 70,
80, 90, or 100 .mu.g.
[0133] Optimal amounts of components for a particular vaccine can
be ascertained by standard studies involving observation of
appropriate immune responses in subjects. For example, in another
embodiment, the dosage for human vaccination is determined by
extrapolation from animal studies to human data. In another
embodiment, the dosage is determined empirically.
[0134] In one embodiment, the dose of the aluminum salt is 10, 15,
20, 25, 30, 50, 70, 100, 125, 150, 200, 300, 500, or 700 .mu.g, or
1, 1.2, 1.5, 2, 3, 5 mg or more. In yet another embodiment, the
dose of alum salt described above is per .mu.g of recombinant
protein.
[0135] According to any of the methods of the present invention and
in one embodiment, the subject is human. In certain embodiments,
the human subject is an infant (less than 1 year of age), toddler
(approximately 12 to 24 months), or young child (approximately 2 to
5 years). In other embodiments, the human subject is an elderly
subject (e.g., >50 years old or >65 years old). The
compositions of this invention are also suitable for use with older
children, adolescents and adults (e.g., aged 18 to 45 years or 18
to 65 years).
[0136] In one embodiment of the methods of the present invention, a
composition of the present invention is administered as a single
inoculation. In another embodiment, the vaccine is administered
twice, three times or four times or more, adequately spaced apart.
For example, the composition may be administered at 1, 2, 3, 4, 5,
or 6 month intervals or any combination thereof. The immunization
schedule can follow that designated for pneumococcal vaccines. For
example, the routine schedule for infants and toddlers against
invasive disease caused by S. pneumoniae is 2, 4, 6 and 12-15
months of age. Thus, in an embodiment, the composition is
administered as a 4-dose series at 2, 4, 6, and 12-15 months of
age.
[0137] The compositions of this invention may also include one or
more proteins from S. pneumoniae. Examples of S. pneumoniae
proteins suitable for inclusion include those identified in
International Patent Application Publication Nos. WO 02/083855 and
WO 02/053761.
Formulations
[0138] The compositions of the invention can be administered to a
subject by one or more methods known to a person skilled in the
art, such as parenterally, transmucosally, transdermally,
intramuscularly, intravenously, intra-dermally, intra-nasally,
subcutaneously, intra-peritonealy, and formulated accordingly.
[0139] In one embodiment, compositions of the present invention are
administered via epidermal injection, intramuscular injection,
intravenous, intra-arterial, subcutaneous injection, or
intra-respiratory mucosal injection of a liquid preparation. Liquid
formulations for injection include solutions and the like.
[0140] The composition of the invention can be formulated as single
dose vials, multi-dose vials or as pre-filled syringes.
[0141] In another embodiment, compositions of the present invention
are administered orally, and are thus formulated in a form suitable
for oral administration, i.e., as a solid or a liquid preparation.
Solid oral formulations include tablets, capsules, pills, granules,
pellets and the like. Liquid oral formulations include solutions,
suspensions, dispersions, emulsions, oils and the like.
[0142] Pharmaceutically acceptable carriers for liquid formulations
are aqueous or non-aqueous solutions, suspensions, emulsions or
oils. Examples of nonaqueous solvents are propylene glycol,
polyethylene glycol, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Examples of oils are those of animal, vegetable, or
synthetic origin, for example, peanut oil, soybean oil, olive oil,
sunflower oil, fish-liver oil, another marine oil, or a lipid from
milk or eggs.
[0143] The pharmaceutical composition may be isotonic, hypotonic or
hypertonic. However it is often preferred that a pharmaceutical
composition for infusion or injection is essentially isotonic, when
it is administrated. Hence, for storage the pharmaceutical
composition may preferably be isotonic or hypertonic. If the
pharmaceutical composition is hypertonic for storage, it may be
diluted to become an isotonic solution prior to administration.
[0144] The isotonic agent may be an ionic isotonic agent such as a
salt or a non-ionic isotonic agent such as a carbohydrate. Examples
of ionic isotonic agents include but are not limited to NaCl,
CaCl.sub.2, KCl and MgCl.sub.2. Examples of non-ionic isotonic
agents include but are not limited to mannitol, sorbitol and
glycerol.
[0145] It is also preferred that at least one pharmaceutically
acceptable additive is a buffer. For some purposes, for example,
when the pharmaceutical composition is meant for infusion or
injection, it is often desirable that the composition comprises a
buffer, which is capable of buffering a solution to a pH in the
range of 4 to 10, such as 5 to 9, for example 6 to 8.
[0146] The buffer may for example be selected from the group
consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate,
phosphate, citrate, carbonate, glycinate, histidine, glycine,
succinate and triethanolamine buffer.
[0147] The buffer may furthermore for example be selected from USP
compatible buffers for parenteral use, in particular, when the
pharmaceutical formulation is for parenteral use. For example the
buffer may be selected from the group consisting of monobasic acids
such as acetic, benzoic, gluconic, glyceric and lactic; dibasic
acids such as aconitic, adipic, ascorbic, carbonic, glutamic,
malic, succinic and tartaric, polybasic acids such as citric and
phosphoric; and bases such as ammonia, diethanolamine, glycine,
triethanolamine, and TRIS.
[0148] Parenteral vehicles (for subcutaneous, intravenous,
intraarterial, or intramuscular injection) include sodium chloride
solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's and fixed oils. Intravenous vehicles include fluid and
nutrient replenishers, electrolyte replenishers such as those based
on Ringer's dextrose, and the like. Examples are sterile liquids
such as water and oils, with or without the addition of a
surfactant and other pharmaceutically acceptable adjuvants. In
general, water, saline, aqueous dextrose and related sugar
solutions, glycols such as propylene glycols or polyethylene
glycol, Polysorbate 80 (PS-80), Polysorbate 20 (PS-20), and
Poloxamer 188 (P188) are preferred liquid carriers, particularly
for injectable solutions. Examples of oils are those of animal,
vegetable, or synthetic origin, for example, peanut oil, soybean
oil, olive oil, sunflower oil, fish-liver oil, another marine oil,
or a lipid from milk or eggs.
[0149] The formulations of the invention may also contain a
surfactant. Surfactants include, but are not limited to: the
polyoxyethylene sorbitan esters surfactants (commonly referred to
as the Tweens), especially PS-20 and PS-80; copolymers of ethylene
oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold
under the DOWFAX.TM. tradename, such as linear EO/PO block
copolymers; octoxynols, which can vary in the number of repeating
ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (TRITON.TM.
X-100, or t-octylphenoxypolyethoxyethanol) being of particular
interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40);
phospholipids such as phosphatidylcholine (lecithin); nonylphenol
ethoxylates, such as the Tergitol.TM. NP series; polyoxyethylene
fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols
(known as BRIJ.TM. surfactants), such as triethyleneglycol
monolauryl ether (BRIJ.TM. 30); and sorbitan esters (commonly known
as the SPANs), such as sorbitan trioleate (Span 85) and sorbitan
monolaurate. A preferred surfactant for including in the emulsion
is PS-80.
[0150] Mixtures of surfactants can be used, e.g. PS-80/Span 85
mixtures. A combination of a polyoxyethylene sorbitan ester such as
polyoxyethylene sorbitan monooleate (PS-80) and an octoxynol such
as t-octylphenoxypolyethoxyethanol (TRITON.TM. X-100) is also
suitable. Another useful combination comprises laureth 9 plus a
polyoxyethylene sorbitan ester and/or an octoxynol.
[0151] Preferred amounts of surfactants (% by weight) are:
polyoxyethylene sorbitan esters (such as PS-80) 0.01 to 1%, in
particular about 0.1%; octyl- or nonylphenoxy polyoxyethanols (such
as TRITON.TM. X-100, or other detergents in the TRITON.TM. series)
0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers
(such as laureth 9) 0.1 to 20%, preferably 0.1 to 10% and in
particular 0.1 to 1% or about 0.5%.
[0152] In certain embodiments, the composition consists essentially
of histidine (20 mM), saline (150 mM) and 0.02% PS-20 or 0.04%
PS-80 at a pH of 5.8 with 250 ug/mL of APA (Aluminum Phosphate
Adjuvant). PS-20 can range from 0.005% to 0.1% (w/v) with the
presence of PS-20 or PS-80 in formulation controlling aggregation
during simulated manufacture and in shipping using primary
packaging. Process consists of combining blend of up to 24
serotypes in histidine, saline, and PS-20 or PS-80 then combining
this blended material with APA and saline with or without
antimicrobial preservatives.
[0153] The choice of surfactant may need to be optimized for
different drug products and drug substances. For multivalent
vaccines having 15 or more serotypes, PS-20 and P188 are preferred.
The choice of chemistry used to make conjugate can also play an
important role in the stabilization of the formulation. In
particular, when the conjugation reactions used to prepare
different polysaccharide protein conjugates in a multivalent
composition include both aqueous solvent and DMSO solvent, has
found that particular surfactant systems provide significant
differences in stability. Improved stability of polysachharide
protein conjugates was seen with polysorbate 20 alone or with
poloxamer 188 in combination with a polyol.
[0154] The exact mechanism of how a specifc detergent protects a
biotherapeutic is poorly understood and cannot be predicted a
priori. Possible stabilization mechanisms include preferential
hydration, preferential exclusion, air/liquid interface competition
between biotherapeutic and surface, surface tension, and/or direct
association of the detergent with the biotherpeutic to mask
hydrophobic patches which serve as seeds for aggregation.
[0155] Poloxamer may also be used in the compositions of the
invention. A poloxamer is a nonionic triblock copolymer composed of
a central hydrophobic chain of polyoxypropylene (poly(propylene
oxide)) flanked by two hydrophilic chains of polyoxyethylene
(poly(ethylene oxide)). Poloxamers are also known by the tradename
Pluronic.RTM.. Because the lengths of the polymer blocks can be
customized, many different poloxamers exist that have slightly
different properties. For the generic term "poloxamer", these
copolymers are commonly named with the letter "P" (for poloxamer)
followed by three digits, the first two digits.times.100 give the
approximate molecular mass of the polyoxypropylene core, and the
last digit.times.10 gives the percentage polyoxyethylene content
(e.g., P407=Poloxamer with a polyoxypropylene molecular mass of
4,000 g/mol and a 70% polyoxyethylene content). For the
Pluronic.RTM. tradename, coding of these copolymers starts with a
letter to define its physical form at room temperature (L=liquid,
P=paste, F=flake (solid)) followed by two or three digits. The
first digit (two digits in a three-digit number) in the numerical
designation, multiplied by 300, indicates the approximate molecular
weight of the hydrophobe; and the last digit.times.10 gives the
percentage polyoxyethylene content (e.g., L61=Pluronic.RTM. with a
polyoxypropylene molecular mass of 1,800 g/mol and a 10%
polyoxyethylene content). See U.S. Pat. No. 3740421.
[0156] Examples of poloxamers have the general formula:
HO(C.sub.2H.sub.4O).sub.a(C.sub.3H.sub.6O).sub.b(C.sub.2H.sub.4O).sub.aH-
,
wherein a and b blocks have the following values:
TABLE-US-00001 Pluronic .RTM. Poloxamer a b Molecular Weight L31 2
16 1100 (average) L35 1900 (average) L44NF 124 12 20 2090 to 2360
L64 2900 (average) L81 2800 (average) L121 4400 (average) P123 20
70 5750 (average) F68NF 188 80 27 7680 to 9510 F87NF 237 64 37 6840
to 8830 F108NF 338 141 44 12700 to 17400 F127NF 407 101 56 9840 to
14600
Molecular weight units, as used herein, are in Dalton (Da) or
g/mol.
[0157] Preferably, the poloxamer generally has a molecular weight
in the range from 1100 to 17,400 Da, from 7,500 to 15,000 Da, or
from 7,500 to 10,000 Da. The poloxamer can be selected from
poloxamer 188 or poloxamer 407. The final concentration of the
poloxamer in the formulations is from 0.001% to 5% weight/volume,
or 0.025% to 1% weight/volume. In certain aspects, the polyol is
propylene glycol and is at final concentration from 1% to 20%
weight/volume. In certain aspects, the polyol is polyethylene
glycol 400 and is at final concentration from 1% to 20%
weight/volume.
[0158] Suitable polyols for the formulations of the invention are
polymeric polyols, particularly polyether diols including, but are
not limited to, propylene glycol and polyethylene glycol,
Polyethylene glycol monomethyl ethers. Propylene glycol is
available in a range of molecular weights of the monomer from
.about.425 to .about.2700. Polyethylene glycol and Polyethylene
glycol monomethyl ether is also available in a range of molecular
weights ranging from .about.200 to .about.35000 including but not
limited to PEG200, PEG300, PEG400, PEG1000, PEG MME 550, PEG MME
600, PEG MME 2000, PEG MME 3350 and PEG MME 4000. A preferred
polyethylene glycol is polyethylene glycol 400. The final
concentration of the polyol in the formulations of the invention
may be 1% to 20% weight/volume or 6% to 20% weight/volume.
[0159] The formulation also contains a pH-buffered saline solution.
The buffer may, for example, be selected from the group consisting
of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate,
citrate, carbonate, glycinate, histidine, glycine, succinate, HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS
(3-(N-morpholino)propanesulfonic acid), MES
(2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffer.
The buffer is capable of buffering a solution to a pH in the range
of 4 to 10, 5.2 to 7.5, or 5.8 to 7.0. In certain aspect of the
invention, the buffer selected from the group consisting of
phosphate, succinate, histidine, MES, MOPS, HEPES, acetate or
citrate. The buffer may furthermore, for example, be selected from
USP compatible buffers for parenteral use, in particular, when the
pharmaceutical formulation is for parenteral use. The
concentrations of buffer will range from 1 mM to 50 mM or 5 mM to
50 mM. In certain aspects, the buffer is histidine at a final
concentration of 5 mM to 50 mM, or succinate at a final
concentration of 1 mM to 10 mM. In certain aspects, the histidine
is at a final concentration of 20 mM.+-.2 mM.
[0160] While the saline solution (i.e., a solution containing NaCl)
is preferred, other salts suitable for formulation include but are
not limited to, CaCl.sub.2, KCl and MgCl.sub.2 and combinations
thereof. Non-ionic isotonic agents including but not limited to
sucrose, trehalose, mannitol, sorbitol and glycerol may be used in
lieu of a salt. Suitable salt ranges include, but not are limited
to 25 mM to 500 mM or 40 mM to 170 mM. In one aspect, the saline is
NaCl, optionally present at a concentration from 20 mM to 170
mM.
[0161] In an embodiment, the formulatons comprise a L-histidine
buffer with sodium chloride.
[0162] In certain embodiments of the formulations described herein,
the polysaccharide-protein conjugates comprise one or more
pneumococcal polysaccharides conjugated to a carrier protein. The
carrier protein can be selected from CRM.sub.197, diphtheria toxin
fragment B (DTFB), DTFBC8, Diphtheria toxoid (DT), tetanus toxoid
(TT), fragment C of TT, pertussis toxoid, cholera toxoid, E. coli
LT, E. coli ST, exotoxin A from Pseudomonas aeruginosa, and
combinations thereof. In one aspect, all of the
polysaccharide-protein conjugates are prepared using aqueous
chemisty. In another aspect, one or more of the polysaccharide
protein conjugates are prepared using DMSO solvent. As an example,
the polysaccharide-protein conjugate formulation can be a 15-valent
pneumococcal conjugate (15vPnC) formulation wherein polysaccharide
protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and
23F are prepared using DMSO solvent and polysaccharide protein
conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F are
prepared using aqueous solvent.
[0163] In another embodiment, the pharmaceutical composition is
delivered in a controlled release system. For example, the agent
can be administered using intravenous infusion, a transdermal
patch, liposomes, or other modes of administration. In another
embodiment, polymeric materials are used; e.g. in microspheres in
or an implant.
[0164] The compositions of this invention may also include one or
more proteins from S. pneumoniae. Examples of S. pneumoniae
proteins suitable for inclusion include those identified in
International Patent Application Publication Nos. WO 02/083855 and
WO 02/053761.
[0165] Having described various embodiments of the invention with
reference to the accompanying description and drawings, it is to be
understood that the invention is not limited to those precise
embodiments, and that various changes and modifications may be
effected therein by one skilled in the art without departing from
the scope or spirit of the invention as defined in the appended
claims.
[0166] The following examples illustrate, but do not limit the
invention.
EXAMPLES
Example 1: Preparation of S. pneumoniae Capsular
Polysaccharides
[0167] Methods of culturing pneumococci are well known in the art.
See, e.g., Chase, 1967, Methods of Immunology and Immunochemistry
1:52. Methods of preparing pneumococcal capsular polysaccharides
are also well known in the art. See, e.g., European Patent No.
EP0497524. Isolates of pneumococcal subtypes are available from the
American Type Culture Collection (Manassas, Va.). The bacteria are
identified as encapsulated, non-motile, Gram-positive,
lancet-shaped diplococci that are alpha-hemolytic on blood-agar.
Subtypes can be differentiated on the basis of Quelling reaction
using specific antisera. See, e.g., U.S. Pat. No. 5,847,112.
[0168] Cell banks representing each of the S. pneumococcus
serotypes present were obtained from the Merck Culture Collection
(Rahway, N.J.) in a frozen vial. A thawed seed culture was
transferred to the seed fermentor containing a pre-sterilized
growth media appropriate for S. pneumoniae. The culture was grown
in the seed fermentor with temperature and pH control. The entire
volume of the seed fermentor was transferred to a production
fermentor containing pre-sterilized growth media. The production
fermentation was the final cell growth stage of the process.
Temperature, pH, and the agitation rate were controlled.
[0169] The fermentation process was terminated via the addition of
an inactivating agent. After inactivation, the batch was
transferred to the inactivation tank where it was held at
controlled temperature and agitation. Cell debris was removed using
a combination of centrifugation and filtration. The batch was
ultrafiltered and diafiltered. The batch was then subjected to
solvent-based fractionations that remove impurities and recover
polysaccharide.
Example 2: Conjugation of Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14,
18C, 19A, 19F, 22F, 23F, and 33F to CRM.sub.197 Using Reductive
Amination in Aqueous Solution
[0170] The different polysaccharide serotypes were individually
conjugated to purified CRM.sub.197 carrier protein using a common
process flow. Polysaccharide was dissolved, size reduced,
chemically activated and buffer-exchanged by ultrafiltration.
Purified CRM.sub.197 was then conjugated to the activated
polysaccharide utilizing NiCl.sub.2 (2 mM) in the reaction mixture,
and the resulting conjugate was purified by ultrafiltration prior
to a final 0.2-micron filtration. Several process parameters within
each step, such as pH, temperature, concentration, and time were
controlled to serotype-specific values in section below.
Polysaccharide Size Reduction and Oxidation
[0171] Purified pneumococcal capsular polysaccharide powder was
dissolved in water, and all serotypes, except serotype 19A, were
0.45-micron filtered. All serotypes, except serotype 19A, were
homogenized to reduce the molecular mass of the polysaccharide.
Serotype 19A was not size reduced due to its relatively low
starting size. Homogenization pressure and number of passes through
the homogenizer were controlled to serotype-specific targets
(150-1000 bar; 4-7 passes) to achieve a serotype-specific molecular
mass. Size-reduced polysaccharide was 0.2-micron filtered and then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane.
[0172] The polysaccharide solution was then adjusted to a
serotype-specific temperature (4-22.degree. C.) and pH (4-5) with a
sodium acetate buffer to minimize polysaccharide size reduction due
to activation. For all serotypes (except serotype 4),
polysaccharide activation was initiated with the addition of a 100
mM sodium metaperiodate solution. The amount of sodium
metaperiodate added was serotype-specific, ranging from
approximately 0.1 to 0.5 moles of sodium metaperiodate per mole of
polysaccharide repeating unit. The serotype-specific charge of
sodium metaperiodate was to achieve a target level of
polysaccharide activation (moles aldehyde per mole of
polysaccharide repeating unit). For serotype 4, prior to the sodium
metaperiodate addition, the batch was incubated at approximately
50.degree. C. and pH 4.1 to partially deketalize the
polysaccharide.
[0173] For all serotypes, with the exception of serotypes 5 and 7F,
the activated product was diafiltered against 10 mM potassium
phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane. Serotypes 5 and 7F were diafiltered
against 10 mM sodium acetate. Ultrafiltration for all serotypes was
conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM.sub.197
[0174] Oxidized polysaccharide solution was mixed with water and
1.5 M potassium phosphate, pH 6.0 or pH 7.0, depending on the
serotype. The buffer pH selected was to improve the stability of
activated polysaccharide during the conjugation reaction. Purified
CRM.sub.197, obtained through expression in Pseudomonas fluorescens
as previously described (WO 2012/173876 A1), was 0.2-micron
filtered and combined with the buffered polysaccharide solution at
a polysaccharide to CRM.sub.197 mass ratio ranging from 0.4 to 1.0
w/w depending on the serotype. The mass ratio was selected to
control the polysaccharide to CRM.sub.197 ratio in the resulting
conjugate. The polysaccharide and phosphate concentrations were
serotype-specific, ranging from 3.6 to 10.0 g/L and 100 to 150 mM,
respectively, depending on the serotype. The serotype-specific
polysaccharide concentration was selected to control the size of
the resulting conjugate. The solution was then 0.2-micron filtered.
Nickel chloride was added to approximately 2 mM using a 100 mM
nickel chloride solution. Sodium cyanoborohydride (2 moles per mole
of polysaccharide repeating unit) was added. Conjugation proceeded
for a serotype-specific duration (72 to 120 hours) to maximize
consumption of polysaccharide and protein.
Reduction with Sodium Borohydride
[0175] Following the conjugation reaction, the batch was diluted to
a polysaccharide concentration of approximately 3.5 g/L, cooled to
2-8.degree. C., and 1.2-micron filtered. All serotypes (except
serotype 5) were diafiltered against 100 mM potassium phosphate, pH
7.0 at 2-8.degree. C. using a 100 kDa NMWCO tangential flow
ultrafiltration membrane. The batch, recovered in the retentate,
was then diluted to approximately 2.0 g polysaccharide/L and
pH-adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4.
Sodium borohydride (1 mole per mole of polysaccharide repeating
unit) was added. 1.5 M potassium phosphate, pH 6.0 was later added.
Serotype 5 was diafiltered against 300 mM potassium phosphate using
a 100 kDa NMWCO tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0176] The batch was then concentrated and diaftiltered against 10
mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4.degree. C.
using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The
retentate batch was 0.2 micron filtered.
[0177] Serotype 19F conjugate was incubated for approximately 7
days at 22.degree. C., diafiltered against 10 mM L-histidine in 150
mM sodium chloride, pH 7.0 at 4.degree. C. using a 100 kDa NMWCO
tangential flow ultrafiltration membrane, and 0.2-micron
filtered.
[0178] The batch was adjusted to a polysaccharide concentration of
1.0 g/L with additional 10 mM L-histidine in 150 mM sodium
chloride, pH 7.0. The batch was dispensed into aliquots and frozen
at .ltoreq.-60.degree. C.
Example 3: Methods for the Conjugation of Serotypes 3, 4, 6A, 6B,
7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F to CRM.sub.197 Using
Reductive Amination in Dimethylsulfoxide
[0179] The different polysaccharide serotypes 3, 4, 6A, 6B, 7F, 9V,
14, 18C, 19A, 19F, 22F, 23F, and 33F were individually conjugated
to the purified CRM.sub.197 carrier protein using a common process
flow. Polysaccharide was dissolved, sized to a target molecular
mass, chemically activated and buffer-exchanged by ultrafiltration.
Activated polysaccharide and purified CRM.sub.197 were individually
lyophilized and redissolved in dimethylsuloxide (DMSO). Redissolved
polysaccharide and CRM.sub.197 solutions were then combined and
conjugated as described below. The resulting conjugate was purified
by ultrafiltration prior to a final 0.2-micron filtration. Several
process parameters within each step, such as pH, temperature,
concentration, and time were controlled to serotype-specific values
in section below.
Polysaccharide Size Reduction and Oxidation
[0180] Purified pneumococcal capsular Ps powder was dissolved in
water, and all serotypes, except serotype 19A, were 0.45-micron
filtered. All serotypes, except serotypes 18C and 19A, were
homogenized to reduce the molecular mass of the Ps. Homogenization
pressure and number of passes through the homogenizer were
controlled to serotype-specific targets (150-1000 bar; 4-7 passes).
Serotype 18C was size-reduced by acid hydrolysis at
.gtoreq.90.degree. C.
[0181] Size-reduced polysaccharide was 0.2-micron filtered and then
concentrated and diafiltered against water using a 10 kDa NMWCO
tangential flow ultrafiltration membrane. A 5 kDa NMWCO membrane
was used for serotype 18C.
[0182] The polysaccharide solution was then adjusted to a
serotype-specific temperature (4-22.degree. C.) and pH (4-5) with a
sodium acetate buffer to minimize polysaccharide size reduction due
to activation. For all serotypes (except serotype 4),
polysaccharide activation was initiated with the addition of a 100
mM sodium metaperiodate solution. The amount of sodium
metaperiodate added was serotype-specific, ranging from
approximately 0.1 to 0.5 moles of sodium metaperiodate per mole of
polysaccharide repeating unit. The serotype-specific charge of
sodium metaperiodate was to achieve a target level of
polysaccharide activation (moles aldehyde per mole of
polysaccharide repeating unit). For serotype 4, prior to the sodium
metaperiodate addition, the batch was incubated at approximately
50.degree. C. and pH 4.1 to partially deketalize the
polysaccharide.
[0183] The activated product was diafiltered against 10 mM
potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow
ultrafiltration membrane, then diafiltered or dialyzed against
water using a 10 kDa NMWCO membrane. A 5 kDa NMWCO membrane was
used for serotype 18C. Ultrafiltration or dialysis for all
serotypes was conducted at 2-8.degree. C.
Polysaccharide Conjugation to CRM.sub.197
[0184] Purified CRM.sub.197, obtained through expression in
Pseudomonas fluorescens as previously described (WO 2012/173876
A1), was diafiltered against 2-5 mM phosphate, pH 7.0 buffer using
a 5 kDa NMWCO tangential flow ultrafiltration membrane and
0.2-micron filtered.
[0185] For serotypes other than serotype 3, the oxidized
polysaccharides were formulated at 6 mg Ps/mL and 5% w/v sucrose
(50 mg sucrose/mL) in water. For serotype 3, the oxidized
polysaccharide was formulated at 2 mg Ps/mL and 10% w/v sucrose
(100 mg sucrose/mL) in water. The protein solution was formulated
at 6 mg Pr/mL with 1% w/v sucrose (10 mg sucrose/mL) in phosphate
buffer.
[0186] Formulated Ps and CRM.sub.197 solutions were individually
lyophilized. Lyophilized Ps and CRM.sub.197 materials were
redissolved in DMSO and combined using a mixing tee. Sodium
cyanoborohydride (1 moles per mole of polysaccharide repeating
unit) was added, and conjugation proceeded for a serotype-specific
duration (1 to 48 hours) to achieve a targeted conjugate size.
Reduction with Sodium Borohydride
[0187] Sodium borohydride (2 moles per mole of polysaccharide
repeating unit) was added following the conjugation reaction. The
batch was diluted into 150 mM sodium chloride, with or without
approximately 0.025% (w/v) polysorbate 20, at approximately
4.degree. C. Potassium phosphate buffer was then added to
neutralize the pH. For serotypes 3, 6A, 6B, 7F, 9V, 18C, 19A, 19F,
22F, 23F, and 33F, the batch was concentrated and diafiltered at
approximately 4.degree. C. against 150 mM sodium chloride, with or
without 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO
tangential flow ultrafiltration membrane.
Final Filtration and Product Storage
[0188] Serotypes 3, 6A, 6B, 7F, 9V, 18C, 19A, 22F, 23F, and 33F
were concentrated and diaftiltered against 10 mM histidine in 150
mM sodium chloride, pH 7.0, with or without 0.015% (w/v)
polysorbate 20, at 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane. The retentate batch was 0.2 micron
filtered.
[0189] Serotype 19F was incubated for approximately 5 days,
diafiltered against 10 mM histidine in 150 mM sodium chloride, pH
7.0 at approximately 4.degree. C. using a 300 kDa NMWCO tangential
flow ultrafiltration membrane, and 0.2-micron filtered.
[0190] Serotypes 3, 6A, 6B, 7F, 9V, 18C, 19A, 19F, 22F, 23F, and
33F were diluted with additional 10 mM histidine in 150 mM sodium
chloride, pH 7.0, dispensed into aliquots and frozen at
.ltoreq.-60.degree. C.
[0191] Serotypes 4 and 14 were dialyzed against 150 mM sodium
chloride at approximately 4.degree. C. using a 300 kDa NMWCO
membrane, 0.2-micron filtered, dispensed into aliquots and frozen
at .ltoreq.-60.degree. C.
Example 4: Analysis of Conjugates
Molecular Weight and Concentration Analysis of Conjugates Using
HPSEC/UV/MALS/RI Assay
[0192] Conjugate samples were injected and separated by high
performance size-exclusion chromatography (HPSEC). Detection was
accomplished with ultraviolet (UV), multi-angle light scattering
(MALS) and refractive index (RI) detectors in series. Protein
concentration was calculated from UV280 using an extinction
coefficient. Polysaccharide concentration was deconvoluted from the
RI signal (contributed by both protein and polysaccharide) using
the do/dc factors which are the change in a solution's refractive
index with a change in the solute concentration reported in mL/g.
Average molecular weight of the samples were calculated by Astra
software (Wyatt Technology Corporation, Santa Barbara, Calif.)
using the measured concentration and light scattering information
across the entire sample peak.
Polysaccharide Degree of Activation Assay
[0193] Conjugation occurs through reductive amination between the
activated aldehydes and mainly lysine residues on the carrier
protein. The level of activation, as represented by moles of
aldehyde per moles of polysaccharide repeat unit, is important to
control the conjugation reactions. An assay for measure the degree
of activation is described in U.S. Patent Application Publication
No. 2017/0021006.
[0194] An internal assay was developed to measure the degree of
activation based on reaction of aldehyde groups (created during
periodate oxidation of the polysaccharide) with thiosemicarbazide
(available from commercial sources).
[0195] Quantification can be achieved by NMR (nuclear magnetic
resonance) or by comparing the derivatized polysaccharide to
appropriate reference standards and/or through the use of
extinction coefficients of the derivative. The use of extinction
coefficients for this assay is similar to its use in
HPSEC/UV/MALS/RI method.
[0196] Generally, the assay can be run under the following for
reaction conditions:
[0197] Time: 0.5 h-35 hr (this is serotype specific, but the
reaction is followed until completion, i.e., plateaus in a time
course)
[0198] Temperature: 15.degree. C.-37.degree. C., preferably around
21-27.degree. C.
[0199] TSC concentration: 1-5 mg/mL
[0200] pH of reaction: pH 3-5.5, preferably 4.0
[0201] For Example 4, polysaccharide was derivatized with 1.25 to
2.5 mg/mL thiosemicarbazide (TSC) at pH 4.0 to introduce a
chromophore (derivatization of activated polysaccharide for
serotypes 1, 5, and 9V uses 1.25 mg/mL TSC). The derivatization
reaction was allowed to proceed to reach a plateau. The actual time
varied depending on reaction speed for each serotype. TSC-Ps was
then separated from TSC and other low molecular weight components
by high performance size exclusion chromatography. The signal was
detected by UV absorbance at 266 nm. The level of activated
aldehyde is calculated against either standard curve injections of
Mono-TSC or directly using predetermined extinction coefficients.
Mono-TSC is a synthesized thiosemicarbazone derivative of
monosaccharide. The aldehyde level is then converted to moles of
aldehyde per mole of repeat unit (Ald/RU) using the Ps
concentration measured by HPSEC/UV/MALS/RI assay.
[0202] Similar derivatization can be conducted with
thiosemicarbazide structural analogs, hydrazides, hydrazine,
semicarbazide, semicarbazide structural analogs, aminooxy compounds
or aromatic amines as long as the derivatives have significant UV
absorbance. The UV absorbance could be from the chromophore
attached to the derivatization agents, or a chromophore generated
as a result of the aldehyde derivatization, as the case of
thiosemicarbazide.
Determination of Lysine Consumption in Conjugated Protein as a
Measure of the Number of Covalent Attachments Between
Polysaccharide and Carrier Protein
[0203] The Waters AccQ-Tag amino acid analysis (AAA) was used to
measure the extent of conjugation in conjugate samples. Samples
were hydrolyzed using vapor phase acid hydrolysis in the Eldex
workstation, to break the carrier proteins down into their
component amino acids. The free amino acids were derivatized using
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The
derivatized samples were then analyzed using UPLC with UV detection
on a C18 column. The average protein concentration was obtained
using representative amino acids other than lysine. Lysine
consumption during conjugation (i.e., lysine loss) was determined
by the difference between the average measured amount of lysine in
the conjugate and the expected amount of lysine in the starting
protein.
Attributes of Conjugates Made Using Reductive Amination in Aqueous
and DMSO Solutionss
[0204] Polysaccharide activation and lysine consumption (i.e.,
lysine loss) results for conjugates generated using the processes
described in Examples 2 and 3 are listed in Table 1. There is a
clear distinction that conjugates made in DMSO (Example 3) had
higher lysine consumption with lower polysaccharide activation than
conjugates made in aqueous solution (Example 2). This suggests that
preparing the conjugates in DMSO solution allows the polysaccharide
to attach to more conjugation sites on the carrier protein with
less activation or destruction to native polysaccharide structures.
As a result, the conjugates on average contain more glycopeptide
per polysaccharide repeating unit due to higher cross-linking in
conjugates prepared in DMSO solution than in aqueous solution. It
is believed that the glycopeptide is the antigenic domain to which
an immune response is generated. Consequently, conjugates generated
in DMSO are expected to be more immunogenic that conjugates
generate in aqueous solution.
[0205] The average molecular weight (Mw) of the conjugates in Table
1 were measured by the HPSEC UV-MALS-RI assay. Conjugates generated
by reductive amination in aqueous solution ranged from 990 to 3410
kDa. Conjugates generated in DMSO were generally larger with sizes
ranging from 1300 to 5822 kDa.
TABLE-US-00002 TABLE 1 Lysine loss for pneumococcal serotype 3, 4,
6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F CRM197 conjugates made
using reductive amination in aqueous solution or in DMSO.
Conjugation Polysaccharide reaction in activation (mole Lysine loss
Conjugate aqueous or DMSO aldehyde/mole repeat (mol/mol Conjugate
Lot # solution unit) protein) Serotype 3- 1 Aqueous 0.10 3.1
CRM.sub.197 2 0.10 2.5 3 0.10 3.1 4 DMSO 0.092 16.3 5 0.053 9.6
Serotype 4- 1 Aqueous 0.43 2.7 CRM.sub.197 2 DMSO 0.25 3.0 Serotype
6A- 1 Aqueous 0.19 4.5 CRM.sub.197 2 DMSO 0.11 9.1 Serotype 6B- 1
Aqueous 0.18 4.6 CRM.sub.197 2 DMSO 0.11 9.6 Serotype 7F- 1 Aqueous
0.26 2.0 CRM.sub.197 2 DMSO 0.22 10.6 Serotype 9V- 1 Aqueous 0.30
4.7 CRM.sub.197 2 DMSO 0.15 7.9 Serotype 14- 1 Aqueous 0.22 6.4
CRM.sub.197 2 DMSO 0.22 12.7 Serotype 18C- 1 Aqueous 0.12 3.5
CRM.sub.197 2 DMSO 0.11 9.2 Serotype 19A- 1 Aqueous 0.38 4.9
CRM.sub.197 2 DMSO 0.14 9.5 Serotype 19F- 1 Aqueous 0.13 2.7
CRM.sub.197 2 DMSO 0.15 9.6 Serotype 22F- 1 Aqueous 0.12 1.7
CRM.sub.197 2 DMSO 0.15 7.2 3 0.15 7.0 Serotype 23F- 1 Aqueous 0.39
3.2 CRM.sub.197 2 DMSO 0.19 10.8 Serotype 33F- 1 Aqueous 0.23 4.5
CRM.sub.197 2 DMSO 0.14 7.0
Quantification of the Extent of Conjugation at Different Sites on
CRM.sub.197
[0206] Polysaccharide can be conjugated either to the amine group
at the N-terminus of the carrier protein or to any of the side
chains of the 39 lysine residues in CRM.sub.197. The amino acid
sequence of CRM.sub.197 is provided in Table 2, where the lysines
(abbreviated as K) are underlined and in bold. To locate and
quantify the extent of polysaccharide conjugation at the different
sites on CRM.sub.197 protein, an LC/UV/MS peptide mapping method
was used. Representative conjugate samples (prepared with DMSO or
aqueous solution) were digested in duplicate with trypsin,
producing tryptic peptides. The mixtures were then separated on a
reversed phase C.sub.18 column and analyzed by UV and mass
spectrometer. A CRM.sub.197 protein sample (not conjugated with a
polysaccharide) was also processed in triplicate at the same time
as a control. Since trypsin cleaves a protein on the C-terminal
side of lysine and arginine residues, conjugation at a lysine
residue makes that site protease resistant. The extent of
conjugation at a particular site was determined by calculating a
decrease of peak intensity of a tryptic peptide as compared to a
CRM.sub.197 control. Depending on the cleavage sites and sequences,
the signal decrease of a particular peptide could be due to
mis-cleavage of the lysines at the preceeding peptide, or
mis-cleavage of the lysine at the end of the peptide, or
conjugation in the middle of the peptide sequence.
[0207] The relative percentages of peptide signal decrease for
serotype 19A conjugates compared to CRM.sub.197 control were
plotted against possible sites of conjugation in FIG. 1. The lysine
locations listed in the x-axis were numbered based on their order
on the CRM.sub.197 protein sequence, and represent possible
conjugation sites of the analyzed peptides. For example, "33" means
the peptide signal decrease was due to conjugation at the 33rd
lysine; and "6, 7" means the peptide signal descrease was due to
conjugation at the 6th, or the 7th, or both lyines. The data in
FIG. 1 suggested that not only the extent of conjugation at each
site was generally higher for conjugates prepared in DMSO compared
to aqueous solution, there were also more sites of conjugation in
DMSO. Those additional conjugation sites include the 29th, 30th,
31st, and 32nd lysines, which were only lysines located in
previously identified common human T-cell peptide epitopes (See
Raju et al., 1995, Eur. J. Immunol. 25:3207-3214, located in
peptide 411-430 and peptide 431-450 of CRM.sub.197 sequence).
Similar results were observed with other serotypes tested.
TABLE-US-00003 TABLE 2 CRM.sub.197 amino acid sequence Amino Acid
Amino Acid Sequence 1-535
JDGGYYGVVN#VIYPHQIVV\#KJWNSJ\YGV#LTNJLTNSNV#JWTJQ\GGGZ
NHI\VWGQN\#GDDJ\VYGQH#QSOVJNDJJY#YNYW\SJOWN#YODONYGQDH
WLNNHOJOVO#WHSOPHTYJW#HHILNUIJGJ#DVUYYOVOSI#DHJVVVYH\L
QQZHTDNDOV#YHOHLQIHWU#JNUJTGDP\H#\PDTDFDJQU#YUUVYJVVOV
FLQOGZGYLU#GNWNWNLHVO#NHKJSLNQNP#VHVSQNWYVH#HNDNT\OHHI
KTWDOHKSHO#VHONWYWJWQ#SYIDJDQ\DD#ZDYQYDTYLG#VHWDGQOHNW
WDDOVLOSJL#JVYPJLDGJD#YKKQWHHLYD#TVLDOVVOPY#DTDLSOYJHO
YGLJIDD\QI#YHVLLQOITY#YKQV\QUSD\#VSJKNWTSIO#KGJ\DYVZQW
YHGVLLUWJI#TJHVJKGLNL#WDHQWSOSLD#JYOOSWLSJN#OGYQNVNWKL
VYQJUNLUPU#FUDLGJGYWI#FUSNVSY\YJ#QJYKDQOKYD#IKUVVVHNLK
VQHLVVGVLJ#YOJ\TNWYGK#WNYQVNOVOI#IHLNV#+ SEQ ID NO: 1,
Example 5: Mouse Immunogenicity Studies Comparing Serotype 3
Ps-CRM.sub.197 Conjugates Prepared in Aqueous Solution Versus
DMSO
[0208] All animal experiments were performed in strict accordance
with the recommendations in the Guide for Care and Use of
Laboratory Animals of the National Institutes of Health. The
protocol was approved by the Institutional Animal Care and Use
Committee (IACUC), MRL, West Point, Pa.
[0209] Eight week old female CD1 mice were housed in micro isolator
cages (n=10/cage) in the animal facility at MRL, West Point, Pa.
Food and water were available ad libitum. Mice (n=10/group) were
intramuscularly (IM) immunized with ST3-CRM.sub.197 conjguates (0.4
.mu.g ST3 polysaccharide), formulated with aluminum phosphate
adjuvant (APA) as described in Table 3. Negative control animals
received APA alone. Immunizations were performed on days 0, 14 and
28. Blood was collected in serum separator tubes (BD, Franklin
Lakes, N.J.) via tail vein on days 6 and 34.
TABLE-US-00004 TABLE 3 Mouse study arms comparing serotype 3
Ps-CRM.sub.197 conjugates prepared in aqueous solution versus DMSO
solution. Arm # Arm Description of Conjugate Description of
Formulation 1 APA Control, no conjugate used 250 .mu.g/mL APA, 20
mM L- histidine, pH 5.8 and 150 mM NaCl with 0.2% w/v PS-20 2
ST3-CRM.sub.197(aqueous)/ Monovalent ST3-CRM.sub.197 0.4 .mu.g
ST3-CRM.sub.197, 250 APA conjugate (Lot #1 in Table 1) .mu.g/mL
APA, 20 mM L- prepared by reductive histidine, pH 5.8 and 150 mM
amination in aqueous solution NaCl with 0.2% w/v PS-20 as described
in Example 2 3 ST3-CRM.sub.197(DMSO)/ Monovalent ST3-CRM.sub.197
0.4 .mu.g ST3-CRM.sub.197, 250 APA conjugate (Lot #4 in Table 1)
.mu.g/mL APA, 20 mM L- prepared by reductive histidine, pH 5.8 and
150 mM amination in DMSO as NaCl with 0.2% w/v PS-20 described in
Example 3
Electrochemiluminescent (ECL) Immunogenicity Assays
[0210] Mouse antibody responses were measured in 96-well
multiplexed electrochemiluminescent assays as described previously
with slight modifications. See Marchese et al., 2009, Clin Vaccine
Immunol 16(3):387-96; Skinner et al., 2011, Vaccine 29(48):8870-6;
and Caro-Aguilar et al., 2017 Vaccine 35(6):865-72. Briefly,
following test sera incubation for 1 hour on Meso-Scale Discovery
plates (Meso Scale Diagnostics, Rockville, Md.) and washing, 25
.mu.l of a 2 .mu.g/ml Sulfo-tag (Meso Scale Diagnostics, Rockville,
Md.) labeled goat anti-mouse IgG was added to each well. Plates
were incubated for 1 hour at room temperature while shaking and
then processed as described previously and read on a MESO Sector
S600.
[0211] The ECL titer was calculated as the reciprocal of the
linearly interpolated dilution corresponding to the cutoff value
(pneumococcal polysaccharide ECL geometric mean signal of
pre-determined positive control pooled mouse sera). Interpolation
was performed using logarithmic scaling for ECL and the dilution.
Titer was then obtained by back-transforming the linearly
interpolated dilution. Titers were extrapolated for samples falling
outside the studied dilution range of 100 to 1,562,500, based on
linear extrapolation (in the log-log scaling) using the intercept
and slope of the last 3 ECL data points for the sample curve
completely above the cutoff line or using the intercept and slope
of the first 2 ECL data points for the sample curve completely
below the cutoff line. Titer was then obtained by back-transforming
the linearly extrapolated dilution.
Opsonophagocytic Killing Assay (OPA)
[0212] Pneumococcal serotype 3 opsonophagocytosis killing assays
(OPA) were performed as described previously with slight
modifications (Caro-Aguilar et al., 2017 Vaccine 35(6):865-72; and
Burton et al., 2006, Clin Vaccine Immunol 13(9):1004-9). Following
incubation of the sera, bacteria, complement and HL-60 cells, 10
.mu.l of the opsonophagocytic reaction was transferred to an
individual well on a Millipore 96-well filter plate containing 200
.mu.l/well of sterile water. The plate was vacuum filtered and 100
.mu.l of Todd Hewett yeast extract (THYE, Teknova) broth was added.
The medium was filtered and the moist plate was placed in a sealed
plastic bag overnight at 27.degree. C. Plate filters were then
stained with 100 .mu.l/well of a 0.1% Coomassie blue solution
(Bio-Rad, Hercules, Calif.). Stain was filtered through the plate,
colonies were destained with Coomassie destaining solution
(Bio-Rad) and vacuum filtered again until dry. Stained bacterial
colonies were counted on a CTL Immunospot reader (Shaker Heights,
Ohio). The OPK titer was defined as the reciprocal of the serum
dilution with at least 50% killing, compared to the average growth
in the complement control (no serum control) wells and was
calculated by linearly interpolating between the consecutive
dilutions whose signals bracket 50% killing.
[0213] Results of pre-immunization and post dose 3 are illustrated
in FIG. 2 and Table 4 for ECL Immunogencity, and in FIG. 3 for OPA.
Both conjugates prepared by processes using aqueous and DMSO
solutions are immunogenic and provide functional killing activities
against the bacteria. Interestingly, conjugate prepared by process
using DMSO solution gave both higher ECL immunogenicity and OPA
responses than conjugate prepared using aqueous solution. The ECL
immunogenicity difference is statistically significant. The GMT
ratio of Arm 3 relative to Arm 2 is 3.41 (with lower and upper 95%
confidence interval of 1.26 and 9.26).
TABLE-US-00005 TABLE 4 Post-Dose 3 ECL immunogenicity results of
mouse study arms comparing serotype 3 Ps-CRM.sub.197 conjugates
prepared in aqueous solution versus DMSO solution. Geometric Lower
95% Upper 95% Mean confidence confidence Arm # Arm Titer, GMT
interval interval 1 APA 368 227 596 2 ST3-CRM.sub.197(aqueous)/APA
355,207 187,905 671,466 3 ST3-CRM.sub.197(DMSO)/APA 1,211,654
719,297 2,041,028
Example 6: Adult Human Immunogenicity Studies Comparing
Pneumococcal Polysaccharide-Protein Conjugates Prepared with
Reductive Amination in Aqueous Solution Versus in DMSO
[0214] The immunogenicity and safety of two 15-valent pneumococcal
conjugate vaccine (PCV15) in healthy Pneumococcal vaccine-naive
adults 50 years of age or older is described in this example.
Trial Design
[0215] A randomized, multi-site, double-blind trial was carried out
to compare the safety, tolerability and immunogenicity of a single
dose of 2 different PCV15 formulations (PCV15-A and PCV15-B) and
Prevnar 13.TM. (Pneumococcal 13-valent Conjugate Vaccine
[Diphtheria CRM.sub.197 Protein], Wyeth Pharmaceuticals Inc., a
subsidiary of Pfizer Inc., Philadelphia, Pa., USA) in adult
subjects 50 years of age or older in good health (any underlying
chronic illness must be documented to be in stable condition), to
be conducted in conformance with Good Clinical Practices.
[0216] A total of 690 healthy Pneumococcal vaccine-naive
individuals, 50 years of age or older, were enrolled, and
randomized into three different vaccination groups: Prevnar 13.TM.,
PCV15-A and PCV15-B with the ratio 1:1:1. Randomization was
stratified by age at study entry (50 to 64 years, 65 to 74 years,
and >75 years).
[0217] PCV15 contained 2 .mu.g/0.5 mL dose of each of the following
serotypes of Pneumococcal polysaccharide conjugated to CRM.sub.197
(1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, 33F), 4
.mu.g/0.5 mL dose of serotype 6B Pneumococcal polysaccharide
conjugated to CRM.sub.197, 125 .mu.g/0.5 mL dose of Aluminum
Phosphate Adjuvant, 20 mM L-histidine, 150 mM Sodium Chloride, pH
5.8. PCV15-A was formulated with 0.2% w/v P188. PCV15-B was
formulated with 0.1% w/v PS-20.
[0218] For PCV15-A , all fifteen polysaccharide serotypes (1, 3, 4,
5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F) were
conjugated to CRM.sub.197 using reductive amination in aqueous
solution as described in Example 2. Attributes for some of these
conjugates (Conjugate Lot #1 materials) are listed in Table 1.
[0219] PCV15-B, serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F were
conjugated to CRM.sub.197 using reductive amination in DMSO
described in Example 3. Attributes for these conjugates (Conjugate
Lot #2 materials) are listed in Table 1. The conjugates for the
remaining serotypes (1, 3, 4, 5, 9V, 14, 22F, and 33F) are the same
conjugates that were used in PCV15-A.
[0220] Both PCV15 formulations had generally comparable safety
profiles to Prevnar 13.TM. based on the cumulative safety
evaluation (data not shown). The serotype-specific IgG GMCs and OPA
GMTs were measured at Day 30. (OPA results not included).
Results
[0221] The IgG Geometric Mean Concentrations (GMCs) and confidence
intervals (CI) are summarized in the Table 6. Serotype 6A, 6B, 7F,
18C, 19A, 19F, and 23F conjugates in PCV15-A and PCV15-B were made
with different conjugation processes as described above. Consistent
with the results shown in Table 4, the immunogenicity responses for
each of the serotypes shown in Table 6 was improved when the
polysaccharide serotypes were conjugated to CRM.sub.197 in DMSO.
The GMCs for serotypes 18C, 19A, 19F, and 23F in PCV15-B were
significantly higher than those in PCV15-A (2-sided alpha=0.05).
These data strongly demonstrate the advantage of conjugating in
DMSO to improve immunogenicity. This discovery that has not been
previously demonstrated for pneumococcal or other conjugate
vaccines.
TABLE-US-00006 TABLE 6 Summary of IgG antibody responses of
PCV15-A, and PCV15-B formulation for serotypes 6A, 6B, 7F, 18C,
19A, 19F, and 23F. Conjugates of these serotypes were made using
reductive amination in aqueous solution (PCV15-A) or by reductive
amination in DMSO (PCV15-B). Estimated PCV15-A (N = 231), PCV15-B
(N = 231), GMC Ratio.dagger. GMC.sup..dagger. (Day 30)
GMC.sup..dagger. (Day 30) [PCV15-B/ Estimated Estimated PCV15-A]
Serotypes n Response n Response (95% CI) .sup..dagger. 6A 217 3.74
217 4.93 1.32 (0.96, 1.81) 6B 217 3.69 217 4.95 1.34 (0.98, 1.85)
7F 217 4.09 217 4.53 1.11 (0.86, 1.43) 18C 217 6.61 217 10.99 1.66
(1.27, 2.18) 19A 217 8.77 217 13.83 1.58 (1.23, 2.02) 19F 217 4.11
217 6.80 1.66 (1.26, 2.17) 23F 217 3.92 217 5.53 1.41 (1.04, 1.91)
.sup..dagger.Estimated GMCs, GMC ratio, and 95% CI are obtained
from a cLDA model. N = Number of subjects randomized and
vaccinated. n = Number of subjects with Day 30 postvaccination
serology results contributing to the analysis. GMC = Geometric Mean
Concentration. CI = Confidence interval
Sequence CWU 1
1
11535PRTArtificial SequenceCRM197 (detoxified variant of diphtheria
toxin) 1Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu
Asn1 5 10 15Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser
Ile Gln 20 25 30Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn
Tyr Asp Asp 35 40 45Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr
Asp Ala Ala Gly 50 55 60Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly
Lys Ala Gly Gly Val65 70 75 80Val Lys Val Thr Tyr Pro Gly Leu Thr
Lys Val Leu Ala Leu Lys Val 85 90 95Asp Asn Ala Glu Thr Ile Lys Lys
Glu Leu Gly Leu Ser Leu Thr Glu 100 105 110Pro Leu Met Glu Gln Val
Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly 115 120 125Asp Gly Ala Ser
Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser 130 135 140Ser Ser
Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser145 150 155
160Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp
165 170 175Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg
Val Arg 180 185 190Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu
Asp Trp Asp Val 195 200 205Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu
Ser Leu Lys Glu His Gly 210 215 220Pro Ile Lys Asn Lys Met Ser Glu
Ser Pro Asn Lys Thr Val Ser Glu225 230 235 240Glu Lys Ala Lys Gln
Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu 245 250 255His Pro Glu
Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val 260 265 270Phe
Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val 275 280
285Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu
290 295 300Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp
Gly Ala305 310 315 320Val His His Asn Thr Glu Glu Ile Val Ala Gln
Ser Ile Ala Leu Ser 325 330 335Ser Leu Met Val Ala Gln Ala Ile Pro
Leu Val Gly Glu Leu Val Asp 340 345 350Ile Gly Phe Ala Ala Tyr Asn
Phe Val Glu Ser Ile Ile Asn Leu Phe 355 360 365Gln Val Val His Asn
Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His 370 375 380Lys Thr Gln
Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr385 390 395
400Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His
405 410 415Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala
Gly Val 420 425 430Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn
Lys Ser Lys Thr 435 440 445His Ile Ser Val Asn Gly Arg Lys Ile Arg
Met Arg Cys Arg Ala Ile 450 455 460Asp Gly Asp Val Thr Phe Cys Arg
Pro Lys Ser Pro Val Tyr Val Gly465 470 475 480Asn Gly Val His Ala
Asn Leu His Val Ala Phe His Arg Ser Ser Ser 485 490 495Glu Lys Ile
His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu 500 505 510Gly
Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser 515 520
525Leu Phe Phe Glu Ile Lys Ser 530 535
* * * * *