U.S. patent application number 13/591687 was filed with the patent office on 2013-03-21 for targeted immune conjugates.
This patent application is currently assigned to AUGMENTA BIOLOGICALS, LLC. The applicant listed for this patent is Paul M. Simon. Invention is credited to Paul M. Simon.
Application Number | 20130071413 13/591687 |
Document ID | / |
Family ID | 38833775 |
Filed Date | 2013-03-21 |
United States Patent
Application |
20130071413 |
Kind Code |
A1 |
Simon; Paul M. |
March 21, 2013 |
TARGETED IMMUNE CONJUGATES
Abstract
Disclosed herein are materials and methods related to vaccines.
Materials and methods for delivery of immunogens to the
reticuloendothelial system via non-circulating lymphoid cells are
provided.
Inventors: |
Simon; Paul M.; (Wilmington,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Simon; Paul M. |
Wilmington |
DE |
US |
|
|
Assignee: |
AUGMENTA BIOLOGICALS, LLC
Wilmington
DE
|
Family ID: |
38833775 |
Appl. No.: |
13/591687 |
Filed: |
August 22, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12306457 |
Sep 11, 2009 |
8318912 |
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13591687 |
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PCT/US07/71875 |
Jun 22, 2007 |
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12306457 |
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60816015 |
Jun 23, 2006 |
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Current U.S.
Class: |
424/178.1 ;
435/252.3; 435/254.2; 435/320.1; 435/328; 530/387.3; 530/391.7;
536/23.4 |
Current CPC
Class: |
A61K 47/6898 20170801;
B82Y 5/00 20130101; C07K 19/00 20130101; A61P 37/02 20180101; A61P
37/04 20180101 |
Class at
Publication: |
424/178.1 ;
435/320.1; 435/252.3; 435/254.2; 435/328; 530/391.7; 530/387.3;
536/23.4 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 1/21 20060101 C12N001/21; C12N 15/62 20060101
C12N015/62; C12N 5/10 20060101 C12N005/10; C07K 19/00 20060101
C07K019/00; A61P 37/02 20060101 A61P037/02; C12N 15/63 20060101
C12N015/63; C12N 1/19 20060101 C12N001/19 |
Claims
1. An immune conjugate comprising: (a) a ligand that binds
specifically to a cell surface molecule on a circulating
non-lymphoid cell of a mammal, wherein said molecule is selected
from the group consisting of glycophorin A, band 3, Ter-119, blood
group antigen H, blood group antigen A, blood group antigen B,
CD41a, CD14, CD56, CD66d, CD83, CMKLR1, and BDCA-4; and (b) an
immunogen coupled to said ligand, wherein said immune conjugate,
when administered to an individual, induces or enhances an immune
response against said immunogen
2. The immune conjugate of claim 1, wherein the ligand is an
antibody.
3. The immune conjugate of claim 2, wherein the antibody is an
anti-TER-119 antibody, an anti-glycophorin A antibody, an anti-band
3 antibody, an anti-blood group antigen A antibody, an anti-blood
group antigen B antibody, an anti-blood group antigen H antibody,
an anti-CD41a antibody, an anti-CD14 antibody, an anti-CD56
antibody, an anti-CD66d antibody, an anti-CD83 antibody, an
anti-CMKLR1 antibody, or an anti-BDCA-4 antibody.
4-5. (canceled)
6. The immune conjugate of claim 1, wherein the ligand is
biotinylated, glycosylated, acetylated, alkylated, isoprenylated,
lipoylated, or phosphorylated.
7-8. (canceled)
9. The immune conjugate of claim 1, wherein the cell-surface
molecule is glycophorin A (CD235A), band 3 (CD233), TER-119, blood
group antigen A, blood group antigen B, or blood group antigen
H.
10-17. (canceled)
18. The immune conjugate of claim 1, wherein the circulating
non-lymphoid cell is a red blood cell.
19-20. (canceled)
21. The immune conjugate of claim 1, wherein the immunogen is a
molecule expressed or released by an infectious agent.
22. The immune conjugate of claim 21, wherein the infectious agent
comprises a virus, viroid, bacterium, fungus, prion or
parasite.
23-29. (canceled)
30. The immune conjugate of claim 29, wherein the immunogen is
influenza A M2 protein or a fragment of influenza A M2 protein.
31. The immune conjugate of claim 30, wherein the fragment of
influenza A M2 comprises the ectodomain peptide M2e.
32-34. (canceled)
35. The immune conjugate of claim 1, wherein the immunogen is
biotinylated, glycosylated, acetylated, alkylated, isoprenylated,
lipoylated, or phosphorylated.
36. The immune conjugate of claim 1, wherein the immunogen is
coupled to the ligand via a covalent bond.
37. The immune conjugate of claim 1, wherein the immunogen and the
ligand comprise a fusion protein.
38. A nucleic acid sequence encoding the fusion protein of claim
37.
39. An expression vector comprising the nucleic acid sequence of
claim 38.
40. A host-cell comprising the expression vector of claim 39.
41. The immune conjugate of claim 1, wherein the immunogen is
coupled to the ligand via a non-covalent bond.
42. The immune conjugate of claim 41, wherein the non-covalent bond
is a biotin-avidin linkage.
43-46. (canceled)
47. The immune conjugate of claim 1, wherein the ligand is an
anti-TER-119 antibody and the immunogen is the ectodomain peptide
of M2e.
48. A composition comprising the immune conjugate of claim 1 and an
adjuvant.
49. A method of inducing or enhancing an immune response against an
immunogen in a mammalian subject, the method comprising
administering a therapeutically effective amount of the immune
conjugate of claim 1, wherein an immune response to the immunogen
is induced in the mammalian subject.
50-53. (canceled)
54. An article of manufacture comprising a measured amount of one
or more immune conjugates and one or more items selected from the
group consisting of packaging material, a package insert comprising
instructions for use, a sterile fluid, and a sterile container.
Description
TECHNICAL FIELD
[0001] This invention relates to materials and methods involved in
immunotherapy.
BACKGROUND
[0002] Vaccination is one of the most important medical
interventions for preventing disease. The purpose of vaccination is
to induce an optimal immune response that provides preventive or
therapeutic benefit to the host. Vaccines typically contain one or
more immunogens (IMGs) that are harmless variants or derivatives of
pathogens, which act to stimulate the immune system to mount
defenses against the actual pathogen. There are many types of
immunogens (IMGs), ranging from attenuated or killed
microorganisms, microbial extracts, whole proteins,
polysaccharides, and peptides. Some IMGs are very effective
inducers of the desired immune response, while others require the
co-administration of non-specific immune stimulants, or adjuvants,
or the coupling of the IMG to a carrier protein or microparticulate
substances. Still other IMGs are inherently poor at inducing
effective immune responses, despite combination with adjuvants and
repeated boosts. Many of these inherently weak immunogens are
involved in diseases e.g., influenza and cancer, that remain major
causes of human morbidity and mortality. There is a continuing need
for effective and more potent vaccines, particularly those with the
capacity to stimulate a robust immune response against weakly
immunogenic targets.
SUMMARY
[0003] Disclosed herein are immune conjugates that are useful for
inducing or enhancing an immune response against an antigen.
Accordingly, disclosed are immune conjugates that include a ligand
which binds specifically to a cell surface molecule on a
circulating non-lymphoid cell, and an immunogen coupled to the
ligand, wherein the immune conjugate, when administered to an
individual, induces or enhances an immune response against the
immunogen. In one embodiment, the immune conjugate includes a
ligand which binds specifically to a cell surface molecule on a
circulating non-lymphoid cell, wherein the molecule is selected
from the group consisting of CR2, glycophorin A, band 3, Ter-119,
blood group antigen H, blood group antigen A, blood group antigen
B, CD41a, CD14, CD56, CD66d, CD83, CMKLR1, and BDCA-4; and an
immunogen coupled to the ligand, where the immune conjugate, when
administered to an individual, induces or enhances an immune
response against the immunogen.
[0004] A circulating non-lymphoid cell can be any cell that
circulates through the body of a mammal in the blood and/or lymph
system; and is not a B lymphocyte or a T lymphocyte. The
circulating non-lymphoid cell can be, for example, a red blood
cell, a platelet, a natural killer cell, a monocyte, a granulocyte
or a plasmacytoid dendritic cell. In one embodiment, the
circulating non-lymphoid cell is a red blood cell. In another
embodiment, the circulating non-lymphoid cell is a platelet. The
circulating non-lymphoid cell can be derived from any mammal, e.g.,
humans, a non-human primates, cattle, horses, pigs, sheep, goats,
deer, elk, dogs, cats, mustelids, rabbits, guinea pigs, hamsters,
rats, or mice.
[0005] The cell-surface molecule can be any molecule that is
differentially expressed on circulating non-lymphoid cells relative
to the levels of the same molecule on a cell type that is not a
circulating non-lymphoid cell. The cell-surface molecule can be a
polypeptide, a carbohydrate, a phospholipid, or a glycolipid.
[0006] In one embodiment, the cell surface molecule can be a
molecule on the surface of a red blood cell, including, for
example, complement receptor 2 (CR2), glycophorin A (CD235A), band
3 (CD233), TER-119, the ABO blood group antigens, e.g., blood group
antigen A, blood group antigen B, blood group antigen H, and
phosphatidyl serine. In another embodiment, the cell surface
molecule can be a molecule on the surface of a platelet, including,
for example, gpIIb/IIIa (CD41a), CD42d, CD61, CD62P(P-selectin) and
CD151. In another embodiment, the cell surface molecule can be a
molecule on the surface of a natural killer (NK) cell, including,
for example, CD56 (NCAM, Leu-19, NKH1). In another embodiment, the
cell surface molecule can be a molecule on the surface of a
monocyte, including, for example, CD14 (LPS-receptor). In another
embodiment, the cell surface molecule can be a molecule on the
surface of a granulocyte, including, for example, CD66d (CGM1, a
member of the CEA family). In another embodiment, the cell surface
molecule can be a molecule on the surface of a plasmacytoid
dendritic cell, including, for example, CMKLR1 (serpentine
chemokine-like receptor 1), BDCA-2 (CD303) and BDCA-4 (CD304).
[0007] The ligand can be any molecule capable of binding a specific
cell surface molecule on a circulating non-lymphoid cell. A ligand
can include, for example, a polypeptide, a carbohydrate, glycolipid
or biomimetic of a polypeptide, carbohydrate or glycolipid, as long
as the ligand binds specifically to a cell surface molecule that is
differentially expressed on a circulating non-lymphoid cell.
[0008] A ligand can be a polypeptide, provided that it is not C3d,
a heat shock protein, muramyl dipeptide, or muramyl tripeptide.
[0009] In one embodiment, a polypeptide ligand can be an antibody.
The antibody can be a monoclonal antibody, a polyclonal antibody, a
holoantibody, a single chain variable fragment immunoglobulin, or a
chimeric molecule that contains the constant region of an
immunoglobulin and cell-binding sequences from a different source
grafted in place of the immunoglobulin variable regions. The
antibody can be, for example, an antibody that recognizes targets
on red blood cells e.g., complement receptor 2 (CR2), glycophorin A
(CD235A), band 3 (CD233), TER-119, blood group antigen A, blood
group antigen B, and blood group antigen H. The antibody can be,
for example, an antibody that recognizes targets on platelets,
e.g., gpIIb/IIIa (CD41a), CD42d, CD61, CD62P(P-selectin) and CD151.
The antibody can be, for example, an antibody that recognizes
targets on monocytes, e.g., CD14. The antibody can be, for example,
an antibody that recognizes targets on granulocytes, e.g., CD66d.
The antibody can be, for example, an antibody that recognizes
targets on NK cells, e.g., CD56. The antibody can be, for example,
an antibody that recognizes targets on plasmacytoid dendritic
cells, e.g., CMKLR1, BDCA-2 (CD303) and BDCA-4 (CD304).
[0010] A ligand can also be a polypeptide that is not an
immunoglobulin, including for example, the erythrocyte-binding
antigen 175 (EBA-175) of Plasmodium falciparum or a fragment of
EBA-175 that binds to a red blood cell, for example, SEQ ID NO:7, a
complement fragment that binds to CR2, for example, C4b, C3b, iC3b,
C1q, C3d or a peptide derived from these complement fragments,
e.g., C3 residues 1201-1214; or a lectin, e.g., a glycoprotein that
recognizes blood group antigen A, blood group antigen B or blood
group antigen H.
[0011] The polypeptide ligand can include post-translational
modifications, e.g., biotinylation, glycosylation, acetylation,
alkylation, isoprenylation, lipoylation, phosphorylation.
[0012] In another embodiment a ligand can also be a carbohydrate or
glycolipid, including, for example, bacterial lipopolysaccharide or
a fragment of it, microbial products bound by Toll-like receptors,
bacterial diacyl and triacyl lipopeptides, lipoteichoic acid or
zymosan.
[0013] In another embodiment, a ligand can be a nucleic acid,
including, for example, single- and double-stranded viral RNA and
CpG DNA.
[0014] The immunogen can be any molecule capable of eliciting a
functional immune response in a mature T or a B lymphocyte or a
precursor of a T or a B lymphocyte. Immunogens can include
polypeptides, carbohydrates, glycolipids, haptens or biomimetics
thereof. The immunogen can be a molecule expressed or released by
an infectious agent, including, for example, viruses, viroids,
bacteria, fungi, prions or parasites. An infectious agent can
include for example, Orthomyxoviridae, e.g., influenza viruses,
including the strain A(H5N1), Rhadboviridae, Hepadnaviridae, e.g.,
hepatitis B, Picornaviridae, e.g., hepatitis A, Flaviviridae, e.g.,
hepatitis C; Retroviridae, e.g., human immunodeficiency viruses
HIV1 and HIV2, Togaviridae, Bunyaviridae, e.g., hantavirus;
Paramyxoviridae, Herpesviridae, Arenaviridae, e.g., lassa virus;
Reoviridae; Bacillus anthracis, Clostridium botulinum, Salmonella
enteriditis, Escherichia coli, including E. coli O157:H7,
Streptococcus pneumoniae, Staphylococcus aureus, Aspergilllus,
Stachybotrys, Candida, Cryptosporidium, Toxoplasma, and Plasmodium
falciparum.
[0015] Immunogens derived from pathogenic organisms can include,
for example, influenza A M2 protein, hepatitis B surface antigen,
HBV preS1 protein, HIV tat, HIV gp120, anthrax protective antigen,
botulinum toxin, and Streptococcus pneumoniae pneumococcal
polysaccharides. An influenza M2 protein antigen can be the
ectodomain peptide M2e, for example SEQ ID NO:1, or a variant of
the ectodomain peptide M2e, for example, SEQ ID NO: 3 or SEQ ID 4.
An HBV preS1 protein can include the preS1 protein peptide 35-49,
for example SEQ ID NO:2.
[0016] The immunogen can also be a molecule expressed by a mammal.
For example, an immunogen can be a molecule whose expression is
correlated with a particular disease for example, cancer or
neurodegenerative disease. The immunogen can be a tumor-associated
antigen (TAA), including for example, MART-1, Muc-1, MAGE, RAGE, or
CEA. In another embodiment, the immunogen can be an antigen that is
involved in the initiation or progression of neurodegenerative
diseases, e.g. Alzheimer's disease and Transmissible Spongiform
Encephalopathies (TSEs), including for example, beta-amyloid, tau
protein, alpha synuclein, or a prion-related protein. In another
embodiment, the immunogen can be a germ cell antigen, including for
example, sperm adhesion molecule 1 (SPAM-1), and human
intra-acrosomal protein. In another embodiment, the immunogen can
be a non-toxic variant of a toxic substance, including for example,
ricin, botulinum toxins A, B, C, D, E, F and G, nicotine, or a drug
of abuse such as an opiate or opiate derivative.
[0017] The ligand and the immunogen are connected by a linker. A
linker can be any reagent, molecule or macromolecule that connects
the ligand and the immunogen such that a) the immune complex is
stable under physiological conditions; b) the connection between
the linker and the ligand does not alter the ability of the ligand
to bind to its target on the surface of a circulating non-lymphoid
cell; and c) the connection between the linker and the immunogen
does not abolish the capacity of the immunogen to induce an
effective immune response in a host against an infectious agent,
cell or molecule on which the immunogen is naturally found.
[0018] In one embodiment, a linker can be a peptide bond. The
ligand and the immunogen can be a fusion polypeptide comprising one
or more amino acid segments from the ligand and one or more amino
acid segments from the immunogen. The amino acid segments of the
ligand can be contiguous with the amino acid segments of the
immunogen or they can be separated by amino acids inserted as a
structural spacer. A spacer segment can be one or more amino acids.
The one or more amino acids can include amino acids that are the
same or that are different. Also encompassed are nucleic acids
comprising a nucleotide sequence that encodes the fusion proteins,
a vector (e.g., a vector that includes a transcriptional regulatory
element (TRE) operably linked to the nucleotide sequence)
containing the nucleic acid, and a cell (e.g., a prokaryotic cell
or a eukaryotic cell) containing the vector.
[0019] In another embodiment, the ligand and immunogen can be
obtained separately, either through chemical synthesis or synthesis
in vivo, purified and then linked non-covalently or covalently. The
non-covalent linkage can be a for example, a biotin-avidin (or
streptavidin) linkage. The covalent linkage can be through a
chemical cross-linking agent, for example, a homobifunctional
cross-linking reagent or a heterobifunctional cross-linking
reagent. In another embodiment, the ligand and the immunogen can be
connected through a linking polymer, including, for example, linear
or branched polymers or co-polymers (e.g., polyalkylene,
poly(ethylene-lysine), polymethacrylate, polyamino acids, poly- or
oligosaccharides, or dendrimers).
[0020] In another embodiment, the ligand and the immunogen can be
connected through a microparticle, including, for example,
micelles, liposomes, fullerenes, nanotubes, or other colloidal
complexes such as lipoproteins. The ligand and the immunogen can be
attached to the linking molecule or microparticle through a
non-covalent high affinity linkage, e.g., avidin-biotin high
affinity binding, adsorbed or incorporated into a hydrophobic
microparticle by hydrophobic affinity, or covalent chemical
cross-linking techniques.
[0021] The immune conjugates provided herein can include one or
more of the same ligands or any combination of different ligands.
The immune conjugates can also include one or more of the same
immunogens or any combination of different immunogens.
[0022] Also provided are methods and materials for inducing or
enhancing an immune response in a mammal, the method comprising
administering to the mammal an effective amount of a composition
comprising an immune conjugate that includes a ligand which binds
specifically to a cell surface molecule on a circulating
non-lymphoid cell, and an immunogen coupled to the ligand, wherein
the immune conjugate, when administered to an individual, induces
or enhances an immune response against the immunogen. In one
embodiment, the immune conjugate includes a ligand which binds
specifically to a cell surface molecule on a circulating
non-lymphoid cell, wherein the molecule is selected from the group
consisting of CR2, glycophorin A, band 3, Ter-119, blood group
antigen H, blood group antigen A, blood group antigen B, CD41a,
CD14, CD56, CD66d, CD83, CMKLR1, and BDCA-4; and an immunogen
coupled to the ligand, where the immune conjugate, when
administered to an individual, induces or enhances an immune
response against the immunogen.
[0023] The mammals can be, for example, humans, non-human primates,
horses, cattle, pigs, sheep, deer, elk, goats, dogs, cats,
mustelids, rabbits, guinea pigs, hamsters, rats, and mice. The
mammal can have, be likely to have, or be at risk for having, an
infectious disease, e.g., a viral disease, a bacterial disease, a
protozooal disease, or a fungal disease. Infectious diseases can
include, for example, influenza, HIV-AIDS, hepatitis, botulism,
smallpox, viral hemorrhagic fevers, gastrointestinal disease
induced by pathogenic forms of E. coli, Salmonella and Shigella,
Staphylococcal infection, trypanosomiasis, and malaria.
Alternatively, the mammal can have, be likely to have, or be at
risk for having, a proliferative cell disease, e.g., a cancer such
as a neural tissue cancer, melanoma, breast cancer, lung cancer, a
gastrointestinal cancer, ovarian cancer, testicular cancer, lung
cancer, prostate cancer, cervical cancer, bladder cancer, vaginal
cancer, liver cancer, renal cancer, bone cancer, a hematological
cell cancer, or a vascular tissue cancer. In another embodiment,
the mammal can have, be likely to have, or be at risk for having, a
neurodegenerative disease, e.g., Alzheimer's disease or a
Transmissible Spongiform Encephalopathy (TSE).
[0024] In another embodiment, methods and materials for inducing or
enhancing an immune response in a mammal against a germ cell
antigen are provided. In a further embodiment, methods and
materials for inducing or enhancing an immune response in a mammal
against toxic substances, e.g., ricin, botulinum toxin, nicotine
and drugs of abuse are provided.
[0025] Also provided are compositions featuring immune conjugates
that include a ligand which binds specifically to a cell surface
molecule on a circulating non-lymphoid cell, and an immunogen
coupled to the ligand, wherein the immune conjugate, when
administered to an individual, induces or enhances an immune
response against the immunogen in a pharmaceutically acceptable
carrier or excipient. In another embodiment, the composition can
include an adjuvant.
[0026] Also provided are articles of manufacture that can include
immune conjugates as described herein. An article of manufacture
can include, for example, one or more immune conjugates. In
addition, an article of manufacture further may include, for
example, packaging materials, instructions for use, buffers or
other control reagents for treating or monitoring the condition for
which prophylaxis or treatment is required.
[0027] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0028] FIG. 1 depicts the electrophoretic mobility of tripartite
immune conjugates containing biotinylated antibody, streptavidin,
and varying amounts of biotinylated M2e peptide (SEQ ID NO:1).
[0029] FIG. 2 depicts the electrophoretic mobility of tripartite
immune conjugates containing biotinylated antibody and a constant
ratio of streptavidin, and biotinylated M2e peptide (SEQ ID
NO:1).
DETAILED DESCRIPTION
[0030] The mammalian immune system mounts two different types of
responses to immunogens (IMGs), humoral and cellular. The humoral
response, mediated by B lymphocytes, defends primarily against
extracellular pathogens through the production of circulating
antibodies that mark foreign cells and molecules for destruction by
other specialized cells and proteins. The cellular response,
mediated by T lymphocytes, defends predominantly against
intracellular pathogens and cancers by directly binding to and
destroying the infected or cancerous cells. Both responses depend
upon specialized cells that internalize through endocytosis,
pinocytosis or phagocytosis, and process IMGs; fragments of the
IMGs are then presented to T lymphocytes, which in turn, help to
trigger B-lymphoctye responses against the immunogens and/or the T
cells to attack the target directly.
[0031] The phagocytic cells that function as antigen-presenting
cells (APCs) are part of the reticuloendothelial system (RES). The
RES is a diffuse system comprised of circulating and tissue-fixed
cells including monocytes, macrophages, dendritic cells, Kupffer
cells in the liver, Langerhans cells in the skin and microglial
cells in the brain Several tissues and organs, by virtue of their
wealth of phagocytic and specialized endothelial cells, comprise
critical locations for the clearance and antigen presentation
functions of the RES. These include the liver, spleen, bone marrow
and lymphatic tissues. The RES plays an important role in clearing
potentially harmful materials from the blood including
micro-organisms, bacterial endotoxins, immune complexes, tumor
cells and senescent and damaged cells of the blood and lymph
systems. The cells that cycle through the RES, e.g. red blood cells
(or erythrocytes), platelets, natural killer cells, monocytes,
granulocytes, and plasmacytoid dendritic cells, serve as carriers
of foreign materials, which may be stripped off in RES organs. If
the load of foreign material on the surface of the circulating
cells is very dense, these cells themselves can be targets for
phagocytic activity. In addition, as circulating cells age, they
acquire surface markers that cause them to be captured by the RES,
removed from circulation and destroyed by phagocytosis, resulting
in the degradation of the circulating cell components and their
surface-bound materials. Once these cells have been phagocytized,
any fragments of macromolecules they originally contained, either
endogenously expressed molecules or exogenous molecules that were
opsonized to the cell surface can potentially be presented to T
lymphocytes. Thus, molecules bound to red blood cells, platelets,
natural killer cells, monocytes, granulocytes, and plasmacytoid
dendritic cells can function as immunogens.
[0032] Disclosed herein are materials and methods for the specific
delivery of immunogens to the reticuloendothelial system via
circulating non-lymphoid cells. In particular, targeted immune
conjugates are provided that specifically bind to circulating
non-lymphoid cells, e.g., red blood cells, platelets, natural
killer cells, monocytes, granulocytes, and plasmacytoid dendritic
cells, that are cleared through the RES. Thus, the circulating
non-lymphoid cells act as vehicles for the specific delivery of
IMGs to the RES and thereby to the immune system. As used herein,
the term "targeting immune conjugate" includes a ligand which binds
specifically to a cell surface molecule on a circulating
non-lymphoid cell coupled to an IMG, where the targeted immune
conjugate, when administered to an individual, induces or enhances
an immune response against the IMG. Also provided are methods of
treatment using targeted immune conjugates. Targeted immune
conjugates provide injectable vaccines for efficient immunization
against weakly immunogenic IMGs.
II. Compositions
[0033] The targeting immune conjugates provided herein have the
general formula:
(L)y-X-(IMG)z
[0034] wherein L is a ligand which binds specifically to a cell
surface molecule on a circulating non-lymphoid cell; X is a linker;
IMG is an immunogen, and y and z are integers having a value of one
or greater than one.
Circulating Non-Lymphoid Cells and Targets
[0035] As used herein, a circulating non-lymphoid cell can be any
cell that a) circulates through the body of a mammal in the blood
and/or lymph system; and b) is not a B lymphocyte or a T
lymphocyte. Thus, circulating non-lymphoid cells include
erythrocytes, i.e., red blood cells; platelets; natural killer
cells; monocytes; granulocytes, i.e., neutrophils, eosinophils, and
basophils; and plasmcytoid dendritic cells.
[0036] B and T lymphocytes are ultimately derived from
hematopoietic stem cells and perform the principal functions of the
immune system. T lymphocytes mature through the thymus and are
generally identified by their expression of CD3 (which is
associated with the T cell receptor) and either CD4 or CD8.
CD8-expressing (or CD8+) T cells are principally involved with
direct cell killing, or cytotoxicity. CD4+ T cells are primarily
regulatory cells which stimulate and suppress immune responses as
needed. B lymphocytes are characterized by their expression of CD19
or CD20, among other surface markers, and they are responsible for
antibody production. B cells are also effective antigen presenting
cells.
[0037] Erythrocytes, also known as red blood cells, are the most
abundant cell type in mammalian blood. They are small disc-shaped,
anucleated, biconcave cells whose primary function is to carry
oxygen and carbon dioxide to and from the tissues. Red blood cells
express a distinctive complement of cell surface markers, including
the human blood group antigens, glycophorin, band 3 and the Lewis
antigens.
[0038] Platelets are derived from megakaryocytes, they are
centrally involved in blood clotting, and can be identified by
their surface expression of CD41a (or gpIIb/IIIa). Natural killer
cells, also referred to as large granular lymphocytes, are derived
from the bone marrow and do not express T-cell antigen receptors
(TCR), the pan-T marker CD3 or surface immunoglobulins (Ig) B cell
receptor, but typically express the surface markers CD16
(Fc.gamma.RIII) and CD56. Monocytes are derived from myeloid stem
cells and are found primarily in the circulation. They are
competent phagocytes. Upon their binding of pathogens and/or
stimulation by various cytokines, monocytes mature into
macrophages, which are even more avid phagocytes and producers of
many cytokines, degradative enzymes and other molecules that
mediate inflammatory reactions. Macrophages are generally found
bound to vascular endothelium or within various tissues. Monocytes
(and macrophages) are characterized by the surface expression of
CD14, among other markers.
[0039] Granulocytes are also derived from myeloid stem cells and
are characterized by the presence of abundant granules in their
cytoplasm; different classes of granulocytes, e.g., eosinophils,
basophils and neutrophils, are distinguished by their ability stain
with eosin, basophilic dyes or neither, respectively. Eosinophils
are involved in defense against parasitic pathogens and allergens;
basophils are also involved in allergic reactions. Neutrophils are
early and aggressive phagocytes at the site of infections and
release products that induce inflammatory reactions.
[0040] Plasmacytoid dendritic cells (pDC) are distinct from myeloid
dendritic cells. Both are found in the circulation and in tissues.
pDC are an important link between the innate and adaptive immune
responses, in particular in mounting anti-viral immune responses.
They produce abundant interferons and can be identified by the
surface marker BDCA-2 (CD303).
[0041] The circulating non-lymphoid cells can be derived from any
mammal, e.g., humans, a non-human primates, cattle, horses, pigs,
sheep, goats, deer, elk, dogs, cats, mustelids, rabbits, guinea
pigs, hamsters, rats, or mice.
[0042] Any cell-surface molecule that is differentially expressed
on circulating non-lymphoid cells relative to the levels of the
same molecule on a cell type that is not a circulating non-lymphoid
cell is a suitable target for the ligand. The cell-surface molecule
can be a polypeptide, a carbohydrate, or a glycolipid. Full-length
molecules, epitopes, analogs, mutants, and functional fragments
thereof are encompassed by this definition. A "functional fragment"
of a molecule is a fragment of the molecule that is smaller
(shorter where the molecule is a polypeptide) than the molecule per
se but has at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95%, 98%, 99%, 99.5%, 100% or even more) of the
ligand-binding activity of the molecule per se.
[0043] Typically, a cell-surface molecule on a circulating
non-lymphoid cell can be classified as being differentially
expressed if the molecule is present at a level that is greater
than the average level observed in cells that are not circulating
non-lymphoid cells if the expression levels differ by at least 50%
(e.g., 50, 100, 200, 300% or more). Any method can be used to
determine whether or not a specific gene product is expressed at a
level that is greater or less than the average level of expression
observed in control cells. The level of expression of a cell
surface polypeptide can be measured using any method such as
immuno-based assays (e.g., immunofluorescence, flow cytometry,
ELISA), western blotting, or polyacrylamide gel electrophoresis
with silver staining Levels of particular carbohydrates or lipids
can be measured by immunodetection e.g., ELISA, flow cytometry, or
immunostaining using fluorochrome- or radioisotope-labeled
antibodies or lectins. In some embodiments, the level of expression
from a particular gene can be measured by assessing the level of
mRNA expression from the gene. Levels of mRNA expression can be
evaluated using, without limitation, northern blotting, slot
blotting, quantitative reverse transcriptase polymerase chain
reaction (RT-PCR), or chip hybridization techniques. Such methods
can be used to determine simultaneously the relative expression
levels of multiple mRNAs.
[0044] Examples of targets on red blood cells include, without
limitation, complement receptor 2 (CR2), glycophorin A (CD235A),
band 3 (CD233), TER-119 (Kina et al., Br. J. Hematol. 109: 280-7,
2000), the ABO blood group antigens, e.g., blood group antigen A,
blood group antigen B, blood group antigen H, and phosphatidyl
serine (Hematology: Basic Principles and Practice, R. Hoffman,
2005, 4.sup.th ed. New York: Churchill-Livingstone). One useful red
blood cell target is TER-119, a region selectively bound by the
antibody, anti-TER-119 and which corresponds to the extracellular
domain of glycophorin A. Suitable platelet targets includes
gpIIb/IIIa (CD41a), CD42d, CD61, CD62P (P-selectin) and CD151.
Natural killer cell targets include, for example, CD56 (NCAM,
Leu-19, HNK1). Monocyte and granulocyte targets include, for
example, CD14 (LPS-receptor) and CD66d (CGM1, a member of the CEA
family), respectively. Plasmacytoid dendritic cell targets include,
for example, CMKLR1 (serpentine chemokine-like receptor 1), BDCA-2
(CD303) and BDCA-4 (CD304) (Kuby Immunology, J. Kuby et al., edt.,
2002, 5.sup.th edition, W.H. Freeman & Co.).
Ligands
[0045] The term "ligand" as used herein refers to a molecule
capable of binding a specific cell surface molecule on a
circulating non-lymphoid cell. A ligand can be a polypeptide, a
carbohydrate, glycolipid or biomimetic of a polypeptide,
carbohydrate or glycolipid, as long as the ligand binds
specifically to a cell surface molecule that is differentially
expressed on a circulating non-lymphoid cell.
[0046] As defined herein, ligands are defined to be "specifically
binding" if: 1) they exhibit a threshold level of binding activity,
and/or 2) they do not significantly cross-react with related target
molecules. The binding affinity of a ligand can be readily
determined by one of ordinary skill in the art, for example, by
Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660-672,
1949). For example, a ligand disclosed herein can bind to its
target with at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold,
103-fold, 104-fold, 105-fold, 106-foldor greater affinity for the
target than for a closely related or unrelated polypeptide. A
ligand can bind its target with high affinity (10.sup.-4M or less,
10.sup.-7M or less, 10.sup.-9M or less, or with subnanomolar
affinity (0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 nM or even
less). Ligands can also be described or specified in terms of their
binding affinity to a target, for example, binding affinities
include those with a Kd less than 5.times.10.sup.-2 M, 10.sup.-2 M,
5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M,
5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M,
5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8M,
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10
M, 5.times.10.sup.-11 M, 10.sup.-11M, 5.times.10.sup.-12 M,
10.sup.-12M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14M, 10.sup.-14 M, 5.times.10.sup.-15 M, or
10.sup.-15 M, or less.
[0047] In some embodiments, the ligands disclosed herein do not
bind to known related molecules. In other embodiments, the ligands
disclosed herein can bind to orthologs, homologs, paralogs or
variants, or combinations and subcombinations thereof, of their
targets.
[0048] As defined herein, a ligand that specifically binds to
circulating non-lymphoid cells is a ligand the binds to circulating
non-lymphoid cells and does not significantly bind to lymphoid
cells. For example, an immune conjugate that includes a ligand that
specifically binds to red blood cells will bind to red blood cells
and not significantly bind to other circulating cells, e.g.,
lymphocytes, platelets, natural killer cells, monocytes,
granulocytes or dendritic cells. In another example, an immune
conjugate that includes a ligand that specifically binds to
platelets will bind to platelets and not significantly bind to
other circulating cells, e.g., lymphoid cells, red blood cells,
natural killer cells, monocytes, granulocytes or dendritic
cells.
[0049] Ligands may be screened against known related target
polypeptides to isolate a ligand that specifically binds the
target. For example, a ligand specific to a target will flow
through an affinity chromatography column comprising other closely
related target molecules adhered to insoluble matrix under
appropriate buffer conditions. Such screening allows isolation of
ligands non-crossreactive to closely related targets (Antibodies: A
Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor
Laboratory Press, 1988; Current Protocols in Immunology, Cooligan
et al. (eds.), National Institutes of Health, John Wiley and Sons,
Inc., 1995). Screening and isolation of specific antibodies is well
known in the art (see, Fundamental Immunology, W. Paul (ed.), Raven
Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98, 1988;
Monoclonal Antibodies: Principles and Practice, Goding, J. W.
(ed.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev.
Immunol. 2: 67-101, 1984). Representative examples of such assays
include: concurrent immunoelectrophoresis, radioimmunoassay (RIA),
radioimmunoprecipitation, flow cytometry, FACS, enzyme-linked
immunosorbent assay (ELISA), dot blot or western blot assay,
inhibition or competition assay, and sandwich assay.
[0050] A ligand can be a polypeptide, provided that it is not C3d,
a heat shock protein, muramyl dipeptide, or muramyl tripeptide. The
term "polypeptide" as used herein refers to a compound of two or
more subunit amino acids, amino acid analogs, or other
peptidomimetics, regardless of post-translational modification,
e.g., phosphorylation or glycosylation. The subunits may be linked
by peptide bonds or other bonds such as, for example, dicysteine,
ester or ether bonds. The term "amino acid" refers to natural
and/or unnatural or synthetic amino acids, including D/L optical
isomers. Full-length proteins, analogs, mutants, and fragments
thereof are encompassed by this definition.
[0051] The amino acid sequence of the ligands disclosed herein can
be identical to the wild-type sequences of appropriate components.
Alternatively, any of the components can contain mutations such as
deletions, additions, or substitutions. All that is required is
that the variant ligand have at least 5% (e.g., 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or even more) of the
ability of the ligand containing only wild-type sequences to
specifically bind the target on the circulating non-lymphoid cell.
Substitutions will preferably be conservative substitutions.
Conservative substitutions typically include substitutions within
the following groups: glycine and alanine; valine, isoleucine, and
leucine; aspartic acid and glutamic acid; asparagine, glutamine,
serine and threonine; lysine, histidine and arginine; and
phenylalanine and tyrosine.
[0052] Any method can be used to make a polypeptide including, for
example, expression by prokaryotic systems, expression by
eukaryotic systems, and chemical synthesis techniques. Any method
can be used to purify a polypeptide including, without limitation,
fractionation, centrifugation, and chromatography, e.g., gel
filtration, ion exchange chromatography, reverse-phase HPLC and
immunoaffinity purification.
[0053] A suitable polypeptide ligand can be an antibody. In one
embodiment, the antibody can be a monoclonal antibody, i.e.,
homogeneous antibodies of identical antigenic specificity produced
by a single clone of antibody-producing cells. In another
embodiment, the antibody can be a polyclonal antibody, i.e.,
heterogenous antibodies that can recognize different epitopes on
the same antigen and that are produced by more than one clone of
antibody producing cells.
[0054] The antibody can include any class of immunoglobulin, e.g.,
IgG, IgA, IgM; and can be derived from any species e.g., humans,
mice, rats, or can be a humanized version of a non-human antibody.
An antibody can include, without limitations, a holoantibody, i.e.,
an antibody that includes one or more of an immunoglobulin monomer
units of two heavy and two light chains, a single chain variable
fragment immunoglobulin, or a chimeric molecule that contains the
constant region of an immunoglobulin and cell-binding sequences
from a different source grafted in place of the immunoglobulin
variable regions. The cell-binding regions can be from a different
antibody, a lectin, a cytokine, a microbial protein fragment or any
other molecule that binds the target cell receptor molecule with
specificity.
[0055] Methods for producing antibodies are well know to those in
the art; Antibodies can be purified by chromatographic methods
known to those of skill in the art, including ion exchange and gel
filtration chromatography (for example, Caine et al., Protein Expr.
Purif. (1996) 8(2):159-166). Alternatively or in addition,
antibodies can be purchased from commercial sources, for example,
Invitrogen (Carlsbad, Calif.); MP Biomedicals (Solon, Ohio); Nventa
Biopharmaceuticals (San Diego, Calif.) (formerly Stressgen);
Serologicals Corp. (Norcross, Ga.).
[0056] The antibody can be, for example, an antibody that
recognizes targets on red blood cells, including for example,
without limitation, glycophorin A (CD235A), band 3 (CD233),
TER-119, blood group antigen A, blood group antigen B, and blood
group antigen H. One useful antibody is anti-TER-119, which
specifically binds to TER-119, a region corresponding to the
extracellular domain of glycophorin A. Other suitable antibodies
include, without limitation, antibodies that recognize targets on
platelets, e.g., gpIIb/IIIa (CD41a), CD42d, CD61, CD62P
(P-selectin) and CD151; monocytes, e.g., CD14; NK cells, e.g.,
CD56; granulocytes, e.g., CD66d; and plasmacytoid dendritic cells,
e.g., CMKLR1, BDCA-2 (CD303) and BDCA-4 (CD304).
[0057] A ligand can also be a polypeptide that is not an
immunoglobulin. One non-immunoglobulin type ligand can be the
erythrocyte-binding antigen 175 (EBA-175) of Plasmodium falciparum,
which specifically binds the red blood cell surface protein band 3,
or a fragment of EBA-175 that binds to a red blood cell, for
example EBA-175 peptide 1085-96, SEQ ID NO:7. Another polypeptide
ligand can also be a complement fragment that binds to CR2, for
example, C4b, C3b, iC3b, C1q or a peptide derived from the
complement fragments, e.g., C3 residues 1201-1214 (Tsokos et al.,
Journal of Immunol. 144: 1640-45, 1990) or residues 727-768
(Becherer, Biochemistry 31(6):1787-94. 1992). A polypeptide ligand
can also be a lectin, e.g. a glycoprotein that recognizes blood
group antigen A, blood group antigen B or blood group antigen
H.
[0058] A ligand can also be a peptidomimetic, a small protein-like
chain containing non-peptidic structural elements that is capable
of mimicking or antagonizing the biological action(s) of a natural
parent peptide. Peptidomimetic compounds are synthetic, non-peptide
compounds having a three-dimensional conformation (i.e., a "peptide
motif") that is substantially the same as the three-dimensional
conformation of a selected peptide. The peptide motif provides the
peptidomimetic compound with the ability to bind the ligand in a
manner qualitatively identical to that of the parent peptide from
which the peptiomimetic was derived. Peptidomimetic compounds can
have additional characteristics that enhance their therapeutic
utility, such as increased prolonged biological half-life.
[0059] The peptidomimetics typically have a backbone that is
partially or completely non-peptide, but with side groups that are
identical to the side groups of the amino acid residues that occur
in the peptide on which the peptidomimetic is based. Several types
of chemical bonds, e.g., ester, thioester, thioamide, retroamide,
reduced carbonyl, dimethylene and ketomethylene bonds, are known in
the art to be generally useful substitutes for peptide bonds in the
construction of protease-resistant peptidomimetics.
[0060] Any peptidomimetic that binds specifically and selectively
to a cell surface molecule that is differentially expressed on a
circulating non-lymphoid cell can be used. Examples of useful
peptidomimetics include those that mimic the ability of antibodies
that recognize, for example, complement receptor 1 (CR1),
complement receptor 2 (CR2), glycophorin A (CD235A), band 3
(CD233), TER-119, blood group antigen A, blood group antigen B, and
blood group antigen H, gpIIb/IIIa (CD41a), CD42d, CD61, CD62P
(P-selectin), CD151, CD14, CD56, CD66d, CMKLR1 and BDCA-2.
[0061] The polypeptide can include post-translational
modifications, i.e., chemical modification of the polypeptide after
its synthesis. Chemical modifications can be naturally occurring
modifications made in vivo following translation of the mRNA
encoding the polypeptide or synthetic modifications made in vitro.
A polypeptide can include one or more post-translational
modifications, in any combination of naturally occurring, i.e., in
vivo, and synthetic modifications made in vitro. Examples of
post-translational modifications include, but are not limited to,
biotinylation, e.g., acylation of lysine or other reactive amino
acid residues with a biotin molecule; glycosylation, e.g., addition
of a glycosyl group to either asparagines, hydroxylysine, serine or
threonine residues to generate a glycoprotein or glycopeptide;
acetylation, e.g., the addition of an acetyl group, typically at
the N-terminus of a polypeptide; alkylation, e.g., the addition of
an alkyl group; isoprenylation, e.g., the addition of an isoprenoid
group; lipoylation, e.g. attachment of a lipoate moeity;
phosphorylation, e.g., addition of a phosphate group to serine,
tyrosine, threonine or histidine.
[0062] A highly suitable post-translational modification can be
biotinylation. Biotin, also known as vitamin H or B7 is a
water-soluble B-complex vitamin that binds avidin or streptavidin
with very high affinity (10.sup.-15M). Both egg avidin and
bacterial streptavidin have 4 biotin-binding sites and thus can
serve to couple several biotinylated ligands or to couple
biotinylated ligand(s) to other biotinylated molecules (e.g.,
IMGs). Polypeptides can be convalently linked to one or more biotin
molecules through primary amines, e.g. lysine and N-terminus),
carboxyl groups found on aspartic- and glutamic-acid residues and
at the C-terminus, sulfhydryl groups, or carbohydrate modifications
on glycoproteins. Methods for derivatizing polypeptides with biotin
are well known in the art and there are many commercial sources for
such reagents (e.g. Pierce, Sigma-Aldrich). Any method of
biotinylation can be used that preserves the ability of the ligand
to bind to its target on the non-circulating lymphoid cell. For
example, desirable biotinlyation methods would modify residues in
the Fc portion of the antibody without compromising the
immunoreativity of the antibody. Polypeptides derivatized with
biotin can then be linked to immunogens through an avidin or
streptavidin molecule.
[0063] A post-translational modification can be glycosylation,
i.e., the addition of saccharides. Glycosylation is typically
classified based on the amino acid through which the saccharide
linkage occurs and can include: N-linked glycosylation to the amide
nitrogen of asparagines side chains, O-linked glycosylation to the
hydroxyloxygen of serine and threonine side chains, and
C-mannosylation.
[0064] A ligand can also be a carbohydrate or glycolipid. Examples
of carbohydrate and glycolipid ligands, include, without
limitation, bacterial lipopolysaccharide (LPS) or a fragment of it,
microbial products bound by Toll-like receptors (TLRs), bacterial
diacyl and triacyl lipopeptides and lipoteichoic acid from
bacteria, and zymosan from yeast cell walls.
[0065] A ligand can also be a nucleic acid. Examples of nucleic
acids include, without limitation, single- and double-stranded RNA
from viruses, and CpG DNA from bacteria or viruses.
Immunogens
[0066] As defined herein, an immunogen is any molecule capable of
eliciting a functional immune response (e.g., a cytotoxic or helper
T cell response or an antibody producing response) in a T or a B
cell.
[0067] As used herein, an "effector T lymphocyte" is a T lymphocyte
having immunological activity. Such immunological activity can be,
without limitation, cytotoxic activity, helper activity,
suppressive activity, immune-deviating activity, inflammatory
activity, or pro-inflammatory activity. As used herein, an
"effector T lymphocyte precursor cell" is a T lymphocyte that,
subsequent to activation, has any of the above immunological
activities. Activation can occur, without limitation, by
recognition of a complex of the relevant immunogenic peptide
epitope and the major histocompatibility complex (MHC) molecule by
the T cell receptor (TCR) on the effector T lymphocyte or by a
non-specific stimulus, e.g., a T cell mitogen such as concanavalin
A. Thus, an effector T lymphocyte cell can be a "virgin" T
lymphocyte that has never previously been activated or a "memory" T
lymphocyte that has previously been activated or the progeny of
such a memory T lymphocyte. As used herein, a "cytotoxic T
lymphocyte" (CTL) is a T lymphocyte that can kill a target cell
expressing on its surface a peptide epitope-MHC molecular complex
for which the TCR of the CTL is specific.
[0068] As used herein, a "CTL cell" is a T lymphocyte that can,
subsequent to activation, kill a target cell expressing on its
surface a peptide epitope-MHC molecular complex for which the TCR
of the CTL is specific. Activation can be, without limitation, by
recognition of the relevant peptide epitope-MHC molecular complex
by a TCR on the CTL or by a non-specific stimulus, e.g., a T cell
mitogen such as concanavalin A. Thus, a CTL cell can be a "virgin"
T lymphocyte that has never previously been activated or a "memory"
T lymphocyte that has previously been activated or the progeny of
such a memory T lymphocyte.
[0069] As used herein, a "helper T lymphocyte" (Th) is a T
lymphocyte that provides helper or regulatory activity in an immune
response. Such an immune response can be, for example, an
antibody-producing response of a B lymphocyte, a response of a CTL
precursor cell, or an inflammatory or pro-inflammatory response of
a variety of leukocyte types. As used herein, a "Th cell" is a T
lymphocyte that, subsequent to activation, provides helper activity
in an immune response such as those listed above for Th. Activation
can be as indicated above for CTL cells. Furthermore, a Th cell can
be a "virgin" T lymphocyte that has never previously been activated
or a "memory" T lymphocyte that has previously been activated or
the progeny of such a memory T lymphocyte.
[0070] As used herein, a B cell is a B lymphocyte that, subsequent
to activation, can produce antibody molecules. Activation of a B
cell can be, without limitation, by recognition of an antigen by an
antigen specific immunoglobulin receptor on the B cell surface or
by a non-specific stimulus, e.g., a B cell mitogen such as
lipopolysaccharide or pokeweed mitogen. Thus, a B cell can be a
"virgin" B lymphocyte that has never previously been activated or a
"memory" B lymphocyte that has previously been activated or the
progeny of such a B lymphocyte.
[0071] As used herein, "antigenic" means capable of being
recognized by an effector lymphocyte or an antibody molecule. Thus
a substance is antigenic if it is recognized by an antigen specific
receptor on, for example, a CTL, a Th, or a B lymphocyte producing
antibody molecules or by an antibody molecule physically
unassociated with a B lymphocyte.
[0072] An immunogen can be a polypeptide, carbohydrate, glycolipid,
hapten or biomimetic thereof. A polypeptide immunogen, as defined
above, can include without limitation, a compound of two or more
subunit amino acids, amino acid analogs, or other peptidomimetics,
regardless of post-translational modification, e.g.,
phosphorylation or glycosylation. The subunits may be linked by
peptide bonds or other bonds such as, for example, ester or ether
bonds. The term "amino acid" refers to natural and/or unnatural or
synthetic amino acids, including D/L optical isomers. Full-length
proteins, analogs, mutants, and fragments thereof are encompassed
by this definition.
[0073] The amino acid sequence of the immunogens disclosed herein
can be identical to the wild-type sequences of appropriate
components. Alternatively, any of the components can contain
mutations such as deletions, additions, or substitutions. All that
is required is that the variant ligand have at least 5% (e.g., 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or even
more) of the ability of the immunogen containing only wild-type
sequences to induce an immune response against the naturally
occurring wild-type immunogen. Substitutions will preferably be
conservative substitutions. Conservative substitutions typically
include substitutions within the following groups: glycine and
alanine; valine, isoleucine, and leucine; aspartic acid and
glutamic acid; asparagine, glutamine, serine and threonine; lysine,
histidine and arginine; and phenylalanine and tyrosine.
[0074] A polypeptide immunogen can include any peptide epitopes of
a variety of lengths, for example, 7-50 (e.g., 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 32, 34, 36, 38, 40, 45, or 50) amino acid residues long. A
polypeptide immunogen can include one or more epitopes, for
example, 1, 2, 4, 6, 10, 20, 30, 50 or more.
[0075] Polypeptide immunogens can include one or more
post-transcriptional modifications as described above in the
Ligands section. An immunogen can be, without limitation,
biotinylated, glycosylated, acetylated, alkylated, isoprenylated,
lipoylated, or phosphorylated.
[0076] An immunogen can also be a molecule that is not a protein,
e.g., a carbohydrate or glycolipid. Examples of carbohydrate and
glycolipid immunogens, include, without limitation, bacterial
lipopolysaccharide (LPS) or a fragment of it, microbial products
bound by various Toll-like receptors (TLRs), bacterial diacyl and
triacyl lopopeptides and lipoteichoic acid from bacteria, and
zymosan from yeast cell walls.
[0077] The immunogen can be present in a killed or attenuated
organism, in a crude cellular extract, a cell lysate or partially
or substantially pure. The term "substantially pure" with respect
to a naturally-occurring immunogen refers to an immunogen that has
been separated from cellular components by which it is naturally
accompanied, such that it is at least 60% (e.g., 70%, 80%, 90%,
95%, or 99%), by weight, free from naturally-occurring organic
molecules with which it is naturally associated. Methods for
purifying immunogens are known to those in the art. For example, in
general, a substantially pure polypeptide will yield a single major
band on a non-reducing polyacrylamide gel.
[0078] The immunogen can be a molecule expressed or released by any
of a wide range of infectious agents, including, without
limitation, viruses, viroids, bacteria, fungi, prions or
parasites.
[0079] For example, viral pathogens can include, without
limitation, influenza viruses, including the strain A(H1N5),
hepatitis viruses (e.g, Hepatitis A, B, C and D), Arenaviruses,
Bunyaviruses, Flaviviruses, Filoviruses, Alphaviruses, (e.g.,
Venezuelan equine encephalitis, eastern equine encephalitis,
western equine encephalitis), Hantaviruses, human immunodeficiency
viruses HIV1 and HIV2, feline immunodeficiency virus, simian
immunodeficiency virus, measles virus, rabies virus, rotaviruses,
papilloma virus, respiratory syncytial virus, Variola, and viral
encephalitides, (e.g., West Nile Virus, LaCrosse, Calif.
encephalitis, VEE, EEE, WEE, Japanese Encephalitis Virus, Kyasanur
Forest Virus). Bacterial pathogens can include, but are not limited
to, Bacillus anthracis, Yersinia pestis, Yersinia enterocolitica,
Clostridium botulinum, Clostridium perfringens Francisella
tularensis, Brucella species, Salmonella spp., including Salmonella
enteriditis, Escherichia coli, including E. coli O157:H7,
Streptococcus pneumoniae, Staphylococcus aureus, Burkholderia
mallei, Burkholderia pseudomallei, Chlamydia spp., Coxiella
burnetii, Rickettsia prowazekii, Vibrio spp., Shigella spp.
Listeria monocytogenes, Mycobacteria tuberculosis, M. leprae,
Borrelia burgdorferi, Actinobacillus pleuropneumoniae, Helicobacter
pylori, Neisseria meningitidis, Bordetella pertussis, Porphyromonas
gingivalis, and Campylobacter jejuni.
[0080] Fungal pathogens can include, without limitation, members of
the genera Aspergilllus, Penecillium, Stachybotrys, Trichoderma,
mycoplasma, Histoplasma capsulatum, Cryptococcus neoformans,
Chlamydia trachomatis, and Candida albicans. Pathogenic protozoa
can include, for example, members of the genera Cryptosporidium,
e.g., Cryptosporidium parvum, Giardia lamblia, Microsporidia and
Toxoplasma, e.g., Toxoplasma brucei, Toxoplasma gondii, Entamoeba
histolytica, Plasmodium falciparum, Leishmania major and Cyclospora
cayatanensis.
[0081] Examples of useful immunogens derived from pathogenic
organisms include, for example, but are not limited to, influenza A
M2 protein, hepatitis B surface antigen, HBV preS1 protein, HIV
tat, HIV gp120, anthrax protective antigen, and botulinum toxin. An
influenza M2 protein antigen can be the ectodomain peptide M2e, for
example, SLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO:1), or a variant of the
ectodomain peptide M2e, for example, SEQ ID NO: 3 or SEQ ID 4. An
HBV preS1 protein can include the preS1 protein peptide 35-49,
e.g., FGANSNNPDWDFNPNKDHWPEANQVGA (SEQ ID NO:2). Examples of useful
non-peptidic immunogens include the pneumococcal polysaccharides
from Streptococcus pneumoniae.
[0082] The immunogen can also be a molecule expressed by a mammal.
For example, an immunogen can be a molecule whose expression is
correlated with a particular disease state, for example, cancer or
neurodegenerative disease.
[0083] Thus, the immunogen can be a tumor-associated antigen (TAA).
As used herein, a TAA is a molecule (e.g., a polypeptide,
carbohydrate or lipid) that is expressed by a tumor cell and either
(a) differs qualitatively from its counterpart expressed in normal
cells, or (b) is expressed at a higher level in tumor cells than in
normal cells. Thus, a TAA can differ (e.g., by one or more amino
acid residues where the molecule is a protein) from, or it can be
identical to, its counterpart expressed in normal cells. Preferably
it is not expressed by normal cells. Alternatively, it is expressed
at a level at least two-fold higher (e.g., a two-fold, three-fold,
five-fold, ten-fold, 20-fold, 40-fold, 100-fold, 500-fold,
1,000-fold, 5,000-fold, or 15,000-fold higher) in a tumor cell than
in the tumor cell's normal counterpart. Examples of relevant
cancers include, without limitation, hematological cancers such as
leukemias and lymphomas, neurological tumors such as astrocytomas
or glioblastomas, melanoma, breast cancer, lung cancer, head and
neck cancer, gastrointestinal tumors such as gastric or colon
cancer, liver cancer, pancreatic cancer, genitourinary tumors such
ovarian cancer, vaginal cancer, bladder cancer, testicular cancer,
prostate cancer or penile cancer, bone tumors, and vascular tumors.
Relevant TAAs include, without limitation, carcinoembryonic antigen
(CEA), RAGE, MART (melanoma antigen), MAGE (melanoma antigen) 1-4,
6 and 12, MUC (mucin) (e.g., MUC-1, MUC-2, etc.), tyrosinase, Pmel
17 (gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase
V intron V sequence), Prostate cancer psm, PRAME (melanoma
antigen), .beta.-catenin, MUM-1-B (melanoma ubiquitous mutated gene
product), GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10,
c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virus nuclear antigen) 1-6,
gp75, human papilloma virus (HPV) E6 and E7, p53, lung resistance
protein (LRP) Bcl-2, prostate specific antigen (PSA), and
Ki-67.
[0084] Other immunogens that can be included in the immune
conjugates disclosed herein are those derived from antigens that
are involved in the initiation or progression of neurodegenerative
diseases, e.g. Alzheimer's disease and Transmissible Spongiform
Encephalopathies (TSEs), e.g., human prion diseases such as
Creutzfeld-Jacob disease (CJD), variant CJD ("mad cow disease`),
Gerstmann-Straussler-Scheinker syndrome (GSS); Fatal familial
Insomnia (FFI); animal prion diseases such as Scrapie in sheep;
bovine spongiform encephalopathy (BSE) in cows; transmissible mink
encephalopathy (TME) in mink; chronic wasting disease (CWD) in elk
and deer.
[0085] As used herein, a "neurodegenerative antigen" is a molecule
(e.g., a polypeptide, carbohydrate or lipid) that is expressed by a
neuronal cell in an individual with a neurodegenerative disease and
either (a) differs qualitatively from its counterpart expressed in
cells from an individual who does not have the neurodegenerative
disease, e.g., the molecule appears in abnormal locations within
the body or is associated with other molecules not normally found
with the antigen in healthy individuals who do not have the
neurodegenerative disease, or (b) is expressed at a higher level in
cells from an individual who does not have the neurodegenerative
disease. Thus, a neurodegenerative antigen can differ (e.g., by one
or more amino acid residues where the molecule is a protein) from,
or it can be identical to, its counterpart expressed in normal
cells. It is preferably not expressed by normal cells.
Alternatively, it is expressed at a level at least two-fold higher
(e.g., a two-fold, three-fold, five-fold, ten-fold, 20-fold,
40-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, or 15,000-fold
higher) in a tumor cell than in the tumor cell's normal
counterpart.
[0086] Examples of neurodegenerative antigens found in Alzheimer's
disease include beta-amyloid, tau protein, alpha synuclein. Other
neurodegenerative disease antigens can be derived from prions. As
defined herein, a prion is small proteinaceous infectious particle
that resists inactivation by procedures that modify nucleic acids.
Prions are encoded by the prion-related protein gene (PrP). Mutant
forms of the PrP protein aggregate as prions which can lead to
fatal neurodegenerative disease. Thus, an immunogen can be a PrP
polypeptide.
[0087] Germ cell immunogens can be useful in the generation of
immune responses that block the function of germ cells, thereby
interfering with conception. Germ cell antigens can include
antigens on sperm cells. Examples include, without limitation sperm
adhesion molecule 1 (SPAM-1), and human intra-acrosomal protein
[0088] An immunogen can also be a non-toxic variant of a toxic
substance (a "toxoid") that can be used to stimulate an immune
response against the harmful form of the toxin. A toxoid can be,
without limitation, a toxin that has been rendered less toxic or
completely non-toxic through treatment with high temperature,
aggregation, chemical reaction (e.g., formalin fixation), coupling
to a carrier molecule, or molecular alteration (e.g., deletion,
augmentation or substitution). A toxoid can be thus derived from a
toxin such as, for example, ricin, anthrax or botulinum toxin types
A, B, C, D, E, F or G.
[0089] An immunogen can also be a substance of abuse such as
nicotine, or an opiate or opiate derivative. Such an immunogen can
induce antibodies capable of binding and neutralizing the
corresponding substance of abuse.
Forms of Immune Conjugates
[0090] The ligand and the immunogen are connected by a linker. A
linker can be any reagent, molecule or macromolecule that connects
the ligand and the immunogen such that a) the immune complex is
stable under physiological conditions; b) the connection between
the linker and the ligand does not alter the ability of the ligand
to bind to its target on the surface of a circulating non-lymphoid
cell; and c) the connection between the linker and the immunogen
does not substantially affect the capacity of the immunogen to
induce an effective immune response in a host against an infectious
agent, cell or molecule on which the immunogen is naturally
found.
Fusion proteins. A linker can be a peptide bond. That is, the
ligand and the immunogen can be a fusion polypeptide comprising one
or more amino acid segments from the ligand and one or more amino
acid segments from the immunogen. The term "amino acid segment" as
used herein refers to a contiguous stretch of amino acids within a
polypeptide. For example, the amino acid residues 30 to 40 within a
100 amino acid polypeptide would be considered an amino acid
segment. An amino acid segment can be a length greater than eight
amino acid residues (e.g., greater than about nine, ten, 15, 20,
25, 30, 40, 50, 75, 100, 150, 200, 500, 1000, or more amino acid
residues). In some embodiments, an amino acid segment can have a
length less than 1000 amino acid residues (e.g., less than 500,
less than 400, less than 350, less than 300, less than 200, or less
than 100 amino acid residues). In other embodiments, an amino acid
segment can have a length from about 20 to about 200 amino acid
residues (e.g., about 30 to about 180 amino acid residues, or about
40 to about 150 amino acid residues).
[0091] The amino acid segments of the ligand can be contiguous with
the amino acid segments of the immunogen or they can be separated
by amino acids inserted as a structural spacer. A spacer segment
can be one or more amino acids. The one or more amino acids can
include amino acids that are the same or that are different. For
example, a spacer can be a repeating series of a neutral amino acid
(e.g., glycine, alanine, valine, isoleucine or leucine) ranging in
number from 1 to 10 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more). Another example of a spacer configuration can be a series of
interspersed amino acids that may be neutral (e.g.,
glycine-alanine-glycine-alanine-glycine-alanine, or
glycine-glycine-glycine-valine-valine-valine) or charged amino
acids (e.g.,
glutamate-glutamate-glutamate-arginine-arginine-arginine, or
aspartate-lysine-aspartate-lysine-aspartate-lysine) or amino acids
with other functional groups (e.g.,
proline-proline-proline-serine-serine-serine or
tyrosine-glutamine-cysteine-methionine-tryptophan) ranging in
number from 1 to 10 or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more). In another embodiment, a spacer configuration can be a
sequence of amino acids derived from a naturally occurring protein
such as the hinge region joining the heavy chain CH1 and CH2
domains of immunoglobulin G.
[0092] A fusion protein can be produced in vitro by continuous
peptide synthesis according to standard chemical methods know to
those in the art. Synthetic polypeptides can also be purchased from
commercial sources.
[0093] A fusion protein can also be produced by recombinant DNA
techniques. Nucleic acid segments encoding the ligand can be
operably linked in the same open reading frame to nucleic acid
sequences encoding the immunogen in a vector that includes the
requisite regulatory elements, e.g., promoter sequences,
transcription initiation sequences, and enhancer sequences, for
expression in prokaryotic or eukaryotic cells. Methods well known
to those skilled in the art can be used to construct expression
vectors containing relevant coding sequences and appropriate
transcriptional/translational control signals. Alternatively,
suitable vector systems can be purchased from commercial
sources.
[0094] Nucleic acid segments encoding ligands and immunogens are
readily available in the public domain. Examples of nucleic acid
segments encoding ligands include, without limitation, the
erythrocyte (glycophorin A)-binding antigen of Plasmodium
falciparum EBA-175 (Bharara et al., Mol. Biochem. Parasitol. 138:
123-9, 2004), or mouse anti-human glycophorin A monoclonal antibody
heavy chain (GenBank accession # AAZ67132) and corresponding light
chain (Genbank accession # AAA21366)). Examples of nucleic acid
segments encoding immunogens include, without limitation, Hepatitis
virus C polyprotein (Genbank public gi number 2654998); influenza
virus A conserved M2 protein ectodomain peptide M2e (Genbank public
gi number gi 78210829; HBV preS1 protein (Genbank public gi number
gi 92111469).
[0095] The terms "nucleic acid" and "polynucleotide" are used
interchangeably herein, and refer to both RNA and DNA, including
cDNA, genomic DNA, synthetic DNA, and DNA (or RNA) containing
nucleic acid analogs. Polynucleotides can have any
three-dimensional structure. A nucleic acid can be double-stranded
or single-stranded (i.e., a sense strand or an antisense strand).
Non-limiting examples of polynucleotides include genes, gene
fragments, exons, introns, messenger RNA (mRNA), transfer RNA,
ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant
polynucleotides, branched polynucleotides, plasmids, vectors,
isolated DNA of any sequence, isolated RNA of any sequence, nucleic
acid probes, and primers, as well as nucleic acid analogs. The
nucleic acid molecules can be synthesized (for example, by
phosphoramidite based synthesis) or obtained from a biological
cell, such as the cell of a mammal. The nucleic acids can be those
of mammal, e.g., humans, a non-human primates, cattle, horses,
pigs, sheep, goats, deer, elk, dogs, cats, mustelids, rabbits,
guinea pigs, hamsters, rats, or mice.
[0096] An "isolated" nucleic acid can be, for example, a
naturally-occurring DNA molecule, provided one of the nucleic acid
sequences normally found immediately flanking that DNA molecule in
a naturally-occurring genome is removed or absent. Thus, an
isolated nucleic acid includes, without limitation, a DNA molecule
that exists as a separate molecule, independent of other sequences
(e.g., a chemically synthesized nucleic acid, or a cDNA or genomic
DNA fragment produced by the polymerase chain reaction (PCR) or
restriction endonuclease treatment). An isolated nucleic acid also
refers to a DNA molecule that is incorporated into a vector, an
autonomously replicating plasmid, a virus, or into the genomic DNA
of a prokaryote or eukaryote. In addition, an isolated nucleic acid
can include an engineered nucleic acid such as a DNA molecule that
is part of a hybrid or fusion nucleic acid. A nucleic acid existing
among hundreds to millions of other nucleic acids within, for
example, cDNA libraries or genomic libraries, or gel slices
containing a genomic DNA restriction digest, is not to be
considered an isolated nucleic acid.
[0097] Isolated nucleic acid molecules can be produced by standard
techniques. For example, polymerase chain reaction (PCR) techniques
can be used to obtain an isolated nucleic acid containing a
nucleotide sequence described herein. PCR can be used to amplify
specific sequences from DNA as well as RNA, including sequences
from total genomic DNA or total cellular RNA. Various PCR methods
are described, for example, in PCR Primer: A Laboratory Manual,
Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory
Press, 1995. Generally, sequence information from the ends of the
region of interest or beyond is employed to design oligonucleotide
primers that are identical or similar in sequence to opposite
strands of the template to be amplified. Various PCR strategies
also are available by which site-specific nucleotide sequence
modifications can be introduced into a template nucleic acid.
Isolated nucleic acids also can be chemically synthesized, either
as a single nucleic acid molecule (e.g., using automated DNA
synthesis in the 3' to 5' direction using phosphoramidite
technology) or as a series of oligonucleotides. For example, one or
more pairs of long oligonucleotides (e.g., >100 nucleotides) can
be synthesized that contain the desired sequence, with each pair
containing a short segment of complementarity (e.g., about 15
nucleotides) such that a duplex is formed when the oligonucleotide
pair is annealed. DNA polymerase is used to extend the
oligonucleotides, resulting in a single, double-stranded nucleic
acid molecule per oligonucleotide pair, which then can be ligated
into a vector. Isolated nucleic acids disclosed herein also can be
obtained by mutagenesis of, e.g., a naturally occurring DNA.
[0098] As used herein, the term "percent sequence identity" refers
to the degree of identity between any given query sequence and a
subject sequence. A subject sequence typically has a length that is
more than 80 percent, e.g., more than 82, 85, 87, 89, 90, 93, 95,
97, 99, 100, 105, 110, 115, or 120 percent, of the length of the
query sequence. A query nucleic acid or amino acid sequence can be
aligned to one or more subject nucleic acid or amino acid sequences
using the computer program ClustalW (version 1.83, default
parameters), which allows alignments of nucleic acid or protein
sequences to be carried out across their entire length (global
alignment). Chema et al., Nucleic Acids Res., 31(13):3497-500
(2003). ClustalW can be run, for example, at the Baylor College of
Medicine Search Launcher site
(searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at
the European Bioinformatics Institute site on the World Wide Web
(ebi.ac.uk/clustalw).
[0099] The term "exogenous" with respect to a nucleic acid
indicates that the nucleic acid is part of a recombinant nucleic
acid construct, or is not in its natural environment. For example,
an exogenous nucleic acid can be a sequence from one species
introduced into another species, i.e., a heterologous nucleic acid.
Typically, such an exogenous nucleic acid is introduced into the
other species via a recombinant nucleic acid construct. An
exogenous nucleic acid can also be a sequence that is native to an
organism and that has been reintroduced into cells of that
organism. An exogenous nucleic acid that includes a native sequence
can often be distinguished from the naturally occurring sequence by
the presence of non-natural sequences linked to the exogenous
nucleic acid, e.g., non-native regulatory sequences flanking a
native sequence in a recombinant nucleic acid construct. In
addition, stably transformed exogenous nucleic acids typically are
integrated at positions other than the position where the native
sequence is found.
[0100] It will be appreciated that a number of nucleic acids can
encode a polypeptide having a particular amino acid sequence. The
degeneracy of the genetic code is well known to the art; i.e., for
many amino acids, there is more than one nucleotide triplet that
serves as the codon for the amino acid.
[0101] A "vector" is a replicon, such as a plasmid, phage, or
cosmid, into which another DNA segment may be inserted so as to
bring about the replication of the inserted segment. Generally, a
vector is capable of replication when associated with the proper
control elements. Suitable vector backbones include, for example,
those routinely used in the art such as plasmids, viruses,
artificial chromosomes, BACs, YACs, or PACs. The term "vector"
includes cloning and expression vectors, as well as viral vectors
and integrating vectors. An "expression vector" is a vector that
includes a regulatory region. Suitable expression vectors include,
without limitation, plasmids and viral vectors derived from, for
example, bacteriophage, baculoviruses, and retroviruses. Numerous
vectors and expression systems are commercially available from such
corporations as Novagen (Madison, Wis.), Clontech (Palo Alto,
Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life
Technologies (Carlsbad, Calif.).
[0102] Vectors typically contain one or more regulatory regions.
The term "regulatory region" refers to nucleotide sequences that
influence transcription or translation initiation and rate, and
stability and/or mobility of a transcription or translation
product. Regulatory regions include, without limitation, promoter
sequences, enhancer sequences, response elements, protein
recognition sites, inducible elements, protein binding sequences,
5' and 3' untranslated regions (UTRs), transcriptional start sites,
termination sequences, polyadenylation sequences, and introns.
[0103] As used herein, the term "operably linked" refers to
positioning of a regulatory region and a sequence to be transcribed
in a nucleic acid so as to influence transcription or translation
of such a sequence. For example, to bring a coding sequence under
the control of a promoter, the translation initiation site of the
translational reading frame of the polypeptide is typically
positioned between one and about fifty nucleotides downstream of
the promoter. A promoter can, however, be positioned as much as
about 5,000 nucleotides upstream of the translation initiation
site, or about 2,000 nucleotides upstream of the transcription
start site. A promoter typically comprises at least a core (basal)
promoter. A promoter also may include at least one control element,
such as an enhancer sequence, an upstream element or an upstream
activation region (UAR). The choice of promoters to be included
depends upon several factors, including, but not limited to,
efficiency, selectability, inducibility, desired expression level,
and cell- or tissue-preferential expression. It is a routine matter
for one of skill in the art to modulate the expression of a coding
sequence by appropriately selecting and positioning promoters and
other regulatory regions relative to the coding sequence.
[0104] The vectors also can include, for example, origins of
replication, scaffold attachment regions (SARs), and/or markers. A
marker gene can confer a selectable phenotype, e.g., antibiotic
resistance, on a cell. In addition, an expression vector can
include a tag sequence designed to facilitate manipulation or
detection (e.g., purification or localization) of the expressed
polypeptide. Tag sequences, such as green fluorescent protein
(GFP), glutathione S-transferase (GST), polyhistidine, c-myc,
hemagglutinin, or Flag.TM. tag (Kodak, New Haven, Conn.) sequences
typically are expressed as a fusion with the encoded polypeptide.
Such tags can be inserted anywhere within the polypeptide,
including at either the carboxyl or amino terminus.
[0105] The expression vectors disclosed herein containing the above
described coding can be used, for example, to transfect or
transduce either prokaryotic (e.g., bacteria) cells or eukaryotic
cells (e.g., yeast, insect, or mammalian) cells. Such cells can
then be used, for example, for large or small scale in vitro
production of the relevant fusion protein by methods known in the
art. In essence, such methods involve culturing the cells under
conditions which maximize production of the fusion protein and
isolating the fusion protein from the cells or from the culture
medium.
Conjugates. In another embodiment, the ligand and immunogen can be
obtained separately, either through chemical synthesis or synthesis
in vivo, purified and then linked non-covalently or covalently. A
useful non-covalent linkage is a biotin-avidin linkage. The binding
of biotin to avidin or streptavidin is essentially irreversible,
with a reported Kd of 10.sup.-15M. The term "biotin-avidin linkage"
as used herein refers to any linkage via biotin or a biotin
derivative or biomimic (e.g., Strep-Tag (IBA, St. Louis, Mo.)) and
avidin or an avidin derivative, streptavidin, or biotin-binding
fragments or subunits of avidin or streptavidin.
[0106] Thus, the biotinylated ligand can be linked to a
biotinylated immunogen via avidin or streptavidin, or
biotin-binding fragments or subunits of avidin or streptavidin.
Methods for forming biotin-avidin linkages are well known to those
in the art. (See for example, Handbook of Affinity Chromatography,
(Chromatographic Sciences Series, vol. 63) ed. T. Kline, ISBN:
0824789393--Marcel Dekker (1993). Avidin and avidin derivatives are
available from commercial sources (Pierce Biotechnology, Rockford,
Ill.; Invitrogen, Carlsbad, Calif.).
[0107] The ligand and the immunogen can also be linked through a
biotin-streptavidin linkage that includes an additional
biotinylated immunoglobulin. For example, a biotinylated ligand can
be linked to an avidin molecule that is bound to a biotinylated
antibody that specifically binds the immunogen.
[0108] Avidin and streptavidin have four biotin-binding sites each.
By varying the relative ratios of the ligand and the immunogens
used in the assembly of the targeted immune conjugates, it is
possible to generate targeted immune conjugates with various ratios
of linker: immunogen. Thus, biotin-avidin heterocomplexes can be
prepared to include 1 molecule of the ligand and 3 molecules of the
immunogen; 2 molecules each of ligand and immunogen; or 3 molecules
of ligand and 1 molecule of immunogen. Assembly of the
biotin-avidin linkages can be performed in any order. The
composition of the assembled immune conjugates can be validated by
SDS-PAGE and western blotting and LC/MS methods.
[0109] Alternative docking pairs of molecules with ultra-high
affinity (10.sup.-10M or more) may be used in place of
biotin-avidin. An example of such a pair is vitamin B12
(cyanocobalamin), which is bound by vitamin B12-binding protein
with a Kd of 10.sup.-10M).
[0110] The ligand and the immunogen can also be synthesized as
separate entities (by either chemical synthetic or recombinant
methods) and then linked together by standard chemical methods
known in the art. Chemical cross-linking agents can be
homo-bifunctional (the same chemical reaction takes place at each
end of the linker) or hetero-bifunctional (different chemical
reactions take place at the ends of the linker). The chemistries
available for such linking reactions include, but are not limited
to, reactivity with sulfhydryl, amino, carboxyl, diol, aldehyde,
ketone, or other reactive groups using electrophilic or
nucleophilic chemistries, as well as photochemical cross-linkers
using alkyl or aromatic azido or carbonyl radicals. An example of a
targeted conjugate coupled via a homobifunctional cross-linking
reagent can be a complex of an anti-band 3 monoclonal antibody as
the red blood cell-targeting component and anthrax protective
antigen as the immunogen linked by disuccinimidyl suberate (DSS,
Pierce, Rockford, Ill.). An example of a targeted conjugate coupled
via a heterobifunctional cross-linking reagent can be an
anti-glycophorin A monoclonal antibody as the targeting component
and a non-toxic fragment of botulinum neurotoxin A as the immunogen
linked by N-succinimidyl 3-[2-pyridyldithio]-propionamido (SPDP).
In this example, the antibody is first derivatized at sulfhydryl
groups with SPDP's pyridyldithio reactivity, followed by the
addition of the toxin, whose amino residues react with SPDP's
succinimidyl groups.
[0111] Examples of chemical cross-linking agents include, without
limitation, glutaraldehyde, carbodiimides, bisdiazobenzidine, and
N-maleimidobenzoyl-N-hydroxysuccinimide ester. Chemical
cross-linkers are widely available from commercial sources (e.g.,
Pierce Biotechnology (Rockford, Ill.); Invitrogen (Carlsbad,
Calif.); Sigma-Aldrich (St. Louis, Mo.); and US Biological
(Swampscott, Mass.)).
[0112] In another embodiment, the ligand and the immunogen can be
connected through a linking polymer. Examples of linking molecules
include, but are not limited to linear or branched polymers or
co-polymers (e.g., polyalkylene, poly(ethylene-lysine),
polymethacrylate, polyamino acids, poly- or oligosaccharides,
dendrimers). The ligand and the immunogen can be attached to the
linking molecule or microparticle through a non-covalent high
affinity linkage, e.g., streptavidin-biotin high affinity binding
or chemical cross-linking techniques as described above.
[0113] For example, a polymer-supported targeted immunogen
conjugate can be formed using a poly(ethylene-lysine) backbone.
Such a linear copolymer backbone can be synthesized using
bis(succinimidyl) poly(ethylene glycol.sub.2000) (Fluka Chemicals)
to react with the .alpha. and .epsilon. amino groups of lysine. The
available carboxyl termini of the lysines can be activated using
(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride) (EDC,
Pierce Biotechnology) in preparation for coupling amine-containing
compounds. The total length of the co-polymer can be determined, in
part, by the duration of the polymerization reaction. The targeting
and IMG units can be combined in various ratios since there are up
to 10 positions in a 2000 dalton co-polymer built with, e.g., PEG
units of 2,000 dalton. Co-polymers using shorter PEG units (e.g.,
500 daltons) can also be synthesized.
[0114] One example of a polymer-based targeted immunogen conjugate
can be the addition of equimolar amounts of a targeting monoclonal
antibody (e.g., anti-CR2) and immunogen (e.g., influenza virus
peptide M2e) to produce a complex with a co-polymeric scaffold
studded with targeting antibody molecules as well as immunogen
molecules.
[0115] In another embodiment, the ligand and the immunogen can be
connected through a microparticle. Examples of linking
microparticles include, but are not limited to, micelles,
liposomes, fullerenes, nanotubes, or other colloidal complexes such
as lipoproteins. Liposomes and micelles can be prepared by methods
described in Lasic DD, 1998, TIBTech 16:307. Fullerenes and
nanotubes can be purchased from American Dye Source
(www.adsdyes.com). Lipoproteins can be purchased from Biodesign
International (www.biodesign.com).
[0116] The ligand and the immunogen can be attached to the linking
molecule or microparticle through a non-covalent high affinity
linkage, e.g., avidin-biotin high affinity binding or chemical
cross-linking techniques as described above.
[0117] Alternatively or in addition, the ligand and/or the
immunogen can be adsorbed or incorporated into a hydrophobic
microparticle by hydrophobic affinity. A ligand and/or and
immunogen with an available hydrophobic domain can spontaneously
associate with a hydrophobic microparticle by hydrophobic
partitioning. The hydrophobic domain on the ligand and/or immunogen
can be a polyamino acid stretch comprised of repeating or mixed
hydrophobic amino acids (e.g., poly-Ala, poly-Gly, poly-Leu,
poly-Ile, or Ala-Gly-Leu-Ile (SEQ ID NO:5), etc.) or a
bilayer-spanning polypeptide from a known trans-membrane protein,
such as membrane IgM), alkyl chains (e.g., fatty acyl), or other
hydrophobic structure (e.g., steroid). Such hydrophobic sequences
can be naturally occurring sequences within the ligand and/or
immunogen. Alternatively, such sequences can be introduced into the
native amino acid sequence of the ligand or immunogen by standard
recombinant DNA technology. The recombinant protein can be
expressed and purified as described above.
[0118] The immune conjugates disclosed herein can include one or
more of the same ligands or any combination of different ligands.
The immune conjugate can also include one or more of the same
immunogens or any combination of different immunogens. Thus, the
immune conjugates can include immunogens that contain multiple
copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10, 30, or more) of a
single antigen or a single copy of multiple (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, 10, 30, or more) antigens or multiple copies
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10, 30, or more) of two or
more antigens (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 10, 30, or
more). The immunogen can contain one or more copies of one or more
peptide epitopes together with one or more copies of any of the
non-peptide epitopes, e.g., post-transcriptional modifications,
carbohydrates, or lipopolysaccharides.
[0119] Further, a targeted immune conjugate can include an
immunogen that includes more than one polypeptide, or any
combination of different polypeptides. It is noted that each
polypeptide in a composition can have an identical amino acid
sequence. In addition, the polypeptides in a composition can
contain different amino acid segments, each of which can act as a
defined immunogenic unit against which an immune response is
desired. Thus, the polypeptides in a composition can contain
different amino acid segments that correspond to any region from a
polypeptide including, without limitation, receptor binding
regions, ligand binding regions, enzyme active sites, enzyme
cleavage sites of polypeptide substrates, antigen-binding regions
of antibodies, and epitopes recognized by antibodies. Typically,
the administration of a polypeptide results in the formation of
antibodies having specificity for an epitope or combination of
epitopes formed by the amino acid segments within one or more of
the polypeptides in the composition.
III. Methods of Use
[0120] The immune conjugates disclosed herein are generally useful
for generating immune responses and as prophylactic vaccines or
immune response-stimulating therapeutics. As used herein,
"prophylaxis" can mean complete prevention of the symptoms of a
disease, a delay in onset of the symptoms of a disease, or a
lessening in the severity of subsequently developed disease
symptoms. As used herein, "therapy" can mean a complete abolishment
of the symptoms of a disease or a decrease in the severity of the
symptoms of the disease.
[0121] The methods disclosed herein can be applied to a wide range
of species, e.g., humans, non-human primates (e.g., monkeys),
horses, cattle, pigs, sheep, deer, elk, goats, dogs, cats,
mustelids, rabbits, guinea pigs, hamsters, rats, and mice. Thus,
they can be used, for example, as vaccines or therapeutic agents
against infectious diseases, including diseases that can
potentially result from bioterrorism attacks. The immune conjugates
can be used in the preparation of a medicament for treatment of an
infectious disease. Infectious diseases can include diseases caused
by any of the pathogens listed herein. Examples include, without
limitation, influenza, HIV-AIDS, hepatitis, botulism, plague,
smallpox, tularemia, viral hemorrhagic fevers, brucellosis,
gastrointestinal disease induced by pathogenic forms of E. coli,
Salmonella and Shigella, glanders, melioidosis, psittacosis, Q
fever, Staph infection, typhus fever, viral encephalitis, water and
foodborne safety threats, cholera, diphtheria, endocarditis,
Legionaire's disease, Listeriosis, periodontal disease,
Asperigillosis, Blastomycosis, histoplasmosius, trypanosomiasis,
malaria, Giardiasis, Schistosomiasis, toxoplasmosis, smallpox, west
Nile virus.
[0122] In addition, the immune conjugates can be useful as both
prophylactics and therapeutics for cancer (e.g., any of those
recited above). The immune conjugates can be employed to stimulate
an immune response against cells in a cancer patient or can be
administered in cases where a subject is at relatively high risk
for a cancer (e.g., lung cancer in a tobacco smoker or melanoma in
a subject with multiple nevi). Moreover, as described above, the
immune conjugates can also be useful in therapy or prophylaxis of
neurodegenerative diseases. Thus the immune conjugates can be
administered to an individual with Alzheimer's disease or TSE or
administered to an individual who is at risk for developing
Alzheimer's disease or TSE.
[0123] Immune conjugates disclosed herein can also be useful as a
contraceptive vaccine, when the immunogen is a germ cell
antigen.
[0124] The immune conjugates disclosed herein can also be useful as
prophylactics and therapeutics against medical conditions that
result from exposure to toxins. Such targeted immune conjugates
that include non-toxic variants of toxic substances, e.g., ricin,
botulinum toxin, nicotine and other drugs, can be used to stimulate
an immune response against the harmful form of the toxin, and thus
protect against or mitigate the potential damage the toxin or drug
may cause.
[0125] The immune conjugates can be administered directly to a
mammal. The immune conjugates can be used in the preparation of a
medicament. Generally, the immune conjugates can be suspended in a
pharmaceutically-acceptable carrier (e.g., physiological saline). A
composition can be made by combining any of the immune conjugates
provided herein with a pharmaceutically acceptable carrier. Such
carriers can include, without limitation, sterile aqueous or
non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents include mineral oil, propylene glycol,
polyethylene glycol, vegetable oils, and injectable organic esters,
for example. Aqueous carriers include, without limitation, water,
alcohol, saline, and buffered solutions. Preservatives, flavorings,
and other additives such as, for example, antimicrobials,
anti-oxidants, chelating agents, inert gases, and the like also may
be present. It will be appreciated that any material described
herein that is to be administered to a mammal can contain one or
more pharmaceutically acceptable carriers.
[0126] Any composition described herein can be administered to any
part of the host's body. A composition can be delivered to, without
limitation, the joints, nasal mucosa, blood, lungs, intestines,
muscle tissues, skin, or peritoneal cavity of a mammal. In
addition, a composition can be administered by intravenous,
intraperitoneal, intramuscular, subcutaneous, intramuscular,
intrarectal, intravaginal, intrathecal, intratracheal, intradermal,
or transdermal injection, by oral or nasal administration, by
inhalation, or by gradual perfusion over time. In a further
example, an aerosol preparation of a composition can be given to a
host by inhalation.
[0127] The dosage required depends on the route of administration,
the nature of the formulation, the nature of the patient's illness,
the subject's size, weight, surface area, age, and sex, other drugs
being administered, and the judgment of the attending physician.
Suitable dosages are in the range of 0.01-1,000 .mu.g/kg. Wide
variations in the needed dosage are to be expected in view of the
variety of immune conjugates available and the differing
efficiencies of various routes of administration. Variations in
these dosage levels can be adjusted using standard empirical
routines for optimization as is well understood in the art.
Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-,
8-, 10-, 20-, 50-, 100-, 150-, or more fold). Encapsulation of the
immune conjugate in a suitable delivery vehicle (e.g., polymeric
microparticles or implantable devices) may increase the efficiency
of delivery.
[0128] The duration of treatment with any composition provided
herein can be any length of time from as short as one day to as
long as the life span of the host (e.g., many years). For example,
an immune conjugate can be administered once a month for three
months or once a year for a period of ten years. It is also noted
that the frequency of treatment can be variable. For example, an
immune conjugate can be administered once (or twice, three times,
etc.) daily, weekly, monthly, or yearly.
[0129] Alternatively or in addition the immune conjugates can be
administered along with an adjuvant. An "adjuvant" is an
immunological compound that can enhance an immune response against
a particular antigen such as a polypeptide. Examples of adjuvants
include alum and other aluminum-based compounds (e.g.,
Al.sub.2O.sub.3). Aluminum-based compounds can be obtained from
various commercial suppliers. Other adjuvants include
immuno-stimulating complexes (ISCOMs) that can contain such
components as cholesterol and saponins; one or more additional
immunostimulatory components, including, without limitation,
muramyldipeptide (e.g., N-acetylmuramyl-L-alanyl-D-isoglutamine;
MDP), monophosphoryl-lipid A (MPL), and formyl-methionine
containing tripeptides such as N-formyl-Met-Leu-Phe. Such compounds
are commercially available from Sigma Chemical Co. (St. Louis,
Mo.), for example. Other adjuvants can include CpG
oligodeoxynucleotides (Coley Pharmaceuticals), QS21 (Cambridge
Biotech) and MF59 (Chiron).
[0130] The compositions provided herein can contain any ratio of
adjuvant to immune conjugate. The adjuvant:immune conjugate ratio
can be 50:50 (vol:vol), for example. Alternatively, the
adjuvant:immune conjugate ratio can be, without limitation, 90:10,
80:20, 70:30, 64:36, 60:40, 55:45, 40:60, 30:70, 20:80, or
90:10.
[0131] An effective amount of any composition provided herein can
be administered to a host. The term "effective" as used herein
refers to any amount that induces a desired immune response while
not inducing significant toxicity in the host. Such an amount can
be determined by assessing a host's immune response after
administration of a known amount of a particular composition. In
addition, the level of toxicity, if any, can be determined by
assessing a host's clinical symptoms before and after administering
a known amount of a particular composition. It is noted that the
effective amount of a particular composition administered to a host
can be adjusted according to a desired outcome as well as the
host's response and level of toxicity. Significant toxicity can
vary for each particular host and depends on multiple factors
including, without limitation, the host's disease state, age, and
tolerance to pain.
[0132] Any method can be used to determine if a particular immune
response is induced. For example, antibody responses against a
particular immunogen can be determined using an immunological assay
(e.g., ELISA or lymphocyte proliferation assay). In such an assay,
the wells of a microtiter plate can be coated with the immunogen
and incubated with serum from a mammal treated with the immune
conjugate designed to produce antibodies against the corresponding
immunogen in that mammal, and the presence or absence of antibodies
against the immunogen can be determined by standard methods know to
those in the art. In addition, clinical methods that can assess the
degree of a particular disease state can be used to determine if a
desired immune response is induced. For example, in a cancer
patient, a reduction in tumor burden can indicate a desired immune
response in a patient treated with a composition designed to
stimulate an immune response against a tumor antigen expressed on
the patient's tumor.
[0133] Alternatively, a polynucleotide containing a nucleic acid
sequence encoding an immune conjugate of interest can be delivered
to an appropriate cell of the animal. This can be achieved by, for
example, the use of a polymeric, biodegradable microparticle or
microcapsule delivery vehicle, sized to optimize phagocytosis by
phagocytic cells such as macrophages. For example, PLGA
(poly-lacto-co-glycolide) microparticles approximately 1-10 .mu.m
in diameter can be used. The polynucleotide is encapsulated in
these microparticles, which are taken up by macrophages and
gradually biodegraded within the cell, thereby releasing the
polynucleotide. Once released, the DNA is expressed within the
cell. A second type of microparticle is intended not to be taken up
directly by cells, but rather to serve primarily as a slow-release
reservoir of nucleic acid that is taken up by cells only upon
release from the micro-particle through biodegradation. These
polymeric particles should therefore be large enough to preclude
phagocytosis (i.e., larger than 5 .mu.m and preferably larger than
20 .mu.m).
[0134] Another way to achieve uptake of the nucleic acid is using
liposomes, prepared by standard methods. The vectors can be
incorporated alone into these delivery vehicles or co-incorporated
with tissue-specific antibodies. Alternatively, one can prepare a
molecular conjugate composed of a plasmid or other vector attached
to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine
binds to a ligand that can bind to a receptor on target cells.
Delivery of "naked DNA" (i.e., without a delivery vehicle) to an
intramuscular, intradermal, or subcutaneous site, is another means
to achieve in vivo expression.
[0135] In the relevant polynucleotides (e.g., expression vectors)
the nucleic acid sequence encoding the fusion protein of interest
with an initiator methionine and optionally a targeting sequence is
operatively linked to a promoter or enhancer-promoter combination.
Promoters and enhancers are described above.
[0136] Polynucleotides can be administered in a pharmaceutically
acceptable carrier. Pharmaceutically acceptable carriers are
biologically compatible vehicles which are suitable for
administration to a human or other mammalian subject, e.g.,
physiological saline. A therapeutically effective amount is an
amount of the polynucleotide which is capable of producing a
medically desirable result (e.g., a T cell response) in a treated
mammal. As is well known in the medical arts, the dosage for any
one patient depends upon many factors, including the patient's
size, body surface area, age, the particular compound to be
administered, sex, time and route of administration, general
health, and other drugs being administered concurrently. Dosages
will vary, but a preferred dosage for administration of
polynucleotide is from approximately 10.sup.6 to 10.sup.12 copies
of the polynucleotide molecule. This dose can be repeatedly
administered, as needed. Routes of administration can be any of
those listed above.
[0137] The immune conjugates provided herein can be administered in
conjunction with other therapeutic modalities to an individual in
need of therapy. The immune conjugates can be given prior to,
simultaneously with or after treatment with other agents. In the
case of infectious disease, the immune conjugates can be
administered in conjunction with any antimicrobial agent, e.g. an
antibiotic, e.g. including, without limitation, aminoglycosides,
cephalosporins, macrolides, penicillins, peptides, quinolones,
sulfonamides, tetracyclines; an antiviral, including without
limitation, amantadine, rimantadine, zanamavir and oseltamivir; an
anti-fungal, including, without limitation, echinocandin,
caspofungin, anidulafungin; or anti-parasitic agent, including,
without limitation, chlorquine, mebendazole, and clotrimazole.
[0138] The immune conjugates can also be used in conjuction with
standard anti-cancer therapies, including, without limitation,
chemotherapy, e.g., alkylating agents, anthracyclines,
cycloskeletal disruptors, topoisomerase inhibitors, nucleotide
analogues, platinum-based agents, retinoids, vinca alkaloids;
radiation therapy, hormone ablation and surgery. The immune
conjugates can also be used in conjuction with other therapueitcs
for neurodegenerative diseases, including donepezil, galantamine,
memantine.
[0139] In vitro application of the immune conjugates can be useful,
for example, in basic scientific studies of immune mechanisms or
for production of activated T cells for use in either studies on T
cell function or, for example, passive immunotherapy.
Articles of Manufacture
[0140] Also disclosed are articles of manufacture that can include
immune conjugates as provided herein. Components and methods for
producing articles of manufacture are well known. An article of
manufacture can include, for example, one or more immune
conjugates. In addition, an article of manufacture further may
include, for example, packaging materials, instructions for use,
buffers or other control reagents for treating or monitoring the
condition for which prophylaxis or treatment is required.
[0141] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
EXAMPLES
Example 1
Assembly of Tripartite Conjugates of the Biotinylated Monoclonal
Antibody Anti-TER-119, Streptavidin and the Biotinylated Peptide
M2e
[0142] Conjugates to deliver the immunogen M2e, to the mouse red
blood cell (mRBC), were prepared by coupling the peptide with the
rat anti-mouse monoclonal antibody anti-TER-119 and using
streptavidin (StAv) as the linker.
[0143] Preparation of Tripartite Immune Conjugates.
[0144] The peptide M2e (SLLTEVETPIRNEWGCRCNDSSD) (SEQ ID NO: 1) was
produced by peptide synthesis (BioWorld, Dublin Ohio) to contain a
biotinylated lysine residue at its carboxy terminus. Anti-TER-119,
a rat anti-mouse IgG2b,.kappa. monoclonal antibody, was purchased
biotinylated from BD-Pharmingen, as was the isotype control,
biotinylated rat IgG2b,.kappa.. The extent of biotinylation of
these products was not available from the manufacturer.
Streptavidin was purchased from Sigma-Aldrich.
[0145] Biotinylated M2e peptide (b-M2e) (SEQ ID NO: 1) was
incubated with StAv in phosphate-buffered saline, 10 mM sodium
phosphate, 140 mM NaCl, pH 7.4 (PBS) at ambient temperature at
various molar ratios. After 30 min incubation biotinylated
anti-TER-119 antibody (b-Ab) was added and the incubation was
continued for an additional 30 min with occasional agitation.
Reaction samples were analyzed by 3 to 8% gradient gel
electrophoresis under native (or non-denaturing) conditions (NDGE,
Invitrogen, tris-glycine running buffer) and visualized by
Coomassie blue staining. The results showed that mixtures of
conjugates of different apparent molecular weights and
stoichiometries were formed caused by differential cross-linking by
StAv between the biotinylated peptide and the Ab. The relative
amounts of reagents used in the reactions are shown in the grids
below the respective NDGE images.
[0146] Characterization of Tripartite Immune Conjugates.
[0147] In the experiment depicted in FIG. 1 the amounts of StAv and
b-Ab were kept constant but the amount of b-M2e was varied as
indicated. Pre-incubation of 100 or 200 pmol b-M2e with 20 pmol
StAv, which is well in excess of the 80 pmol required to saturate
the StAv biotin-binding sites, resulted in formation of
StAv(M2e).sub.4. This conjugate migrated faster than free StAv and
similarly to unreacted b-M2e (lanes 10-12 compared to lane 3).
[0148] Because the mobility of reagents in NDGE is not solely
determined by their molecular weight but also by their conformation
and charge, StAv migrated considerably faster than Ab, to a
location on the gel that was slightly above the peptide b-M2e.
Addition of b-M2e peptide to StAv, while increasing its MW, caused
a shift in the complex's migration downward, to the position of
unreacted b-M2e (lanes 3 and 6). That this was the effect of
binding of b-M2e to StAv was demonstrated by the reaction in lane
4, in which excess non-biotinylated peptide M2e did not alter the
mobility of StAv). When b-M2e+StAv mixtures containing an excess of
b-M2e were combined with b-Ab there was no shift in the migration
of the Ab (lanes 11 and 12, compared to lane 5).
[0149] The admixture of 50 pmol or less b-M2e, which was below the
theoretical 80 pmol needed to saturate 20 pmol StAv, did shift the
mobility of the added b-Ab upward indicating the presence of
Ab-StAv.sub.n, where n represents the number of biotin residues per
Ab molecule. In addition, the laddering pattern in lanes 7-10,
indicated the presence of possible higher order complexes such as
Ab-StAv-Ab, Ab-StAv2-Ab2, Ab-StAv-Ab-StAv-Ab, etc. Useful
tripartite complexes were those that did not include unreacted Ab;
these complexes had a mobility that was shifted upward from that of
the unconjugated antibody, but not to a degree that prevented the
complexes from entering the gel, e.g.,
in lanes 9-10, where the ratio of tetravalent StAv:b-M2e was
approximately 1:3.
[0150] Conditions for the formation of the Ab-StAv-M2e tripartite
immune conjugates were analyzed in the experiment shown in FIG. 2.
The concentration of b-Ab was held constant at 12.5 pmol, and
different amounts of StAv-b-M2e conjugates pre-formed at a 1:3
ratio were added. Complexes of various stoichiometries were seen in
all combinations, with the higher order complexes seen where StAv
was in excess over b-Ab (lanes 6-9). FIG. 2 also showed that in the
1:3 StAv: b-M2e mixtures there were some StAv molecules that had a
minimum of 2 free biotin-binding sites which were responsible for
cross-linking b-Ab molecules (lanes 5-9). This experiment also
indicated that the antibody molecules contained more than one
biotin residue. If b-Ab were mono-biotinylated, the reaction with
free StAv would lead to a preponderance of Ab tetramers, with
lesser amounts of trimers, dimmers and monomers. Lane 3 shows that
this is not the case. Instead there were more types of complexes
without apparent preponderance of tetramers.
[0151] One objective of these experiments was to define conditions
that produced tripartite conjugates with a maximum number of M2e
peptides per Ab molecule. The stoichiometric ratios of Ab:StAv:M2e
in the experiments described above are listed in Table 1.
TABLE-US-00001 TABLE 1 Molar ratios of the components in tripartite
conjugate reactions depicted in FIGS. 1 and 2 (normalized to the
concentration of Ab). FIG. 1 FIG. 2 lane 8 9 10 11 12 5 6 7 8 9 Ab
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 StAv 1.3 1.3 1.3 1.3 1.3
0.8 1.6 2.0 2.4 3.2 M2e 0.7 1.7 3.3 6.7 13.3 2.4 5.4 6.0 7.2
9.6
[0152] The conditions in which most of the Ab was shifted by the
reaction, but the complexes formed were not too large to diffuse
into the gel are found in FIG. 1, lanes 9 and 10 and FIG. 2, lanes
6, 7 and 8. The immune conjugates that included the highest
proportion of peptides per Ab (6 or 7.2) were those shown in lane 7
or 8 of FIG. 2.
Example 2
Analysis of Anti M2e Activity in Mice Challenged with the Immune
Conjugate Biotinylated Rat Anti-Mouse TER-119
Mab-Streptavidin-Biotinylated M2e Peptide
[0153] Mice are challenged intraperitoneally with an immune
conjugate of biotinylated rat anti-mouse TER-119
Mab-streptavidin-biotinylated M2e peptide ((SEQ ID NO: 1)) and
their serum antibody response to M2e (SEQ ID NO: 1) is assayed.
[0154] M2e peptide (SEQ ID NO: 1) with a C-terminal addition of
lysyl-.epsilon.-N-biotin or PGGG (SEQ ID NO:6) is synthesized by
solid phase peptide synthesis (Bio-World). This peptide is coupled
to streptavidin (StAv) (Sigma-Aldrich) at a peptide:StAv molar
ratio of 3:1 by mixing with b-Ab, as described in Example 1.
Identical tripartite conjugate is prepared using biotinylated rat
IgG (BD Pharmingen) as a control. To prepare TER-1,9-StAv and rat
IgG-StAv conjugates the StAv is first reacted at a biotin:StAv
molar ratio of 3:1 to occupy 3 of the 4 biotin-binding sites (BBSs)
on each StAv molecule. In groups receiving uncoupled StAv, it is
first reacted with a minimum of 4 molar equivalents of biotin to
saturate all the BBSs. M2e peptide conjugated to keyhole limpet
hemocyanin (KLH) is produced by Bio-World.
[0155] Groups of 5 mice each (Balb/C, female, 6-8 weeks old) are
injected intravenously (i.v.) in the tail vein with a range of free
and conjugated peptide doses (0.01 to 10 .mu.g equivalent of
peptide in 50 .mu.l PBS). Since StAv and TER-119 (a rat IgG) are
also immunogenic in mice, Ab titers against them alone or in
RBC-targeted conjugates are also evaluated. Groups 1-6 are the
minimum set to analyze the relative anti-M2e responses. Groups 7-15
include additional controls to further analyze the anti-StAv and
anti-rat IgG responses, as well as the effect of simply mixing the
various components, instead of coupling them into targeted
conjugates. The experimental design is depicted in the table
below:
TABLE-US-00002 TABLE 2 Experimental design: Example 2. Group number
Ligand Linker Immunogen 1 Biotinylated Anti- streptavidin
Biotinylated M2e TER119 2 Biotinylated Rat streptavidin
Biotinylated M2e IgG 3 -- streptavidin Biotinylated M2e 4 KLH
Chemical conjugate M2e 5 -- -- M2e 6 (PBS -- -- -- injected only) 7
Biotinylated Anti- streptavidin -- TER119 8 Biotinylated Rat
streptavidin -- IgG 9 Biotinylated Anti- streptavidin M2e TER119 10
Biotinylated Rat streptavidin M2e IgG 11 -- streptavidin M2e 12
Biotinylated Anti- -- M2e TER119 13 Biotinylated Rat -- M2e IgG 14
Biotinylated Anti- -- -- TER119 15 Biotinylated Rat -- -- IgG
[0156] The mice are boosted with a repeat i.v. injection after 2
and again after 4 weeks. Blood samples (.about.100 .mu.l) are
collected retro-orbitally prior to and every week after the primary
injection for 5 weeks. Blood samples are chilled on ice and allowed
to clot overnight. The tubes are centrifuged and serum is collected
and stored at -20.degree. C.
[0157] Mouse IgG titers against all 3 antigens (Ags), M2e, StAv and
rat IgG, are determined by ELISA. ELISA plates (Immulon, VWR) are
coated with 100 .mu.l Ag at 10 .mu.g/ml in sodium carbonate
(Na.sub.2CO.sub.3) buffer, 50 mM, pH 9.6 overnight at 4.degree. C.
Plates are washed and blocked with 200 .mu.l/well of 3% BSA in PBS
containing 0.05% Tween-20 (PBST) for 1.5 hours at room temperature
(RT). Wash 3.times. with 200 .mu.l/well of PBST. Dispense 100
.mu.l/well of individual mouse sera at 1:50 dilution in PBST in
duplicates and prepare serial dilutions in 1:3 X increments. Allow
mouse antisera to bind for 1.5 hours at RT and wash the plates as
above. Add 100 .mu.l/well goat anti-mouse IgG conjugated with
horseradish peroxidase (diluted per vendor instructions, Sigma),
incubate for 1 hour at RT, and wash as above. Add 100 .mu.l/well
substrate (SureBlue, KPL) and read absorbance at 405 nm. Positive
control Abs are: Mouse Mab 14C2 which recognizes the N-terminal
ectodomain epitope of M2 (Abcam), mouse Mab anti-StAv (Abcam) and
mouse polyclonal anti-rat IgG (H & L chains) (Invitrogen).
Example 3
Analysis of Anti M2e Activity in Mice Challenged with the Immune
Conjugate Biotinylated Rat Anti-Mouse Ter-119
Mab-Streptavidin-Biotinylated M2e Peptide (SEQ ID NO: 1):
Comparison of Different Routes of Administration
[0158] The effect of the route of administration on the serum
antibody response to M2e peptide in mice challenged with an immune
conjugate of biotinylated rat anti-mouse TER-119 Mab+
streptavidin-biotinylated M2e peptide is evaluated by comparing
serum titers of anti-M2e antibodies in mice immunized
intraperitoneally, intravenously, subcutaneously and
intramuscularly. The full Mab-StAv-IMG conjugate is compared to the
non-targeted StAv-IMG partial complex, as well as to PBS vehicle.
The dose will be 1.0 .mu.g equivalent of M2e peptide (SEQ ID NO:
1). Immunizations and ELISA analysis are performed as described in
Example 2.
Example 4
Analysis of Anti-M2e Fecal IgG and IgA Activity in Mice Challenged
with the Immune Conjugate Biotinylated Rat Anti-Mouse TER-119
Mab-Streptavidin-Biotinylated M2e Peptide (SEQ ID NO: 1)
[0159] Mice are dosed with TER-119-StAv-M2e conjugate and controls
as depicted in Table 2 and following the dosing schedule in Example
2:
TABLE-US-00003 TABLE 3 Experimental design: Example 4. Group number
Ligand Linker Immunogen 1 Biotinylated Anti- streptavidin
Biotinylated M2e TER119 2 Biotinylated Rat streptavidin
Biotinylated M2e IgG 3 -- streptavidin Biotinylated M2e 4 KLH
Chemical conjugate M2e 5 -- -- M2e 6 (PBS -- -- -- injected
only)
Feces are collected at the times described in Example 2. Soybean
trypsin inhibitor (0.1 mg/ml in PBS) is added to every 0.1 g of
feces, which are vortexed in a mini-beadbeater (Biospec Products)
for 10 sec at 2500 rpm, and debris is removed by centrifugation at
9000.times.g at 4.degree. C. for 15 min. The supernatant is assayed
for anti-M2e IgG titer in the ELISA set-up described in Example 2,
but also for IgA by adding anti-IgA 2.sup.nd antibody (goat
anti-mouse IgA conjugated with horseradish peroxidase
(Sigma-Aldrich).
Example 5
Analysis of Anti-M2e Activity in Mice Challenged with the Immune
Conjugate Biotinylated Rat Anti-Mouse CD21/CD35
Mab-Streptavidin-Biotinylated M2e Peptide (SEQ ID NO: 1)
[0160] Mice are injected with an immune conjugate of biotinylated
rat anti-mouse CD21/CD35 Mab+streptavidin-biotinylated M2e peptide
(influenza) and their serum antibody response to M2e is assayed.
The anti-CD21/CD35 antibody is purchased from eBioscience. The
conjugate is prepared according to the method described in Example
2. Groups of 5 mice each (Balb/C, female, 6-8 weeks old) are
injected intravenously (i.v.) with a range of free and conjugated
peptide doses (0.01 to 10 .mu.g equivalent of peptide in 50 .mu.l
PBS) according to the experimental design in shown in Table 4
below.
TABLE-US-00004 TABLE 4 Experimental design: Example 5. Group number
Ligand Linker Immunogen 1 Biotinylated Anti- streptavidin
Biotinylated M2e CD21/CD35 2 Biotinylated Rat streptavidin
Biotinylated M2e IgG 3 -- streptavidin Biotinylated M2e 4 KLH
Chemical conjugate M2e 5 -- -- M2e 6 (PBS -- -- -- injected only) 7
Biotinylated Anti- streptavidin -- CD21/CD35 8 Biotinylated Rat
streptavidin -- IgG 9 Biotinylated Anti- streptavidin M2e CD21/CD35
10 Biotinylated Rat streptavidin M2e IgG 11 -- streptavidin M2e 12
Biotinylated Anti- -- M2e CD21/CD35 13 Biotinylated Rat -- M2e IgG
14 Biotinylated Anti- -- -- CD21/CD35 15 Biotinylated Rat -- --
IgG
[0161] Immunizations and ELISA analysis will be performed as
described in Example 2.
Example 6
Analysis of Anti-M2e Activity in Mice Challenged with the Immune
Conjugate Biotinylated Plasmodium Falciparum EBA-175 Peptide
1085-1096 (SEQ ID NO: 7) (pEBA)+Streptavidin-Biotinylated M2e
Peptide
[0162] Since Plasmodium falciparum EBA-175 peptide 1085-1096 binds
human, but not mouse RBCs, the mouse experiments are performed in
human GphA-transgenic mice (see Auffrey, et al., 2001, Blood
97:2872-2878. Glycophorin A dimerization and band 3 interaction
during erythroid membrane biogenesis: in vivo studies in human
glycophorin A transgenic mice). Alternately, the corresponding
binding protein derived from other species of Plasmodium, e.g., P.
bergheii or P. yoelii, which bind mouse RBCs is used (referred to
as pEBA.sub.m). Complexes are assembled and immunizations and ELISA
analysis are performed as described in Example 2.
Example 7
Analysis of Anti-M2e Activity in Mice Challenged with the Synthetic
Peptide pEBA-Glycine-Glycine-Glycine-M2e Peptide
[0163] Mice are injected intravenously with a synthetic peptide of
the sequence pEBA.sub.m-175 SEQ ID NO: 7 linked via three glycine
residues to the M2e peptide (SEQ ID NO: 1) described in Example 1
above and according to the scheme depicted in Table 5. Each mouse
receives a range of free and conjugated peptide doses (0.01 to 10
.mu.g equivalent of M2e peptide in 50 .mu.A PBS) intravenously and
blood samples are collected and analyzed according to the schedule
in Example 2.
TABLE-US-00005 TABLE 5 Experimental Design: Example 7. Group number
Ligand Linker immunogen 1 pEBA.sub.m Gly-Gly-Gly M2e 2 pEBA.sub.m
(scrambled) Gly-Gly-Gly M2e 3 KLH Chemical conjugate M2e 4 -- --
M2e 5 (PBS injected -- -- -- only)
Example 8
Analysis of Anti-HBV peptide (preS1 Amino Acids 34-59 (SEQ ID
NO:2), or pHBV) Activity in Mice Challenged with the Immune
Conjugate Biotinylated Rat Anti-Mouse TER-119
Mab+Streptavidin-Biotinylated pHBV
[0164] Mice are challenged with immune conjugates in which the
immunogen is an HBV (hepatitis B virus) peptide preS1 amino acids
34-59 (Hu W G, et al. 2005. World J Gastroenterol. 11:2088-2094.
Identification of the immunogenic domains in HBsAg preS1 region
using overlapping preS1 fragment fusion proteins) or pHBV coupled
to biotinylated TER-119 Mab. Experimental groups, immunizations and
ELISA analysis are as described in Example 2.
Example 9
Analysis of Anti-M2e Activity in Mice Challenged with the Immune
Conjugate Biotinylated Mouse Anti-Mouse Band 3
Mab-Streptavidin-Biotinylated M2e Peptide (SEQ ID No:1)
[0165] Mouse anti-mouse band 3 Mabs have been produced in several
labs from NZB mice. The immune conjugate, biotinylated mouse
anti-mouse band 3 Mab-streptavidin-biotinylated M2e peptide, is
produced according to the method in Example 1 with an IgG
anti-mouse band-3 Mabs (e.g., 34-3C or class-switched 4C8
(Fossati-Jimack, 2002, J Autoimmun 18:17-25. Selective increase of
autoimmune epitope expression on aged erythrocytes in mice:
implications in anti-erythrocyte autoimmune responses.)), and is
evaluated according to the protocol in Example 2.
Example 10
Optimization of the Degree of Biotinylation of the Targeting
Mabs
[0166] Establish the optimal number of StAv-IMG units to couple to
the biotinylated Mab. Maximize IMG load while retaining full target
binding capacity of the Mab.
[0167] The FluoReporter Biotin Quantitation Kit (Invitrogen) is
used for estimating the molar ratio of biotin:protein. Mab
anti-TER-119 is biotinylated using amino-reactive (Pierce EZ-link
NHS biotin) and carbohydrate-reactive (Pierce EZ-link
hydrazide-biotin) reagents at various levels by adding a range of
biotinylation molar excess factors (e.g., 3.times., 5.times.,
10.times., 50.times., 250.times.). Add a 5.times. molar excess StAv
per mole of biotinylated Mab to saturate all biotinylated sites.
Test functional integrity of the biotinylated Mab preparations by
binding to mouse RBCs. Use goat anti-rat IgG conjugated with
fluoresceine isothiocyanate (FITC) to detect RBC-bound TER-119
using flow cytometry. Select the condition that results in the
highest degree of biotinylation of the TER-119 Mab while retaining
maximum RBC-binding. Use un-biotinylated TER-119 Mab and goat
anti-rat IgG-FITC as the reference.
Example 11
Determination of the Optimal Stoichiometry of Mab-StAv-IMG
[0168] The effect of loading 1, 2 or 3 IMGs per StAv and the effect
of binding 1 or more of these StAv-IMG conjugates per targeting Mab
is compared. The optimal construct is identified based on the
elicited response in vivo (see Example 2). Prepare
anti-TER-119+StAv+M2e conjugates at various ratios (e.g., 1:1:1,
1:1:3, 1:2:2, 1:2:6, 1:3:3, 1:3:9) and administer them to mice as
in Example 2 (always administer 1.0 .mu.g equivalent of M2e).
Compare the resulting anti-M2e titers.
Example 12
Comparison of Monovalent, Divalent and Trivalent Peptide-Mediated
Targeting of IMG to RBC
[0169] Biotinylated pEBA.sub.m (SEQ ID NO:7) (see Example 7) is
used as the targeting ligand and biotinylated M2e is used as the
IMG. The following constructs are prepared: 1pEBA.sub.m:1StAv:3M2e,
2pEBA.sub.m:1StAv:2M2e and 3pEBA.sub.m:1StAv:1M2e. Inject mice with
each of the constructs containing equal equivalents of M2e (1.0
.mu.g). Evaluate the elicited immune response according to the
methods in Example 2.
Example 13
Evaluation of the Efficacy of Immunizing with a Construct in which
the Targeting Component and the IMG are Covalently Bound to a
Common Polymer Backbone
[0170] Synthesize each component peptide (e.g., pEBA.sub.m (SEQ ID
NO:7) and M2e (SEQ ID NO:1)) with an added Lys whose
.alpha.-carboxyl group is joined to the peptide's C-terminus,
leaving the .alpha. and .epsilon. amino groups of Lys free.
Co-polymerize various ratios of the components with
bis-succinimidyl poly(ethylene glycol), or BS-PEG (mean molecular
weight 2,000 da). The free .alpha. and .epsilon. amino groups of
Lys are linked to the reactive PEG units to create a co-polymer
that has the pattern PEG-P-PEG-P-PEG-P-PEG . . . , where P is
either peptide pEBA.sub.m or M2e in a random distribution. The
ratio of pEBA.sub.m to M2e is a function of the ratio of added
peptides to the polymerization reaction. Prepare co-polymer
backbone targeted IMG constructs in which the Mab:IMG ratios are
0:10 (un-targeted multivalent IMG control), 1:9, 3:7, 5:5, 7:3 and
9:1. Evaluate their efficacy by injecting 1.0 .mu.g equivalents of
M2e as described in Example 2.
Example 14
Preparing Targeted Constructs Using a Microparticulate Linking
Component
[0171] Liposomes containing a biotinylated lipid in the bilayer
(e.g., phosphatidyl ethanolamine reacted with succinimidyl-biotin)
are prepared. The exposed biotin groups are used to bind StAv and
then the StAv's biotin-binding sites are loaded with biotinylated
targeting and IMG components at various ratios. The comparatively
large liposomal surface permits adding many more units of each
component without causing steric hindrance. In order to prevent
StAv from cross-linking the exposed biotin groups on the liposomal
surface, pre-load 3 mole equivalents of either targeting or IMG
units (or combinations of these) per mole of StAv, and then add
this conjugate to the liposomes. Administer and analyze as in
Example 2.
Example 15
Preparing Targeted Constructs Using a Microparticulate Linking
Component and a Mixture of IMG's
[0172] Biotinylate a mixture of IMGs from a relevant source (e.g.,
the multiple strain variants of a multivalent pneumococcal
polysaccharide vaccine or the various proteins or peptides from a
viral vaccine) using a suitable reactive biotin reagent (in the
case of oligosaccharides use a biotinylation reagent with a
hydrazide reactive terminus (EZ-link hydrazide biotin, Pierce), and
in the case of peptidic preparations use one with a terminus
containing either a succinimidyl group for reaction with amines, a
maleimidyl group for reaction with sulfhydryls, or an amine
terminus reactive with carboxyls in the presence of ethylenediamine
carbodiimide (EDC)). Add such a mixture of biotinylated IMGs to a
targeting conjugate consisting of StAv bound to a biotinylated Mab,
a biotinylated targeting peptide, such that the StAvs have
remaining unoccupied biotin-binding sites. These are produced by
reacting a large molar excess of StAv with the biotinylated
targeting component. The biotinylated IMG mixture can also be
reacted with liposomes bearing surface StAv molecules bound to
lipid-bound biotin (e.g., biotinylated phosphatidyl ethanolamine),
with sufficient unoccupied biotin-binding sites remaining on the
bound StAv molecules. Administer and analyze as in Example 2.
Example 16
Using Anti-Band 3 Mab for Targeting IMGs to Senescent RBCs
[0173] Approximately 1% of RBCs are senescent and destined for
removal from the circulation by phagocytosis in the RES. Their
clearance is mediated by pre-existing natural IgG Abs (Nabs). These
Nabs have low affinity for band 3. They can only bind their target
Ag on the RBC firmly if the band 3 molecules are clustered in the
RBC membrane, through augmented avidity. Band 3 clustering occurs
as a result of cumulative oxidative damage in the course of the
life of the cells. Use a slightly higher affinity anti-band 3 Mab,
sufficiently high to out-compete the Nabs, but not so high as to
bind all RBCs. The Mab can be a humanized IgG, for targeting IMGs
to senescent RBCs.
[0174] To develop a human anti-band 3 Mab, immunize a mouse with
human band 3 protein or with human red blood cells (which contain a
senescent subpopulation) and fuse the spleen cells with a myeloma
fusion partner (e.g., SP2/0). Plate fused cells at 100,000
cells/well in hybridoma selection medium (HAT). After 3 weeks
screen supernatants of wells with hybridoma clones by ELISA on
wells coated with band 3 protein extracellular domain. Subclone
hybridomas that react specifically with human band 3 until the
cultures are monoclonal. The resulting murine Mab can be
"humanized" by methods familiar to those skilled in the art (e.g.,
Recombinant Antibodies (1999), Breitling, F. and Diibel, S. (eds.),
John Wiley & Sons). Alternately, human anti-band 3 Mabs can be
produced by fusion of normal human donor B cells, among which are
found the cells producing anti-band 3 Nabs. Enrich B cells (the
CD19+ or CD20+ subpopulation) from the peripheral blood mononuclear
fraction by immunomagnetic purification (StemCell Sciences). Fuse
the B cells by electrofusion (CytoPulse) to a suitable fusion
partner myeloma or heteromyeloma cell line (e.g., K6H6/A5 or A6
(ATCC) or SP2-IL6-TERT (Dessain S K, et al., 2004, J Immunol
Methods 291:109-122. High efficiency creation of human monoclonal
antibody-producing hybridomas.)) to form hybridomas. Plate fused
cells at 20,000 cells/well in hybridoma selection medium (HAT).
After 3 weeks screen supernatants of wells with hybridoma clones by
ELISA on wells coated with band-3 protein extracellular domain.
Subclone band 3-specific IgG hybridomas. Purify the antibodies and
use them to prepare an immune conjugate as described in Example 1.
Use biotinylated influenza M2e peptide as the IMG and couple to the
Mab with a StAv bridge. Assay in mice according to the method in
Example 2.
Sequence CWU 1
1
13123PRTInfluenza A virus 1Ser Leu Leu Thr Glu Val Glu Thr Pro Ile
Arg Asn Glu Trp Gly Cys 1 5 10 15 Arg Cys Asn Asp Ser Ser Asp 20
227PRTHepatitis B virus 2Phe Gly Ala Asn Ser Asn Asn Pro Asp Trp
Asp Phe Asn Pro Asn Lys 1 5 10 15 Asp His Trp Pro Glu Ala Asn Gln
Val Gly Ala 20 25 323PRTInfluenza A virus 3Ser Leu Leu Thr Glu Val
Glu Thr Leu Ile Arg Asn Glu Trp Gly Cys 1 5 10 15 Arg Cys Asn Asp
Ser Ser Asp 20 423PRTInfluenza A virus 4Ser Leu Leu Thr Glu Val Glu
Thr His Ile Arg Asn Glu Trp Gly Cys 1 5 10 15 Arg Cys Asn Asp Ser
Ser Asp 20 54PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Ala Gly Leu Ile 1 64PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Pro
Gly Gly Gly 1 711PRTPlasmodium falciparum 7Leu Lys Ser His Met Asn
Arg Glu Ser Asp Asp 1 5 10 86PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Gly Ala Gly Ala Gly Ala 1 5
96PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Gly Gly Gly Val Val Val 1 5 106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Glu
Glu Glu Arg Arg Arg 1 5 116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Asp Lys Asp Lys Asp Lys 1 5
126PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Pro Pro Pro Ser Ser Ser 1 5 135PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Tyr
Gln Cys Met Trp 1 5
* * * * *