U.S. patent application number 16/566787 was filed with the patent office on 2020-09-10 for use of an antibody specific for bdca 2 for ligation and removal of dendritic cells in the treatment systemic lupus erythematosis.
This patent application is currently assigned to Miltenyi Biotec GmbH. The applicant listed for this patent is Miltenyi Biotec GmbH. Invention is credited to David William Buck, Andrzej Dzionek, Juergen Schmitz.
Application Number | 20200283499 16/566787 |
Document ID | / |
Family ID | 1000004853202 |
Filed Date | 2020-09-10 |
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United States Patent
Application |
20200283499 |
Kind Code |
A1 |
Schmitz; Juergen ; et
al. |
September 10, 2020 |
Use of an antibody specific for BDCA 2 for ligation and removal of
dendritic cells in the treatment systemic lupus erythematosis
Abstract
The invention provides humanized antibody and other
antigen-binding fragments that are specific for the newly
identified dendritic cell marker BDCA-2. These agents may be used
for treatment of conditions caused or mediated by dendritic cells,
including but not limited to systemic lupus erythematosus (SLE).
The invention includes pharmaceutical compositions that contain a
means for specifically binding BDCA-2, methods of preparing
therapeutic products, and their use in therapy. Methods are also
provided for screening, manufacture, and use of specific antibodies
that identify other dendritic cell markers.
Inventors: |
Schmitz; Juergen; (Bergheim,
DE) ; Buck; David William; (Mayfield, GB) ;
Dzionek; Andrzej; (Koeln, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Miltenyi Biotec GmbH |
Bergisch Gladbach |
|
DE |
|
|
Assignee: |
Miltenyi Biotec GmbH
Bergisch Gladbach
DE
|
Family ID: |
1000004853202 |
Appl. No.: |
16/566787 |
Filed: |
September 10, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13454008 |
Apr 23, 2012 |
10407486 |
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16566787 |
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11393155 |
Mar 29, 2006 |
8183039 |
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13454008 |
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09714712 |
Nov 15, 2000 |
7030228 |
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11393155 |
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60197205 |
Apr 13, 2000 |
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60196824 |
Apr 11, 2000 |
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60180775 |
Feb 7, 2000 |
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60179003 |
Jan 28, 2000 |
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60167076 |
Nov 23, 1999 |
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60165555 |
Nov 15, 1999 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/7056 20130101;
C07K 16/2851 20130101; Y10S 435/81 20130101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; C07K 16/28 20060101 C07K016/28 |
Claims
1.-8. (canceled)
9. A pharmaceutical composition for treating an inflammatory
disorder in a subject in need thereof, wherein the composition
comprises an effective amount of a means for binding blood
dendritic cell antigen 2 (BDCA-2) combined in a formulation with a
pharmaceutically acceptable excipient, wherein the composition is
formulated and prepared in a manner so as to be suitable for
administration to a human, and wherein the amount of the means for
binding BDCA-2 and the formulation of the composition configure the
composition to be effective for treating the inflammatory disorder
without causing undue side effects in the subject.
10. The pharmaceutical composition of claim 9, wherein the means
for binding BDCA-2 is an antibody or an antigen binding fragment
that is specific for BDCA-2.
11. The pharmaceutical composition of claim 9, wherein the means
for binding blood dendritic cell antigen 2 (BDCA-2) is a humanized
monoclonal antibody that is specific for BDCA-2.
12. The pharmaceutical composition of claim 9, wherein the
inflammatory disorder is an autoimmune disease.
13. The pharmaceutical composition of claim 9, wherein the
inflammatory disorder is systemic lupus erythematosus (SLE).
14. The pharmaceutical composition of claim 9, wherein the BDCA-2
is encoded by exons 1-6; exons 1 and 3-6; exons 1-2 and 4-6; or
exons 1-3 and 5-6 of SEQ ID NO:1.
15. A product comprising a pharmaceutical composition according to
claim 9, packaged with information about the use of the composition
for treatment of systemic lupus erythematosus (SLE).
16. A method of treating an inflammatory condition in a patient in
need thereof, comprising administering to the patient a
pharmaceutical composition according to claim 9.
17. The method of claim 15, wherein the means for binding blood
dendritic cell antigen 2 (BDCA-2) is a humanized monoclonal
antibody that is specific for BDCA-2.
18. The method of claim 15, wherein the inflammatory condition is
systemic lupus erythematosus (SLE).
19. A method of removing dendritic cells from a subject in need
thereof, comprising contacting the dendritic cells in the subject
with a means for binding blood dendritic cell antigen 2
(BDCA-2).
20. The method of claim 19, wherein the contacting is consequent to
administering the means for binding BDCA-2 to the subject by
intravenous, subcutaneous, or intramuscular injection.
21. A method of treating systemic lupus erythematosus (SLE) in a
subject in need thereof, comprising removing dendritic cells from
the subject according to the method of claim 19.
22. The method of claim 21, wherein the means for binding BDCA-2 is
a humanized antibody that is specific for BDCA-2.
23. A method of ligating BDCA-2 protein on a dendritic cell,
comprising: contacting the dendritic cell with an antibody or
antigen-binding fragment under conditions where the antibody or
antigen-binding fragment will ligate to BDCA-2 protein on the
dendritic cell; wherein said antibody or antigen-binding fragment
comprises a polypeptide domain that specifically binds a BDCA-2
protein encoded by SEQ ID NO:1.
24. A method of determining whether an antibody or antigen binding
fragment can specifically recognize a dendritic cell or a dendritic
cell marker and is therefore suitable for compounding as a
pharmaceutical composition according to claim 2, wherein the method
comprises contacting the antibody or antigen binding fragment with
BDCA-2 protein in vitro, and determining that the antibody or
antigen binding fragment can specifically recognize a dendritic
cell or a dendritic cell marker if it binds to the BDCA-2 protein
in preference to other proteins.
25. A method of preparing a pharmaceutical composition according to
claim 9, comprising: obtaining an antibody that specifically binds
BDCA-2; obtaining a pharmaceutically acceptable excipient; and
combining a predetermined amount of the antibody with a
predetermined amount of the excipient in a formulation that is
suitable for administration to a human; wherein the amount of the
antibody and the formulation of the composition configure the
composition to be effective for treating the inflammatory disorder
without causing undue side effects in a subject in need
thereof.
26. The method of claim 25, wherein the antibody is a humanized
monoclonal antibody and the inflammatory condition is systemic
lupus erythematosus (SLE).
27. The method of claim 25, wherein the antibody is a humanized
from of an antibody selected from AC144, AD5-13A11, and
AD5-5B8.
28. A method of manufacturing a pharmaceutical product, comprising
obtaining a pharmaceutical composition that has been prepared
according to the method of claim 25, and packaging the composition
with information about the use of the antibody or antigen binding
fragment in the treatment of systemic lupus erythematosus (SLE).
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/454,008, filed Apr. 23, 2012, now U.S. Pat.
No. 10,407,486; which is a divisional of U.S. patent application
Ser. No. 11/393,155, filed Mar. 29, 2006, now U.S. Pat. No.
8,183,039; which claims priority to U.S. patent application Ser.
No. 09/714,712, filed Nov. 15, 2000, now U.S. Pat. No. 7,030,228;
and claims the benefit of U.S. Provisional Application Ser. No.
60/197,205, filed Apr. 13, 2000; U.S. Provisional Application Ser.
No. 60/196,824, filed Apr. 11, 2000; U.S. Provisional Application
Ser. No. 60/180,775, filed Feb. 7, 2000; U.S. Provisional
Application Ser. No. 60/179,003, filed Jan. 28, 2000; U.S.
Provisional Application Ser. No. 60/167,076, filed Nov. 23, 1999;
and U.S. Provisional Application Ser. No. 60/165,555, filed Nov.
15, 1999.
[0002] The above listed applications are hereby incorporated herein
in their entirety for all purposes.
TECHNICAL FIELD
[0003] The present invention relates to antibodies and derivatives
thereof specific for subpopulations of dendritic cells (DCs).
Compositions and methods of use thereof are also provided including
isolation and purification of DCs and subpopulations thereof and
antibody- or ligand-mediated immunotherapy. The invention also
provides substantially isolated DC subpopulations. Methods of use
thereof are also provided including DC-based immunotherapy,
characterization of various diseases and in vivo numeric DC
expansion for instance with flt3-Ligand.
BACKGROUND OF THE INVENTION
[0004] The hematopoietic development of dendritic cells (DCs),
potent antigen presenting cells (APCs) is distinct and may follow
several precursor pathways some closely linked to monocytes. DCs
may be derived from a lymphoid precursor. Thomas et al. (1993) J.
Immunol. 150:821-834. Like in blood, there may be three distinct
subsets of DCs present in the thymus: 1) plasmacytoid CD4+CD11c-
DCs; 2) CD4+CD11c+ DCs and 3) interdigitating DCs. It has been
proposed that thymic DCs and T cells arise from a common stem cell.
Thomas et al. (1996) Stem Cells 14:196-206.
[0005] Generation of large numbers of DCs for potential clinical
use has recently been accomplished through the in vitro culturing
of progenitors with. cytokines. Various strategies have been
adopted to introduce antigens into dendritic cells so that they may
be more effectively presented to T cells in the context of
costimulation. It has also been shown that dendritic cells can
influence the T cell response to antigen to follow either a humoral
or systemic pathway.
[0006] T cells are unable to respond to unprocessed proteins,
rather, they require accessory cells to present antigen as peptide
epitopes displayed on the cell surface in conjunction with MHC
molecules. Antigens generated endogenously in the cell cytoplasm
are typically presented in the Class I pathway and stimulate
cytotoxic T lymphocyte (CTL) reactions while exogenous protein is
process in WIC Class LE compartments and induce helper (CD4) T cell
responses. The stimulation of naive T cells requires the presence
of costimulatory molecules that act as secondary signals in the
activation of primary immunity. APCs such as B cells and
macrophages are typically incapable of inducing primary responses.
In contrast, dendritic cells drive their potency from the
constitutive unregulated expression of costimulatory, adhesion and
MHC Class I and H molecules essential for the initiation of
effective cellular immunity. For review see, Avigan (1999) Blood
Rev. 13:51-64.
[0007] DCs are APC that are essential for initiation of primary
immune responses and the development of tolerance. DCs express MHC,
necessary for stimulation of naive T cell populations. The
hematopoietic development of DCs is distinct and may follow several
precursor pathways, some of which are closely linked to monocytes.
See, for review, Avigan (1999) Blood Rev. 13:51-64. Different DC
subsets have distinct developmental pathways. The emerging concept
is that one DC subset has regulatory functions that may contribute
to the induction of tolerance to self-antigens. Austyn (1998) Curr.
Opin. Hematol. 5:3-15. Conversely, DCs, or a subset thereof, may
also be involved in the induction of immune responses to
self-proteins. It is thought that certain autoimmune responses may
be due to microenvironmental tissue injury followed by local DC
activation and subsequent interaction with T cells to initiate an
immune response. Ibrahim et al. (1995) Immunol. Today
16:181-186.
[0008] The ability of DCs to initiate T cell responses is being
used in DC cancer vaccines. Hart et al. (1999) Sem. Hematol.
36::21-25. For instance, DCs are generated in vitro from CD34.sup.+
cells or monocytes, pulsed with tumor-derived peptides or proteins
and returned to the patient to act as APCs in cancer-specific T
cell induction. Brugger et al. (1999) Ann. N.Y. Acad. Sci.
872:363-371. Animal models have demonstrated that DC tumor vaccines
reverse T cell anergy and result in subsequent tumor rejection.
Avigan (1999); see also, Tarte et al. (1999) Leukemia 13:653-663;
Colaco (1999) Molec. Med. Today 5:14-17; Timmerman et al. (1999)
Ann. Rev. Med. 50:507-529; Hart et al. (1999) Semin. Hematol.
36:21-25; Thurnher et al. (1998) Urol. hit. 61:67-71; and Hermans
et al. (1998) N.Z. Med. J. 111:111-113. One approach has been to
increase DCs in vivo by administration of flt-Ligand. This has the
effect of compensating for VEGF-induced DC suppression. Ohm et al.
(1999) J. Immunol. 163:3260-3268. DCs have been proposed for use as
adjuvants in vaccination and in recombinant vaccines. Fernandez et
al. (1998) Cyto. Cell. Mol. Ther. 4:53-65; and Gilboa et al. (1998)
Cancer Immunol. Immunother. 46:82-87. DC have also been proposed
for use in enhancing immunity after stem cell transplantation.
Brugger et al. (1999) Ann. NY Acad. Sci. 363-371. DCs play a number
of potential roles in immunology. For instance, DCs are involved in
human immunodeficiency virus (HIV) infection. Zoeteweij et al.
(1998) J. Biomed. Sci. 5:253-259. DCs have also been proposed as
suitable for use in HIV therapy. Weissman et al. (1997) Clin.
Microbiol. Rev. 10:358-367.
[0009] Additional immunologic functions are related to DCs such as
differential induction of Th1 or Th2 responses, autoimmune
reactions and allergies. Rissoan et al. (1999) Science
283:1183-1186; Hermans et al. (1998) NZ Med. J. 111:111-113; and De
Palma et al. (1999) J. Immunol. 162:1982-1987.
[0010] Increased levels of circulating. IFN-.alpha. and of
IFN-.alpha. inducing factor (something like a complex of anti-DNA
antibody and DNA) are found in SLE patients and correlate to
disease activity. Furthermore, patients with non-autoimmune
disorders treated with IFN-.alpha. frequently develop
autoantibodies and occasionally SLE. Several papers from Ronnblom
et al. (1999) Clin. Exp. Immunol. 115: 196-202; (1999) J. Immunol.
163: 6306-6313; and (2000) J. Immunol. 165: 3519-3526) show that
IFN-.alpha. inducing factors derived from patients induce secretion
of IFN-.alpha. in PBMC from healthy donors and they selectively
activate natural IFN-.alpha. producing cells (NIPC plasmacytoid
DC).
[0011] Studies on DC's in blood have been hampered by scarcity of
the cells and the relative lack of DC-specific cell surface
markers. Methods for DC isolation are based on either maturational
change after a short culture period, like the acquisition of low
buoyant density or the expression of DC activation/maturation
antigens (CD83, CMRF-44 and CMRF-56). Young et al. (1988) Cell
Immunol. 111:167; Van Voorhis et al. (1982) J. Exp. Med. 155:1172;
Zhou et al. (1995) J. Immunol. 154:3821-3835; Fearnley et al.
(1997) Blood 89:3708-3716; Mannering et al. (1988) J. Immunol. Met.
219:69-83; Hock et al. (1999) Tiss. Antigens 53:320-334; and Hock
et al. Immunol. 83:573-581.
[0012] Functional CD1a.sup.+ DCs are typically generated ex vivo
from monocytes and from CD34.sup.+ hematopoietic progenitor cells.
Bender et al. (1996) J. Immunol. Met. 196:121-135; Pickl et al.
(1996) J. Immunol. 157:3850-3859; Romani et al. (1994) J. Exp. Med.
180:83-93; Sallusto et al. (1994) J. Exp. Med. 179:1109-1118; Caux
et al. (1992) Nature 360:258-261; Mackensen et al. (1995) Blood
86:2699-2707; Szabolcs et al. (1995) J. Immunol. 154:5851-5861;
Herbst et al. (1996) Blood 88:2541-2548; de Wynter et al. (1998)
Stem Cells 16:387-396; Strunk et al. (1996) Blood
87:1292-1302--U.S. Pat. Nos. 6,010,905; and 6,004,807. It is not
known if DCs generated in vitro from monocytes and hematopoietic
progenitor cells retain or obtain all of the characteristics of in
vivo. DCs.
[0013] In addition, several attempts to generate mAb specific for
human DC have failed, yielding only mAb that bind antigens
expressed by both DC and other leukocytes. Human DC share a large
number of immunogenic cell surface structures with other blood
cells, including HLA molecules, CD18, CD29, CD31, CD43, CD44, CD45,
CD54, and CD58. These antigens may dominate the immune response to
injected DC to a level where B cells with specificity for
DC-specific antigens are not at all or only very rarely represented
among B cells that have the capability to fuse with myeloma
cells.
[0014] Many investigators have tried to overcome this problem by
injecting adult mice with non-DC and cyclophosphamide, in order to
ablate B cells with specificity for shared antigens, or by
injecting neonatal mice with non-DC, in order to tolerize B cells
with specificity for shared antigens. O'Doherty et al. (1993) Adv.
Exp. Med. Biol. 329:165-172; and Yamaguchi et al. (1995) J.
Immunol. Meta 181:115-124.
[0015] A mAb designated CMRF44 has been used to monitor DCs in stem
cell transplant patients. Fearnley et al. (1999) Blood 93:728-736.
These CMRF44.sup.+ cells were proposed to be suitable for use in
initiating, maintaining and directing immune responses. Fearnley et
al. (1997). DCs have been isolated most often by using a
combination of cell surface markers. For instance; U.S. Pat. No.
5,972,627 describes "hematopoietic cells enriched for human
hematopoietic dendritic progenitor cells" as having "at least 80%
expressing CD34, CD45RA, and CD10 but not CD19, CD2, CD3, CD4, CD8,
CD20, CD14, CD15, CD16 CD56 and glycophorin."
[0016] Isolation of DCs from blood relies on a multitude of
immunophenotypic criteria, like the absence of a panel of leukocyte
lineage (lin)-specific antigens (e.g. CD3, CD14, CD19 and CD56) and
the presence of HLA-DR, CD4 or CD33. Romani et al. (1996) J.
Immunol. Met. 196:137-151; Thomas et al. (1993) J. ImmunoL
150:821-834; Thomas et al. (1994) J. Immunol. 153:401.6-4028;
O'Doherty et al. (1994) Immunol. 82:487-493; O'Doherty et al.
(1993) J. Exp. Med. 178:1067-1076; Nijman et al. (1995) J. Exp.
Med. 182:163-174; Ferbas et al. (1994) J. Immunol. 152:4649-4662;
Heufler et al. (1996) Eur. J. Imrnunol. 26:659-668; Ito et al.
(1999) J. Immunol. 163:1409-1419; Cella et al. (1999) Nature Med.
5:919-923; Robinson et al. (1999) Eur. J. Immunol. 29:2769-2778;
Olweus et al. (1997) Proc. Natl. Acad: Sci. USA 94:12551-12556;
Robert et al. (1999) J. Exp. Med. 189:627-636; and Kohrgruber et
al. (1999) J. Immunol. 163:3250-3259.
[0017] From analyses of DC isolated-from non-cultured blood it
became evident that blood DC are not a homogeneous cell population
but a mixture of at least two populations. Thomas et al. (1994);
O'Doherty et al. (1994); Ito et al. (1999); Cella et al. (1999);
Robinson et al. (1999); Olweus et al. (1997); Kohrgruber et al.
(1999); Strobl et al. (1998) J. Immunol. 161:740-748; and Rissoan
et al. (1999) Science 283:1183-1186. The first blood DC
subpopulation is CD123.sup.bright CD11c.sup.-DC, which possesses a
plasmacytoid morphology and potent T cell stimulatory function. The
second blood DC subpopulation is CD123.sup.dim CD11 C.sup.bright,
which is rather monocytoid in appearance, expresses CD45RO and
spontaneously develops into typical mature DCs even when cultured
without any exogenous cytokines. Plasmacytoid CD123.sup.bright
CD11c.sup.-DC display some features, like the expression of the
pre-T cell receptor a chain, which indicate that they may arise
from lymphoid precursors. Strobl et al. (1998); Rissoan et al.
(1999); and Bruno et al. (1997) J. Exp. Med. 185:875-884.
CD123.sup.dim CD11c.sup.bright DC display all the criteria of
myeloid DCs., O'Doherty et al. (1994); and Ito et al. (1999).
Robinson et al. (1999); Kohrgruber et al. (1999); and Strobl et al.
(1998). DCs resembling plasmacytoid CD123.sup.bright CD11c.sup.-DC
have been detected in the T cell-rich areas of lymphoid tissue and
were initially erroneously designated plasmacytoid T cells or
plasmacytoid monocytes due to their morphology and phenotype.
Grouard et al. (1997) J. Exp. Med. 185:1101-1111; Lennert et al.
(1975) Lancet 1:1031-1032; Lennert et al. (1984) in Leukocyte
Typing. Human Leukocyte differentiation antigens detected by
monoclonal antibodies. Bernard et al. eds. Springer-Verlag, Berlin;
and Facchetti et al. (1988) Am. J. Pathol. 133:15. DCs resembling
CD123.sup.dim CD11c.sup.bright blood DC have been found in the dark
and light zone of germinal centers. Grouard (1996) Nature
384:364-367.
Splice Variants
[0018] Estimates of the total number of expressed genes range from
40,000 to more than 150,000. This number is not an accurate
reflection of the number of proteins encoded since, in many cases,
more than one splice variant from the mRNAs (transcriptome)
produced from these genes. Estimates again vary, but perhaps as
many as 500,000 different mRNAs are produced in the human. It is
estimated that at least 30% of the human genes have several splice
variants. Mironov et. al. (1999) Genome Research 9:1288-1293).
These numbers are believed by some to be conservative. Similar
numbers are believed to be true for mouse and rat and alternative
splicing occurs also in lower organisms, such as Drosophila
melanogaster and Caenorhabditis elegans. Proteins translated from
different splice variants can have significantly different
functions, as evidenced by a growing number of research papers.
Different splice variants may be expressed in different tissues,
different developmental stages and different disease states.
C-Type Lectins
[0019] C-type lectins are a family of glycoproteins that exhibit
amino acid sequence similarities in their carbohydrate recognition
domains (CRD) and that bind to selected carbohydrates in a
Ca.sup.2+-dependent manner. C-type lectins have been subdivided
into four categories (Vasta et al., 1994; and Spiess 1990). The
first group comprises type II membrane-integrated proteins, such as
asialoglycoprotein receptors, macrophage galactose and N-acetyl
glucosamine (G1cNac)-specific lectin, and CD23 (FcsRII). Many
members in this group exhibit specificity for galactose/fucose,
galactosamine/GalNac or GlcNac residues. The second group includes
cartilage and fibroblast proteoglycan core proteins. The third
group includes the so-called "collectins" such as serum
mannose-binding proteins, pulmonary surfactant protein SP-A, and
conglutinin. The fourth group includes certain adhesion molecules
known as LEC-CAMs (e.g., Mel-14, GMEP-140; and ELAM-1).
[0020] C-type lectins are known to function as agglutinins,
opsonins, complement activators, and cell-associated recognition
molecules (Vasta et al. 1994; Spiess 1990; and Kery 1991). For
instance, macrophage mannose receptors serve a scavenger function
(Shepherd et al., 1990), as well as mediating the uptake of
pathogenic organisms, including Pneumocystis carinii (Ezekowitz et
al. 1991) and Candida albicans (Ezekowitz et al. 1990). Serum
mannose-binding protein mimics Clq in its capacity to activate
complement through the classical pathway. Genetic mutations in this
lectin predispose for severe recurrent infections, diarrhea, and
failure to thrive (Reid et al. 1994). Thus, C-type lectins exhibit
diverse functions with biological significance.
[0021] Carbohydrate moieties do not necessarily serve as "natural"
ligands for C-type lectins. For example, CD23
(FC.sub..epsilon.RII), which belongs to the C-type lectin family as
verified by its binding of Gal-Gal-Nac (Kijimoto-Ochiai et al.
1994) and by its CRD sequence, is now known to recognize IgE in a
carbohydrate-independent manner; an enzymatically deglycosylated
form of Ty as well as recombinant (non-glycosylated) IgE produced
in E. coli both bind to CD23 (Vercelli et al. 1989). Thus, some
C-type lectins recognize polypeptide sequences in their natural
ligands.
[0022] Several C-type lectins have been identified on the surface
of DCs. First, Jiang et al. cloned the protein recognized by the
NLDC-145 mAb, one of the most widely used mAb against murine DC
(Jiang et al., 1995). This protein, now termed DEC-205, was found
to be a new member of the C-type lectin family, one that contains
ten distinct CRD. Second, Sallusto et al. reported that human DC
express macrophage mannose receptors (MMR), which also contain
multiple-CRD (Sallusto et al., 1995). Both receptors have been
proposed to mediate endocytosis of glycosylated molecules by DC,
based on the observations that: a) polyclonal rabbit antibodies
against DEC-205 not only bound to DEC-205 on DC surfaces, but were
subsequently internalized; b) these DC activated effectively a T
cell line reactive to rabbit IgG; and c) internalization of
FITC-dextran by DC was blocked effectively with mannan, a mannose
receptor competitor (Jiang et al. 1995; and Sallusto et al. 1995).
With respect to cell type specificity, DEC-205 is now known to be
also expressed, albeit at lower levels, by B cells and epithelial
cells in thymus, intestine, and lung (Witmer-Pack et al. 1995; and
Inaba et al. 1995) and MMR. is also expressed even more abundantly
by macrophages (Stahl 1992). Other have also been found on DC
surfaces, these include DCIR, MDL-1, NURPIA, Dectin-1, Dectin-2,
CLEC-1, CLEC-2, Langerin; and DC-sign.
Allergies
[0023] Allergic responses, including those of allergic asthma and
allergic rhinitis, are characterized by an early phase response,
which occurs within seconds to minutes of allergen exposure and is
characterized by infiltration of eosinophils into the site of
allergen exposure. Specifically, during the early phase of the
allergic response, activation of Th2-type lymphocytes stimulates
the production of antigen-specific IgE antibodies, which in turn
triggers the release of histamine and other mediators of
inflammation from mast cells and basophils: During the late phase
response, IL-4 and IL-5 production by CD4.sup.+ Th2 cells is
elevated. These cytokines appear to play a significant role in
recruiting eosinophils into the site of allergen exposure, where
tissue damage and dysfunction result.
[0024] Currently, antigen immunotherapy for allergic disorders
involves the subcutaneous injection of small, but gradually,
increasing amounts, of antigen in a process called desensitization
therapy. Antigen immunotherapy is merely palliative and, at
present, not curative. Weber (1997) JAMA 278:1881-1887; Stevens
(1998) Acta Clinica Beligica 53:66-72; and Canadian Society of
Allergy and Clinical Immunology (1995) Can. Med. Assoc. J.
152:1413-1419.
[0025] Many patients who begin the therapy do not complete the
regimen, and if injections are missed for over a week, the patient
must begin the entire treatment regimen again. A variety of
antigens have been identified and produced by recombinant means.
For reviews, see Baldo et al. (1989) Allergy 44:81-97; Baldo (1991)
Cum. Opin. Immunol. 3:841-850; Blaser (1994) Ther. Umsch 51:19-23;
and Valenta et al. (1996) Adv. Exp. Med. Bio. 409:185-196.
[0026] Antigen immunotherapy treatments present the risk of
inducing potentially lethal IgE-mediated anaphylaxis and do not
address the cytokine-mediated events of the allergic late phase
response. This therapy has been described as "having the potential
for misadventure." Weber (1997). Another significant problem with
antigen immunotherapy is that the risk of adverse reactions,
especially anaphylaxis, significantly reduces the dosage of antigen
both with respect to the amount given per administration and the
amount given over a period of time. Thus, traditional allergy
immunotherapy is protracted and thus time-consuming, inconvenient,
and expensive.
[0027] An alternative approach for treatment of IgE-associated
disorders such as allergies involves administration of compounds
that inhibit histamine release. Many such drugs are available as
over-the-counter remedies. Other drugs include an anti-IgE binding
antibody. However, a drawback of this approach is that it merely
masks the symptoms, while not providing any kind of permanent cure
or protection.
BRIEF SUMMARY OF THE INVENTION
[0028] The invention relates to methods of enriching for
hematopoietic cell populations enriched in DCs and subsets thereof.
Compositions enriched for the cells and populations of cells
obtained therefrom are also provided by the invention. Methods of
making genetically modified DCs are also provided. Compositions of
genetically modified DCs are also provided. Methods of use of the
cells are also included. Antigen-binding fragments specific for
BDCA:2 and BDCA-3 and the antigens recognized thereby are also
provided.
[0029] The invention encompasses antigen,binding fragments specific
for a subset of DCs specifically recognized by an antibody
designated AC144; AD5-1311, AD5-20E5, AD5-17F6, AD5-4B8, AD5-5E8,
AD5-14H12 or AD5-8E7. The invention encompasses antigen-binding
fragments specific for an epitope of an antigen designated BDCA-2
(SEQ ID NO:2). The invention encompasses antigen-binding fragments
specific for an epitope of an antigen designated BDCA-3.
[0030] The invention encompasses a substantially isolated or
concentrated DC population or subpopulation specifically recognized
by an antigen-binding fragment of the invention. These
antigen-binding fragments can be any one of AC144, AD5-1311,
AD5-20E5, AD5-17F6, AD5-4B8, AD5-5E8, AD5-14H12 or AD5-8E7 or
antigen-binding fragments specific for BDCA-1, BDCA-2, BDCA-3 or
BDCA-4. Antigen-binding fragments recognizing neuropilin-1 also
recognize BDCA-4 and are suitable for use herein.
[0031] The invention further encompasses populations or
subpopulations of DCs wherein substantially all of the cells
express or are isolated, concentrated or enumerated on the basis of
expression of at least one of BDCA-1, BDCA-2, BDCA-3 and BDCA-4.
These cells can be suspended in any physiologically acceptable
excipient. Preferably, the excipient is pharmacologically
acceptable.
[0032] The invention further encompasses methods for obtaining
compositions of hematopoietic cells enriched for DCs by separating
a mixture of human hematopoietic cells into a fraction wherein at
least 80% of the cells in the fraction are BDCA-1.sup.+.
[0033] The invention further encompasses methods for obtaining
compositions of hematopoietic cells enriched for DCs by separating
a mixture of human hematopoietic cells into a fraction wherein at
least 80% of the cells in the fraction are BDCA-2.sup.+.
[0034] The invention further encompasses methods for obtaining
compositions of hematopoietic cells enriched for DCs by separating
a mixture of human hematopoietic cells into a fraction wherein at
least 80% of the cells in the fraction are BDCA-3.sup.+.
[0035] The invention further encompasses methods for obtaining
compositions of hematopoietic cells enriched for DCs by separating
a mixture of human hematopoietic cells into a fraction wherein at
least 80% of the cells in the fraction are BDCA-4.sup.+.
[0036] The invention further encompasses methods for isolating a
substantially pure subset of DCs by a) obtaining a mixture of human
hematopoietic cells; and b) substantially isolating cells from the
mixture specifically recognized by an antigen-binding fragment
specific for the antigen designated BDCA-2.
[0037] The invention further encompasses methods for isolating a
substantially pure subset of DCs by a) obtaining a mixture of human
hematopoietic cells; and b) substantially isolating cells from the
mixture specifically recognized by an antigen-binding fragment
specific for the antigen designated BDCA-3.
[0038] The invention further encompasses methods for isolating a
substantially pure subset of DCs by a) obtaining a mixture of human
hematopoietic cells; and b) substantially isolating cells from the
mixture specifically recognized by an antigen-binding fragment
specific for the antigen designated BDCA-4.
[0039] The invention further encompasses methods for enumerating
DCs by: a) obtaining a mixture of cells; and b) labeling the cells
with an antigen-binding fragment specific for any one or more of
the antigens BDCA-1, BDCA-2, BDCA-3, and BDCA-4.
[0040] The invention further encompasses methods of modulating the
immune capacity of DCs by: isolating a substantially pure
population or subpopulation of DCs; and modulating the calcium
mobilization of the DCs.
[0041] The invention further encompasses methods of screening for
test agents for the presence of pharmaceutically effective agents
by isolating a substantially pure population or subpopulation of
DCs with an antigen-binding fragment specific for any one or more
of the antigens BDCA-1, BDCA-2, BDCA-3, and BDCA-4; screening the
isolated cells with test agents; monitoring the response of the
cells to the agents; comparing the response of the cells to the
agents to cells exposed to a control agent; and determining whether
the test agent-modulated any one immunologic properties of the
isolated cell.
[0042] The invention further encompasses methods of modulating an
immunologic property of DCs by altering the ability of the DC to
Mobilize calcium.
[0043] The invention further encompasses immunogenic and
immunomodulating compositions of DCs preferably in a
physiologically acceptable excipient.
[0044] The invention further encompasses methods of treating a
physiologic condition by administering to a subject in need thereof
an effective amount of immunogenic or immunomodulating compositions
of DCs.
[0045] The invention further encompasses methods of producing DC
cytokines by isolating a substantially pure population or
subpopulatiori of DCs with an antigen-binding fragment specific for
any one or more of BDCA-1, BDCA-2, BDCA-3, and BDCA-4; and
isolating cytokines from the cells or cellular products or
supernatants.
[0046] The invention further encompasses methods of modulating DC
cytokine production by isolating a substantially pure population or
subpopulation of DCs with an antigen-binding fragment specific for
any one or more of BDCA-1, BDCA-2, BDCA-3, and BDCA-4; and treating
the cells with agents that modulate DC cytokine production.
[0047] The invention further encompasses methods of modulating in
vivo DC cytokine production by administering to a subject in need
thereof an effective amount of an agent that modulates DC cytokine
production.
[0048] The invention further encompasses methods of generating
antibodies specific for an antigen by administering to a subject in
need thereof an effective amount of a substantially pure population
or subpopulation of DCs loaded with the antigen and isolated with
an antigen-binding fragment specific for any one or more of BDCA-1,
BDCA-2, BDCA-3, and BDCA-4 wherein the DCs are modulated to induce
a Th2 response.
[0049] The invention further encompasses methods of generating a T
cell or humoral immune response specific for an antigen by
administering to a subject in need thereof an effective amount of a
substantially pure population or subpopulation of DCs loaded with
the antigen and isolated with an antigen-binding fragment specific
for any one or more of BDCA-I, BDCA-2, BDCA-3, and BDCA-4 wherein
the cells are modulated to induce a Th1 response.
[0050] The invention further encompasses polypeptides prepared by
expressing, in a recombinant host cell, the polypeptides and
purifying the expressed polypeptide away from total recombinant
host cell components, wherein the polypeptide contains about 5
contiguous amino acid residues from SEQ ID NO:2.
[0051] The invention further encompasses of purified polypeptides
and compositions thereof, wherein the polypeptide contains about 5
contivous amino acid residues from SEQ ID NO:2.
[0052] The invention further encompasses fusion proteins of a
polypeptide amino-acid sequence linked to a polypeptide amino acid
sequence that is not SEQ ID NO: 2, wherein the amino acid sequence
contains about 5 contiguous amino acid residues from SEQ ID
NO:2.
[0053] The invention further encompasses polypeptides containing at
least one splice variant of BDCA-2.
[0054] The invention further encompasses a polynucleotide or a
complement thereof encoding at least 5 contiguous amino acid
residues of BDCA-2, a splice variant or a fragment thereof.
[0055] The invention further encompasses recombinant host cells
containing a polynucleotide or a complement thereof encoding at
least 5 contiguous amino acid residues of BDCA-2, a splice variant
or a fragment thereof.
[0056] The invention further encompasses a method of inhibiting an
interaction of a DC with a T cell by contacting a composition
containing DC and T cells with an effective amount of an agent that
inhibits the interaction of BDCA-2, BDCA-3, or BDCA-4 with the T
cell.
[0057] The invention further encompasses a method of treating
inflammation by administering to a subject in need thereof an
amount of an agent that inhibits the interaction of BDCA-2, BDCA-3,
or BDCA-4 with the T cell effective to reduce inflammation in the
subject.
[0058] The invention further encompasses a method of suppressing
the expression of BDCA-2 in a cell by expressing a BDCA-2 antisense
polynucleotide in the cell.
[0059] The invention further encompasses a transgenic animal
containing the polynucleotide or a complement thereof encoding at
least 5 contiguous amino acid residues of BDCA-2, a splice variant
or a fragment thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] FIGS. 1A and 1B are multi-panel figures showing dot plots
from the flow cytometric analysis of peripheral blood mononuclear
cells (PBMC) isolated by Ficoll-Paque density gradient
centrifugation. In FIG. 1, expression of BDCA-2, BDCA-3 and CD1c
(BDCA-1) on PBMC is shown.
[0061] FIG. 1A shows staining of PBMC with FITC-conjugated mAb
against BDCA-2 (AC144), BDCA-3 (ADS-5E8) and CD1c (ADS-8E7), and
PE-conjugated mAb against the TCRal3 heterodimer, CD14, CD19 and
CDS6, respectively. The numbers indicate the percentage of cells in
the respective quadrant. Propidium iodide fluorescence and light
scatter signals were used for gating of live cells.
[0062] FIG. 1B shows the scatter profile of (a) PBMC, (b) gated
BDCA 2.sup.+ cells, (c) gated. BDCA-3.sup.+ cells and (d) gated
CD1c.sup.+ cells.
[0063] FIG. 2 shows that BDCA-2, BDCA-3, BDCA-4 and CD1c (BDCA-1)
are expressed on three distinct blood DC subsets. Blood DC were
isolated from PBMC by depletion of CD3, CD11b and CD16 positive
cells followed by enrichment of CD4 positive cells. The purity of
blood DC is demonstrated by light-scatter properties (upper-left
dotplot) and anti-HLA-DR-Cy5 vs. anti-Lin-FITC
(anti-TCR.alpha..beta., CD14, CD19 and CD56) staining (upper-middle
dotplot). Note that only few lin.sup.+ cells are present.
Expression of BDCA-2, BDCA-3, BDCA-4 and CD1c on blood DC is
characterized in a series of two-color stainings with PE- and
FITC-conjugated mAb against CD11c, CD123 and the antigens
themselves. Note that BDCA-2, BDCA-3, BDCA-4 and CD1c are
exclusively expressed on only one of three distinct blood DC
subsets each. The subsets are defined according to staining of
blood DC with CD123-PE vs. CD11c-FITC (upper left. dotplot):
CD11c.sup.-CD123.sup.brightblood DC; CD11 C.sup.bright
CD123.sup.dim blood DC; and CD11c.sup.dimCD123.sup.- blood DC.
[0064] FIG. 3 is a multi-panel figure that depicts expression of
BDCA-4 on PBMC. Shown is a two-color staining of PBMC with
FITC-conjugated MAB against BDCA-2 (AC144) and PE-conjugated mAB
against BDCA-4 (AD5-17F6). Note that a few single positive
(BDCA-2.sup.+BDCA-4- and BDCA-2-BDCA-4.sup.+) PBMC are detected
[0065] FIGS. 4A and 4B show expression of BDCA-2, BDCA-3 and BDCA-4
on purified blood DC after various periods of culture in the
presence of IL-3. Purified blood DC were cultured for 0 h, 1 h, 3
h, 6 h, 9 h, 12 h, 18 h, 24 h, 36 h, and 48 h in the presence of
rIL-3 and then flow cytometrically analyzed for the expression of
CD11c, BDCAj-3, BDCA-2 and BDCA-4. (A) Histograms show staining of
gated CD11c.sup.- and CD11c.sup.+ blood DC with PE-conjugated anti
BDCA-2 mAB (AC144) and anti-BDCA-4 mAB (AD5-17F6) (bold lines), and
PE-conjugated isotype-matched control mAB (faint lines),
respectively. Dot plots show staining of blood DC with CD11c-PE vs.
anti BDCA-3 (AD5-5E8) biotin/streptavidin-APC. (B) Diagrams show
mean fluroescence intensity (MFI) values for anti-BDCA-2-PE, anti
BDCA-4-PE, and anti-BDCA-3 biotin/streptavidin-APC staining of
CD11c.sup.- (.tangle-solidup.) and CD11c.sup.+ (.box-solid.) DC,
respectively. For BDCA-2 and BDCA-4, MR values were calculated by
subtracting the values obtained with isotype control mAb from the
values obtained with the AC144 and ADS-17F6, respectively. For
BDCA-3, MFI values are calculated by subtracting the values
obtained without any staining mAb (autofluorescence) from the
values obtained with AD5-5E8.
[0066] FIG. 5 shows the amino acid sequence of one isoform of
BDCA-2 with all six exons being expressed (SEQ ID. NO:2).
[0067] FIG. 6 shows that BDCA-1-specific mAb AD5-8E7 blocks binding
of the CD1c mAb M241 to MOLT-4 cells. MOLT-4 cells were
pre-incubated with saturating amounts of AD5-8E7 mAb (bold line) or
an isotope control mAb (faint line) and then stained with
PE-conjugated CD1c mAb (M241).
[0068] FIGS. 7A and 7B show expression of BDCA-2, BDCA-3 and BDCA-4
on Mo-DC and CD34.sup.+ cell-derived DC (CD34-DC). CD14.sup.+
monocytes and CD34.sup.+ hematopoietic progenitor cells were
immunomagnetically purified via direct magnetic labeling with CD14
and CD34 mAb-conjugated microbeads, respectively. Purified
monocytes were cultured for 7 d in the presence of rGM-CSF and
rIL-4, and purified CD34-DC were cultured for 11 d in the presence
of rflt3-ligand, rTGF-.beta.1, rTNF-.alpha., rSCF and rGM-CSF.
After the culture period, cells were stained with CD1a-FITC,
CD1c-PE (AD5-8E7), anti-BDCA-2-PE (AC114), anti-BDCA-3-PE (AD5-5E8)
and anti-BDCA-4-PE (AD5-17F6). Histograms show staining of (A)
Mo-DC and (B) CD34-DC (bold lines), respectively. The faint lines
show staining with isotype control mAb. Except for the left-most
histogram (CD1a staining), gated CD1a.sup.+ cells are shown in
(B).
[0069] FIG. 8 shows that culturing of anti-BDCA-2 mAb-labeled
BDCA-2.sup.+ cells results in rapid mAb internalization. PBMC were
labeled at 4.degree. C. with FITC-conjugated anti-BDCA-2 mAb
(AC144, IgG1), incubated at 37.degree. C. for the time periods
indicated, and were then stained at 4.degree. C. with PE-conjugated
rat anti-mouse IgG1 mAb (X56) and Cy5-conjugated CD123 mAb (AC145,
IgG2a). Shown are MEI values of anti-BDCA-2-FITC (.box-solid.) and
rat anti-mouse IgG1 mAb-PE (.tangle-solidup.) staining of gated
BDCA-2.sup.+CD123.sup.+ cells.
[0070] FIG. 9 is a mult-panel drawing that shows the morphology of
immunomagnetically purified CD1c.sup.+, BDCA-2.sup.+ and
BDCA-3.sup.+ blood DC. CD1c.sup.+, BDCA-2.sup.+ and BDCA-3.sup.+
cells were isolated from PBMC by indirect magnetic labeling with
PE-conjugated primary mAb (ADS-8E7, AC144 and AD5-5E8) and anti-PE
mAb-conjugated microbeads followed by enrichment of labeled cells
by MACS. The dotplots show staining of PBMC with HLA-DR-FITC and
the PE-conjugated mAb before (left dotplots) and after (right
dotplots) magnetic enrichment of CD1c.sup.+ (upper dotplots)
BDCA-2.sup.+ (middle dotplots) and BDCA-3.sup.+ (lower dotplots)
cells, respectively. The three pictures on the right side show May
Grunwald/Giemsa staining of isolated CD1c.sup.+ (upper picture),
BDCA-2.sup.+ (middle picture) and BDCA-3.sup.+ cells after
cytocentrifugation. Note that small lymphocytes can be seen in the
picture of the enriched CD1c.sup.+ cells. These are CD1c.sup.+ B
cells.
[0071] FIGS. 10A to 10E show up-regulation of MHC class II, CD83
and co-stimulatory molecules on CD1c, BDCA-2.sup.+ and BDCA-3.sup.+
blood DC upon culturing. Purified CD1c.sup.+ (A), BDCA-2.sup.+ (C)
and BDCA-3.sup.+ (B) were cultured for 1 day in medium (CD1c.sup.+
and BDCA-3.sup.+ BDC) or for 2 days in medium with rIL-3 and
anti-CD40 mAb on CD32-transfected L cells (BDCA-2.sup.+DC),
respectively. "Immature" Mo-DC (D) were generated by culturing of
monocytes for 7 days in medium in the presence of rGM-CSF and
rIL-4. "Mature" Mo-DC (E) were generated by culturing of immature
Mo-DC for another 3 days in medium in the presence of TNF.alpha..
The histograms show cell staining with CD1a-FITC, CD80-PE, CD83-PE,
CD86-PE and HLA-DR-PE, respectively (bold lines). The faint lines
show cell staining with isotype and fluorochrome-matched control
mAb.
[0072] FIG. 11 shows endocytic capacity of freshly isolated
CD1c.sup.+, BDCA-2.sup.+ and BDCA-3.sup.+ blood DC in comparison
with purified CD3.sup.+ T cells. Isolated CD1c.sup.+DC
(.tangle-solidup.), BDCA-2.sup.+ BDC (.tangle-solidup.),
BDCA-3.sup.+DC (.box-solid.) and CD3.sup.+ T cells (*) were
incubated at 37.degree. C. in medium with 1 mg/ml Lucifer Yellow
(LY) for 0, 15, 45 and 75 min, washed three times in ice cold PB
S/EDTA/BSA and were then analyzed by flow Oytometry. Shown are the
MFI values for LY fluorescence after subtracting the MFI values,
which are obtained upon incubation at 4.degree. C. in the absence
of LY.
[0073] FIG. 12 depicts the cDNA sequence of BDCA-2 (SEQ ID
NO:1).
[0074] FIGS. 13A to 13E show intracellular Ca.sup.2+ mobilization
is induced in immunomagnetically purified BDCA-2.sup.+BDCA-4.sup.+
blood DC (A, B) and BDCA-2-transfected U937 cells (D), but not in
non-transfected U937 cells (E) via anti-BDCA-2 mAb alone (A) and or
anti-BDCA-2 plus crosslinking secondary mAb (B, D, E). Ligation of
BDCA-4 on immunomanetically purified BDCA-2.sup.+BDCA-4.sup.+ BDC
with anti-BDCA-4 mAb and cross-linking secondary mAb does not
induce intracellular Ca.sup.2+ mobilization. Shown is the
Ca.sup.2+-dependent 405 nm1525 nm ratio of Indo-1-fluorescence
(Y-axis) against time (X-axis, a value of 1024 corresponds to
204.80 sec). A is BDCA-2.sup.+BDCA-4.sup.+ blood DC, anti-BDCA-2
(AC144, IgG1). B is BDCA-2+ BDCA-4+ blood DC, anti-BDCA-2 (AC144,
IgG1) plus rat anti-mouse IgG1 (X56). C is BDCA-2+ BDCA-4+ blood
DC, anti-BDCA-4 (AD5-17F6, IgG1) plus rat anti-mouse IgG1 (X56). D
is BDCA-2 transfected U937 cells, anti-BDCA-2 (AC144, IgG1) plus
rat anti-mouse IgG1 (X56). E is non-transfected U937 cells,
anti-BDCA72 (AC144, IgG1) plus rat anti-mouse IgG1 (X56).
[0075] FIGS. 14A and 14B show ligation of BDCA-2 but not of BDCA-4
with a specific mAb followed by a secondary cross-linking mAb
inhibits secretion of type I interferon by plasmacytoid
BDCA-2.sup.-BDCA-4.sup.+DC from blood or tonsils in response to
stimulation with influenza virus strain PR8. Plasmacytoid
BDCA-2.sup.-BDCA-4.sup.+DC from freshly isolated blood (A) or
tonsils (B) were cultured for 24 hours in the presence of IL-3
alone (control); IL-3, anti-BDCA-2 mAb and rat anti-mouse IgG1 mAb
(AC144.sup.+RamG1); IL-3; anti-BDCA-2 mAb, rat anti-mouse IgG1 mAb,
and influenza virus strain PR8 (AC144.sup.+RamG1.sup.+FLU); IL-3
and influenza virus strain PR8 (FLU); IL-3, anti-cytokeratin mAb,
rat anti-mouse IgG1 mAb, and influenza virus strain PR8
(CK3.sup.+RamG1.sup.+FLU); IL-3, anti-BDCA-4 mAb, rat anti-mouse
IgG1 mAb, and influenza virus strain PR8
(17F6.sup.+RamG1.sup.+FLU). Secreted type I interferon (U/ml) in
the culture supernatants was measured by a bioassay with reference
to a standard type I interferon (U/ml) curve.
[0076] FIG. 15 shows presentation of anti-BDCA-2 mAb (AC144, IgG1)
to a T cell clone specific for mouse IgG1 by isolated BDCA-2- and
BDCA-4-expressing plasmacytoid DC. BDCA-2.sup.+BDCA-4.sup.+
plasmacytoid DC present anti-BDCA-2 mAb (AC144, IgG1, .box-solid.)
to T cells much more efficiently than anti-ILT-3 mAb (ZM3.8, IgG1,
.tangle-solidup.) and anti-cytokeratin mAb (CK3-11D5, IgG1
.circle-solid.).
[0077] FIGS. 16A and 16B show expression of BDCA-2 and BDCA-4 on
tonsillar plasmacytoid CD123.sup.+DC.
[0078] FIG. 17 shows that neuropilin-1 (GenBank Accession No.
003873) is immunoprecipitated from cell lysates of
neuropilin-1-transfected PEA cells (NP), but not of non-transfected
PAE cells (P) with the anti-BDCA-4 mAb AD5-17F6 (anti-NRP-1 (ML)).
Precipitated proteins were analyzed by SDS-PAGE and Western
blotting with the BDCA-4-specific mAb AD5-17F6 (ML) or an
neuropilin-1-specific mAb from Shay Soker, Children's Hospital,
Boston, Mass. (S).
[0079] FIG. 18 shows ligation of BDCA-2 but not of BDCA-4 with a
specific mAb followed by a secondary cross-linking mAb inhibits
secretion of INF-.alpha. by plasmacytoid BDCA-2.sup.+BDCA-4.sup.+DC
from blood or tonsils in response to stimulation with poly I:C.
Plasmacytoid BDCA-2.sup.+BDCA-4.sup.+DC from blood were cultured
with 10 .mu.g/ml of AC144 mAb (2 and 4) or mouse IgG1 mAb (CF6B,
anti-TPO, 1 and 3) at 37.degree. C. for 30 min.
[0080] FIG. 19 shows an analysis of human multiple tissue cDNA
panels from CLONTECH (lane 1: heart; lane 2: brain; lane 3:
placenta; lane 4: lung; lane 5: liver; lane 6: skeletal muscle;
lane 7: kidney; lane 8: pancreas; lane 9: spleen; lane 10: thymus;
lane 11: testis; lane 12: ovary; lane 13: small intestine; lane 14:
lymph node; lane 15: bone marrow; lane 16: fetal liver; lane 17:
tonsil) and an analysis of cDNAs prepared from different
populations of blood leukocytes (lane 18: T cells; lane 19: B
cells; lane 20: NK cells; lane 21: monocytes; lane 22:
CD11c.sup.bright CD123.sup.low BDC; lane23: CD11c-CD123.sup.bright
plasmacytoid DC) for BDCA-2 cDNA. The control is G3PDH.
[0081] FIG. 20 shows the splice variants-of the BDCA-2 transcript.
Splice variants were analyzed by RT-PCR using the specific primers
for BDCA-2 used in expression analysis. The amplified fragments
were cloned to plasmid vectors and sequenced.
[0082] FIG. 21 shows the splice variants of Dectin-2
transcripts.
[0083] FIGS. 22A, 22B, 22C, and 22D show an alignment of the mRNA
sequences of BDCA-2 (SEQ ID NO:1) and mouse Dectin-2 (SEQ ID NO:3)
with the precise positions of the deduced introns indicated.
[0084] FIG. 23 shows the alignment of the amino acid sequences of
human BDCA-2 (SEQ ID NO:2), human DCLR (SEQ ID NO:5) and mouse
Dectin-2 (SEQ ID NO:4). In FIG. 23, * represents identical
conserved residues in all the aligned sequences, : represents
conserved substitutions, . represents semi-conserved substitutions,
shaded areas denote the conserved carbohydrate recognition domain
(CRD), italics show putative transmembrane domains. The following
symbols highlight residues strongly conserved between C-type
lectins in the CRD:
TABLE-US-00001 H hydrophobic A Aliphatic C Cysteine G Glycine E
glutamic acid W tryptophan .DELTA. aromatic amino acid + residues
involved in calcium-dependent binding of carbohydrates +P++ region
determining carbohydrate-binding specificity
[0085] FIG. 24 shows BDCA-3 immunoprecipitated from cell lysates of
surface biotinylated HD-MY-Z cells with the BDCA-3-specific mAb
AD5-14H12 (IgG1). For control of specificity, the CD19-specific mAb
5J25-C1 (IgG1) was used. Precipitated proteins were analyzed by
SDS-PAGE (4-12%) and Western blotting with streptavidin-peroxidase.
Note that the BDCA-3-specific mAb AD5-14H12 specifically
immunoprecipitates a cell surface protein of about 100 kD from
HD-MY-Z cells. Thus, BDCA-3 has an apparent molecular weight of 100
kD.
[0086] Sequence identifiers are assigned as follows:
[0087] SEQ ID NO: 1 refers to human BDCA-2 cDNA sequence.
[0088] SEQ ID NO: 2 refers to mouse BDCA-2 amino acid sequence.
[0089] SEQ ID NO: 3 refers to mouse Dectin-2 cDNA sequence.
[0090] SEQ ID NO: 4 refers to mouse Dectin-2 cDNA sequence.
[0091] SEQ ID NO: 5 refers to human DCIR amino acid sequence.
[0092] SEQ ID NO: 6 refers to basic unit of a linking peptide
(GGGGS).
[0093] SEQ ID NO: 7 refers to BDCA-2 forward primer (ttgaaagaac
cacaccccga, aagt).
[0094] SEQ ID NO: 8 refers to BDCA-2 reverse primer (tagctttcta
caacggtgga tgcc).
[0095] SEQ ID NO: 9 refers to BDCA-2 ASN glycosylation domain
(NCSV).
[0096] SEQ ID NO: 10 refers to BDCA-2 ASN glycosylation domain
(NSSY).
[0097] SEQ ID NO: 11 refers to BDCA-2 ASN glycosylation domain
(NVTF).
[0098] SEQ ID NO: 12 refers to Dectin-2 ASN glycosylation domain
(NESL).
[0099] SEQ ID NO: 13 refers to DOR ASN glycosylation domain
(NESS).
[0100] SEQ ID NO: 14 refers to BDCA-2 cAMP- and cGMP-dependent
protein kinase phosphorylation site domain (KRLS).
[0101] SEQ ID NO: 15 refers to DCIR cAMP- and cGMT-dependent
protein kinase phosphorylation site domain (KKTT).
[0102] SEQ ID NO: 16 refers to BDCA-2 Casein kinase II
phosphorylation site domain (TREE).
[0103] SEQ ID NO: 17 refers to BDCA-2 Casein kinase II
phosphorylation site domain (SSEE):
[0104] SEQ ID NO: 18 refers to Dectin Casein kinase II
phosphorylation site domain (STKE).
[0105] SEQ ID NO: 19 refers to Dectin Casein kinase II
phosphorylation site domain (STSE).
[0106] SEQ ID NO: 20 refers to Dectin Casein kinase II
phosphorylation site domain (TEAE).
[0107] SEQ ID NO: 21 refers to Dectin Casein kinase II
phosphorylation site domain (SICE).
[0108] SEQ ID NO: 22 refers to DCIR Casein kinase ITphosphorylation
site domain (TYAE).
[0109] SEQ ID NO: 23 refers to DCIR Casein kinase II
phosphorylation site domain (TTKE).
[0110] SEQ ID NO: 24 refers to DCIR Casein kinase II
phosphorylation site domain (TILE).
[0111] SEQ ID NO: 25 refers to DCIR Casein kinase II
phosphorylation site domain (SWQD).
[0112] SEQ ID NO: 26 refers to DCIR Casein kinase II
phosphorylation site domain (SEKD).
[0113] SEQ ID NO: 27 refers to DCIR Casein kinase II
phosphorylation site domain (TQEE).
[0114] SEQ ID NO: 28 refers to DCIR Casein kinase II
phosphorylation site domain (SDPE).
[0115] SEQ ID NO: 29 refers to DCIR Casein kinase II
phosphorylation site domain (SVCE).
[0116] SEQ ID NO: 30 refers to BDCA Tyrosine kinase phosphorylation
site domain (KLREYQQY).
[0117] SEQ ID NO: 31 refers to mouse Dectin Tyrosine kinase
phosphorylation site domain (RRLYELHTY).
[0118] SEQ ID NO: 32 refers to BDCA-2 Amidation site domain
(GGRR).
[0119] SEQ ID NO: 33 refers to mouse Dectin N-myristylation site
(GVCWTL).
[0120] SEQ ID NO: 34 refers to mouse Dectin N-myristylation site
(GTMVSE).
[0121] SEQ ID NO: 35 refers to mouse Dectin N-myristylation site
(GCCPNH).
[0122] SEQ ID NO: 36 refers to DCIR. N-myristylation site
(GINTAS).
[0123] SEQ ID NO: 37 refers to consensus immunoreceptor
tyrosine-based inhibitory motif ITIM motif, (I/V)XYXX(L/V).
[0124] SEQ ID NO: 38 refers to the ITIM motif in DCIR (ITYAEV).
DETAILED DESCRIPTION OF THE INVENTION
[0125] The invention relates to methods of enriching for cell
populations enriched in DCs and subsets thereof. Compositions
enriched for the DCs and populations of cells obtained therefrom
are also provided by the invention. Methods and compositions for
modified cells are also provided. Compositions of modified cells,
including genetically modified cells are also provided. Methods of
use of the cells both modified and non-modified are provided.
Antigen-binding fragments and the antigens recognized thereby are
also provided.
[0126] Described herein is a panel of new mAb raised against
immunomagnetically purified CD4.sup.+lin.sup.- DC that identify
three DC antigens: BDCA-2, BDCA-3 and BDCA-4. BDCA-2 and BDCA-3 are
novel. In the case of BDCA-4, while not previously described as a
DC-specific antigen, the antigen has been identified as
neuropilin-1, a receptor for the collapsin/semaphorin family that
mediates neuronal cell guidance. He et al. (1997) Cell
90:739-751.
[0127] In non-cultured human blood, expression of BDCA-2 and BDCA-4
is strictly confined to plasmacytoid CD123.sup.brightCD11c.sup.-DC,
whereas expression of BDCA-3 is restricted to a small population of
CD123.sup.-CD11c.sup.dim DC. This BDCA-3.sup.+DC population shares
many immunophenotypic features with classical
CD123.sup.dimCD11c.sup.brightDC, but, unlike
CD123.sup.dimCD11c.sup.dim DC, BDCA-3 DC lack expression of CD1c
(BDCA-1), CD2 and several of the Fc receptors.
[0128] The unpurified source of DCs may be any known in the art,
such as the bone marrow, fetal, neonate or adult or other
hematopoietic cell source, e.g., fetal liver, peripheral blood or
umbilical cord blood tonsil, lymph node, nasal membrane, spleen,
skin, airway epithelia, lung, liver gut, Peyers patches, etc. DCs
can also be isolated from cultured cells such as DCs derived from
progenitor cells. Various techniques can be employed to separate
the cells. For instance, negative selection methods can remove
non-DCs initially. mAbs are particularly useful for identifying
markers associated with particular cell lineages and/or stages of
differentiation for both positive and negative selections.
[0129] If desired, a large proportion of terminally differentiated
cells can be initially removed using a relatively crude negative
separation. For example, magnetic bead separations can be used
initially to remove large numbers of irrelevant cells. At least
about 80%, usually at least 70% of the total cells will be removed
prior to isolation of DCs. Preferably, the DC are directly isolated
from the cell source by positive selection.
[0130] Procedures for separation include, but are not limited to,
density gradient centrifugation; rosetting; coupling to particles
that modify cell density; magnetic separation with antibody-coated
magnetic beads or antibody-coated fero fluids (nonoporticles);
affinity chromatography; cytotoxic agents joined to or used in
conjunction with a mAb, including, but not limited to, complement
and cytotoxins; and panning with antibody attached to a solid
matrix, e.g. plate, elutriation or any other convenient
technique.
[0131] Techniques providing accurate separation and analysis
include, but are not limited to, magnetic bead separation and flow
cytometry, which can have varying degrees of sophistication, e.g.,
a plurality of color channels, low angle and obtuse light
scattering detecting channels, impedance channels, etc.
[0132] The cells can be selected against dead cells, by employing
dyes associated with dead cells such as propidium iodide (PI).
Preferably, the cells are collected in a medium comprising 2%
serum, such as fetal calf serum (FCS) or, human serum albumin (HSA)
or any other suitable, preferably sterile, isotonic medium. For
physiologic indications, HAS is preferred. Genetic modification of
the cells can be accomplished at any point during their maintenance
by transducing a substantially homogeneous cell composition with a
recombinant DNA construct, transfected with RNA, cell fusion,
loading with antigens and various methods known in the and/or
described herein.
[0133] For modification of the cells, a retroviral vector can be
employed, however any other suitable vector, delivery system or
cellular modification can be used. These include, e.g., adenovirus,
adeno-associated virus, artificial chromosomes, derived from yeast
and RNA derived from an antigen source such as a tumor. The genetic
modification, if any, need not be permanent as mature DCs have a
limited lifetime. Genetic approaches are used to express foreign
(tumor, viral, parasitic, etc.) antigens or autoantigens in DCs in
order to induce immunity or tolerance. The longevity of the
modification can also be controlled by suicide genes to limit
therapy (as with T cells).
[0134] Methods of transduction include any known in the art
including, without limitation, direct co-culture of the cells with
producer cells, e.g., by the method described by Bregni et al.
(1992) Blood 80:1418-1422, or culturing with viral supenatant alone
with or without appropriate growth factors and polycations, e.g.,
by the method described by Xu et al. (1994) Exp. Hemat. 22:223-230;
and Hughes et al. (1992) J. Clin. Invest. 89:1817.
[0135] Upon reintroduction of the modified cells expressing or
loaded with an antigen so as to present the antigen, into the host,
T cells are activated, anergized or deleted and are specifically
directed against the antigen. Generally, suitable antigens include
those expressed by virally infected cells, or cancer cells,
bacteria, yeast, protozoan, autoantigens (tolerogens) and
allergens. More specifically, suitable antigens include, but are
not limited to, viral proteins, proteins of cancer cells,
tissue-specific proteins or tolerogenic proteins. "Induction" of T
cells can include inactivation of antigen-specific T cells such as
by deletion or anergy. Inactivation is particularly useful to
establish or reestablish tolerance such as in organ transplantation
and autoimmune disorders respectively. The modified DCs can be
administered by any method known in the art including, but not
limited to, intravenously, subcutaneously, intranodally and
directly to the thymus. Preferably, administration is intravenous
(IV).
[0136] Often, cell immunotherapy involves removal of bone marrow
leukopheresis harvests, or other source of cells from a human host,
isolating the cells from the source. Meanwhile, the host may be
treated to partially, substantially or completely ablate native
hematopoietic capability if hematopoietic stem cell transplantation
is to occur. The isolated cells can be modified during this period
of time, so as to provide for cells having the desired
modification. In the case of complete hematopoietic ablation, stem
cell augmentation will also be required. The cells or modified
cells can then be restored to the host to provide for the new
capability. The methods of cell removal, host ablation and
stem/progenitor cell repopulation are known in the art.
[0137] The modified cells can be administered in any
physiologically acceptable vehicle, normally intravascularly,
intranodal and subcutaneously. Usually, at least 1.times.10.sup.5
cells will be administered, preferably 1.times.10.sup.6 or more.
The cells can be introduced by injection, catheter, or the like. If
desired, factors can also be included, including, but not limited
to, interleukins, e.g. IL-2, LL-3, IL-4, IL-12, and flt-Ligand, as
well as the other interleukins, the colony stimulating factors,
such as G-, M- and GM-CSF, interferons, e.g.
.gamma.-interferon.
[0138] The term "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to polymers of amino acid residues
of any length. The polymer can be linear or branched, it can
comprise modified amino acid residues or amino acid analogs, and it
can be interrupted by chemical moieties other than amino acid
residues. The terms also encompass an amino acid polymer that has
been modified naturally or by intervention; including, but not
limited to, disulfide bond formation, glycosylation, lipidation,
acetylation, phosphorylation, or any other manipulation or
modification, such as conjugation with a labeling or bioactive
component. Unless stated or implied otherwise, the term
antigen-binding fragment includes any polypeptide monomer or
polymer with immunologic specificity, including the intact
antibody, and smaller and larger functionally equivalent
polypeptides, as described herein.
1. Antigen-Binding Fragments and Compositions Thereof
[0139] This invention encompasses antigen-binding fragments that
specifically recognize DCs. That is, the antigen is found on DCs
such that antigen-binding fragments that recognize the antigen
preferentially recognize or bind to DCs or a subset thereof. Or, as
with BDCA-4, the antigen may be found on other cell types; but
within hematopoietic cells, the antigen is predominately present on
DCs.
[0140] The invention further encompasses a composition of matter
comprising an isolated antigen-binding fragment that binds
specifically to at least one DC antigen. Preferably, the
antigen-binding fragment is or is derived from a mAb designated
AC144, AD5-13A11, AD5-20E5, AD5-17F6, AD5-4B8, AD5-5E8, AD5-14H12
and AD5-8E7. Table 1 shows the antigen and epitope recognized by
each mAb and the isotype of the mAbs specific for DC.
TABLE-US-00002 TABLE 1 CD11c.sup.bright CD11c.sup.low CD11c.sup.-
CD123.sup.low CD123.sup.- CD123.sup.bright Other Antigen Antibody
Epitope Isotype DC DC DC leukocytes CD1c ADS-8E7 1A IgG2a + - - B
cell subset BDCA-2 AC144 2A IgG1 - - + BDCA-2 AD5- 2A IgG2a - - + -
13A11 BDCA-2 AD5-5B8 2A IgG1 - - + - BDCA-3 AD5-5E8 3A IgG1 - + - -
BDCA-3 AD5- 3B IgG1 - + - - 14H12 BDCA-4 AD5-17F6 4A IgG1 - - +
-
[0141] In non-cultured human blood, BDCA-2 and BDCA-4 are expressed
by a CD123.sup.brightCDC11c.sup.-DC population. This DC population
is now commonly referred to as plasmacytoid DC. Using BDCA-2 or
BDCA-4 as a surface marker for immunomagnetic isolation and/or flow
cytometric identification of plasmacytoid DC, the results presented
herein on frequency, immunophenotype, morphology, endocytic
capacity, and maturation of these cells, were completely consistent
with previous reports, where a large panel of leukocyte antigens
was used. This clearly illustrates that both antigens are useful
markers for plasmacytoid DC in non-cultured human blood. Stainings
of tonsillar cells show (FIG. 16) that the T cell zone associated
plasmacytoid DC in peripheral lymphoid organs can also be
discriminated from other lymphoid tissue-associated DC populations,
such as germinal center DC, interdigitating DC and follicular DC
based on the expression of BDCA-2 and BDCA-4.
[0142] Unlike BDCA-2, BDCA-4 is also expressed on several in vitro
differentiated DC populations: (1) in contrast to BDCA-2, BDCA-4 is
expressed on both Mo-DC and CD34-DC; (2) whereas expression of
BDCA-2 is completely down-regulated on plasmacytoid DC once they
have undergone IL-3-mediated maturation in culture, expression of
BDCA-4 is in fact up-regulated on cultured plasmacytoid DC; and (3)
in contrast to BDCA-2, BDCA-4 becomes expressed within 12 h by a
majority of cultured CD11c.sup.+DC, whereby it is unclear whether
this is only true for the larger CD1c.sup.+CD11c.sup.bright
population or also true for the smaller
CD1c.sup.-CD11C.sup.dimCD123.sup.-population. The finding that no
other BDCA-4.sup.+ cells than plasmacytoid DC are present in
non-cultured human blood, in fact, indicates that no counterparts
of the in vitro differentiated BDCA-4.sup.+DC populations mentioned
above are present in blood.
[0143] Cross-linking of BDCA-2 by means of anti-BDCA-2 mAb induces
rapid internalization of the antigen-Ab complex. In analogy to
other endocytic receptors on DC that are down-regulated upon
maturation, like Langerin. Valladeau et al. (2000) Immunity
12:71-81. Therefore, BDCA-2 may be a receptor with antigen-capture
function. BDCA-2 is a C-type lectin, is rapidly internalized after
ligation (FIG. 8), and BDCA-2 ligand(s) are processed and presented
to T cells (FIG. 15). Thus, like DEC-205, BDCA-2 has an antigen
uptake and presentation function for ligands to T cells.
[0144] Expression of BDCA-3 is restricted to a small population of
CD1c.sup.-CD11c.sup.dimCD123.sup.-DC in non-cultured human blood.
With respect to phenotype, morphology, endocytic capacity, and
maturation requirements, this DC population is quite similar to the
CD1c.sup.+CD1.sup.brightCD123.sup.dim DC population. However, apart
from BDCA-3 and CD1c expression themselves, the immunophenotypic
analysis has revealed some striking differences: in contrast to
CD1c.sup.+ BDC, BDCA-3.sup.+ BDC do not express the Fc receptors
CD32, CD64 and EcoRI, and they do not express CD2. The lack of Fc
receptor expression indicates that BDCA-3.sup.+ BDC, unlike
CD1c.sup.+ BDC do not have the capability of Ig-mediated antigen
uptake. Fanger et al. (1996) J. Immunol. 157:541-548; Fanger et al.
(1997) J. Immunol. 158:3090-3098; and Maurer et al. (1996) J.
Immunol. 157:607-616. As shown herein, BDCA-3 is a 100 kD
protein.
[0145] There is evidence that CD1c.sup.+CD11c.sup.bright DC, in
contrast to CD1c.sup.-CD11c.sup.dim DC, have the capacity to
acquire Langerhans cell characteristics (expression of Lag antigen,
E-cadherin and Langerin, and presence of Birbeck granules) when
cultured with GM-CSF, IL-4 and TGF-.beta.1. If BDCA-3.sup.+DC and
CD1c.sup.+DC represent maturational stages of the same cell type,
this would indicate that BDCA-3.sup.+DC have either already lost or
not yet acquired the capacity to differentiate into Langerhans
cells.
[0146] In contradiction to the results presented herein, Ito et al.
(1999) reported that CD1c.sup.+CD1.sup.bright DC, unlike
CD1c.sup.-CD11c.sup.dim DC, express CD1a. Two mAb BL-6 and B-B5
were used for staining of CD and that a difference in staining
intensity was actually observed when the two mAb were compared
(staining with B-B5 was probably brighter). As shown herein,
staining of DC was clearly negative using optimal titers of the
CD1a mAb BL-6 and HI149, but positive using B-B5. Moreover, B-B5,
unlike BL-6 and HI149, stained a high proportion of CD19.sup.+ B
cells in blood. Thus, the staining pattern of B-B was quite
reminiscent of a CD mAb rather than a CD mAb and, in fact, CD1c mAb
AD5-8E7 inhibits binding of B-B5 to MOLT-4 cells. Therefore, we
conclude that B-5 recognizes CD1c and that CD1c.sup.+DC do not
express CD1a.
[0147] Staining of cD1c.sup.+DC for CD1c, CD2 and CD14 revealed
that a minor proportion of DC expresses CD14 to a variable degree
and that the level of CD1c as well as CD2 expression on these cells
is inversely proportional to the level of CD14 expression. This
observation is in accordance with a linear differentiation model,
where CD1c.sup.+CD2.sup.+CD11c.sup.brightCD14.sup.-DC are the
progeny of CD14.sup.+CD1c.sup.-CD2.sup.-monocytes rather than the
progeny of a common precursor of both cell types. This concept
finds further support by the observation that a considerable
proportion of CD14.sup.+ monocytes already express very low levels
of CD2 and have the capacity to rapidly differentiate into mature
DC with typical dendritic morphology and potent T cell stimulatory
function when cultured with GM-CSF and IL-4. Crawford et al. (1999)
J. Immunol. 163:5920-5928.
[0148] The use of CD1c (BDCA-1), BDCA-2, BDCA-3 and BDCA-4 mAb
provides a convenient and efficient way to rapidly detect,
enumerate and isolate DC populations from PBMC, leukapheresis
material, whole blood, tonsil, etc., without apparent functional
perturbation. This is a valuable aid for their further functional
and molecular characterization and can be useful in elucidating
their interrelationships. Furthermore, the ability, to easily
isolate DC populations to homogeneity greatly facilitates their
clinical use. The antigen-binding fragments are also useful in
detecting, enumerating and/or isolating DCs from tissues, both
non-hematopoietic tissues (including, without limitation, airway
epithelia, skin, gut, lung, and liver) and hematopoietic tissues
(including, without limitation, tonsil, spleen, lymph node and
thymus).
[0149] Hybridomas secreting the antibodies are also encompassed by
the invention as are other cells expressing antigen-binding
fragments thereof. Also encompassed by the invention are any
antigen-binding fragments that specifically recognize BDCA-2, or
BDCA-3 or BDCA-4. As seen from Table 1 and the Examples provided
herein, multiple types of mAbs can be produced which specifically
recognize these antigens. As also seen from the results presented
herein, the antigen-binding fragments need not recognize the same
epitope on the same antigen. All such antigen-binding fragments and
compositions thereof are encompassed by the invention.
[0150] The term "antigen-binding fragment" includes any moiety that
binds preferentially to a DC or a sub-population thereof. Suitable
moieties include, without limitation, oligonucleotides known as
aptomers that bind to desired target molecules (Hermann and Pantel
(2000) Science 289:820-82), carbohydrates, lectins, Ig fragments as
Fab, F(ab').sub.2, Fab', scFv (both monomer and polymeric forms)
and isolated H and L chains. An antigen-binding fragment retains
specificity of the intact Ig, although avidity and/or affinity can
be altered.
[0151] Certain compounds, compositions and methods described herein
relate generally to antibodies and derivatives thereof which having
provided the antigenic determinants herein, can be generated
routinely by standard immunochemical techniques. These include, but
are not limited to, antigen-binding fragments coupled to another
compound, e.g. by chemical conjugation, or associated with by
mixing with an excipient or an adjuvant. Specific conjugation
partners and methods of making them are described herein and known
in the art.
[0152] Antigen-binding fragments (also encompassing "derivatives"
thereof) are typically generated by genetic engineering, although
they can be obtained alternatively by other methods and
combinations of methods. This classification includes, but is not
limited to, engineered peptide fragments and fusion peptides.
Preferred compounds include polypeptide fragments containing the
anti-DC CDRs, antibody fusion proteins containing cytokine effector
components, antibody fusion proteins containing adjuvants or drugs,
antibody fusion proteins containing tumor cell-derived antigens,
viral antigens, bacterial antigens, parasite antigens, yeast
antigens, autoantigens or antigenic peptides (T cell epitopes)
derived therefrom, and, single chain V region proteins.
Antigen-binding fragments are considered to be of human origin if
they are isolated from a human source, and used directly or cloned
and expressed in other cell types and derivatives thereof or whole
human chromosomes or portions thereof (such as mice with human
chromosomes encoding V.sub.H, D.sub.H, J.sub.H, V.sub.L, JL.sub.L,
C.sub.H, C.sub.L gene segments).
[0153] A "fusion polypeptide" is a polypeptide comprising
contiguous peptide regions in a different position than would be
found in nature. The regions can normally exist in separate
proteins and are brought together in the fusion polypeptide; they
can normally exist in the same protein but are placed in a new
arrangement in the fusion polypeptide; or they can be synthetically
arranged. For instance, the invention encompasses recombinant
proteins (and the polynucleotides encoding the proteins or
complementary thereto) that are comprised of a functional portion
of an antigen-binding fragment and another peptide such as a toxin.
Methods of making these fusion proteins are known in the art and
are described for instance in WO93/07286.
[0154] A "functionally equivalent fragment" of a polypeptide varies
from the native sequence by any combination of additions,
deletions, or substitutions while preserving at least one
functional property of the fragment relevant to the context in
which it is being used.
[0155] The antigen-binding fragments are useful in palliating the
clinical conditions related to immunologic disorders. The invention
further comprises polypeptide derivatives of the antigen-binding
fragments and methods for using these compositions in diagnosis,
treatment, and manufacture of novel reagents.
[0156] The invention also encompasses antigen-binding fragments
conjugated to a chemically functional moiety. Typically, the moiety
is a label capable of producing a detectable signal. These
conjugated antigen-binding fragments are useful, for example, in
detection systems such as quantitation of DCs in various tissues,
in various diseases, after stem cell transplantation, and after
immunoablative therapy like chemotherapy and radiation, and imaging
of DCs for instance in following chemotherapy or autoimmune
therapy. Such labels are known in the art and include, but are not
limited to, radioisotopes, enzymes, fluorescent compounds,
chemiluminescent compounds, bioluminescent compounds, substrate
cofactors and inhibitors and magnetic particles. For examples of
patents teaching the use of such labels, see, for instance U.S.
Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;
4,275,149; and 4,366,241. The moieties can be covalently linked,
recombinantly linked, or conjugated (covalently or non-covalently)
through a secondary reagent, such as a second antibody, protein A,
or a biotin-avidin complex.
[0157] Other functional moieties include, without limitation,
signal peptides, agents that enhance immunologic reactivity, agents
that facilitate coupling to a solid support, vaccine carriers,
bioresponse modifiers, paramagnetic labels and drugs. Signal
peptides include prokaryotic and eukaryotic forms. Agents that
enhance immunologic reactivity include, but are not limited to,
bacterial superantigens and adjuvants. Agents that facilitate
coupling to a solid support include, but are not limited to,
biotin, avidin or derivatives thereof. Immunogen carriers include,
but are not limited to, any physiologically acceptable buffer.
Bioresponse modifiers include, but are not limited to, cytokines,
particularly tumor necrosis factor (TNF), IL-2, interleukin-4
(IL-4), GM-CSF; IL-12, TGF-.beta. and certain interferons, and
chemokines (MIP-3.beta., SDF-1, Lymphotactin, DC-CK1, Eotaxins,
IP-10, TARC, Rantes, MIP-1x, M1P-1B, SLC, 1-TAC, MIG, MDC, MCP-1,
TCA-3, MCP-2, -3, -1. See also, U.S. Pat. No. 5,750,119; and WO
patent publibations: 96/10411; 98/34641; 98/23735; 98/34642;
97/10000; 97/10001; and 97/06821. Such, chemokines may be useful to
attract other cells such as T cells.
[0158] A "signal peptide" or "leader sequence" is a short amino
acid sequence that directs a newly synthesized protein through a
cellular membrane, usually the endoplasmic reticulum (ER) in
eukaryotic cells, and either the inner membrane or both inner and
outer membranes of bacteria. Signal peptides are typically at the
N-terminus of a polypeptide and are removed enzymatically between
biosynthesis and secretion of the polypeptide from the cell or
through the membrane of the ER. Thus, the signal peptide is not
present in the secreted protein but is present only during protein
production.
[0159] Immunotoxins, including single chain conjugates, can be
produced by recombinant means. Production of various immunotoxins
is well known in the art, and methods can be found, for example, in
"Monoclonal Antibody-toxin Conjugates: Aiming the Magic Bullet,"
Thorpe et al. (1982) Monoclonal Antibodies in Clinical Medicine,
Academic Press, pp. 168-190; Vitatta (1987) Science 238:1098-1104;
and Winter and Milstein (1991) Nature 349:293-299. Suitable toxins
include, but are not limited to, ricin, radionuclides, pokeweed
antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin
A chain, fungal toxins such as fungal ribosome inactivating
proteins such as gelonin, restrictocin and phospholipase enzymes.
See, generally, "Chimeric Toxins," Olsnes and Pihl, Pharmac. Ther.
15:355-381 (1981); and "Monoclonal Antibodies for Cancer Detection
and Therapy," eds. Baldwin and Byers, pp. 159-179, 224-266,
Academic Press (1985).
[0160] The chemically functional moieties can be made recombinantly
for instance by creating a fusion gene encoding the antigen-binding
fragment and functional regions from other genes (e.g. enzymes). In
the case of gene fusions, the two components are present within the
same gene. Alternatively, antigen-binding fragments can be
chemically bonded to the moiety by any of a variety of well known
chemical procedures. For example, when the moiety is a protein, the
linkage can be by way of homo- or hetero-bifunctional cross
linkers, e.g., SPDP, SMCC, carbodiimide glutaraldehyde, or the
like. The moieties can be covalently linked, or conjugated, through
a secondary reagent, including, but not limited to, a second
antibody, protein A, or a biotin-avidin complex. Paramagnetic
moieties and the conjugation thereof to antibodies are well-known
in the art. See, e.g., Miltenyi et al. (1990) Cytometry
11:231-238.
[0161] Here, we overcame problems described in the art (O'Doherty
et. al. (1993); and Yamaguchi et al. (1995)) with a recently
described contralateral footpad immunization procedure. Yin et al.
(1997) Blood 90:5002-5012. This system utilizes naive
antigen-specific T and B cells which continuously recirculate among
peripheral lymphoid organs as long as they do not encounter
antigen, but become immediately retained within a peripheral
lymphoid organ for several days, if not weeks, once they are
activated by antigen. Picker et al. (1992) Annu. Rev. Immunol.
10:561-591; Butcher et al. (1996) Science 272:60-66; Bradley et al.
(1996) Curr. Opin. Immunol. 8:312:320; Watson et al. (1998) Cell.
Adhes. Commun. 6:105-110; Kearney et al. (1994) Immunity 1:327-339;
Jacob .et al. (1992) J. Exp. Med. 176:679-687; Ridderstaad et al.
0998) J. Immunol. 160:4688-4695; and Tarlinton (1998) Cum Opin.
Immunol. 10:245-251. In the examples provided herein, the left
footpads of mice were injected on days -3, 0, 4, 7, 11, and 14 with
Bristol-8 B lymphoblastoma cells, while the right footpads were
injected with DC on days 0, 4, 7, 11, and 14. Naive B an T cells
with specificity for shared antigens, e.g. HLA class II molecules,
should become activated by Bristol-8 cells between d -3 and 0 in
the left popliteal lymph node and thereupon be retained there,
while all lymphocytes with specificity for antigens unique to DC
should remain available for activation after d 0 in the right
popliteal lymph node.
[0162] This immunization technique was combined with a powerful
procedure for rapid isolation of large numbers of DC and permitted
production a. panel of mAb that recognize three novel DC antigens:
BDCA-2, BDCA-3 and BDCA-4. The use of antigens in producing
additional DC-specific antibodies allows more traditional methods
of antibody production to be used with a greater chance of
success.
[0163] Methods of antibody production and isolation are well known:
in the art. See, for example; Harlow and Lane (1988) Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York. General
antibody purification methods include, but are not limited to, salt
precipitation (for example, with ammonium sulfate); ion exchange
chromatography (for example, on a cationic or anionic exchange
column run at neutral pH and eluted with step gradients of
increasing ionic strength); gel filtration chromatography
(including gel filtration HPLC); and chromatography on affinity
resins such as protein A, protein G, hydroxyapatite, or anti-Ig.
Antigen-binding fragments can also be purified on affinity columns
comprising DCs or an antigenic portion thereof. Preferably
fragments are purified using Protein-A-CL-Sepharose.TM. 4B
chromatography followed by chromatography on a DEAE-Sepharose.TM.
4B ion exchange column.
[0164] The invention also encompasses hybrid antibodies, in which
one pair of H and L chains is obtained from a first antibody, while
the other pair of H and L chains is obtained from a different
second antibody: For purposes of this invention, one pair of L and
H chains is from anti-DC antibody. In one example, each L-H chain
pair binds different epitopes of a DC-specific antigen. Such
hybrids can also be formed using humanised H or L chains. The
invention also encompasses other bispecific antibodies such as
those containing two separate antibodies covalently linked through
their constant regions.
[0165] Other antigen-binding fragments encompassed by this
invention are antibodies in which the H or L chain has been
modified to provide additional properties. For instance, a change
in amino acid sequence can result in reduced immunogenicity of the
resultant polypeptide. The changes range from changing one or more
amino acid residues to the complete redesign of a region such as a
C region domain. Typical changes include, but are not limited to,
those related to complement fixation, interaction with membrane
receptors, and other effector functions. A recombinant antibody can
also be designed to aid the specific delivery of a substance (such
as a cytokine) to a cell. Also encompassed by the invention are
peptides in which various Ig domains have been placed in an order
other than that which occurs in nature.
[0166] The size of the antigen-binding fragments can be only the
minimum size required to provide: a desired function. It can
optionally comprise additional amino acid sequence, either native
to the antigen-binding fragment, or from. a heterologous source, as
desired. Anti-DC antigen-binding fragments can contain only 5
consecutive amino acid residues from an antibody V region sequence.
Polypeptides comprising 7 amino acid residues, more preferably
about 10 amino acid residues, more preferably about 15 amino acid
residues, more preferably about 25 amino acid residues, more
preferably about 50 amino acid residues, more preferably about 75
amino acid residues from the antibody L or H chain V region are
also included. Even more preferred are polypeptides, comprising the
entire antibody L or H chain V region.
[0167] Substitutions can range from changing or modifying one or
more amino acid residue to complete redesign of a region, such as
the V region. Amino acid residue substitutions, if present, are
preferably conservative substitutions that do not deleteriously
affect folding or functional properties of the peptide. Groups of
functionally related amino acid residues within which conservative
substitutions can be made are glycine/alanine;
valine/isoleucine/leucine; asparagine/glutamine; aspartic
acid/glutamic acid; serine/threonine/methionine; lysine/arginine;
and phenylalanine/tyrosine/tryptophan. Antigen-binding fragments
can be glycosylated or unglycosylated, can be modified
post-translationally (e.g., acetylation, and phosphorylation) or
can be modified synthetically (e.g., the attachment of a labeling
group).
[0168] Polypeptide derivatives comprising both an L chain and an H
chain can be formed as separate L and H chains and then assembled,
or assembled in situ by an expression system for both chains. Such
expression systems can be created by transfecting with a plasmid
comprising separate transcribable regions for the L and H chain, or
by co-transfecting the same cell with plasmids for each chain. In a
third method, a suitable plasmid with an H chain encoding region is
transfected into an H chain loss mutant.
[0169] H chain loss mutants can be obtained by treating anti-DC
antibody producing cells with fluorescein-labeled rabbit anti-mouse
IgG (H chain specific, DAKO Corporation, Carpinteria, Calif.)
according to the supplier's instruction. The stained and unstained
cell populations are analyzed by flow cytometry. Unstained cells
are collected in a sterilized tube and placed in 96-well plates at
1 cell/well by limiting dilution. Culture supernatants are then
assayed by ELISA using goat anti-mouse IgG (H chain specific) and
goat anti-mouse kappa. Clones having a kappa-positive, IgG-negative
phenotype are subcloned at least 3 times to obtain stable
anti-DC.sup.(-H) mutants. mRNA from putative H chain loss mutants
can be isolated and the sequence of the L chain V region cDNA
determined. Reverse PCR of the mRNA for the V.sub.H is performed
with 2 sets of 5'- and 3'-primers, and used for cloning of
anti-DC.sup.(-H) cDNA. An H chain loss mutant yields no detectable
DNA band with these primers. Transfection of the cells proceeds
with a suitable H chain plasmid.
[0170] Another antigen-binding fragment derivative encompassed by
this invention is an antibody in which the constant region of the H
or L chain has been modified to provide additional properties. For
instance, a change in amino acid sequence can result in altered
immunogenicity of the resultant polypeptide. The changes range from
one or more amino acid residues to the complete redesign of
constant region domain. Changes contemplated affect complement
fixation, interaction with membrane receptors, and other effector
functions. A recombinant antibody can also be designed to aid the
specific delivery of a substance (such as a lymphokine or an
antigen or an antigenic peptide derived from a tumor, virus,
parasite or bacteria, or tolerogen (autoantigen)) to a cell. Also
encompassed by the invention are proteins in which various Ig
domains have been placed in an order other than that which occurs
in nature.
[0171] The invention also encompasses single chain V region
fragments ("scFv") of anti-DC antibodies. Single chain V region
fragments are made by linking L and/or H chain V regions by using a
short linking peptide. Bird et al. (1988) Science 242:423-426. Any
peptide having sufficient flexibility and length can be used as a
linker in a scFv. Usually the linker is selected to have little to
no immunogenicity. An example of a linking peptide is (GGGGS).sub.3
(SEQ ID NO:6), which bridges approximately 3.5 nm between the
carboxy terminus of one V region and the amino terminus of another
V region. Other linker sequences can also be used, and can provide
additional functions, such as a for attaching to a drug or solid
support or specific delivery of a substance (such as a lymphokine
or an antigen or an antigenic peptide derived from a tumor, virus,
parasite or bacteria, or tolerogen (autoantigen)) to a cell.
[0172] All or any portion of the H or L chain can be used in any
combination. Typically, the entire V regions are included in the
scFv. For instance, the L chain V region can be linked to the H
chain V region. Alternatively, a portion of the L chain V region
can be linked to the H chain V region, or portion thereof Also
contemplated are scFvs in which the H chain V region is from an
antibody described herein, and the L chain V region is from another
Ig. A biphasic, scFv can be made in which one component is an
antigen-binding fragment and another component is a different
polypeptide, such as a T cell epitope.
[0173] The scFvs can be assembled in any order, for example,
V.sub.H-(linker)-V.sub.L or V.sub.L-(linker)-V.sub.H, There can be
a difference in the level of expression of these two configurations
in particular expression systems, in which case one of these forms
can be preferred. .Tandem scFvs can also be made, such as
(X)-(linker)-(X)-(linker)-(X), in which X are scFvs, or
combinations thereof with other polypeptides. In another
embodiment, single chain antibody polypeptides have no linker
polypeptide, or just a short, inflexible linker. Possible
configurations are V.sub.L-V.sub.H and V.sub.H-V.sub.L. The linkage
is too short to permit interaction between V.sub.L and V.sub.H
within the chain, and the chains form homodimers with a
V.sub.L/V.sub.H antigen-binding site at each end. Such molecules
are referred to as "diabodies."
[0174] ScFvs can be produced recombinantly or synthetically. For
synthetic production of scFv, an automated synthesizer can be used.
For recombinant production of scFv, a suitable plasmid-containing
polynucleotide that encodes the scFv can be introduced into a
suitable host cell, either eukaryotic, such as yeast, plant, insect
or mammalian cells, or prokaryotic, such as Escherichia coli, and
the expressed protein can be isolated using standard protein
purification techniques. ScFvs can also be obtained from a phage
display library.
[0175] A particularly useful system for the production of scFvs is
plasmid pET-22b(+) (Novagen, Madison, Wis.). Escherichia coli
pET-22b(+) contains a nickel ion binding domain consisting of 6
sequential histidine residues, which allows the expressed protein
to be purified on a suitable affinity resin. Another example of a
suitable vector is pcDNA3 (Invitrogen, San Diego, Calif.).
[0176] Conditions of gene expression preferably ensure that the
scFv assumes optimal tertiary structure. Depending on the plasmid
used (especially promoter activity), and the host cell, it can be
necessary to modulate production rate. For instance, use of a
weaker promoter, or expression at lower temperatures, can be
necessary to optimize production of properly folded scFv in
prokaryotic systems; or, it can be used to express scFv in
eukaryotic cells.
[0177] The invention also encompasses polymeric forms of
antigen-binding fragments, containing a plurality of DC-specific
antigen-binding fragments. One embodiment is a linear polymer of
antigen-binding fragments, optionally conjugated to carrier. These,
linear polymers can comprise multiple copies of a single
antigen-binding fragment polypeptide, or combinations of different
polypeptides, and can have tandem polypeptides, or polypeptides
separated by other amino acid sequences.
[0178] Another embodiment is multiple antigen peptides (MAPs). MAPs
have a small immunologically inert core having radially
branching-lysine dendrites, onto which a number of antigen-binding
fragment polypeptides are covalently attached. See for instance,
Posnett et al. (1988) J. Biol. Chem. 263:1719-1725; and Tam (1989)
Met. Enz. 168:7-15. The result is a large macromolecule having a
high molar ratio of antigen-binding fragment polypeptides to core.
MAPs are efficient immunogens and useful antigens for immunoassays.
The core for creating MAPs can be made by standard peptide
synthesis techniques, or obtained commercially (Quality Controlled
Biochemicals, Inc., Hopkinton, Mass.). A typical core matrix is
made up of three levels of lysine and eight amino acid
residues.
[0179] The invention further includes anti-idiotypic
antigen-binding fragments to the DC-specific antigen-binding
fragments of the invention. Such anti-idiotypes can be made by any
method known in the art.
[0180] Cancer patients are often immunosuppressed and tolerant to
some tumor-associated antigens (TAA). Triggering an active immune
response to such TAA represents an important challenge in cancer
therapy. Immunization with a given antigen generates an immune
response including a CTL response, preferably a strong CTL
response. The production of antibodies against the antigen can be
helpful if the tumor cells are killed by ADCC (antibody-dependent
cellular cytotoxicity). The invention encompasses the use of DCs
identified and isolated by use of the antigen-binding fragments of
the invention in inducing specific immune responses by methods
known in the art. The immune responses can be specific to any
antigen including, without limitation, those associated with
cancer, infectious viruses, infectious bacteria, infectious
parasites, infectious yeast, and autoimmune diseases (the induce
tolerance). The ability to isolate subpopulations that are uniquely
suited to inducing such a response results in preparations of DCs
that are more effective than mixtures of subpopulations. Hybrid
cells (e.g. DC/tumor cell) could also be used as cancer-specific
therapy. Modified cells (including, without limitation, activated,
in vitro matured, modulated with respect to their T helper cell
polarizing capacity (Th1 v Th2 v Th3/Th-R), and modulated with
respect to their T cell stimulating or anergizing or deleting
capacity) are likewise encompassed by the invention and include,
but are not limited to, genetically modified or transfected cells
and cells that have been incubated with peptides or proteins
suitable for antigen presentation or for internalization.
Subpopulations include, without limitation, a particular
differentiation stage within one lineage and a separate lineage of
differention.
[0181] The invention further provides DCs, subpopulations thereof
and mixtures thereof. The cells are selected using the
antigen-binding fragments provided herein by any separation method
known in the art. Compositions comprising the isolated cells are
also encompassed by the invention. These include pharmaceutical and
therapeutic compositions and any other composition containing the
isolated cells. The DCs subpopulations isolated by the methods
described herein are preferably substantially homogeneous. That is,
cells isolated by a BDCA-specific antigen-binding fragments are
preferably more than about 80% BDCA.sup.+, more preferably more
than about 90% BDCA.sup.+ and most preferably more than about 95%
BDCA.sup.+. Of course, subsequent combinations of the cells with
other DCs, or other hematopoietic cells can decrease the percentage
of BDCA.sup.+ cells, such combinations are also encompassed by the
invention.
[0182] Likewise, the DCs obtained by the methods described herein
are suitable for use in any method of treatment known in the art
include references here. DCs altered to achieve these methods are
also encompassed by the invention. These methods include, but are
not limited to:
[0183] a) therapy with isolated DCs to induce specific T cell
tolerance (killing or anergy instead of stimulation) in autoimmune
diseases, allergies, graft versus host disease (GvHD), allograft
rejection. For instance, DCs specific for such T cells can be
modified to, contain lysis, inactivating or death-inducing moieties
SQ as to specifically a. target the T cells involved in the
unwanted immune response for instance by antigen labeling or
genetic modification such as by CD95L transfection. DC specificity
for T cells is primarily caused by presentation of the appropriate
T cell epitopes (peptides) via MTIC I and IL The particular subsets
of DCs with tolerance-inducing functions can be administered
directly to the patient. Peripheral tolerance can be mediated by
DCs modified to induce deletion (killing), anergy and
suppression/regulation of T cells; [0184] b) immunomodulation
therapy with isolated DCs to induce particular cytokine expression
profiles in specific T cells. This is particularly useful to
influence production of Th1 (cytokines for specific inflammatory
immune responses), Th2 (cytokines for specific humoral immune
responses) or Th3 (cytokines for specific immunosuppression)
cytokines. In the case of allergies and asthma for instance,
induction of a Th1 response may reduce or eliminate the
symptom-producing Th2 response;
[0185] c) therapy with DCs presenting antigens including, but not
limited to, tumor antigens, viral antigens and cellular
antigens;
[0186] d) therapy with DCs (with or without presenting antigens)
and various cofactors including, but not limited to cytokines,
costimulatory molecules and effector molecules in amounts and under
conditions sufficient to modulate the immune response; and [0187]
e) stimulating T cells in vitro to obtain antigen-specific T
cells.
[0188] The antigen-binding fragments described herein are also
suitable for a. number of methods of treatment. These include, but
are not limited to:
[0189] a) antibodies mimicking the ligand- or ligand-mediated
immunotherapy--for instance of DCs involved in autoimmunity or in
vivo targeting of antigens or nucleic acids (viruses, plasmid DNA,
RNA etc) to DCs for optimal and selective uptake/transfection.
BDCA-2 may be particularly useful in this context as it appears to
be a molecule with antigen uptake and processing function; and
[0190] b) immunomonitoring: e.g. enumeration and characterization
of BDCA-2.sup.+, BDCA-3.sup.+ and BDCA-4.sup.+DCs in various
diseases and upon mobilization e.g. with a proliferation inducing
ligand, e.g. flt3-Ligand or G-CSF.
[0191] Any carrier not harmful to the host can be used for the DCs.
Suitable carriers are typically large, slowly metabolized
macromolecules such as proteins; polysaccharides (such as latex
functionalized Sepharose, agarose, cellulose, cellulose beads and
the like); polymeric amino acid residues (such as polyglutamic
acid, polylysine, and the like); amino acid copolymers; and
inactive virus particles or attenuated bacteria, such as
Salmonella.
2. Methods of Obtaining Additional DC-Specific Antigen-Binding
Fragments
[0192] The invention encompasses methods of obtaining DC-specific
antigen-binding fragments.
[0193] Methods of generating new DC-specific antigen-binding
fragments, as detailed below, include, but are not limited to: 1)
employing phage display techniques by which cDNA encoding antibody
repertoires are preferably amplified from lymphocyte or spleen RNA
using PCR and oligonucleotide primers specific for V regions; 2)
immunizing mammals with the antigen and generating polyclonal or
mAbs; and 3) employing phage display to make antibodies without
prior immunization by displaying on phage, very large and diverse V
gene repertoires. See, generally Hoogenboom et al. (1998)
Immunotechnol. 4:1-20. Preferably, for therapeutic purposes, if
non-human antigen binding fragments are to be used, these can be
humanized by any method known in the art.
[0194] The method described by Medez et al. (1997) Nature Genetics
18:410 can be used. Briefly, purified antigen; is used to immunize
transgenic mice lacking the native murine antibody repertoire and
instead having most of the human antibody V-genes in the germ line
configuration. Human antibodies are subsequently produced by the
murine B cells. The antibody genes are recovered from the B cells
by PCR library selection or classic hybridoma technology.
[0195] Alternatively, antibodies can be obtained from mice (such
as, BALB/c) after injection with purified DC-specific antigen. mAbs
are generated using standard hybridoma technology. Maiti et al.
(1997) Biotechnol. Int. 1:85-93 (human hybridornas); and Kohler and
Milstein (1975) Nature 256:495-497 (mouse hybridomas). Murine
antibodies can be subsequently humanized for instance by the
methods described by Rosok et al. (1996) J. Biol. Chem.
271:22611-22618; Baca et al. (1997) J. Biol. Chem. 272:10678-10684;
Rader et al. Proc. Natl. Acad. Sci. USA 95:8910-8915; and Winter
and Milstein (1991) Nature 349:293-299.
[0196] A phage display approach can also be used to rapidly
generate human antibodies against DCs. This approach can employ the
method described by Henderikx et al. (1998) Cancer Res. 58:4324-32.
Antibody fragments displayed on phage are selected from a large
naive phage antibody/fragment library containing different single
chain autibodies by separating those that bind to immobilized
antigen or DCs. Human antibody fragments are selected from naive
repertoires constructed either from germline V-domains or
synthesized with many mutations (mutations are targeted either by
homologous gene re-assortments or error prone PCR) in both the
framework and CDR regions. Antigen-binding fragments specifically
reactive with DCs can be identified by screening against tumor and
normal cells as described herein in order to identify DC-specific
antigen-binding fragments.
[0197] The invention also encompasses methods of identifying
antigen-binding fragments specific for a DCs by generating a
suitable phage display library; isolating DC-specific antigens;
screening the phage display library with the antigens according to
standard immunochemical techniques to obtain phage that display an
antigen-binding-fragment that binds specifically to DCs; or
screening the phage display library obtained for DC specific
antigen-binding fragments, by screening against DCs and other,
related cells such as APCs and selecting the phage that bind
preferentially to DCs. Methods of generating antigen-binding
fragments by phage display are well known in the art. Hoogenboom et
al. (1998).
[0198] Lymphocyte (PBL) or spleen RNA is typically used to make
antibody fragment repertoires. Mutagenesis using homologous
reassortment or error prone PCR increases the diversity. Any method
known in the art can be used.
[0199] Repertoires of antibody genes can be amplified from
immunized mice or humans using PCR and scFv or Fab antibody
fragments obtained thereby can be cloned and expressed on the
surface of bacteriophage. The antibody gene repertoires are
amplified from lymphocyte or spleen RNA using PCR and
oligonucleotide primers specific for host animal-specific V
regions. Phage display can also be used to make antibodies without
prior immunization by displaying very large and diverse V gene
repertoires on phage. The natural V gene repertoires present in PBL
are isolated by PCR amplification and the VII and VL regions are
spliced together at random using PCR. Mutations can be targeted to
the V-domain genes by homologous gene reassortments or error-prone
PCR. Zhao et al. (1998) Nat. Biotechnol. 15:258; and Hoogenboom et
al. (1998). Totally synthetic human libraries can also be created
and used to screen for DC-specifics, antibody fragments. Regardless
of the method used to operate the phage display library, each
resulting phage has a functional antibody fragment displayed on its
surface and contains the gene encoding the antibody fragment in the
phage genome. See, e.g. WO97/02342.
[0200] Affinity chromatography in which binding antibodies can be
subtracted from non-binding antibodies has been established for
sometime. Nissim et al. (1994) EMBO J. 13:692-698; and Vaughan et
al. (1996) Nat. Biotechnol. 14:309-314. Critical parameters
affecting success are the number and affinity of antibody fragments
generated against a particular antigen. Until recently, the
production of large, diverse libraries remained somewhat difficult.
Historically, scFv repertoires have been assembled directly from VH
and VL RT-PCR products. RNA availability and the efficiency of
RT-PCR were limiting factors of the number of V genes available.
Also, assembly required ligating three fragments, namely VH and VL
and the linker regions. Marks et al. (1991) J. Mol. Biol.
222:581-597.
[0201] An improved library construction method uses cloned VH and
VL gene repertoires in separate plasmid vectors to provide a stable
and limitless supply of material for scFv assembly. Sheets et al.
(1998) Proc. Natl. Acad. Sci. USA 95:6175-6162. Also, the
efficiency is increased by having DNA encoding the linker region at
the 5' end of the VL library. Therefore there are only two
fragments to be ligated instead of three.
[0202] Anti-DC-antigen-binding fragments can also be derived or
manipulated. For example, the immunogenic activity of the V regions
of the L and H chains can be screened by preparing a series of
short polypeptides that together span the entire V region amino
acid sequence. Using a series of polypeptides of 20 or 50 amino
acid residues in length, each V region can be surveyed for useful
functional properties. It is also possible to carry out a computer
analysis of a protein sequence to identify potentially immunogenic
polypeptides. Such peptides can then be synthesized an tested.
[0203] The invention further encompasses various adaptations of
antigen-binding fragments described herein combined in various
fashions to yield other anti-DC antigen-binding fragments with
desirable properties. For instance, antigen-binding fragments with
modified residues can be comprised in MAPs. In another example, an
scEv is fused to a cytokine, such as IL-2. All such combinations
are encompassed by this invention.
[0204] The antigen-binding fragments can be made by any suitable
procedure, including proteolysis, recombinant methods or chemical
syntheses. These methods are known in the art and need not be
described in detail. Examples of proteolytic enzymes include, but
are not limited to, trypsin, chymotrypsin, pepsin, papain, V8
protease, subtilisin, plasmin, and thrombin. Intact antigen-binding
fragments can be incubated with one or more proteases
simultaneously or sequentially. Alternatively, or in addition,
intact antibody can be treated with disulfide reducing agents.
Peptides can then be separated from each other by techniques known
in the art, including but not limited to, gel filtration
chromatography, gel electrophoresis, and reverse-phase HPLC.
[0205] Anti-DC antigen-binding fragments can also be made by
expression from a polynucleotide encoding the peptide, in a
suitable expression system by any method known in the art.
Typically, polynucleotides encoding a suitable polypeptide are
ligated into an expression vector under control of a suitable
promoter and used to genetically alter the intended host cell. Both
eukaryotic and prokaryotic host systems can be used. The
polypeptide is then isolated from lysed cells or from the culture
medium and purified to the extent needed for its intended use.
Examples of prokaryotic host cells appropriate for use with this
invention include E. coli, Bacillus subtilis and any other suitable
host cell. Examples of eukaryotic host cells include, but are not
limited to yeast, avian, insect, plant, and animal cells such as
COST, HeLa, and CHO cells.
[0206] Optionally, matrix-coated channels or beads and cell
co-cultures can be included to enhance growth of antigen-binding
fragment producing cells. For the production of large amounts of
mAbs, it is generally more convenient to obtain ascitic fluid. The
method of raising ascites generally comprises injecting hybridoma
cells into an immunologically naive, histocompatible or
immunotolerant mammal, especially a mouse. The mammal can be primed
for ascites production by prior administration of a suitable
composition; e.g., Pristane. The ascitic fluid is removed from the
animal and processed to isolate antibodies.
[0207] Alternatively, antigen-binding fragments can be chemically
synthesized using amino acid sequence data and other information
provided in this disclosure, in conjunction with standard methods
of protein synthesis. A suitable method is the solid phase
Merrifield technique. Automated peptide synthesizers are
commercially available, such as those manufactured by Applied
Biosystems, Inc. (Foster City, Calif.).
[0208] Another method of obtaining anti-DC antigen-binding
fragments is to immunize suitable host animals with BDCA-2, BDCA-3
and/or BDCA-4 and follow standard methods for polyclonal or mAb
production and isolation. mAbs thus produced can be "humanized" by
methods known in the art. The invention thus encompasses humanized
mAbs.
[0209] In "humanized" antibodies at least part of the sequence has
been altered from its initial form to render it more like human
Igs. In one version, the H chain and L chain C regions are replaced
with human sequence. This is a fusion polypeptide comprising an
anti-DC V region and a heterologous Ig (C) region. In another
version, the CDR regions comprise anti-DC amino acid sequences,
while the V framework regions have also been converted human
sequences. See, for example, EP 0329400. In a third version, V
regions are humanized by designing consensus sequences of human and
mouse V regions, and converting residues outside the CDRs that are
different between the consensus sequences.
[0210] In making humanized antibodies, the choice of framework
residues can aid in retaining high binding affinity. In principle,
a framework sequence from any human antibody can serve as the
template for CDR grafting; however, it has been demonstrated that
straight CDR replacement into such a framework can lead to
significant loss of antigen binding affinity. Glaser et al. (1992)
J. Immunol. 149:2606; Tempest et al. (1992) Biotechnol. 9:266; and
Shalaby et al. (1992) J. Exp. Med. 17:217. The more homologous a
human antibody is to the original murine antibody, the less likely
that the human framework will introduce distortions into the murine
CDRs that could reduce affinity. Based on a sequence homology
search against an antibody sequence database, the human antibody
IC4 provides good framework homology to muM4TS.22, although other
highly homologous human antibodies are suitable as well, especially
.kappa. L chains from human subgroup I or H chains from human
subgroup III. Kabat et al. (1987). Various computer programs such
as ENCAD predict the ideal sequence for the V region. Levitt et al.
(1983) J. Mol. Biol. 168:595. The invention thus encompasses human
antibodies with different V regions. It is within the skill of one
in the art to determine suitable V region sequences and to optimize
these sequences. Methods for obtaining antibodies with reduced
immunogenicity are also described in U.S. Pat. No. 5,270,202 and EP
699,755.
[0211] In certain applications, such as when an antigen-binding
fragment or DCA is expressed in a suitable storage medium such as a
plant seed, the antigen-binding fragment can be stored without
purification. Fiedler et al. (1995) Biotechnol. 13:1090-1093. For
most applications, it is generally preferable that the polypeptide
is at least partially purified from other cellular constituents.
Preferably, the peptide is at least about 50% pure as a weight
percent of total protein. More preferably, the peptide is at least
about 50-75% pure. For clinical use, the peptide is preferably at
least about 80% pure.
[0212] If the peptides are to be administered to an individual,
preferably it is at least 80% pure, more preferably at least 90%
pure, even more preferably at least 95% pure and free of pyrogens
and other contaminants. In this context, the percent purity is
calculated as a weight percent of the total protein content of the
preparation, and does not include constituents which are
deliberately added to the composition purification.
[0213] The invention also encompasses methods of detecting,
enumerating and/or identifying DCs and subsets thereof, in a
biological sample and measuring antigens such as soluble BDCA-2,
BDCA-3 or BDCA-4 and/or DCs in body fluids. The methods include
obtaining a biological sample, contacting the sample with an
antigen-binding fragment described herein under conditions that
allow antibody-antigen-binding and detecting binding, if any, of
the antibody to the sample as compared to a control, biological
sample.
[0214] After a biological sample is suitably prepared, for instance
by enriching for DC concentration or antigen concentration, it is
mixed with excess antigen-binding fragments under conditions that
permit formation of a complex between DCs or antigen and the
antibody. The amount of complex formed or the number of complex
bearing DCs then determined, and eventually compared with complexes
formed with standard samples containing known amounts of target
antigen in the range expected or known DC concentrations. Complex
formation can be observed by immunoprecipitation or nephelometry,
but it is generally more sensitive to employ a reagent labeled with
such labels as radioisotopes like .sup.125I, enzymes like
peroxidase and .beta.-galactosidase, or fluorochromes like
fluorescein. Methods of detecting cells and antigens are well known
in the art. For cell detection, flow cytometry is particularly
useful, with antigen, ELISA is preferred.
[0215] The specific recognition of an anti-DC antigen-binding
fragment to an antigen can be tested by any immunoassay known in
the art. Any form of direct binding assay is suitable. In one such
assay, one of the binding partners, the antigen or the putative
antigen-binding fragment, is labeled. Suitable labels include, but
are not limited to, radioisotopes such as .sup.125I, enzymes such
as peroxidase, fluorescent labels such as fluorescein, and
chemiluminescent labels. Typically, the other binding partner is
insolubilized (for example, by coating onto a solid phase such as a
microtiter plate) to facilitatexemoval of unbound soluble binding
partner. After combining the labeled binding partner with the
unlabeled binding partner, the solid phase is washed and the amount
of bound label is determined.
[0216] When used for immunotherapy, the antigen-binding fragments
described herein can be unlabeled or labeled with a therapeutic
agent as described herein and as known in the art. These agents can
be coupled either directly or indirectly to the antigen-binding
fragments of the invention. One example of indirect coupling is by
use of a spacer moiety. These spacer moieties, in turn, can be
either insoluble or soluble (Diener et al. (1986) Science 231:148)
and can be selected to enable drug release at the target site.
Examples of therapeutic agents that can be coupled to
antigen-binding fragments for immunotherapy include, but are not
limited to, antigens, including tumor antigens, viral antigens,
bacterial antigens, parasite-derived antigens and autoantigens,
bioresponse modifiers, drugs, radioisotopes, lectins, and toxins:
Bioresponse modifiers include cytokines and chemokines which
include, but are not limited to, IL-2, IL-3, IL-4, G-CSF, GM-CSF,
IL-10, IL-12, TGF-.beta., MTP-AB, SDF-1, Lymphotactin, DC-CK1,
Eotoxins, IP-10, TARC, Rantes, MIP-1.alpha., MPP-1.beta., SLC,
ITAC, MIE, MDC, MCP-1, TCA-3, MCP-2, -3, -4 and interferons.
Interferons with which antigen binding' fragments can be labeled
include IFN-.alpha., IFN-.beta., and IFN-.gamma. and their
subtypes.
[0217] In using radioisotopically conjugated antigen-binding
fragments for immunotherapy, certain isotypes can be more
preferable than others depending on such factors as isotype
stability and emission. If desired, cell population recognition by
the antigen-binding fragment can be evaluated by the in vivo
diagnostic techniques described below. In general, .alpha. and
.beta. particle-emitting radioisotopes are preferred in
immunotherapy. For example, a high energy .beta. emitter capable of
penetrating several millimeters of tissue, such as .sup.90Y, can be
preferable. On the other hand, a short range, high energy a
emitter, such as .sup.212Bi, can be preferable. Examples of
radioisotopes which can be bound to the antigen-binding fragments
of the invention for therapeutic purposes include, but are not
limited to, .sup.125I, .sup.131I, .sup.90Y, .sup.67Cu, .sup.212Bi,
.sup.211At, .sup.212Pb, .sup.47Sc, .sup.109Pd, and .sup.188Re.
[0218] Lectins are proteins, usually isolated from plant material,
which bind to specific sugar moieties. Many lectins are also able
to agglutinate cells and stimulate lymphocytes. However, ricin is a
toxic lectin which has been used immunotherapeutically. This is
preferably accomplished by binding the .alpha. peptide chain of
ricin, which is responsible for toxicity, to the antibody molecule
to enable site specific delivery of the toxic effect.
[0219] Toxins are poisonous substances produced by plants, animals,
or microorganisms that, in sufficient dose, are often lethal.
Diphtheria toxin is a substance produced by Cotynebacterium
diphtheria which can be used therapeutically. This toxin consists
of an .alpha. and .beta. subunit which under proper conditions can
be separked. The toxic A chain component can be bound to an
antigen-binding fragment described herein and used for site
specific delivery to a specific subset of DCs.
[0220] Recombinant methods are well known in the art. The practice
of the invention employs, unless otherwise indicated, conventional
techniques of molecular biology (including recombinant techniques),
microbiology, cell biology, biochemistry and immunology, which are
within the skill of the art. Such techniques are explained fully in
the literature, such as, "Molecular Cloning: A Laboratory Manual",
second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis"
(Gait, ed., 1984); "Animal Cell Culture" (Freshney, ed., 1987);
"Methods in, Enzymology" (Academic Press, Inc.); "Handbook of
Experimental Immunology" (Wei & Blackwell, eds.); "Gene
Transfer Vectors for Mammalian Cells" (Miller & Calos, eds.,
1987); "Current Protocols in Molecular Biology" (Ausubel et al.,
eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al.,
eds., 1994); and "Current Protocols in Immunology" (Coligan et al.,
eds., 1991). These techniques are applicable to the production of
the polynucleotides and polypeptides, and, as such, can be
considered in making and praCticing the invention. Particularly
useful techniques are discussed in the sections that follow.
[0221] The invention provides various polynucleotides encoding BDCA
antigens. The invention also encompasses polynucleotides encoding
for functionally equivalent variants and derivatives of these
antigens and functionally equivalent fragments thereof that can
enhance, decrease or not significantly affect properties of the
polypeptides encoded thereby. These functionally equivalent
variants, derivatives, and fragments may display the ability to
specifically bind to their respective antibodies. For instance,
changes that will not significantly affect properties of the
encoded polypeptide include, but are not limited to changes in a
DNA sequence that do not change the encoded amino acid sequence, as
well as those that result in conservative substitutions of amino
acid residues, one or a few amino acid residue deletions or
additions, and substitution of amino acid residues by amino acid
analogs. Conservative substitutions are those which conservative
amino acid substitutions are glycine/alanine;
valinensoleucine/leucine; asparagine/glutamine; aspartic
acid/glutamic acid; serine/threonine/methionine; lysine/arginine;
and phenylalanine/tyrosine/tryptophan.
[0222] The polynucleotides of the invention can comprise additional
sequences, such as additional encoding sequences within the same
transcription unit, controlling elements such as promoters,
ribosome binding sites, and polyadenylation sites, additional
transcription units under control of the same or a different
promoter, sequences that permit cloning, expression, and
transformation of a host cell, and any such construct as can be
desirable to provide embodiments of this invention.
[0223] The invention encompasses a polynucleotide of at least about
15 consecutive nucleotides, preferably at least about 20
nucleotides, more preferably at least about 25 consecutive
nucleotides, more preferably at least about 35 consecutive
nucleotides, more preferably at least about 50 consecutive
nucleotides, even more preferably at least about 75 nucleotides,
still more preferably at least about 100 nucleotides, still more
preferably at least about 200 nucleotides, and even more preferably
at least about 300 nucleotides that forms a stable hybrid with a
polynucleotide. encoding BDCA-2 and BDCA-3, preferably the cDNA
sequence found in FIG. 12. Any set of conditions can be used for
this test, provided at least one set exists where the test
polynucleotide demonstrates the required specificity.
[0224] Hybridization reactions can be performed under conditions of
different "stringency". Conditions that increase stringency of a
hybridization reaction are published. See, for example, Sambrook
and Maniatis. Examples of relevant conditions include (in order of
increasing stringency): incubation temperatures of 25.degree. C.,
37.degree. C., 50.degree. C. and 68.degree. C.; buffer
concentrations of 10.times.SSC, 6.times.SSC, 1.times.SSC,
0.1.times.SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer)
and their equivalent using other buffer systems; formamide
concentrations of 0%, 25%, 50%, and 75%; incubation times from 5
minutes to 24 hours; 1, 2, or more washing steps; wash incubation
times oil, 2, or 15 minutes; and wash solutions of 6.times.SSC,
1.times.SSC, 0.1.times.SSC, or deionized water.
[0225] The invention also provides polynucleotides encoding the
BDCA polypeptides. Preferably, the polypeptides are or are derived
from those in. FIG. 12.
[0226] The invention also provides polynucleotides covalently
linked with a detectable label. Such polynucleotides are useful,
for example, as probes for detection of related nucleotide
sequences.
[0227] The polynucleotides of this invention can be obtained using
chemical synthesis, recombinant cloning methods, PCR, or any
combination thereof. Methods of chemical polynucleotide synthesis
are well known in the art and need not be described in detail
herein. One of skill in the art can use the sequence data provided
herein to obtain a desired polynucleotide by employing a DNA
synthesizer or ordering from a commercial service.
[0228] Alternatively, nucleotides encoding BDCAs and the peptides
encoded thereby can be obtained from a producing cell line, cloning
vector, or expression vector. RNA or DNA encoding the desired
sequence can be isolated, amplified, and processed by standard
recombinant techniques. Such techniques include digestion with
restriction nucleases, and amplification by polymerase chain
reaction (PCR), or .a suitable combination thereof. PCR technology
is described in U.S. Pat. Nos. 4,683,195; 4,800,159; 4,754,065; and
4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et
al. eds., Birkauswer Press, Boston (1994). Isolation and
purification of the peptides encoded thereby can be by any method
known in the art.
[0229] Polynucleotides comprising a desired sequence can be
inserted into a suitable vector, the vector in turn can be
introduced into a suitable host cell for replication and
amplification. Polynucleotides can be inserted into host cells by
any means known in the art. Cells are transformed by introducing an
exogenous polynucleotide by direct uptake, endocytosis,
transfection, f-mating or electroporation. Once introduced, the
exogenous polynucleotide can be maintained within the cell as a
non-integrated vector (such as a plasmid) or integrated into the
host cell genome. Amplified DNA can be isolated from the host cell
by standard methods. See, e.g., Sambrook et al. (1989). RNA can
also be obtained from transformed host cell, it can be obtained by
using a DNA-dependent RNA polymerase.
[0230] The present invention further includes a variety of vectors
comprising a polynucleotide encoding BDCA-2 and/or BDCA-3. These
vectors can be used for expression of recombinant polypeptides as
well as a source of BDCA-encoding polynucleotides.' Cloning vectors
can be used to obtain replicate copies of the polynucleotides, or
for storing the polynucleotides in a depository for future
recovery. Expression vectors (and host cells containing these
expression vectors) can be used to obtain polypeptides produced
from the polynucleotides they contain. They can also be used where
it is desirable to express BDCA-2 and/or BDCA-3 in an individual
and thus have intact cells capable of synthesizing the polypeptide,
such as in gene therapy. Suitable cloning and expression vectors
include any known in the art e.g., those for use in bacterial,
mammalian, yeast and insect expression systems. Specific vectors
and suitable host cells are known in the art and are not described
in detail herein. See e.g. Gacesa and Ramji, Vectors, John Wiley
& Sons (1994).
[0231] Cloning and expression vectors typically contain a
selectable marker (for example, a gene encoding a protein necessary
for the survival or growth of a host cell transformed with the
vector), although such a marker gene can be carried on another
polynucleotide sequence co-introduced into the host cell. Only
those host cells into which a selectable gene has been introduced
will grow under selective conditions. Typical selection genes
either: (a) confer resistance to antibiotics or other toxic
substances, e.g., ampicillin, neomycin, methotrexate; (b)
complement auxotrophic deficiencies; or (c) supply critical
nutrients not available from complex media. The choice of the
proper marker gene will depend on the host cell, and appropriate
genes for different hosts are known in the art. Vectors also
typically contain a replication system recognized by the host.
[0232] Suitable cloning vectors can be constructed according to
standard techniques, or can be selected from a large number of
cloning vectors available in the art. While the cloning vector
selected can vary according to the host cell intended to be used,
useful cloning vectors will generally have the ability to
self-replicate, can possess a single target for a particular
restriction endonuclease, or can carry genes for a marker that can
be used in selecting clones containing the vector. Suitable
examples include plasmids and bacterial viruses, e.g., pUC18, mp18,
mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle
vectors such as pSA3 and pAT28. These and many other cloning
vectors are available from commercial vendors such as BioRad,
Stratagene, and Invitrogen.
[0233] EXpression vectors generally are replicable polynucleotide
constructs that contain a polynucleotide encoding a BDCA of
interest. The polynucleotide encoding BDCA is operatively linked to
suitable transcriptional controlling elements, such as promoters,
enhancers and terminators. For expression (i.e., translation), one
or more translational controlling elements are also usually
required, such as ribosome binding sites, translation initiation
sites, and stop codons. These controlling elements (transcriptional
and translational) can be derived from a gene encoding a BDCA, or
they can be heterologous (i.e., derived from other genes or other
organisifs). A polynucleotide sequente encoding a signal peptide
can also be included to allow a BDCA to cross or lodge in cell
membranes' or be secreted from the cell. A number of expression
vectors suitable for expression in eukaryotic cells including
yeast, avian, and mammalian cells are known in the art. One example
of an expression vector is pcDNA3 (Invitrogen, San Diego, Calif.),
in which transcription is driven by the cytomegalovirus (CMV) early
promoter/enhancer. This vector also contains recognition sites for
multiple restriction enzymes for insertion of the polynucleotide of
interest. Another example of an expression vector (system) is the
baculovirus/insect system. Other suitable for use in
antibody-targeted gene therapy comprising a polynucleotide encoding
a BDCA. Suitable systems are described for instance by Brown et al.
(1994) Virol. 198:477-488; and Miyamura et al. (1994) Proc. Natl.
Acad. Sci. USA 91:8507-8511.
[0234] The vectors containing the polynucleotides of interest can
be introduced into the host cell by any of a number of appropriate
means, including electroporation, transfection employing calcium
chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection; and
infection. The choice of means of introducing vectors or
polynucleotides encoding BDCAs will often depend on features of the
on the host cell.
[0235] Once introduced into a suitable host cell, expression of a
BDCA can be determined using any assay known in the art. For
example, the presence thereof can be detected by RIA or ELISA of
the culture supernatant (if the polypeptide is secreted) or cell
Iysates.
[0236] A vector of this invention can contain one or more
polynucleotides encoding a BDCA. It can also contain polynucleotide
sequences encoding other polypeptides that enhance, facilitate, or
modulate the desired result, such as cytokines, including, but not
limited to, IL-2, IL-4, GM-CSF, TNF-.alpha. and IFN-.gamma.. Also
embodied in this invention are vaccinia vectors encoding for
recombinant BDCAs.
[0237] Other embodiments of this invention are host cells
transformed with polynucleotides encoding BDCAs and vectors
comprising the polynucleotide sequences, as described above. Both
prokaryotic and eukaryotic host cells can be used.
[0238] Prokaryotic hosts include, but are not limited to, bacterial
cells, for example E. coli and mycobacteria. Eukaryotic hosts
include, but are not limited to, yeast, insect, avian, plant and
mammalian cells. Host systems are known in the art and need not be
described in detail herein. Examples of a mammalian host cells
include, but are not limited to, CHO and NSO, obtainable from the
European Collection of Cell Cultures (England). Transfection of NSO
cells with a plasmid, for example, which is driven by a CMV
promoter, followed by amplification of this plasmid in using
glutamine synthetase provides a useful system for protein
production. Cockett et al. (1990) Bio/Technology 8:662-667.
[0239] The host cells of this invention can be used, inter alia, as
repositories of polynucleotides encoding BDCAs, or as vehicles for
production thereof. They can be used also as vehicles for in vivo
expression of BDCAs.
[0240] The polynucleotides of this invention have several uses.
They are useful, for example, in expression systems for the
production of BDCA. They are also useful as hybridization probes to
assay for the presence of polynucleotides encoding BDCAs or related
sequences in a sample using methods well known to those in the art.
Further; the polynucleotides are also useful as primers to effect
amplification of desired polynucleotides. The polynucleotides of
this invention are also useful in pharmaceutical compositions
including vaccines and for gene therapy.
[0241] The polynucleotides can also be used as hybridization probes
for detection of BDCA-encoding sequences. Suitable hybridization
samples include cells transformed ex vivo for use in gene therapy.
In one illustration, DNA or RNA is extracted from a sample, and
optionally run on a gel and/or digested with restriction nucleases.
The processed sample polynucleotide is typically transferred to a
medium suitable for washing. The sample polynucleotide is then
contacted with the BDCA-encoding polynucleotide probe under
conditions that permit a stable duplex to form if the sample
contains a complementary polynucleotide sequence. Any stable
duplexes formed are detected by any suitable means. For example,
the polynucleotide probe can be supplied in labeled form, and label
remaining with the sample after washing will directly reflect the
amount of stable duplex formed. In a second illustration,
hybridization is performed in situ. A suitably prepared tissue
sample is overlaid with a labeled probe to indicate the location of
BDCA-encoding sequences.
[0242] A short polynucleotide can also be used as a primer for a
PCR reaction, particularly to amplify a longer sequence comprising
a region hybridizing with the primer. This can be conducted
preparatively, in order to produce polynucleotide for further
genetic manipulation. It can also be conducted analytically, to
determine whether a BDCA-encoding polynucleotide is present, for
example, in a sample of diagnostic interest.
[0243] Another use of the polynucleotides is in vaccines and gene
therapy. The general principle is to administer the polynucleotide
so that it either promotes or attenuates the expression of the
polypeptide encoded thereby. Thus, the present invention includes
methods of inducing an immune response and methods of treatment
comprising administration of an effective amount of polynucleotides
encoding BDCAs to an individual. In these methods, a polynucleotide
encoding BDCA is administered to an individual, either directly or
via cells transfected with the polynucleotide. Preferably, the
polynucleotide is in the form of a circular pla.smid, preferably in
a supercoiled configuration. Preferably, the polynucleotide is
replicated inside a cell. Thus, the polynucleotide is operatively
linked to a suitable promoter, such as a heterologous promoter that
is intrinsically active in cells of the target tissue type.
Preferably, once in cell nuclei, plasmids persist as circular
non-replicating episomal molecules. In vitro mutation can be
carried out with plasmid constructs to encode, for example,
molecules with greater affinity and/or avidity.
[0244] To determine whether plasmids containing BDCA
polynucleotides are capable of expression in eukaryotic cells,
cells such as COS-7, CHO, or HeLa can be transfected with the
plasmids. Expression is then determined by immunoassay; for
example, by Western blot. Smaller BDCAs can be detected, for
example, by constructing the plasmid so that the resultant
polypeptide is fused with a tag, such as a target epitope or enzyme
label. Further characterization of the expressed polypeptide can be
achieved by purifying the peptide and then conducting one of the
functional assays described herein.
[0245] In one mode of gene therapy, the polynucleotides of this
invention are used for genetically altering cells ex vivo. In this
strategy, cells removed from a donor or obtained from a cell line
are transfected or transduced with BDCA vectors, and then
administered to a recipient. Suitable cells for transfection
include peripheral blood mononuclear cells.
[0246] In another mode of gene therapy, the polynucleotides of this
invention are used for genetically altering cells in vivo. The
purpose can include, but is not limited to, treating various types
of cancer.
[0247] The polynucleotides can also be used to produce cells that
do not express BDCA-2, and transgenic animals expressing
BDCA-2.
[0248] Also obtained from the invention are cells engineered not to
express or to express at significantly reduced levels BDCA-2. Such
cells may be produced by selecting a cell, preferably a DC, and
providing to the cell an expression construct comprising a
polynucleotide encoding a BDCA-2 gene wherein the polynucleotide is
positioned antisense to and operatively linked to a promoter. The
expression of such a polynucleotide effectively produces a cell
deficient in BDCA-2.
[0249] In other embodiments the present invention provides a method
for the preparation of recombinant host cells that produce
significantly reduced amounts or even "knockout" the production of
BDCA-2. These recombinant host cells can be prepared by using one
or more means that are well known to those of skill in the art. For
example, gene expression can, be inhibited by the incorpOration of
constructs for antisense DNA or RNA into the genome. Deletions or
mutations of the endogenous BDCA-2 genes can render them
nonfunctional. Nucleic acids encoding ribozymes--RNA-cleaving
enzymes--that specifically cleave BDCA-2 mRNA can be introduced
into the recombinant host cells.
[0250] The term "knockout" refers to partial or complete
suppression of the expression of at least a portion of a protein
encoded by an endogenous DNA sequence, e.g. BDCA-2, in a cell. The
term "knockout construct" refers to a nucleic acid sequence that is
designed to decrease or suppress expression of a protein encoded by
endogenous DNA sequences in a cell.
[0251] These recombinant constructs can be incorporated into
knockout mammals such that the production of BDCA-2 is suppressed
in DCs. The preparation of knock out and transgenic animals is well
known to those of skill in the art and is described in U.S. Pat.
Nos. 5,434,340, 5,530,179 and 5,557,032.
[0252] The invention further provides methods for producing animals
and the animals so produced that over-express BDCA-2. These methods
generally comprise introducing animal cells into an animal, the
animal cells having been treated in vitro to insert therein a DNA
segment encoding a BDCA-2 polypeptide, the animal cells expressing
in vivo in the animal BDCA-2.
D. Kits
[0253] The invention encompasses kits containing anti-DC-specific
antigen-binding fragments, for measuring BDCA-2 iuncluding soluble
BDCA-2, including isoforms thereof, in serum and other sources.
Diagnostic procedures using the kits can be performed by diagnostic
laboratories, experimental laboratories, practitioners, or private
individuals. The clinical sample is optionally pre-treated for
enrichment of the target being tested for. The user then applies a
reagent contained in the kit in order to detect the changed level
or alteration in the diagnostic component.
[0254] Optionally, the reagent can be conjugated with a label to
permit detection of any complex formed with the target in the
sample. In another option, a second reagent is provided that is
capable of combining with the first reagent after it has found its
target and thereby supplying the detectable label. For example,
labeled. anti-murine IgG can be provided as a secondary reagent.
Labeled avidin is a secondary reagent when the primary reagent has
been conjugated to biotin.
[0255] The kits can be employed on a variety of biological samples
including, both liquid samples, cell suspensions and tissue
samples. Suitable assays that can be supplied in kit form include
those described herein.
[0256] Each reagent is supplied in a solid form
or.dissolved/suspended in a liquid buffer suitable for inventory
storage and later for exchange or addition into the reaction medium
when the test is performed. Suitable packaging is provided. The kit
can optionally provide additional components that are useful in the
procedure. These optional components include, but are not limited
to, buffers, capture reagents, developing reagents, labels,
reacting surfaces, means for detection, control samples,
instructions, and interpretive information.
E. Therapeutic Compositions
1. Compositions of Matter
[0257] The preparation of pharmaceutical compositions described
herein is conducted in accordance with generally accepted
procedures for the preparation of pharmaceutical preparations. See,
for example, Remington's Pharmaceutical Sciences 18th Edition
(1990), E.W. Martin ed., Mack Publishing Co., PA. Depending on the
intended use and mode of administration, it can be desirable to
process the active ingredient further in the preparation of
pharmaceutical compositions. Appropriate processing can include
sterilizing, mixing with appropriate non-toxic and non-interfering
components, dividing into dose units, and enclosing in a delivery
device. In one embodiment, the therapeutic compositions contain
DCs, subpopulations thereof or mixtures thereof. In another
embodiment, the compositions contain the antigen-binding fragments
described herein. Preferably, the antigen-binding fragments are, or
are derived from, the mAbs listed in Table 1. Preferably the DC
compositions contain DCs isolated with one of these antigen-binding
fragments.
[0258] (a) General Modes of Administration
[0259] Pharmaceutical compositions of the invention are
administered by a mode appropriate for the form of composition.
Typical routes include intravenous, subcutaneous, intramuscular,
intraperitoneal, intradermal, oral, intranasal, intradermal, and
intrapulmonary (i.e., by aerosol). Pharmaceutical compositions for
human use are typically administered by a parenteral route, most
typically intravenous, subcutaneous, intramuscular. Although not
required, pharmaceutical compositions are preferably supplied in
unit dosage form suitable for administration of a precise amount.
Also contemplated by this invention are slow release or sustained
release forms, whereby a relatively consistent level of the active
compound are provided over an extended period.
[0260] (b) Liquid Formulations
[0261] Liquid pharmaceutically acceptable compositions can, for
example, be prepared by dissolving or dispersing a polypeptide or
polynucleotide embodied herein in a liquid excipient, such as
water, saline, aqueous dextrose, glycerol, or ethanol. The
composition can optionally also contain other medicinal agents,
pharmaceutical agents, carriers, and auxiliary substances such as
wetting or emulsifying agents, and pH buffering agents.
Compositions for injection can be supplied as liquid solutions or
suspensions, as emulsions, or as solid forms suitable for
dissolution or suspension in liquid prior to injection.
[0262] Pharmaceutical compositions for oral, intranasal, or topical
administration can be supplied in solid, semi-solid or liquid
forms, including tablets, capsules, powders, liquids, and
suspensions. For administration via the respiratory tract, a
preferred composition is one that provides a solid, powder, or
liquid aerosol when used with an appropriate aerosolizer
device.
[0263] The invention also encompasses compositions comprising
liposomes with membrane bound peptide to specifically deliver the
liposome to the area of the tumor or neoplastic cells or to the
immune system. These liposomes can be produced such that they
contain, in addition to peptide, immunotherapeutic agents such as
those described above which would then be released at the
recognition site. Wolff et al. (1984) Biochem. Biophys. Acta
802:259. Another such delivery system utilizes chimeric parvovirus
B19 capsids for presentation of the antigen-binding fragments.
Brown et al. (1994) Virol. 198:477-488; and Miyamura et al. (1994)
Proc. Natl. Acad. Sci. USA 91:8507-8511. Such chimeric systems are
encompassed for use herein.
[0264] Compositions embodied in this invention can be assessed for
their efficacy in a number of ways. Accordingly, test compounds are
prepared as a suitable pharmaceutical composition and administered
to test subjects. Initial studies are preferably done in small
animals such as mice or rabbits, optionally next in non-human
primates and then ultimately in humans. Immunogenicity is
preferably tested in individuals without a previous antibody
response. A test composition in an appropriate test dose is
administered on an appropriate treatment schedule. It can be
appropriate to compare different doses and schedules within the
predicted range. The dosage ranges for the administration of
antigen-binding fragments are large enough to produce the desired
effect in which the symptoms of the disease are ameliorated without
causing undue side effects such as unwanted cross-reactions and
anaphylactic reactions. Generally, the dosage will vary with the
age, condition, sex and extent of the disease in the patient and
can be determined by one of skill in the art. The dosage can be
adjusted by the individual physician in the event of any
complication. Generally, when the compositions are administered
conjugated with therapeutic agents, lower dosages, comparable to
those used for in vivo immunodiagnostic imaging, can be used.
2. Antigen-Binding Fragments
[0265] The invention encompasses pharmaceutical compositions
containing the antigen-binding fragments described herein. Such
pharmaceutical compositions are useful for inducing or aiding an
immune response and treating neoplastic diseases, or including
tolerance and treating autoimmune diseases, (GvHD, allogaft
rejection, allergen, etc.) either alone or in conjunction with
other forms of therapy, such as chemotherapy, radiotherapy or
immune therapies described in WO98/23735; WO98/34642; WO97/10000;
WO97/10001; and WO97/06821. Other methods of treatment are
described herein and/or known in the art. Suitable diseases
include, without limitation, viral, parasitic, bacterial, fungal,
neoplastic and autoimmune.
[0266] In a murine breast cancer model, Flt3-Ligand (Flt3-L), a
stimulatory "cytokine for a variety of hematopoietic lineages,
including DCs and B cells, has been used in conjunction with murine
breast cancer cells as a vaccine. Chen et al. (1997) Cancer Res.
57:3511-6. DCs can also be loaded with or transduced to express
tumor antigens; these cells are then used as adjuvants to tumor
vaccination. DCs present tumor-associated antigens endogenously to
the afferent lymphatic system in the appropriate MHC-restricted
context Wan et al. (1997) Hum. Gene Ther. 8:1355-63; Peiper et al.
(1997) Surgery 122:235-41; and Smith et al. (1997) Int. Immunol.
9:1085-93. Current melanoma vaccines manipulate antigen
presentation networks and combine the best cellular and antibody
anti-tumor immune response effective in mediating tumor protective
immunity. These therapies have caused regression, delayed disease
progression or an improvement in survival in some cases--with a
paucity of side effects. Kuhn et al. (1997) Dermatol. Surg.
23:649-54. Melanoma vaccines are also reviewed in Conforti et al.
(1997) J. Surg. Oncol. 66:55-64.
[0267] Vaccines can be packaged in pharmaceutically acceptable
carriers, admixed with adjuvants or other components (such as
cytokines) as known in the art. Vaccines for veterinarian use are
substantially similar to that in humans with the exception that
adjuvants containing bacteria and bacterial components such as
Freund's complete or incomplete adjuvants, are allowed in the
formulations.
F. Methods of Treatment
[0268] Also included in this invention are methods for treating a
variety of disorders as described herein and/or known in the art.
The methods comprise administering an amount of a pharmaceutical
composition containing a composition of the invention in an amount
effective to achieve the desired effect, be it palliation of an
existing condition or prevention of recurrence. For treatment of
cancer, the amount of a pharmaceutical composition administered is
an amount effective in producing the desired effect. An effective
amount can be provided in one or a series of administrations. An
effective amount can be provided in a bolus or by continuous
perfusion. Suitable active agents include the anti-neoplastic
drugs, bioresponse modifiers and effector cells such as those
described by Douillard et al. (1986) Hybridomas (Supp. 1:5139).
[0269] Pharmaceutical compositions and treatment modalities are
suitable for treating a patient by either directly or indirectly
eliciting an immune response against neoplasia. An "individual,"
"patient" or "subject" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to:
humans, wild animals, feral animals, farm animals, sport animals,
and pets. A "cancer subject" is a mammal, preferably a human,
diagnosed as having a malignancy or neoplasia or at risk
thereof.
[0270] As used herein, "treatment" refers to clinical intervention
in an attempt to alter the disease course of the individual or cell
being treated, and can be performed either for prophylaxis or
during the course of clinical pathology. Therapeutic effects of
treatment include without limitation, preventing occurrence or
recurrence of disease, alleviation of symptoms, diminishment of any
direct or indirect pathological consequences of the disease,
preventing metastases, decreasing the rate of disease progression,
amelioration or palliation of the disease state; and remission or
improved prognosis.
[0271] The "pathology" associated with a disease condition is any
condition that compromises the well-being, normal physiology--or
quality of life of the affected individual. This can involve, but
is not limited to, destructive invasion of affected tissues into
previously unaffected areas, growth at the expense of normal tissue
function, irregular or suppressed biological activity, aggravation
or suppression of an inflammatory or immunologic response,
increased susceptibility to other pathogenic organisms or agents,
and undesirable clinical symptoms such as pain, fever, nausea,
fatigue, mood alterations, and such other disease-related features
as can be determined by an attending physician.
[0272] An "effective amount" is an amount sufficient to effect a
beneficial or desired clinical result upon treatment. An effective
amount can be administered to a patient in one or more doses. In
terms of treatment, an effective amount is an amount that is
sufficient to palliate, ameliorate, stabilize, reverse or slow the
progression of the disease, or otherwise reduce the pathological
consequences of the disease. The effective amount is generally
determined by the physician on a case-by-case basis and is within
the skill of one in the art. Several factors are typically taken
into account when determining an appropriate dosage to achieve an
effective amount. These factors include age, sex and weight of the
patient, the condition being treated, the severity of the condition
and the form and effective concentration of the antigen-binding
fragment administered.
[0273] The term "immunomodulatory" or "modulating an immune
response" as used herein includes immunostimulatory as well as
immunosuppressive effects. Immunostimulatory effects include, but
are not limited to, those that directly or indirectly enhance
cellular or humoral immune responses. Examples of immunostimulatory
effects include, but are not limited. to, increased
antigen-specific antibody production; activation or proliferation
of a lymphocyte population such as NK cells, CD4.sup.+ cells,
CD8.sup.+ cells; macrophages and the like; increased synthesis of
cytokines or chemokines including, but not limited to, IL-J, IL-2,
IL-4, IL-5, IL-6, IL-12, interferon, TNF-.alpha., IL-10, TGF-.beta.
and the like. Immunosuppressive effects include those that directly
or indirectly decrease cellular or humoral immune responses.
Examples of immunosuppressive effects include, but are not limited
to, a reduction in antigen-specific antibody production such as
reduced IgE production; activation of lymphocyte or other cell
populations that have immunosuppressive activities such as those
that result in immune tolerance; and increased synthesis of
cytokines that have suppressive effects toward certain cellular
functions including, but not limited to IL-10 and TGF-.beta.. One
example of this is ITN-.gamma., which appears to block IL-4 induced
class switch to IgE and IgG1, thereby reducing the levels of these
antibody subclasses.
[0274] Suitable human subjects for cancer therapy further comprise
two treatment groups, which can be distinguished by clinical
criteria. Patients with "advanced disease" or "high tumor burden"
are those who bear a clinically measurable tumor. A clinically
measurable tumor is one that can be detected on the basis of tumor
mass (e.g., by palpation, CAT scan, sonogram, mammogram or X-ray;
positive biochemical or histopathologic markers on their own are
insufficient to identify this population). A pharmaceutical
composition embodied in this invention is administered to these
patients to elicit an anti-tumor response, with the objective of
palliating their condition. Ideally, reduction in tumor mass occurs
as a result, but any clinical iniprovement constitutes a benefit.
Clinical improvement includes decreased risk or rate of progression
or reduction in pathological consequences of the tumor.
[0275] A second group of suitable subjects is known in the art as
the "adjuvant group." These are individuals who have had a history
of cancer, but have been responsive to another mode of therapy. The
prior therapy can have included (but is not restricted to, surgical
resection, radiotherapy, and traditional chemotherapy. As a result,
these individuals have no clinically measurable tumor. However,
they are suspected of being at risk for progression of the disease,
either near the original tumor site, or by metastases.
[0276] "Adjuvant" as used herein has several meanings, all of which
will be clear depending on the context in which the term is used.
In the context of a pharmaceutical preparation, an adjuvant is a
chemical or biological agent given in combination (whether
simultaneously or otherwise) with, or recombinantly fused to, an
antigen to enhance immunogenicity of the antigen. For review see,
Singh et al. (1999) Nature Biotech. 17:1075-1081. Isolated DCs have
also been suggested for use as adjuvants. Compositions for use
therein are included in this invention. In the context of cancer
diagnosis or treatment, adjuvant refers to a class of cancer
patients with no clinically detectable tumor mass, but who are
suspected of risk of recurrence.
[0277] This group can be further subdivided into high-risk and
low-risk individuals. The subdivision is made on the basis of
features observed before or after the initial-treatment. These
features are known in the clinical arts, and are suitably defined
for each different cancer. Features typical of high-risk subgroups
are those in which the tumor has invaded neighboring tissues, or
who show involvement of lymph nodes.
[0278] Another suitable group is those with a genetic
predisposition to cancer but who have not yet evidenced clinical
signs of cancer. For instance, women testing positive for a genetic
mutation associated with breast cancer, but still of childbearing
age, can wish to receive one or more of the antigen-binding
fragments described herein in treatment prophylactically to prevent
the occurrence of cancer until it is suitable to perform preventive
surgery.
[0279] Human cancer patients, including, but not limited to,
glioblastoma, melanoma, neuroblastoma, adenocarcinoma, glioma, soft
tissue sarcoma, and various carcinomas (including small cell lung
cancer) are especially appropriate subjects. Suitable carcinomas
further include any known in the field of oncology, including, but
not limited to, astrocytoma, fibrosarcoina, myxosarcoma,
liposarcoma, oligodendroglioma, ependymoma, medulloblastoma,
primitive neural ectodermal tumor (PNET), chondrosarcoma,
osteogenic sarcoma, pancreatic ductal adenocarcinoma, small and
large cell lung adenocarcinomas, chordoma, angiosarcoma,
endotheliosarcoma, squamous cell carcinoma,
bronchoalveolarcarcinoma, epithelial adenocarcinoma, and liver
metastases thereof, lymphangiosarcoma, lymphangioendotheliosarcoma,
hepatoma, cholangiocarcinoma, synovioma, mesothelioma, Ewing's
tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma,
sweat gland carcinoma, papillary carcinoma, sebaceous gland
carcinoma, papillary adenocarcinoma, cystacienocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, bileduct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, testicular tumor, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, neuroblastoma, retinoblastoma,
leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, and
heavy chain disease, breast tumors such as ductal and lobular
adenocarcinoma, squamous and adenocarcinomas of the uterine cervix;
uterine and ovarian epithelial carcinomas, prostatic
adenocarcinomas, transitional squamous cell carcinoma of the
bladder, B and T cell lymphomas (nodular and diffuse) plasmacytoma,
acute and chronic leukemias, malignant melanoma, soft tissue
sarcomas and leiomyosarcomas.
[0280] The patients can have an advanced form of disease, in which
case the treatment objective can include mitigation or reversal of
disease progression, and/or amelioration of side effects. The
patients can have a history of the condition, for which they have
been treated, in which case the therapeutic objective will
typically include a decrease or delay in the risk of
recurrence.
[0281] Autoimmune disorders are the caused by a misdirected immune
response resulting in self-destruction of a variety of cells,
tissues and organs. The cause of these disorders is unknown.
Recognition of self through the MHC is known to be of importance in
an immune response. However, prevention of an autoimmune response
and the cells responsible for autoimmunity are not well
understood.
[0282] Autoimmunity results from a combination of factors,
including genetic, hormonal, and environmental influences. Many
autoimmune disorders are characterized by B cell hyperactivity,
marked by proliferation of B cells and autoantibodies and by
hypergammaglobulinemia. B cell hyperactivity is probably related to
T cell-abnormalities. Hormonal and genetic factors strongly
influence the incidence of autoimmune disorders; for example, lupus
erythematosus predominantly affects women of child-bearing age, and
certain HLA haplotypes are associated with an increased risk of
specific autoimmune disorders.
[0283] Common autoimmune disorders include, but are not limited to,
rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic
arthritis, ankylosing spondylitis, Sjogren's syndrome, lupus
erythematosus, Goodpasture's syndrome, Reiter's syndrome,
scleroderma, vasculitis, polymyositis and dermatomyositis. Many of
these conditions include aberrant inflammatory reactions related to
the immunologic disorders. The DCs described herein are suitable
for use in treatment of these disorders particularly when used to
inactivate or induce tolerogenization in T cells involved in the
disorder. Methods of treatment are known in the art. As discussed
herein, one or more of the subsets of DCs obtained by the methods
described herein are suitable for use in treatment of
autoimmunity.
[0284] "Immunologic activity" of an antigen-binding fragment refers
to specifically binding the antigen which the intact antibody
recognizes. Such binding can or can not elicit an immune response.
A specific immune response can elicit antibody, B cell responses, T
cell responses, any combination thereof, and effector functions
resulting therefrom. Included, without limitation, are the
antibody-mediated functions ADCC and complement-mediated cytolysis
(CDC). The T cell response includes, without limitation, T helper
cell function, cytotoxic T cell function, inflammation/inducer T
cell function, and T cell mediated immune suppression. A compound
(either alone or in combination with a carrier or adjuvant) able to
elicit either directly or indirectly, a specific immune response
according to any of these criteria is referred to as "immunogenic."
Antigen-binding fragment "activity" or "function" refers to any of
the immunologic activities of an antibody, including detection,
amelioration or palliation of cancer.
[0285] An "immune response" refers to induction or enhancement of
an immunologic response to malignant or diseased tissue,
disease-causing agents and other foreign agents to which the body
is exposed. Immune responses can be humoral, as evidenced by
antibody production; and/or cell-mediated, as evidenced by
cytolytic responses demonstrated by such cells as natural killer
cells or cytotoxic T lymphocytes (CTLs) and the cytokines produced
thereby. Immune responses can be monitored by a mononuclear cell
infiltrate at the site of infection or malignancy. Typically, such
monitoring is by histopathology. A "cancer-specific immune
response" is one that occurs against the malignancy but not against
non-cancerous cells. The treatments described herein" typically
induce or augment a cell-mediated immune response but can also
induce or augment an antibody-mediated immune response. The
treatments can also influence the type of immune response to the
antigen.
[0286] The compositions according to the invention are also
suitable for use in inducing an antigen-specific Th1 immune
response. Stimulating a Th1-type immune response can be measured in
a host treated in accordance with the invention and can be
determined by any method known in the art including, but not
limited to, a reduction in levels of IL-4 measured before and after
antigen challenge; or detection of lower (or--even absent) levels
of IL-4 in a treated host as compared to an-antigen-primed, or
primed and challenged, control treated without the compositions of
the invention; an increase in levels of IL-12, IL-18 and/or IFN
(.alpha., .beta. or .gamma., preferably IFN-.gamma. in a treated
host as compared to an antigen-primed or primed and challenged
control; IgG2a antibody production in a treated host as compared to
an untreated control; a reduction in levels of antigen-specific IgE
as measured before and after antigen challenge or detection of
lower (or even absent) levels of antigen-specific IgE in a treated
host as compared to an antigen primed or primed and challenged
untreated host. A variety of these determinations can be made by
measuring cytokines made by APCs and/or lymphocytes; preferably
DCsand/or T cells, in vitro or ex vivo using methods described
herein and known in the art. Methods to determine antibody
production include any known in the art.
[0287] The Th1 biased cytokine induction produces enhanced cellular
immune responses, such as those performed by NK cells, cytotoxic
killer cells, Th1 helper and memory cells. These responses are
particularly beneficial for use in protective or therapeutic
vaccination against viruses, fungi, protozoan parasites, bacteria,
allergic diseases and asthma, as well as tumors.
[0288] The invention further includes down-regulation of type I
interferon production via ligation of BDCA-2, down-regulation of
Th1 immune responses via ligation of BDCA-2, and polarization of an
immune response to Th2 via ligation of BDCA-2. These indications
can be reversed by interfering with ligation of BDCA-2. The
invention further encompasses screening for suitable moieties for
interfering with ligation of BDCA-2 and compositions of these
moieties.
[0289] When antigen-binding fragments are used in combination with
various therapeutic agents, the administration of both usually
occurs substantially contemporaneously. The term "substantially
contemporaneously" means that they are administered reasonably
close together with respect to time. The administration of the
therapeutic agent can be daily, or at any other suitable interval,
depending upon such factors, for example, as the nature of the
ailment, the condition of the patient and half-life of the
agent.
[0290] Therapeutic compositions can be administered by injection or
by gradual perfusion over time. The antigen-binding fragments can
be administered intravenously, intraperitoneally, intra-muscularly,
subcutaneously, intracavity, intranodal, intrathecally or
transdermally, alone or in combination with other therapeutic
agents.
[0291] Another method of administration is intralesionally, for
instance by injection directly into the tumor. Intralesional
administration of various forms of immunotherapy to cancer patients
does not cause the toxicity seen with systemic administration of
immunologic agents. Fletcher et al. (1987) Lymphokine Res. 6:45;
Rabinowich et al. (1987) Cancer Res. 47:173; Rosenberg et al.
(1989) Science 233:1318; and Pizz et al. (1984) J. Int. Cancer
34:359.
[0292] Further, it can be desirable to administer the compositions
locally to the area in need of treatment; this can be achieved by,
for example, local infusion during surgery, by injection, by means
of a catheter, or by means of an implant, the implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as silastic membranes, or fibers. A suitable such membrane is
Gliadel.RTM. provided by Guilford sciences.
[0293] The fact that ligation of BDCA-2 with anti-BDCA-2 monoclonal
antibody (AC144) induces intracellular Ca2.sup.+ mobilization
indicates that plasmacytoid DC (and all other cells which express
BDCA-2) can be functionally modulated by triggering of BDCA-2
signaling or inhibition of triggering of BDCA-2 signaling.
Regarding functional modulation of DC, the following aspects are
encompassed by the claims: [0294] A) Induction and down-regulation
of CD4.sup.+ and CD8.sup.+ T cells responses. [0295] B)
Polarization of the immune response towards tolerance or immunity
[0296] C) Polarization of CD4.sup.+ T cell responses towards Th1
cell development, Th2 cells development or Th3/T-regulatory-1
CD4.sup.+ T cell development. The latter down-regulate immune
responses, possibly via secretion of TGF-.beta. and/or IL-10.
[0297] D) DC are usually thought of as antigen-presenting cells for
T cells. However, recent studies from several laboratories have
shown that they have important roles in B-cell activation and
regulation of antibody synthesis. B cell responses can therefore be
modulated via BDCA-2 on DCs. The same can also be true for NK cell
responses.
[0298] As type I interferon can induce Th1 type immune responses in
humans (Parronchi et al. (1996) Eur. J. Immunol. 26:697-703),
triggering of BDCA-2 polarizes CD4.sup.+ T cell responses towards
Th2 cell development, whereas inhibition of BDCA-2 signaling
polarizes CD4.sup.+ T cell responses towards Th1 cell development.
The invention thus encompasses the polarization of CD4.sup.+ T cell
responses towards Th2 or Th1 cell development by triggering of
BDCA-2 signaling or inhibition of triggering of BDCA-2 signaling,
respectively.
[0299] All publications cited herein are hereby incorporated herein
by reference in their entirety. The following examples are provided
to illustrate, but not limit, the invention.
Example 1
Generation of DC-Specific mAb
[0300] Five 6-8 week old female Balb/c mice (Simonsen Laboratories,
Gilroy, Calif.) were inoculated with approximately 5.times.10.sup.5
to 1.times.10.sup.6 purified HLA-DR.sup.+lin.sup.- blood DC under
anesthesia on d 0, 4, 7, 11, and 14 in the right hand footpad, and
approximately 1.times.10.sup.6 HLA-A2.sup.+ Bristol-8 B
lymphoblastoma cells in the left hand footpad on d -3, 0, 4, 7, 11,
and 14. Both cell types were incubated with 1:100 PHA (Gibco/BRL,
Gaithersburg, Md.) for 10 min at room temperature and washed with
PBS before injection. This treatment provides non-specific adjuvant
effects and obviates the need for adjuvants such as Freund's
adjuvant.
[0301] On d 15, one day after the fifth injection of
HLA-DR.sup.+lin.sup.- DC, the mouse right hand popliteal lymph
nodes were removed. A lymphocyte suspension was prepared and the
cells were fused to SP2/0 Ag14 myeloma cells using a modification
of the method described by Kohler and Milstein (1975) Nature
256:495. Fused cells were plated on 96-well plates in DMEM
supplemented with 20% FCS (HyClone, Logan, Utah), 2 mmol/L
Lglutamine, 15 mmol/L Hepes, 10.sup.4 mmol/L hypoxanthine
(Gibco/BRL), and placed in a 37.degree. C. incubator with 9%
CO.sub.2.
[0302] When visible hybridoma colonies were apparent, supernatants
from these wells were screened by' flow cytometry for antibody
secretion and for non-reactivity (<1% positive cells) to PBMC.
Briefly, a mixture of rat anti-mouse kappa mAb-conjugated
polystyrene beads (2.5 .mu.m in diameter, Interfacial Dynamics
Corp., Portland, Oreg.) and PBMC was incubated with 50 .mu.l
hybridoma supernatant for 20 min at room temperature. The bead/cell
mixture was then washed twice with PBS, pH 7.4, containing 5 mmol/L
EDTA and 0.5% BSA (PBS/EDTA/BSA), and binding of mouse IgM, IgG1,
IgG2a and IgG2b from the supernatants to the beads and the test
cells was detected by staining with PE conjugated rat anti-mouse
IgM mAb (clone X54, BD Biosciences, San Jose, Calif.), rat
anti-mouse IgG1 mAb (clone X56, BD Biosciences) and rat anti-mouse
IgG2 mAb (clone X57, BD Biosciences). PBMC and polystyrene beads
can easily be discriminated in the flow cytometric analysis by
scatter signals.
[0303] Culture supernatants which fulfilled the screening criteria
of the first round were then screened by flow cytometric analysis
for reactivity to a significant proportion of blood DC. Briefly, a
mixture of rat anti-mouse mAb-conjugated polystyrene beads and
enriched blood DC (PBMC depleted of B cells, T cells and monocytes)
was incubated with 50 .mu.l hybridoma supernatant for 20 min at
room temperature. The mixture was then washed twice with
PBS/EDTA/BSA, and stained with PE-conjugated rat anti-mouse IgM
mAb, rat anti-mouse IgG1 mAb, and rat anti-mouse. TgG2 mAb to
detect binding of mouse IgM, IgG1, IgG2a and IgG2b from the
supernatants to the beads and the enriched blood DC. For
discrimination of HLA-DR.sup.+DC from HLA-DR.sup.- cells in the
flow cytometric analysis, the bead/cell mixture was washed once,
free binding sites of the PE-conjugated rat anti-mouse IgG2 mAb and
the bead-conjugated rat anti-mouse .kappa. mAb were saturated by
incubation with 100 .mu.g/ml mouse IgG2a for 5 min at room
temperature, and the mixture was counter-stained with
anti-HLA-DR-FITC (cloneAC122, IgG2a).
[0304] Selected hybridoma cells were expanded in culture, stocks
were frozen in liquid nitrogen, subclones were established by
limiting dilution, and series of positive subclones were also
frozen in liquid nitrogen. The isotype of the mAb was determined by
the ISOTYPE Ab-STAT Kit (SangStat Medical Corp., Palo Alto,
Calif.).
[0305] For mAb production, hybridoma cells were either grown as an
ascites tumor in Balb/c mice, with collection of mAb-rich ascites
fluid, or in cell culture (roller culture or hollow-fiber culture),
with collection of mAb-rich culture supernatant. Pure IgG mAb was
prepared from ascites fluid or cell culture supernatant by Protein
A affinity chromatography followed in some cases by hydrophobic
interaction chromatography and stored in PBS with 5 mmol/L EDTA and
0.05% sodium azide at 4.degree. C. Purified mAb were conjugated to
FITC (Sigma, St. Louis, Mo.), PE (Cyanotech Corp., Kailua Kona,
Hi.), Cy5 (Amersham Life Science Inc., Arlington Heights, Ill.),
APC (Europa Bioproducts Ltd., Cambridge, UK), biotin (Pierce,
Rockford, Ill.) and colloidal super-paramagnetic beads
(approximately 50 nm in diameter, Miltenyi Biotec GmbH, Bergisch
Gladbach, Germany) according to standard techniques. Hermanson
(1996) Bioconjugate Techniques. Academic Press Inc., San Diego, 785
pp.; Aslam et al. (1998) Bioconjugation: protein coupling
techniques for the biomedical sciences. Macmillan Reference Ltd.,
London, 833 pp.; and Kantor et al. (1997) Magnetic cell sorting
with colloidal superparamagnetic particles. In, Cell Separation
Methods and Applications. Recktenwald et al. Eds. Marcel Dekker
Inc. New York, pp. 153-173.
Cell Preparations
[0306] Buffy coats from normal healthy volunteers were obtained
from the Institute for Transfusionmedicine, Hospital Merheim,
Cologne, Germany. PBMC were prepared from buffy coats by standard
Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient
centrifugation.
[0307] Peripheral blood leukocytes were prepared from buffy coats
by lysis of erythrocytes in isotonic ammonium chloride buffer (155
mmol/L NH.sub.4Cl, 10 mmol/L KHCO.sub.3 and 0.1 mM EDTA). Hansel et
al. (1991) J. Immunol. Met. 145:105-110. CD4.sup.+lin.sup.- blood
DC were isolated from PBMC by two-step immunomagnetic cell sorting
(MACS) as described in detail elsewhere. Robert et al. (1999); and
Miltenyi et al. (1999) High gradient magnetic cell sorting. In,
Flow cytometry and cell sorting. Ed., Radbruch. Springer-Verlag,
Berlin. pp. 218-247. Briefly, monocytes, T cells, and NK cells were
depleted using rnAb against CD3 (Clone BW264/56), CD11b (clone
M1/70.15.11.5), CD16 (Clone VEP-13) and in a few experiments a
poorly defined antigen expressed on B cells and monocytes (clone
L179). From the depleted cell fraction, blood DC were then enriched
to high purity using an antibody against CD4 (M-T321). To screen
hybridoma culture supernatants (see above), blood DC were merely
partially enriched by immunomagnetic depletion of T cells, B cells
and monocytes based on CD3 and L179 antigen expression.
[0308] CD1c.sup.-, BDCA-2.sup.-, and BDCA-3-expressing cells were
isolated from PBMC or tonsils by indirect magnetic labeling with
PE- or FITC-conjugated mAb (AD5-8E7, AC144 and AD6-5E8,
respectively) as primary reagent and anti-PE or anti-FITC
mAb-conjugated microbeads (Miltenyi Biotec GmbH) as secondary
reagent, and enrichment of labeled cells by MACS. In some
experiments, BDCA-3.sup.+ cells were isolated based on direct
magnetic labeling with anti-BDCA-3 mAb (AD5-5E8)-conjugated
microbeads. Highly pure CD1c.sup.+ blood DC without contaminating
CD1c.sup.+ B cells were obtained by immunomagnetic depletion of
CD18+ B cells using CD19 mAb-conjugated microbeads (Miltenyi Biotec
GmbH) followed by immunomagnetic enrichment of CD1c.sup.+ cells.
Basophils were purified from PBMC by immunomagnetic depletion of
non-basophils based on indirect magnetic labeling of CD3-, CD7-,
CD14-, CD15-, CD36-, CD45RA-, and HLA-DR-expressing cells with a
magnetic labeling kit (Miltenyi Biotec). CD14.sup.+ monocytes,
CD34.sup.+ hematopoietic progenitor cells and CD3.sup.+ T cells
were immunomagnetically purified based on direct magnetic labeling
with CD14, CD34 and CD3 mAb-Conjugated microbeads (Miltenyi Biotec
GmbH), respectively.
Cell Culturing
[0309] For generation of "immature" monocyte-derived DC (Mo-DC),
purified CD14.sup.+ monocytes were cultured at a cell density of
5.times.10.sup.5 to 1.times.10.sup.6 cells/ml in medium [RPMI 1640
(Gibco/BRL) supplemented with 2 mmol/L L-glutamine, 10% FCS
(Sigma), 100 mmol/L sodium pyruvate (Gibco/BRL), 100 U/ml
penicillin (Gibco/BRL), and 100 pg/ml streptomycin (Gibco/BRL)] at
37.degree. C. in a humidified 5% CO.sub.2-containing atmosphere in
the presence of 500-1000 U/ml rIL-4 (PeproTech, Rocky Hill, N.J.)
and 100 ng/ml rGM-CSF (PeproTech) for 7 d. For generation of
"mature" Mo-DC, "immature" Mo-DC were washed once and cultured in
medium in the presence of 20 ng/ml TNF-.alpha. (PeproTech) for
another 3 d. For generation of CD34.sup.+ hematopoietic progenitor
cell-derived DC (CD34-DC), purified CD34.sup.+ cells were cultured
at a cell density of 5.times.10.sup.4 cells/ml in medium in the
presence of 100 ng/ml rFlt3-Ligand (PeproTech), 0.5 ng/mL
rTGF-.beta.1 (PeproTech), 10 ng/ml rTNF-.alpha., 20 ng/ml rSCF
(PeproTech) and 100 ng/ml rGM-CSF for 11 d. Freshly isolated
CD4.sup.+lin.sup.- blood DC were cultured at a cell density of
5.times.10.sup.5 to 1.times.10.sup.6 cells/ml in medium in the
presence of 10 ng/ml rIL-3 (PeproTech) for up to 48 h. Isolated
CD1c-, BDCA-2-, and BDCA-3-expressing DC were cultured at a cell
density of 5.times.10.sup.5 to 1.times.10.sup.6 cells/ml in medium
without any cytokines or in the presence of 10 ng/ml rIL-3, 20
ng/ml IL-4 (PeproTech) and 100 ng/ml GM-CSF for up to 48 h.
Example 2
Flow Cytometric Analysis of Blood DCs
[0310] A FACScalibur (BD Biosciences) was used for one-, two-,
three- or four-color flow cytometry. Data of 5.times.10.sup.3 to
2.times.10.sup.5 cells per sample were acquired in list mode and
analyzed using CellQuest software (BD Biosciences).
[0311] The following mAb (clone names) were used in this study for
flow cytothetry: CD1a (HI149), CD10 (HI10a), CD11a (G43-25B), CD11c
(B-1y6), CD25 (M-A261), CD27 (M-T271), CD32 (FL18.26), CD38 (HIT2),
CD40 (5C3), CD43 (IG10), CD54 (HA58), CD62L (Dreg 56), CD64 (10.1),
CD69 (FN50), CD98 (UM7F8), anti-HLA-DQ (TU169), and
anti-TCR.alpha..beta. TIOB9.1A-31 from PharMingen, San Diego,
Calif.; CD2 (S5.2), CD8 (SK1), CD13 (L138), CD14 (MFP9), CD19
(SJ25-C1), CD33 (P67.6), CD34 (8G12), CD45RO (UCHL-1), CD56
(NCAM16.2), CD71 (L01.1), CD123 (9F5), anti-IgD (TA4.1), anti-mouse
IgG1 (X56), anti-mouse IgG2 (X57), and anti-mouse IgM (X54) from BD
Biosciences; CD5 (CLB-T11/11, 6G4), CD7 (CLB-T-3A1/1, 7F3), CD16
(CLB-FcR gran/1, 5D2), CD45RA (F8-11-13), CD80 (CLB-DALI) from CLB,
Amsterdam, Netherlands; CD18 (7E4), CD23 (9P25); CD58 (AICD58),
CD77 (38.13), CD83 (HB I5A), CD86 (HA5.2B7), CD116 (SC06) from
Coulter Immunotech, Marseilles, France; CD4 (M-T321), CD11b
(M1/70.15.11.5), CD14 (TOK4), CD15 (VIMC6), anti-HLA-DR (910/D7),
anti-AC133 (AC133/1), and anti-TCRal3 (BW242/412) from Miltenyi
Biotec GmbH, CD36 (AC106), CD123 (AC145), anti-HLA-DR (AC122 and
AC123) and anti-GPA (AC107) from Amcell, Sunnyvale, Calif.; CD1c
(M241) from Ancell, Bayport, Minn.; polyclonal anti-IgG, anti-IgM
(SA-DA4), polyclonal anti-kappa, and polyclonal anti-lambda from
Southern Biotechnology Associates, Birmingham, Ala.; CD61 (VIPL2)
from W. Knapp, Institute of Immunology, University of Vienna,
Vienna, Austria; CD44 (IM7) from J. Moll, Forschungszentrum
Karlsruhe, Karlsruhe, Germany; CD20 (H147) from Caltag
Laboratories, Burlingame, Calif.; anti-CLA (HECA-452) from E.
Butcher, Department of Pathology, Stanford University, Stanford,
Calif.; anti-FI.sub..epsilon.RI (15-1) from J. P. Kinet, Molecular
Allergy and Immunology Section, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Rockville, Md.;
CD11c (Ki-M1) from M. R. Parwaresch, Department of Pathology,
Christian Albrechts University, Kiel, Germany; CMRF-44 and CMRF-56
from D. N. Hart, Mater Medical Research Institute, Mater
Misericordiae Hospitals, South Brisbane, Queensland, Australia; and
anti-HLA-A, --B, -C(W6/32) from Sigma.
[0312] All antibodies were used as FITC-, PE-, biotin- or
Cy5-conjugated mAb. For indirect immunofluorescent staining with
biotinylated mAb, streptavidin-APC (BD Biosciences) was used. To
exclude dead cells in the flow cytometric analysis, cells were
stained with propidium iodide. To minimize Fc receptor-mediated mAb
binding, cells were stained in most experiments in the presence of
FcR-blocking reagent (Miltenyi Biotec GmbH) containing human
IgG.
Microscopic Analysis
[0313] Cells were spun down on slides in a cytocentrifuge (Cytospin
3, Shandon, Pittsburgh, Pa.). For fluorescence microscopy, slides
were air dried overnight after cytocentrifugation and mounted with
Fluoromount G (Southern Biotechnology Associates). For May
GrunwaldlGiemsa staining, slides were air dried for at least 2 h
after cytocentrifugation, stained in May GrunwaldlGiemsa solution
(Merck, Darmstadt, Germany) for 2 min at room temperature, rinsed
thoroughly in distilled water, stained in Giemsa solution (Merck)
for 15 min at room temperature, washed repeatedly in distilled
water, and air dried for at least 2 h. A Zeiss Axioscop, microscope
(Zeiss, Oberkochen, Germany) was used for analysis. Digital
pictures were made using the Xillix Microlmager Mll400-12X (Xillix,
Vancouver, Canada).
Example 3
Cross-Inhibition, Co-Capping and Co-Internalization Analysis
[0314] To analyze whether two different mAb clones recognize the
same (or a closely related) antigen epitope, cross-inhibition
binding assays were performed. Between 1.times.10.sup.6 and
2.times.10.sup.6 cells were pre-incubated with one of the two mAb
clones at a concentration of about 100 pg/ml for 10 min at
4.degree. C., and then stained with a PE-conjugate of the other mAb
clone at its optimal titer for another 5 min at 4.degree. C. PBMC
were used to analyze cross-inhibition of BDCA-2-, BDCA-3-, and
BDCA-4-specific mAb clones and MOLT-4 cells were used to analyze
cross-inhibition of CD1c-specific mAb clones. Cell staining was
analyzed by flow cytometry.
[0315] To ascertain whether AD5-5E8 and AD5-14H12 recognize the
same antigen (or the same antigen-complex) a co-capping assay was
performed. Briefly, BDCA-3-expressing cells were isolated from PBMC
by indirect magnetic labeling with PE-conjugated AD5-14HI2 mAb and
anti-PE mAb-conjugated microbeads, and isolated cells were
incubated for 30 min at 37.degree. C. to induce capping of the
mAb-antigen complex. Afterwards, cells were washed with ice cold
PBS/EDTA/BSA supplemented with 0.1% sodium azide
(PBS/EDTAJBSA/azide), and stained with FITC-conjugated AD5-5E8 mAb
in PBS/EDTA/BSA/azide for 10 min at 4.degree. C. Cell staining was
analyzed by fluorescence microscopy.
[0316] A co-internalization assay was used to investigate whether
AC144 and AD5-17F6 recognize the same antigen (or the same
antigen-complex). Briefly, 1.times.10.sup.6 PBMC were incubated
with 50 pg/ml AC144 mAb for 15 min at room temperature in PBS/BSA,
washed twice in PBS/BSA, and then incubated in cell culture medium
at 37.degree. C. for 30 min. To analyze whether AC144 mAb is
internalized upon culturing, aliquots of the cells were stained
before and after the culture period with rat anti-mouse IgG1-PE. To
determine whether all AC144 mAb-binding sites were saturated with
unconjugated AC144 mAb before culturing and whether any free
binding sites reappear after culturing, aliquots of the cells were
stained before and after the culture period with AC144-PE. To
analyze whether AD5-17F6 antigen is co-internalized, aliquots were
stained before and after the culture period with AD5-17F6-PE. All
cells were counter stained with CD123-FITC and HLA-DR-Cy5 to be
able to gate on CD123.sup.brightHLA-DR.sup.+ plasmacytoid DC in the
flow cytometric analysis.
Example 4
Endocytosis Assay
[0317] To assess endocytosis of blood DC subsets, purified
CD1c.sup.+, BDCA-2.sup.+ and BDCA-3.sup.+blood DC, and (as control)
purified CD3.sup.+ T cells were incubated at 37.degree. C. in
medium with 1 mg/ml Lucifer yellow (LY) for 0, 15, 45, and 75 min.
Afterwards, cells were washed three times in ice cold PBS/EDTA/BSA
and analyzed by flow cytometry,
Example 5
Reactivity of Isolated Blood DCs with Non-Cultured Blood Cells
[0318] According to their reactivity with blood cells, the mAb
listed in Table 1 could be divided into four groups: (1) AC144,
AD5-13A11 and ADB-4B8; (2) AD5-17F6; (3) AD5-5E8 and AD5-14H12; and
(4) AD5-8E7.
[0319] The mAb of the first group, AC144, AD5-13A11 and ADB-4B8,
stain approximately 0.41.+-.0.17% (n=10) of all PBMC (FIG. 1A). In
a dot plot of forward and side scatter signals, these rare cells
constitute a homogeneous cell population that is located between
small resting lymphocytes and monocytes (FIG. 1B). Accordingly,
these rare cells do not express the .alpha..beta. T cell receptor
(TCR.alpha..beta.), CD14, CD19 and CD56 (FIG. 1A), lineage markers
which are expressed on T cells, monocytes, B cells and NK cells,
respectively. Staining of highly purified blood DC (>95%
HLA-DR.sup.+, TCR.alpha..beta..sup.-, CD14.sup.-, CD19.sup.-and
CD56.sup.-) reveals that the mAb of the first group are reactive
with CD11c.sup.-CD123.sup.bright blood DC (FIG. 2) but not reactive
with CD11c.sup.+ blood DC. To analyze whether all of them react
with a single antigen, we performed two-color stainings and
cross-inhibition studies. The results show that all mAb of this
group recognize a single epitope of the same antigen. This antigen
was named BDCA-2.
[0320] As shown in FIG. 3, the mAb of the second group, AD5-17F6,
recognizes the same cells among PBMC as AC144, one of the
BDCA-2-specific mAb of the first group. Nevertheless, AD5-17F6
stains an antigen which is different from BDCA-2. This was
unequivocally demonstrated by co-internalization experiments, where
AD5-17F6 showed surface staining with equal intensity before and
after anti-BDCA-2 mAb-mediated internalization of BDCA-2, and by
staining of DC after culture, where AC144 mAb and AD5-17F6 mAb
showed entirely different staining patterns (FIG. 4). The antigen
recognized by AD5-17F6 was named BDCA-4 and is identical to
neuropilin-1. He et al. (1997).
[0321] The mAb of the third group, AD5-5E8 and AD5-14H12, stain
approximately 0.04.+-.0.01% (n=10) of all PBMC (FIG. 1A). According
to scatter signals (FIG. 1B) and counterstaining with mAb against
the TCR.alpha..beta., CD14, CD19 and CD56 (FIG. 1A), these cells
are distinct from lymphocytes and monocytes and slightly larger
than the cells recognized by the antibodies of the first group.
Accordingly, staining of blood DC shows that a different subset is
recognized by AD5-5E8 and ADS-14H12, namely
CD11c.sup.dimCD123.sup.-blood DC (FIG. 2). According to two-color
stainings, cross-blocking studies and co-capping experiments both
mAb appear to recognize two spatially unrelated epitopes of the
same antigen. We named this antigen BDCA-3.
[0322] The fourth group, mAb AD5-8E7, reacts with up to
2.39.+-.0.96% (n=10) of unfractionated PBMC (FIG. 1A).
Light-scatter analysis (FIG. 1B) and counter-staining of the
lineage markers TCR.alpha..beta., CD14, and CD19 reveal that the
mAb is not reactive to"T cells and monocytes, but is reactive to a
major subset of small resting CD19.sup.+ B cells. Staining of
purified DC shows that AD5-8E7, in addition to B cells, stains a
third subset of blood DC distinct from those subsets recognized by
the mAb of the first and the second group, namely CD 1
1c.sup.brightCD123,.sup.d" blood DC. A significant proportion of
the CD11C.sup.brightCD123.sup.dim blood DC expresses CD56 (see
below). For this reason, some AD5-8E7-reactive PBMC stain for CD56
(FIG. 1A). AD5-8E7 is not reactive to purified NK cells. The
antigen recognized by AD5-8E7 was initially named BDCA-1 as it
appeared to be a new antigen. However, it later transpired that
AD5-8E7 completely blocks binding of the CD1c mAb M241 to MOLT-4
cells (FIG. 6). Thus, the antigen recognized by AD5-8E7 is
CD1c.
[0323] None of the mAb listed in Table 1 is reactive with
granulocytes, platelets, erythrocytes, purified basophils and
purified CD34.sup.+ hematopoietic progenitor cells.
Example 6
Expression of BDCA-2, BDCA-3 and BDCA-4 on Cultured Blood DC, Mo-DC
and CD34-DC
[0324] Freshly isolated plasmacytoid CD11c.sup.-blood DC depend on
IL-3 for survival and maturation, whereas survival and maturation
of CD11c.sup.+ blood DC is far less cytokine-dependent. Expression
of BDCA-2, BDCA-3 and BDCA-4 on CD1c.sup.-and CD11e blood DC was
analyzed after 0 h, 1 h, 3 h, 6 h, 9 h, 12 h, 18 h, 24 h, 36 h, and
48 h of culture of total blood DC in the presence of rIL-3. The
results are shown in FIG. 4. Expression of BDCA-2 is completely
down-regulated within 48 h on CD11c.sup.-blood DC. In contrast,
BDCA-4 is even further up-regulated on CD11c.sup.-blood DC and,
unlike BDCA-2, is also expressed to a high level on most, if not
all, CD11c.sup.+DC. Expression of BDCA-3 is rapidly induced on
CD11c.sup.-blood DC, reaching the highest expression level after 24
h. Thereafter, BDCA-3 expression appears to be down-regulated
again. Analyzing the expression of BDCA-3 on CD11c.sup.+ blood DC
is complicated by the fact that BDCA-3.sup.-CD11c.sup.bright and
BDCA-3.sup.+CD11c.sup.dim subsets are present at the onset of the
culture: Expression of BDCA-3 remains unchanged at least until 6 h
of culture on the BDCA-3.sup.+CD11c.sup.bright blood DC population,
and is induced within 3 h on at least some cells of the
BDCA-3.sup.-CD11c.sup.bright blood DC subset. Expression of BDCA-2,
BDCA-3 and BDCA-4 on Mo-DC and CD34.sup.-DC.
[0325] Functional CD1a.sup.+DC were generated ex vivo from
monocytes and from CD34.sup.+ hematopoietic progenitor cells.
Bender et al. (1996); Pickl et al. (1996; Romani et al. (1994);
Sallusto et al. (1994); Caux et al. (1992); Mackensen et al.
(1995); Szabolcs et al. (1995); Herbst et al. (1996); de Wynter et
al. (1998); and Strunk et al. (1996). FIG. 7 shows that Mo-DC,
which were generated by culturing monocytes for 7 d in the presence
of rGM-CSF and IL-4 and CD34-DC, generated by culturing CD34.sup.+
hematopoietic progenitor cells for 11 d in the presence of
rFlt3-Ligand, rTGF-.beta.1, rTNF-.alpha., rSCF and rGM-CSF, express
CD1a, CD1c and BDCA-4, but neither BDCA-2 nor BDCA-3.
Example 7
Internalization of BDCA-2 Upon Anti-BDCA-2 mAb-Mediated
Cross-Linking
[0326] The possibility that 37.degree. C. incubation of anti-DCA-2
mAb-labeled BDCA-2.sup.+ cells results in mAb internalization was
addressed by staining of PBMC with FITC-conjugated AC144 mAb
(IgG1). Then, following incubation at 37.degree. C., remaining cell
surface associated mAb was detected by staining with PE-conjugated
rat anti-mouse IgG1 mAb. As shown in FIG. 8, when cells were
incubated at 37.degree. C., the intensity of the rat anti-mouse
IgG1-PE staining decreases extremely rapidly to background levels.
In contrast, the intensity of the AC144-FITC staining decreases
only temporarily to a level of approximately 50%, but thereafter
nearly returns to the pre-incubation level. This demonstrates that
BDCA-2 is internalized upon anti-BDCA-2 mAb cross-linking, with
kinetics similar to receptor-mediated endocytosis. The transient
decrease in AC144-FITC staining intensity is probably due to
patching and capping of the BDCA-2/anti-BDCA-2 mAb complex before
endocytosis.
Example 8
Morphology of Isolated CDIc.sup.+, BDCA-2.sup.+ and BDCA-3.sup.+
Blood DC
[0327] CD1c.sup.+, BDCA-2.sup.+ and BDCA-3.sup.+ cells were
isolated from PBMC by indirect magnetic labeling with PE-conjugated
primary mAb and anti-PE Ab-conjugated microbeads and enrichment of
labeled cells by MACS (FIG. 9). On May Grunwald/Giemsa staining of
cytocentrifuge slides (FIG. 9), freshly isolated BDCA-2-expressing
cells display the typical lymphoplasmacytoid morphology of
CD11c.sup.-CD4.sup.+lin.sup.-DC from blood and tonsils: that is,
medium-sized round cells with oval or indented nuclei. In contrast,
both freshly isolated CD1c.sup.+blood DC as well as freshly
isolated BDCA-3.sup.+ blood DC display the typical morphological
characteristics of CD11c.sup.+CD4.sup.+lin.sup.- DC from blood or
tonsils: that is, less rounded cells with short cell processes and
more hyperlobulated nuclei. In addition to CD1c.sup.+ BDC,
CD1c.sup.+ B cells with the typical morphology of small resting
lymphocytes can be seen on the cytocentrifuge slides of isolated
CD1c.sup.+PBMC. Highly pure CD1c.sup.+ BDC are obtained if, prior
to the enrichment of CD1c.sup.+ cells, CD19.sup.+ B cells are
magnetically depleted from PBMC.
Example 9
Surface Phenotype of CD1c.sup.+, BDCA-2.sup.+ and BDCA-3.sup.+
Blood DC
[0328] The phenotype of BDCA-2.sup.+ and BDCA-3.sup.+ blood DC was
analyzed by two-color irnmunofluorescence with PE- and
FITC-conjugated mAb. For analysis of CD1c.sup.+ blood DC,
three-color stainings were performed using CD19-Cy5 for exclusion
of B cells. The results of the phenotypic analysis are shown in
Table 2 and can be summarized as follows: none of the blood DC
subsets express CD1a, CD8, CD15, CD16, CD19, CD20, CD23, CD25,
CD27, CD34, CD61, CD69, CD7.1, CD77, CD80, CD83, glycophorin A
(GPA), TCRal3, AC133, IgD, IgM and the CMRF-56 antigen. All blood
DC subsets express CD43, CD44, CD54 and MHC class I molecules at a
similar level. BDCA-2.sup.+ blood DC differ from the other two
subsets in that they do not express CD13, CD40, CD45RO, CD56, but
CD45RA and little amounts of CD10, and in that they express lower
levels of CD18, CD38, CD58, CD98, CD116 and CLA, but higher levels
of CD4. CD1c.sup.+ blood DC differ from the other two subsets in
that they express CD2, higher levels of MHC class11 molecules, but
lower levels of CD62L, and in that they express the Fc receptors
CD32, CD64 and Fc.sub..epsilon.R1. Probably due to the Fc
receptor-expression, CD1c.sup.+ blood DC are also positive for IgG,
kappa and lambda. Furthermore, some CD1c.sup.+DC are positive for
CD14 and CD11b, whereby the level of expression inversely
correlates with the level of both CD1c and CD2 expression.
BDCA-3.sup.+ blood DC differ from the other two subsets in that
they express CD36 at a much lower level and in that they appear to
express low levels of CDS. Finally, apart from CD11c and CD123, at
least one additional antigen, CD33, is useful for discrimination of
all three subsets: CD33 is expressed at low levels on
BDCA-2.sup.+DC, at intermediate levels on BDCA-3.sup.+DC and at
high levels on CD1c.sup.+DC.
TABLE-US-00003 TABLE 2 Antigen Clone BDCA-2.sup.+ BDCA-3.sup.+
CD1c.sup.+ CD1a HI149 - - - CD1c M241 - - + CD2 S5.2 -/minor
subset+ - + CD4 M-T321 ++ + + CD5 CLB-T1/1, 6G4 - -/+ -/+ CD7
CLB-T3AI, 7F3 -/minor subset+ - + CD8 SK1 - - - CD10 HI1Oa -/+ - -
CD11a G43-25B + ++ + CD11b M1/7O.15.11.5 - - -/+ CD11c Ki-M1 - + ++
CD13 L138 - + + CD14 TUK4 - - -/+ CD15 VIMC6 - - - CD16 CLB-FcR
Gran/1 - - - CD18 7E4 + ++ ++ CD19 SJ25-C1 - - - CD20 HI47 - - -
CD23 9P25 - - - CD25 M-A251 - - - CD27 M-T271 - - - CD32 FL18.26
(2003) - - + CD33 P67.6 -/+ + ++ CD34 8G12 - - - CD36 AC106 + -/+ +
CD38 HIT2 + ++ ++ CD40 FC3 - -/+ -/+ CD43 1G10 + + + CD44 IM7 + + +
CD45RA F8-11-13 + - - CD45R0 UCHL-1 - + + CD54 HA58 + + + CD56
NCAM16.2 - -/subset+ -/subset+ CD58 AICD58 + ++ ++ CD61 VIPL2 - - -
CD62L DREG56 - + + CD64 10.1 ++ ++ + CD69 FN50 - - - CD71 LO1.1 - -
- CD77 38.13 - - - CD80 DAL-1 - - - CD83 HB15A - - - CD86 HA5.2B7 +
++ +++ CD98 HIM6 ++ +++ +++ CD116 SC06 + ++ ++ CD123 AC145 ++ - +
HLA-DR AC122 + + ++ HLA-DQ TU169 + + ++ HLA-A, B, C W6/32 + + + GPA
AC107 - - - TCR.alpha..beta. T10B9.1A-31 - - - AC133 AC133 - - -
Fc.sub..epsilon.R I 15-1 - - + IgD TA4.1 - - - IgG Polyclonal - - +
IgM SA-DA4 - - - Kappa Polyclonal - - + Lambda Polyclonal - - + CLA
HECA-452 +++ +++ +++ CMRF44 CMRF44 - - -/minor subset+ CMRF56
CMRF56 - - -
Example 10
Expression of MHC Class H, CD83 and Co-Stimulatory Molecules on
CD1c.sup.+, BDCA-2.sup.+ and BDCA-3.sup.+ Blood DC after
Culture
[0329] Freshly isolated CD1c.sup.+ blood DC and BDCA-3.sup.+ blood
DC were cultured for 1 d in medium without any supplemented
cytokines and freshly isolated BDCA-2.sup.+ blood DC were cultured
for 2 d in medium supplemented with IL-3 and CD40 mAb on
CD32-transfected fibroblasts. After the culture period, cells were
analyzed for the expression of CD1a, CD80, CD83, CD86 and HLA-DR.
For comparison, so-called "immature" Mo-DC, generated by culturing
of monocytes for 7 d in the presence of GM-CSF and IL-4, and
so-called "mature" Mo-DC, generated by culturing of "immature"
Mo-DC for 3 din the presence of TNF-.alpha., were also included.
Sallusto et al. (1995) J. Exp. Med. 182:389-400; and Sallusto et
al. (1998) J. Immunol. 28:2760-2769. As shown in FIG. 10, in
contrast to "immature" Mo-DC and "mature" Mo-DC, none of the blood
DC subsets expresses CD1a after the culture period. However, the
costimulatory molecules CD80 and CD86, the DC activation antigen
CD83 (Zhou et al. (1995); Zhou et al. (1992) J. Immunol.
149:735-742; and Zhou et al. (1996) Proc. Nati. Acad. Sci. USA
93:2588-2592), and HLA-DR molecules are up-regulated upon culturing
on all three blood DC subsets to a similar level as compared to
mature Mo-DC. The result's were not significantly different in
another experiment in which all three blood DC subsets were
cultured for 2 d in medium supplemented with IL-3, IL-4 and GM-CSF.
As has been previously shown for CD11c.sup.-CD4.sup.+lin.sup.-DC
from blood and tonsils, BDCA-2.sup.+ blood DC rapidly die when
cultured in medium without IL-3.
Example 11
Endocytic Capacity of Freshly Isolated CD1c.sup.+, BDCA-2.sup.+ and
BDCA-3.sup.+ Blood DC
[0330] The endocytic capacity of purified CD1c.sup.+, BDCA-2.sup.+
and BDCA-3.sup.+ blood DC, and, as a control, of purified CD3.sup.+
T cells was examined by culturing the cells at 37.degree. C. in the
presence of LY and analyzing the uptake of LY after various periods
of time by flow cytometry. As shown in FIG. 11, unlike purified
CD3.sup.+ T cells, purified CD1c.sup.+ blood DC, BDCA-3.sup.+ blood
DC, and to some extent also BDCA-2.sup.+ blood DC have the ability
to endocytose LY. Similar results were obtained using FITC-Dextran.
The endocytic capacities of all blood DC populations are much lower
if compared with Mo-DC.
[0331] The amino acid sequence of BDCA-4 was obtained by purifying
the antigen with AD5-17F6 mAb (AD5-17F6 affinity column) and
analyzing the purified antigen by MALDI TOF mass spectrometry.
BDCA-4 is identical to neuropilin-1. He et al. (1997).
Example 12
Ligation of BDCA-2 with Anti-BDCA-2 Monoclonal Antibody (AC144)
Induces Intracellular Ca.sup.2+ Mobilization, Whereas Ligation of
BDCA-4 (Neuropilin-1) with Anti-BDCA-4 does not Induce Ca.sup.2+
Mobilization
Materials and Methods:
[0332] Measurement of cytosolic calcium in BDCA-2.sup.+BDCA-4.sup.+
BDC and BDCA-2-transfected or non-transfected U937 cells.
BDCA-2.sup.+BDCA-4.sup.+ blood DC and BDCA-2-transfected or
non-transfected U937 cells were loaded with Indo-1 AM (Sigma, St.
Louis, Mo.) as described by Valitutti et al. (1993) Eur. J.
Immunol. 23:790-795. Anti-BDCA-2 (AC144, IgG1) or anti-BDCA-4
(AD5-17F6, IgG1) mAb were added to freshly isolated
BDCA-2.sup.+BDCA-BDC and BDCA-2-transfected or non-transfected U937
cells, respectively, followed or not followed by rat anti-mouse
IgG1 mAb (X56) as cross-linker. Cells were analyzed on a flow
cytofluorimeter to detect Ca.sup.2+ fluxes. Only live (based on
scatter criteria) and Indo-1-labeled cells (based on 405 nm versus
525 nm emission spectra) were included in the analysis.
[0333] FIG. 13 shows intracellular mobilization is induced in
immunomagnetically purified BDCA-2.sup.2+DCA-4.sup.+ BDC (A, B) and
BDCA-2-transfected U937 cells (D), but not in nori-transfected U937
cells (E) via anti-BDCA-2 mAb alone (A) and or anti-BDCA-2 plus
crosslinking secondary mAb (B, D, E).
[0334] Ligation of BDCA-4 on immunomagnetically purified
BDCA-2.sup.+BDCA-4.sup.+ BDC with anti-BDCA-4 mAb and cross-linking
secondary mAb does not induce cytosolic Ca.sup.2+-mobilization.
Shown is the Ca.sup.2+-dependent 405 nm/525 nm ratio of
Indo-1-fluorescence (Y-axis) against time (X-axis, a value of 1024
corresponds to 204.80 sec).
[0335] As shown in FIG. 13, ligation of surface BDCA-2 on
plasmacytoid BDC (FIGS. 13A and B) and BDCA-2-transfected U937
cells (FIG. 13D) with a specific mAb (AC144, IgG1) followed (FIGS.
13B and D) or not followed (FIG. 13A) by a secondary cross-linking
mAb (rat anti-mouse IgG1, X56) elicited a rapid and transient rise
in cytosolic calcium concentration. On the contrary, incubation of
plasmacytoid DC with anti-BDCA-4 mAb (AD5-17F6) followed by a
secondary cross-linking mAb (rat anti-mouse IgG1, X56) (FIG. 14C),
or of non-transfected U937 cells with anti-BDCA-2 mAb (AC144, IgG1)
followed by a secondary cross-linking mAb (rat anti-mouse IgG1,
X56) (FIG. 13E) did not induce a rapid and transient rise in
cytosolic calcium concentration.
Example 13
Production of Type I Interferon by Purified BDCA-2 BDCA-4.sup.+ BDC
in Response to Viral Stimulation (Influenza Virus Strain PR8) is
Inhibited by Triggering of BDCA-2 with Anti-BDCA-2 mAb
[0336] CD4.sup.+CD123.sup.brightCD11c.sup.- plasmacytoid DC were
shown to be the chief type I interferon producers in response to
enveloped viruses, bacteria, and tumor cells. Fitgerald-Bocarsly et
al. (1993) Pharmacol. Ther. 60:39-62; Siegal et al. (1999) Science
284:1835-1837; Cella et al. (1999) Nature Med. 5:919-923. For, this
reason, they have also been called natural type I interferon
producing cells (NIPC). Plasmacytoid DC express BDCA-2 and BDCA-4.
As shown in FIG. 14, ligation of surface BDCA-2 on plasmacytoid DC
with a specific mAb followed by a secondary cross-linking mAb (rat
anti-mouse IgG1, X56), inhibits secretion of type I interferon by
immunomagnetically purified plasmacytoid BDCA-2.sup.+BDCA-4.sup.+DC
from blood or tonsils in response to stimulation with influenza
virus strain PR8 (5 HAU/ml). The level of type I interferon
production in cultures with anti-BDCA-2, influenza virus and
cross-linking mAb (FIG. 14, AC144.sup.+RamG1-FLU) is much lower as
in cultures with influenza virus alone (FIG. 14, FLU), or with an
isotype control mAb (anti-cytokeratin mAb CK3-11D5, IgG1),
influenza virus and cross-linking mAb (FIG. 14, CK3+RamG1+FLU).
[0337] Conversely, ligation of surface BDCA-4 on plasmacytoid DC
with a specific mAb followed by a secondary cross-linking mAb (rat
anti-mouse IgG1, X56), does not inhibit secretion of type I
interferon by immunomagnetically purified plasmacytoid
BDCA-2.sup.+BDCA-4.sup.+DC from blood or tonsils in response to
stimulation with influenza virus strain PR8 (5 HAU/ml). The level
of type I interferon production in cultures with anti-BDCA-4,
influenza virus and cross-linking mAb (FIG. 14,
17F6.sup.+RamG1.sup.+FLU) is the same as in cultures with an
isotype control mAb (anti-cytokeratin mAb CK3-11D5, IgG1),
influenza virus and cross-linking mAb (FIG. 14, CK3+RamG1+FLU).
Materials and Methods:
[0338] BDCA-2- and BDCA-4-expressing plasmacytoid DC were isolated
from PBMC (FIG. 14A) or tonsillar cells (FIG. 14B) by direct
magnetic labeling with anti-BDCA-4 (AD5-17F6)-conjugated microbeads
and enrichment of labeled cells by MACS. Isolated BDCA-2- and
BDCA-4-expressing plasmacytoid DC were cultured for 24 hours in
medium in the presence of: a) IL-3 alone (FIG. 14, Control); b)
IL-3, anti-BDCA-2 mAb (AC144, IgG1) and rat anti-mouse IgG1 mAb
(FIG. 14, AC144+RamG1); c) IL-3, anti-BDCA-2 mAb (AC144, IgG1), rat
anti-mouse IgG1 mAb, and influenza virus strain PR8 (FIG. 14,
AC144+RamG1+FLU); d) IL-3 and influenza virus strain PR8 (FIG. 14,
FLU); e) IL-3, anti-cytokeratin mAb.(CK3-11D5, IgG1), rat
anti-mouse IgG1 mAb, and influenza virus strain PR8 (FIG. 14,
CK3+RamG1+FLU); and f) IL-3, anti-BDCA-4 mAb (AD5-17F6), rat
anti-mouse IgG1 mAb, and influenza virus strain PR8 (FIG. 14
17F6.sup.+RamG1.sup.+FLU).
[0339] Secreted type I interferon in the culture supernatants was
measured by evaluating inhibition of Daudi cell proliferation
(Nederman et al. (1990) Biologicals 18:29-34) with reference to a
standard IFN-.alpha. curve.
[0340] Regarding the inhibition of type I interferon production by
BDCA-2.sup.+BDCA-4.sup.+ plasmacytoid DC, increased levels of
circulating type I interferon and of type I interferon inducing
factor (something like a complex of anti-DNA antibody and DNA) are
found in SLR patients and correlate to disease activity.
Furthermore, patients with non-autoimmune disorders treated with
type I interferon frequently develop autoantibodies and
occasionally SLE. Several papers from Ronnblom et al. (1999) Clin.
Exp. Immunol. 115: 196.202; (1999) J. Immunol. 163: 6306-6313; and
(2000) J. Immunol. 165: 3519-3526) show that type I interferon
inducing factors derived from patients induce secretion of type I
interferon in PBMC from healthy donors and they selectively
activate natural type I interferon producing cells
(NIPC=plasmacytoid DC).
[0341] The findings presented herein that ligation of BDCA-2
suppresses.the production of type I interferon induced by viral
stimulation show that binding to BDCA-2 can be applied to treat the
disease not just by ligation of BDCA-2 but also by depleting
NIPC(=BDCA-2+ BDCA-4+ plasmacytoid DC). The invention thus further
encompasses in vivo, in vitro and ex vivo depletion of NIPC. Such
depletion is suitable for use in treatment or prophylaxis of
autoimmune diseases.
[0342] FIG. 14 shows ligation of BDCA-2 but not of BDCA-4 with a
specific mAb followed by a secondary cross-linking mAb inhibits
secretion of type I interferon by plasmacytoid
BDCA-2.sup.+BDCA-4.sup.+DC from blood or tonsils in response to
stimulation with influenza virus strain PR8. Plasmacytoid
BDCA-2.sup.+DCA-4.sup.+DC from blood (A) or tonsils (B) were
cultured for 24 hours in the presence of IL-3 alone (control);
IL-3, anti-BDCA-2 mAb and rat anti-mouse IgG1 mAb (AC144+RamG1);
IL-3, anti-BDCA-2 mAb, rat anti-mouse IgG1 mAb, and influenza virus
strain PR8 (AC144+RamG1+FLU); IL-3 and influenza virus strain PR8
(FLU); IL-3, anti-cytokeratin mAb, rat anti-mouse IgG1 mAb, and
influenza virus strain PR8 (CK3+RamG1+FLU); anti-BDCA-4 mAb, rat
anti-mouse IgG1 mAb, and influenza virus strain PR8
(17F6.sup.+RamG1.sup.+FLU). Secreted type I interferon (U/ml) in
the culture supernatants was measured by a bioassay with reference
to a standard type I interferon curve.
Example 14
BDCA-2 is not Only Able to Endocytose a Ligand, but Also to Deliver
it to an Antigen-Processing and Loading Compartment, and to Present
it to CD.sup.4+ Class II-Restricted T Cells
Materials and Methods:
[0343] BDCA-2- and BDCA-4-expressing plasmacytoid DC were isolated
from PBMC by direct magnetic labeling with anti-BDCA-4
(ADS-17F6)-conjugated microbeads and enrichment of labeled cells by
MACS. Isolated BDCA-2- and BDCA-4-expressing plasmacytoid DC were
co-cultured with 4.times.10.sup.4 cells/well of the B13 T cell
clone (Lanzavecchia et al. (1988) J. Exp. Med. 167:345-352) in
96-well flat-bottom microplates in the presence of IgG1 mAbs (0.2
.mu.g/ml). mAbs used in the assay were the following: AC144
(anti-BDCA-2, IgG1), ZM3.8 (anti-ILT3, IgG1) and CK3-11D5
(anti-cytokeratin, IgG1). After 48 hours, the cultures were pulsed
with (.sup.3H)thymidine (1 Ki/well), and the radioactivity
incorporated was measured after additional 16 hours. (3H)Thymidine
uptake (cpm) was plotted against the number of isolated BDCA-2- and
BDCA-4-expressing plasmacytoid DC in the cultures (FIG. 15).
[0344] FIG. 15 shows presentation of anti-BDCA-2 mAb (AC144, IgG1)
to a T cell clone specific for mouse IgG1 by isolated BDCA-2- and
BDCA-4-expressing plasmacytoid DC. BDCA-2.sup.+DCA-4.sup.+
plasmacytoid DC present anti-BDCA-2 mAb (AC144, IgG1, .box-solid.)
to T cells much more efficiently than anti-ILT-3 mAb (ZM3.8, IgG1,
.tangle-solidup.) and anti-cytokeratin mAb (CK3-11D5, IgG1,
.circle-solid.).
[0345] Incubation of anti-BDCA-2 mAb (AC144, IgG1)-labeled
BDCA-2.sup.+BDCA-4.sup.+ plasmacytoid DC at 37.degree. C. results
in extremely rapid internalization of the anti-BDCA-2 mAb/BDCA-2
complexes on the cell surface (see FIG. 8). Here, it is shown that
the anti-BDCA-2 mAb (AC144, IgG1) accesses an antigen-processing
and loading compartment and peptides derived from the antibody are
efficiently presented to a CD4.sup.+ class II-restricted T cell
clone (B13) specific for a mouse IgG1 peptide epitope. The
presentation of the anti-BDCA-2 mAb was compared to that of an IgG1
mAb that binds to a receptor (ILT3) known to be capable of
targeting its ligand(s) into processing and peptide-loading
compartments, and to that of an IgG1 mAb that does not bind to a
cell-surface molecule on BDCA-2.sup.+DCA-4.sup.+ plasmacytoid DC
(anti-cytokeratin mAb CK3-11D5, IgG1), but can be taken up in the
fluid phase. As shown in FIG. 15, BDCA-2.sup.+DCA-4.sup.+
plasmacytoid DC presented anti-BDCA-2 mAb (AC144) to T cells much
more efficiently than the anti-ILT-3 mAb and the anti-cytokeratin
mAb.
Example 15
In Tonsillar Cells, Expression of BDCA-2 is Restricted to
CD123.sup.+ T Cell-Zone Associated Plasmacytoid DC, Whereas BDCA-4
May Also be Expressed at Low Levels on a Few Other Cells
[0346] FIG. 16 shows expression of BDCA-2 and BDCA-4 on tonsillar
plasmacytoid CD123.sup.+DC. Shown are two-color stainings of
tonsillar cells with a FITC-conjugated mAb against BDCA-2 (AC144)
and a PE-conjugated mAb against CD123 and BDCA-4 (AD5-17F6),
respectively. Note that expression of BDCA-2 is restricted to
CD123bright plasmacytoid DC, whereas BDCA-4 is also expressed at
low levels on a few other cells.
Example 16
BDCA-4 mAb (A115-17F6) Recognizes Neuropilin-1
[0347] Neuropilin-1 is a receptor for the collapsin/semaphorin
family that mediates neuronal cell guidance. Neuropilin-1 is also
expressed by endothelial and tumor cells as an isoform-specific
receptor for vascular endothelial growth factor. However, it was
not known before, that neuropilin-1 is expressed on, plasmacytoid
DC in blood and tonsils and that it represents an excellent marker
for plasmacytoid DC at least in fresh non-cultured blood.
Material and Methods:
[0348] Neuropilin-1 was immunoprecipitated from cell lysates of
non-transfected PAE cells (P) and neuropilin-1-transfected PEA
cells (NP) (Soker et al. (1998) Cell 92:735-745) using the
anti-BDCA-4 mAb AD5-17F6 (anti-NRP-1 (ML)). Precipitated proteins
were analyzed by SDS-PAGE and Western blotting with the
BDCA-4-specific mAb AD5-17F6 (ML) or a neuropilin-1-specific mAb
from Shay Soker, Children's Hospital, Boston, Mass. (S).
[0349] FIG. 17 shows that neuropilin-1 was immunoprecipitated from
cell lysates of neuropilin-1-transfected PEA cells (NP) but not of
non-transfected PAE cells (P) with the anti-BDCA-4 mAb AD5-17F6
(anti-NRP-1 (ML)). Precipitated proteins were analyzed by SDS-PAGE
and Western blotting with the BDCA-4-specific mAb AD5-17F6 (ML) or
a neuropilin-1-specific mAb from Shay Soker, Children's Hospital,
Boston, Mass. (S).
[0350] Note that the BDCA-4-specific mAb AD5-17F6
immunoprecipitates a specific band of about 130-140 kDa from
neuropilin-1-transfected PEA cells (NP), but not from
non-transfected PAE cells (P). The band can be detected with the
neuropilin-1-specific mAb from Shay Soker (S) but not with the
anti-BDCA-4 mAb AD5-17F6 (ML). Thus, our anti-BDCA-4 mAb AD5-17F6
recognizes the-native form of neuropilin-1 in standard
immunoprecipitation experiments, but fails to detect the denatured
form of neuropilin-1 when used in SDS-PAGE/Western blotting
experiments.
[0351] Interestingly, neuropilin-1 is also expressed by endothelial
and tumor cells as an isoform-specific receptor for vascular
endothelial growth factor (VEGF). More interestingly, several
papers (Gabrilovich et al. (1996) Nature Med. 2:1267; Nature Med.
2: 1096-103; Gabrilovich et aL (1999) Clin. Cancer Res. 5: 2963-70;
Ohm et al. (1999). J. Immunol. 163: 3260-8; Oyama et al. (1998) J.
Immunol. 160:1224-32; Gabrilovich et al. (1998). Blood 92:4150-66;
Ishida et al. (1998) J. Immunol. 161:4842-51) have shown that VEGF
produced by a large percentage of tumors decreases DC generation
and function in vivo. It is not clear whether these effects on DCs
are mediated by neuropilin-1 (BDCA-4), but the invention
encompasses neuropilin-1-mediated functional modulation of DCs.
Example 17
Production of Type I Interferon (IFN-.alpha.) by Purified
BDCA-2.sup.+BDCA-4.sup.+ BDC in Response to Stimulation with Poly
I:C is Inhibited by Triggering of BDCA-2 with Anti-BDCA-2 mAb
[0352] CD4.sup.+CD123.sup.brightCD11c.sup.-plasmacytoid DC were
shown to be the chief type I interferon producers in response to
enveloped viruses, bacteria, and tumor cells. Fitzgerald-Bocarsly
et al. (1993) Pharmacol. Ther. 60:39-62; Siegal et al. (1999)
Science 284: 1835-1837; Cella et al. (1999) Nature Med. 5:919-923.
For this reason, they have also been called natural type I
interferon producing cells (NIPC). Plasmacytoid DC express BDCA-2
and BDCA-4. As shown in FIG. 19, ligation of surface BDCA-2 on
plasmacytoid DC with a specific mAb followed by a secondary
cross-linking mAb (goat anti-mouse IgG), inhibits secretion of
IFN-.alpha. by immunomagnetically purified plasmacytoid
BDCA-2.sup.+DCA-4.sup.+DC from blood or tonsils in response to
stimulation with poly1:C. The level of 1FN-a production in cultures
with anti-BDCA-2, poly I:C and cross-linking mAb (FIG. 18,
AC144+Goat anti-mouse IgG+Poly I:C) is lower as in cultures with
mouse IgG1, poly I:C and cross-linking mAb (FIG. 18, Mouse
IgG1+Goat anti-mouse IgG+Poly I:C).
Materials and Methods:
[0353] CD11c CD123.sup.bright plasmacytoid DC were separated from
human peripheral blood mononuclear cells using BDCA-4 microbeads.
CD11c.sup.- C123.sup.bright plasmacytoid DC (1.times.10.sup.6
cells/ml) were incubated with 10 .mu.g/ml of AC144 mAb or mouse
IgG1 mAb (CF6B, anti-TPO) in RPMI, 10% FCS, 10 mM HEPES, 50.mu.,M
2-ME, 20 .mu.g/ml gentamicin at 37.degree. C. for 30 min. 20
.mu.g/ml of goat anti-mouse IgG (Chemicon International) were added
and cells were again incubated at 37.degree. C. for 30 min. These
cells were cultured with or without 20 .mu.g of poly I:C (Sigma) at
37.degree. C. for 24 hours. Culture supernatants were harvested and
EFN-a concentrations were determined by ELISA (Endogen). The
sensitivity of the assay is 3 pg/ml.
[0354] FIG. 18 shows ligation of BDCA-2 but not of BDCA-4 with a
specific mAb followed by a secondary cross-linking mAb inhibits
secretion of IFN-.alpha. by plasmacytoid BDCA-2.sup.+BDCA-4.sup.+DC
from blood or tonsils in response to stimulation with poly I:C.
Plasmacytoid BDCA-2.sup.+BDCA-4.sup.+DC from blood were cultured
with 10 .mu.g/ml of AC144 mAb (2 and 4) or mouse IgG1 mAb (CF6B,
anti-TPO, 1 and 3) at 37.degree. C. for 30 min. 20 .mu.g/ml of goat
anti-mouse IgG were added and the cells were again incubated at
37.degree. C. for 30 min. These cells were cultured with (3 and 4)
or without (1 and 2) 20 ug of poly I:C at 37.degree. C. for 24
hours. Culture supernatants were harvested and IFN-.alpha.
concentrations were determined by ELISA.
Example 18
BDCA-2 mRNA Expression Analysis by RT-PCR in Various Tissues and
Purified Blood Cell Populations
Nucleic and Amino Acid Sequences
[0355] The cDNA encoding BDCA-2 was obtained by expression cloning
in COS cells. FIG. 5 shows the amino acid sequence of BDCA-2 (the
isoform with all six exons expressed). BDCA-2 is a novel C-type
lectin type II membrane protein. Such lectins are described, for
instance in Bates et al. (1999) J. Immunol. 163:1973-1983.
Comparison of BDCA-2 to known C-type lectins is shown in Example
20.
[0356] FIG. 19 shows on analysis of human multiple tissue cDNA
panels from Clonetech (lane 1: heart; lane 2: brain; lane 3:
placenta; lane 4: lung; lane 5: liver; lane 6: skeletal muscle;
lane 7: kidney; lane 8: pancreas; lane 9: spleen; lane 10: thymus;
lane 11: testis; lane 12: ovary; lane 13: small intestine; lane 14:
lymph node; lane 15: bone marrow; lane 16: fetal liver; lane 17:
tonsil) and on analysis of cDNAs prepared from different
populations of blood leukocytes (lane 18: T cells; lane 19: B
cells; lane 20: NK cells; lane 21: monocytes; lane 22:
CD11c.sup.brightCD123.sup.lowDC; lane23: CD11c-CD123.sup.bright
plasmacytoid BDC) for the presence of BDCA-2 cDNA.
[0357] All cDNAs were normalized to the mRNA expression level of
several different housekeeping genes (glyceraldehyde-3-phosphate
dehydrogenase, phospholipase A2, a-tubulin, and .beta.-actin).
Normalization ensures an accurate assessment of tissue specificity
and relative abundance of target mRNAs. The same amount of cDNA
(about 50 pg) was used for each RT-PCR reaction. RT-PCR reactions
were performed with specific primers for BDCA-2 (forward:
5'-TTGAAAGAACCACACCCCGAAAGT (SEQ ID NO:7) and reverse:
5'-TAGCTTTCTACAACGGTGGATGCC (SEQ ID NO:8)) and primers for the four
housekeeping genes (CLONTECH) mentioned above using AdvanTaq Plus
DNA Polymerase (CLONTECH).
[0358] Cycle conditions were as follows: 94.degree. C. for 30 sec
and 68.degree. C. for 2 min. 34 cycles were used for BDCA-2 and 38
cycles for glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Note
that BDCA-2 mRNA signals are only detected in CDI 1c
CD123.sup.bright plasmacytoid DC. If four more PCR cycles were used
for amplification of BDCA-2 cDNA (38 cycles instead of 34 cycles),
weak signals were also detected in pancreas, testis, ovary, bone
marrow and tonsil. With cDNA from testis (38 PCR cycles), signals
of shorter transcripts (splice variants) were more prominent as
compared to the signals from CD11c.sup.-CD123.sup.bright
plasmacytoid DC and signals from the full-length transcript were
actually only detectable with even more PCR cycles.
Example 19
Exon/Intron Structure of BDCA-2 and Splice Variants of BDCA-2
[0359] The information on the splice variants of BDCA-2 was
obtained by RT-PCR amplification of mRNA from plasmacytoid DC with
primers complementary to mRNA sequences in front of the start codon
(forward primer: 5'-TTGAAAGAACCACACCCCGAAAGT (SEQ II) NO:7)) and
behind the stop codon (reverse primer:
5'-TAGCTTTCTACAACGGTGGATGCC(SEQ ID NO:8)), cloning of the resulting
fragments in plasmids and sequencing of the inserts. The results
are shown in FIG. 20. For comparison, splice variants of mouse
dendritic cell-associated C-type lectin 2 (pectin-2) are shown in
FIG. 21.
[0360] FIGS. 22(A), 22(B), 22(C), and 22(D) show an alignment of
the mRNA sequences of BDCA-2 and mouse Dectin-2 with the positions
of the deduced introns being indicated. Table 3 shows the
parameters of the exons.
TABLE-US-00004 TABLE 3 Number of amino acid amino acid residues
residues Exon mRNA encoded encoded 0 0-361 0 1 362-522 1-10 10 2
523-615 11-41 31 3 616-726 42-78 37 4 727-872 78-127 49 5 873-988
128-166 39 6 989-1283 167-213 47
[0361] The positions of the introns are based on Homo sapiens
Chromosome 12 Clone RP11-277J24, Working Draft Sequence, 21
unordered pieces (GenBank Accession Number AC006.517) and the rules
for splicing of transcripts. The intron/exon makeup of BDCA-2 is
similar to that of Dectin-2.
[0362] At least four splice variants of BDCA-2 are produced. These
are an mRNA encoding a protein with all six exons; an mRNA encoding
a protein containing exons 1, 3, 4, 5, and 6 and an mRNA encoding a
protein containing exons 1, 2, 4, 5, and 6 and an mRNA encoding a
protein containing exons 1, 2, 3, 5, and 6.
Example 20
BDCA-2 Homology and Protein Domains
[0363] An alignment of the amino acid sequences of human BDCA-2,
human DCIR (dendritic cell immunoreceptor), and mouse Dectin-2
(dendritic cell-associated C-type lectin-2) is shown in FIG.
23.
[0364] Human DCIR (GenBank Accession Number AJ133532) is the
molecule with the highest homology to BDCA-2 among human molecules
(see Bates et al. (1999) J. Immunol. 163:1973) with about 51% of
the aa being identical over a stretch of 191 aa.
[0365] Mouse Dectin-2 (GenBank Accession Number AF240357) is most
likely the murine homolog of human BDCA-2 (see Ariizumi et al.
(2000) J. Biol. Chem. 16:11957; WO 98/28332; PCT/US97/23761; and
U.S. Pat. No. 6,046,158) with about 51% of the aa being identical
over a stretch of 211 aa.
[0366] BDCA-2 (213 aa), DCIR (237 aa) and Dectin-2 (209 aa) are all
type II membrane glycoproteins of the calcium-dependent (C-type)
lectin family. Each of the molecules contains a putative
cytoplasmic domain (BDCA-2: aa 1-21; DCIR: aa 1-44; Dectin-2: aa
1-17), a putative transmembrane domain (BDCA-2: aa 22-41; DCIR:
44-69; Dectin-2: 18-40), and a putative extracellular domain
(BDCA-2: aa 42-213; DCIR: 70-237; Dectin-2: 40-209). Within the
putative extracellular domain, each of the molecules contains a
single carbohydrate recognition domain (CRD) at the COOH-terminal
end (BDCA-2: aa 83-206; DOR: 106-230; Dectin-2: 79-202). FIG. 23
shows the alignment of human 13DCA-2, human DCIR and mouse
Dectin-2.
[0367] Putative protein domains/motifs as found using the PROSLUE
database are shown in Table 4.
TABLE-US-00005 TABLE 4 Domain BDCA-2 ASN glycosylation 110-113 NCSV
(SEQ ID NO: 9) 137-140 NSSY (SEQ ID NO: 10) 164-167 NVTF (SEQ. ID
NO: 11) cAMP- and cGMP- dependent protein 53-56, KRLS (SEQ ID NO:
14) kinase phosphorylation site 135-138 SQK Protein Kinase C
phosphorylation site 51-53 TVK 107-109 SQK Casein kinase ft
phosphorylation site 123-126 TREE (SEQ ID NO: 16) 187-190 SSEE (SEQ
ID. NO: 17) Tyrosine kinase phosphorylation site 57-64 KLREYQQY
(SEQ ID NO: 30) Amidation site 148-151 GGRR (SEQ ID NO: 32)
N-rnyristylation site C-type lectin domain signature 180-206 Domain
Dectin-2 ASN glycosylation 131-134 NESL (SEQ ID NO: 12) cAMP- and
cGMP- dependent protein kinase phosphorylation site Protein Kinase
C phosphorylation site 15-17 TLR 49-51 SRR 72-74 SEK 94-96 STK
Casein kinase II phosphorylation site 94-97 STKE (SEQ ID NO: 18)
101-104 STSE (SEQ ID NO: 19) 119-122 TEAE (SEQ ID NO: 20) 200-203
SI.CE (SEQ ID NO: 21) Tyrosine kinase phosphorylation site 50-58
RRLYELHTY (SEQ ID NO: 31) Amidation site N-myristylation site 11-16
GVCWTL (SEQ ID NO: 33) 68-73 GTMVSE (SEQ ID NO: 34) 77-82 GCCPNH
9SEQ ID NO: 35) C-type lectin domain signature 11-17 176-202 Domain
DCIR ASN glycosylation 185-188 NESS (SEQ ID NO: 13) CamP- and cGMP-
dependent protein 78-81 KKTT (SEQ ID NO: 15) kinase phosnhorylation
site Protein Kinase C phosphorylation site 80-82 TTK 130-132 SEK
211-213 SPK Casein kinase H phosphorylation site 1-9 TYAE (SEQ ID
NO: 22) 80-83 TTKE (SEQ ID NO: 23) 87-90 TTLE (SEQ ID NO: 24)
126-129 SWQD (SEQ ID NO: 25) 130-133 SEKD (SEQ ID NO: 26) 146-149
TQEE (SEQ ID NO: 27) 168-171 SDPE (SEQ ID NO: 28) 228-231 SVCE (SEQ
ID NO: 29) Tyrosine kinase phosphorylation site Amidation site
N-myristylation site 20-25 GINTAS (SEQ ID NO: 36) C-type lectin
domain signature 203-230
[0368] BDCA-2 contains three putative N-glycosylation sites (aa
110-113 NCSV; aa 137-140 NSSY; an 164-167 NVTF), whereas Dectin-2
(aa 131-134 NESL) and DCIR (aa 185-188 'NESS) contain only one
putative N-glycosylation site. All the putative phosphorylation
sites of BDCA-2 and Dectin-2 are located in the putative
extracellular dothain. Thus, it is rather unlikely that they become
phosphorylated by intracellular kinases. Like many C-type lectins
(e.g. CD94, Ly-49, and NKG2) that are encoded in the natural killer
gene complex, DCIR contains the consensus immunoreceptor
tyrosine-based inhibitory motif (ITIM motif; (I/V)XYXX(LN) (SEQ ID
NO:37)) in the cytoplasmic domain (aa 5-10 ITY AEV (SEQ ID NO:38)).
Interestingly, this ITIM motif is not found in the relatively short
cytoplasmic tail of BDCA-2 and Dectin-2 (BDCA-2: 21 aa; Dectin-2:
17 aa).
Example 21
BDCA-3 Protein Analysis
[0369] BDCA-3-expressing HD-MY-Z cells were stimulated for 24 hours
with 10 ng/ml PMA (Sigma) and 0.5 mg/ml Ionomycin to up-regulate
BDCA-3-expression. 3.times.10.sup.7 PMA/Ionomycin stimulated
HD-MY-Z cells were surface biotinylated by incubation for 15
minutes at 4.degree. C. with 1 mg/ml Sulfo-NHS-LC-Biotin (Pierce),
and washed twice. Cells were resuspended in 50 mM Tris-HCL pH 8.0
supplemented with 10% sucrose and proteinase inhibitors
(Phenyhnethylsulfonylfluoride, Pepstatin A, Leupeptin, and
Aprotinin from Serva) and at 0.degree. C. ultrasonified (5.times.4
seconds, 70% output). Sonified cells were centrifuged at
900.times.g at 4.degree. C. for 10 minutes to remove nuclei and
intact cells. The supernatant was centrifuged at 30,000.times.g at
4.degree. C. for 2 hours to obtain purified cell membranes.
Membranes were lysed by incubation in 50 mM Tris-HCl pH 8.0, 150 mM
NaCl supplemented with proteinase inhibitors and 1% NP-40 for 1
hour at 0.degree. C. Non-solubilized membrane fragments were
removed by centrifugation at 30,000.times.g at 4.degree. C. To the
supernatant, MnCl.sub.2 and CaCl.sub.2 were added to a final
concentration of 1 mM each. The lysate was adsorbed onto a ConA
Sepharose column (1 ml), and bound proteins were eluted with 10 ml
elution buffer (0.5 M D(+) Mannose, 20 mM Tris-HCl pH 7.4, 0.5 M
NaCl, 1% NP-40) and concentrated to a volume of 1 ml using
Cetriprep-10 centrifugal concentrators (Amicon).
[0370] The proteins were pre-cleared by incubation with 150 .mu.l
anti-NIP mAb-conjugated MicroBeads (Miltenyi Biotec) for 30 minutes
at 4.degree. C. and .mu.MACS column separation. For specific
immunoprecipitation of BDCA-3, proteins were either incubated with
2 .mu.g of the NIP-conjugated BDCA-3-specific mAb AD5-14H12 (IgG1)
or for control of specificity with 2 .mu.g of the NIP-conjugated
CD19-specific mAb SJ25-C1 (IgG1) as primary reagent for 14 hours at
4.degree. C., and with anti-NIP mAb-conjugated MicroBeads as
secondary reagent for 3 hours at 4.degree. C. Precipitated proteins
were isolated by .mu.MACS column separation. Retained proteins were
eluted with 70 .mu.l SDS-PAGE buffer containing DTT. Precipitated
proteins were analyzed by SDS-PAGE (4-12%) and Western blotting
with streptavidin-peroxidase.
[0371] The results in FIG. 24 show that the BDCA-3-specific mAb
AD5-14H12 specifically immunoprecipitates a cell surface protein of
about 100 kD from HD-MY-Z cells. Thus, BDCA-3 has an apparent
molecular weight of 100 kD.
[0372] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
and understanding, it will be apparent to those skilled in the art
that certain changes and modifications can be practiced. Therefore,
the description and examples should not be construed as limiting
the scope of the invention, which is delineated by the appended
claims.
Sequence CWU 1
1
3811312DNAHomo sapiens 1cagtgattct cgtgcctcag cctcctgagt agccgaaatt
acagacgtgt gccaccatgc 60ttggctaatt ttttggattt ttagtagaga tggggtttca
ctatgttggc caggctagtc 120ttgaactcct ggcctgaagc aatccgccca
cctcagcctc ccaaagtgct gagattatag 180gcacgagcca ctacacctgg
ccacaaaatt ctttaaagaa gccaatccca tcctccctca 240agagccaagg
ggccacctca ccctcttgtt acagcagatc ctgcctccac agtcaccctg
300ctcccaagtg caacctctgt ctgaccctgc atggtgtgcg gtgccctcct
gcctcaggcc 360gcgaagaagg atctaagggc ttggcttgtt tgaaagaacc
acaccccgaa agtaacatct 420ttggagaaag tgatacaaga gcttctgcac
ccacctgata gaggaagtcc aaagggtgtg 480cgcacacaca atggtgcctg
aagaagagcc tcaagaccga gagaaaggac tctggtggtt 540ccagttgaag
gtctggtcca tggcagtcgt atccatcttg ctcctcagtg tctgtttcac
600tgtgagttct gtggtgcctc acaattttat gtatagcaaa actgtcaaga
ggctgtccaa 660gttacgagag tatcaacagt atcatccaag cctgacctgc
gtcatggaag gaaaggacat 720agaagattgg agctgctgcc caaccccttg
gacttcattt cagtctagtt gctactttat 780ttctactggg atgcaatctt
ggactaagag tcaaaagaac tgttctgtga tgggggctga 840tctggtggtg
atcaacacca gggaagaaca ggatttcatc attcagaatc tgaaaagaaa
900ttcttcttat tttctggggc tgtcagatcc agggggtcgg cgacattggc
aatgggttga 960ccagacacca tacaatgaaa atgtcacatt ctggcactca
ggtgaaccca ataaccttga 1020tgagcgttgt gcgataataa atttccgttc
ttcagaagaa tggggctgga atgacattca 1080ctgtcatgta cctcagaagt
caatttgcaa gatgaagaag atctacatat aaatgaaata 1140ttctccctgg
aaatgtgttt gggttggcat ccaccgttgt agaaagctaa attgattttt
1200taatttatgt gtaagttttg tacaaggaat gcccctaaaa tgtttcagca
ggctgtcacc 1260tattacactt atgatataat ccaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aa 13122213PRTHomo sapiens 2Met Val Pro Glu Glu Glu Pro
Gln Asp Arg Glu Lys Gly Leu Trp Trp1 5 10 15Phe Gln Leu Lys Val Trp
Ser Met Ala Val Val Ser Ile Leu Leu Leu 20 25 30Ser Val Cys Phe Thr
Val Ser Ser Val Val Pro His Asn Phe Met Tyr 35 40 45Ser Lys Thr Val
Lys Arg Leu Ser Lys Leu Arg Glu Tyr Gln Gln Tyr 50 55 60His Pro Ser
Leu Thr Cys Val Met Glu Gly Lys Asp Ile Glu Asp Trp65 70 75 80Ser
Cys Cys Pro Thr Pro Trp Thr Ser Phe Gln Ser Ser Cys Tyr Phe 85 90
95Ile Ser Thr Gly Met Gln Ser Trp Thr Lys Ser Gln Lys Asn Cys Ser
100 105 110Val Met Gly Ala Asp Leu Val Val Ile Asn Thr Arg Glu Glu
Gln Asp 115 120 125Phe Ile Ile Gln Asn Leu Lys Arg Asn Ser Ser Tyr
Phe Leu Gly Leu 130 135 140Ser Asp Pro Gly Gly Arg Arg His Trp Gln
Trp Val Asp Gln Thr Pro145 150 155 160Tyr Asn Glu Asn Val Thr Phe
Trp His Ser Gly Glu Pro Asn Asn Leu 165 170 175Asp Glu Arg Cys Ala
Ile Ile Asn Phe Arg Ser Ser Glu Glu Trp Gly 180 185 190Trp Asn Asp
Ile His Cys His Val Pro Gln Lys Ser Ile Cys Lys Met 195 200 205Lys
Lys Ile Tyr Ile 21031227DNAMus musculusCDS(146)..(775)Coding
sequence of mouse Dectin-2 3cattggcccg ctctgtggca tttaactcaa
gtgtgtgtgg aagttgattc tgaactctgg 60cctctttgac agaagccagg tccctgagtc
gtattttgga gacagatgca agaaacccct 120gaccttctga acatacacct caaca atg
gtg cag gaa aga caa tcc caa ggg 172 Met Val Gln Glu Arg Gln Ser Gln
Gly 1 5aag gga gtc tgc tgg acc ctg aga ctc tgg tca gct gct gtg att
tcc 220Lys Gly Val Cys Trp Thr Leu Arg Leu Trp Ser Ala Ala Val Ile
Ser10 15 20 25atg tta ctc ttg agt acc tgt ttc att gcg agc tgt gtg
gtg act tac 268Met Leu Leu Leu Ser Thr Cys Phe Ile Ala Ser Cys Val
Val Thr Tyr 30 35 40caa ttt att atg gac cag ccc agt aga aga cta tat
gaa ctt cac aca 316Gln Phe Ile Met Asp Gln Pro Ser Arg Arg Leu Tyr
Glu Leu His Thr 45 50 55tac cat tcc agt ctc acc tgc ttc agt gaa ggg
act atg gtg tca gaa 364Tyr His Ser Ser Leu Thr Cys Phe Ser Glu Gly
Thr Met Val Ser Glu 60 65 70aaa atg tgg gga tgc tgc cca aat cac tgg
aag tca ttt ggc tcc agc 412Lys Met Trp Gly Cys Cys Pro Asn His Trp
Lys Ser Phe Gly Ser Ser 75 80 85tgc tac ctc att tct acc aag gag aac
ttc tgg agc acc agt gag cag 460Cys Tyr Leu Ile Ser Thr Lys Glu Asn
Phe Trp Ser Thr Ser Glu Gln90 95 100 105aac tgt gtt cag atg ggg gct
cat ctg gtg gtg atc aat act gaa gcg 508Asn Cys Val Gln Met Gly Ala
His Leu Val Val Ile Asn Thr Glu Ala 110 115 120gag cag aat ttc atc
acc cag cag ctg aat gag tca ctt tct tac ttc 556Glu Gln Asn Phe Ile
Thr Gln Gln Leu Asn Glu Ser Leu Ser Tyr Phe 125 130 135ctg ggt ctt
tcg gat cca caa ggt aat ggc aaa tgg caa tgg atc gat 604Leu Gly Leu
Ser Asp Pro Gln Gly Asn Gly Lys Trp Gln Trp Ile Asp 140 145 150gat
act cct ttc agt caa aat gtc agg ttc tgg cac ccc cat gaa ccc 652Asp
Thr Pro Phe Ser Gln Asn Val Arg Phe Trp His Pro His Glu Pro 155 160
165aat ctt cca gaa gag cgg tgt gtt tca ata gtt tac tgg aat cct tcg
700Asn Leu Pro Glu Glu Arg Cys Val Ser Ile Val Tyr Trp Asn Pro
Ser170 175 180 185aaa tgg ggc tgg aat gat gtt ttc tgt gat agt aaa
cac aat tca ata 748Lys Trp Gly Trp Asn Asp Val Phe Cys Asp Ser Lys
His Asn Ser Ile 190 195 200tgt gaa atg aag aag att tac cta tga
gtgcctgtta ttcattaata 795Cys Glu Met Lys Lys Ile Tyr Leu
205tctttaaagt tcagacctac caagaagcca taacttcttg gcctgtacat
ctgacagagg 855ccgttctttt cctagccact attctttact caaacagaat
gagccctttc tccttctgat 915ggttagagtt ttgtcaactt gacacaaact
agagtcacct ggggagtagg atcttcagct 975aaggaattgc ctctgtcagc
ttgaccagtc agcatgtctg ggggcatttt cttgattaat 1035gattgttgta
agagggtcca ggtggtaagc aaaggtgtta aacccatgaa gagcaagcca
1095gggagcatca tccatccatc tctgccctca ggtttctgcc ccagggtctt
gccctggttt 1155ctttctatga actgctgtta cttgaaagta taagatgaat
aaacaatttc atccaaaaaa 1215aaaaaaaaaa aa 12274209PRTMus musculus
4Met Val Gln Glu Arg Gln Ser Gln Gly Lys Gly Val Cys Trp Thr Leu1 5
10 15Arg Leu Trp Ser Ala Ala Val Ile Ser Met Leu Leu Leu Ser Thr
Cys 20 25 30Phe Ile Ala Ser Cys Val Val Thr Tyr Gln Phe Ile Met Asp
Gln Pro 35 40 45Ser Arg Arg Leu Tyr Glu Leu His Thr Tyr His Ser Ser
Leu Thr Cys 50 55 60Phe Ser Glu Gly Thr Met Val Ser Glu Lys Met Trp
Gly Cys Cys Pro65 70 75 80Asn His Trp Lys Ser Phe Gly Ser Ser Cys
Tyr Leu Ile Ser Thr Lys 85 90 95Glu Asn Phe Trp Ser Thr Ser Glu Gln
Asn Cys Val Gln Met Gly Ala 100 105 110His Leu Val Val Ile Asn Thr
Glu Ala Glu Gln Asn Phe Ile Thr Gln 115 120 125Gln Leu Asn Glu Ser
Leu Ser Tyr Phe Leu Gly Leu Ser Asp Pro Gln 130 135 140Gly Asn Gly
Lys Trp Gln Trp Ile Asp Asp Thr Pro Phe Ser Gln Asn145 150 155
160Val Arg Phe Trp His Pro His Glu Pro Asn Leu Pro Glu Glu Arg Cys
165 170 175Val Ser Ile Val Tyr Trp Asn Pro Ser Lys Trp Gly Trp Asn
Asp Val 180 185 190Phe Cys Asp Ser Lys His Asn Ser Ile Cys Glu Met
Lys Lys Ile Tyr 195 200 205Leu5237PRTHomo sapiens 5Met Thr Ser Glu
Ile Thr Tyr Ala Glu Val Arg Phe Lys Asn Glu Phe1 5 10 15Lys Ser Ser
Gly Ile Asn Thr Ala Ser Ser Ala Ala Ser Lys Glu Arg 20 25 30Thr Ala
Pro His Lys Ser Asn Thr Gly Phe Pro Lys Leu Leu Cys Ala 35 40 45Ser
Leu Leu Ile Phe Phe Leu Leu Leu Ala Ile Ser Phe Phe Ile Ala 50 55
60Phe Val Ile Phe Phe Gln Lys Tyr Ser Gln Leu Leu Glu Lys Lys Thr65
70 75 80Thr Lys Glu Leu Val His Thr Thr Leu Glu Cys Val Lys Lys Asn
Met 85 90 95Pro Val Glu Glu Thr Ala Trp Ser Cys Cys Pro Lys Asn Trp
Lys Ser 100 105 110Phe Ser Ser Asn Cys Tyr Phe Ile Ser Thr Glu Ser
Ala Ser Trp Gln 115 120 125Asp Ser Glu Lys Asp Cys Ala Arg Met Glu
Ala His Leu Leu Val Ile 130 135 140Asn Thr Gln Glu Glu Gln Asp Phe
Ile Phe Gln Asn Leu Gln Glu Glu145 150 155 160Ser Ala Tyr Phe Val
Gly Leu Ser Asp Pro Glu Gly Gln Arg His Trp 165 170 175Gln Trp Val
Asp Gln Thr Pro Tyr Asn Glu Ser Ser Thr Phe Trp His 180 185 190Pro
Arg Glu Pro Ser Asp Pro Asn Glu Arg Cys Val Val Leu Asn Phe 195 200
205Arg Lys Ser Pro Lys Arg Trp Gly Trp Asn Asp Val Asn Cys Leu Gly
210 215 220Pro Gln Arg Ser Val Cys Glu Met Met Lys Ile His Leu225
230 23565PRTArtificial sequenceSynthetic polypeptide 6Gly Gly Gly
Gly Ser1 5724DNAArtificial sequenceSynthetic DNA primer 7ttgaaagaac
cacaccccga aagt 24824DNAArtificial sequenceSynthetic DNA primer
8tagctttcta caacggtgga tgcc 2494PRTHomo sapiens 9Asn Cys Ser
Val1104PRTHomo sapiens 10Asn Ser Ser Tyr1114PRTHomo sapiens 11Asn
Val Thr Phe1124PRTMus musculus 12Asn Glu Ser Leu1134PRTHomo sapiens
13Asn Glu Ser Ser1144PRTHomo sapiens 14Lys Arg Leu Ser1154PRTHomo
sapiens 15Lys Lys Thr Thr1164PRTHomo sapiens 16Thr Arg Glu
Glu1174PRTHomo sapiens 17Ser Ser Glu Glu1184PRTMus musculus 18Ser
Thr Lys Glu1194PRTMus musculus 19Ser Thr Ser Glu1204PRTMus musculus
20Thr Glu Ala Glu1214PRTMus musculus 21Ser Ile Cys Glu1224PRTHomo
sapiens 22Thr Tyr Ala Glu1234PRTHomo sapiens 23Thr Thr Lys
Glu1244PRTHomo sapiens 24Thr Thr Leu Glu1254PRTHomo sapiens 25Ser
Trp Gln Asp1264PRTHomo sapiens 26Ser Glu Lys Asp1274PRTHomo sapiens
27Thr Gln Glu Glu1288PRTHomo sapiens 28Lys Leu Arg Glu Tyr Gln Gln
Tyr1 5294PRTHomo sapiens 29Ser Val Cys Glu1304PRTHomo sapiens 30Ser
Val Cys Glu1319PRTMus musculus 31Arg Arg Leu Tyr Glu Leu His Thr
Tyr1 5324PRTHomo sapiens 32Gly Gly Arg Arg1336PRTMus musculus 33Gly
Val Cys Trp Thr Leu1 5346PRTMus musculus 34Gly Thr Met Val Ser Glu1
5356PRTMus musculus 35Gly Cys Cys Pro Asn His1 5366PRTHomo sapiens
36Gly Ile Asn Thr Ala Ser1 5376PRTArtificial sequenceSynthetic
polypeptideMISC_FEATURE(1)..(1)Xaa is Ile or
ValMISC_FEATURE(2)..(2)Xaa is any amino acidMISC_FEATURE(4)..(5)Xaa
is any amino acidMISC_FEATURE(6)..(6)Xaa is Leu or Val 37Xaa Xaa
Tyr Xaa Xaa Xaa1 5386PRTHomo sapiens 38Ile Thr Tyr Ala Glu Val1
5
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