U.S. patent application number 11/040774 was filed with the patent office on 2005-06-30 for antibodies to mammalian langerhans cell antigen and their uses.
This patent application is currently assigned to Schering Corporation. Invention is credited to Clair, Valerie, Duvert-Frances, Valerie, Lebecque, Serge J.E., Pin, Jean-Jacques, Saeland, Sem, Valladeau, Jenny.
Application Number | 20050142619 11/040774 |
Document ID | / |
Family ID | 34426566 |
Filed Date | 2005-06-30 |
United States Patent
Application |
20050142619 |
Kind Code |
A1 |
Duvert-Frances, Valerie ; et
al. |
June 30, 2005 |
Antibodies to mammalian langerhans cell antigen and their uses
Abstract
Purified mammalian DC cell surface protein, designated Langerin,
nucleic acids encoding Langerin, and antibodies which specifically
bind Langerin.
Inventors: |
Duvert-Frances, Valerie;
(Tasin La Demi-Lune, FR) ; Pin, Jean-Jacques;
(Saint Bonnet de Mure, FR) ; Valladeau, Jenny;
(Lyon, FR) ; Clair, Valerie; (Lyon, FR) ;
Saeland, Sem; (Lyon, FR) ; Lebecque, Serge J.E.;
(Civrieux d'Azergues, FR) |
Correspondence
Address: |
SCHERING-PLOUGH CORPORATION
PATENT DEPARTMENT (K-6-1, 1990)
2000 GALLOPING HILL ROAD
KENILWORTH
NJ
07033-0530
US
|
Assignee: |
Schering Corporation
|
Family ID: |
34426566 |
Appl. No.: |
11/040774 |
Filed: |
January 21, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11040774 |
Jan 21, 2005 |
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09787192 |
Mar 15, 2001 |
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6878528 |
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09787192 |
Mar 15, 2001 |
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PCT/US99/22269 |
Sep 23, 1999 |
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Current U.S.
Class: |
435/7.2 ;
435/334; 530/388.26; 536/23.53 |
Current CPC
Class: |
C07K 16/2851 20130101;
C07K 14/705 20130101 |
Class at
Publication: |
435/007.2 ;
530/388.26; 435/334; 536/023.53 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567; C07H 021/04; C07K 016/40; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 18, 1999 |
EP |
99400394 |
Sep 25, 1998 |
EP |
98402374 |
Claims
1. An isolated antibody which selectively binds a mammalian
polypeptide, said polypeptide characterized by: a) binds with
selectively to DCGM4 monoclonal antibody; b) natural form of said
polypeptide has a molecular weight by SDS PAGE of .about.40 kD; and
c) expressed on the cell surface of Langerhans cells.
2. The antibody of claim 1 which is a monoclonal antibody.
3. A hybridoma that produces a monoclonal antibody of claim 2.
4. The hybridoma of claim 3, which has ATCC Accession No.
HB-12576.
5-15. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions which function
in controlling physiology, development, and differentiation of
mammalian cells, e.g., cells of a mammalian immune system. In
particular, it provides antibodies, e.g., agonists and antagonists,
which regulate cellular physiology, development, differentiation,
or function of various cell types, including hematopoietic cells,
and particularly dendritic cells e.g., Langerhans cells.
BACKGROUND OF THE INVENTION
[0002] Dendritic cells (DC) are antigen-presenting cells that are
required for initiation of a specific immune response. See, e.g.,
Banchereau and Steinman (1998) Nature 392: 242-52. One type of DC
is exemplified by Langerhans cells (LC), immature DC cells that
reside in non-lymphoid tissue, such as the epidermis, and whose
primary function is to capture antigen. See, e.g., Steinman, et al.
(1995) J. Exp. Med. 182: 283-288.
[0003] Antigen capture is achieved primarily through specialized
surface-membrane endocytic structures or through macropinocytosis,
thus permitting Langerhans cells to concentrate solutes which are
present in large volumes of fluid. See, e.g., Sallusto, et al.
(1995) J. Exp. Med. 182: 389-400. Concomitant with processing of
antigen in specialized organelles of the endocytic pathway, DC,
such as Langerhans cells, migrate to secondary lymphoid tissue and
undergo a number of phenotypic modifications. These maturation
events ultimately translate into highly efficient presentation of
processed antigen, by appropriate MHC molecules of the DC, to T
cells.
[0004] In particular, the maturation process of the DC Langerhans
cell includes loss of adhesion receptors such as E-cadherin, and
the disappearance of Birbeck granules (BG), which are
characteristic for LC. Conversely, upon acquisition of
antigen-presentation function, costimulatory receptors such as the
CD80 and CD86 molecules are upregulated on DC to permit T cell
activation. Maturation events of DC can be reconstituted in vitro
by TNF-.alpha. and CD40-ligand which mimic, respectively, the
response to pro-inflammatory cytokines following encounter with
pathogen, and the response to contact with T cells in secondary
lymphoid tissue. See, e.g., Caux, et al. (1996) J. Exp. Med. 184:
695-706.
[0005] Thus, it is apparent that the highly specialized and
anatomically localized functions of DC are controlled by tight
regulation of the expression of a number of key molecules.
[0006] Recently, culture systems have become available to obtain
large numbers of DC when cultured in the presence of cytokines.
Using such culture methods, DC can be obtained for in vitro study
from CD34.sup.+ hematopoietic progenitor cells (HPC) present in
cord blood when the HPC are co-cultured with TNF-.alpha. and GM-CSF
(such cultures are referred to as CD34-derived DC), or from
peripheral blood monocytes when they are co-cultured with GM-CSF
and IL-4 (such cultures are referred to as monocyte-derived DC).
See, e.g., Caux, et al. (1992) Nature 392: 258-261; Chapuis, et al.
(1997) Eur. J. Immunol. 27: 431-441; Romani, et al. (1994) J.
Invest. Dermatol. 93: 600-609.
[0007] These methods permit either, the in vitro (see, e.g., Caux,
et al. (1996) J. Exp. Med. 184: 695-706), or, the ex-vivo (from
various organs; see e.g., Grouard, et al. (1996) Nature 384:
364-367; O'Doherty, et al. (1994) Immunol. 82: 487-493; Zhou (1995)
J. Immunol. 154: 3821-3835) isolation of phenotypically and
functionally distinct DC subpopulations.
[0008] Consequently, it would be of great benefit to possess novel
reagents capable of identifying markers that are expressed and
associated with different DC subpopulations. These markers are
detectable using antibodies, e.g., monoclonal or polyclonal. Such
markers would permit the monitoring, characterization, and/or
isolation of defined subsets of immature DCs by facilitating, e.g.,
cell-sorting and functional studies. Thus, needs exist for tools
which permit a better understanding of the molecules involved in DC
maturation, antigen presentation, and the mechanisms of DC
interaction with other molecules, cells, and tissues. The present
invention fulfills such needs by providing useful reagents and
compositions involved in DC maturation and function.
SUMMARY OF THE INVENTION
[0009] The present invention is based, in part, upon the discovery
of an antibody which defines and recognizes a novel cell antigen
found on dendritic cells (DC) and Langerhans cells (LC). This
monoclonal antibody is designated DCGM4 and the antigen it
recognizes has been designated Langerin. The invention embraces
this antibody and methods for its use. In addition, the invention
is directed to antigen recognized by this antibody, along with
variants of these proteins, e.g., mutations (muteins) of the
natural sequence, species and allelic variants, fusion proteins,
chemical mimetics, and other structural or functional analogs.
Various uses of these different antibodies and protein compositions
are also provided.
[0010] The present invention provides antibody which binds
specifically to a mammalian Langerin. In preferred embodiments the
mammal is a primate; or the antibodies are a monoclonal antibody,
interfere with binding of DCGM4 to the Langerin, or are detectably
labeled, e.g., with a fluorescent or enzymatic label.
[0011] The invention also provides methods of detecting a mammalian
Langerin, comprising binding DCGM4 to Langerin. In various
embodiments, the antibody is a labeled antibody or is immobilized
to a solid substrate; the Langerin is expressed on a cell surface;
the detecting allows isolation of a cell which comprises a nucleic
acid which expresses Langerin; or the detecting further allows
purification of Langerin. The invention also embraces a kit for
detecting Langerin with a compartment containing an antibody. In
preferred embodiments, the kit is a fluorescence immunoassay
kit.
[0012] The present invention further provides methods of modulating
an immune function modulated by a cell comprising contacting said
cell with an antibody described herein. For instance, the
modulation can be blocking DC cell maturation or function.
[0013] Also embraced herein are methods for analyzing a DC cell
population, comprising measuring the presence of Langerin.
Typically, the measuring is a quantitative determination, e.g., by
measuring binding of an antibody to Langerin.
[0014] The invention also provides substantially pure mammalian
Langerin antigens. Human Langerin and mouse Langerin are
specifically described. Langerin can be purified by, for example,
immunoaffinity, e.g., using an antibody which binds specifically to
a mammalian Langerin. A preferred antibody for such is DCGM4. Along
with full length Langerin, the invention provides fragments which
express an immunological epitope of said Langerin or modulate an
immune response, e.g., a response mediated by a DC cell, including
a Langerin.sup.+ cell.
[0015] The invention further provides genomic DNA of human
langerin.
DETAILED DESCRIPTION OF THE INVENTION
[0016] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety including all figures, graphs, and drawings.
[0017] General
[0018] The present invention provides antibodies which recognize a
mammalian protein that exhibits properties characteristic of
functionally significant DC expressed molecules. The antibody is
exemplified in one embodiment by a monoclonal antibody designated
DCGM4.
[0019] The mammalian protein defined, e.g., selectively recognized,
by the antibody DCGM4 is designated Langerin. The natural Langerin
protein mediates various physiological responses leading to
biological or physiological responses in target cells. In
particular, Langerhans DC cells are responsible for antigen
presentation, e.g., presentation of haptens in hypersensitivity
reactions. The DCGM4 antibody modulates various immunological
responses which affect DC maturation and antigen presentation.
[0020] Langerin is a 40 kDa N-glycosylated protein.
Immunoprecipitation with DCGM4 from DC extracts and, subsequent
elution with SDS-PAGE sample buffer yielded a homogeneous band of
40-42 kDa molecular mass. If DTT was omitted all along the
purification steps, the profile was not modified on the gel,
suggesting that Langerin is present at the cell membrane as a
single chain or as an homodimer with non-covalent association. Two
dimensional analysis confirmed the molecular mass of the molecule
and indicated a pI of 5.2-5.5. Finally, Langerin is a glycoprotein,
and most of the carbohydrate constituents were removed by
N-glycosylase treatment.
[0021] A gene encoding human langerin has now been cloned. The
nucleotide sequence is showing in SEQ ID NO:1. The predicted amino
acid sequence is shown in SEQ ID NO: 2. The predicted protein is a
type II membrane lectin, with a calcium-dependent carbohydrate
recognition domain. Amino acid homology with the Kupffer receptor
of mouse, rat and chicken was found and provides support that
langerin functions as a langerhans cell specific receptor for
antigen capture. The carbohydrate recognition domain motif EPN
supports that the sugar moieities are probably mannose and
glucose.
[0022] Identifying characteristics that permit one of ordinary
skill in the art to distinguish Langerin from other proteins are
listed below.
[0023] binds with specificity to DCGM4 monoclonal antibody
[0024] N-glycosylated form of protein .about.40 kD, both reduced or
non-reduced with a pI of 5.2-5.5 and lacking interchain disulfide
bonds
[0025] expressed on subset of DC cells
[0026] normally expressed on the cell surface of Langerhans
cells
[0027] after binding with DCGM4, Langerin is transported within
endocytic coated pits and participates in cytomembrane sandwiching;
subsequently Langerin is associated with Birbeck granules.
[0028] Cells of the clonally derived hybridoma cell line designated
DCGM4 from which mAb DCGM4 has been purified have been deposited
with the American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas Va., USA, on Sep. 22, 1998 under ATCC Accession
Number No. HB-12576.
[0029] It is to be understood that this invention is not limited to
the particular methods, compositions and antibodies described
herein, as such methods, compositions and antibodies may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which is only limited by the appended claims.
[0030] II. Antibodies
[0031] Antibodies can be raised to the various mammalian, e.g.,
primate Langerin proteins and fragments thereof, both in naturally
occurring native forms and in their recombinant forms, the
difference being that antibodies to the active Langerin are more
likely to recognize epitopes which are only present in the native
conformations. Denatured antigen detection can also be useful in,
e.g., Western analysis. Anti-idiotypic antibodies are also
contemplated, which would be useful as agonists or antagonists of a
natural Langerin protein or an antibody.
[0032] Antibodies, including binding fragments and single chain
versions, against predetermined fragments or the whole of the
protein (e.g., a protein having an amino acid sequence shown in SEQ
ID NO: 2) can be raised by immunization of animals. Monoclonal
antibodies are prepared from cells secreting the desired antibody.
These antibodies can be screened for binding to normal or defective
protein, or screened for agonistic or antagonistic activity. These
monoclonal antibodies will usually bind with at least a K.sub.D of
about 1 mM, more usually at least about 300 .mu.M, typically at
least about 100 .mu.M, more typically at least about 30 .mu.M,
preferably at least about 10 .mu.M, and more preferably at least
about 3 .mu.M or better.
[0033] The antibodies, exemplified by the DCGM4, including antigen
binding fragments, can have significant diagnostic or therapeutic
value. They can be potent antagonists that bind to Langerin and
inhibit binding partner interaction or inhibit the ability of the
interaction to mediate a biological response. They also can be
useful as non-neutralizing antibodies and can be coupled to toxins
or radionuclides so that when the antibody binds to the antigen, a
cell expressing it, e.g., on its surface, is killed. Further, this
antibody can be conjugated to drugs or other therapeutic agents,
either directly or indirectly by means of a linker, and may effect
drug targeting.
[0034] The antibodies of this invention can also be useful in
diagnostic applications. As capture or non-neutralizing antibodies,
they might bind Langerin without inhibiting Langerin function on
cells of the immune system, for example immature DC. As
neutralizing antibodies, they can be useful in competitive binding
assays. They will also be useful in detecting or quantifying
Langerin protein or its binding partners. They may be used as
reagents for Western blot analysis, or for immunoprecipitation or
immunopurification of the Langerin protein. They will also be
useful in evaluating cell populations to determine, e.g., the
physiological state of an immune system.
[0035] Antigen fragments may be joined to other materials,
particularly polypeptides, as fused or covalently joined
polypeptides to be used as immunogens. The antigen may be purified
as described below, including immunoaffinity methods using
antibodies, e.g., DCGM4. An antigen and its fragments may be fused
or covalently linked to a variety of carriers, such as keyhole
limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See,
e.g., Microbiology, Hoeber Medical Division, Harper and Row, 1969;
Landsteiner (1962) Specificity of Serological Reactions, Dover
Publications, New York; and Williams, et al. (1967) Methods in
Immunology and Immunochemistry, Vol. 1, Academic Press, New York,
each of which are incorporated herein by reference, for
descriptions of methods of preparing polyclonal antisera. A typical
method involves hyperimmunization of an animal with the antigen.
The blood of the animal is then collected shortly after the
repeated immunizations and the gamma globulin is isolated.
[0036] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies may be found in, e.g., Stites, et al. (eds.)
Basic and Clinical Immunology (4th ed.), Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.) Academic Press, New York; and particularly in Kohler and
Milstein (1975) in Nature 256: 495-497, which discusses one method
of generating monoclonal antibodies. Each of these references is
incorporated herein by reference. Summarized briefly, this method
involves injecting an animal with an immunogen. The animal is then
sacrificed and cells taken from its spleen, which are then fused
with myeloma cells. The result is a hybrid cell or "hybridoma" that
is capable of reproducing in vitro. The population of hybridomas is
then screened to isolate individual clones, each of which secrete a
single antibody species to the immunogen. In this manner, the
individual antibody species obtained are the products of
immortalized and cloned single B cells from the immune animal
generated in response to a specific site recognized on the
immunogenic substance.
[0037] Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides or, alternatively,
selection of libraries of antibodies in phage or similar vectors.
See, Huse, et al. (1989) "Generation of a Large Combinatorial
Library of the Immunoglobulin Repertoire in Phage Lambda," Science
246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546, each
of which is hereby incorporated herein by reference. The
polypeptides and antibodies of the present invention may be used
with or without modification, including chimeric or humanized
antibodies. Frequently, the polypeptides and antibodies will be
labeled by joining, either covalently or non-covalently, a
substance which provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and are reported
extensively in both the scientific and patent literature. Suitable
labels include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent moieties, chemiluminescent moieties,
magnetic particles, and the like. Patents teaching the use of such
labels include 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. Also, recombinant
immunoglobulins may be produced, see Cabilly, U.S. Pat. No.
4,816,567; or made in transgenic mice, see Mendez, et al. (1997)
Nature Genetics 15: 146-156. These patents are incorporated herein
by reference.
[0038] The antibodies of this invention can also be used for
affinity chromatography in isolating the protein. Columns can be
prepared where the antibodies are linked to a solid support, e.g.,
particles, such as agarose, Sephadex, or the like, where a cell
lysate may be passed through the column, the column washed,
followed by increasing concentrations of a mild denaturant, whereby
the purified Langerin protein will be released. Alternatively, the
antibody may be used to quantitate and identify fractionated
samples containing the antigen. Standard protein purification
procedures, e.g., chromatography, will be used to enrich and purify
Langerin protein with, e.g., ELISA assays, to identify fractions
where Langerin separates.
[0039] Purified protein will be sequenced. The sequence will allow
selection of oligonucleotide sequences useful as primers or probes.
Alternatively, the sequence allows production of polypeptide
segments for making additional antibodies. In addition, purified
protein may be used for immunization, allowing production of
polyclonal or monoclonal antibodies.
[0040] The antibodies may also be used to screen expression
libraries for particular expression products. Usually the
antibodies used in such a procedure will be labeled with a moiety
allowing easy detection of presence of antigen by antibody binding.
This will allow isolation of a cell which expresses a nucleic acid,
e.g., a vector, encoding the antigen by, e.g., fluorescence
activated cell sorting (FACS) analysis, and enrichment.
Alternatively, an affinity method using antibodies of this
invention can be used to immobilize and separate cells expressing
the Langerin, e.g., encoded on a vector.
[0041] Antibodies raised against each Langerin protein will also be
useful to raise anti-idiotypic antibodies. These will be useful in
detecting or diagnosing various immunological conditions related to
expression of the respective antigens.
[0042] III. Purified Langerin Protein
[0043] Human Langerin protein can be isolated from natural sources
using standard biochemical purification techniques and/or by use of
the antibody to determine the presence of the antigen in particular
fractionation procedures. The purified proteins allow both sequence
determination and preparation of peptides to generate further
antibodies to recognize such segment. As used herein, Langerin
shall encompass, when used in a protein context, a protein which,
in a natural state, exhibits the properties listed above, or a
significant fragment of such a protein. e.g., a protein having an
amino acid sequence set forth in SEQ ID NO: 2 or a subsequence
thereof. It also refers to a mammalian, e.g., primate, derived
polypeptide which exhibits similar biological function or interacts
with Langerin protein specific binding components. These binding
components, e.g., antibodies, typically bind to a Langerin protein
with high affinity, e.g., at least about 100 nM, usually better
than about 30 nM, preferably better than about 10 nM, and more
preferably at better than about 3 nM. One such preferred binding
component is the antibody DCGM4.
[0044] The purified protein or peptide fragments are useful for
generating antibodies by standard methods, as described below.
Synthetic peptides or purified protein can be presented to an
immune system to generate a specific binding composition, e.g.,
monoclonal or polyclonal antibodies. See, e.g., Coligan (1991)
Current Protocols in Immunology Wiley/Greene; and Harlow and Lane
(1989) Antibodies: A Laboratory Manual Cold Spring Harbor
Press.
[0045] The term polypeptide, as used herein, includes a significant
fragment or segment, and encompasses a stretch of amino acid
residues of at least about 8 amino acids, generally at least 10
amino acids, more generally at least 12 amino acids, often at least
14 amino acids, more often at least 16 amino acids, typically at
least 18 amino acids, more typically at least 20 amino acids,
usually at least 22 amino acids, more usually at least 24 amino
acids, preferably at least 26 amino acids, more preferably at least
28 amino acids, and, in particularly preferred embodiments, at
least about 30 or more amino acids. Preferably, the fragment
exhibits a biological property in common with the full length
Langerin, e.g., immunological activity, including sharing of an
epitope.
[0046] Substantially pure, in the polypeptide context, typically
means that the protein is free from other contaminating proteins,
nucleic acids, and other biologicals derived from the original
source organism. Purity may be assayed by standard methods, and
will ordinarily be at least about 40% pure, more ordinarily at
least about 50% pure, generally at least about 60% pure, more
generally at least about 70% pure, often at least about 75% pure,
more often at least about 80% pure, typically at least about 85%
pure, more typically at least about 90% pure, preferably at least
about 95% pure, more preferably at least about 98% pure, and in
most preferred embodiments, at least 99% pure. The analysis may be
weight or molar percentages, evaluated, e.g., by gel staining,
spectrophotometry, or terminus labeling.
[0047] A binding composition or agent refers to molecules that bind
with specificity to Langerin protein, e.g., in a ligand-receptor
type fashion, an antibody-antigen interaction, or compounds, e.g.,
proteins which specifically associate with Langerin protein, e.g.,
in a natural physiologically relevant protein-protein interaction,
either covalent or non-covalent. The molecule may be a polymer, or
chemical reagent. This implies both binding affinity and binding
specificity or selectivity. A functional analog may be a protein
with structural modifications, or may be a wholly unrelated
molecule, e.g., which has a molecular shape which interacts with
the appropriate binding determinants. The proteins may serve as
agonists or antagonists of a receptor, see, e.g., Goodman, et al.
(eds. 1990) Goodman & Gilman's: The Pharmacological Bases of
Therapeutics (8th ed.) Pergamon Press, Tarrytown, N.Y.
[0048] Soluble fragments of both the antibodies and Langerin
antigens are provided by the invention. Solubility of a polypeptide
or fragment depends upon the environment and the polypeptide. Many
parameters affect polypeptide solubility, including temperature,
electrolyte environment, size and molecular characteristics of the
polypeptide, and nature of the solvent. Typically, the temperature
at which the polypeptide is used ranges from about 4.degree. C. to
about 65.degree. C. Usually the temperature at use is greater than
about 18.degree. C. and more usually greater than about 22.degree.
C. For diagnostic purposes, the temperature will usually be about
room temperature or warmer, but less than the denaturation
temperature of components in the assay. For therapeutic purposes,
the temperature will usually be body temperature, typically about
37.degree. C. for humans, though under certain situations the
temperature may be raised or lowered in situ or in vitro.
[0049] The electrolytes will usually approximate in situ
physiological conditions, but may be modified to higher or lower
ionic strength where advantageous. The actual ions may be modified,
e.g., to conform to standard buffers used in physiological or
analytical contexts.
[0050] The size and structure of the polypeptide should generally
be in a substantially stable state, and usually not in a denatured
state. The polypeptide may be associated with other polypeptides in
a quaternary structure, e.g., to confer solubility, or associated
with lipids or detergents in a manner which approximates natural
lipid bilayer interactions.
[0051] The solvent will usually be a biologically compatible
buffer, of a type used for preservation of biological activities,
and will usually approximate a physiological solvent. Usually the
solvent will have a neutral pH, typically between about 5 and 10,
and preferably about 7.5. On some occasions, a detergent will be
added, typically a mild non-denaturing one, e.g., CHS or CHAPS, or
a low enough concentration as to avoid significant disruption of
structural or physiological properties of the antigen.
[0052] Solubility is reflected by sedimentation measured in
Svedberg units, which are a measure of the sedimentation velocity
of a molecule under particular conditions. The determination of the
sedimentation velocity was classically performed in an analytical
ultracentrifuge, but is typically now performed in a standard
ultracentrifuge. See, Freifelder (1982) Physical Biochemistry (2d
ed.), W.H. Freeman; and Cantor and Schimmel (1980) Biophysical
Chemistry, parts 1-3, W.H. Freeman & Co., San Francisco; each
of which is hereby incorporated herein by reference. As a crude
determination, a sample containing a putatively soluble polypeptide
is spun in a standard full sized ultracentrifuge at about 50K rpm
for about 10 minutes, and soluble molecules will remain in the
supernatant. A soluble particle or polypeptide will typically be
less than about 30S, more typically less than about 15S, usually
less than about 10S, more usually less than about 6S, and, in
particular embodiments, preferably less than about 4S, and more
preferably less than about 3S.
[0053] A Langerin protein that specifically binds to or that is
specifically immunoreactive with an antibody generated against a
defined immunogen, such as an immunogen consisting of the Langerin
protein of the invention is typically determined in an immunoassay.
The immunoassay typically uses a polyclonal antiserum which was
raised, e.g., to a Langerin protein of the invention. This
antiserum is selected to have low crossreactivity against other
potential Langerin family members, e.g., allelic variants of
Langerin proteins preferably from the same species, and any such
crossreactivity is removed by immunoabsorption prior to use in the
immunoassay.
[0054] To produce antisera for use in an immunoassay, the Langerin
protein of the invention is isolated as described herein. For
example, after determining the Langerin nucleic acid sequence using
the methods described herein, a recombinant protein may be produced
in a mammalian cell line. An appropriate host, e.g., an inbred
strain of mice such as Balb/c, is immunized with the selected
protein, typically using a standard adjuvant, such as Freund's
adjuvant, and a standard mouse immunization protocol (see Harlow
and Lane, supra). Alternatively, a synthetic peptide derived from a
Langerin nucleic acid sequence can be conjugated to a carrier
protein and subsequently used an immunogen. Polyclonal sera are
collected and titered against the immunogen protein in an
immunoassay, e.g., a solid phase immunoassay with the immunogen
immobilized on a solid support. Polyclonal antisera with a titer of
10.sup.4 or greater are selected and tested for their cross
reactivity against other Langerin variants e.g., Langerin allelic
variants, using a competitive binding immunoassay such as the one
described in Harlow and Lane, supra, at pages 570-573. Preferably
at least two Langerin variants are used in this determination.
These Langerin family members can be produced as recombinant
proteins and isolated using standard molecular biology and protein
chemistry techniques as described herein.
[0055] Immunoassays in the competitive binding format can be used
for the crossreactivity determinations. For example, the Langerin
protein of the invention can be immobilized to a solid support.
Proteins added to the assay compete with the binding of the
antisera to the immobilized antigen. The ability of the above
proteins to compete with the binding of the antisera to the
immobilized protein is compared to a Langerin protein. The percent
crossreactivity for the above proteins is calculated, using
standard calculations. Those antisera with less than 10%
crossreactivity with each of the proteins listed above are selected
and pooled. The cross-reacting antibodies are then removed from the
pooled antisera by immunoabsorption with the above-listed
proteins.
[0056] The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second Langerin protein to the immunogen protein (e.g., a Langerin
allelic variant). To make this comparison, the two proteins are
each assayed at a wide range of concentrations and the amount of
each protein required to inhibit 50% of the binding of the antisera
to the immobilized protein is determined. If the amount of the
second protein required is less than twice the amount of the
protein of the selected protein or proteins that is required, then
the second protein is said to specifically bind to an antibody
generated to the immunogen.
[0057] It is understood that the Langerin protein of the invention
is not only the protein characterized herein, but also to other
Langerin proteins that are allelic, non-allelic, or species
variants. It is also understood that the terms include nonnatural
mutations introduced by deliberate mutation using conventional
recombinant technology such as single site mutation, or by excising
short sections of DNA encoding the respective proteins, or by
substituting new amino acids, or adding new amino acids to a
Langerin sequence. Such minor alterations typically will
substantially maintain the immunoidentity of the original molecule
and/or its biological activity. Thus, these alterations include
proteins that are specifically immunoreactive with a designated
naturally occurring Langerin protein. The biological properties of
altered proteins can be determined by expressing the protein in an
appropriate cell line and measuring the appropriate effect, e.g.,
upon DC cells in vitro. Particular protein modifications considered
minor include conservative substitution of amino acids with similar
chemical properties. By aligning a protein optimally with the
protein of the Langerins and by using conventional immunoassays as
described herein to determine immunoidentity, one can determine the
protein compositions of the invention.
[0058] IV. Physical Variants
[0059] This invention also encompasses proteins or peptides having
substantial amino acid sequence homology with a natural mammalian
Langerin protein or an antibody described above. The variants
include species and allelic variants. A person having ordinary
skill in the art will recognize that much of the following
discussion of variants will apply to variants of both the Langerin
antigen and the antibodies which recognize it.
[0060] Amino acid sequence homology, or sequence identity, is
determined by optimizing residue matches, if necessary, by
introducing gaps as required. This changes when considering
conservative substitutions as matches. Conservative substitutions
typically include substitutions within the following groups:
glycine, alanine; valine, isoleucine, leucine; aspartic acid,
glutamic acid; asparagine, glutamine; serine, threonine; lysine,
arginine; and phenylalanine, tyrosine. Homologous amino acid
sequences are typically intended to include natural allelic and
interspecies variations in each respective protein sequence.
Typical homologous proteins or peptides will have from 25-100%
homology (if gaps can be introduced), to 50-100% homology (if
conservative substitutions are included) with the amino acid
sequence of the Langerin protein. Homology measures will be at
least about 35%, generally at least 40%, more generally at least
45%, often at least 50%, more often at least 55%, typically at
least 60%, more typically at least 65%, usually at least 70%, more
usually at least 75%, preferably at least 80%, and more preferably
at least 80%, and in particularly preferred embodiments, at least
85% or more. See also Needleham, et al. (1970) J. Mol. Biol. 48:
443-453; Sankoff, et al. (1983) Chapter One in Time Warps, String
Edits, and Macromolecules: The Theory and Practice of Sequence
Comparison Addison-Wesley, Reading, Mass.; and software packages
from IntelliGenetics, Mountain View, Calif.; and the University of
Wisconsin Genetics Computer Group, Madison, Wis.; each of which is
incorporated herein by reference.
[0061] An isolated DNA, isolated as described below, encoding a
Langerin protein can be readily modified by nucleotide
substitutions, nucleotide deletions, nucleotide insertions, and
inversions of nucleotide stretches. These modifications result in
novel DNA sequences which encode these antigens, their derivatives,
or proteins having similar physiological, immunogenic, or antigenic
activity. These modified sequences can be used to produce mutant
antigens or to enhance expression. Enhanced expression may involve
gene amplification, increased transcription, increased translation,
and other mechanisms. Such mutant Langerin protein derivatives
include predetermined or site-specific mutations of the respective
protein or its fragments. "Mutant Langerin protein" encompasses a
polypeptide otherwise falling within the homology definition of the
human Langerin protein as set forth above, but having an amino acid
sequence which differs from that of Langerin protein as found in
nature, whether by way of deletion, substitution, or insertion. In
particular, "site specific mutant Langerin protein" generally
includes proteins having significant homology with a natural
protein with properties described herein, and/or sharing various
biological activities, e.g., antigenic or immunogenic, with those
sequences, and in preferred embodiments include, e.g., singly
substituted natural forms of the proteins. Similar concepts apply
to different Langerin proteins, particularly those found in various
mammals, e.g., primates, including human. As stated before, it is
emphasized that the descriptions are generally meant to encompass
all mammalian Langerin proteins.
[0062] Although site specific mutation sites are predetermined,
mutants need not be site specific. Langerin protein mutagenesis can
be conducted by making amino acid insertions or deletions.
Substitutions, deletions, insertions, or any combinations may be
generated to arrive at a final construct. Insertions include amino-
or carboxy-terminal fusions. Random mutagenesis can be conducted at
a target codon and the expressed mutants can then be screened for
the desired activity. Methods for making substitution mutations at
predetermined sites in DNA having a known sequence are well known
in the art, e.g., by M13 primer mutagenesis or polymerase chain
reaction (PCR) techniques. See also Sambrook, et al. (1989) and
Ausubel, et al. (1987 and Supplements).
[0063] The mutations in the DNA normally should not place coding
sequences out of reading frames and preferably will not create
complementary regions that could hybridize to produce secondary
mRNA structure such as loops or hairpins.
[0064] The present invention also provides recombinant proteins,
e.g., heterologous fusion proteins using segments from these
proteins. A heterologous fusion protein is a fusion of proteins or
segments which are naturally not normally fused in the same manner.
Thus, the fusion product of an immunoglobulin with a Langerin
polypeptide is a continuous protein molecule having sequences fused
in a typical peptide linkage, typically made as a single
translation product and exhibiting properties derived from each
source peptide. A similar concept applies to heterologous nucleic
acid sequences.
[0065] In addition, new constructs may be made from combining
similar functional domains from other proteins. For example,
antigen-binding or other segments may be "swapped" between
different new fusion polypeptides or fragments. See, e.g.,
Cunningham, et al. (1989) Science 243: 1330-1336; and O'Dowd, et
al. (1988) J. Biol. Chem. 263: 15985-15992, each of which is
incorporated herein by reference. Thus, new chimeric polypeptides
exhibiting new combinations of specificities will result from the
functional linkage of biologically relevant domains and other
functional domains.
[0066] The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22: 1859-1862, will produce
suitable synthetic DNA fragments. A double stranded fragment will
often be obtained either by synthesizing the complementary strand
and annealing the strand together under appropriate conditions or
by adding the complementary strand using DNA polymerase with an
appropriate primer sequence, e.g., PCR techniques.
[0067] V. Functional Variants
[0068] The blocking of physiological response mediated by Langerin
proteins may result from the inhibition of binding of the antigen
to its natural binding partner, e.g., through competitive
inhibition. Thus, in vitro assays of the present invention will
often use isolated protein, membranes from cells expressing a
recombinant membrane associated Langerin protein, soluble fragments
comprising binding segments, or fragments attached to solid phase
substrates. These assays will also allow for the diagnostic
determination of the effects of either binding segment mutations
and modifications, or protein mutations and modifications, e.g.,
analogs. In particular, the Langerin is expressed on DC clones, but
the antigen is lost after DC cell maturation.
[0069] This invention also contemplates the use of competitive drug
screening assays, e.g., where neutralizing antibodies to antigen or
binding partner fragments compete with a test compound for binding
to the protein. In this manner, the antibodies can be used to
detect the presence of any polypeptide which shares one or more
antigenic binding sites of the protein and can also be used to
occupy binding sites on the protein that might otherwise interact
with a binding partner.
[0070] Additionally, neutralizing antibodies against the Langerin
protein and soluble fragments of the antigen which contain a high
affinity binding site, can be used to inhibit antigen function in
cells or tissues, e.g., cells or tissues experiencing abnormal or
undesired physiology. "Derivatives" of the Langerin antigens, and
of antibodies, include amino acid sequence mutants, glycosylation
variants, and covalent or aggregate conjugates with other chemical
moieties. Covalent derivatives can be prepared by linkage of
functionalities to groups which are found in the Langerin amino
acid side chains or at the N- or C-termini, by means which are well
known in the art. These derivatives can include, without
limitation, aliphatic esters or amides of the carboxyl terminus, or
of residues containing carboxyl side chains, O-acyl derivatives of
hydroxyl group-containing residues, and N-acyl derivatives of the
amino terminal amino acid or amino-group containing residues, e.g.,
lysine or arginine. Acyl groups are selected from the group of
alkyl-moieties including C3 to C18 normal alkyl, thereby forming
alkanoyl aroyl species. Covalent attachment to carrier proteins may
be important when immunogenic moieties are haptens.
[0071] In particular, glycosylation alterations are included, e.g.,
made by modifying the glycosylation patterns of a polypeptide
during its synthesis and processing, or in further processing
steps. Particularly preferred means for accomplishing this are by
exposing the polypeptide to glycosylating enzymes derived from
cells which normally provide such processing, e.g., mammalian
glycosylation enzymes. Deglycosylation enzymes are also
contemplated. Also embraced are versions of the same primary amino
acid sequence which have other minor modifications, including
phosphorylated amino acid residues, e.g., phosphotyrosine,
phosphoserine, or phosphothreonine.
[0072] A major group of derivatives are covalent conjugates of the
Langerin protein or fragments thereof with other proteins or
polypeptides. These derivatives can be synthesized in recombinant
culture such as N- or C-terminal fusions or by the use of agents
known in the art for their usefulness in cross-linking proteins
through reactive side groups. Preferred antigen derivatization
sites with cross-linking agents are at free amino groups,
carbohydrate moieties, and cysteine residues.
[0073] Fusion polypeptides between the Langerin proteins and other
homologous or heterologous proteins are also provided. Homologous
polypeptides may be fusions between different surface markers,
resulting in, e.g., a hybrid protein exhibiting receptor binding
specificity. Likewise, heterologous fusions may be constructed
which would exhibit a combination of properties or activities of
the derivative proteins. Typical examples are fusions of a reporter
polypeptide, e.g., luciferase, with a segment or domain of amino
acids of the antigen, e.g., a binding segment, so that the presence
or location of the fused antigen may be easily determined. See,
e.g., Dull, et al. U.S. Pat. No. 4,859,609, which is hereby
incorporated herein by reference. Other gene fusion partners
include bacterial .beta.-galactosidase, trpE, Protein A,
.beta.-lactamase, alpha amylase, alcohol dehydrogenase, and yeast
alpha mating factor. See, e.g., Godowski, et al. (1988) Science
241: 812-816.
[0074] The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22: 1859-1862, will produce
suitable synthetic DNA fragments. A double stranded fragment will
often be obtained either by synthesizing the complementary strand
and annealing the strand together under appropriate conditions or
by adding the complementary strand using DNA polymerase with an
appropriate primer sequence.
[0075] Such polypeptides may also have amino acid residues which
have been chemically modified by phosphorylation, sulfonation,
biotinylation, or the addition or removal of other moieties,
particularly those which have molecular shapes similar to phosphate
groups. In some embodiments, the modifications will be useful
labeling reagents, or serve as purification targets, e.g., affinity
ligands.
[0076] Fusion proteins will typically be made by either recombinant
nucleic acid methods or by synthetic polypeptide methods.
Techniques for nucleic acid manipulation and expression are
described generally, for example, in Sambrook, et al. (1989)
Molecular Cloning: A Laboratory Manual (Cur. ed.), Vols. 1-3, Cold
Spring Harbor Laboratory, which are incorporated herein by
reference. Techniques for synthesis of polypeptides are described,
e.g., in Merrifield (1963) J. Amer. Chem. Soc. 85: 2149-2156;
Merrifield (1986) Science 232: 341-347; and Atherton, et al. (1989)
Solid Phase Peptide Synthesis: A Practical Approach, IRL Press,
Oxford; and Dawson, et al. (1994) Science 266: 776-779; each of
which is incorporated herein by reference.
[0077] This invention also contemplates the use of derivatives of
the Langerin proteins other than variations in amino acid sequence
or glycosylation. Such derivatives may involve covalent or
aggregative association with chemical moieties. These derivatives
generally fall into the three classes: (1) salts, (2) side chain
and terminal residue covalent modifications, and (3) adsorption
complexes, for example with cell membranes. Such covalent or
aggregative derivatives are useful as immunogens, as reagents in
immunoassays, or in purification methods such as for affinity
purification of antigens or other binding proteins. For example, a
Langerin antigen can be immobilized by covalent bonding to a solid
support such as cyanogen bromide-activated Sepharose, by methods
which are well known in the art, or adsorbed onto polyolefin
surfaces, with or without glutaraldehyde cross-linking, for use in
the assay or purification of anti-Langerin protein antibodies or
other binding partner. Langerin antigens can also be labeled with a
detectable group, for example radioiodinated by the chloramine T
procedure, covalently bound to rare earth chelates, or conjugated
to another fluorescent moiety for use in diagnostic assays.
Purification of Langerin protein may be effected by immobilized
antibodies or binding partners.
[0078] A solubilized Langerin antigen or fragment of this invention
can be used as an immunogen for the production of antisera or
antibodies specific for the protein or fragments thereof. The
purified antigen can be used to screen monoclonal antibodies or
binding fragments prepared by immunization with various forms of
impure preparations containing the protein. In particular, antigen
binding fragments of natural antibodies are often equivalent to the
antibodies themselves. Purified Langerin protein can also be used
as a reagent to detect any antibodies generated in response to the
presence of elevated levels of the protein or cell fragments
containing the antigen, both of which may be diagnostic of an
abnormal or specific physiological or disease condition.
Additionally, antigen fragments may also serve as immunogens to
produce further antibodies of the present invention, as described
immediately below. For example, this invention contemplates
antibodies raised against amino acid sequences from proteins having
properties described in Table 1, or fragments of them. In
particular, this invention contemplates antibodies having binding
affinity to or being raised against specific fragments which are
predicted to lie outside of the lipid bilayer, e.g., either
extracellular or intracellular domain structures.
[0079] The invention also provides means to isolate a group of
related antigens displaying both distinctness and similarities in
structure, expression, and function. Elucidation of many of the
physiological effects of the antigens will be greatly accelerated
by the isolation and characterization of distinct species variants.
In particular, the present invention provides useful probes for
identifying additional homologous genetic entities in different
species.
[0080] Isolated genes will allow transformation of cells lacking
expression of a corresponding Langerin protein, e.g., either
species types or cells which lack corresponding antigens and should
exhibit negative background biological activity. Expression of
transformed genes will allow isolation of antigenically pure cell
lines, with defined or single specie variants. This approach will
allow for more sensitive detection and discrimination of the
physiological effects of Langerin protein. Subcellular fragments,
e.g., cytoplasts or membrane fragments, can also be isolated and
used.
[0081] Dissection of the critical structural elements which effect
the various physiological or differentiation functions provided by
the proteins is possible using standard techniques of modern
molecular biology, particularly in comparing members of a related
class. See, e.g., the homolog-scanning mutagenesis technique
described in Cunningham, et al. (1989) Science 243: 1339-1336; and
approaches used in O'Dowd, et al. (1988) J. Biol. Chem. 263:
15985-15992; and Lechleiter, et al. (1990) EMBO J. 9: 4381-4390;
each of which is incorporated herein by reference.
[0082] In particular, functional domains or segments can be
substituted between species variants or related proteins to
determine what structural features are important in both binding
partner affinity and specificity, as well as signal transduction.
An array of different variants will be useful to screen for
molecules exhibiting combined properties of interaction with
different species variants of binding partners.
[0083] Antigen internalization may occur under certain
circumstances, and interaction between intracellular components and
"extracellular" segments of proteins involved in interactions may
occur. The specific segments of interaction of Langerin with other
intracellular components may be identified by mutagenesis or direct
biochemical means, e.g., cross-linking or affinity methods.
Structural analysis by crystallographic or other physical methods
will also be applicable. Further investigation of the mechanism of
biological function will include study of associated components
which may be isolatable by affinity methods or by genetic means,
e.g., complementation analysis of mutants.
[0084] Further study of the expression and control of Langerin will
be pursued. The controlling elements associated with the antigens
may exhibit differential developmental, tissue specific, or other
expression patterns. Upstream or downstream genetic regions, e.g.,
control elements, are of interest.
[0085] Structural studies of the antigen will lead to design of new
variants, particularly analogs exhibiting agonist or antagonist
properties on binding partners. This can be combined with
previously described screening methods to isolate variants
exhibiting desired spectra of activities.
[0086] Expression in other cell types will often result in
glycosylation differences in a particular antigen. Various species
variants may exhibit distinct functions based upon structural
differences other than amino acid sequence. Differential
modifications may be responsible for differential function, and
elucidation of the effects are now made possible.
[0087] Thus, the present invention provides important reagents
related to antigen-binding partner interaction. Although the
foregoing description has focused primarily upon the human Langerin
protein, those of skill in the art will immediately recognize that
the invention encompasses other closely related antigens, e.g.,
other primate species or allelic variants, as well as variants and
other members of the family.
[0088] VI. Nucleic Acids
[0089] The properties of natural Langerin protein disclosed herein
allow for purification of the protein. This provides means to
isolate nucleic acids encoding such proteins, and determination of
the sequence provides a handle to other members of the Langerin
family. Such nucleotide sequences and related reagents are useful
in constructing a DNA clone useful for expressing Langerin protein,
or, e.g., isolating a homologous gene from another natural source,
including other members of the family. Typically, the sequences
will be useful in isolating other genes, e.g., allelic variants or
alternatively spliced isoforms, from human.
[0090] For example, a specific binding composition such as DCGM4
could be used for screening of an expression library made from a
cell line which expresses a Langerin protein. The screening can be
standard staining of surface expressed protein, or by panning.
Screening of intracellular expression can also be performed by
various staining or immunofluorescence procedures. The binding
compositions could be used to affinity purify or sort out cells
expressing the Langerin protein.
[0091] This invention contemplates use of isolated DNA or fragments
to encode a biologically active Langerin protein or Langerin
polypeptide. A nucleotide sequence encoding human langerin is shown
in SEQ ID NO: 1. In addition, this invention covers isolated or
recombinant DNA which encodes a biologically active Langerin
protein or Langerin polypeptide and which is capable of hybridizing
under appropriate conditions with the DNA sequences. Said
biologically active Langerin protein or Langerin polypeptide can be
a full length antigen, or fragment thereof. Further, this invention
covers the use of isolated or recombinant DNA, or fragments
thereof, which encode proteins which are homologous to a Langerin
protein or which were isolated using cDNA encoding a Langerin
protein as a probe. The isolated DNA can have the respective
regulatory sequences in the 5' and 3' flanks, e.g., promoters,
enhancers, poly-A addition signals, and others.
[0092] An "isolated" nucleic acid is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially separated from
other components which naturally accompany a native sequence, e.g.,
ribosomes, polymerases, and flanking genomic sequences from the
originating species. The term "isolated" nucleic acid embraces a
nucleic acid sequence which has been removed from its naturally
occurring environment, and it also includes recombinant or cloned
DNA isolates and chemically synthesized analogs or analogs
biologically synthesized by heterologous systems. A substantially
pure molecule includes isolated forms of the molecule.
Alternatively, a purified species may be separated from host
components from a recombinant expression system.
[0093] An isolated nucleic acid will generally be a homogeneous
composition of molecules, but will, in some embodiments, contain
minor heterogeneity. This heterogeneity is typically found at the
polymer ends or portions not critical to a desired biological
function or activity.
[0094] A "recombinant" nucleic acid is defined either by its method
of production or its structure. In reference to its method of
production, e.g., a product made by a process, the process is use
of recombinant nucleic acid techniques, e.g., involving human
intervention in the nucleotide sequence, typically selection or
production. Alternatively, it can be a nucleic acid made by
generating a sequence comprising fusion of two fragments which are
not naturally contiguous to each other, but is meant to exclude
products of nature, e.g., naturally occurring mutants. Thus, for
example, products made by transforming cells with any unnaturally
occurring vector is encompassed, as are nucleic acids comprising
sequence derived using any synthetic oligonucleotide process. Such
is often done to replace a codon with a redundant codon encoding
the same or a conservative amino acid, while typically introducing
or removing a sequence recognition site. Alternatively, synthetic
oligonucleotides are used to join together nucleic acid segments of
desired functions to generate a single genetic entity comprising a
desired combination of functions not found in the commonly
available natural forms. Restriction enzyme recognition sites are
often the target of such artificial manipulations, but other site
specific targets, e.g., promoters, DNA replication sites,
regulation sequences, control sequences, or other useful features
may be incorporated by design. A similar concept is intended for a
recombinant polypeptide, e.g., a fusion polypeptide. Specifically
included are synthetic nucleic acids which, by genetic code
redundancy, encode polypeptides similar to fragments of these
antigens, and fusions of sequences from various different species
variants.
[0095] A significant "fragment" in a nucleic acid context is a
contiguous segment of at least about 17 nucleotides, generally at
least 20 nucleotides, more generally at least 23 nucleotides,
ordinarily at least 26 nucleotides, more ordinarily at least 29
nucleotides, often at least 32 nucleotides, more often at least 35
nucleotides, typically at least 38 nucleotides, more typically at
least 41 nucleotides, usually at least 44 nucleotides, more usually
at least 47 nucleotides, preferably at least 50 nucleotides, more
preferably at least 53 nucleotides, and in particularly preferred
embodiments will be at least 56 or more nucleotides.
[0096] A DNA which codes for a Langerin protein will be
particularly useful to identify genes, mRNA, and cDNA species which
code for related or homologous proteins, as well as DNAs which code
for homologous proteins. There should be homologues in other
mammals, e.g., primates. Various Langerin proteins should be
homologous and are encompassed herein. However, even proteins that
have a more distant evolutionary relationship to the antigen can
readily be isolated under appropriate conditions using these
sequences if they are sufficiently homologous. Primate Langerin
proteins are of particular interest.
[0097] This invention further covers recombinant DNA molecules and
fragments having a DNA sequence identical to or highly homologous
to the isolated DNAs set forth herein. In particular, the sequences
will often be operably linked to DNA segments which control
transcription, translation, and DNA replication. Alternatively,
recombinant clones derived from the genomic sequences, e.g.,
containing introns, will be useful for transgenic studies,
including, e.g., transgenic cells and organisms, and for gene
therapy. See, e.g., Goodnow (1992) "Transgenic Animals" in Roitt
(ed.) Encyclopedia of Immunology Academic Press, San Diego, pp.
1502-1504; Travis (1992) Science 256: 1392-1394; Kuhn, et al.
(1991) Science 254: 707-710; Capecchi (1989) Science 244:1288;
Robertson (ed. 1987) Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach IRL Press, Oxford; Rosenberg (1992) J. Clinical
Oncology 10: 180-199; and Cournoyer and Caskey (1993) Ann. Rev.
Immunol. 11: 297-329; each of which is incorporated herein by
reference.
[0098] Homologous nucleic acid sequences, when compared, exhibit
significant similarity. The standards for homology in nucleic acids
are either measures for homology generally used in the art by
sequence comparison or based upon hybridization conditions. The
hybridization conditions are described in greater detail below.
[0099] Substantial homology in the nucleic acid sequence comparison
context means either that the segments, or their complementary
strands, when compared, are identical when optimally aligned, with
appropriate nucleotide insertions or deletions, in at least about
50% of the nucleotides, generally at least 56%, more generally at
least 59%, ordinarily at least 62%, more ordinarily at least 65%,
often at least 68%, more often at least 71%, typically at least
74%, more typically at least 77%, usually at least 80%, more
usually at least about 85%, preferably at least about 90%, more
preferably at least about 95 to 98% or more, and in particular
embodiments, as high at about 99% or more of the nucleotides.
Alternatively, substantial homology exists when the segments will
hybridize under selective hybridization conditions, to a strand, or
its complement, typically using a sequence as described. Typically,
selective hybridization will occur when there is at least about 55%
homology over a stretch of at least about 14 nucleotides,
preferably at least about 65%, more preferably at least about 75%,
and most preferably at least about 90%. See, Kanehisa (1984) Nuc.
Acids Res. 12: 203-213, which is incorporated herein by reference.
The length of homology comparison, as described, may be over longer
stretches, and in certain embodiments will be over a stretch of at
least about 17 nucleotides, usually at least about 20 nucleotides,
more usually at least about 24 nucleotides, typically at least
about 28 nucleotides, more typically at least about 40 nucleotides,
preferably at least about 50 nucleotides, and more preferably at
least about 75 to 100 or more nucleotides.
[0100] Stringent conditions, in referring to homology in the
hybridization context, will be stringent combined conditions of
salt, temperature, organic solvents, and other parameters,
typically those controlled in hybridization reactions. Stringent
temperature conditions will usually include temperatures in excess
of about 30.degree. C., more usually in excess of about 37.degree.
C., typically in excess of about 45.degree. C., more typically in
excess of about 55.degree. C., preferably in excess of about
65.degree. C., and more preferably in excess of about 70.degree. C.
Stringent salt conditions will ordinarily be less than about 1000
mM, usually less than about 500 mM, more usually less than about
400 mM, typically less than about 300 mM, preferably less than
about 200 mM, and more preferably less than about 150 mM. However,
the combination of parameters is much more important than the
measure of any single parameter. See, e.g., Wetmur and Davidson
(1968) J. Mol. Biol. 31: 349-370; Walker (ed. 1988) New Nucleic
Acid Techniques Humana Press, Clifton, N.J.; Ross (ed. 1998)
Nucleic Acid Hybridization; Wiley, New York; or see, e.g., recent
hybridization protocols on the Internet in "A Selection of
Molecular Biology Protocols--A list of sites with protocols," by
Griffin (1996) Analytical Biochemistry 239: 120-122.
[0101] VII. Making Langerin Protein; Mimetics
[0102] Langerin may be isolated from natural sources using standard
methods of protein biochemistry. The DCGM4 antibody may be used to
track the purification process, or in various immunoaffinity
methods. Isolated protein may be used as starting material for
derivatization or modification. The protein may be used in native
or denatured forms.
[0103] DNA which encodes the Langerin protein or fragments thereof
can be obtained by chemical synthesis, screening cDNA libraries, or
by screening genomic libraries prepared from a wide variety of cell
lines or tissue samples. In particular, the DCGM4 antibody may be
used to expression clone the nucleic acid encoding the protein
antigen.
[0104] This DNA can be expressed in a wide variety of host cells
for the synthesis of a full-length protein or fragments which can
in turn, for example, be used to generate polyclonal or monoclonal
antibodies; for binding studies; for construction and precision of
modified molecules; and for structure/function studies. Each
antigen or its fragments can be expressed in host cells that are
transformed or transfected with appropriate expression vectors.
These molecules can be substantially purified to be free of protein
or cellular contaminants, other than those derived from the
recombinant host, and therefore are particularly useful in
pharmaceutical compositions when combined with a pharmaceutically
acceptable carrier, buffer, and/or diluent. The antigen, or
portions thereof, may be expressed as fusions with other
proteins.
[0105] Expression vectors are typically self-replicating DNA or RNA
constructs containing the desired antigen gene or its fragments,
usually operably linked to suitable genetic control elements that
are recognized in a suitable host cell. These control elements are
capable of effecting expression within a suitable host. The
specific type of control elements necessary to effect expression
will depend upon the eventual host cell used. Generally, the
genetic control elements can include a prokaryotic promoter system
or a eukaryotic promoter expression control system, and typically
include a transcriptional promoter, an optional operator to control
the onset of transcription, transcription enhancers to elevate the
level of mRNA expression, a sequence that encodes a suitable
ribosome binding site, and sequences that terminate transcription
and translation. Expression vectors also usually contain an origin
of replication that allows the vector to replicate independently of
the host cell.
[0106] The vectors of this invention contain DNA which encodes a
Langerin protein, or a fragment thereof, preferably encoding a
biologically active polypeptide. The sequence set forth in SEQ ID
NO: 1 may advantageously be used to prepare Langerin protein. The
DNA can be under the control of a viral promoter and can encode a
selection marker. This invention further contemplates use of such
expression vectors which are capable of expressing eukaryotic cDNA
coding for a Langerin protein in a prokaryotic or eukaryotic host,
where the vector is compatible with the host and where the
eukaryotic cDNA coding for the antigen is inserted into the vector
such that growth of the host containing the vector expresses the
cDNA in question. Usually, expression vectors are designed for
stable replication in their host cells or for amplification to
greatly increase the total number of copies of the desirable gene
per cell. It is not always necessary to require that an expression
vector replicate in a host cell, e.g., it is possible to effect
transient expression of the antigen or its fragments in various
hosts using vectors that do not contain a replication origin that
is recognized by the host cell. It is also possible to use vectors
that cause integration of a Langerin gene or its fragments into the
host DNA by recombination, or to integrate a promoter which
controls expression of an endogenous gene e.g., see WO 96/29411
(incorporated herein by reference including all figures and
drawings) describing such technology.
[0107] Vectors, as used herein, comprise plasmids, viruses,
bacteriophage, integratable DNA fragments, and other vehicles which
enable the integration of DNA fragments into the genome of the
host. Expression vectors are specialized vectors which contain
genetic control elements that effect expression of operably linked
genes. Plasmids are the most commonly used form of vector but all
other forms of vectors which serve an equivalent function and which
are, or become, known in the art are suitable for use herein. See,
e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors: A
Laboratory Manual, Elsevier, N.Y.; and Rodriquez, et al. (eds.
1988) Vectors: A Survey of Molecular Cloning Vectors and Their
Uses, Buttersworth, Boston, Mass.; which are incorporated herein by
reference.
[0108] Transformed cells include cells, preferably mammalian, that
have been transformed or transfected with vectors containing a
Langerin nucleic acid sequence, typically constructed using
recombinant DNA techniques. Transformed host cells usually express
the antigen or its fragments, but for purposes of cloning,
amplifying, and manipulating its DNA, do not need to express the
protein. This invention further contemplates culturing transformed
cells in a nutrient medium, thus permitting the protein to
accumulate in the culture. The protein can be recovered, either
from the culture or from the culture medium.
[0109] For purposes of this invention, DNA sequences are operably
linked when they are functionally related to each other. For
example, DNA for a presequence or secretory leader is operably
linked to a polypeptide if it is expressed as a preprotein or
participates in directing the polypeptide to the cell membrane or
in secretion of the polypeptide. A promoter is operably linked to a
coding sequence if it controls the transcription of the
polypeptide; a ribosome binding site is operably linked to a coding
sequence if it is positioned to permit translation. Usually,
operably linked means contiguous and in reading frame, however,
certain genetic elements such as repressor genes are not
contiguously linked but still bind to operator sequences that in
turn control expression.
[0110] Suitable host cells include prokaryotes, lower eukaryotes,
and higher eukaryotes. Prokaryotes include both gram negative and
gram positive organisms, e.g., E. coli and B. subtilis. Lower
eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and
species of the genus Dictyostelium. Higher eukaryotes include
established tissue culture cell lines from animal cells, both of
non-mammalian origin, e.g.; insect cells, and birds, and of
mammalian origin, e.g., human, primates, and rodents.
[0111] Prokaryotic host-vector systems include a wide variety of
vectors for many different species. As used herein, E. coli and its
vectors will be used generically to include equivalent vectors used
in other prokaryotes. A representative vector for amplifying DNA is
pBR322 or many of its derivatives. Vectors that can be used to
express the Langerin proteins or its fragments include, but are not
limited to, such vectors as those containing the lac promoter
(pUC-series); trp promoter (pBR322-trp); Ipp promoter (the
pIN-series); lambda-pP or pR promoters (pOTS); or hybrid promoters
such as ptac (pDR540). See Brosius, et al. (1988) "Expression
Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters",
in Rodriguez and Denhardt. (eds.) Vectors: A Survey of Molecular
Cloning Vectors and Their Uses, Buttersworth, Boston, Chapter 10,
pp. 205-236, which is incorporated herein by reference.
[0112] Lower eukaryotes, e.g., yeasts and Dictyostelium, may be
transformed with vectors encoding Langerin proteins. For purposes
of this invention, the most common lower eukaryotic host is the
baker's yeast, Saccharomyces cerevisiae. It will be used to
generically represent lower eukaryotes although a number of other
strains and species are also available. Yeast vectors typically
consist of a replication origin (unless of the integrating type), a
selection gene, a promoter, DNA encoding the desired protein or its
fragments, and sequences for translation termination,
polyadenylation, and transcription termination. Suitable expression
vectors for yeast include such constitutive promoters as
3-phosphoglycerate kinase and various other glycolytic enzyme gene
promoters or such inducible promoters as the alcohol dehydrogenase
2 promoter or metallothionine promoter. Suitable vectors include
derivatives of the following types: self-replicating low copy
number (such as the YRp-series), self-replicating high copy number
(such as the YEp-series); integrating types (such as the
YIp-series), or mini-chromosomes (such as the YCp-series).
[0113] Higher eukaryotic tissue culture cells are the preferred
host cells for expression of the functionally active Langerin
protein. In principle, any higher eukaryotic tissue culture cell
line is workable, e.g., insect baculovirus expression systems,
whether from an invertebrate or vertebrate source. However,
mammalian cells are preferred, in that the processing, both
cotranslationally and posttranslationally. Transformation or
transfection and propagation of such cells has become a routine
procedure. Examples of useful cell lines include HeLa cells,
Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell
lines, insect cell lines, bird cell lines, and monkey (COS) cell
lines. Expression vectors for such cell lines usually include an
origin of replication, a promoter, a translation initiation site,
RNA splice sites (if genomic DNA is used), a polyadenylation site,
and a transcription termination site. These vectors also usually
contain a selection gene or amplification gene. Suitable expression
vectors may be plasmids, viruses, or retroviruses carrying
promoters derived, e.g., from such sources as from adenovirus,
SV40, parvoviruses, vaccinia virus, or cytomegalovirus.
Representative examples of suitable expression vectors include
pCDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol. 5:
1136-1142; pMClneo Poly-A, see Thomas, et al. (1987) Cell 51:
503-512; and a baculovirus vector such as pAC 373 or pAC 610.
[0114] It will often be desired to express a Langerin protein
polypeptide in a system which provides a specific or defined
glycosylation pattern. In this case, the usual pattern will be that
provided naturally by the expression system. However, the pattern
will be modifiable by exposing the polypeptide, e.g., an
unglycosylated form, to appropriate glycosylating proteins
introduced into a heterologous expression system. For example, the
Langerin protein gene may be co-transformed with one or more genes
encoding mammalian or other glycosylating enzymes. Using this
approach, certain mammalian glycosylation patterns will be
achievable or approximated in prokaryote or other cells.
[0115] The Langerin protein, or a fragment thereof, may be
engineered to be phosphatidyl inositol (PI) linked to a cell
membrane, but can be removed from membranes by treatment with a
phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl inositol
phospholipase-C. This releases the antigen in a biologically active
form, and allows purification by standard procedures of protein
chemistry. See, e.g., Low (1989) Biochim. Biophys. Acta 988:
427-454; Tse, et al. (1985) Science 230: 1003-1008; and Brunner, et
al. (1991) J. Cell Biol. 114: 1275-1283.
[0116] Fragments or derivatives of Langerin can be prepared by
conventional processes for synthesizing peptides. Solid phase and
solution phase syntheses are both applicable to the foregoing
processes. These include processes such as are described in
Bodanszky (1993) Principles of Peptide Synthesis; Springer-Verlag;
Bodanszky (1994) The Practice of Peptide Synthesis;
Springer-Verlag; Jones (1992) Amino Acid and Peptide Synthesis;
Oxford University Press; Atherton and Sheppard (1989) Solid Phase
Peptide Synthesis: A Practical Approach; IRL Press New York;
Stewart and Young (1984) Solid Phase Peptide Synthesis, Pierce
Chemical Co., Rockford, Ill.; and chemical ligation, e.g., Dawson,
et al. (1994) Science 266: 776-779, a method of linking long
synthetic peptides by a peptide bond; each of which is incorporated
herein by reference. For example, an azide process, an acid
chloride process, an acid anhydride process, a mixed anhydride
process, an active ester process (for example, p-nitrophenyl ester,
N-hydroxysuccinimide ester, or cyanomethyl ester), a
carbodiimidazole process, an oxidative-reductive process, or a
dicyclohexylcarbodiimide (DCCD)/additive process can be used. Solid
phase and solution phase syntheses are both applicable to the
foregoing processes.
[0117] The Langerin protein, fragments, or derivatives are suitably
prepared in accordance with the above processes as typically
employed in peptide synthesis, generally either by a so-called
stepwise process which comprises condensing an amino acid to the
terminal amino acid, one by one in sequence, or by coupling peptide
fragments to the terminal amino acid. Amino groups that are not
being used in the coupling reaction are typically protected to
prevent coupling at an incorrect location.
[0118] If a solid phase synthesis is adopted, the C-terminal amino
acid is bound to an insoluble carrier or support through its
carboxyl group. The insoluble carrier is not particularly limited
as long as it has a binding capability to a reactive carboxyl
group. Examples of such insoluble carriers include halomethyl
resins, such as chloromethyl resin or bromomethyl resin,
hydroxymethyl resins, phenol resins,
tert-alkyloxycarbonyl-hydrazidated resins, and the like.
[0119] An amino group-protected amino acid is bound in sequence
through condensation of its activated carboxyl group and the
reactive amino group of the previously formed peptide or chain, to
synthesize the peptide step by step. After synthesizing the
complete sequence, the peptide is split off from the insoluble
carrier to produce the peptide. This solid-phase approach is
generally described by Merrifield, et al. (1963) in J. Am. Chem.
Soc. 85: 2149-2156, which is incorporated herein by reference.
[0120] The prepared protein and fragments thereof can be isolated
and purified from the reaction mixture by means of peptide
separation, for example, by extraction, precipitation,
electrophoresis and various forms of chromatography, and the like.
The Langerin of this invention can be obtained in varying degrees
of purity depending upon its desired use. Purification can be
accomplished by use of the protein purification techniques
disclosed herein or by the use of the antibodies herein described
in immunoabsorbant affinity chromatography. This immunoabsorbant
affinity chromatography is carried out by first linking the
antibodies to a solid support and then contacting the linked
antibodies with solubilized lysates of appropriate source cells,
lysates of other cells expressing the protein, or lysates or
supernatants of cells producing Langerin protein as a result of DNA
techniques, see below.
[0121] VIII. Utility
[0122] The present invention provides reagents which will find use
in diagnostic applications as described elsewhere herein, e.g., in
the general description for physiological or developmental
abnormalities, or below in the description of kits. The antigen
will be useful in both diagnostic methods, for isolating various
cell types, e.g., Langerhans cells, or for forensic methods to
determine various species sources of biological samples.
[0123] This invention provides reagents with significant
therapeutic value. Langerin (naturally occurring or recombinant),
fragments thereof, muteins, and antibodies, along with compounds
identified as having binding affinity to Langerin or antibodies,
find use in the treatment of conditions exhibiting abnormal
expression of Langerin of its ligands or binding agents. Such
abnormality will typically be manifested by immunological
disorders. Additionally, this invention provides therapeutic value
in various diseases or disorders associated with abnormal
expression or abnormal triggering of response to the ligand or
binding agent. The Langerin ligands or binding agents are suspected
to be involved in maturational development of DC. Langerin may also
be used as a receptor to target antigen to dendritic cells to
elicit therapeutically relevant immune responses.
[0124] Recombinant Langerins, muteins, agonist or antagonist
antibodies thereto, or antibodies can be purified and then
administered to a patient. These reagents can be combined for
therapeutic use with additional active ingredients, e.g., in
conventional pharmaceutically acceptable carriers or diluents,
along with physiologically innocuous stabilizers and excipients.
These combinations can be sterile, e.g., filtered, and placed into
dosage forms as by lyophilization in dosage vials or storage in
stabilized aqueous preparations. This invention also contemplates
the use of antibodies or binding fragments thereof which are not
complement binding.
[0125] Using Langerin or fragments thereof to screen for binding
partner or for compounds having binding affinity to Langerin
antigen can be performed including the isolation of associated
compounds. Subsequent biological assays can then be utilized to
determine if a putative ligand or binding agent can provide
competitive binding, which can block intrinsic stimulating
activity. Langerin fragments can be used as a blocker or antagonist
in that it blocks the activity of ligand or binding agent. This
invention further contemplates the therapeutic use of antibodies to
Langerins as antagonists. This approach will be particularly useful
with other Langerin protein species variants and other members of
the family.
[0126] The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, reagent physiological life,
pharmacological life, physiological state of the patient, and other
medicants administered. Thus, treatment dosages should be titrated
to optimize safety and efficacy. Typically, dosages used in vitro
may provide useful guidance in the amounts useful for in situ
administration of these reagents. Animal testing of effective doses
for treatment of particular disorders will provide further
predictive indication of human dosage. The actual dosage of
reagent, formulation or composition that modulates an immunological
disorder depends on many factors, including the size and health of
an organism, however, one of ordinary skill in the art can use
teachings describing methods and techniques for determining
clinical dosages. See, e.g., Spilker (1984) Guide to Clinical
Studies and Developing Protocols. Raven Press Books, Ltd., New
York, esp. pp. 7-13, 54-60; Spilker (1991) Guide to Clinical
Trials, Raven Press, Ltd., New York, esp. pp. 93-101; Craig and
Stitzel (eds. 1986) Modern Pharmacology, 2d ed., Little, Brown and
Co., Boston, esp. pp. 127-33; Speight (ed. 1987) Avery's Drug
Treatment: Principles and Practice of Clinical Pharmacology and
Therapeutics, 3d ed., Williams and Wilkins, Baltimore, esp. pp.
50-56; Tallarida, et al. (1988) Principles of General Pharmacology,
Springer-Verlag, New York, esp. pp. 18-20; Gilman, et al. (eds.)
Goodman and Gilman's: The Pharmacological Bases of Therapeutics,
latest ed., Pergamon Press; Remington's Pharmaceutical Sciences,
latest ed., Mack Publishing Co., Easton, Pa.; and Rich, et al.
(1998) Clinical Immunology: Principles and Practice Vols. I &
II, Mosby, St. Louis, Mo., each of which is hereby incorporated
herein by reference in its entirety including all drawings and
figures. Methods for administration are discussed therein and
below, e.g., for oral, intravenous, intraperitoneal, or
intramuscular administration, transdermal diffusion, and others.
Pharmaceutically acceptable carriers will include water, saline,
buffers, and other compounds described, e.g., in the Merck Index,
Merck & Co., Rahway, N.J. Because of the likely high affinity
binding, or turnover numbers, between a putative ligand or binding
agent and its binding partner, low dosages of these reagents would
be initially expected to be effective. Thus, dosage ranges would
ordinarily be expected to be in amounts lower than 1 mM
concentrations, typically less than about 10 .mu.M concentrations,
usually less than about 100 nM, preferably less than about 10 pM
(picomolar), and most preferably less than about 1 fM (femtomolar),
with an appropriate carrier. Slow release formulations, or slow
release apparatus will often be utilized for continuous
administration.
[0127] Other abnormal developmental conditions are known in the
cell types shown to possess Langerin antigen, e.g., DC cells and
epithelial cells of the tonsil. See Brew (ed.) The Merck Manual of
Diagnosis and Therapy Merck & Co., Rahway, N.J.; and Thorn, et
al. Harrison's Principles of Internal Medicine McGraw-Hill, N.Y.
These problems may be susceptible to prevention or treatment using
compositions provided herein.
[0128] Langerins, fragments thereof, and antibodies or its
fragments, antagonists, and agonists, may be administered directly
to the host to be treated or, depending on the size of the
compounds, it may be desirable to conjugate them to carrier
proteins such as ovalbumin or serum albumin prior to their
administration. Therapeutic formulations may be administered in
many conventional dosage formulations. While it is possible for the
active ingredient to be administered alone, it is preferable to
present it as a pharmaceutical formulation. Formulations comprise
at least one active ingredient, as defined above, together with one
or more acceptable carriers thereof. Each carrier must be both
pharmaceutically and physiologically acceptable in the sense of
being compatible with the other ingredients and not injurious to
the patient. Formulations include those suitable for oral, rectal,
nasal, or parenteral (including subcutaneous, intramuscular,
intravenous and intradermal) administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by methods well known in the art of pharmacy. See, e.g., Gilman, et
al. (eds. 1990) Goodman and Gilman's: The Pharmacolocical Bases of
Therapeutics, 8th Ed., Pergamon Press; and Reminaton's
Pharmaceutical Sciences, current ed., Mack Publishing Co., Easton,
Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms:
Parenteral Medications Dekker, NY; Lieberman, et al. (eds. 1990)
Pharmaceutical Dosage Forms: Tablets Dekker, NY; and Lieberman, et
al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems
Dekker, NY. The therapy of this invention may be combined with or
used in association with other therapeutic agents.
[0129] Both the naturally occurring and the recombinant forms of
the Langerin specific antibodies and the Langerin proteins of this
invention are particularly useful in kits and assay methods which
are capable of screening compounds for binding activity. Several
methods of automating assays have been developed in recent years so
as to permit screening of tens of thousands of compounds in a short
period. See, e.g., Fodor, et al. (1991) Science 251: 767-773, which
is incorporated herein by reference and which describes means for
testing of binding affinity by a plurality of defined polymers
synthesized on a solid substrate. The development of suitable
assays can be greatly facilitated by the availability of large
amounts of purified, soluble Langerin protein as provided by this
invention.
[0130] This invention is particularly useful for screening
compounds by using recombinant antigen in any of a variety of drug
screening techniques. The advantages of using a recombinant protein
in screening for specific ligands or binding agents include: (a)
improved renewable source of the antigen from a specific source;
(b) potentially greater number of antigen molecules per cell giving
better signal to noise ratio in assays; and (c) species variant
specificity (theoretically giving greater biological and disease
specificity). The purified protein may be tested in numerous
assays, typically in vitro assays, which evaluate biologically
relevant responses. See, e.g., Coligan Current Protocols in
Immunology; Hood, et al. Immunology Benjamin/Cummings; Paul (ed.
1996) Fundamental Immunology 3d ed, Raven Press, NY; and Methods in
Enzymology Academic Press. This will also be useful in screening
for a ligand which binds a Langerin, e.g., from an interacting
cell.
[0131] One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing the Langerin antigens. Cells
may be isolated which express an antigen in isolation from other
functionally equivalent antigens. Such cells, either in viable or
fixed form, can be used for standard protein-protein binding
assays. See also, Parce, et al. (1989) Science 246: 243-247; and
Owicki, et al. (1990) Proc. Nat'l Acad. Sci. USA 87: 4007-4011;
which are incorporated herein by reference and describe sensitive
methods to detect cellular responses. Competitive assays are
particularly useful, where the cells (source of Langerin protein)
are contacted and incubated with a labeled binding partner or
antibody having known binding affinity to the ligand, such as
.sup.125I-antibody, and a test sample whose binding affinity to the
binding composition is being measured. The bound and free labeled
binding compositions are then separated to assess the degree of
antigen binding. The amount of test compound bound is inversely
proportional to the amount of labeled receptor binding to the known
source. Numerous techniques can be used to separate bound from free
antigen to assess the degree of binding. This separation step could
typically involve a procedure such as adhesion to filters followed
by washing, adhesion to plastic followed by washing, or
centrifugation of the cell membranes. Viable cells could also be
used to screen for the effects of drugs on Langerin protein
mediated functions, e.g., second messenger levels, i.e., Ca.sup.++;
cell proliferation; inositol phosphate pool changes; and others.
Some detection methods allow for elimination of a separation step,
e.g., a proximity sensitive detection system. Calcium sensitive
dyes will be useful for detecting Ca.sup.++ levels, with a
fluorimeter or a fluorescence cell sorting apparatus.
[0132] Another method utilizes membranes from transformed
eukaryotic or prokaryotic host cells as a source of Langerin
protein. These cells are stably transformed with DNA vectors
directing the expression of a membrane associated Langerin protein,
e.g., an engineered membrane bound form. Essentially, the membranes
would be prepared from the cells and used in a receptor/ligand type
binding assay such as the competitive assay set forth above.
[0133] Still another approach is to use solubilized, unpurified or
solubilized, purified Langerin protein from transformed eukaryotic
or prokaryotic host cells. This allows for a "molecular" binding
assay with the advantages of increased specificity, the ability to
automate, and high drug test throughput.
[0134] Another technique for drug screening involves an approach
which provides high throughput screening for compounds having
suitable binding affinity to Langerin and is described in detail in
Geysen, European Patent Application 84/03564, published on Sep. 13,
1984, which is incorporated herein by reference. First, large
numbers of different small peptide test compounds are synthesized
on a solid substrate, e.g., plastic pins or some other appropriate
surface, see Fodor, et al. (1991). Then all the pins are reacted
with solubilized, unpurified or solubilized, purified Langerin
binding composition, and washed. The next step involves detecting
bound binding composition.
[0135] Rational drug design may also be based upon structural
studies of the molecular shapes of the Langerin protein and other
effectors or analogs. Effectors may be other proteins which mediate
other functions in response to antigen binding, or other proteins
which normally interact with the antigen, e.g., Langerin ligand.
One means for determining which sites interact with specific other
proteins is a physical structure determination, e.g., x-ray
crystallography or 2 dimensional NMR techniques. These will provide
guidance as to which amino acid residues form molecular contact
regions. For a detailed description of protein structural
determination, see, e.g., Blundell and Johnson (1976) Protein
Crystallography, Academic Press, New York, which is hereby
incorporated herein by reference.
[0136] Purified Langerin protein can be coated directly onto plates
for use in the aforementioned drug screening techniques. However,
non-neutralizing antibodies to these ligands can be used as capture
antibodies to immobilize the respective ligand on the solid
phase.
[0137] IX. Kits and Quantitation/Detection
[0138] Both naturally occurring and recombinant forms of the
Langerin molecule of the invention are particularly useful in kits
and assay methods. For example, these methods would also be applied
to screening for binding activity, e.g., ligands or binding agents
for these proteins. Several methods of automating assays have been
developed in recent years so as to permit screening of tens of
thousands of compounds per year. See, e.g., a BIOMEK automated
workstation, Beckman Instruments, Palo Alto, Calif., and Fodor, et
al. (1991) Science 251: 767-773, which is incorporated herein by
reference. The latter describes means for testing binding by an
agent by a plurality of defined polymers synthesized on a solid
substrate. The development of suitable assays to screen for a
ligand or binding agent or agonist/antagonist homologous proteins
can be greatly facilitated by the availability of large amounts of
purified, soluble Langerin in an active state such as is provided
by this invention.
[0139] Purified Langerin can be coated directly onto plates for use
in the aforementioned ligand or binding agent screening techniques.
However, non-neutralizing antibodies to these proteins can be used
as capture antibodies to immobilize the respective Langerin protein
on the solid phase, useful, e.g., in diagnostic uses.
[0140] This invention also contemplates use of Langerin, fragments
thereof, peptides, and their fusion products in a variety of
diagnostic kits and methods for detecting the presence of the
Langerin protein or its ligand or binding agent. Alternatively, or
additionally, antibodies against the Langerin molecules may be
incorporated into the kits and methods. Typically the kit will have
a compartment containing either a Langerin protein, fragment,
peptide or gene segment or a reagent which recognizes one or the
other of these. Typically, recognition reagents, in the case of a
protein or fragment thereof, would be a receptor or antibody, or in
the case of a gene segment, would usually be a hybridization
probe.
[0141] A preferred kit for determining the concentration of
Langerin in a sample would typically comprise a labeled compound,
e.g., ligand, binding agent, or antibody, having known binding
affinity for Langerin, a source of Langerin (naturally occurring or
recombinant) as a positive control, and a means for separating the
bound from free labeled compound, for example a solid phase for
immobilizing the Langerin in the test sample. Compartments
containing reagents, and instructions, will normally be
provided.
[0142] This invention also contemplates use of Langerin proteins,
fragments thereof, peptides, their fusion products, and binding
compositions in a variety of diagnostic kits and methods for
detecting the presence of a binding composition. Typically the kit
will have a compartment containing either a defined Langerin
peptide or gene segment or a reagent which recognizes one or the
other, e.g., antigen fragments or antibodies. See, e.g., Chen
(ed.)(1987) Immunoassay: A Practical Guide Academic Press, Orlando,
Fla.; Price and Newman (eds.)(1991) Principles and Practice of
Immunoassay Stockton Press, New York; and Ngo (ed.)(1988)
Nonisotopic Immunoassay Plenum Press, NY.
[0143] A kit for determining the binding affinity of a test
compound to a Langerin protein would typically comprise a test
compound; a labeled compound, for example an antibody having known
binding affinity for the antigen; a source of Langerin protein
(naturally occurring or recombinant); and a means for separating
bound from free labeled compound, such as a solid phase for
immobilizing the antigen. Once compounds are screened, those having
suitable binding affinity to the antigen can be evaluated in
suitable biological assays, as are well known in the art, to
determine whether they exhibit similar biological activities to the
natural antigen. The availability of recombinant Langerin protein
polypeptides also provide well defined standards for calibrating
such assays.
[0144] One method for determining the concentration of Langerin
protein in a sample would typically comprise the steps of: (1)
preparing membranes from a sample comprised of a membrane bound
Langerin protein source; (2) washing the membranes and suspending
them in a buffer; (3) solubilizing the antigen by incubating the
membranes in a culture medium to which a suitable detergent has
been added; (4) adjusting the detergent concentration of the
solubilized antigen; (5) contacting and incubating said dilution
with radiolabeled antibody to form complexes; (6) recovering the
complexes such as by filtration through polyethyleneimine treated
filters; and (7) measuring the radioactivity of the recovered
complexes.
[0145] Antibodies, including antigen binding fragments, specific
for the Langerin protein or fragments are useful in diagnostic
applications to detect the presence of elevated levels of Langerin
protein and/or its fragments. Such diagnostic assays can employ
lysates, live cells, fixed cells, immunofluorescence, cell
cultures, tissue samples, body fluids, and further can involve the
detection of antigens related to the protein in serum, or the like.
Diagnostic assays may be homogeneous (without a separation step
between free reagent and protein-protein complex) or heterogeneous
(with a separation step). Various commercial assays exist, such as
radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),
enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique
(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the
like. For example, unlabeled antibodies can be employed by using a
second antibody which is labeled and which recognizes the antibody
to a Langerin protein or to a particular fragment thereof. Similar
assays have also been extensively discussed in the literature. See,
e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH,
and Coligan (ed. 1991) and periodic supplements, Current Protocols
In Immunology Greene/Wiley, New York.
[0146] Anti-idiotypic antibodies may have similar use to diagnose
presence of antibodies against a Langerin protein, as such may be
diagnostic of various abnormal states. For example, overproduction
of Langerin protein may result in production of various
immunological reactions which may be diagnostic of abnormal
physiological states associated with Langerhans cells (e.g., atopic
dermatitis, eosinophilic granuloma, histiocytosis, systemic
sclerosis and Langerhans cell granulomatosis). Also, see, e.g.,
Rich, et al. (1998) Clinical Immunology: Principles and Practice
Vols. I & II, Mosby, St. Louis, Mo.
[0147] Frequently, the reagents for diagnostic assays are supplied
in kits, so as to optimize the sensitivity of the assay. For the
subject invention, depending upon the nature of the assay, the
protocol, and the label, either labeled or unlabeled antibody, or
labeled Langerin protein is provided. This is usually in
conjunction with other additives, such as buffers, stabilizers,
materials necessary for signal production such as substrates for
enzymes, and the like. Preferably, the kit will also contain
instructions for proper use and disposal of the contents after use.
Typically the kit has compartments for each useful reagent.
Desirably, the reagents are provided as a dry lyophilized powder,
where the reagents may be reconstituted in an aqueous medium
providing appropriate concentrations of reagents for performing the
assay.
[0148] Many of the aforementioned constituents of the drug
screening and the diagnostic assays may be used without
modification or may be modified in a variety of ways. For example,
labeling may be achieved by covalently or non-covalently joining a
moiety which directly or indirectly provides a detectable signal.
In these assays, the antigen, test compound, Langerin protein, or
antibodies thereto can be labeled either directly or indirectly.
Possibilities for direct labeling include label groups: radiolabels
such as .sup.125I, enzymes (U.S. Pat. No. 3,645,090) such as
peroxidase and alkaline phosphatase, and fluorescent labels (U.S.
Pat. No. 3,940,475) capable of monitoring the change in
fluorescence intensity, wavelength shift, or fluorescence
polarization. Both of the patents are incorporated herein by
reference. Possibilities for indirect labeling include
biotinylation of one constituent followed by binding to avidin
coupled to one of the above label groups.
[0149] There are also numerous methods of separating the bound from
the free antigen, or alternatively the bound from the free test
compound. The Langerin protein can be immobilized on various
matrices followed by washing. Suitable matrices include plastic
such as an ELISA plate, filters, and beads. Methods of immobilizing
the Langerin protein to a matrix include, without limitation,
direct adhesion to plastic, use of a capture antibody, chemical
coupling, and biotin-avidin. The last step in this approach
involves the precipitation of protein-protein complex by any of
several methods including those utilizing, e.g., an organic solvent
such as polyethylene glycol or a salt such as ammonium sulfate.
Other suitable separation techniques include, without limitation,
the fluorescein antibody magnetizable particle method described in
Rattle, et al. (1984) Clin. Chem. 30: 1457-1461, and the double
antibody magnetic particle separation as described in U.S. Pat. No.
4,659,678, each of which is incorporated herein by reference.
[0150] The methods for linking proteins or their fragments to the
various labels have been extensively reported in the literature and
do not require detailed discussion here. Many of the techniques
involve the use of activated carboxyl groups either through the use
of carbodiimide or active esters to form peptide bonds, the
formation of thioethers by reaction of a mercapto group with an
activated halogen such as chloroacetyl, or an activated olefin such
as maleimide, for linkage, or the like. Fusion proteins will also
find use in these applications.
[0151] Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence
of a Langerin protein. These sequences can be used as probes for
detecting levels of antigen message in samples from patients
suspected of having an abnormal condition, e.g., an immunological
disorder. The preparation of both RNA and DNA nucleotide sequences,
the labeling of the sequences, and the preferred size of the
sequences has received ample description and discussion in the
literature. Normally an oligonucleotide probe should have at least
about 14 nucleotides, usually at least about 18 nucleotides, and
the polynucleotide probes may be up to several kilobases. Various
labels may be employed, most commonly radionuclides, particularly
.sup.32P. However, other techniques may also be employed, such as
using biotin modified nucleotides for introduction into a
polynucleotide. The biotin then serves as the site for binding to
avidin or antibodies, which may be labeled with a wide variety of
labels, such as radionuclides, fluorescers, enzymes, or the
like.
[0152] Alternatively, antibodies may be employed which can
recognize specific duplexes, including DNA duplexes, RNA duplexes,
DNA-RNA hybrid duplexes, or DNA-protein duplexes. The antibodies in
turn may be labeled and the assay carried out where the duplex is
bound to a surface, so that upon the formation of duplex on the
surface, the presence of antibody bound to the duplex can be
detected. The use of probes to the novel anti-sense RNA may be
carried out in any conventional techniques such as nucleic acid
hybridization, plus and minus screening, recombinational probing,
hybrid released translation (HRT), and hybrid arrested translation
(HART). This also includes amplification techniques such as
polymerase chain reaction (PCR).
[0153] Diagnostic kits which also test for the qualitative or
quantitative presence of other markers are also contemplated.
Diagnosis or prognosis may depend on the combination of multiple
indications used as markers. Thus, kits may test for combinations
of markers. See, e.g., Viallet, et al. (1989) Progress in Growth
Factor Res. 1: 89-97.
[0154] X. Isolating Langerin Specific Binding Partners
[0155] The description of the Langerin protein herein provides
means to identify Langerin ligands or binding agents, as described
above. Such a ligand or binding agent should bind specifically,
e.g., selectively, to the Langerin or fragment thereof with
reasonably high affinity. Typical ligand or binding agent binding
constants will be at least about 30 mM, e.g., generally at least
about 3 mM, more generally at least about 300 .mu.M, typically at
least about 30 .mu.M, 3 .mu.M, 300 nM, 30 nM, etc. Various
constructs are made available which allow either labeling of
Langerin to detect its ligand or binding agent. For example,
directly labeling Langerin, fusing onto it markers for secondary
labeling, e.g., FLAG or other epitope tags, etc., will allow
detection of a binding agent or ligand. This can be histological,
as an affinity method for biochemical purification, or labeling or
selection in an expression cloning approach. A two-hybrid selection
system may also be applied making appropriate constructs with the
available Langerin sequences. See, e.g., Fields and Song (1989)
Nature 340: 245-246.
[0156] The Langerin protein should interact with a ligand based,
e.g., upon its similarity in structure and function to other cell
markers exhibiting developmental and cell type specificity of
expression. Methods to isolate a ligand are made available by the
ability to make purified Langerin for screening programs. Soluble
or other constructs using the Langerin sequences provided herein
will allow for screening or isolation of Langerin specific
ligands.
[0157] Generally, descriptions of Langerins will be analogously
applicable to individual specific embodiments directed to Langerin
and/or Langerin reagents and compositions.
[0158] Without further elaboration, it is believed that a person of
ordinary skill in the art can, using the preceding description,
utilize the present invention to its fullest extent. The following
examples are put forth solely for the purpose of illustration as to
make and use the present invention, and are not intended, nor
should they be construed, to limit the scope of what the inventors
regard as their invention. Unless indicated otherwise below, parts
are parts by weight, molecular weight is weight average molecular
weight, temperature is in degrees Centigrade, and pressure is at or
near atmospheric.
EXAMPLES
[0159] General Methods
[0160] Some of the standard methods are described or referenced,
e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory
Manual Cold Spring Harbor Laboratory, Cold Spring Harbor Press;
Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed.), vols 1-3, CSH Press, NY; Ausubel, et al., Biology, Greene
Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987 and
Supplements) Current Protocols in Molecular Biology Greene/Wiley,
New York; Innis, et al. (eds. 1990) PCR Protocols: A Guide to
Methods and Applications Academic Press, N.Y.; all of which are
each incorporated herein by reference. Methods for protein
purification include such methods as ammonium sulfate
precipitation, column chromatography, electrophoresis,
centrifugation, crystallization, and others. See, e.g., Ausubel, et
al. (1987 and periodic supplements); Deutscher (1990) "Guide to
Protein Purification," Methods in Enzymology vol. 182, and other
volumes in this series; Coligan, et al. (1995 and supplements)
Current Protocols in Protein Science John Wiley and Sons, New York,
N.Y.; Matsudaira (ed. 1993) A Practical Guide to Protein and
Peptide Purification for Microsequencing, Academic Press, San
Diego, Calif.; and manufacturer's literature on use of protein
purification products, e.g., Pharmacia, Piscataway, N.J., or
Bio-Rad, Richmond, Calif. Combination with recombinant techniques
allow fusion to appropriate segments, e.g., to a FLAG sequence or
an equivalent which can be fused via a protease-removable sequence.
See, e.g., Hochuli (1989) Chemische Industrie 12: 69-70; Hochuli
(1990) "Purification of Recombinant Proteins with Metal Chelate
Absorbent" in Setlow (ed.) Genetic Engineering, Principle and
Methods 12: 87-98, Plenum Press, N.Y.; and Crowe, et al. (1992)
QIAexpress: The High Level Expression & Protein Purification
System QUIAGEN, Inc., Chatsworth, Calif.: which are incorporated
herein by reference.
[0161] FACS analyses are described in Melamed, et al. (1990) Flow
Cytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro
(1988) Practical Flow Cytometry Liss, New York, N.Y.; and Robinson,
et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New
York, N.Y.
Example 1
Dendritic Cell Clones
[0162] Collection and Purification of Cord Blood CD34.sup.+ HPC
[0163] Umbilical cord blood samples were obtained according to
standard institutional guidelines for human samples. Cells bearing
CD34 antigen were isolated from mononuclear fractions by positive
selection with Minimacs separation columns (Miltenyi Biotec,
Bergish Gladbach, Germany), using an anti-CD34 mAb (Immu-133.3,
Immunotech, Marseille, France) and goat anti-mouse IgG-coated
microbeads (Miltenyi Biotec). In all experiments, isolated cells
were 80-99% CD34.sup.+ as judged by staining with an anti-CD34
mAb.
[0164] Hematopoietic Factors
[0165] Recombinant human (rh) GM-CSF (specific activity:
2.times.10.sup.6 U/mg; Schering-Plough Research Institute,
Kenilworth, N.J.) was used at 100 ng/ml (200 U/ml); rhTNF-.alpha.
(specific activity: 2.times.10.sup.7 U/mg; Genzyme, Boston, Mass.)
was used at 2.5 ng/ml (50 U/ml); rhSCF (specific activity:
4.times.10.sup.5 U/mg; R&D, Abington, UK) was used at 25 ng/ml;
rhIL-4 (specific activity: 107 U/mg; Schering-Plough Research
Institute) was used at 5 ng/ml; and rhTGF-.beta.1 (R&D) was
used at 1 ng/ml.
[0166] Dendritic Cell Generation from CD34.sup.+ HPC
[0167] Cultures of CD34.sup.+ HPC were established as described in
Caux, et al. (1992) Nature 392: 258-261, in the presence of GM-CSF,
SCF, and TNF-.alpha., in endotoxin-free medium consisting of RPMI
1640 (Gibco BRL, Gaithersburg, Md.) supplemented with 10%
heat-inactivated fetal bovine serum (FBS; Flow Laboratories,
Irvine, UK), 10 mM Hepes, 2 mM L-glutamine, 5.times.10.sup.-5 M
2-mercaptoethanol and gentamicin (80 .mu.g/ml) (referred to as
complete medium).
[0168] CD34.sup.+ cells were seeded in 25 to 75 cm.sup.2 culture
flasks (Corning, N.Y.; NY) at 2.times.10.sup.4 cells/ml. Optimal
conditions were maintained by splitting cultures at day 4 with
medium containing fresh GM-CSF and TNF-.alpha.. In some
experiments, TNF-.alpha. was replaced at day 7 by TGF-.beta.. In
other experiments, DC were activated at day 9 or day 12 with murine
Ltk.sup.- fibroblastic L cells stably transfected with the human
CD40 ligand (L) gene (cell line established by Dr. C. van Kooten
(van Kooten, et al. (1994) Eur J Immunol 24: 787-92). Briefly,
10.sup.5 irradiated CD40L L cells (7,500 rads) were seeded together
with 5 10.sup.5 CD34-derived DC in presence of GM-CSF.
[0169] Isolation of Epidermal Cells
[0170] Epidermal cell suspensions were obtained from normal skin of
patients undergoing reconstructive plastic surgery of the breast.
Skin was split-cut with a keratome set and the dermo-epidermal
slices treated for 18 h at 4.degree. C. with 0.05% trypsin (Sigma)
in Hank's balanced salt solution without Ca.sup.2+ and Mg.sup.2+
(Seromed, Biochrom KG, Berlin, FRG). The epidermis was detached
from the dermis with fine forceps. Epidermal sheet cell suspensions
were obtained by subsequent tissue dislocation and filtration
through sterile gaze. Enrichment of Langerhans cells was obtained
by density gradient centrifugation on Lymphoprep (Nycomed Pharma,
Oslo, Norway).
Example 2
Antibodies and Flow Cytometry
[0171] For single cell staining, cells were labeled using the
following mAbs: anti-E-cadherin (SHE 79.7; Takara, Shiga, Japan),
anti-MHC class II (HLA-DR) (Becton Dickinson), and anti-Lag, all
revealed by FITC-conjugated goat anti-mouse immunoglobulin (Dako,
Glostrup, Denmark). For double staining, cells were labeled with
mAb DCGM4 revealed by phycoerythrin(PE)-conjugated goat anti-mouse
immunoglobulin (Dako), and after saturation in 5% mouse serum, with
FITC-conjugated anti-CD1a (Ortho, Raritan, N.J.). Negative controls
were performed with unrelated murine mAbs. Fluorescence was
determined with a FACSCAN flow-cytometer (Becton Dickinson). For
intracytoplasmic phenotyping, cells were stained in PBS, 0.3%
Saponin (Sigma) and 5% BSA, using the same procedure.
Example 3
Biochemistry
[0172] Proteins were extracted from CD34-derived DC supplemented
with TGF-.beta. by addition, to a frozen pellet of 100
.mu.l/10.sup.7 cells, of 50 mM Tris-HCL pH 8 buffer with 150 mM
NaCl, 5 mM EDTA, 1% Triton X100 and protease inhibitor (complete
Mini, Boeringer Mannheim). After 1 h at 4.degree. C., samples were
centrifuged to remove cellular debris. Supernatants were then
incubated 1 h at 4.degree. C. with DCGM4 covalently linked to
Dynabeads M-450 Sheep anti-mouse magnetic beads (Dynal, Oslo,
Norway). Beads were washed with extraction buffer by using a Dynal
magnetic particle-concentrator and boiled in the presence of 50
.mu.l SDS-PAGE sample buffer or resuspended in 100 .mu.l of 0.5 M
Glycine, 0.15 M NaCl pH 2.3 for 4 minutes. Then, supernatant was
neutralized with 3.5 .mu.l of saturated Tris solution. SDS-PAGE
analysis was performed with a PhastSystem in a 10-15% gradient gel
(Pharmacia Biotech), and gels were stained with Coomassie R250. 2-D
analysis was performed on a Multiphor II flat bed system with
Immobiline DryStrip pH 3-10 and Excelgel SDS 8-18% for the second
dimension (Pharmacia Biotech), and gels were silver stained.
Dialyzed samples of proteins eluted from IgG linked to Dynabeads
were digested with N-glycosidase F (Boehringer-Mannheim) at
37.degree. C. overnight. Five microliters of original samples or 10
microliters of digested samples were deposited on nitrocellulose,
and treated with DIG glycan detection kit
(Boehringer-Mannheim).
Example 4
Purification of Langerin
[0173] DCGM4 expressing cells are used for large scale protein
extraction. The protein is gently solubilized, and the resulting
Langerin is purified using standard methods of protein
purification. Chromatographic methods are used, and immunoaffinity
techniques can be applied. The Langerin protein is followed by SDS
PAGE and/or immunoassays. Diagnostic methods are used to ensure
that the protein is substantially pure.
[0174] Purified protein is used for protein microsequencing. See,
e.g., Matsudaira (ed. 1993) A Practical Guide to Protein and
Peptide Purification for Microsequencing, Academic Press, San
Diego, Calif. Sequence data is used to search sequence databases,
e.g., GENBANK, to find natural genes encoding the Langerin.
Alternatively, the sequence data is useful for isolating a nucleic
acid encoding Langerin using, e.g., degenerate PCR primers,
etc.
[0175] Protein will also be used to raise additional antibodies.
Such antibodies may be polyclonal or monoclonal. The protein can be
used to assay and determine titers and affinity. Standard methods
of immunization are available, as described above.
Example 5
Internalization Assay
[0176] CD34-derived DC supplemented with TGF-.beta. were generated
as detailed above and internalization was performed as described
(Cella, et al. (1997) J. Exp. Med. 185: 1743-51). One aliquot of
cells was fixed with RPMI, 0.1% glutaraldehyde for 5 min. at room
temperature and another aliquot was used without fixation. Both
samples were stained with mAb DCGM4 or mAb DCGM1 for 40 min. on
ice, and incubated with biotin-labeled F(ab').sub.2 goat anti-mouse
IgG (Jackson ImmunoResearch Laboratories, PA) for 1 h on ice. Cells
were then placed in a 37.degree. C. water-bath for various time
periods, cooled on ice and stained with PE-conjugated streptavidin
(Becton Dickinson). After washing, cells were analyzed by FACS. The
measure of internalization is given by the percentage decrease of
cell-surface median fluorescence intensity (MFI) as compared to
control samples kept at 4.degree. C. The percentage decrease of MFI
observed in fixed cells was taken as measure of the off-rate of the
antibody at 37.degree. C. Linear regression analysis of the plots
of log.sub.10 (percentage of median fluorescence) vs. time was
performed and rates of ingestion (k) and half-life (t.sub.1/2) of
membrane-bound complexes were calculated from the slope (m) of the
resulting straight line using the relationships k(%/min)=-2,303
m.times.100, and t.sub.1/2 (min)=log2/m (Leslie, E J I 1980). In
this mAb DCGM1 which recognizes the macrophage mannose receptor was
generated by Applicant, and used as positive control for
receptor-mediated endocytosis.
Example 6
Generation and Characterization of DCGM4 Monoclonal Antibodies
[0177] BALB/c mice (Iffa Credo, Les Oncins, France) were immunized
with three intraperitoneal injections of CD34-derived DC (10.sup.6
cells) with Freund's adjuvant (Sigma Chemical Co., St. Louis, Mo.).
Three days after the final injection, splenocytes were fused with
the murine myeloma cell line SP2, using polyethylene glycol-1000
(Sigma). Hybrid cells were placed in 96-well Falcon tissue culture
plates (Falcon, Lincoln Park, N.J.) and fed with DMEM F12 (Life
Technologies, Gaithersburg, Md.) supplemented with streptomycin
(100 .mu.g/ml), penicillin (100 U/ml), glutamine (2 mM), 10% horse
serum (Life Technologies), 1% culture medium additive (CRTS, Lyon,
France), 10.sup.-5 M azaserine (Sigma), and 5.times.10.sup.-5 M
hypoxanthine. Supernatants were screened for reactivity with
CD34-derived DC and three unrelated cell types, namely peripheral
blood polynuclear cells, T lymphocytes activated with PHA, and the
myeloid cell line KG1 (ATCC; Rockville, Md.). Selected hybridomas
were cloned by limiting dilution and ascites were produced in
BALB/c mice. MAb DCGM4 was purified by anion-exchange
chromatography on DEAE A50 (Pharmacia Biotech, Uppsala, Sweden) and
coupled with fluorescein and biotin using standard procedures. Ig
isotype was determined by ELISA using a mouse hybridoma subtyping
kit (Boehringer-Mannheim, Mannheim, Germany).
Example 7
Immunohistology
[0178] Microscope slides of acetone-fixed cryocut tissue sections
or cell cytospin preparations were incubated with mAbs for 60 min.,
and subsequently with biotinylated sheep anti-mouse Ig (The Binding
Site, Birmingham, UK) for 30 min. Following incubation with
streptavidin coupled to alkaline phosphatase (Biosource, Calif.,
USA) for 30 min., enzyme activity was developed using Fast Red
substrate (Dako). Double staining, with mouse IgG1 antibody DCGM4
and IgG2b anti-CD1a (Immunotech) were revealed by sheep anti-mouse
IgG1 (The Binding Site) followed by mouse anti-alkaline
phosphatase-alkaline phosphatase complexes (Dako) (APAAP
technique), and biotinylated sheep anti-mouse IgG2b (The Binding
Site) followed by ExtrAvidin-peroxydase (Sigma). The binding of
goat anti-sIgD-biotin and DCGM4-biotin (for double staining with
Lag) were directly revealed by ExtrAvidin-peroxidase. Alkaline
phosphatase activity and peroxidase activity were respectively
demonstrated using Fast Blue substrate (Sigma) and
3-amino-ethylcarbazole (Sigma).
Example 8
Electron Microscopy
[0179] Langerhans cell-enriched epidermal cell suspensions were
incubated with control mouse IgG1 (Sigma), anti-CD1a (DMC1 mAb;
REF), or DCGM4 for 1 h at 4.degree. C. After washing, cells were
incubated with a goat anti-mouse IgG conjugated with colloidal gold
particles of 5 nM (GAM-nM) (Amersham, address, France) for 30 min.
at 4.degree. C. Cells were either fixed immediately for 18 hours
with 2% glutaraldehyde in cacodylate buffer, followed by washing
for at least 24 h in cacodylate buffer with sucrose or warmed up to
37.degree. C. or room temperature before fixation. Samples were
post-fixed for 1 hour with 1% osmium in cacodylate buffer with
sucrose, dehydrated, and embedded in Epoxy resin. Ultrathin
sections were post-stained with uranyl acetate and lead citrate,
and examined on a JEOL 1200 EX electron microscope (CMEABG,
Universit de Lyon, Lyon, France). A quantitative evaluation of
cell-surface antigen density was performed according to Lafferty,
et al. (1981) J. Histochem Cytochem 29: 49-56. The number of gold
granules bound along the cell membrane was counted and total
circumference of the cell was measured on micrographs with a
minimop morphometric analyzer (Zeiss). Results were expressed as
number of gold granules per 100 .mu.m of cell membrane. For each
group of cells, counts were obtained from at least 20-30 cell
sections. Mean and standard deviation were subsequently
determined.
Example 9
Confocal Microscopy
[0180] Intracellular immunofluorescence staining was performed as
previously described by Winzler, et al. (1997) J. Exp. Med. 185:
317-28. Cells on polylysine coated coverslips and fixed for 15 min.
with 4% paraformaldehyde, were washed in 10 mM glycine, then
permeabilized with 0.5% saponin, 0.2% BSA for 30 min. Coverslips
were incubated for 30 min. at room temperature with anti-LAMP-1
(Pharmingen, San Diego, Calif.), anti-HLA-DR (Becton Dickinson) or
anti-Lag (Kashishara, et al. J Invest Dermatol 87: 602-607) at a
final concentration of 5 .mu.g/ml in permeabilization medium. After
three washes, cells were incubated for 30 min. with secondary
labeled antibody (donkey anti-mouse coupled to Texas-red (Vector
Laboratories, Burlingame, Calif.), washed, and incubated with mouse
preimmune serum for 30 min., washed again, post-fixed with 2%
paraformaldehyde, and finally incubated for 30 min. with mAb DCGM4
coupled to fluorescein (FITC). After washing, coverslips were
mounted onto glass slides with fluoromount (Southern Biotechnology
Associates Inc., Birmingham, Ala.). Confocal microscopy was
performed using Confocal Laser Scanning Microscope TCS 4D (Leica
Lasertechnik GmbH, Heidelberg, Germany) interfaced with an
argon/krypton ion laser and with fluorescence filters and detectors
allowing to simultaneously record FITC and Texas-red markers
Rovere, et al. (1998) Proc. Nat. Acad. Sci. USA 95: 1067-1072.
Example 10
Selection of Monoclonal Antibody DCGM4 Reactive Against
Langerin
[0181] Supernatants from 854 hybridomas were screened for
reactivity on DC obtained from CD34.sup.+ HPC cultured 12 days in
GM-CSF and TNF-.alpha.. In parallel, the supernatants were assayed
for reactivity on three unrelated cell types, namely peripheral
blood polynuclear cells, T lymphocytes activated with PHA and the
myeloid cell line KG1. Supernatant from one hybridoma, designated
DCGM4 was found to react only with a minor subset of DC and not
with the other cell types of the differential screening.
[0182] The hybridoma was cloned by limiting dilution. The antibody
was produced in ascites and subsequently purified using DEAE
chromatography. MAb DCGM4 was found to be of IgG1/.kappa. isotype
as determined by ELISA. As the observed reactivity restricted to a
DC subset appeared of particular interest, mAb DCGM4 was selected
for further studies. Finally, early experiments indicating that LC
stained positive, we termed Langerin the antigen recognized by mAb
DCGM4.
Example 11
Langerin is Selectively Expressed on Langerhans-Type Immature
Dendritic Cells
[0183] CD34.sup.+ HPC cultured with a combination of GM-CSF and
TNF-.alpha. for 12 days differentiate into CD1a.sup.+ DC (Caux, et
al. (1996) J Exp Med 184: 695-706). The expression of Langerin
during such cultures was investigated. Langerin is expressed by a
subset of CD1a.sup.+ dendritic cells derived from cord blood
CD34.sup.+ HPC. Kinetics of CD1a and Langerin expression during
culture of CD34.sup.+ HPC in GM-CSF plus TNF-.alpha. was determined
at various time points, cells were recovered and double-labeled
using anti-CD1a-FITC and DCGM4 plus anti-mouse IgG-PE. The results
are representative of 5 experiments.
[0184] No staining was detected at day 0 or day 6, indicating that
CD34.sup.+ HPC and their immediate progeny do not express Langerin.
The antigen appeared at day 7, on a small subset of the CD1a.sup.+
cells. Between day 7 to day 12, Langerin expression reached a
maximum, staining between 15 to 35% of CD1a.sup.+ cells. The
dendritic nature of the Langerin-expressing cells in the cultures
was confirmed on cytospin preparations of DCGM4.sup.+ FACS-sorted
cells.
[0185] Caux, et al. (1996) J Exp Med 184: 695-706 have further
shown that CD34.sup.+ HPC differentiate along two independent
pathways from distinct precursor subsets, identified by mutually
exclusive expression of CD1a and CD14 at early time points during
the culture (day 5-7). When such precursors were separated by
FACS-sorting at day 6 and cultured with GM-CSF and TNF-.alpha. for
6 more days, Langerin was found mostly expressed on the
CD1a-derived DC (40%) as compared to the CD14-derived DC (16%). The
CD1a-derived DC have been shown to display features that are
associated with Langerhans cells, including the presence of Birbeck
granules.
[0186] Next, the in situ distribution of Langerin was examined by
immunohistological analysis of various human tissues. Langerin is
selectively expressed by LC-like DC. Immunohistological analysis of
Langerin expression was made on sections of skin, tonsil and lung.
Within the epidermis, Langerin was only found on LC, which also
stained with anti-Lag and anti-CD1a antibodies. In skin, staining
with mAb DCGM4, or mAb DCGM4 plus anti-Lag or anti-CD1a, showed
positive Langerin expression by LC. In tonsil, the
Langerin-positive cells were found in the epithelium (e.g., on
follicular mantle B cells). A few cells were occasionally stained
in the T cell areas, but never observed in germinal centers
Langerin.sup.+ cells were also notably present in lung epithelium.
In lung, mAb DCGM4 and counterstaining with hematoxylin showed
LC-like Langerin.sup.+ cells only in bronchiolar epithelium. The
Langerin.sup.+ DC showed dendritic morphology. No staining was
detected with control mAbs. These results were representative of 5
experiments.
[0187] By contrast to CD34-derived DC, mAb DCGM4 did not react with
DC obtained from peripheral blood monocytes cultured 6 days with a
combination of GM-CSF and IL-4, thus, further confirming the
restriction of Langerin expression. Likewise, Langerin was neither
detected in ex-vivo purified DC isolated from peripheral blood, nor
in germinal center DC isolated from tonsils.
[0188] Finally, mAb DCGM4 was analyzed for reactivity on a panel of
different hematopoietic-derived cell types. Langerin was neither
detected in ex-vivo isolated T lymphocytes, B lymphocytes,
monocytes or granulocytes, nor in myeloid (HL60, KG1, U937, THP1)
or lymphoid (Jurkat, JY, PREALP) cell lines.
[0189] Taken together, the above data suggest that Langerin
expression is restricted to an immature DC compartment, and that it
is subsequently lost upon DC maturation.
Example 12
Langerin Expression is Upregulated by TGF-.beta. and Decreased
Following CD40 Activation
[0190] Since mAb DCGM4 was found to react selectively with LC-like
immature DC, it was investigated whether factors that influence DC
maturation would affect the expression levels of Langerin.
[0191] Studies in vitro and in vivo have shown that TGF-.beta.
plays an essential role in LC development. Therefore, the effect of
TGF-.beta. on Langerin expression by in vitro derived DC was
evaluated.
[0192] To do so, cord blood CD34.sup.+ HPC were cultured for 12
days in GM-CSF and TNF-.alpha. in absence or presence of TGF-.beta.
from day 7 to day 12. Subsequently, the DC were cultured with
L-cells transfected with CD40L for 2 days. Cells were processed for
staining without or after pretreatment with 0.1% saponin, using
mAbs revealed by FITC-conjugated anti-mouse Ig. The results are
representative of more than 5 experiments.
[0193] CD34-derived DC were supplemented with TGF-.beta. for the
last three days of culture (day 9-12). This resulted in strong
upregulation of Langerin expression. In addition to increasing the
proportion of Langerin.sup.+ cells, TGF-.beta. raised the mean
number of surface-membrane molecules per cell (93.times.10.sup.3
instead of 33.times.10.sup.3 without TGF-.beta.). The effect of
TGF-.beta. was predominantly exerted on the CD14-derived DC subset,
normally devoid of Langerhans markers such as the Birbeck granule
associated antigen Lag. Langerin expression is not restricted to
the plasma membrane, but is also detected intracellulary following
membrane permeabilization. Levels of intracellular Langerin were
also markedly enhanced by TGF-.beta.. It was also found that
although TGF-.beta. also increased the expression of the LC markers
Lag and E-cadherin, Lag was never detected at the cell-surface.
[0194] Removal of TNF-.alpha., a cytokine known to induce DC
maturation, for the last three days of culture, upregulated
Langerin expression whether in the presence or absence of
TGF-.beta.. In line with this result, a strong decrease in Langerin
surface-membrane expression was found associated with an increase
of HLA-DR following activation with CD40L, a signal which triggers
the maturation of DC including upregulation of costimulatory
molecules. Altogether, these results confirm that TGF-.beta. which
induces an LC phenotype, upregulates Langerin expression, whereas
signals that trigger DC maturation decrease Langerin
expression.
Example 13
Intracellular Langerin and Lag Co-Localize in Immature DC, but
Dissociate Upon Activation
[0195] A unique feature of Langerhans cells is the presence of
intracytoplasmic Birbeck granules (BG). As Langerin was found to be
selectively expressed in Langerhans-type DC, its relationship with
Lag antigen was further examined. Thus, CD34-derived DC
supplemented with TGF-.beta., and which contain a high proportion
of BG.sup.+ cells as detected by electron microscopy, were analyzed
by double fluorescence staining and confocal microscopy.
CD34-derived DC were supplemented with TGF-.beta. from day 7 to day
9 of culture. Double-color confocal laser scanning microscopy was
performed at day 9 and after two subsequent days of CD40L
activation. Langerin-positive and Lag-positive vesicles were found
co-localized in day 9 immature DC. After CD40L activation,
Langerin.sup.+Lag.sup.- staining was observed near the nucleus. No
co-localization was detected between Langerin and HLA-DR or LAMP-1,
irrespective of CD40L activation. Langerin and Lag were found to
display a remarkable intracellular co-localization. Strikingly
however, localization of the two markers segregated upon activation
with CD40L. Thus, Langerin was found in the absence of Lag in close
proximity to the nucleus, whereas a co-localized expression of the
two markers only remained immediately beneath the surface
membrane.
[0196] These results indicate that Langerin is distinct from Lag,
and that Langerin is routed towards a deep cellular compartment
during DC activation. Finally, Langerin was never found to
co-localize with the lysosomal marker LAMP-1, nor with HLA-DR,
irrespective of activation by CD40 crosslinking.
Example 14
Langerin is Associated with Endocytic Structures and Birbeck
Granules
[0197] Since Langerin is expressed at the cell surface (as detected
by FACS in the absence of membrane permeabilization) electron
microscopy was performed on epidermal cell suspensions to analyze
its precise distribution. Langerhans cells are easily recognizable
in such suspensions, by their folded nuclei, lack of keratin
filaments, lack of desmosomes and melanosomes, and the presence of
characteristic BG.
[0198] An epidermal cell suspension was obtained as described
above. mAbs were revealed by 5 nm gold-labeled goat anti-mouse
IgG1. CD1a is homogeneously distributed at the cell surface,
whereas Langerin is often associated with areas of membrane
thickening. Cytomembrane sandwiching structures and coated pits
were visualized upon staining with DCGM4 at 4.degree. C. Upon
incubation at 37.degree. C., cytoplasmic gold particle-containing
coated vesicles and Birbeck granules were seen. DCGM4 staining and
gold particles were revealed on the luminal side of BG. These
results were representative of 3 experiments on different skin
samples.
[0199] Staining at 4.degree. C. with DCGM4 and 5 nm gold particles
confirmed that Langerin is clearly associated with the cell surface
in LC, although at a lower density than CD1a (161.5.+-.97.1 versus
1589.1.+-.418.8 gold granules/100 .mu.m membrane). In addition to
single gold particles, a spontaneous clustering was observed with
DCGM4, even though the antibody was ultracentrifuged. An
isotype-matched control mouse IgG1 did not bind to the LC
(1.3.+-.3.4 granules/100 .mu.m). No labeling of DCGM4 was observed
on the keratinocytes or melanocytes present in the epidermal cell
suspensions.
[0200] As compared to the rather homogeneous distribution of CD1a,
Langerin was not randomly distributed at the cell surface, but was
often associated with particular areas of membrane thickening.
Furthermore, DCGM4 induced typical endocytic coated pits at the
cell membrane at 4.degree. C. The number of coated pits was
significantly enhanced (an average 3.6 times) by DCGM4, as compared
to staining with anti-CD1a or control mouse IgG1. Notably, the
coated pits induced during DCGM4 staining contained gold particles.
Furthermore, when cells were allowed to warm up before fixation,
DCGM4 staining was observed inside coated vesicles already after 2
min. at 37.degree. C. These data demonstrate that Langerin is
associated with structures characteristic of early steps of
receptor-mediated endocytosis.
[0201] Staining with DCGM4 at 4.degree. C. also resulted in the
formation of cytomembrane sandwiching structures at the cell
surface. Consistently, following DCGM4 at 4.degree. C., gold
labeled BG were seen in continuity with the cell membrane, and
found inside the cytoplasm when cells were warmed up to 37.degree.
C. for 2 min. The BG were labeled in their central striated lamella
or in their bulb.
[0202] Taken together, these results indicate that Langerin is
associated with endocytic structures and can also gain access to
Birbeck granules from the cell membrane.
Example 15
Langerin Mediates Rapid Internalization in DC
[0203] To further examine the role of Langerin in endocytosis, we
analyzed its capacity to internalize DCGM4 as a ligand.
[0204] CD34-derived DC supplemented with TGF-.beta. were labeled at
4.degree. C. with mAbs and subsequent F(ab').sub.2 biotinylated
secondary antibody. Cells were incubated at 37.degree. C. for time
periods indicated, and internalization measured as decreased
cell-surface-bound antibody determined by FACS analysis using
PE-conjugated streptavidin. MAb DCGM4 is rapidly internalized at
37.degree. C., with similar kinetics as an anti-mannose-receptor
mAb (positive control). In fixed cells, no decrease of cell-surface
fluorescence was detected. Results were analyzed as the percentage
decrease of mean fluorescence intensity (MFI), as compared to
control samples kept at 4.degree. C.
[0205] It was found that the antibody was very rapidly internalized
by DC at 37.degree. C., but not at 4.degree. C. Approximately 75%
of surface-membrane bound DCGM4 was internalized already within one
minute at 37.degree. C., with similar kinetics to that of
anti-mannose-receptor mAb DCGM1, used as positive control for
receptor-mediated endocytosis. In a representative experiment, the
half-life (t.sub.1/2) of DCGM4 at the cytomembrane was calculated
to be 4.5 minutes at 37.degree. C., with an internalization rate of
k=15.3%/min. The rapid disappearance of mAb DCGM4 from the
cell-surface was not due to antibody dissociation, as no decrease
in fluorescence was observed in glutaraldehyde-fixed DC incubated
at 37.degree. C. Finally, Langerin did not display a mannose-type
receptor specificity, as DCGM4 failed to inhibit uptake of
Dextran-FITC at 37.degree. C. and binding of the antibody was not
inhibited by mannan at 4.degree. C.
[0206] These results are in accordance with the above electron
microscopy analysis and demonstrate that Langerin is implicated in
a rapid endocytosis process by DC.
Example 16
Triggering of Cell-Surface Langerin Results in Birbeck Granule
Formation
[0207] As triggering of cell surface Langerin resulted in the
formation of CMS and the detection of gold-labeled BG as visualized
by electron microscopy, the potential role of Langerin cell surface
in BG formation was further investigated. An epidermal cell
suspension was obtained as described above. Cells were incubated
with an excess of mAb DCGM4 or anti-CD1a at 4.degree. C., and
processed immediately for electron microscopy, or left at room
temperature for 5 min. before fixation. LC incubated with DCGM4
displayed a striking accumulation of Birbeck granules in the
perinuclear region. The effect is predominantly observed at
4.degree. C., as subsequent warming up results in vacuolization.
Treatment with anti-CD1a mAb failed to induce significant changes
in the LC cytoplasm, whether in cells kept at 4.degree. C. or
brought up to room temperature.
[0208] This treatment resulted in a considerable increase of
densely packed BG in the LC cytoplasm, with a marked accumulation
in the perinuclear region around the Golgi. The BG displayed an
elongated, round, or irregularly-shaped expanded portion, in
addition to their typical rod-shaped part. Moreover, the cytoplasm
of DCGM4-treated LC was filled with numerous rounded or elongated
vesicles.
[0209] When LC were warmed up to room temperature (5 min.)
following incubation with excess DCGM4, increased fusion was
observed between single and short rod-shaped BG and large vesicular
components. Moreover, numerous vesicles of various sizes and shapes
occupied a considerable volume of the LC cytoplasm.
[0210] In contrast to DCGM4, incubation of epidermal cell
suspensions with an excess anti-CD1a mAb only led to the formation
of some small vesicles but no other significant changes in the LC
cytoplasm. Notably, no accumulation of perinuclear BG was observed,
even when cells were allowed to warm up before fixation. Similarly,
incubation with a control mouse IgG1 or with anti-E-cadherin mAb
did not modify the BG granules.
[0211] Taken together, these data demonstrate that cell-surface
Langerin actively participates in BG formation, resulting in their
perinuclear accumulation.
Example 17
Langerin is a 40 kDa N-glycosylated Protein
[0212] Immunoprecipitation with DCGM4 from DC extracts and,
subsequent elution with SDS-PAGE sample buffer yielded a
homogeneous band of 40-42 kDa molecular mass. SDS-PAGE analysis of
immunopurified Langerin in non-reducing and reducing conditions was
carried out. If DTT was omitted all along the purification steps,
the profile was not modified on the gel, suggesting that Langerin
is present at the cell membrane as a single chain or as an
homodimer with non covalent association.
[0213] 2-D analysis of immunopurified Langerin was conducted
establishing the molecular mass of the molecule and indicating that
Langerin had a pI of 5.2-5.5. Finally, a dot-blot analysis of
Langerin using (1) creatinase from E. coli as non-glycosylated
control, (2) transferrin as positive N-glycosylated control, (3)
N-glycosidase, (4) Langerin, and (5) N-glycosidase-treated
Langerin, demonstrated that Langerin is a glycoprotein, and that
most of the carbohydrate constituents were removed by N-glycosylase
treatment.
Example 18
Isolating a Nucleic Acid Encoding a Langerin
[0214] Numerous methods are available to isolate a gene encoding a
purified protein, especially where antibodies which recognize the
protein exist. One method is to determine methods for purification
of the protein and subsequently to determine the peptide sequences.
Given sufficient sequence information, and using redundant
oligonucleotides, PCR or hybridization techniques will allow for
isolation of genes encoding Langerin proteins.
[0215] Another alternative is to generate additional antibodies to
Langerin proteins, which may be isolated by immunoaffinity methods
using the DCGM4 antibodies. See above. These antibodies are
applicable in "panning" techniques, such as described by Seed and
Aruffo (1987) Proc. Nat'l Acad. Sci. USA 84: 3365-3369. Phage
expression techniques are also applicable to screen cDNA libraries
derived from appropriate DC or T cell subpopulations enriched for
Langerin expression. Glycosylation interference with antibody
recognition will be generally less problematic in the phage
selection systems. Cell sorting techniques on a mammalian
expression library are applicable also.
[0216] Another method for screening an expression library is to use
antibody to screen successive subpopulations of libraries. The
following provides one method of screening using small populations
of cells on slides stained by a specific labeling composition,
e.g., an antibody.
[0217] For example, on day 0, precoat 2-chamber permanox slides
with 1 ml per chamber of fibronectin, 10 ng/ml in PBS, for 30 min.
at room temperature. Rinse once with PBS. Then plate COS cells at
2-3.times.10.sup.5 cells per chamber in 1.5 ml of growth media.
Incubate overnight at 37.degree. C.
[0218] On day 1 for each sample, prepare 0.5 ml of a solution of 66
.mu.g/ml DEAE-dextran, 66 .mu.M chloroquine, and 4 .mu.g DNA in
serum free DME. For each set, a positive control is prepared, e.g.,
of human IL-10-FLAG cDNA construct at 1 and 1/200 dilution, and a
negative mock. Rinse cells with serum free DME. Add the DNA
solution and incubate 5 h at 37.degree. C. Remove the medium and
add 0.5 ml 10% DMSO in DME for 2.5 min. Remove and wash once with
DME. Add 1.5 ml growth medium and incubate overnight.
[0219] On day 2, change the medium. On days 3 or 4, the cells are
fixed and stained. Rinse the cells twice with Hank's Buffered
Saline Solution (HBSS) and fix in 4% paraformaldehyde (PFA)/glucose
for 5 min. Wash 3.times. with HBSS. The slides may be stored at
-80.degree. C. after all liquid is removed. For each chamber, 0.5
ml incubations are performed as follows. Add HBSS/saponin (0.1%)
with 32 .mu.l/ml of 1M NaN.sub.3 for 20 min. Cells are then washed
with HBSS/saponin 1.times.. Soluble antibody, e.g., DCGM4, is added
to cells and incubate for 30 min. Wash cells twice with
HBSS/saponin. Add second antibody, e.g., Vector anti-mouse
antibody, at 1/200 dilution, and incubate for 30 min. Prepare ELISA
solution, e.g., Vector Elite ABC horseradish peroxidase solution,
and preincubate for 30 min. Use, e.g., 1 drop of solution A
(avidin) and 1 drop solution B (biotin) per 2.5 ml HBSS/saponin.
Wash cells twice with HBSS/saponin. Add ABC HRP solution and
incubate for 30 min. Wash cells twice with HBSS, second wash for 2
min., which closes cells. Then add Vector diaminobenzoic acid (DAB)
for 5 to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2
drops of H.sub.2O.sub.2 per 5 ml of glass distilled water.
Carefully remove chamber and rinse slide in water. Air dry for a
few minutes, then add 1 drop of Crystal Mount and a cover slip.
Bake for 5 min. at 85-90.degree. C.
[0220] Alternatively, the Langerin proteins are used to affinity
purify or sort out cells expressing the ligand. See, e.g.,
Sambrook, et al. (supra) or Ausubel, et al., (supra) both of which
are incorporated herein by reference.
Example 19
Human Genomic Langerin
[0221] The genomic sequence (SEQ ID NO: 3) and the exon-intron
organization of human Langerin has now been determined. The genomic
sequence includes the Langerin cDNA sequence (SEQ ID NO: 1) as 6
exons separated by 5 introns. The nucleotide sequences of exons 1-6
are provided in SEQ ID NOS 4-9 respectively. This 10,663 nucleotide
genomic sequence includes 3251 and 1808 nucleotides at the 5' and
3' ends of exons 1 and 6 respectively, and is predicted to include
the sequence of the human Langerin promoter.
[0222] Exon 1 (SEQ ID NO: 4) corresponds to positions 3252-3371 of
SEQ ID NO: 3. Exon 2 (SEQ ID NO: 5) corresponds to positions
3467-3583 of SEQ ID NO: 3. Exon 3 (SEQ ID NO: 6) corresponds to
positions 5051-5425 of SEQ ID NO: 3. Exon 4 (SEQ ID NO: 7)
corresponds to positions 6020-6171 of SEQ ID NO: 3. Exon 5 (SEQ ID
NO: 8) corresponds to positions 7252-7370 of SEQ ID NO: 3. Exon 6
(SEQ ID NO: 9) corresponds to positions 7871-8855 of SEQ ID NO:
3.
[0223] Human Langerin is a 328 amino acid transmembrane type II
Ca++ dependent lectin. Amino-acid translation of the 6 exons shows
that exon. 1 encodes the NH2-terminal portion of the intracellular
domain (24 amino acids), while both the membrane-proximal portion
of the intracellular domain (19 amino acids) and the entire
transmembrane domain (20 amino acids) are encoded by exon 2. Exon 3
encodes the entire membrane-proximal non-carbohydrate recognition
portion of the extracellular domain (125 amino acids). The
extracellular carbohydrate-recognition domain (CRD) is encoded
jointly by exons 4 (51 amino acids), 0.5 (39 amino acids), and the
beginning of exon 6 (50 amino acids) the latter terminating the
COOH-portion of the molecule. Notably, the CRD of Langerin shares
the three exon structure of other type II transmembrane lectins
including the Kuppfer cell receptors, rat hepatic lectin, and the
CD23 low-affinity IgE receptor, with the coding sequences
interrupted by introns at analogous positions.
[0224] The availability of the genomic Langerin sequence now allows
for the identification of human Langerin promoter elements,
expected to be situated within the 500-600 nucleotides 5' of the
ATG initiation codon at position 48 of exon 1. By analogy, in the
rat Kuppfer cell receptor, homologous to human Langerin, the
transcription initiation site is situated 51 bases upstream from
the ATG translation initiation codon. Two nucleotide sequences have
been identified 5' to the start of transcription, that are
candidates for regulation of gene expression. One is a 54-base
tandomly repeated sequence from nucleotides -151 to -98 and -97 to
-44, and the other is a heptanucleotide sequence (GAGGCAG) that is
repeated 4 times in 380 bases 5' to the start site (G. W. Hoyle and
R. L. Hill, J. Biol Chem, 1991, 266: 1850-1857).
[0225] Human Langerin is specifically expressed in Langerhans
cells, a subtype of immature dendritic cells specialized in capture
and processing of antigen. Thus, it will be feasible to construct
DNA sequences encoding antigens of choice for targeting selective
expression in Langerhans cells under control of the Langerin
promoter elements.
[0226] Human Langerin sequence enables the identification of the
mouse homolog Langerin genomic sequence. This information will be
key to the construction of mice deficient in the Langerin gene, to
further explore the function of Langerin and of the Birbeck
granules, the unique endocytic organelles of Langerhans cells that
are induced by engagement of Langerin.
[0227] The human Langerin sequence also enables the sequencing of
possible mutations in the Langerin gene in patients suffering from
immunological disorders of unknown etiology.
Example 20
Chromosomal Mapping of Human Langerin
[0228] Chromosomal localization was performed by radiation hybrid
(RH) mapping (D. R. Cox et al., Science, 1990, 250: 245-250), using
the Stanford G3 Radiation Hybrid panel (Research Genetics, Inc.,
Huntsville, Ala.). Briefly, two oligonucleotides (U863 and L1180)
used to amplify Langerin cDNA by PCR reaction were selected as they
also amplfied a stretch of human genomic DNA. PCR reactions were
carried out with U863/L1180 on the Stanford G3 panel of 83 clones
covering the human genome. PCR data were submitted to the Stanford
Human Genome Center RHserver (LIENHYPERTEXTE
mailto:rhserver@paxil.stanford.edu rhserver@paxil.stanford.edu) for
mapping using the RHMAP statistical program (RHMAP).
[0229] Results from the RH Server indicated as closest matches the
markers SHGC-58922 (LOD score: 8.67) and SHGC-12714 (LOD score
7.74). Both markers are located on chromosome 2, linked to the GDB
locus D2S292 (SHGC source AFH203yb6.dagger.: SHGC-1610
microsatellite marker). Genes mapping near the D2S292 locus
include.dagger.: transforming growth factor-alpha precursor (2p13),
dynactin (2p13), gamma actin enteric smooth muscle form (2p13),
annexin IV (2p13), MAD (2p12-13), alpha-CP1, early growth response
4, nucleolysin TIA-1, protein tyrosine phosphatase P, protein
kinase C substrate 80K-H, glutamine-fructose 6-phosphate
transaminase, retinoic acid-responsive protein, RAB.1A, and
pleckstrin. The results localize the gene of human Langerin to
chromosome 2p13, in the vicinity of the D2S292 locus.
[0230] The localization of human Langerin will permit to survey
immunological genetic disorders for a potential association with
the chromosome 2p13 region.
Example 21
Identification of Mouse Langerin
[0231] Using the nucleotide sequence of the human Langerin, a
search was performed in the NCBI database. This permitted to
identify two mouse ESTs (AA764540 and AA423304) displaying
considerable homology to the human sequence. These ESTs were then
used to extend the sequence, using RACE-PCR on a mouse lung cDNA
library: The entire coding region, with the exception of the
extremity of the 5' end was determined. Nine nucleotides completing
the sequence were subsequently identified by sequencing a mouse
cosmid clone. A contig of 1756 nucleotides was obtained and is
shown in SEQ ID NO: 10.
[0232] An open-reading frame of 978 nucleotides was identified
(positions 266-1243 in the contig of SEQ ID NO: 10), encoding a
predicted transmembrane type II Ca++ dependent lectin of 326 amino
acids (SEQ ID NO: 11). The mouse amino acid sequence presents
considerable homology (66.6%) with human Langerin (alignement shown
in Table 1), with conservation of the key structural features of
the molecule (ie. intracytoplasmic proline-rich motif as potential
signal transduction site, and extracellular
carbohydrate-recognition domain (CRD) with an EPN motif as found in
human Langerin and indicative of mannose-binding specificity).
These data indicate that the above identified mouse molecule is the
homolog of human Langerin.
[0233] The mouse Langerin protein has been succesfully expressed in
mammalian cells (murine COP5 fibroblasts), which have been used for
immunization of mice to produce monoclonal antibodies (mAbs). A
number of mAbs recognizing mouse Langerin protein have been
isolated 7 of these mAbs display crossreactivity on human Langerin.
The epitopes recognized by the cross-reactive mAbs have all been
mapped to the intracytoplasmic domain using truncated forms of
recombinant human Langerin protein. This is consistent with the
remarkably high conservation
1TABLE 1 1 2 3 4 5 6 7 8 9
[0234] in the cytoplasmic tail (first 30 residues) of human and
mouse Langerin (see Table 1).
[0235] Availability of the sequence encoding mouse Langerin will
permit the performance of studies (RT-PCR, Northerns, in situ
hybridization) to determine the expression pattern of Langerin in
various tissues. Furthermore, the current sequence can be extended
to identify the genomic DNA in order to construct mice deficient in
the Langerin gene. Such animals should be highly valuable to
further understand the role of Langerin in the regulation of the
immune response. MAbs directed against mouse Langerin will be
useful to study expression of the protein in mouse tissues. Also,
mAb can be injected in mice to analyze the impact of triggering
Langerin on the dendritic cell system in vivo. These studies will
be important to complement efforts to target antigen (ie. tumor
antigens) to dendritic cells in vivo via their cell-surface
Langerin with the aim of inducing protective antitumor immunity.
Targeting experiments can, for instance, be performed by injecting
mice (prior of after challenge with tumor) with mAb against mouse
Langerin to which tumor antigen has been coupled chemically.
[0236] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled.
Sequence CWU 1
1
12 1 1547 DNA Homo sapiens CDS (56)..(1039) 1 ctggaaaggg cagccagaag
cacctgtgct cccaggataa gggtgagcac tcagg atg 58 Met 1 act gtg gag aag
gag gcc cct gat gcg cac ttc act gtg gac aaa cag 106 Thr Val Glu Lys
Glu Ala Pro Asp Ala His Phe Thr Val Asp Lys Gln 5 10 15 aac atc tcc
ctc tgg ccc cga gag cct cct ccc aag tcc ggt cca tct 154 Asn Ile Ser
Leu Trp Pro Arg Glu Pro Pro Pro Lys Ser Gly Pro Ser 20 25 30 ctg
gtc ccg ggg aaa aca ccc aca gtc cgt gct gca tta atc tgc ctg 202 Leu
Val Pro Gly Lys Thr Pro Thr Val Arg Ala Ala Leu Ile Cys Leu 35 40
45 acg ctg gtc ctg gtc gcc tcc gtc ctg ctg cag gcc gtc ctt tat ccc
250 Thr Leu Val Leu Val Ala Ser Val Leu Leu Gln Ala Val Leu Tyr Pro
50 55 60 65 cgg ttt atg ggc acc ata tca gat gta aag acc aat gtc cag
ttg ctg 298 Arg Phe Met Gly Thr Ile Ser Asp Val Lys Thr Asn Val Gln
Leu Leu 70 75 80 aaa ggt cgt gtg gac aac atc agc acc ctg gat tct
gaa att aaa aag 346 Lys Gly Arg Val Asp Asn Ile Ser Thr Leu Asp Ser
Glu Ile Lys Lys 85 90 95 aat agt gac ggc atg gag gca gct ggc gtt
cag atc cag atg gtg aat 394 Asn Ser Asp Gly Met Glu Ala Ala Gly Val
Gln Ile Gln Met Val Asn 100 105 110 gag agc ctg ggt tat gtg cgt tct
cag ttc ctg aag tta aaa acc agt 442 Glu Ser Leu Gly Tyr Val Arg Ser
Gln Phe Leu Lys Leu Lys Thr Ser 115 120 125 gtg gag aag gcc aac gca
cag atc cag atc tta aca aga agt tgg gaa 490 Val Glu Lys Ala Asn Ala
Gln Ile Gln Ile Leu Thr Arg Ser Trp Glu 130 135 140 145 gaa gtc agt
acc tta aat gcc caa atc cca gag tta aaa agt gat ttg 538 Glu Val Ser
Thr Leu Asn Ala Gln Ile Pro Glu Leu Lys Ser Asp Leu 150 155 160 gag
aaa gcc agt gct tta aat aca aag atc cgg gca ctc cag ggc agc 586 Glu
Lys Ala Ser Ala Leu Asn Thr Lys Ile Arg Ala Leu Gln Gly Ser 165 170
175 ttg gag aat atg agc aag ttg ctc aaa cga caa aat gat att cta cag
634 Leu Glu Asn Met Ser Lys Leu Leu Lys Arg Gln Asn Asp Ile Leu Gln
180 185 190 gtg gtt tct caa ggc tgg aag tac ttc aag ggg aac ttc tat
tac ttt 682 Val Val Ser Gln Gly Trp Lys Tyr Phe Lys Gly Asn Phe Tyr
Tyr Phe 195 200 205 tct ctc att cca aag acc tgg tat agt gcc gag cag
ttc tgt gtg tcc 730 Ser Leu Ile Pro Lys Thr Trp Tyr Ser Ala Glu Gln
Phe Cys Val Ser 210 215 220 225 agg aat tca cac ctg acc tcg gtg acc
tca gag agt gag cag gag ttt 778 Arg Asn Ser His Leu Thr Ser Val Thr
Ser Glu Ser Glu Gln Glu Phe 230 235 240 ctg tat aaa aca gcg ggg gga
ctc atc tac tgg att ggc ctg act aaa 826 Leu Tyr Lys Thr Ala Gly Gly
Leu Ile Tyr Trp Ile Gly Leu Thr Lys 245 250 255 gca ggg atg gaa ggg
gac tgg tcc tgg gtg gat gac acg cca ttc aac 874 Ala Gly Met Glu Gly
Asp Trp Ser Trp Val Asp Asp Thr Pro Phe Asn 260 265 270 aag gtc caa
agt gcg agg ttc tgg att cca ggt gag ccc aac aat gct 922 Lys Val Gln
Ser Ala Arg Phe Trp Ile Pro Gly Glu Pro Asn Asn Ala 275 280 285 ggg
aac aat gaa cac tgt ggc aat ata aag gct ccc tca ctt cag gcc 970 Gly
Asn Asn Glu His Cys Gly Asn Ile Lys Ala Pro Ser Leu Gln Ala 290 295
300 305 tgg aat gat gcc cca tgt gac aaa acg ttt ctt ttc att tgt aag
cga 1018 Trp Asn Asp Ala Pro Cys Asp Lys Thr Phe Leu Phe Ile Cys
Lys Arg 310 315 320 ccc tat gtc cca tca gaa ccg tgacaggaca
ggctcccaag ctcactcttt 1069 Pro Tyr Val Pro Ser Glu Pro 325
gagctccaac gcttgttaaa catgaggaaa tgcctctttc ttccccagac tccaggatga
1129 ctttgcacgt taatttttct tgcttcaaaa ttgtcccaca gtggcattct
ggagtccgtc 1189 tgtcttggct ggaaattctc tgacgtcttg gaggcagctg
gaatggaaag gagaattcag 1249 gttaaagtgg gaggggtggg tagagaggat
ttagaagttc caattgccct gctaaggagg 1309 atcaagaccc gtaatccggc
acaacaccct ggggttttcc actctttcag agaaacctca 1369 gcttcatcac
atcaaagtta ctccagagca accaagcaat tctcctgata ttgtcatcca 1429
gggcttttct tggccaaacc ccctagaatt tccatgtctc tgcttagctg tgctggcagc
1489 tagcagctgg ctgtgtttgc agtgcaaata gctctgttct tggaaatcct
gctcatgg 1547 2 328 PRT Homo sapiens 2 Met Thr Val Glu Lys Glu Ala
Pro Asp Ala His Phe Thr Val Asp Lys 1 5 10 15 Gln Asn Ile Ser Leu
Trp Pro Arg Glu Pro Pro Pro Lys Ser Gly Pro 20 25 30 Ser Leu Val
Pro Gly Lys Thr Pro Thr Val Arg Ala Ala Leu Ile Cys 35 40 45 Leu
Thr Leu Val Leu Val Ala Ser Val Leu Leu Gln Ala Val Leu Tyr 50 55
60 Pro Arg Phe Met Gly Thr Ile Ser Asp Val Lys Thr Asn Val Gln Leu
65 70 75 80 Leu Lys Gly Arg Val Asp Asn Ile Ser Thr Leu Asp Ser Glu
Ile Lys 85 90 95 Lys Asn Ser Asp Gly Met Glu Ala Ala Gly Val Gln
Ile Gln Met Val 100 105 110 Asn Glu Ser Leu Gly Tyr Val Arg Ser Gln
Phe Leu Lys Leu Lys Thr 115 120 125 Ser Val Glu Lys Ala Asn Ala Gln
Ile Gln Ile Leu Thr Arg Ser Trp 130 135 140 Glu Glu Val Ser Thr Leu
Asn Ala Gln Ile Pro Glu Leu Lys Ser Asp 145 150 155 160 Leu Glu Lys
Ala Ser Ala Leu Asn Thr Lys Ile Arg Ala Leu Gln Gly 165 170 175 Ser
Leu Glu Asn Met Ser Lys Leu Leu Lys Arg Gln Asn Asp Ile Leu 180 185
190 Gln Val Val Ser Gln Gly Trp Lys Tyr Phe Lys Gly Asn Phe Tyr Tyr
195 200 205 Phe Ser Leu Ile Pro Lys Thr Trp Tyr Ser Ala Glu Gln Phe
Cys Val 210 215 220 Ser Arg Asn Ser His Leu Thr Ser Val Thr Ser Glu
Ser Glu Gln Glu 225 230 235 240 Phe Leu Tyr Lys Thr Ala Gly Gly Leu
Ile Tyr Trp Ile Gly Leu Thr 245 250 255 Lys Ala Gly Met Glu Gly Asp
Trp Ser Trp Val Asp Asp Thr Pro Phe 260 265 270 Asn Lys Val Gln Ser
Ala Arg Phe Trp Ile Pro Gly Glu Pro Asn Asn 275 280 285 Ala Gly Asn
Asn Glu His Cys Gly Asn Ile Lys Ala Pro Ser Leu Gln 290 295 300 Ala
Trp Asn Asp Ala Pro Cys Asp Lys Thr Phe Leu Phe Ile Cys Lys 305 310
315 320 Arg Pro Tyr Val Pro Ser Glu Pro 325 3 10663 DNA Homo
sapiens 3 tccctgtccc cactccgcaa tttggtgttc actctacttt cctccatgac
ttgactgagg 60 gacttgggtg actcctccct ggagatgact cttggcactg
ctggagcatt tcatacaacc 120 caggtctctg ttccagtctt gttgattaaa
cccatggttc tcctccaagc ataaggctgg 180 gcccaaccat agtctcaacc
agagaaaggg aaaaagtgac caagctgttt tcttcctcca 240 tccttatcca
gagaaaatat tagaacccca ggaacacagg aagaggagaa actaaaaaaa 300
tgagcacaga cttcccgttc tcatcacagc ggtgattttt ttggtcacta ggtctggcca
360 gagttcctct tctgtttgga agagcagggc attgtctgag tgcccgggag
aactgcccac 420 cttcctgact ggaaagtgtg gccaaggcac ctgcctgctt
ctctgtcttt tctcctgcca 480 ggccaggtgc cagatagagc atccagaggt
ttgcacagga gaggtgctca ggaaagacct 540 gtagatgagc atgataatag
gaagatggct tctgactccc tccctctcca caaagtggga 600 aatgaaactt
cctatggttt gggactttta catccacctc ctctgaaacc ccagaaggcc 660
cacagcccac ggattccatg ccttttgctc agcttcctca tgctggaaca gcctttcgcc
720 tagcggtagc tattctacct acctcagcct gcatttccat cacttggagt
aaatgctccc 780 tgctccagcc ccttcctatg ccatgggcca gccgctaagg
cttcctcatc ctttcctggg 840 accaatgaga gggcatctgg ggggctctat
aaagtataac tcataccaca aaccagcaga 900 gaaaacccaa cttcctgacc
gagcggaaat ggacaagaca cttcccaggc acactgtgtg 960 gcggcttctt
tcccgcaaat gcctcaggag gagacatgga cccctgcaag gccccttcta 1020
ccagggtctt gagtaatttg gattcttctc tatccccagg cccagaaata ggacaggcta
1080 tggatgaagg gccaggagcc aggagcagaa gggcagagat gattctggag
ttaggctggg 1140 tcccaagcat gagagtccag aagtggaaca ttctaatcct
gtctaagccc tgatggaacc 1200 aaggctctgc aaccacacga gccacctgaa
gagagtagga atcttcatgg acacccagag 1260 gcaggcagtg atgtggagcc
aggccctcat attcgggact atcaaatgtt aaaatttaga 1320 gcagacagca
ggcctggaag ttacagacag gctgggtagg gaagcgatgg gacagaacaa 1380
atgagacacc atctttgcta cccagtttcc ttcccagagc ttcccagtgt ggggtcctct
1440 ctgggttccc tcatccagtc cgagaagcca ctccagcctg aggcccggtt
agctggagag 1500 agagcatcaa gggcctgtaa ctacaatgag ctgcacatag
gcatgggctg agcgtgcagc 1560 cactatcctc ccaagtacca aaggtggcag
tagcccctga aagcataggt agatatttcc 1620 ttggccctgt taacttaacc
tctaatcctg gtccctgata caggctgtca tccagctcca 1680 ctggctcagc
ctcagcaggc ccaggacagc tttcttttcc tgcggccaga gcttttgttc 1740
attcctgggt aaggagtaaa tcacacttcc ccttatgctt ttgcattgaa gatgaatgag
1800 aacttctgga gacagtggaa aaaagcagca gcttctagaa ggcagatccc
aggccccaga 1860 ctgtgtaaat gtctgagtgg catgatcagc tatcaagtct
ccaagtcact taccattcac 1920 acatccatcc atgcatcctc tatccatcca
ttcatccatt cacccatcct ccaattatct 1980 ggaccaattt ccttcttttt
tctttccttc cctccctctt tcctctcctc tttcctatat 2040 tccaagaaat
atttgtctag agtctgccat gtgccaaatt tctcagattt ggcagacttc 2100
ttcaaagcat gagaagccgc ccaggagaaa atttagtcct tcattgagcc caaatcagct
2160 cttctgtgcc ctgagcttct tgcacatatg gaactctcta tttaggacta
caaagaagga 2220 gagaggaggt gcggacagag agagaggaga gagaaacaga
acgattccta gtctgaggtt 2280 gctgagcgcc cctacttttt ttgaaaatct
cacatggtgc aggaagcagg gaggagaacc 2340 caagtcttag gtttacactt
tgaatctcca agttgcatat tataaaggga tattgtatca 2400 gccaagatgc
attaataaca ggaagcagga tattctattg caatggttgc ctatttggta 2460
tctctgcttc caggcttctc cccaccagtc tgctcctcac caggatcaga gtgatgtttc
2520 tacagtgtcc ctgtccttgt ctctctcctg cctgaagtcc ttaattggcc
ccatgtcacc 2580 cacaagccag aggtccagct ccttgacatg atcagaagga
tcttcatcat cagaccccag 2640 gtgcctcttc aaccccgtgt gcatcccctt
cctgttggaa tgtatatttt acattccagc 2700 aatattgcaa ctatttacaa
tttgccaagc actccatgct atcttatgct ccatcctttg 2760 gcatatgctt
atctttctac tggatttttt tttttcccat tactccattt catatgcttc 2820
tttgaactgg gcatatttgt ctcttgtgct catcaccttg caatttattt gctcatgtct
2880 gtctttccta ccagactatg agctgcttgg tgcaaggact gcgagttatt
catcactgtg 2940 gccctatgcc tggtagagca tcagtaccta gaaggcactc
agcctgtatt tgtggggtga 3000 atggatgggt ggatggatga cgagagtctt
acaagagaaa tgggataggt ttgggacaag 3060 atggttaatg tatccatgta
acagaccccc agagaagaca acaaatggcc tcttcctgaa 3120 agctcagact
tctgaggatg ggagtaagcc agacaaggta tctagtcagg aatagggaag 3180
ttgggatgat atggtgacct gctgtgggac tgacttcctg tttcctctag ataagagccc
3240 ttggagagac aggcagccag aagcacctgt gctcccagga taagggtgag
cactcaggat 3300 gactgtggag aaggaggccc ctgatgcgca cttcactgtg
gacaaacaga acatctccct 3360 ctggccccga gcaagccaca tcgctgctga
gaacctgctc cgtgttctgt gtgcaaacct 3420 gccctttgct gctccttcaa
cacacatttt cttcttcttc caacagagcc tcctcccaag 3480 tccggtccat
ctctggtccc ggggaaaaca cccacagtcc gtgctgcatt aatctgcctg 3540
acgctggtcc tggtcgcctc cgtcctgctg caggccgtcc tttgtaagtc ctcatgtttc
3600 atcgtctggg cttagcccct ctctgtgcca gccggctccc ttcagatcga
gaccacttcc 3660 ctgctctccg ggtttctcct cctgtggctt tttcatttgt
ctccttcctc ctctttccat 3720 gtgcagtaac aggctgtgct gcccccagta
cggtgagctg atgctctttc cctcccaatt 3780 tctgggagat tattgggatt
agcatttgca cattggtgct caaggataca gtctttgtcc 3840 tcaggaaaca
ttgatctagt tttacatcct gtgctatttt ctccccgtcc accccccacc 3900
aatctgagct ctgctccttg gatctagagc cttgtggaca gtccttgaca gtgcaatgtc
3960 tcctgaccct gtgaaggacc gagcctcatg tatcattggc cccagctcac
cagatggaga 4020 gcctggccaa cgagccaaca gatctccatg actcagtccc
ctctccccag gactcgttgg 4080 tgctgtcttc attctcctgt ccctgtgaag
atcacatcta gggaggcttc ctgctcattt 4140 gatgttgcat ggtttatctt
tcttcctttt ctggctctgt caggcttcat ggcctttgct 4200 gctggcagag
ccttcttcct cctgccaggg ctccaggaag cagagcaaag ggacccaaga 4260
agctgttggg tttttttttc tccctctgtg gcctcgggac atatttgggc ttagacttgt
4320 aggctctgag cagagtcccc cttgccccat cctagacccc tggcctctaa
catcgcattt 4380 tcctcagggc tcgcctttga gcctcttggt ttatctttga
tcctctctct ggtacccagg 4440 cctgggacct gagcagaatg tgaaagtggg
tggggcaagg gaaggggaga aacagttttg 4500 taatctcctc ttccatgttc
tctggagaag ccacttccag attagtggct ggttcttccc 4560 atggtcacag
aggggcccat ggacagatgt gggggagtgg tgctgtccta gcagatggcc 4620
actgcagggg tttctgaaaa cagagggatg gcaaccaagg ggtggggtct agggggaatc
4680 aagttctggg gacagtggtg gggctcatgg agggcaccct ttatcaaatg
ttccctgaac 4740 actcagaaaa ttcaggaatg gtttctaact cctgcttctg
cttgcctgtg aaatcttttc 4800 acagagagcc tgtgttttat caatctcccc
attatctgta tccacctgtg ttcctggcac 4860 atggtaggtg cccattgcac
gtttgttgta cgttaatgaa tgattggagg gttggggtgg 4920 cccattggac
tgtcttggtt ctttgggaag cttcagccta ttccttccct tcctttgatc 4980
aacctgacaa cacccccact cctgtccctg ggactcccct cagctgacct cctgactttc
5040 tcaatcccag atccccggtt tatgggcacc atatcagatg taaagaccaa
tgtccagttg 5100 ctgaaaggtc gtgtggacaa catcagcacc ctggattctg
aaattaaaaa gaatagtgac 5160 ggcatggagg cagctggcgt tcagatccag
atggtgaatg agagcctggg ttatgtgcgt 5220 tctcagttcc tgaagttaaa
aaccagtgtg gagaaggcca acgcacagat ccagatctta 5280 acaagaagtt
gggaagaagt cagtacctta aatgcccaaa tcccagagtt aaaaagtgat 5340
ttggagaaag ccagtgcttt aaatacaaag atccgggcac tccagggcag cttggagaat
5400 atgagcaagt tgctcaaacg acaaagtaag tgactcagaa aattacattg
aagctgacca 5460 gtggcccatg ggatcttacc tgtcccagac ctgaggccat
tgggctggtg ggttggggag 5520 gagagtgggg gcaaaagagg ggcagccatg
ggctaggaag ttaaggagag agggcttgag 5580 gttggggagg acttaggggc
tgttaggaga aaagagacca gggtccagct agagctccca 5640 cacaaaagtg
cagaatgtaa aagcattagg ggatgtccac cctggcccac acctagtcat 5700
ttcccatcaa gttcctttct agagtccagg ggctcagcca cttgtcatgg ccgatggagg
5760 gttgcttcct catcatgggg aagacactct ttgtccaacc tcttgcatta
taacctctcc 5820 agtcccagag actctattag tctctgtctg actttcagga
tttgaaagag tgtccctaat 5880 ctcctataca aggacccaag gacaccagcg
cacagctcca tttgctgctg tctctgagac 5940 ctcattcaag tgcccccacc
aagccagcat cccaagaaat caagaatacc agcgttcact 6000 tttacctctt
gttctctaga tgatattcta caggtggttt ctcaaggctg gaagtacttc 6060
aaggggaact tctattactt ttctctcatt ccaaagacct ggtatagtgc cgagcagttc
6120 tgtgtgtcca ggaattcaca cctgacctcg gtgacctcag agagtgagca
ggtgagtgct 6180 gtgcctatgg gctctgtgaa gggggcgtat gagcactggg
ccagggagga tgggcaagat 6240 tatactgcgt gaacaaaaat ccccaaatat
tgatgaccta atgaagaagg attggttctc 6300 agtagcatgt cgaataaggg
ccggcaaggg ggctgtgctc actgtggcta ctcaaggacc 6360 caggcccaag
gagtcttcat cttaatatgt ctccacaatt gctggggcag gaaaggggaa 6420
cttgacatat tgtgcaaagc ttctgcccag atttaacata cctcattttt gcttacattt
6480 cactagctta agttatgtta attaagttaa gtaagttatg cagtactcct
ggattgggag 6540 gatcctggat tccagtactc ctggaacagg gcagagagga
tctactttcc atgtgcccag 6600 aaaaaaagaa atatttgtga acagccttaa
tgattccaca tgactcaaga agtctcctgc 6660 ctggtgaggc agaaattggg
caggcccttt tcatctggga ggtgggatag cagagcaggt 6720 cagagcctgg
gctctggcgt agttctagag cctgaacctt gccatctaac tagtcctagc 6780
agcttgggtg gaatacccaa cttcactggg caaacttcac tggccctcac ttgatgaaac
6840 atgcatggtg atggcacctg cctcagagga aaggagagaa tgcatatgga
cgactcagca 6900 cagtgccgta tgtggattca gggctcattt aattcaggta
ttatcatatg aaccattctc 6960 ttgcggtccg tgctctggag ttcagctgag
gccttcctgt gcttcagcac ctgcttcctg 7020 agtggcagaa aggcttgagt
cctgagcttg ttagctgcag agcagggaca catcataatc 7080 tggaagatga
aatctgggct ctgggcaagg gcaggaagaa gcttgagagg ccagtttgtg 7140
cagcgcatct gtgggtcagg gctgtcactg agcgcaggtg aagaacaccc agagacagat
7200 gatcaagctc caagtgtggc cgcacctctg cttatcctgt ctttcctaca
ggagtttctg 7260 tataaaacag cggggggact catctactgg attggcctga
ctaaagcagg gatggaaggg 7320 gactggtcct gggtggatga cacgccattc
aacaaggtcc aaagtgtgag gtaagcccct 7380 ggagccctcc gtgccagcct
gactttcccc ggccatggcc agggcatgaa gggagtgggg 7440 gcgatgttcc
ccatgagaca gggtttctga ttcttccctg tcttagagtg acaggaacat 7500
tgcaaccaag atcgagcaca accctgtcac caactggctg tggacctgag ccctccacgc
7560 cctctggggt ttggcaacaa ggccttctac ctggccagct tcagggatct
tgtcatgagt 7620 ctaggtcttc acagtgtggg tttgtgtagg gacttgaaag
tggtgggttg gtttggcctg 7680 gacttggggc atgtgaaagc ttagaggtcg
aagtctcacc agtccccttc ctctgaggct 7740 tgggtgcaga catttgctat
gccattccct aggacaaaag cttgggttga gttaactcat 7800 ttcttcactg
gaaataagtt ctttttgatt ttccactttg taaatccatc tttttccccg 7860
ctcttggtag gttctggatt ccaggtgagc ccaacaatgc tgggaacaat gaacactgtg
7920 gcaatataaa ggctccctca cttcaggcct ggaatgatgc cccatgtgac
aaaacgtttc 7980 ttttcatttg taagcgaccc tatgtcccat cagaaccgtg
acaggacagg ctcccaagct 8040 cactctttga gctccaacgc ttgttaaaca
tgaggaaatg cctctttctt ccccagactc 8100 caggatgact ttgcacgtta
atttttcttg cttcaaaatt gtcccacagt ggcattctgg 8160 agtccgtctg
tcttggctgg aaattctctg acgtcttgga ggcagctgga atggaaagga 8220
gaattcaggt taaagtggga ggggtgggta gagaggattt agaagttcca attgccctgc
8280 taaggaggat caagacccgt aatccggcat aacaccctgg ggttttccac
tctttcagag 8340 aaacctcagc ttcatcacat caaagttact ccagagcaac
caagcaattc tcctgatatt 8400 gtcatccagg gcttttcttg gccaaacccc
ctagaatttc catgtctctg cttagctgtg 8460 ctggcagcta gcagctggct
gtgtttgcag tgcaaatagc tctgttcttg gaaatcctgc 8520 tcatggtatg
tccccagtgg tttcttcatc cacatcatct aaagcctgaa cccgttcttc 8580
tctggttcaa gtcagtggct gacacggact tgtatctcct tcagagctcg gctggcaccc
8640 agcctccctt ctccttccac tcccttagta cactggagtg ccgagccctg
ccttccaccc 8700 agcgtccatc cagcccctgt cctcacctct ccggcacctc
ctcctccttc tgcatttcct 8760 atcttcctgt gtcttgtgca tgggaagcag
ccttcagtgc cttcatgaat tcaccttcca 8820 gcttcctcag aataaaatgc
tgcctgggtc aaggactcac tccaagtgca ctttttcatt 8880 tctggttgtc
caggtgaata tgtgggaaag gcagtctcct ctggtggaca tgaagttcta 8940
gggtatcctc aggaaacatc tggggagtca aaaataacaa ggactgggga agttccagtc
9000 ctggaaatgc cacaaaatgt gaccagtact tatctctagt ttttattaaa
gtagagcaag 9060 gtctccaatg tcacgatctt ggtgatcttt cttcttgttt
actgcacaat cttctagtct 9120 atagctcaat tcccaagaac aagtctcagc
aggttcccca ctcttcacag agacccagtt 9180 ccacaggcat cagttccaaa
tcccaagtcc agtggctgaa gctggaatcc aggcagcagc 9240 caccacagag
aggagaggag ggtggagtga gcacaggtct tcattaaggt cctcaggaaa 9300
agatgcttcc ttaaataact gtaaccagca gtgtgttgtt ctgggtgcaa atgggtcaca
9360 gctgagggca caggcttgta ttgtaagacc tgaaatacca cgtgctgctg
tgacatttta 9420 tgcctcacag ggccccaaag acctaaccct gagttccctg
cctctcacca gatatatcct 9480 tgccctcggt ccccacctgg ctaatttcct
atcatctgga ccagctgcat gccacccagt 9540 tttctactta atgggtttca
cttctctgcc agcctgagaa actattcaaa caagccaatc 9600 acatcctcct
acaggaatcc ggggcatctc atccttttat tactacaagg cctgcctccc 9660
acagccctgg ctggttcact ctgctcctga gggtgacccc atgtggccct gtgtggctta
9720 tgatatcttt ccccagtaga ctgtatttgt gactagtaaa ctgctgccag
tctcacctgc 9780 acagtgtcaa atgtcttgtt ttggccatct tgtcctattt
agagcagggg atccctccct 9840 caccaatgga ggaaatggga ggtgacaaga
acaaggctgg tcggggatct ctggttggtt 9900 ttggcaaaga gatgagctgg
gaaaatcaga ccatttctct gggaaagaat ttgaaccagg 9960 aaatagcaag
aggatgaggc tgttaacaaa aggaagttga gctggaaggc actgagttaa 10020
gagaaaggct ggaggggccg tcacgtggca ttggaagaaa ctagcaatga gcagaagcta
10080 tgaggcaggg gaaagacatg aatacaggcc tggcatggtg gctcatgcct
ataatcccag 10140 cactttggga ggctgaagtg ggtgggtcac ctgaggtcag
gagttcgaga cagcctggcc 10200 aacatggtga aaccccatct ctactaaaaa
tacaaaaatt agccgggcat ggtggtgggc 10260 acctgtaatt ccaactactt
gggagactga ggcaggagaa ttgcttgaat ctgggaggca 10320 gaggttgcag
tgagctgaga tcccaccact gcactccagc ctgggcaaca gggcaagact 10380
ttgtttcaaa aaaaaagaag tgactgcaga ggattatagt tggcagagaa aagagaacgg
10440 ctcagaggag tcgcaatgga ggtcccggag ggcagcctga agggctccgg
ctgctcccgt 10500 tcccagggct gcctcagatc ctcccagccc ttctgatcct
cctggtttct gtgcatgggg 10560 accttacgag gctgtgctcc tgaccccaac
cattgctttt tcttgaaact gaaagagcct 10620 gagtcagtga ggatgtgttt
ttatctggag tctgtgcccc agc 10663 4 120 DNA Homo sapiens 4 ggcagccaga
agcacctgtg ctcccaggat aagggtgagc actcaggatg actgtggaga 60
aggaggcccc tgatgcgcac ttcactgtgg acaaacagaa catctccctc tggccccgag
120 5 117 DNA Homo sapiens 5 agcctcctcc caagtccggt ccatctctgg
tcccggggaa aacacccaca gtccgtgctg 60 cattaatctg cctgacgctg
gtcctggtcg cctccgtcct gctgcaggcc gtccttt 117 6 375 DNA Homo sapiens
6 atccccggtt tatgggcacc atatcagatg taaagaccaa tgtccagttg ctgaaaggtc
60 gtgtggacaa catcagcacc ctggattctg aaattaaaaa gaatagtgac
ggcatggagg 120 cagctggcgt tcagatccag atggtgaatg agagcctggg
ttatgtgcgt tctcagttcc 180 tgaagttaaa aaccagtgtg gagaaggcca
acgcacagat ccagatctta acaagaagtt 240 gggaagaagt cagtacctta
aatgcccaaa tcccagagtt aaaaagtgat ttggagaaag 300 ccagtgcttt
aaatacaaag atccgggcac tccagggcag cttggagaat atgagcaagt 360
tgctcaaacg acaaa 375 7 152 DNA Homo sapiens 7 atgatattct acaggtggtt
tctcaaggct ggaagtactt caaggggaac ttctattact 60 tttctctcat
tccaaagacc tggtatagtg ccgagcagtt ctgtgtgtcc aggaattcac 120
acctgacctc ggtgacctca gagagtgagc ag 152 8 119 DNA Homo sapiens 8
gagtttctgt ataaaacagc ggggggactc atctactgga ttggcctgac taaagcaggg
60 atggaagggg actggtcctg ggtggatgac acgccattca acaaggtcca aagtgcgag
119 9 985 DNA Homo sapiens 9 gttctggatt ccaggtgagc ccaacaatgc
tgggaacaat gaacactgtg gcaatataaa 60 ggctccctca cttcaggcct
ggaatgatgc cccatgtgac aaaacgtttc ttttcatttg 120 taagcgaccc
tatgtcccat cagaaccgtg acaggacagg ctcccaagct cactctttga 180
gctccaacgc ttgttaaaca tgaggaaatg cctctttctt ccccagactc caggatgact
240 ttgcacgtta atttttcttg cttcaaaatt gtcccacagt ggcattctgg
agtccgtctg 300 tcttggctgg aaattctctg acgtcttgga ggcagctgga
atggaaagga gaattcaggt 360 taaagtggga ggggtgggta gagaggattt
agaagttcca attgccctgc taaggaggat 420 caagacccgt aatccggcac
aacaccctgg ggttttccac tctttcagag aaacctcagc 480 ttcatcacat
caaagttact ccagagcaac caagcaattc tcctgatatt gtcatccagg 540
gcttttcttg gccaaacccc ctagaatttc catgtctctg cttagctgtg ctggcagcta
600 gcagctggct gtgtttgcag tgcaaatagc tctgttcttg gaaatcctgc
tcatggtatg 660 tccccagtgg tttcttcatc cacatcatct aaagcctgaa
cccgttcttc tctggttcaa 720 gtcagtggct gacacggact tgtatctcct
tcagagctcg gctggcaccc agcctccctt 780 ctccttccac tcccttagta
cactggagtg ccgagccctg ccttccaccc agcgtccatc 840 cagcccctgt
cctcacctct ccggcacctc ctcctccttc tgcatttcct atcttcctgt 900
gtcttgtgca tgggaagcag ccttcagtgc cttcatgaat tcaccttcca gcttcctcag
960 aataaaatgc tgcctgggtc aagga 985 10 1756 DNA Mus musculus CDS
(266)..(1243) 10 ggcctctttg ctcgtttggt gtcttcagtt aagatgatta
aaatgtctgt gtaccaggga 60 agtagtcagg tggcttcctc atcaaagccc
aattcttagt caaaaggatg aagctgtggc 120 ctctgcctgg ctaatcgtct
aagccaagaa ctgggaactt gggcatgaca aagtggcctg 180 ctttttggct
catttcccgt ttctttctgg atgaaaaggt ccttggggag acggatattc 240
agcttttcct aagccagagg cagag atg ttg gag gag gct ccc gaa gcg cac 292
Met Leu Glu Glu Ala Pro Glu Ala His 1 5 ttc aca gtg gac aaa cag aac
atc tct ctc tgg cct cga gag cct cct 340 Phe Thr Val Asp Lys Gln Asn
Ile Ser Leu Trp Pro Arg Glu Pro Pro 10 15 20 25 ccc aag caa gat ctg
tct cca gtt ctg agg aaa cct ctc tgt atc tgc 388 Pro Lys Gln Asp Leu
Ser Pro Val Leu Arg Lys Pro Leu Cys Ile Cys 30 35 40 gtg gcc ttc
acc tgc ctg gca ttg gtg ctg gtc acc tcc att gtg ctt 436 Val Ala Phe
Thr Cys Leu Ala Leu Val Leu Val Thr Ser Ile Val Leu 45 50 55 cag
gct gtt ttc tat cct agg ttg atg ggc aaa ata ttg gat gtg aag 484 Gln
Ala Val Phe Tyr Pro Arg Leu Met Gly Lys Ile Leu Asp Val Lys 60 65
70 agt gat gcc cag atg ttg aaa ggt cgt gtg gac aac atc agc acc ctg
532 Ser Asp Ala Gln Met Leu Lys Gly Arg Val Asp Asn Ile Ser Thr Leu
75 80 85 ggt tct gat ctt aag act gaa aga ggt cgt gtg gac gat gct
gag gtt 580 Gly Ser Asp Leu Lys Thr Glu Arg Gly Arg Val Asp Asp Ala
Glu Val 90 95 100 105 cag atg cag ata gtg aac acc acc ctc aag agg
gtg cgt tct cag atc 628 Gln Met Gln Ile Val Asn Thr Thr Leu Lys Arg
Val Arg Ser Gln Ile 110 115 120 ctg tct ttg gaa acc agc atg aag ata
gcc aat gat cag ctc ctg ata 676 Leu Ser Leu Glu Thr Ser Met Lys Ile
Ala Asn Asp Gln Leu Leu Ile 125 130 135 tta aca atg agc tgg gga gag
gtt gac agt ctc agt gcc aaa atc cca 724 Leu Thr Met Ser Trp Gly Glu
Val Asp Ser Leu Ser Ala Lys Ile Pro 140 145 150 gaa ctg aaa aga gat
ctg gat aaa gcc agc gcc ttg aac aca aag gtc 772 Glu Leu Lys Arg Asp
Leu Asp Lys Ala Ser Ala Leu Asn Thr Lys Val 155 160 165 caa gga cta
cag aac agc ttg gag aat gtc aac aag ctg ctc aaa caa 820 Gln Gly Leu
Gln Asn Ser Leu Glu Asn Val Asn Lys Leu Leu Lys Gln 170 175 180 185
cag agt gac att ctg gag atg gtg gct cga ggc tgg aag tat ttc tcg 868
Gln Ser Asp Ile Leu Glu Met Val Ala Arg Gly Trp Lys Tyr Phe Ser 190
195 200 ggg aac ttc tat tac ttt tca cgc acc cca aag acc tgg tac agc
gca 916 Gly Asn Phe Tyr Tyr Phe Ser Arg Thr Pro Lys Thr Trp Tyr Ser
Ala 205 210 215 gag cag ttc tgt att tct aga aaa gct cac ctg acc tca
gtg tcc tca 964 Glu Gln Phe Cys Ile Ser Arg Lys Ala His Leu Thr Ser
Val Ser Ser 220 225 230 gaa tcg gaa caa aag ttt ctc tac aag gca gca
gat gga att cca cac 1012 Glu Ser Glu Gln Lys Phe Leu Tyr Lys Ala
Ala Asp Gly Ile Pro His 235 240 245 tgg att gga ctt acc aaa gca ggg
agc gaa ggg gac tgg tac tgg gtg 1060 Trp Ile Gly Leu Thr Lys Ala
Gly Ser Glu Gly Asp Trp Tyr Trp Val 250 255 260 265 gac cag aca tca
ttc aac aag gag caa agt agg agg ttc tgg att cca 1108 Asp Gln Thr
Ser Phe Asn Lys Glu Gln Ser Arg Arg Phe Trp Ile Pro 270 275 280 ggt
gaa ccc aac aac gca ggg aac aac gag cac tgt gcc aat atc agg 1156
Gly Glu Pro Asn Asn Ala Gly Asn Asn Glu His Cys Ala Asn Ile Arg 285
290 295 gtg tct gcc ctg aag tgc tgg aac gat ggt ccc tgt gac aat aca
ttt 1204 Val Ser Ala Leu Lys Cys Trp Asn Asp Gly Pro Cys Asp Asn
Thr Phe 300 305 310 ctt ttc atc tgc aag agg ccc tac gtc caa aca act
gaa tgacagatct 1253 Leu Phe Ile Cys Lys Arg Pro Tyr Val Gln Thr Thr
Glu 315 320 325 ggcctgagct cggcatctgt ggggcaacag tgacctggct
gaagagatgt ctctctccct 1313 gaggctccaa gattgctctg tacttacgtt
tttttcttgc ttgaaaattg tcccaaacac 1373 agcctgtggt ctttctgtct
tggctggcag ttctctgctc ctggaggcct tggaggagct 1433 tgggttaaac
gggtgaggac ctgaaaaggg tgtagcagtc cttactgccc aggcgaggca 1493
ggtcagcaca ccaaacaggt tgtttagatt ttcctgatcc ttctcagaag ccttggctga
1553 ccatataaaa gctacattca aatatgacca gtatttgagg agacagacat
gcccaaattt 1613 aaccatgata caatttatac aacatgtatt agaacacctc
atggtatgtt caaaatagta 1673 aatatgttgt ttttatgtgc ctattgcaaa
taaatgtaaa gacttaaaaa aaaaaaaaaa 1733 aaaaaaaaaa aaaaaaaaaa aaa
1756 11 326 PRT Mus musculus 11 Met Leu Glu Glu Ala Pro Glu Ala His
Phe Thr Val Asp Lys Gln Asn 1 5 10 15 Ile Ser Leu Trp Pro Arg Glu
Pro Pro Pro Lys Gln Asp Leu Ser Pro 20 25 30 Val Leu Arg Lys Pro
Leu Cys Ile Cys Val Ala Phe Thr Cys Leu Ala 35 40 45 Leu Val Leu
Val Thr Ser Ile Val Leu Gln Ala Val Phe Tyr Pro Arg 50 55 60 Leu
Met Gly Lys Ile Leu Asp Val Lys Ser Asp Ala Gln Met Leu Lys 65 70
75 80 Gly Arg Val Asp Asn Ile Ser Thr Leu Gly Ser Asp Leu Lys Thr
Glu 85 90 95 Arg Gly Arg Val Asp Asp Ala Glu Val Gln Met Gln Ile
Val Asn Thr 100 105 110 Thr Leu Lys Arg Val Arg Ser Gln Ile Leu Ser
Leu Glu Thr Ser Met 115 120 125 Lys Ile Ala Asn Asp Gln Leu Leu Ile
Leu Thr Met Ser Trp Gly Glu 130 135 140 Val Asp Ser Leu Ser Ala Lys
Ile Pro Glu Leu Lys Arg Asp Leu Asp 145 150 155 160 Lys Ala Ser Ala
Leu Asn Thr Lys Val Gln Gly Leu Gln Asn Ser Leu 165 170 175 Glu Asn
Val Asn Lys Leu Leu Lys Gln Gln Ser Asp Ile Leu Glu Met 180 185 190
Val Ala Arg Gly Trp Lys Tyr Phe Ser Gly Asn Phe Tyr Tyr Phe Ser 195
200 205 Arg Thr Pro Lys Thr Trp Tyr Ser Ala Glu Gln Phe Cys Ile Ser
Arg 210 215 220 Lys Ala His Leu Thr Ser Val Ser Ser Glu Ser Glu Gln
Lys Phe Leu 225 230 235 240 Tyr Lys Ala Ala Asp Gly Ile Pro His Trp
Ile Gly Leu Thr Lys Ala 245 250 255 Gly Ser Glu Gly Asp Trp Tyr Trp
Val Asp Gln Thr Ser Phe Asn Lys 260 265 270 Glu Gln Ser Arg Arg Phe
Trp Ile Pro Gly Glu Pro Asn Asn Ala Gly 275 280 285 Asn Asn Glu His
Cys Ala Asn Ile Arg Val Ser Ala Leu Lys Cys Trp 290 295 300 Asn Asp
Gly Pro Cys Asp Asn Thr Phe Leu Phe Ile Cys Lys Arg Pro 305 310 315
320 Tyr Val Gln Thr Thr Glu 325 12 328 PRT Artificial majority
sequence between human and mouse Langerin 12 Met Leu Val Glu Glu
Glu Ala Pro Asp Ala His Phe Thr Val Asp Lys 1 5 10 15 Gln Asn Ile
Ser Leu Trp Pro Arg Glu Pro Pro Pro Lys Ser Gly Leu 20 25 30 Ser
Leu Val Leu Gly Lys Thr Leu Thr Val Arg Ala Ala Leu Ile Cys 35 40
45 Leu Ala Leu Val Leu Val Ala Ser Val Val Leu Gln Ala Val Leu Tyr
50 55 60 Pro Arg Leu Met Gly Thr Ile Leu Asp Val Lys Ser Asp Ala
Gln Leu 65 70 75 80 Leu Lys Gly Arg Val Asp Asn Ile Ser Thr Leu Gly
Ser Asp Leu Lys 85 90 95 Thr Glu Ser Gly Gly Val Asp Ala Ala Gly
Val Gln Ile Gln Ile Val 100 105 110 Asn Thr Ser Leu Gly Arg Val Arg
Ser Gln Ile Leu Ser Leu Glu Thr 115 120 125 Ser Val Glu Ile Ala Asn
Ala Gln Leu Leu Ile Leu Thr Arg Ser Trp 130 135 140 Gly Glu Val Ser
Ser Leu Ser Ala Gln Ile Pro Glu Leu Lys Ser Asp 145 150 155 160 Leu
Asp Lys Ala Ser Ala Leu Asn Thr Lys Val Gln Gly Leu Gln Gly 165 170
175 Ser Leu Glu Asn Val Ser Lys Leu Leu Lys Gln Gln Ser Asp Ile Leu
180 185 190 Glu Val Val Ala Gln Gly Trp Lys Tyr Phe Ser Gly Asn Phe
Tyr Tyr 195 200 205 Phe Ser Leu Ile Pro Lys Thr Trp Tyr Ser Ala Glu
Gln Phe Cys Val 210 215 220 Ser Arg Asn Ala His Leu Thr Ser Val Ser
Ser Glu Ser Glu Gln Glu 225 230 235 240 Phe Leu Tyr Lys Ala Ala Gly
Gly Leu Ile His Trp Ile Gly Leu Thr 245 250 255 Lys Ala Gly Ser Glu
Gly Asp Trp Ser Trp Val Asp Asp Thr Ser Phe 260 265 270 Asn Lys Val
Gln Ser Ala Arg Phe Trp Ile Pro Gly Glu Pro Asn Asn 275 280 285 Ala
Gly Asn Asn Glu His Cys Gly Asn Ile Lys Ala Ser Ala Leu Gln 290 295
300 Ala Trp Asn Asp Gly Pro Cys Asp Asn Thr Phe Leu Phe Ile Cys Lys
305 310 315 320 Arg Pro Tyr Val Gln Ser Thr Glu 325
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