Title: Nucleic acids encoding a cytokine receptor complex
Abstract: Nucleic acids encoding mammalian cytokine receptor, e.g., for cytokine IL-B50, purified proteins and fragments thereof. Antibodies, both polyclonal and monoclonal, are also provided. Methods of using the compositions for both diagnostic and therapeutic utilities are described.
Patent Number: 6,890,734 Issued on 05/10/2005 to Reche-Gallardo, et al.
| Inventors:
|
Reche-Gallardo; Pedro A. (Boston, MA);
Soumelis; Vassilli (Paris, FR);
Liu; Yong-Jun (Palo Alto, CA);
de Waal Malefyt; Rene (Sunnyvale, CA);
Bazan; J. Fernando (Palto Alto, CA);
Kastelein; Robert A. (Redwood City, CA)
|
| Assignee:
|
Schering Corporation (Kenilworth, NJ)
|
| Appl. No.:
|
008566 |
| Filed:
|
November 8, 2001 |
| Current U.S. Class: |
435/69.1; 435/71.1; 435/71.2; 435/252.3; 435/254.11; 435/320.1; 435/325; 435/471 |
| Intern'l Class: |
C12N 005/10; C12N015/12; C12N015/63; C12N015/64 |
| Field of Search: |
435/691,711,712,325,320.1,471,252.3,254.11
|
References Cited [Referenced By]
| Foreign Patent Documents |
| WO 99 4753/8 | Sep., 1999 | WO.
| |
| WO 00 1736/2 | Mar., 2000 | WO.
| |
Other References
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P.C.L. Beverley & B. Grubeck-Loebenstein, Vaccine, 18(16):1721-1724, Feb.
25, 2000. "Is Immune senescence reversible?".
Laurel M. Bolin, et al., J. Neurosci, 17(14):5493-5502, Jul. 15, 1997.
"HNMP-1: a novel hematopoietic and neural membrane protein differentially regulated
in neural development and injury".
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"Differential cytokine and chemokine gene expression by human NK cells following
activation with IL-18 or IL-15 in combination with IL-12: implication for the innate
immune response".
Norimitsu Kadowaki, et al., J Exp Med, 192(2):219-226, Jul. 17, 2000.
"Natural interferon alpha/beta-producing cells link innate and adaptive immunity".
Brandley A. Katz, Biomol Eng, 16(1-4):57-65, Dec. 31, 1999. "Streptavidin-binding
and -dimerizing ligands discovered by phage display, topochemistry, and structure-based design".
Anthony D. Kelleher & Sarah L. Rowland-Jones, Curr Opin Immunol, 12(4):370-374,
Aug. 2000. "Functions of tetramer-stained HIV-specific CD4(+) and CD8(+) T cells".
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approaches in retrovirus-mediated expression screening".
Bruce Koppelman, et al., Immunity, 7(6):861-871, Dec. 1997. "Interleukin-10
down-regulates MHC class II alphabeta peptide complexes at the plasma membrane
of monocytes by affecting arrival and recycling".
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Aug. 1998. "HLA-peptide tetrameric complexes".
Linda S. Park, et al., J Exp Med, 192(5):659-670, Sep. 4, 2000. "Cloning
of the murine thymic stromal lymphopoietin (TSLP) receptor: Formation of a functional
heteromeric complex requires interleukin 7 receptor".
Anne Puel & Warren J. Leonard. Curr Opin Immunol, 12(4):468-73, Aug. 2000.
"Mutations in the gene for the IL-7 receptor result in T(-)B(+)NK(+) severe combined
immunodeficiency disease".
John E. Sims. et al., J Exp Med, 192(5):671-680, Sep. 4, 2000. "Molecular
cloning and biological characterization of a novel murine lymphold growth factor".
Angela Stoddart, et al., Immunol Rev, 175:47-58, Jun. 2000. "The role
of the preBCR, the interleukin-7 receptor, and homotypic interactions during B-cell development".
T.A. Waldmann, Ann Oncol, Suppl 1:101-106, 2000. "T-cell receptors for
cytokines: targets for immunotherapy of leukemia/lymphoma".
Pandey, A. et al., "Cloning of a receptor subunit required for signaling by
thymic stromal lymphopoietin", Nature Immunology, United States Jul. 2000,
vol. 1, No. 1, Jul. 2000, pp. 59-64, XP008014551, ISSN 1529-2908, abstract: figures. 1,4,5,7.
Levin, S. D. et al., "Thymic stromal lymphopoeitin(TSLP): A
cytokine that promotes the development of IgM+ B cells in vitro and signals via
a novel mechanism", FASEB Journal, vol. 13, No. 4, Part 1, Mar. 12, 1999, p.
A322, XP008014578, Annual Meeting of the Professional Research Scientists for Experimental
Biology 99, Washington DC, USA, Apr. 17-21, 1999, ISSN 0892-6638, the whole document.
|
Primary Examiner: Mertz; Prema
Attorney, Agent or Firm: Ching; Edwin P., Mohan-Peterson; Sheela
Parent Case Text
This application claims benefit of U.S. Provisional Patent Applications No.
60/298,268, filed Jun. 14, 2001, and No. 60/247,218, filed Nov. 10, 2000.
Claims
1. An isolated or purified expression vector comprising a first polynucleotide
operably linked to a first promoter, wherein the first polynucleotide encodes a
polypeptide of the amino acid sequence sot forth in SEQ ID NO: 2, and a second
polynucleotide operably linked to a second promoter, wherein the second polynucleotide
encodes a polypeptide of the amino acid sequence as set forth in SEQ ID NO: 4.
2. An isolated or purified host cell comprising the expression vector of claim 1.
3. A method of making a heterodimeric receptor complex of the polypeptides of
amino acid sequences as set forth in SEQ ID NO: 2 and SEQ ID NO; 4, comprising
culturing the host cell of claim 2 under conditions suitable for expression of
the heterodimeric receptor complex.
4. The method of claim 3, wherein the heterodimeric receptor complex, when expressed,
binds a polypeptide of the amino acid sequence as set forth in residues 1-131 of
SEQ ID NO: 6.
5. An isolated or purified host cell transfected with a first expression vector
comprising a nucleic acid encoding a polypeptide of the amino acid sequence set
forth in SEQ ID NO: 2, and a second expression vector comprising a nucleic acid
encoding a polypeptide of the amino acid sequence set forth in SEQ ID NO: 4.
6. The host cell of claim 5, wherein the first expression vector and the second
expression vector are retroviral vectors.
7. The host cell of claim 6, wherein the retroviral vectors are pMX retroviral vectors.
8. A method of making a heterodimeric receptor complex comprising culturing the
host cell of claim 5 under conditions suitable for the expression of the heterodimeric
receptor complex.
9. The method of claim 8, wherein the heterodimeric receptor complex, when expressed,
binds a polypeptide of the amino acid sequence set forth in SEQ ID NO: 8.
Description
FIELD OF THE INVENTION
The present invention relates to compositions and methods for affecting mammalian
physiology, including hematopoietic cell proliferation or immune system function.
In particular, it provides methods of using nucleic acids, proteins, and antibodies
which regulate development and/or the immune system; and provides functional details
on ligand-receptor pairing. Diagnostic and therapeutic uses of these materials
are also disclosed.
BACKGROUND OF THE INVENTION
Recombinant DNA technology refers generally to techniques of integrating
genetic information from a donor source into vectors for subsequent processing,
such as through introduction into a host, whereby the transferred genetic information
is copied and/or expressed in the new environment. Commonly, the genetic information
exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA)
coding for a desired protein product. The carrier is frequently a plasmid having
the capacity to incorporate cDNA for later replication in a host and, in some cases,
actually to control expression of the cDNA and thereby direct synthesis of the
encoded product in the host.
For some time, it has been known that the mammalian immune response is based
on a series of complex cellular interactions, called the "immune network". Recent
research has provided new insights into the inner workings of this network. While
it remains clear that much of the immune response does, in fact, revolve around
the network-like interactions of lymphocytes, macrophages, granulocytes, and other
cells, immunologists now generally hold the opinion that soluble proteins, known
as lymphokines, cytokines, or monokines, play critical roles in controlling these
cellular interactions. Thus, there is considerable interest in the isolation, characterization,
and mechanisms of action of cell modulatory factors, an understanding of which
will lead to significant advancements in the diagnosis and therapy of numerous
medical abnormalities, e.g., immune system disorders.
Lymphokines apparently mediate cellular activities in a variety of ways.
They have been shown to support the proliferation, growth, and/or differentiation
of pluripotent hematopoietic stem cells into vast numbers of progenitors comprising
diverse cellular lineages which make up a complex immune system. Proper and balanced
interactions between the cellular components are necessary for a healthy immune
response. The different cellular lineages often respond in a different manner when
lymphokines are administered in conjunction with other agents.
Cell lineages especially important to the immune response include two classes
of lymphocytes: B-cells, which can produce and secrete immunoglobulins (proteins
with the capability of recognizing and binding to foreign matter to effect its
removal), and T-cells of various subsets that secrete lymphokines and induce or
suppress the B-cells and various other cells (including other T-cells) making up
the immune network. These lymphocytes interact with many other cell types. Monocytes
are precursors of macrophages which, with dendritic cells, are functionally important
in their roles as processors and presenters of antigen, an important step in initiation
of an immune response.
IL-7 is a cell modulatory factor which affects hematopoietic cell growth and/or
differentiation. See, e.g., Mire-Sluis and Thorpe (1998)
Cytokines Academic
Press, San Diego; Thomson (ed. 1998)
The Cytokine Handbook (3d ed.) Academic
Press, San Diego; Metcalf and Nicola (1995)
The Hematopoietic Colony Stimulating
Factors Cambridge University Press; and Aggarwal and Gutterman (1991)
Human
Cytokines Blackwell.
Research to better understand and treat various immune disorders has been
hampered by the general inability to maintain cells of the immune system in vitro.
Immunologists have discovered that culturing many of these cells can be accomplished
through the use of T-cell and other cell supernatants, which contain various growth
factors, including many of the lymphokines.
From the foregoing, it is evident that the understanding of the signal transduction
pathways and identification of components in such pathways should contribute to
new therapies for a wide range of degenerative or abnormal conditions which directly
or indirectly involve development, differentiation, or function, e.g., of the immune
system and/or hematopoietic cells. Furthermore, soluble regulatory molecules, including
cytokines, are known to sometimes act outside the immune system with effects on
physiology (leptin), morphogenesis, and tissue and skeletal remodeling (RANKL).
Thus, the discovery and understanding of novel cytokine-like molecules and their
receptors which enhance or potentiate the beneficial activities of other lymphokines
would be highly advantageous.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery of the receptor complex
for the cytokine ligand IL-B50. Moreover, identification of the components allows
for the identification of cell types and stages which express the necessary components
to be responsive to ligand. This provides insights and capacity to predict the
physiological and immunological role of the cytokine.
The present invention provides, e.g., methods of producing a ligand:receptor
complex, comprising contacting: a substantially pure or recombinant mammalian IL-B50
with a receptor comprising the IL-7Rα. or the Rδ2 subunit; a mammalian
IL-B50 with a receptor comprising a substantially pure or recombinant IL-7Rα
subunit; or a mammalian IL-B50 with a receptor comprising a substantially pure
or recombinant Rδ2 subunit; which contacting thereby allows the complex to
form. In preferred embodiments, the mammalian IL-B50 is primate IL-B50, such as
human IL-B50; the complex formation results in signal transduction, STAT activation,
or TARC expression; the receptor is on a cell; the receptor comprises both IL-7Rα
and Rδ2 subunit; the complex formation results in a physiological change
in the cell expressing the receptor; the contacting is in combination with a proliferative
agent, cytokine, or chemokine; the contacting allows quantitative detection of
the ligand; or receptor is on a hematopoietic cell, including a lymphoid lineage
cell, a myeloid cell such as a monocyte, or dendritic cell.
Another method is provided for modulating physiology or development of an
IL-7Rα or Rδ2 expressing cell comprising contacting the cell to an
exogenous agonist or antagonist of a mammalian IL-B50. Various embodiments include
those wherein: the antagonist is an antibody which neutralizes the mammalian IL-B50,
a mutein of the IL-B50; or an antibody which binds to IL-7Rα or Rδ2
or a complex of both; or the physiology is selected from proliferation, lymphoid
lineage cell development, antigen presentation, or production of inflammatory mediators,
including cytokines, chemokines, or adhesion molecules; or the cell is a hematopoietic
cell. Other embodiments include those wherein: the antagonist is an antibody and
the physiology is hematopoietic cell proliferation; the agonist is IL-B50 and the
physiology is hematopoietic cell differentiation; the physiology is antigen presentation;
or the modulating is blocking, and the physiology is lymphoid lineage cell proliferation.
Other embodiments provide methods of modulating a signal to a cell mediated
by IL-B50 comprising contacting the cell to an administered agonist or antagonist
of IL-B50. These include those wherein the modulating is inhibiting, and the signal
is a proliferation signal, the antagonist is a neutralizing antibody to IL-7Rα
or the Rδ2 subunit or a complex comprising the subunits; the agonist or antagonist
is administered in combination with another antagonist or agonist of IL-B50; the
agonist or antagonist is administered in combination with a growth factor, cytokine,
chemokine, or immune adjuvant; or the contacting is with another anti-proliferative
agent or treatment.
Other methods include those of selectively labeling a population of cells,
the method comprising contacting the cells with an antibody which binds: IL-7Rα;
Rδ2; or a complex comprising one of the subunits; thereby resulting in the
identification of cells expressing the subunit or complex. Certain embodiments
include those wherein: the contacting results in modulation of STAT activation;
the labeling allows purification of IL-7Rα or Rδ2 subunit expressing
cells; or the labeling allows depletion of IL-7Rα or Rδ2 subunit expressing
cells. Also provided are populations of cells made by the methods, including those
which are prepared by Fluorescent Activated Cell Sorting.
The invention further provides methods of testing a compound for ability to affect
receptor-ligand interaction, the method comprising comparing the interaction of
a receptor complex comprising IL-7Rα and/or Rδ2 subunit with IL-B50
in the presence and absence of the compound. In certain embodiments, the compound
is an antibody which binds one of: IL-7Rα; Rδ2 subunit; a receptor
comprising IL-7R and/or Rδ2; or IL-B50.
Certain compositions are provided, e.g., an isolated or recombinant protein
complex comprising: at least 15 contiguous amino acid residues of SEQ ID NO: 2
and at least 15 contiguous amino acid residues of SEQ ID NO: 4; at least two distinct
segments of at least 8 contiguous amino acid residues of SEQ ID NO: 2 and at least
two distinct segments of at least 8 contiguous amino acid residues of SEQ ID NO:
4; or at least one segment at least 21 contiguous nucleotides of SEQ ID NO: 1 and
at least one segment at least 21 contiguous nucleotides of SEQ ID NO: 3. In preferred
embodiments, one of the segments of SEQ ID NO: 2 is from the extracellular portion
of the sequence; one of the segments of SEQ ID NO: 4 is from the extracellular
portion of the sequence; or the polypeptide comprises the mature SEQ ID NO: 2 and
the mature SEQ ID NO: 4 sequences.
Nucleic acid embodiments include an isolated or recombinant polynucleotide
encoding described components of the complex, wherein: the polynucleotide comprises
a deoxyribonucleotide; the polynucleotide comprises a ribonucleotide; or at least
one of the segments is operably linked to a promoter.
Antibodies are also provided which recognize epitopes presented by the
complex, e.g., a binding compound comprising an antigen binding portion from an
antibody which binds with selectivity to a polypeptide comprising at least 12 contiguous
amino acid residues of SEQ ID NO: 2 and at least 12 contiguous amino acid residues
of SEQ ID NO: 4.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-1E show the nucleotide and amino acid sequences (see SEQ ID NO: 1
and 2) of a primate, e.g., human, IL-7Rα; predicted signal cleavage site indicated.
FIGS. 2A-2E show the nucleotide and amino acid sequences (see SEQ ID NO: 3
and 4) of a primate, e.g., human, Rδ2; predicted signal cleavage site indicated.
FIGS. 3A and 3B show the amino acid and nucleotide sequences, respectively,
of primate, e.g., human, IL-B50; predicted signal cleavage position indicated.
FIGS. 4A-4E show expression levels of hIL-7Rα (FIGS. 4A-4B),
Rδ2 (hTSLPR, FIGS. 4C-4D), and IL-B50 in various tissues and
cell types. Expression levels were normalized and expressed as femtograms mRNA
per 50 ng total cDNA.
FIG. 5 shows the induction of TARC by IL-B50. Human CD11c+ DC were cultured
in the absence or the presence of IL-B50 (50 ng/ml) and the production of TARC
was determined in the culture supernatant by ELISA.
FIG. 6A depicts a culture of sorted CD11c+ DC after 24 h in medium alone. DC
form small and irregular clumps with a dark center, indicating the presence of
dying cells.
FIG. 6B depicts a culture of sorted CD11c+ DC from the same donor as in FIG.
6A, treated with 15 ng/ml of IL-B50. DC form larger and round clumps with fine
dendrites visible at the periphery, indicating the maturation of the DC.
FIG. 7 shows the surface phenotype of CD11c+ DC after 24 h of culture with and
without (medium alone) IL-B50 and shows the upregulation of HLA-DR, as well as
the costimulatory molecules CD40, CD80 and CD86. Results shown are from one representative
of four independent experiments.
FIGS. 8A-8C show the surface phenotype of DC after treatment with medium alone,
IL-B50, CD40-ligand (CD40L), IL-7 and LPS. IL-B50 is more potent than CD40-ligand
and IL-7 in upregulating costimulatory molecules CD40 and CD80.
FIG. 9 shows the results of a T cell proliferation assay using CD11c+ DC matured
for 24 h in medium or with IL-B50 (15 ng/ml) and cocultured with 5×10
4
allogenic CD4+CD45RA+ naïve T cells at increasing DC/T cell ratios.
Proliferation was assessed on day 6 by measuring [
3H]thymidine incorporation.
Each point represents the mean [
3H]thymidine incorporation of triplicate
cocultures. Vertical bars indicate the SD. DC alone (□) were used as a control
and did not significantly proliferate. Results shown are from one representative
of the two independent experiments.
FIG. 10 shows the results of a similar experiment as described for FIG. 9, using
DC matured in medium, IL-B50, CD40-ligand (CD40L), IL-7 and LPS.
FIGS. 11A-11E show the production of various cytokines (expressed as pg/ml)
by naïve CD4 T cells cocultured with DC matured in medium alone, IL-B50, CD40-ligand
(CD40L), IL-7 and LPS. FIG. 11A shows the effect on the production of IL-4; FIG.
11B shows the effect on the production of IL-13; FIG. 11C shows the effect on the
production of IFN-γ, FIG. 11D shows the effect on the production of IL-10
and FIG. 11E shows the effect on the production of TNF-α.
FIG. 12 shows the effect of DCs treated with medium alone, IL-B50, IL-7, and
CD40-ligand on CD8 T cell expansion.
FIG. 13 compares expression of perforin by human naïve CD8 T cells induced
by DCs treated with medium alone, IL-B50 or CD40-ligand.
FIGS. 14A-14C show the results of a comparison of IL-B50 with GM-CSF, IL-7,
CD40-ligand (CD40L) and medium alone as a control, to stimulate human DCs to produce
mRNA for various cytokines and chemokines. FIG. 14A shows effects on IL-1α,
IL-1β, IL-6, IL-12p40 and TNF-α. FIG. 14B shows effects on TARC, MDC
and MIP3-β. FIG. 14C shows effects on MCP-1, MCP-4, Rantes and MIG.
FIG. 15 shows the effect of IL-B50 on the induction of IL12p75 protein.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
OUTLINE
I. General
II. Activities
III. Nucleic acids
A. encoding fragments, sequence, probes
B. mutations, chimeras, fusions
C. making nucleic acids
D. vectors, cells comprising
IV. Proteins, Peptides
A. fragments, sequence, immunogens, antigens
B. muteins
C. agonists/antagonists, functional equivalents
D. making proteins
V. Making nucleic acids, proteins
VI. Antibodies
A. polyclonals
B. monoclonal, Kd
C. anti-idiotypic antibodies
D. hybridoma cell lines
VII. Kits and Methods to quantify ligand/receptor
A. ELISA
B. assay mRNA encoding
C. qualitative/quantitative
D. kits
VIII. Therapeutic compositions, methods
A. combination compositions
B. unit dose
C. administration
IX. Receptors
I. General
Before the present compositions, formulations, and methods are described,
it is to be understood that this invention is not limited to the particular methods,
compositions, and cell lines described herein, as such methods, compositions, and
cell lines may, of course, vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments.
As used herein, including the appended claims, singular forms of words such as
"a," "an," and "the" include their corresponding plural referents unless the context
clearly dictates otherwise. Thus, e.g., reference to "an organism" includes one
or more different organisms, reference to "a cell" includes one or more of such
cells, and reference to "a method" includes reference to equivalent steps and methods
known to a person of ordinary skill in the art, and so forth.
Unless otherwise defined, technical and scientific terms used herein have
the same meaning as commonly understood by a person of ordinary skill in the art
to which this invention belongs. Although methods and materials similar or equivalent
to those described herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. Publications, patent
applications, patents, and other references discussed above are provided solely
for their disclosure prior to the filing date of the present application. Nothing
herein is to be construed as an admission that the invention is not entitled to
antedate any such disclosure by virtue of its prior invention. Publications, patent
applications, patents, and other references mentioned herein are incorporated by
reference in their entirety including all figures and drawings.
The present invention is based on the discovery of the receptor subunits for
the IL-B50 cytokine. This allows advantageous coproduction of the subunits on vectors,
production of fusion proteins, and antibody preparations which recognize epitopes
resulting from the interaction of the subunit components into a functional unit.
IL-7 biology is reasonably well described. See, e.g., Stoddart, et al. (2000)
Immunol. Rev. 175:47-58; Puel and Leonard (2000)
Curr. Opin. Immunol.
12:468-473; Akashi, et al. (2000)
Curr. Opin. Immunol. 12:144-150; Watanabe,
et al. (1999)
Immunol. Res. 20:251-259; Waldmann (2000)
Ann. Oncol. 11
Suppl 1:101-106; Beverley and Grubeck-Loebenstein (2000)
Vaccine 18:1721-1724;
Aspinall and Andrew (2000)
Vaccine 18:1629-1637; Appasamy (1999)
Cytokines
Cell Mol. Ther. 5:25-39; Hofmeister, et al. (1999)
Cytokine Growth Factor
Rev. 10:41-60; Or, et al. (1998)
Cytokines Cell Mol. Ther. 4:287-294;
Akashi, et al. (1998)
Immunol. Rev. 165:13-28; and Offner and Plum (1998)
Leuk. Lymphoma 30:87-99. Moreover, since the IL-7 receptor and the WL-B50
receptor share one subunit, the signaling pathways and biology should significantly
overlap. This is similar to the GM/IL-3/IL-5 family, which is one of the first
groups whose overlapping biologies were explained by the sharing of receptor subunits.
Additionally, recognition of the receptor subunits provides the opportunity
to determine cell types and developmental stages where the functional receptor
components are coordinately expressed. This provides the opportunity to determine
what cell types are likely to respond to ligand, and the resulting biological functions
mediated by those cells provides suggestions as to the physiological effects mediated
by the ligand. This leads to better understanding of therapeutic uses of the ligand
or blocking ligand:receptor interaction and signaling.
Some of the standard methods applicable 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 periodic supplements)
Current Protocols in Molecular Biology,
Greene/Wiley, New York; each of which is incorporated herein by reference. See
also U.S. Ser. No. 09/130,972, which is incorporated herein by reference.
A complete nucleotide (SEQ ID NO: 1) and corresponding amino acid sequence (SEQ
ID NO: 2) of a primate, e.g., human, IL-7Rα. coding segment is shown in FIG.
1; similarly for Rδ2 in FIG. 2. FIG. 3 provides sequence of primate,
e.g., human, IL-B50. See U.S. Ser. No. 09/399,492, which is incorporated herein
by reference.
For the IL-7Rα subunit, notable predicted features include, e.g., CK2 phosphorylation
sites at about S24-E27, T47-E50, S187-D190, S214-E217, S255-D258, S282-D285, S355-D358,
and S423-E426; peroxisomal motif at about S133-L135; PKC phosphorylation sites
at about T76-K78, T97-K99, S165-K167, S315-K317, S364-R366, and S373-K375; receptor
cyt2 motif at about G195-S201; cAMP kinase motif at about f342-S346; cAMP kinase
motif at about R343-S346 or R343-L367; GSK3 motifs at about T129-S133, S201-Y205,
T208-n212, S324-p330, T337-f341, S363-S367, and T413-S417; N glycosylation sites
at about N29-S31, N45-T47, N131-S133, N162-S164, N212-S214, N213-S215, N275-S277,
N353-S355, and N392-T394; tyrosine kinase motif at about E14-Y18; cAMP PK sites
at about K94-T97, K84-S87, R343-S346, Cas phos sites at about T208-K210, S215-E217,
T262-E264, S331-D333, T337-E339, and S367-D369 cyt C me sites at about T76-F79,
C98-I101, Q136-Y139, W244-R247, H259-T261 and C267-P270; histone methylation sites
at about F85-L88, Q172-L175, and P270-N273; myristoly sites at about G322-S326,
G352-A256, and G389-S392; Phos2 sites at about K184/S187, R343/S345, R343/S346,
and R371/S373; and PKC phos sites at about T76-K78, T97-K99, S165-K167, S315-K317,
S364-R366, and S373-K375.
Regions of particular interest from the Rδ2 sequence include predictions
of, e.g., CK2 phosphorylation sites at about T115-D118, S120-D123, S132-D135, S140-E143,
S242-D245, S284-E287, and T297-E300; peroxisomal localization motifs at about S218-F220
and A272-L274; PKC phosphorylation sites at about S76-R78, S99-K101, and S300-R302;
tyr phosphorylation sites at about R39/D43/Y46 and K166/E169/Y172; cAMP kinase
sites at about i77-T81 and R78-T82; Ca++ kinase site at about R78-h82; GSK3 sites
at about T23-S27, S99-v103, T113-S117, S132-t136, T136-S140, T199-p203, T201-p205,
T297-S301, and S301-1305; SigPase sites at about A195-A197 an A278-A280; Tyr kinase
site at about D70-Y74; cAMP PK sites at about R78-T81, K96-S99, K157-S160, and
K312-S315; Ca++ phosphatase sites at about T149-E151 and T309-E311; cyt C Me site
at about V235-F238; myristoly sites at about F3-G7 and G329-T333; N glycosylation
sites at about N25-S27, N33-T35, N79-T81, and N147-T149; phos2 sites at about K28/S30,
K96/S98, K96/S99, R104/S106, K206/S208, and K312/S315; PKC phosphorylation sites
at about S76-R78, S99-K101, and S301-R303; SPKK sites at about S99-h103 and S301-m303;
and Tyr Kinase sites at about R39-Y46.
In the IL-B50 sequence, the region from K97-K103 is known to be subject to is
proteolysis, and mutations may be targeted to that region to protect the ligand
from proteolytic degradation. Thus, pharmacokinetic properties of the ligand may
be modified, especially for the indications described herein.
Segments with boundaries adjacent these positions will be particularly useful,
as will polynucleotides encoding such segments. Mutagenesis in these regions will
be used to determine structure-activity relationship, particularly with the receptor,
as provided herein.
As used herein, the term "primate IL-7Rα" shall be used to describe a protein
comprising a protein or peptide segment having or sharing the amino acid sequence
shown in FIGS. 1A-1E (SEQ ID NO:2), or a substantial fragment thereof; but distinct
from rodent sequences. The invention also includes protein variations of the IL-7Rα
allele whose sequence is provided, e.g., a mutein agonist or antagonist. Typically,
such agonists or antagonists will exhibit less than about 10% sequence differences,
and thus will often have between 1- and 11-fold substitutions, e.g., 2-, 3-, 5-,
7-fold, and others. It also encompasses allelic and other variants, e.g., natural
polymorphic variants, of the protein described. "Natural" as used herein means
unmodified by artifice, found, e.g., in natural sources. Typically, it will bind,
when in a functional receptor complex, to ligand 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. The term shall also be used
herein to refer to related naturally occurring forms, e.g., alleles, polymorphic
variants, and metabolic variants of the primate protein. Corresponding meanings
apply to uses of terms related to the Rδ2 sequences.
This invention also encompasses proteins or peptides having amino acid sequence
homology with combinations of amino acid sequences presented in FIGS. 1 and 2.
In particular, it will include fusion constructs or proteins comprising segments
from both of the sequences provided.
A substantial polypeptide "fragment", or "segment", is 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.
Sequences of segments of different proteins can be compared to one another over
appropriate length stretches.
Amino acid sequence homology, or sequence identity, is determined by optimizing
residue matches, if necessary, by introducing gaps as required. See, e.g., 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 (GCG), Madison, Wis.; each of which is incorporated herein by reference.
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 intended to include natural allelic and interspecies variations
in the cytokine sequence. Typical homologous proteins or peptides will have from
50-100% homology (if gaps can be introduced), to 60-100% homology (if conservative
substitutions are included) with an amino acid sequence segment of FIGS. 1 or 2.
Homology measures will be at least about 70%, generally at least 76%, more generally
at least 81%, often at least 85%, more often at least 88%, typically at least 90%,
more typically at least 92%, usually at least 94%, more usually at least 95%, preferably
at least 96%, and more preferably at least 97%, and in particularly preferred embodiments,
at least 98% or more. The degree of homology will vary with the length of the compared
segments. Homologous proteins or peptides, such as the allelic variants, will share
most biological activities with the embodiments shown in FIGS. 1 and 2. As used
herein, the term "biological activity" is used to describe, without limitation,
effects on immune or other cell proliferation, differentiation, induction of cytokines
or chemokines (e.g., TARC, PARC, MDC, MIP3-b, etc.), effects on STAT3 and/or STAT5
mediated signal transduction, antigen presentation effects, changes in cell surface
molecule expression, and Th2 specific activities.
The terms ligand, agonist, antagonist, and analog of these receptors, particularly
the functional complex, include molecules that modulate the characteristic cellular
responses to the IL-B50, as well as molecules possessing the more standard structural
binding competition features of ligand-receptor interactions, e.g., where the receptor
is a natural receptor or an antibody. The cellular responses likely are mediated
through binding of IL-B50 to cellular receptors, as described. Also, a ligand is
a molecule which serves either as a natural ligand to which said receptor, or an
analog thereof, binds, or a molecule which is a functional analog of the natural
ligand. The functional analog may be a ligand with structural modifications, or
may be a wholly unrelated molecule which has a molecular shape which interacts
with the appropriate ligand binding determinants. The ligands may serve as agonists
or antagonists, see, e.g., Goodman, et al. (eds.) (1990)
Goodman & Gilman's:
The Pharmacological Bases of Therapeutics, Pergamon Press, New York.
Rational drug design may also be based upon structural studies of the molecular
shapes of a receptor or antibody and other effectors or ligands. Effectors may
be other proteins which mediate other functions in response to ligand binding,
or other proteins which normally interact with the receptor. 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.
II. Activities
The IL-B50 proteins have a number of different biological activities based on
coexpression of IL-7Rα and Rδ2, e.g., in the immune system, and include
proliferative, developmental, or physiological functions, in particular of lymphoid
lineage cells, e.g., macrophages or dendritic cells. The IL-B50 proteins are homologous
to other IL-7 ligand family proteins, but each have structural differences. For
example, human IL-B50 shows 43% amino acid sequence identity to mouse TSLP. Additionally,
the human receptor subunit Rδ2 displays 39% amino acid sequence identity
to mouse TSLPR.
The mouse IL-B50 molecule has the ability to stimulate TARC production and various
Th2 specific cytokines. The signaling pathway seems to use STAT3 and/or STAT5,
and sends proliferation or differentiation signals. Differentiation tends to result
in limitation of proliferation, and vice versa. Differentiation typically results
in changes in cell surface marker expression
As shown herein, human IL-B50 improves dendritic cell survival in cultures, upregulates
the expression of costimulatory molecules and adhesion molecules, including HLA-DR,
CD40, CD80, CD86, CD11a, CD18 and CD83, induces dendritic cells to produce the
chemokines TARC, PARC and MDC, and strongly promotes the capacity of dendritic
cells to induce naive T cells to proliferate and to produce cytokines IL-4, IL-13,
and TNF-alpha. Additionally, IL-B50 has a synergistic effect with CD40-ligand and
LPS in activated dendritic cells to upregulate costimulatory molecules CD40, CD80
and CD86. IL-B50 also strongly enhances CD40-ligand-induced production of IL-12
by dendritic cells.
TARC, PARC and MDC are all notably ligands for CCR4, a chemokine receptor predominantly
found on Th2-type lymphocytes. Thus, IL-B50 can activate myeloid cells, such as
monocytes, to release chemokines that may attract effector cells with a Th2 phenotype.
As shown in the examples below, IL-B50-induced expression of TARC was very strong
in the CD11c subset of DCs. This subset, representing less than 1% of mononuclear
cells in the blood, normally differentiates into mature DCs in response to inflammatory
stimuli. The expression of TARC in these cells was accompanied by a dramatic enhancement
of their maturation as evidenced by the strong induction of the costimulatory molecules
CD40 and CD80. These results indicate that this DC subset stimulated with IL-B50
could be a potent inducer of primary T-cell-mediated immune responses. Indeed,
CD11+ DCs cultured in the presence of IL-B50 are much more potent in their capacity
to elicit the proliferation of naïve T cells as compared to DC cultured in medium.
Dendritic cells are professional antigen presenting cells, which are capable
of inducing primary antigen-specific T cell-mediated immune responses. Dendritic
cells play a critical role in initiating immune responses against tumors and infectious
microorganisms. Dendritic cells are also involved in autoimmune diseases, allergic
diseases, graft-versus-host disease and rejection of solid organ transplants. Therefore,
enhancing dendritic cell function allows for treatment of tumors and infectious
diseases. Similarly, blocking dendritic cell function provides therapies for autoimmune
diseases, allergic diseases, graft-versus-host diseases and transplantation associated rejection.
Thus, IL-B50 may be used in enhancing dendritic cell function in treating cancers
and infectious diseases and IL-B50 antagonists may be used in blocking the function
of dendritic cells in treating autoimmune diseases, allergic diseases, graft-versus-host
diseases and transplantation associated rejection. The elucidation of the IL-B50
receptor subunits, therefore, allows for the identification of agonists and antagonists
of ILB-50 for use in treating the aforementioned diseases.
The present disclosure also describes new assays for activities described for
these molecules. Corresponding activities should be found in other mammalian systems,
including primates. The new IL-7-like molecules produced by recombinant means exhibit
a biological activity of modulating lymphoid lineage cells. Furthermore, there
is substantial likelihood of synergy with other IL-7 related agonists or antagonists.
It is likely that the receptors, which are expected to include multiple different
polypeptide chains, exhibit species specificity for their corresponding ligands.
III. Nucleic Acids
This invention contemplates use of isolated nucleic acid or fragments, e.g.,
which encode these or closely related proteins, or fragments thereof, e.g., fusion
proteins or coordinately expressed or combination expression constructs.
The term "isolated nucleic acid or fragments" as used herein means a nucleic
acid, e.g., a DNA or RNA molecule, that is not immediately contiguous with sequences
present in the naturally occurring genome of the organism from which it is derived.
Thus, the term describes, e.g., a nucleic acid that is incorporated into a vector,
such as a plasmid or viral vector; a nucleic acid that is incorporated into the
genome of a heterologous cell (or the genome of homologous cell, but at a site
different from that at which it normally occurs); and a nucleic acid that exists
as a separate molecule, e.g., a DNA fragment produced by PCR amplification or restriction
enzyme digestion, or an RNA molecule produced by in vitro transcription. The term
also describes a recombinant (e.g., genetically engineered) nucleic acid that forms
part of a hybrid gene encoding additional polypeptide sequences that can be used,
e.g., in the production of a fusion protein. In addition, this invention embodies
any engineered or nucleic acid molecule created by artifice that encodes a biologically
active protein or polypeptide having characteristic IL-B50 receptor activity. Typically,
the nucleic acid is capable of hybridizing, under appropriate conditions, with
nucleic acid sequence segments shown in FIGS. 1 and 2. Further, this invention
covers the use of isolated or recombinant nucleic acid, or fragments thereof, which
encode proteins having fragments which are homologous to the newly disclosed receptor
complex proteins. The isolated nucleic acids can have the respective regulatory
sequences in the 5′ and 3′ flanks, e.g., promoters, enhancers, poly-A
addition signals, and others from the natural gene.
An "isolated" nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer,
which is substantially pure, e.g., separated from other components which naturally
accompany a native sequence, such as ribosomes, polymerases, and flanking genomic
sequences from the originating species. The term embraces a nucleic acid sequence
which has been removed from its naturally occurring environment, and includes recombinant
or cloned DNA isolates, which are thereby distinguishable from naturally occurring
compositions, and chemically synthesized analogs or analogs biologically synthesized
by heterologous systems. A substantially pure molecule includes isolated forms
of the molecule, either completely or substantially pure.
An isolated nucleic acid will generally be a homogeneous composition of molecules,
but will, in some embodiments, contain heterogeneity, preferably minor. This heterogeneity
is typically found at the polymer ends or portions not critical to a desired biological
function or activity.
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 this intervention involves
in vitro manipulation, although under certain circumstances it may involve more
classical animal breeding techniques. 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 as found in their natural state. Thus, e.g., 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 a process 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 restriction enzyme sequence recognition site. Alternatively, the process is performed
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, e.g., encoding a fusion protein. 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, e.g., fusion, polypeptide. This will include a dimeric
repeat. Specifically included are synthetic nucleic acids which, by genetic code
redundancy, encode similar combination polypeptides to fragments of the receptor
subunits and fusions of sequences from various different receptors or related molecules,
e.g., growth factors.
A "fragment" in a nucleic acid context is a contiguous segment of at least about
17 nucleotides, generally at least 21 nucleotides, more generally at least 25 nucleotides,
ordinarily at least 30 nucleotides, more ordinarily at least 35 nucleotides, often
at least 39 nucleotides, more often at least 45 nucleotides, typically at least
50 nucleotides, more typically at least 55 nucleotides, usually at least 60 nucleotides,
more usually at least 66 nucleotides, preferably at least 72 nucleotides, more
preferably at least 79 nucleotides, and in particularly preferred embodiments will
be at least 85 or more nucleotides including, e.g., 100, 150, 200, 250, etc. Typically,
fragments of different genetic sequences can be compared to one another over appropriate
length stretches, particularly defined segments such as the domains described below.
A nucleic acid which codes for an IL-B50 receptor complex will be particularly
useful to identify genes, mRNA, and cDNA species which code for itself or closely
related proteins, as well as DNAs which code for polymorphic, allelic, or other
genetic variants, e.g., from different individuals or related species. Preferred
probes for such screens are those regions of the interleukin which are conserved
between different polymorphic variants or which contain nucleotides which lack
specificity, and will preferably be full length or nearly so. In other situations,
polymorphic variant specific sequences will be more useful.
This invention further covers recombinant nucleic acid molecules and fragments
having a nucleic acid sequence identical to or highly homologous to the isolated
DNA set forth herein. In particular, the sequences will often be operably linked
to DNA segments which control transcription, translation, and DNA replication.
These additional segments typically assist in expression of the desired nucleic
acid segment.
Homologous nucleic acid sequences, when compared to one another or to
the sequences shown in FIGS. 1 or 2, 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.
Comparative hybridization conditions are described in greater detail below.
Substantial identity 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 60% of the nucleotides, generally at least 66%, ordinarily
at least 71%, often at least 76%, more often at least 80%, usually at least 84%,
more usually at least 88%, typically at least 91%, more typically at least about
93%, preferably at least about 95%, more preferably at least about 96 to 98% or
more, and in particular embodiments, as high at about 99% or more of the nucleotides,
including, e g., segments encoding structural domains such as the segments described
below. Alternatively, substantial identity will exist when the segments will hybridize
under selective hybridization conditions, to a strand or its complement, typically
using a sequence derived from the sequences depicted in FIGS. 1 and 2. Typically,
selective hybridization will occur when there is at least about 55% homology over
a stretch of at least about 14 nucleotides, more typically at least about 65%,
preferably at least about 75%, and more preferably at least about 90%. See, Kanehisa
(1984)
Nuc. Acids Res. 12:203-213. 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, generally at least about 20 nucleotides,
ordinarily at least about 24 nucleotides, usually at least about 28 nucleotides,
typically at least about 32 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.
Stringent conditions, in referring to homology in the hybridization context,
will be stringent combined conditions of salt, temperature, organic solvents, and
other parameters typically controlled in hybridization reactions. Stringent temperature
conditions will usually include temperatures in excess of about 30 degrees C, more
usually in excess of about 37 degrees C., typically in excess of about 45 degrees
C, more typically in excess of about 55 degrees C., preferably in excess of about
65 degrees C, and more preferably in excess of about 70 degrees C. Stringent salt
conditions will ordinarily be less than about 500 mM, usually less than about 400
mM, more usually less than about 300 mM, typically less than about 200 mM, preferably
less than about 100 mM, and more preferably less than about 80 mM, even down to
less than about 20 mM. Certain detergents or destabilizing reagents may be added,
e.g., formamide at 50%, etc. 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, which is hereby incorporated herein by reference.
The isolated DNA 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 this protein or its derivatives.
These modified sequences can be used to produce mutant proteins (muteins) or to
enhance the expression of variant species. Enhanced expression may involve gene
amplification, increased transcription, increased translation, and other mechanisms.
Such mutant receptor-like derivatives include predetermined or site-specific mutations
of the protein or its fragments, including silent mutations using genetic code
degeneracy. "Mutant IL-B50 receptor" as used herein encompasses a polypeptide otherwise
falling within the homology definition of the receptor as set forth above, but
having an amino acid sequence which differs from that of other IL-7 receptor-like
proteins as found in nature, whether by way of deletion, substitution, or insertion.
In particular, "site specific mutant IL-B50 receptor" encompasses a protein having
substantial homology with a protein shown in FIGS. 1 and 2, and typically shares
most of the biological activities of the form disclosed herein.
Although site specific mutation sites are predetermined, mutants need not
be site specific. Mammalian IL-B50 receptor mutagenesis can be achieved by making
amino acid insertions or deletions in the gene, coupled with expression. 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 mammalian IL-B50 receptor
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. See also Sambrook, et al. (1989) and
Ausubel, et al. (1987 and periodic Supplements).
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.
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.
Polymerase chain reaction (PCR) techniques can often be applied in mutagenesis.
Alternatively, mutagenesis primers are commonly used methods for generating defined
mutations at predetermined sites. See, e.g., Innis, et al. (eds. 1990)
PCR Protocols:
A Guide to Methods and Applications Academic Press, San Diego, Calif.; and
Dieffenbach and Dveksler (eds. 1995)
PCR Primer: A Laboratory Manual Cold
Spring Harbor Press, CSH, N.Y.
IV. Proteins, Peptides
As described above, the present invention encompasses primate IL-B50 receptor,
e.g., whose sequences are disclosed in FIGS. 1 and 2, and described above. Allelic
and other variants are also contemplated, including, e g., fusion proteins combining
portions of such sequences with others, including epitope tags and functional domains.
The present invention also provides recombinant proteins, e.g., heterologous
fusion proteins using segments from these primate 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 a growth factor receptor with a
cytokine receptor is a continuous protein molecule having sequences fused in a
typical peptide linkage, typically made as a single translation product and exhibiting
properties, e.g., antigenicity, derived from each source peptide. A similar concept
applies to heterologous nucleic acid sequences.
In addition, new constructs may be made from combining similar functional or
structural
domains from other related proteins, e.g., growth factors or other cytokines. For
example, receptor-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 receptor-binding specificities. For example, the ligand binding domains from
other related receptor molecules may be added or substituted for other domains
of these or related proteins. The resulting protein will often have hybrid function
and properties. For example, a fusion protein may include a labeling epitope which
may serve to provide diagnostic locatability of the fusion protein for histology
or other methods.
Candidate fusion partners and sequences can be selected from various sequence
data bases, e.g., GenBank, c/o IntelliGenetics, Mountain View, Calif.; and BCG,
University of Wisconsin Biotechnology Computing Group, Madison, Wis., which are
each incorporated herein by reference.
"Derivatives" of the mammalian IL-B50 receptor include amino acid sequence
mutants, glycosylation variants, metabolic derivatives and covalent or aggregative
conjugates with other chemical moieties. Covalent derivatives can be prepared by
linkage of functionalities to groups which are found in the WL-B50 receptor amino
acid side chains or at the N- or C-termini, e.g., by means which are well known
in the art. These derivatives can include
