JP2002541210A - Novel use of mammalian OX2 protein and related reagents - Google Patents

Abstract

  (57) [Summary] Compositions and methods for using the mammalian ligand OX2 to treat an abnormal physiological condition in an individual. The method comprises administering a therapeutically effective amount of OX2, alone or in combination with other therapeutic reagents; or an OX2 antagonist. In one embodiment, the method of the present invention for modulating leukocyte trafficking or activation in an animal comprises administering to the myeloid lineage cells in the animal the following: a) an agonist of the mammalian OX2 protein; Contacting a therapeutic amount of the antagonist.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to methods of using proteins that function in controlling the physiology, development, and differentiation of mammalian cells (eg, cells of the mammalian immune or nervous system). In particular, the present invention relates to proteins and mimetics that regulate the physiology, development, differentiation, or function of cells of various cell types, including hematopoietic cells or neural cells.
mimetics).

BACKGROUND OF THE INVENTION The vertebrate immune system consists of many organs and several different cell types. 2
One major cell type includes the myeloid and lymphoid lineages. Of the lymphoid cell lineages, B cells were originally characterized as differentiating in fetal liver or adult bone marrow, and T cells were originally characterized as differentiating in the thymus. Another cell type is the mononuclear phagocyte, a cell line that is widely distributed through most tissues. Phagocytes play a role in inflammation, host defense, and response to a range of self and foreign substances. For example, Paul (ed., 19
97) Fundamental Immunology (4th edition) Raven
See Press, New York.

[0003] In many aspects of the development or regulation of an immune response or cell differentiation, soluble or membrane proteins play an important role in regulating cellular interactions. These proteins also mediate the activity of cells in many ways. These proteins have in many cases been shown to regulate the proliferation, growth, and differentiation of hematopoietic stem cells to a large number of progenitors that make up the lineage that can be responsible for the immune response.
Others are important mediators of intercellular signaling, often as receptors or ligands. These are also of considerable importance in immunological responses and physiology.

[0004] However, cellular molecules represented by differences in the stage of development of cells in these maturation pathways have not yet been completely identified. In addition, the role and mechanism of action of signaling molecules that induce, maintain or regulate the various physiological, developmental, or proliferative states of these cells is poorly understood. Obviously, the immune system and their response to various stresses are medicinal (eg, injuries, infectious diseases, cancer related responses and treatments, and cellular or other substances following allergic and transplant rejection responses). Substance clearance). For example, Thor
n et al., Harrison's Principles of Internal
Medicine McGraw / Hill, New York; Ziegl
(Ed., 1997) Growth Factors and Wound
Healing; Basic Science and Potential
Clinical Applications Springer Verla
g; Clark (ed., 1996) The Molecular and Cel
lulu Biology of Wound Repair Plenum
And Peacock (1984) Wound Repair Sounde.
See rs.

[0005] Medical science relies, for the most part, on the proper recruitment or suppression of the immune system in effecting the cure of an inadequate or inappropriate physiological response to environmental factors. However, the lack of understanding of how to regulate or differentiate the immune system has hindered the ability to beneficially modulate the immunological mechanisms (ie, response to biological damage) to a biological challenge. Thus, medical conditions characterized by abnormal or inappropriate regulation of the development or physiology of the associated cells have remained unmanageable. The discovery and characterization of specific regulatory pathways and their physiological effects contribute to the development of therapeutics for a wide range of degenerative or other conditions that affect biological systems, immune cells and other cell types. The present invention provides solutions to some of these and many other problems.

SUMMARY OF THE INVENTION The present invention is based, in part, on the discovery of the physiological role of the ligand OX2 (also referred to herein as the OX2 protein) in various models of the immune response. In particular, the role of ligand OX2 has been elucidated in pathways involved in infectious diseases, hematopoietic development and viral infection.

The present invention provides a method of modulating leukocyte trafficking or activation in an animal,
The method includes contacting a myeloid lineage cell (eg, a monocyte / macrophage) in an animal with a therapeutic amount of an agonist of a mammalian OX2 protein; or an antagonist of a mammalian OX2 protein. Preferred embodiments include when the mammalian OX2 protein is a primate protein; and / or the antagonist is an antibody that binds to mammalian OX2. In certain embodiments, when the myeloid lineage cells (eg, monocytes / macrophages) include macrophages, microglia, granulocytes or dendritic cells, or the animal comprises
Infectious state, inflammatory state, leukocyte proliferative state (leukoproliferative)
), A neurodegenerative condition, or a sign or symptom of a post-traumatic condition. Preferred embodiments include when the sign or symptom is nervous tissue; lymphoid tissue; bone marrow tissue; pancreas; gastrointestinal tissue; thyroid tissue; muscular tissue; or skin or collagenous tissue.

[0008] Other methods include when the modulation is to inhibit the function of white blood cells; and / or when an agonist is administered. Preferably, the agonist is mammalian OX2. In certain embodiments, the animal is an autoimmune; inflammatory condition; tissue-specific autoimmunity; degenerative autoimmunity; rheumatoid arthritis; atherosclerosis; multiple sclerosis; vasculitides; Hypersensitivity; skin transplantation; transplantation; spinal cord injury; stroke; neurodegeneration; or experiencing signs or symptoms of ischemia. Administration can be in combination with an agonist or antagonist of an anti-inflammatory cytokine; an analgesic; an anti-inflammatory; or a steroid.

[0009] Various other methods are provided, wherein the modulating is to enhance the function of white blood cells, and / or the antagonist is administered. Preferably, the antagonist is: an antibody that binds to mammalian OX2; or a mutein of mammalian OX2 that competes with mammalian OX2 for binding to the OX2 receptor, but does not substantially transmit a signal. In various embodiments, the method comprises:
Applies when experiencing signs or symptoms of infection, wound healing or clot formation. Administration is often performed in combination with an angiogenic factor; a growth factor (including FGF or PDGF); an antibiotic or antiviral reagent; or a clotting factor.

For example, various methods of modulating leukocyte activation in a tissue are provided, comprising a myeloid or monocyte / macrophage lineage cell in the tissue and an agonist of a mammalian OX2 protein; Contacting with an animal OX2 protein antagonist. Often, the modulation is to inhibit white blood cells, and the contact is with the agonist. Administration is often performed in combination with an agonist or antagonist of an anti-inflammatory cytokine; an analgesic; an anti-inflammatory; or a steroid. Alternatively, modulation is enhancing and contacting is with the antagonist. Administration can be in combination with an angiogenic factor; a growth factor (including FGF or PDGF); an antibiotic or antiviral agent; or a clotting factor.

(I. General) The OX2 antigen is a monoclonal antibody (mAb) MRC OX2
Was first characterized using For example, McMaster and William
ms (1979) Eur. J. Immunol. 9: 426-433; Barc
lay (1981) Immunology 44: 727-736; Barcl
ay (1981) Immunology 42: 593-600; Bukovs
(1984) Immunology 52: 631-640; and W.
ebb and Barclay (1984) J. Am. Neurochem. 43:10
See 61-1067. The use of this antibody in immunohistochemical (IHC) staining of tissue sections or cell suspensions for flow cytometry allows the OX2 antigen to be expressed in a wide variety of cells (eg, neurons, vascular endothelium, B cells,
Activated T cells, follicular dendritic cells, interdigitating dendritic cells, smooth muscle cells and trophoblasts have been shown to be expressed. further,
Human OX2 is known to be expressed in normal brain and by B cells. McCaughan et al. (1987) Immunogenetics 2
5: 329-335. MRC OX2 (Clark et al. (1985) EMBO J
. 4: 113-118), the characterization of the rat protein recognized by O
X2 was shown to consist of approximately 248 amino acids including two extracellular immunoglobulin (Ig) domains, a transmembrane domain and a short C-terminal cytoplasmic tail. This molecule is glycosylated through six N-linked glycosylation sites, of which three
One is in the N-terminal V-like Ig domain, and the other is in the C2-like Ig domain near the membrane. This makes OX2 an Ig superfamily (
IgSF) and enter CD2, CD48, CD58, CD80, CD86, CD9
Form a subgroup of small IgSF molecules with molecules like 0 and CD147. Interestingly, CD90 is also highly expressed by neurons. (1977) Cold Spring Harb. S
ymp. Quant. Biol. 41 Part 1: 51-61. Furthermore, that OX2 is a structural homolog of CD80 and CD86 (Borriello et al. (1)
997). Immunol. 158: 4548-4554) and that the OX2 gene is closely linked to the genes encoding CD80 and CD86 on chromosome 16 in mice (Borriello et al. (1998) Mamm. G).
enome 9: 114-118). Both CD80 and CD86 serve as ligands in a process known as co-stimulation, and therefore OX2 appears to act as a ligand. The OX2 antigen is hereinafter referred to as OX2 protein or ligand OX.
Called 2. The binding partner is called the OX2 receptor, which has not been fully characterized.

[0012] To identify the receptor for OX2 (OX2R), Barclay
Group prepared a multivalent reagent using rat OX2-rat CD4 fusion protein conjugated to fluorescent beads. This reagent was shown to bind to mouse and rat peritoneal macrophages. And this coupling is due to the mAb MRCO
It can be blocked by X88. Preston et al. (1997) Eur. J.
Immunol. 27: 1911-1918. This mAb was shown to bind to macrophages isolated from both the peritoneum and spleen, and in IHC on spleen sections, staining was found in areas known to contain a high proportion of macrophages .

[0013] Defective or exacerbated activation of macrophages contributes to the pathogenesis of a wide variety of immunological and other diseases. For example, McGee et al.
Oxford Textbook of Pathology, Oxf
ord University Press, Oxford; Lewis and McGee (ed., 1992) The Macrophage, IRL Pre.
ss, Oxford; and Bock and Good (ed., 1997) T
he Molecular Basis of Cellular Defen
See ce Mechanisms, Wiley & Sons.

[0014] The distribution of OX2 is consistent with the hypothesis that OX2 relays signals through the OX2R to macrophages and possibly to other cells of the bone marrow or monocyte-macrophage lineage. In this scenario, for example, the expression of OX2 on neurons is
One can establish a method of direct access to native macrophages of the brain, called microglia, which can express OX2R. Since they are from the monocyte-macrophage lineage. Perry and Gordon (1988) Trend
s Neurosci. 11: 273-277. MRC in brain section IHC
Using the OX88 mAb, it was not possible to identify molecules on microglia. However, this negative result can be caused by the fact that MRC OX88 is IgM, an antibody isotype commonly known to have low affinity.

To study the biological role of OX2, and in particular, to study whether the OX2-OX2R interaction is involved in regulating macrophage function, the mouse O
An X2 genomic clone was isolated from the C57BL / 6 genomic library. This allows the construction of a targeting vector, which can be used to construct C57BL
/ 6 knockout (KO) mice were generated by targeted disruption of the OX2 gene by homologous recombination in ES cells. Homozygous KO mice were bred and developed normally, but initial examination of internal organs showed anatomical abnormalities in some lymphoid tissues. These included swollen red pulp of the spleen and failure to separate mesenteric lymph nodes from the swollen marginal sinus. Both of these changes can contribute to expanded macrophages and, at least in the spleen, to an expanded granulocyte population. These results indicate that, even in steady state, OX2 is in myeloid cells (
It shows that the number of macrophages, for example, and their activation can be regulated, probably by OX2R ligation.

[0016] OX2 KO mice are now used in studies of myeloid cell or macrophage function, particularly monocyte / macrophage lineage activity, by applying a model system for the activation of cells of these cell lines. obtain. The first model system used for this purpose is the paradigm for microglial activation in the brain through nerve injury. Street and Graeber (1993)
Glia 7: 68-74. This model accounts for the fact that transection of the facial nerve, which directs motility behavior in the facial region, triggers microglial activation in the facial nucleus of the brainstem where motor neurons are located after 4-7 days. use. In OX2 KO mice, this activation occurs much earlier than in normal mice, already two days after surgery. This activation is achieved by the expression of the activation marker DAP12, as indicated by the IHC.

The results of both steady state and facial nerve transection are consistent with the hypothesis that ligation of OX2R on macrophages by OX2 results in downstream regulatory signals. This hypothesis has been studied in more detail and in vivo activation of cells of the monocyte-macrophage lineage (eg, intraperitoneal injection with LPS and quantification of serum levels of TNF).
Can be studied in different model systems. LPS in OX2 KO mice
The TNF response to challenge can be more robust, and macrophages in these mice lack certain downstream regulatory mechanisms.

If the postulated role of preserving the OX2-OX2R interaction is true, manipulating this interaction can have important clinical implications. In settings where macrophage activation is desired (eg, wound healing, some aspects of healing in CNS injury, etc.), inhibition of OX2 or use of an OX2R antagonist is beneficial. Release from typical inhibition causes a rapid or more pronounced activation. Enhanced granulocyte activation is also beneficial for controlling bacterial infections.

Conversely, in situations where macrophage activation is to be suppressed (eg, inflammation as seen in rheumatoid arthritis), activation of OX2R by agonists (eg, a multivalent form of recombination that can crosslink OX2R) Soluble OX2) may be useful. This delays active release or avoids release therefrom.

The following description, for exemplary purposes, relates to a primate (eg, human) or rodent (eg, mouse or rat) ligand OX2, but also related embodiments from other species Applicable to Thus, conditions known to be mediated by macrophages or conditions associated with macrophage function can be modulated using these reagents.

II. Nucleic Acids A general description of nucleic acids, manipulation of nucleic acids, and uses of nucleic acids (including, for example, complementary and antisense nucleic acids) can be found in the following: NCBI Entrez Accession Numbers (“
MRC OX-2 ”)

[0022]

[Table 1] (Each of which is incorporated by reference). Further aspects will be apparent to one of skill in the art in view of the teachings provided herein.

III. Purified Ligand OX2 Protein A general description of proteins and polypeptides in a pharmaceutical or biochemical context can be found, for example, in the following:

[0024]

[Table 2] Can be found in Specific descriptions of OX2 are described, for example, in WO 97/21450 (human); P41217 (human); P04218 (rat); and AAC15911.
NCBI Entrez registration number including (mouse) (search for MRC OX-2)
Is found in Recombinant methods for producing this protein are well known. Preparation of fragments by synthetic methods or by biochemical cleavage of natural or recombinant forms is available.

(IV. Production of OX2 Protein; Mimic) DNA encoding the ligand OX2 protein or a fragment thereof can be obtained by chemical synthesis, screening of a cDNA library, or genome prepared from a wide variety of cell lines or tissue samples. It can be obtained by screening libraries.

This DNA is, for example, as follows:

[0027]

[Table 3] (Each of which is incorporated herein by reference) can be expressed in a wide variety of expression systems.

[0028] Now that various ligand OX2 proteins have been characterized, fusion polypeptides, fragments thereof, or derivatives thereof can be prepared by conventional processes for synthesizing peptides. These include:

[0029]

[Table 4] (Each of which is incorporated herein by reference). Further aspects will be apparent to one of skill in the art in view of the teachings provided herein.

V. Physical Variants Proteins or peptides having substantial amino acid sequence homology to the amino acid sequence of the OX2 protein are also contemplated. Variants include species or allelic variants. Homology or sequence identity can be determined, for example, by:

[0031]

[Table 5] Is defined as

[0032] The isolated DNA encoding the OX2 protein can be obtained, for example, from Sambrook.
(1989); Ausubel et al. (1987 and addenda); Cunning.
Ham et al. (1989) Science 243: 1330-1336; O'Do
wd et al. Biol. Chem. 263: 15985-15992
And Carruthers (1981) Tetra. Letts. 22: 1
859-1862, each of which can be readily modified as described herein. Further methods will be apparent to one of skill in the art in light of the teachings provided herein.

VI. Functional Variants Inhibition of the physiological response to the ligand OX2 protein may be due to inhibition of binding of the ligand to its natural binding partner by a variant of native OX2, or OX2
Can result from inhibition of binding of the antibody to Methods for producing such variants include, for example:

[0034]

[Table 6] (Each of which is incorporated herein by reference). Further methods will be apparent to one of skill in the art in light of the teachings provided herein.

VII. Antibodies Antibodies are directed against various ligand OX2 proteins (including species or allelic variants) and fragments thereof, both in their naturally occurring forms and in their recombinant forms. Can be triggered. In addition, antibodies can be raised against the ligand OX2 protein in either the activated or inactivated form. Anti-idiotype antibodies are also contemplated. Methods of making antibodies and binding compositions, and their use, are described, for example, in:

[0036]

[Table 7] U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939
No. 3,996,345; No. 4,277,437; No. 4,
275,149; and 4,366,241; and Cabilly.
No. 4,816,567, each of which is incorporated herein by reference. Further methods will be apparent to one of skill in the art in light of the teachings provided herein.

VIII. Uses Mammalian OX2 reagents have a variety of therapeutic uses, for example, for the treatment of conditions or diseases where bone marrow or macrophage cell function or dysfunction is involved. These include, for example, wound healing, some aspects of healing CNS injuries, and inflammation as seen in rheumatoid arthritis. Administration of an effective amount of ligand OX2 will typically be at least about 100 ng / kg body weight;
At least about 1 μg / kg body weight; and often about 1 / kg body weight
less than 10 mg; or preferably less than about 10 mg / kg body weight. An effective amount adjusts the symptoms, or the onset of symptoms, typically to at least about 10%; usually to at least about 20%; preferably to at least about 30%; or more preferably to at least about 50%. The present invention provides a reagent. This reagent, for example,
In the general description of physiological or developmental abnormalities, or in the following description of the diagnostic kit, find use in further diagnostic and therapeutic applications as described elsewhere herein. For example, Berkow (ed.) The Merck Manual of Diagnostics and T
herpy, Merck & Co. Rahway, N .; J. Thorn et al.
Harrison's Principles of Internal Me
dicine McGraw-Hill, NY; Gilman et al. (eds., 1990)
) Goodman and Gilman's; The Pharmacolo
Gical Bases of Therapeutics 8th edition. , Per
gammon Press; (1990) Remington's Pharma
therapeutic Sciences (18th edition) Mack Publisher
ng Co. , Easton, Penn; Langer (1990) Science.
ce 249: 1527-1533; Merck Index, Merck & C
o. , Rahway, New Jersey; Avis et al. (Eds., 1993) Ph.
armaceuticalDosage Forms: Parental
Medicines, 2nd edition, Dekker, NY; Lieberman, et al. (Eds., 1990) Pharmaceutical Dosage Forms: T
Ablets Second Edition, Dekker, NY; Lieberman et al. (eds. 199
0) Pharmaceutical Dosage Forms: Disper
se Systems Dekker, NY: Fodor, et al. (1991) Sc.
issue 251: 767-773, Colligan Current Pr
ocotols in Immunology; Hood et al., Immunology.
gy Benjamin / Cummings; Paul (eds.) Fundamen
tal Immunology; Methods in Enzymology
Academic Press; Parce et al. (1989) Science.
246: 243-247; Owicki et al. (1990) Proc. Nat'l
Acad. Sci. USA 87: 4007-4011; and Blunde.
II and Johnson (1976) Protein Crystallog.
raphy, Academic Press, New York (each,
(Incorporated herein by reference). Further uses will be apparent to those skilled in the art in light of the teachings provided herein.

IX. Kit The present invention also contemplates the use of ligand OX2 protein, fragments thereof, and peptides. And their fusion products and related reagents can also be
low and Lane (1988) Antibodies: A Labor
atrial Manual CSH; U.S. Patent No. 3,645,090; U.S. Patent No. 3,940,475; Rattle et al. (1984) Clin. Chem.
30: 1457-1461; U.S. Pat. No. 4,659,678; and Via
llet et al. (1989) Progress in Growth Factor.
Res. 1: 89-97, each of which is useful in various diagnostic kits and methods for detecting the presence of the binding composition as described herein.

The broad scope of the present invention is best understood by reference to the following examples, which are not intended to limit the invention to particular embodiments.

EXAMPLES I. General Methods Several standard methods are described, for example, in Maniatis et al. (1982) Mole.
cultural Cloning; A Laboratory Manual Co
ld Spring Harbor Laboratory, Cold Spr
ing Harbor Press; Sambrook et al. (1989) Mole.
cultural Cloning: A Laboratory Manual (2nd
Edition) Volumes 1-3, CSH Press, NY; Ausubel et al. (1987 and addendum) Current Protocols in Molecular B
iology, Greene / Wiley, New York; or Inn
(ed., 1990) PCR Protocols: A Guide to
Methods and Applications Academic Pr
ess, N.M. Y. It is described in. Methods for protein purification include ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and the like. See, eg, Ausubel et al. (1987 and periodic addendum);
Oligan et al. (eds. 1995 and periodic addendum) Current Protocol
ols in Protein Science Wiley &Sons; De
utscher (1990) "Guide to Protein Purif"
ication "Methods in Enzymology, Vol. 182,
And other volumes in this series; and manufacturers in the use of protein purification products (eg, Pharmacia, Piscataway, NJ or B
io-Rad, Richmond, CA). Combination with recombinant techniques allows fusion with a suitable segment (eg, FLAG sequence or equivalent) that can be fused via a protease-removable sequence. For example, Hochuli (1990) "Purification of Reco"
mbinant Proteins with Metal Chelate
Absorbent "Setlow (eds.) Genetic Engineeri
ng, Prinple and Methods 12: 87-98, Ple
num Press, N.M. Y. And Crowe et al. (1992) QIAexp.
ress: The High Level Expression & Pro
tein Purification System QUIAGEN, Inc
. , Chatsworth, CA.

FACS analysis was performed using the method of Melamed et al. (1990) Flow Cytometry.
y and Sorting Wiley-Liss, Inc. , New Yo
rk, NY; Shapiro (1988) Practical Flow Cy
Tomory Lis, New York, NY; and Robinson
(1993) Handbook of Flow Cytometry Me.
described in Wiley-Lis, New York, NY.

II. Isolation of DNA Clones Encoding Ligand OX2 Protein Isolation of the human ligand OX2 was performed as described by McCaughan et al. (1987) Immun.
genetics 25: 329-335. Standard hybridization methods can be used, or PCR primers can be constructed to isolate clones. Entr for both nucleotide and amino acid sequences
The ez registration number is provided above.

Various cells are screened using appropriate probes for high level message expression. And the expression distribution is public. Appropriate cells are selected as a source for cDNA cloning using, for example, standard methods of PCR or hybridization.

Using standard PCR techniques, the OX2 gene sequence, or
OX2 or fragment is amplified from cDNA derived from RNA. Appropriate primers are selected from the sequences described and full-length clones are isolated. Various combinations of primers of various lengths and potentially sequence differences can be prepared. The full-length clone can be used as a hybridization probe to screen for other homologous genes. This employs stringent or low stringency hybridization conditions.

In another method, libraries are screened using oligonucleotides. In combination with the polymerase chain reaction (PCR) technique, the correct clone is selected from the library using the synthetic oligonucleotide in the appropriate direction as a primer.

III. Large Scale Production of OX2 For in vitro or in vivo biological assays, for example, 1% Nut
lidoma HU (Boehringer Mannheim, Mannhe
transformed CO grown in RPMI medium supplemented with B. im, Germany).
The OX2 or OX2-E tag is produced in large quantities using S-7 cells and subsequently purified. An adenovirus expression system may be used.

[0047] Recombinant proteins can be purified using standard procedures. Affinity chromatography of the epitope-tagged fusion protein may be utilized.

IV. Purification of Antibodies Specific for OX2 Inbred Balb / c mice, eg, 1 ml of purified OX2, were added on day 0, in complete Freund's adjuvant, and on days 15 and 22. Immunize by emulsification in incomplete Freund's adjuvant. 0.5 ml of purified OX
2 is boosted by intravenous administration.

For example, hybridomas are produced using the non-secreting myeloma cell line SP-2 / 0-Ag8 and polyethylene glycol 1000 (Sigma, St. Louis, MO) as the fusion factor. Hybridoma cells were transferred to a 96-well Falc
on tissue culture plates (Becton Dickinson, NJ) and 80 μg / ml gentamicin, 2 mM glutamine, 10% horse serum (Gi
bco, Gaithersburg, MD), 1% ADCM (CRTS, Lyo
n, France) 10 ^-5 M Azaserine (Sigma, St. Louis, MO)
) And DMEM F12 (Gibc) supplemented with 5 × 10 ⁻⁵ M hypoxanthine
o, Gaithersburg, MD). For the production of antibodies against OX2, for example, by immunocytochemistry (ICC) using acetone-fixed OX2-transformed COS-7 cells and / or by ELISA using OX2 purified from COS-7 supernatant as coating antigen. Screen the supernatant. Aliquots of positive cell clones were expanded for 6 days and cryopreserved,
And pristane (2,6,10,14-tetramethylpentadecane, Sigma
a, ST. (Louis, MO) treated with ascites from Balb / c mice (15 days prior to receiving an intraperitoneal injection of pristane). Approximately 10 ⁵ hybridoma cells in 1 ml of PBS are administered intraperitoneally, and 10 days later, ascites is collected from each mouse.

After this ascites centrifugation, ammonium sulfate precipitation and 20 mM Tris p
Zephyr-D silicon column (IBF Seprac) equilibrated with H8.0
or) by anion exchange chromatography above. The protein is eluted with a NaCl gradient (NaCl over 0-1M). Two ml fractions can be collected and tested for the presence of anti-OX2 antibody by ELISA. Fractions with specific anti-OX2 activity are pooled, dialyzed and frozen.

(V. Production of OX2-Deficient Mice) OX2 knockout (KO) mice were prepared according to the method of Galli-Taliadoros et al. mmunol. Methods 181: 1-15; Korn
or et al. (1997) Eur. J. Immunol. 27: 2600-2609;
And Lemckert et al. (1997) Nucl. Acids Res. 25:
Made essentially according to the procedure described in 917-918. Briefly, C57
The BL / 6 genomic library was screened using the PCR fragment of mouse OX2 cDNA as a probe. The isolated genomic clone contained an approximately 16 kB insert from which a 9.5 kB Sall fragment was subcloned into pBluescript. This clone is
Portions of intron I, exon II (encoding a signal peptide), intron II, exon III (encoding a V-like lg domain), intron II
I, exon IV (encoding a C2-like lg domain), and intron IV
Code part. From this clone, a targeting construct was created by replacing the Ncol fragment encoding the C-terminal portion of the V-like lg domain with a neomycin cassette and shortening the upstream portion of this clone,
It contained only the 3 'portion of the exon encoding the signal peptide. C57
ES cell line derived from BL / 6J mouse (Bruce 4: Galli-Taliad)
oros et al. (1995) J. Mol. Immunol. Methods 181: 1-1
5 and Lemcket et al. (1997) Nucl. Acids. Res. 25:
917-918) were transfected by electroporation and G418 resistant colonies were isolated and screened for homologous recombination by PCR and Southern blot. One homologous recombinant from 1,000 clones was isolated and used to generate chimeric mice.
Lemckert et al. (1997) Nucl. Acids. Res. 25: 917
See -918. Male chimeras were bred to female wild-type C57BL / 6J mice and progeny with black coat color (indicating germline transmission) were screened for the presence of the target allele. Heterozygous F1 mice were cross-bred to obtain heterozygous knockout mice. Using this knockout mouse, pure C57BL / 6. OX2-/-bleeding colonies were established. Gender-matched wild-type C57BL / 6J mouse generation (age a sex-mat)
(child wild type C57BL / 6J mice) was used as a control in all studies.

VI. Initial Observations on OX2-/-(Knockout Mice) Analysis of OX2 KO versus wild-type (wt) mice included a macroscopic analysis of organ structure. At the macroscopic level, the organ structures appeared normal, except for the mesenteric lymph nodes (MLN), which were "fused" together into one long tubular structure. In wt mice, normal MLN structure is characterized by distinct lymph nodes connected by lymphatic vessels in a "one-pearl" configuration. The spleen was slightly enlarged, as were some lymph nodes. Differences became more apparent at the histological level upon staining for various leukocyte antibodies. In particular, the red pulp of the spleen of OX2 KO mice was enlarged (but not edematous) and appeared to be filled with F4 / 80 + cells (ie, actually macrophages). Subpopulations of metallophilic macrophages (MOMA-1 +) surrounding B cell vesicles of the spleen were also
Increased by a factor of two. Gr-1 + cells (eg, granulocytes) also have about two factors,
There were more in the spleen of OX2 KO mice. The white pulp region was of normal size. Thus, there appears to be a relative expansion of myeloid lineage cells (including macrophages) in the spleen that could allow for an increase in size. MLN "
The "vessel" consists of distinct lymph node structures that are distinct, but each is attached (fused) together with what is a paracortical or subcapsular area from the enlarged cortex. And this is also F4 / 8
Positive for 0 + / MOMA-1 + macrophages. The cells appeared to be growing and activated and were MHC class II +.

Dissection of CNS (stained for microglia), resident CNS macrophages (with antibodies against Mac-1 (CD11b)) from wild-type (wt) and OX2 KO mice. The major findings were: (i) In the spinal cord, the number of microglia was OX up to about 20% compared to wt mice.
It appears to be increased in 2 KO mice.

(Ii) Small loci of microglia, even structures that occasionally mimic microglia clustering associated with neuritic amyloid plaques, were observed in the OX2 KO spinal cord. Such loci are never found in the normal healthy wt CNS and show microglial activation, proliferation or clustering. The level of CD45 expression is generally increased on microglia of OX2 KO mice.
CD45 is usually low in normal microglia, but is increased upon activation. See Sedgwick et al. (1991) Proc. Nat'l Acad. Sci.
ScL USA 88: 7438-7442; Sedgwick et al. Ex
p. Med. 177: 1145-1152; Ford et al. (1995) J. Mol. Imu
nol. 154: 4309-4321; Ford et al. Exp. M
ed. 184: 1737-1745; Sedgwick and Hickkey, K.
eane and Hickkey (ed.) Immunology of the Ne
rvous System Oxford Press, New York; and Sedgwick et al. Immunol. 160: 5320-
See 5330.

These findings are consistent with the view that OX2 deficiency, in this case on neurons, leads to some dysregulation of resident macrophages, which are resident microglia of the CNS. .

The collective implication from our studies in OX2 KO mice is that deficiency of this molecule generally releases myeloid cells, and especially macrophage lineage cells, from normal regulation even at steady state. Met. Thus, in situations where macrophage activation and proliferation is enhanced (eg, in pathological conditions), OX2 deficiency can lead to a greater or more rapid increase in macrophage activation.

To test this hypothesis, a model of macromerge activation was chosen. First
Was a facial nerve transection model to study CNS macrophage (microglia) activation in the downstream facial nuclei following transection of the facial nerve. Stre
See it and Graeber (1993) Glia 7: 68-74. This model shows that the damaging effect of nerve transection on neurons (which are OX2-positive) leads to a subsequent response by microglia (which are OX2-R-positive) in the facial nucleus This is appropriate in the case of the present invention which is known to be This response can be tested by immunohistological assessment of the facial nucleus.

According to this hypothesis, in the absence of OX2 on neurons in OX2 KO mice, microglial responses are expected to be more rapid and of greater magnitude. Indeed, this was found, in particular, two days after transection, microglial activation was already evident in OX2 KO, but not in wt mice. In addition, 4 between the wt and OX2 KO mice
Day differences were more apparent. By day 7, microglial activation was the same in both types of mice.

This experiment demonstrates that OX from non-macrophage lineage cells (in this case, neurons)
Two signals provide direct evidence that they are involved in macrophage regulation.

In a second model, mice were parenterally injected with lipopolysaccharide (LPS), which is known to induce rapid macrophage activation. Quantification of serum TNF production within 90 minutes is useful as a measure of macrophage activation. OX
2 KO mice should respond to much lower doses of LPS and with increased production of TNF. This correlation has been confirmed; in certain cases, TNF production in these mice is 2-4 times greater than in wild-type mice.
It was twice as expensive. OX2 KO mice show an earlier and more accelerated onset of EAE compared to wt mice. The disease is ultimately less severe than the wt mouse, so that, similar to microglia, the onset is faster, but does not ultimately exceed that in the wt mouse. Elimination of the OX2 interaction with its reporter enhances the macrophage response, leading to more serious or faster onset of disease. The opposite effect is
Typically, it is therapeutically desired.

Thus, when macrophages are stimulated within or outside the CNS,
The OX2-negative environment leads to increased macrophage activity and function.

[0062] All references cited herein are incorporated by reference in their entirety.

Many modifications and variations of this invention can be made without departing from its spirit and scope, which will be apparent to those skilled in the art. The specific embodiments described herein are provided by way of illustration only, and the invention is to be limited only by the appended claims, along with the full scope of equivalents to which such claims are entitled. Should be restricted.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61K 39/395 A61P 9/00 9/10 45/06 17/00 A61P 9/00 19/00 9/10 19/02 17/00 25/00 19/00 25/28 19/02 29/00 25/00 101 25/28 31/00 29/00 37/02 101 37/08 31/00 A61K 37/02 37 / 02 37/24 37/08 37/465 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW) , SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, J, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, HR, HU, ID, IL, IN, IS, JP, KG, KR, KZ, LC, LK, LR, LT, LU, LV, MA, MD, MG, MK , MN, MX, NO, NZ, PL, PT, RO, RU, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UZ, VN, YU, ZA (72) Inventors Hoek, Robert M. United States California 94040, Mountain View, Number 308, Mount Vernon Court 1943 (72) Inventor Sedgewick, Jonathan Dee. United States California 94301, Palo Alto, North California Avenue 365F Term (Reference) 4C084 AA02 AA17 AA24 BA08 BA23 BA44 CA17 CA53 CA56 CA59 DA45 DB52 DC50 MA02 NA05 NA14 ZA022 ZA082 ZA362 ZA452 ZA892 ZA962 ZB072 ZB112 ZB132 CC EE01 EE03 GG01 4C086 AA01 AA02 DA08 MA03 MA04 MA07 NA05 NA14 ZA02 ZA08 ZA36 ZA45 ZA89 ZA96 ZB07 ZB11 ZB13 ZB15 ZB32

Claims (20)

[Claims] 1. A method of modulating leukocyte trafficking or activation in an animal, the method comprising the steps of: (a) an agonist of a mammalian OX2 protein; or b) a mammal. Contacting a therapeutic amount of an OX2 protein antagonist. 2. The method of claim 1, wherein: a) the mammalian OX2 protein is a primate protein; b) the antagonist is an antibody that binds to the mammalian OX2. Or c) the cells are monocyte / macrophage lineage cells. 3. The method of claim 2, wherein said myeloid lineage cells comprise monocytes, macrophages, microglia or dendritic cells. 4. The method of claim 1, wherein said animal exhibits signs or symptoms of an inflammatory, infectious, leukocyte proliferative, neurodegenerative, or post-traumatic condition. 5. The method of claim 4, wherein the sign or symptom is a sign or symptom in nervous tissue; lymph tissue; bone marrow tissue; pancreas; gastrointestinal tissue; thyroid tissue; muscular tissue; or skin or collagenous tissue. The described method. 6. The method of claim 1, wherein the modulating is inhibiting the function of the white blood cell.
The method of claim 1. 7. The method of claim 6, wherein administration is said agonist. 8. The method of claim 7, wherein said agonist is said mammalian OX2. 9. The method of claim 9, wherein the animal is an autoimmune; inflammatory condition; infection; tissue-specific autoimmunity; degenerative autoimmunity; rheumatoid arthritis; atherosclerosis; multiple sclerosis; vasculitis; The method of claim 7, wherein the patient experiences signs or symptoms of a skin transplant; transplantation; spinal cord injury; stroke; neurodegeneration; or ischemia. 10. The method according to claim 7, wherein the administration is in combination with: a) an agonist or antagonist of an anti-inflammatory cytokine; b) an analgesic; c) an anti-inflammatory; or d) a steroid. . 11. The method of claim 1, wherein said modulating is enhancing the function of said white blood cells. 12. The method of claim 11, wherein administration is said antagonist. 13. The method of claim 13, wherein the antagonist comprises: a) an antibody that binds to the mammalian OX2; or b) a compound that competes with the mammalian OX2 for binding to the OX2 receptor, but does not substantially transmit a signal. 13. The method of claim 12, which is a mammalian OX2 mutein. 14. The method of claim 12, wherein said animal experiences signs or symptoms of wound healing or clot formation. 15. The method of claim 12, wherein the administration is in combination with: a) an angiogenic factor; b) a growth factor comprising FGF or PDGF; c) an antibiotic; or d) a clotting factor. the method of. 16. A method of regulating the activation of leukocytes in a tissue, comprising the steps of: (a) an agonist of a mammalian OX2 protein; or b) a mammalian OX2 protein. Contacting an antagonist of the method. 17. The method of claim 16, wherein said modulating is inhibiting said white blood cells, and said contacting is contacting with said agonist. 18. The method of claim 17, wherein the administration is in combination with: a) an agonist or antagonist of an anti-inflammatory cytokine; b) an analgesic; c) an anti-inflammatory; or d) a steroid. . 19. The method of claim 16, wherein said modulation is enhancing and said contacting is with said antagonist. 20. The method of claim 19, wherein the administration is in combination with: a) an angiogenic factor; b) a growth factor comprising FGF or PDGF; c) an antibiotic; or d) a clotting factor. the method of.