CZ365098A3 - Isolated and recombinant nucleic acid, bas-1 protein or peptide thereof and preparation, antibody, expression vector, host cell as well as processes of their use - Google Patents

Abstract

The purification and isolation of a gene which encodes a human B-cell antigen. Nucleic acids, proteins, antibodies, and other reagents useful in modulating development of cells, e.g., lymphoid, are provided, along with methods for their use.

Description

CELL AND METHODS OF USING THEM ^: . ¹

AREA OF TECHNOLOGY • · • · · • · ·

The present invention relates to a number of biological agents that are used to modulate human*.··.

• · · immune responses. In particular, it is focused on the substances and methods used, e.g. in the interaction of B and T*' cells.

STATE OF THE ART

Leukemias, lymphomas, carcinomas and other malignancies are described, for example, in Wilson et al. (eds.) Harrison's Principles of Internal medicine, McGraw-Hill, New York, pp. 1599 - 1612. One example of such diseases, malignant lymphomas are neoplastic transformed cells that predominantly occur in lymphoid tissues (eg Nadler L.M. in Harrisn). 90% of non-Hodgkins lymphomas, of which approximately 30,000 new cases occur in the United States each year, are B cell lymphomas. Less than 25% of these cases are treated. Another example is leukemia, of which approximately 30,000 new cases occur annually in the United States. Therefore, there is a need to treat B and T cell lymphomas, leukemias, cancers and other malignancies more effectively.

The growth of normal resting B cells (also referred to as "B lymphocytes") involves two distinct pathways. First, quiescent cells activate the transition from the Go phase to the Gi phase of the cell cycle. See, for example, Alberts et al. (eds. 1989) Molecular Biology of the Cell, Garland Publ., NY and Darnell et al. (1990) Molecular Cell Biology, Freeman, NY. Furthermore, activated cells induce proliferation. See, for example, Paul, ed. (1989) Fundamental Immunology, ^2nd ed., Raven Press, NY and third edition. Several factors have been identified that induce B cell growth, including interleukin-1, (IL1), IL-2, IL-4, IL-10, and IL 13. In addition, antibodies against certain B cell surface molecules have been found to promote B cell proliferation. UT cells (also referred to as "T lymphocytes") are also induced to proliferate by certain factors that include phytohemagglutinin, anti-T cell receptor monoclonal antibodies, anti-CD3 monoclonal antibodies, and others. A number of studies implicate the CD40 molecule as a growth factor receptor and an important regulator of human B cell proliferation and development. B lymphocytes are activated by the interaction of the CD40 molecule on the surface of B lymphocytes with a ligand, thereby transiently expressing it in activated helper T cells.

·· · • * · · · ·

CD40 is a membrane glycoprotein expressed in normal B lymphocytes and B cellsK ϊ'ζ • · · malignant tumors, interstitial cells, follicular dendritic cells, thymic · • · · · epithelial cells and other cancers. Human CD40 was first identified in 198^ _<>ee · • · as a monoclonal antibody epitope that is almost exclusively expressed in B lymphocytes and is therefore used as a marker for B cells. The human CD40 antigen is a 45 to 50 kD glycoprotein.

• · ·

Anti-CD40 antibodies cause a strong costimulatory signal for B cell proliferation ····* • · · induced by phorbolmyristic acid acetate (PMA), anti-CD20 antibodies or anti-.í..' immunoglobulin antibodies. In addition, anti-human CD40 antibodies with IL-40 on activated B cells also cause a variety of effects, including short-term replication, induction of IgE synthesis, and long-term proliferation when cultures are further supplemented with CD32-transfected L cells.

B7 (CD80) and B70 (CD86) are a second “group” of molecules that mediate tight B and T cell interaction. These molecules interact with the ligands CD28 on B cells and CTLA-4 on T cells. These interactions are the main costimulatory signals of both B and T cell activation.

Over the past 15 years, it has become clear that these two pairs of surface molecules have a fundamental function in the activation of T and B cells. A number of in vitro and in vivo experiments have been demonstrated where these two pairs of molecules play an important role in immunosuppression. See, for example, Banchereau et cil. (1994) Ann. Roar. Immunol. 12: 881-922, van Kooten et al. (1996) Adv. Immunol. 61:1-77, Linsley and Ledbetter (1993) Ann. Roar. Immunol. 11: 191–212.

Immunosuppression is a very important immunological intervention to protect and treat autoimmune diseases and graft rejection during transplantation. Immunosuppression can be achieved by a) antiproliferative drugs (cyclophosphamide, 15-deoxystergualine, etc.), b) glucocorticosteroids, c) inhibitors of intracellular signaling pathways (e.g. cyclosporine), d) immunosuppressive cytokines (e.g. TGF-β, IL-10), e) specific tolerance induction by antigens and f) inhibition of cell surface molecules involved in the activation of T and B lymphocytes, such as CD40/CD40 ligand and B7/B70 with CD28/CTLA-4.

In 1995, another molecule, called RP105, was cloned from mouse spleen cells.

E.g. Miyake et al. (1995) "RP105, a novel B cell surface molecule implicated in B cell activation, is a member of the leucine-rich repeat protein family" J. Immunol. 154: 3333-3340. Monoclonal antibody against RP105 induces robust proliferation of murine B cells and protects murine B cells against radiation-induced apoptosis in a manner similar to anti-CD40 antibody or CD40-ligand. See Miyake et al. (1994) "Murine B cell proliferation and protection from apoptosis with an antibody against a 105 kDa molecule: Unresponsiveness of X-linked immunodeficient B cells" J. Exp. Copper. 180: 1217–1224.

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RP105 and its putative ligand may be a third pair of molecules that play an important role in the activation of ;*· • · · and B cells. However, until now, the human sequence was not available, nor were its structure and activities · ^J ···· determined. The present invention provides this and many other practices in use. ·....* • · · • · · ·

SUMMARY OF THE INVENTION • · · • · · • · ·· • · • · ·

The present invention deals with components selected from the group consisting of: isolated ····* or recombinant nucleic acid encoding human BAS-1 protein or a fragment thereof, highly purified BAS-1 protein or its peptide, a fusion protein containing protein sequences BAS-1, and from an antibody raised against recombinant or purified monkey BAS-1 protein.

In the case of an isolated or recombinant nucleic acid, the acid may comprise the sequence defined in SEQ ID NO: 1.

In the case of proteins, the protein may be highly purified BAS-1 protein or a peptide thereof, may be selected from the group consisting of: a primate protein or peptide comprising a human protein, a protein or peptide comprising at least one polypeptide segment defined in SEQ ID NO: 2, a protein or peptide that exhibits a post-translationally modified pattern different from the native BAS-1 protein and a protein lacking the intracellular domain of BAS-1, and may be a substance containing the protein and a pharmaceutically acceptable carrier.

Examples of an antibody include those wherein the BAS-1 protein is a monkey protein comprising one of the human proteins, the antibody is raised against a peptide of the sequence defined in SEQ ID NO: 2, the antibody exhibits a Kd of at least about 10 μΜ, the antibody is a monoclonal antibody, or the antibody is labeled. Other cases include those where the antibody induces robust B cell proliferation and or protects B cells against radiation- or steroid hormone-induced apoptosis.

The present invention also includes a kit that contains, e.g., a nucleic acid encoding a BAS-1 protein or peptide, a highly purified BAS-1 protein or fragment, or an antibody or receptor that specifically binds the BAS-1 protein. A kit that comprises a nucleic acid comprises the coding sequence defined in SEQ ID NO: 1. For a kit that comprises a protein or peptide, the polypeptide is often selected from the group consisting of: a protein or peptide from a primate comprising a human protein, a protein or peptide comprising at least one polypeptide segment defined in SEQ ID NO: 2 and a protein or peptide that exhibits a post-translationally modified pattern different from the native BAS-1 protein.

Another kit may include an antibody or receptor where the BAS-1 protein is a monkey protein containing one of the human proteins. Antibody formation is induced against the peptide sequence

·· · defined in SEQ NO; 2, the antibody exhibits a Kd of at least about 10 μΜ and is a monoclonal ·'· • · · antibody or is labeled. · • · · ·

The invention also provides a method of screening a sample of a binding partner for BAS-1 protein....' • · comprising the process of producing purified or recombinant BAS-1 protein and screening?..·.· sample for specific binding of a binding partner to BAS-1 protein. In some cases • · · the sample contains proteins from a T cell, a dendritic cell including a follicular dendritic cell*··· • · · or contains a stromal cell including a fibrous cell, an endothelial cell, and an epithelial····' cell. In other cases, the binding partner is an antibody and the sample is a hybridoma supernatant.

In another aspect, the present invention includes a method of modulating the physiology or development of a cell comprising contacting the cell with an agonist or antagonist of a BAS-1 protein. The physiology is selected from: immunosuppression, activation of cytotoxic death, modulation of cytokine production, or lymphoma growth. The antagonist is often an antibody against the monkey BAS-1 protein. And the method may include precisely in combination with a signal mediator through the antigen receptor, the CD40:CD40 ligand pathway or the CD28/CTLA-4:B7/B70 pathway, e.g. anti-CD3, anti-CD40, anti-CD40 ligand, anti-CD28, antiCTLA-4, anti B7, anti B70 or a soluble part of a suitably surface labeled molecule.

I. In general

The present invention provides the amino acid sequence and DNA sequence of a human protein that exhibits the properties of an activated antigen. This protein is designated BAS-1 (B cell Activation and Survival antigen-1). The primate sequence described here was not found until after the mouse gene was described. See Miyake et al. (1995) J. Immunol. 154: 3333-3340. Similar protein sequences in other primate species could also be applicable. The examples below are, in exemplary cases, focused on the described human BAS-1 wild-type allele, but are equally applicable to allelic and or polymorphic variants, e.g. from other individuals, as well as spliced variants, e.g. wild species.

These genes allow the isolation of other monkey genes encoding proteins similar to this one, further expanding the group beyond the specific cases described. The method is described in detail below.

Human BAS-1 is so named for its biological activity. A monoclonal antibody against the murine homolog of RP105 induces robust proliferation of murine B cells and protects murine B cells against radiation-induced apoptosis in a manner similar to anti-CD40 antibody or CD40-ligand.

• 000

000 ·0 0 0000 0000 00 0 0000 0 0000000 0 0 « 000 000 000 0000 0 ·

0 0 0

Human BAS-1 cDNA was isolated using a nucleic acid sequence based on; ;·;

0 0 sequence of mouse RP105. Analysis of the respective encoded protein suggests that human BAS-1

0 0 0 belongs to a group of proteins containing a leucine-rich stretch. Other representatives of this group include 5 Ϊ

CD14-LPS receptor, FSH/LH/TSH receptors and neurotropic receptors. They are assumed to be;* . ;

0 0 0 these receptors involved in signal transduction.

0 0

Human BAS-1 cDNA was derived using the sequences of several regions of mouse RP105. Comparison*.!..'

0 open reading frame homology between mouse and human BAS-1 shows approximately 67% ·.;..' DNA sequence homology and approximately 73% amino acid sequence identity to mouse RP105.

Human BAS-1 may have a significant role in the external immune system, eg in developmental regulation in other cells. See, eg, Gilbert (1991) Developmental Biology ( ^3rd ed.), Sinauer Associates, Sunderland, MA, Browder et al. (1991) Developmental Biology ( ^3rd ed.), Saunders, Philadelphia, PA., Russo et al. (1992) Development: The Molecular Genetic Approach, Springer-Verlag, New York, NY and Wilkins (1993) Genetic Analysis of Animal Development (2 ^nd ed.) Wiley-Liss, New York, NY Identification of a ligand for human BAS-1 may answer some of the questions raised by this observation.

II. Purified human BAS-1 protein

SEQ ID NO: 1 describes the nucleotide sequence of the cDNA and the corresponding amino acid sequence. The amino acid sequence is also given by SEQ ID NO: 2. We believe that the signal sequence runs from Met (1) to Val (20), the N-terminus, which contains two potential N-linked glycosylated sites on the asparagine (residues 34, 53), runs from Cys (28) to Leu (58), a tandem repeat of a leucine-rich region that contains 9 potential sites for N-linked glycosylation on asparagine (residues 70, 78, 201, 234, 244, 394, 402, 451, and 573 ), goes from Thr (55) to Tyr (592). The C-flanking region runs from Leu (574) to Cys (625), the transmembrane region runs from Ala (629) to Val (650), the intracellular region, which contains two potentially phosphorylated tyrosine sites (652 and 658), runs from Lys (651) to Phe (661).

The following table shows the alignment of the leucine-rich repeats of the human BAS-1 protein described in SEQ ID NO: 2.

Table 1

C I I consensus: lúQíIZXLXXNXIŮCXLX^

1: 55-78 TEFLEFSFNFLPTIHNRTFSRIiMN » · φ · » φ φ φ φ«

I Φ Φ φ · φ

ΦΦΦΦ φφφ • ·

2: 79-102 LTFI®LTR£QIW,XPEDTFQSHHQ Φ 3: 103-126 LSTLVLTGNPLIFMAETSLNGPKS ΦΦΦΦ φ Φ 4: 127-150 LKHLFLIQTGISNLEFIPVHNLEN ♦ Φ ΦΦΦΦ 5: 151-174 LESLYLGSNHISSIKFPKDFPARN φ φ φ φ φ 6: 175-198 LKVLDFQNNAIHYISREDMRSLEQ φ φ φ φ φ 7: 199-223 AINLSLNFNGNN-VKGIELGAFDSTV φ φ φ φ φ 8: 224-246 FQSLNFGGTPNL-SVIFNGLQNST φ φ φ φ··· 9: 247-275 TQSLWLGTFEDIDDEDISSAMLKGLCEMS φ · φ φ · 10; 276-299 VESLNLQEHRFSDISSTTFQCFTQ ΦΦΦΦ 11: 300-322 LQELDLTATHLKGLPSGMKG-LNL 12: 323-346 LKKLVLSWHFDQLCQISAANFPS 13: 347-371 LTHLYIRGN-VKKLHLGVGC -leklgn 14: 372-397 LQTLDLSHNDIEASDCCSLQ-LKNLSH 15: 398-421 LQTLNLSHNEPLGLQSQAFKECPQ 16: 422-446 LELLDIíAFTRLHINAPQSPFQNLHF 17: 447-470 LQV33NLTYQFLDTSNQHLLAGLPV 18: 471-497 LRHLNLKGNHFQDGTITKTNLLQTVGS 19: 498-521 LEVLILSSCGLLSIDQQAFHSLGK 20: 522-544 MSHVDLSHNSLTCDSIDSLSHLK 21: 545-568 GIYLNLAANSINIISPRLLPILSQ 22: 569-592 QSTINLSHNPLDCTCSNIHF-LTWY

In the context of a protein, the term "human BAS-1" as used herein refers to a protein having the amino acid sequence described in SEQ ID NO: 2. The present invention also includes proteins comprising a large fragment thereof, e.g., mutants and polymorphic variants, together with the obtained a human polypeptide that exhibits identical biological functions or interactions with binding components specific to human BAS-1. These binding moieties typically bind to human BAS-1 with high affinity, eg, at least 100 nM, usually better than 30 nM, more often better than 10 nM, and most often better than 3 nM. Homologous proteins are found in non-human species such as primates.

As used herein, the term "polypeptide" refers to a fragment or segment and includes a stretch of amino acid residues of at least 8 amino acids, generally at least 10 amino acids, most generally at least 12 amino acids, often at least 14 amino acids, more often at least 16 amino acids, typically at least 20 amino acids, usually at least 22 amino acids, more often at least 24 amino acids, preferably at least 26 amino acids, more often at least 28 amino acids, and partially preferred at least 30 or more amino acids, e.g. 33, 37, 41, 45, 49, 53, 57, etc.

The term "ligand" or "binding component" refers to molecules that specifically bind to BAS-1, eg, an antibody-antigen. Other interactions include e.g. the ligand-receptor type, or with substances that bind to BAS-1, e.g. in a protein-protein interaction, either covalently or non-covalently • ·

9 9 • · · · · 9

99 9999 • 9 binding. A molecule can be a polymer or a chemical agent. No other indication as to tcj

9 9 appears BAS-1 in ligand-receptor interaction either ligand or receptor, not shown, except that the interaction *

9 shows specific affinity. A functional analogue can be a ligand with structural modifications...'

9 or a completely different molecule in such a molecular form that interacts with a suitable ligand binding determinant. Ligands can serve as agonists or antagonists, see e.g. .♦

9 9

Goodman et al. (eds.) (1990) Goodman and Gilmarís: The Pharmacological Bases of**

9 9

Therapeutics (8 ^th ed.), Pergamon Press. *.and·.*

The solubility of the polypeptide or fragment depends on the environment and the polypeptide. Solubility is influenced by many factors, such as temperature, electrolyte environment, size and molecular properties of the polypeptide, and solvent origin. Typically, the temperature at which the polypeptide is used is between 4°C and 65°C. Usually, the temperature used is higher than 18 °C, and it is more common when it is higher than 22 °C. Room temperature or warmer is usually used for diagnostic purposes, but a temperature lower than the denaturation temperature of the components is used in the determination. Body temperature is usually used for therapeutic purposes, typically 37°C in humans, although in certain situations it may be higher or lower in situ or in vitro.

Electrolytes usually closely approximate physiological conditions, but may be modified to a higher or lower ionic strength that is preferred. Specific ions can be modified to adapt to standard buffers used in physiological or analytical conditions. The size and structure of the polypeptide should usually be in a fairly stable state, not in a denatured form. A polypeptide may be linked to other polypeptides to form a quaternary structure, eg to allow solubility, or linked to lipids or detergents in a manner that is similar to a natural lipid bilayer. In particular, it appears that the natural protein is often linked to the lipid by a PI bond.

The solvent is usually a biologically compatible buffer used to preserve biological activities and is usually conditioned with a physiological solvent. The solvent normally has a neutral pH between 5 and 10, but preferably around 7.5. In some cases, a mild non-denaturing detergent, e.g. CHS (cholesteryl hemisuccinate) or CHAPS (3-([3-cholamidopropyl] dimethylammonio)-1-propanesulfonate) is added or is added at a sufficiently low concentration not to disturb the tertiary structure of the protein.

Solubility is expressed by sedimentation measured in Sedberg units, which are a measure of the sedimentation rate of a molecule under specific conditions. Sedimentation velocity determination was classically performed in an analytical ultracentrifuge, but is now commonly performed in a standard ultracentrifuge. See Freifelder (1982) Physical Biochemistry ( ^2nd ed.), WH Freeman and Cantor and Schimel (1980) Biophysical Chemistry, Parts 1 to 3, WH Freeman & Co., San Francisco.

0000 «« 0 0 00

In an approximate determination, a sample containing a supposedly soluble polypeptide is curled in a standard ;*í

0 0 ultracentrifuge at 50K rpm for 10 minutes and soluble molecules remain in the supernatant. Soluble *

0 particle or polypeptide will be less than 30S, usually less than 15S, usually less than 10S, part<5.·..*

0 less than 6S, especially more often less than 4S and most often less than 3S.

III. Physical variants ··”

0 0 0 0 0 0000

The present invention also includes proteins or peptides having a substantially identical amino acid sequence, such as the amino acid sequence of human BAS-1. It contains, for example, one-, two- and three-hairpin conservative substitutions. More preferably, the substitution is outside the conservative cysteines and occasionally outside the region of the helical structural domain. Such variants can be used to generate specific antibodies and often share many or all biological properties.

Amino acid sequence identity is determined by optimizing residual matching portions. These will change at any time due to conservative substitutions. Conservative substitutions commonly include substitutions in the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine and phenylalanine, tyrosine. Similar amino acid sequences are believed to include natural allelic variants in individual protein sequences. Common homologous proteins or peptides may have 85 to 100% identity (if gaps are provided) and up to 90 to 100% (if conservative substitutions are included) to the amino acid sequence of human BAS-1. The degree of agreement is at least about 85%, generally about 87%, often at least about 89%, commonly at least about 91%, usually at least about 93%, more often at least about 95%, preferably at least about 97% and most often at least about 98% and especially best at least around 99% or more. See also Needleham et al. (1970) 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, MA and software by IntelliGenetics, Mountain View, CA and University of Wisconsin Genetics Computer Group, Madison, WI.

Isolated human BAS-1 DNA can be easily modified by nucleotide substitution, nucleotide deletion, insertion and inversion of the nucleotide stretch. These modifications will result in new DNA sequences that encode useful antigens, their derivatives, or proteins with similar or opposite activity. These modified sequences can be used to generate mutant antigens or enhance expression. Enhancement of expression may involve gene amplification, increased transcription, translation, and other mechanisms. Mutant BAS-1 derivatives contain predetermined or site-specific mutations of the respective protein or its ·· · ··· · · · · ♦ · • · · · φ » · · · · · • * ···· · · · · ··· ··· ···· ······· · · · · ·· · · · · · · · «9 · * ···· fragments. "Mutant BAS-1" comprises a polypeptide that otherwise shares an important feature of human BAS-1; ;*· ·· · as above but has an amino acid sequence different from native BAS-1, which is · ···· caused by deletion, substitution or insertion. In particular, a "site-specific BAS-1 mutant"is....' • · defined as being homologous to the antigen described in SEQ ID NO: 2 and thus sharing with this..·,; antigen a number of biological activities. Similar concepts apply to various BAS-1 proteins, ^J 9 9 · , . Q r · · 9 9 9 especially those found in various other primates. As previously noted and emphasized, the examples generally list other BAS-1 proteins, which are not strictly limited to the particular human cases discussed.

Although the sites of site-specific mutations are predetermined, mutants need not be site-specific. Mutagenesis of human BAS-1 can be caused by amino acid insertion or deletion. Substitutions, deletions, insertions, or other combinations may be generated to achieve the final form. The insertion involves joining the N- and C-termini. Random mutagenesis can lead to a target codon and then the expressed mutants can be examined for the desired activity. Methods of substitution mutations at predetermined sites of DNA of known sequence are well known in the art, e.g. M13 primer mutagenesis. See also Sambrook et al. (1989) and Ausubel et al. (1987 and supplement).

Mutation of DNA would not normally move the coding sequence out of reading frames and better not create a complementary region that could hybridize to form a secondary mRNA structure such as loops or hairpins.

The present invention also includes recombinant proteins, eg, heterologous fusion proteins using segments from these proteins. Heterologous protein fusion is the fusion of proteins or segments that do not normally fuse in the same way. Thus, the fusion product of immunoglobulin with BAS-1 polypeptide is a protein molecule with a sequence that is formed by a characteristic peptide bond. The molecule is typically formed as a simple translation product and exhibits properties derived from both peptide sources. A similar concept is also applied to heterologous nucleic acid sequences.

In addition, a new construct can be created by combining similar functional domains from other proteins. For example, ligand binding segments or other segments can be "swapped" with different new fusion polypeptides or fragments. See, for example, Cunningham et al. (1989) Science 243: 1330-1336 and O'Dowd et al. (1988) J. Biol. Chem. 263: 15985-15992. Novel chimeric polypeptides exhibit novel combinations of specificities resulting from the functional linkage of ligand-binding specificities and other functional domains.

By the phosphoramidite method described by Breaucage and Carruthers (1981) Tetra. Lets. 22: 1859 1862, suitable synthetic DNA fragments are produced. A double-stranded fragment is often obtained either

4444

44 • · · 44 4 4444

4444 44 4 4444

4 4444 4 4 4 · · 444 444

444 4444 4 4 ?

4 44 44 44 44 * by synthesis of the complementary strand and at the same time under appropriate conditions temperature hybridization of the strand or by adding the complementary strand to a suitable primer using DNA polymerase. · ····

4444 444 • · 4 4

4 • 444 •

4 4 4 • 4

With 4 4 4 4

IV. Functional variants ..

4 «

4 4

4 4 4 44

4 4

Blockade of the physiological response to BAS-1 antigens may result from inhibition of ligand binding to BAS-1, probably competitive inhibition. Thus, isolated protein, membranes from cells expressing recombinant BAS-1, soluble fragments containing the ligand-binding segments of these antigens, or fragments fixed to solid phase substrates will often be used in the in vitro assay of the present invention. These assays will also allow for diagnostic assays to be influenced by either mutations and modifications of the binding segment or mutations and modifications of the ligand, e.g. ligand analogs.

The invention also contemplates the use of competitive drug screening where, for example, neutralizing antibodies against antigens or antigenic fragments compete with the test substance for protein binding. Thus, antibodies can be used to detect the presence of certain polypeptides that share one or more antigen binding sites and can also be used to occupy binding sites on a protein that may otherwise be occupied by a ligand.

In addition, neutralizing antibodies against BAS-1 and a soluble fragment thereof that contains a high-affinity ligand binding site can be used to inhibit ligand function in tissues, e.g., tissues undergoing abnormal physiology.

"Derivatives" of BAS-1 antigens include amino acid mutants, glycosylated variants, and covalent conjugates or conjugates linked to other chemical components. Covalent derivatives can be prepared by linking functional groups located at the amino acid site of the BAS-1 antigen chains or at the N- or C-terminus of the polypeptides in a manner well known in the art. These derivatives may include, without limitation, aliphatic esters or amides at the C-terminus of polypeptides or residues containing a carboxyl site of the chains, Oacyl derivatives of hydroxyl group containing residues, and N-acyl derivatives of the N-terminus of amino acids or amino group containing residues, e.g., lysine or arginine. Acyl groups are derived from alkyl groups containing C ₃ to C 18 alkyl, forming alkanoylaroyl species. In particular, glycosyl modifications are included, eg, modification of the glycosyl pattern of polypeptides during its synthesis and processing or other methods. A particularly suitable method for this embodiment is the action of glycosylation enzymes obtained from cells that usually ensure this process, e.g. human glycosylation enzymes. Deglycosylation enzymes are also being studied.

4444 ·· « • 4 4 • 4 4 · • · 4 9 ·· · 4 ·· 4

4« k·*·

4 « • 4 44 «4 44

4 4 4 • 4 4 4

444 444

4

44

4444 444 • · 4 4

4

The invention also includes a variant of the same primary amino acid sequence that is slightly modified and contains phosphorylated amino acid residues, eg phosphotyrosine, phosphoserine or phosphothreonine. ·

The main group of derivatives are covalent conjugates of BAS-1 antigens or its fragments with other polypeptide proteins. These derivatives can be synthesized in recombined

4444 •

4444 4 »

4

44

4

4

4444 culture, such as N- and C-terminal fusion or by using reagents known in the art for their utility in cross-linking proteins at reactive site groups. More suitable sites for derivatization of ligands with cross-linking agents are on free amino groups, hydrocarbon moieties and cysteine residues.

Fusion polypeptides between BAS-1 antigens and other homologous and heterologous proteins have also been performed. Homologous proteins can be associated with different surface patterns, for example a hybrid protein exhibits the ligand specificity of one or more pattern proteins. Similarly, a heterologous fusion can be created that exhibits a combination of the properties and activities of the derived proteins. A typical example is the fusion of a reporter polypeptide, eg, luciferase, with a segment or domain of antigens, eg, a ligand-binding segment, so that the presence or location of the desired ligand can be more easily determined. See, e.g., Duli et al., U.S. Patent No. 4,859,609, which is cited herein. Other gene fusion partners include bacterial B-galactosidase, trpE, protein A, β-lactamase, α-amylase, alcohol dehydrogenase, and yeast and mating factor. See, for example, Godowski et al. (1988) Science 241: 812-816.

By the phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Lets. 22: 1859 1862 suitable synthetic DNA fragments are produced. A double-stranded fragment is often obtained either by synthesis of the complementary strand and simultaneously, under appropriate conditions, thermal hybridization of the strand or by adding the complementary strand to a suitable primer using DNA polymerase.

Such polypeptides may have amino acid residues that are chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, especially those whose molecular shape is similar to a phosphate group. In some cases, the modifications will be suitable labeled substances or will serve as purification targets, eg ligand affinity.

Fusion of proteins can generally occur either by recombinant nucleic acid methods or by polypeptide synthesis. Nucleic acid manipulation methods and expression methods are generally described in, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual ( ^2nd ed), Vols. 1 - 3, Cold Spring Harbor Laboratory. Methods of synthesizing polypeptides are described, for example, in Merrifield (1963) J. Amer. Chem. Soc. 85: 2149-2156, Merrifield (1986) Science 232:

4

4444 ♦ » ···♦ ···· » · « ··«« * ····· · * · . * ·.· ··· • · · · · · . · * ·· « ·· ·· ·· ·· **·.

341-347 and Atherton et al. (1989) Solid Phase Peptide Synthesis: A Practical Approach, IRE...* Press, Oxford. ·. J ·

This invention also describes the use of derivatives of BAS-1 antigens other than the v.λ variants.

• · amino acid sequence or glycosylation. Such derivatives may be with chemical moieties.

• · covalently linked or assembled into clusters. These derivatives generally belong to three classes: (1) salts'··'·· (2) chain site and terminal parts of covalent modifications and (3) adsorption complexes, eg» ·**· • · · · with cell membranes. Such covalent and stacked derivatives are used as; ,··, • · · immunogens, substances in immunological assays or purification methods, such as affinity "" purification of ligands or binding ligands. For example, BAS-1 antigen can be, by methods well known in the art, immobilized by covalent binding to a solid surface such as cyanogen bromide-activated Sepharose or by adsorption to polyolefin surfaces with or without glutaraldehyde cross-linking, for the determination or purification of anti-BAS-1 antibodies or of its ligands. For diagnostic assays, BAS-1 antigens can also be labeled with a detectable group, eg, radioiodinated chloramine T, covalently bound to exceptional soil chelates or conjugated to other fluorescent moieties.

The soluble BAS-1 antigen, in the present invention, can be used as an immunogen to generate an antiserum or antibody specific to the antigens or some part thereof. Purified antigens can be used to examine monoclonal antibodies or antigen-binding fragments that have been prepared by immunization of various types of contaminated preparations containing proteins. The term "antibodies" includes in particular antigen-binding fragments of natural antibodies. Purified BAS-1 antigen can also be used as an agent to detect antibodies raised in response to the presence of elevated levels of BAS-1 or antigen-containing cellular components under abnormally diagnostic or specifically physiological or disease conditions. Additionally, in the present invention, BAS-1 fragments can serve as immunogens to generate antibodies, as described below. For example, the present invention examines antibodies raised against the amino acid sequences or encoded by the nucleotide sequence described in SEQ ID NO: 1 or fragments thereof. The present invention contemplates antibodies that have binding affinity to or are raised against specific fragments predicted to lie outside the region of the lipid bilayer. In addition, a number of constructs can arise from the fusion of membrane-accumulated segments with otherwise extracellular free parts of molecules.

The present invention explores the isolation of other similar species. It is highly likely that allelic species exist in different individuals, eg, greater than 90 to 97% similarity to the case previously described is described here.

4 4 4 »» 4··* • 4 4 • · 4 4 ·

4« 44

The invention also provides methods for isolating groups of related antigens with different and similar...* · 4 · structure, expression and function. The elucidation of the many physiological effects of antigens will be greatly accelerated by the isolation and characterization of distinct species of the opposite parts of antigens. Submitted.^..

• · the invention presents in particular the use of nucleotide probes to identify other homologous ch****.

4 genetic entities in different species.

The isolated genes allow the transformation of cells lacking BAS-1 expression, eg other .**.

4 4 4 species types or cells that lack the corresponding antigens and show negative activity. .··.

· ·

Expression of the transformed genes allows the isolation of antigenically pure cell lines, with '*' defined or single-species variants. This approach allows for much more sensitive detection and discrimination of the physiological effects of any ligands. Subcellular fragments, eg cytoplasts or membrane fragments, can be isolated and used.

Dissecting the critical structural parts that influence the many different functions provided by the ligands allows the use of standard methods of modern molecular biology, especially in the comparison of members of similar classes. See, e.g., the method of homolog sensing mutagenesis described by Cunningham et al. (1989) Science 243: 1339-1336 and the procedures used in O'Dowd et al. (1988)

J. Biol. Chem. 263: 15985-15992 and Lechleiter et al. (1990) EMBO J. 9: 4381-4390.

Ligand binding segments can be substituted with species variants to determine what structural features are important in both ligand binding affinities and specificities. A number of different BAS-1 variants will be used to screen for ligands exhibiting combined interaction properties with other species variants.

Intracellular function would likely involve antigenic segments that are normally available in the cytosol. However, antigen internalization can occur under certain circumstances and interactions between intracellular components and designated "extracellular" segments can occur. Specific binding segments of BAS-1 antigens to other intracellular components may be identified by mutagenesis or directly biochemically, eg, cross-linking or affinity methods. Structural analysis by crystallography or other physical methods will also be applied. Further investigation of the mechanism of signal transduction may involve the study of accumulated components that can be isolated by affinity methods.

Furthermore, the expression and control of BAS-1 antigens will be studied. Controlled antigen-associated components show different developmental, tissue-specific, or other expression patterns. Upstream and downstream genetic regions are of interest, eg component control.

The study of the structure of BAS-1 antigens leads to the construction of new ligands, especially analogs showing agonistic and antagonistic properties. This can be combined with previously described and monitored methods to isolate ligands with the desired spectrum of activities.

• · · 0 ·· ···· ·· · • · «· ·* • · · · · · · • · · 0 · * « 0*00 • ······· · 0 0 0 * 0 > · 0 * • · · 0 · 0 0 0 0 ·· * ·· ·· ·· ♦· ···· • · ·

Expression in other cell types will often cause different •

glycosylation. A number of species may have different functions based on different structure versus amino acid sequence. Different modification may be responsible for different functions and it is now possible to elucidate these effects.

Important developmental antigens and substances dependent on them are highlighted in the present invention. Although the above examples focus primarily on human BAS-1, those skilled in the art will immediately recognize that the invention includes other BAS-1 antigens, e.g. primates and other mammals. .

V. Antibodies

Antibodies can be raised against a number of allelic variants of BAS-1 antigens and their fragments, both in the originally occurring form and in their recombined form. Additionally, antibodies can be raised against BAS-1 in either its active or inactive form. Anti-idiotype antibodies are often observed.

Antibodies that contain binding fragments and single-chain forms against predefined fragments of BAS-1 are created by immunization of animals by conjugates of fragments with immunogenic proteins. Monoclonal antibodies are prepared from cells secreting the desired antibodies. These antibodies can be monitored for binding to normal and defective BAS-1 or ligand activity of agonists or antagonists. These monoclonal antibodies are usually bound with an sKd of at least more than 1 mM, more commonly at least more than 300 μΜ, typically more than 30 μΜ, more preferably more than 10 μΜ and most preferably more than 3 μΜ, e.g. 1 μΜ, 300 nM, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 300 pM, 100 pM, 30 pM, etc.

These antibodies containing antigen-binding fragments can be of significant importance in diagnostics and therapy. They may be potent antagonists that bind to BAS-1 and inhibit ligand binding or the ability of the ligand to elicit a biological response. They can also be used as antibodies without neutralizing effects and attached to toxins or radionuclides so that the antibody binds to the antigen and the cell itself is destroyed. Furthermore, these antibodies can be conjugated with drugs or other therapeutic agents, either directly or indirectly through the linker.

The antibodies of the present invention can be used in diagnostics. As captured or non-neutralizing antibodies, they can bind to BAS-1 without inhibitory ligand binding. As neutralizing antibodies they can be used in competitive binding assays. They can also be used to detect or quantify BAS-1 or its ligands.

ΦΦ 4 » 4 • · · · • Φ φ φ · Φ

ΦΦΦ • 4 4

Φ

Φ

Φ

4» ·«/*·

Φ»

Φ · Φ Φ

Φ Φ Φ ·

Φ φ* Φ φφφ

Φ Φ *»· ·· • ΦΦΦ

BAS-1 fragments can be bound to other materials, especially polypeptides, thus being fused*

Φ Φ Φ 4 or covalently linked polypeptides and used as immunogens. BAS-1 and its fragments mohofl..* ;

Φ be fused or covalently linked to a number of immunogens, such as the hemocyanin of the cogs^····.

• · bovine serum albumin, tetanus toxoid, etc. See Microbiology, Hoeber Medical Division, Harper*'”.

Φ Φ and Row, 1969, Landsteiner (1962) Specificity of Serological Reactions, Dover Publications,·· ” New York, and Williams et al. (1967) Methods in Immunology and Immunochemistry Vol. 1. ·”.

• φ Φ Φ

Academie Press, New York, for a description of methods for preparing polyclonal antisera. Typical method .**.

Φ φ Φ refers to hyperimmunization of animals with antigens. The animal's blood is then collected shortly after the immunization is repeated and the gamma-globulin is isolated. Or cells can be harvested to form hybridomas.

In some examples, it is desired to prepare monoclonal antibodies from other mammalian hosts, such as mice, rats, monkeys, humans, etc. A description of methods for preparing monoclonal antibodies is found, e.g., in Stites et al. (eds.) Basic and Clinical Immunology (4 ^th ed.), Lange Medical Publications, Los Altos, CA and cited herein, Harlow and Lané (1988) Antibodies: A Laboratory Manual, CSH Press, Goding (1986) Monoclonal Antibodies: Principles and Practice ( ^2nd ed.) Academie Press, New York, and in part in Kohler and Milstein (1975) in Nature 256: 495-497, where one method of generating monoclonal antibodies is discussed. Briefly summarized, these methods relate to injecting animals with an immunogen. The animal is then sacrificed and cells from the spleen are collected and combined with myelocytes. The result is a hybrid cell or "hybridoma" capable of in vitro reproduction. The hybridoma population is then monitored by isolating individual clones, each of which secretes one type of antibody to the immunogen. The individual types of antibodies obtained in this way are products of still living and cloned individual B cells from immune animals, which arose in response to a specific site recognized by an immunogenic substance.

Other suitable techniques involve the in vitro display of lymphocytes for antigenic polypeptides or the selection of an antibody library in phage or similar vectors. See Huse et al. (1989) "Generation of Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda", Science 246: 1275-1281 and Ward et al. (1989) Nature 341:544-546. The polypeptides and antibodies of the present invention may be used as modified or unmodified, including chimeric or humanized antibodies. Polypeptides and antibodies are more often labeled with a substance, either covalently or non-covalently bound, that exhibits a detectable signal. A wide spectrum of labeled substances and conjugation techniques are known and are mentioned numerously in the scientific and patent literature. Suitable labeled agents include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, etc. Uses of these labeled agents include these U.S. Pat. Patent No. 3,817,837;

«· ·♦ ♦· ··*· ·· ·· • · · · · φ < · * · • · » · · · · · · · · • · ···· · · · · · «·» · ·· • · · ···· · · ♦ · ♦ ·♦ ·· ·· ····

3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,145 and 4,366,241. Recombinant immunoglobulins can also be···· ¹ • · · produced, see Cabilly US Patent No. 4,816,567. *..* I

The antibodies of this invention can also be used to isolate proteins by affinity chromatography.····, • ·

Columns can be prepared when the antibodies are bound to a solid surface, e.g. to Z'**.

• · · particles such as agarose, SEPHADEX, etc. After the cell lysate passes through the column, the column is'* '* washed, followed by increasing concentrations of a mild denaturing agent, releasing the ·**· • ··· purified BAS-1 protein . Antibodies can also be used to monitor the expression of libraries • · · · for specific expression products. Typically, antibodies are used in such cases where they are labeled with groups that easily detect the presence of an antigen by binding to the antibody.

Antibodies obtained against the BAS-1 antigen will also be used to obtain anti-idiotype antibodies. This will be beneficial to detect or determine various immunological conditions related to the expression of relevant antigens.

VI. Nucleic acids

The human BAS-1 probe or fragments thereof will be identified or nucleic acids encoding homologous proteins from other species or other similar proteins from the same or other species will be isolated.

The present invention contemplates the use of isolated DNA or fragments encoding the biologically active respective BAS-1 polypeptide. In addition, the invention relates to isolated or recombinant DNA that encodes a biologically active protein or polypeptide that is capable of hybridizing under appropriate conditions with the DNA sequence described herein. Said biologically active protein or polypeptide may be the entire BAS-1 or a fragment and has the amino acid sequence encoded by the nucleic acid shown in SEQ ID NO: 1. The present invention further provides the use of isolated or recombinant DNA or fragments that encode a protein that is homologous to to BAS-1 or that were isolated using cDNA encoding human BAS-1 as a probe. Isolated DNA can have its own regulatory sequence at the 5' and 3' ends, eg promoters, enhancers, polyadenylation signals and others.

An "isolated" nucleic acid is a nucleic acid, e.g., RNA, DNA, or mixed polymer, that is completely separated from other components immediately adjacent to the original sequence, e.g., ribosomes, polymerases, and flanking genomic sequences from the original species. The invention includes nucleic acid that is taken from the environment where it originally occurred and contains recombinant or cloned DNA isolates and chemically synthesized analogs or ···· ·· ·· • · · · « • · · · «

analogues biologically synthesized by heterologous systems. A substantially pure molecule contains····' ^J · · · · • 9 · isolated forms of the molecule. ·..· i

An isolated nucleic acid will generally be a homogeneous molecule, but in some cases it will be····.

• · have less diversity. Heterogeneity is usually found at the polymer ends or parts,!*”.

• · • 9 9 which are not demanding on the required biological function or activity. ·· A "recombinant" nucleic acid is defined either by the method of its production or by its • · · · structure. In the citation for the method of production, e.g. the product was obtained by the method, the method is .”.

• · · · using recombinant nucleic acid techniques, eg involving human intervention in the nucleotide sequence. Alternatively, a nucleic acid can be made by generating a sequence containing two linked fragments that are not initially close to each other, but have been noted not to admit natural products, eg, naturally occurring mutants. Thus, eg, products produced by transformation of cells with any non-naturally occurring vector, such as nucleic acids containing a sequence produced using some synthetic oligonucleotide methods. This is often done to relocate a codon with a redundant codon encoding the same or conservative amino acid, while typically introducing or relocating, eg, a restriction or recognition site sequence. Thus, the joining of nucleic acid segments of desired functions is performed to create simple genetic entities containing the desired combination of functions not found in generally available natural forms. Restriction enzymes of recognition sites are often the target of such manipulations, to the exclusion of other sites of specific targets, e.g. promoters,

DNA replication sites, regulatory sequences, control sequences, or other characters used may be included in the plan.

A similar concept is intended for recombinants, e.g. fusion, polypeptide. In particular, synthetic nucleic acids that encode similar polypeptides to fragments of these antigens and sequence fusions of a number of different species by redundant genetic engineering are included.

A "fragment" in the context of a nucleic acid is a cognate segment of at least 17 nucleotides, typically at least 20 nucleotides, more commonly at least about 23 nucleotides, commonly at least 26 nucleotides, more commonly at least 29 nucleotides, often at least 32 nucleotides, more commonly at least about 35 nucleotides, typically at least 38 nucleotides, more typically at least around 41 nucleotides, usually at least 44 nucleotides, more usually at least 47 nucleotides, better at least 50 nucleotides, more preferably at least 53 nucleotides and especially suitable is at least 56 or more nucleotides, e.g. 60, 75,

100, 150, 200, 250, 300 etc.

In particular, the DNA that encodes the BAS-1 protein will be used to identify genes, mRNAs and cDNAs that encode similar or homologous antigens, as well as DNA that encodes homologous ·· · ·· ···· ·· · ·· ·· ·· ” ··*« • 9 proteins from different species. The various BAS-1 proteins should have similar sequences and are described here····* • ♦ ·. However, identical proteins that have an evolutionarily different relationship to BAS-1 may be..· í *

easily isolated using these sequences if they show sufficient similarity. Monkey BAS;····

9 proteins have a specific meaning. From * ’ ’, • 9

9 9

This invention further describes recombinant DNA molecules and fragments with a DNA sequence identical to or highly homologous to the isolated DNAs disclosed herein. Especially the budoij sequence ·”·

9 99 often functionally linked to DNA segments that are responsible for controlling transcription • · · · translation and DNA replication. Or recombinant clones obtained from genomic sequences, i.e. containing introns, will be used for transgenic studies, including, for example, transgenic cells and organisms and for gene therapy. See, e.g., Goodnow (1992) "Transgenic Animals" in Roitt (ed.) Encyclopedia of Immunology Academie Press, San Diego, 1502-1504, Travis (1992) Science 256: 1392-1394, Kuhn et al. (1991) Science 254: 707-710, Capecchi (1989) Science 244:

1288, Robertson (1987) (ed.) Teratocarcinomas and Embryonic Stem Cells: A Practical Approach IRL Press, Oxford, and Rosenberg (1992) J. Clinical Oncology 10: 180-199.

Homologous nucleic acid sequences that are compared show significant sequence similarity. Standards for homology in nucleic acids are either measured for homology commonly used in the art by sequence comparison or are based on hybridization conditions. Hybridization conditions are detailed below.

Nucleic acid sequence comparison shows substantial identity, meaning that the compared segments or their complementary strands are identical when optimally aligned by appropriate nucleotide insertions or deletions, in at least 50% of the nucleotides, generally at least 56%, typically at least 62%, more typically at least 65%, often at least 68%, more often at least 71%, typically at least 74%, more typically at least 77%, typically at least 80%, more typically at least 85%, better at least 90%, more preferably at least 95 to 98% or more, and in some cases more than 99% or more nucleotides. Or substantial identity occurs if the segments will hybridize under selective hybridization conditions to the strand or a complementary stretch thereof, typically the sequence used is selected from SEQ ID NO: 1. Typically, selective hybridization can occur when at least 55% homology is to a region of at least 14 nucleotides, better when the homology is at least about 65%, more preferably at least about 75% and most preferably at least about 90%. See Kanehisa (1984) Nuc. Acid Res. 12: 203-213. The length of homology compared as described can be a longer region and in some cases the region will be at least 17 nucleotides, typically at least 20 nucleotides, more typically at least 24 nucleotides, typically at least 28 nucleotides, more typically at least 40 nucleotides, preferably at least about • · · · · • · · ·· · ·· • · · nucleotides and much more preferably at least 75 to 100 or more nucleotides, e.g. 125, 150, 200*····* 250, 300 etc.' .Her

Strict conditions, in reference to identity in connection with hybridization, will be strict combined····.

• · conditions of salts, temperature, organic solvents and other parameters typically controlled^'”.

• · · in hybridization reactions. Strict temperature conditions will typically include temperatures above** '*°C, typically above 37°C, typically above 45°C, more typically above 55°C, preferably above 65°(· ·”· ···· and more preferably above 70° C. The strict salt content will usually be less than 500 mM, usually .*'.

• · Φ » less than 350 mM, more usually less than 200 mM, typically less than 150 mM, more preferably less than 100 mM and more preferably less than 50 mM. However, the combination of parameters is much more important than the measurement of individual parameters. See, e.g., Wetmur and Davidson (1968)

J. Mol. Biol. 31:349 -370.

BAS-1 protein from other human subjects can be cloned and isolated by hybridization or PCR. An alternative preparation of an antibody preparation that exhibits lower allelic specificity can be used in expression cloning. Allelic variants can be characterized using, e.g., a combination of redundant PCR and sequencing analysis, e.g., using defined primers to provide information on allelic variation in the human population.

VII. Preparation of BAS-1, mimetics

DNA that encodes the BAS-1 antigen or fragments thereof can be obtained by chemical synthesis, screening of cDNA libraries, or screening of gene libraries prepared from a wide variety of cell lines or tissue samples.

This DNA can be expressed in a wide variety of host cells to synthesize the entire antigen or its fragments, which can be further used, e.g., for the generation of polyclonal or monoclonal antibodies, for binding studies, for the construction and expression of modified molecules, and for structural studies and functions. Each antigen or fragment thereof can be expressed in host cells that are transformed or transfected with appropriate expression vectors. These molecules can be completely purified so that they do not contain any proteins or cellular contaminants, e.g. contaminants from the recombinant host, and thus, after combining with pharmaceutically acceptable carriers and or solvents, they are used especially in pharmaceutical preparations. The antigen or parts thereof can be expressed in fusions with other proteins.

Expression vectors are typically self-replicating DNA or RNA constructs that contain the desired antigenic gene or fragments thereof, usually operably linked to appropriate ··· · · · · · · ···· · · · ···· • · ··· · · · · · · ··· ···

0 0 ···· · · ·· 0 ·· ·· ·· ·*·♦·· • · genetically controlled elements that can be recognized in a suitable host cell. These control···»* ^J · 0 · 0 0 0 0 elements are capable of efficient expression in a suitable host. Specific types of control elements..· !

required for efficient expression depends on the type of host cell used. They can usually

0 genetically controlled elements contain a prokaryotic promoter system or eukaryoticZ”*.

0 0 promoter expression control system and typically include a transcriptional promoter, an optional·* ** operator to control transcription initiation, transcriptional enhancers to increase the expression level of ·''· 0000 mRNA, a sequence encoding an appropriate ribosome binding site, and a transcription termination sequence and .·* ♦ 0000 translation. Expression vectors also usually contain an origin of replication, which allows the vector to replicate independently of the host cell.

The vectors of the present invention contain DNA that encodes a human BAS-1 antigen or a fragment thereof encoding a biologically active polypeptide. The DNA may be under the control of a viral promoter and may encode a selection pattern. The present invention further includes the use of such expression vectors that are capable of expressing a eukaryotic cDNA encoding a human BAS-1 antigen in a prokaryotic or eukaryotic host, wherein the vector is compatible with the host and wherein the eukaryotic cDNA encoding the antigen is inserted into the vector such that the growth of the host containing the vector expresses the respective cDNA. Typically, expression vectors are designed for stable replication in their host cells or for amplification to increase the total number of copies of the desired gene per cell. It is not always necessary to require the expression vector to replicate in the host cell, eg it is possible to transiently express antigens or fragments thereof in different hosts using vectors that do not contain an origin of replication recognized by the host cell. It is also possible to use vectors that cause integration of the human BAS-1 gene or fragments thereof into host DNA by recombination.

Vectors used herein include plasmids, viruses, bacteriophages, integrable DNA fragments, and others that allow the DNA fragments to be integrated into the host genome. Expression vectors are specialized vectors that contain genetic control elements that cause the expression of usefully linked genes. Plasmids are the most commonly used form of vectors, but all other forms of vectors that provide equivalent functionality and that are or become known in the art are suitable for use herein. See, for example, Powels et al. (1985 and Supplements) Cloning Vectors: A Laboratory Manual, Elsevier, N.Y. and Rodriquez et al. (1988) (eds.) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston,

MA.

Transformed cells are cells, preferably mammalian, that have been transformed or transfected with human BAS-1 vectors created using recombinant DNA techniques. Transformed host cells usually express the antigen or its fragments, but for the purpose of cloning,

4 4 4 ·· 4 4 • · 4 4 4 4 * · 4 4 4 4

44,444,444

4 4 4 4 ·♦ ·· ·· 44»· DNA amplification and manipulation may not express protein. This invention further includes

4 4 cultivation of transformed cells in a nutrient medium, thus enabling the accumulation of protein in the culture*.·* í

The protein can be obtained either from the culture or from the culture medium. .··*'.

4

For the purpose of this invention, DNA sequences are operably linked if they are "operably".

4 4 similar to each other. For example, DNA is usefully linked** ** to polypeptides for presequencing or secretion vesicles if it is expressed as a preprotein or participates in the assembly of polypeptides n$ ·”;

4444 cell membrane or in the secretion of polypeptides. The promoter is operably linked to the coding ·**.

44 4 sequence if it controls the transcription of polypeptides. A ribosome binding site is operably linked to a coding sequence if it is positioned so that translation can occur. Usually, as used, contiguous means contiguous, but in the reading frame certain genetic elements, such as repressor genes, are not contiguous but still bound to operator sequences that turn on controlled expression.

Suitable host cells are prokaryotes, lower and higher eukaryotes. Prokaryotes include both gram-positive and gram-negative organisms, such as E. coli and B. subtilis. Lower eukaryotes are yeasts, such as S. cerevisiae and Pichia and species of the genus Dictyostelium. Higher eukaryotes include named tissue cell cultures from animal cells, both of non-mammalian origin, e.g., insect, bird, and mammalian, e.g., human, monkey, and rat origin.

Prokaryotic host vector systems contain a wide spectrum of vectors of many different species. As used herein, E. coli and its vectors will generally be used to include equivalent vectors used in other prokaryotes. A representative vector for DNA amplification is pBR322 or many of its derivatives. Vectors that can be used to express human BAS-1 antigens or fragments thereof include, but are not limited to, for example, vectors that contain the lac promoter (pUC species), the trp promoter (pBR322-trp), the Ipp promoter (pIN species), λ-ρΡ or pR promoters (pOTS) or hybrid promoters such as bird (pDR540).

See Brosius et al. (1988) "Expression Vectors Employing Lambda-, trp-, lac-, and IPP-derived Promoters" in Rodriguez and Denhardt (eds.) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston, Chapter 10, p .205 to 236.

Lower eukaryotes such as yeast and Dictyostelium can be transformed with human BAS-1 antigen containing vectors. The aim of this invention is that the most common host of lower eukaryotes is the yeast Saccharomyces cerevisae. This will be used generally to represent the lower eukaryotes, although a number of other phyla and species are also available. Yeast vectors mainly consist of an origin of replication (preferably integrative type), a selection gene, a promoter, DNA encoding the desired protein or its fragment, and sequences for translation termination, polyadenylation and transcription termination. Suitable yeast expression vectors are

91

9 9 • 9 9

9 9 9 4

9999

9999 9 9 9 9 9

9

9

9999

9999 9 9

9

99 both constitutive promoters and 3-phosphoglycerate kinase and a number of other glycolytic enzyme promoters or both inducible promoters and the alcohol dehydrogenase 2 promoter or the metallothionein promoter. Suitable promoters include derivatives of the following types, self-replicating low-copy (such as YRp species), self-replicating high-copy (such as

YEp), integrative types (such as Yip species) or minichromosomes (such as YCp species). Suitable host cells for the expression of functionally active human BAS-1 antigen hcj protein are tissue cell cultures of higher eukaryotes. In essence, many tissue cell cultures of higher eukaryotes, e.g., insect baculovirus expression systems, whether from vertebrates or invertebrates, work. However, in both co-translational and post-translational procedures, mammalian cells are preferred.

Transformation or transfection and propagation of such cells has become a routine method. Examples of cell lines used are HeLa cells, Chinese hamster ovary (CHO) cell line, baby rat kidney (BRK) cell line, insect cell line, bird cell line and mouse cell line (COS). Expression vectors for such cell lines typically contain an origin of replication, a promoter, a translation initiation site, an RNA splice site (if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection or amplification gene. Suitable expression vectors may be plasmids, viruses or retroviruses carrying promoters obtained, for example, from such sources as adenoviruses, SV40, parvoviruses, vaccinia virus or cytomegalovirus. A representative example of suitable expression vectors are pCDNAl, pCD see Okayama et al. (1985) Mol. Cell Biol. 5: 1136 1142, pMClneo Poly-A see Thomas et al. (1987) Cell 51: 503-512 and a baculovirus vector such as pAC 373 or pAC 610.

It is often desired that the human BAS-1 antigen polypeptide be expressed in a system that gives a specific or defined glycosylation pattern. In such a case, the usual pattern will naturally be equipped with an express system. However, the pattern will be modifiable so that the polypeptide, e.g. non-glycosylated form, is acted upon by suitable glycosylation proteins indicated in the heterologous expression system. For example, BAS-1 antigen genes can be cotransformed with one or more genes encoding mammalian or other glycosylation enzymes. Using this approach, certain mammalian glycosylation patterns will be achievable or approximated in prokaryotic or other cells.

BAS-1 antigen could also be produced in a phosphatidylinositol (PI)-bound form, but can be removed from membranes using a phosphatidylinositol-cleaving enzyme, eg, phosphatidylinositol phospholipase-C. Thus, the antigen is released in a biologically active form and enables purification by standard biochemical methods. See, e.g., Low (1989) Biochim. Biophys. Acta

9

9

9999 99

9

9

9 9 9 • •9 99 99*9 99 44

9Q 999 9999999

9 9 9 99 9 9999

9 9999 999 99 999 999

999 9999 9 9

9 99 99 99 99

988: 427-454, Tse et al. (1985) Science 230: 1003-1008 and Brunner et al. (1991) J. Cell Bioí··;;' • 9 4

114: 1275 - 1283. Alternatively, purified segments can be constructed in the sequence, eg, at the N^ ··* · terminus or C-terminus to aid in the purification or detection of the protein product. Тр····.

• 9 means that removal of such segments can also be engineered, e.g., by protease cleaves.!**'*, sites. *..*4

This entire sequence is now known, and thus human BAS-1 antigens, fragments or their derivativesj

9444 can be prepared by conventional procedures for peptide synthesis. They also include how to

94 9 are described in Stewart and Young (1984) Solid Phase peptide Synthesis, Pierce Chemical Co., Rockford, IL, Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis, SpringerVerlag, New York and Bodanszky (1984) The Principles of peptide Synthesis, Springer-Verlag,

New York. For example, the azide method, the hydrochloric acid method, the anhydride method, the mixed anhydride method, the active ester method (eg, pnitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester), the carboxyimidazole method, the redox method, or the dicyclohexylcarbodiimide (DCCD)/additive method may be used. Solid phase and solution syntheses are applicable for the previously mentioned methods.

Human BAS-1 antigens, fragments or derivatives are conveniently prepared in accordance with the above-mentioned methods that are typically used in peptide synthesis, generally by any so-called stepwise method that involves the condensation of amino acids at the terminal amino acid in a sequence or by the pairing of peptide fragments at the terminal amino acid. Amino groups not used for pairing must be protected to avoid pairing in the wrong region.

If solid-phase synthesis is chosen, the C-terminus of the amino acid is bound to an insoluble carrier or support by its carboxyl group. The insoluble carrier is not particularly limited as long as it has a binding capacity to a reactive carboxyl group. Examples of such insoluble carriers are halo methyl resins such as chloromethyl resin or bromomethyl resin, hydroxymethyl resins, phenolic resins, tar. alkyloxycarbonylhydrazide resins, etc.

The protected amino group of an amino acid is bound in sequence by condensing its activated carboxyl group and the reactive amino group of a previously formed peptide or chain to sequentially synthesize the peptide. After the synthesis of the complete sequence, the peptide is detached from the insoluble carrier and the peptide itself is formed. Solid phase recovery is generally described by Merrifield et al. (1963) in J. Am. Chem. Soc. 85: 2149–2156.

The prepared antigen and its fragments can be isolated and purified from the reaction mixture by separation of peptides, e.g. extraction, precipitation, electrophoresis and a number of different chromatographies, etc.

00

0 0 ·

0 0 0

000,000

0 ·· ·· 0··0 ·· 0

0 0

0 0 0

000000

0 0 ·

l* 0090

0 0

The human BAS-1 antigens of the present invention can be obtained in various degrees of purity ····* • · * depending on the desired use. Purification can be achieved by the techniques disclosed herein..* í protein purification or by using the antibodies described herein in aminoabsorption affinity····,

0 chromatography. This aminoabsorption affinity chromatography is performed first by binding!***.

0 9 antibodies to a solid support and then combining the bound antibodies with soluble lysates of small'* ” lung cancer cells, lysates of other cells expressing BAS-1 antigens or lysates

0000 or supernatants of cells producing BAS-1 antigens as a result of DNA techniques, see below. ·

VIII. Use

The present invention provides agents that find use in diagnostics as described elsewhere herein, eg, the general description of developmental and physiological abnormalities or in the description of diagnostic kits below.

This invention also provides substances of important therapeutic value. Human BAS-1 (naturally occurring or recombinant), fragments and antibodies thereof, together with identified agents having binding activity to human BAS-1, could be used to treat a condition associated with an abnormal B cell response, including abnormal proliferation, e.g. .cancer conditions or degenerative conditions. Abnormal proliferation, regeneration, degeneration and atrophy can be modulated by appropriate therapeutic treatment using the components provided herein. For example, a disease or disorder associated with abnormal expression or abnormal action of BAS-1 could potentially be targeted by agonists or antagonists of the antigen. BAS-1 likely plays a role in the activation or regulation of B cells that influence immunological responses, eg, autoimmune disorders or allergic responses.

Among other things, the BAS-1:BAS-1 binding partner interaction may be involved in the T:B cell interaction that allows activation, proliferation and/or differentiation of naïve B cells, as observed in the hyper-IgM syndrome. Thus, the treatment may cause some kind of interference with the BAS1:BAS-1 binding partner of signal transduction. Blockage of the signal can be caused by, for example, soluble BAS-1 or an antibody to BAS-1.

The BAS-1:BAS-1 binding partner interaction may also be required in the initiation and/or regulation of robust proliferation of B cell blasts and centroblasts in the dark region of the germinal centers. See, e.g., Banchereau and Rosset (1992) Adv. Immunol. 125: 868-877. Cell surface interactions involved in signaling to initiate and/or regulate the somatic Ig mutational process may also require BAS-1 and may be regulated by agonistic or antagonistic interventions.

φφ φφφφ

ΦΦ φ φφ φφ φ φ φ φ φ φ φφφφ φφ « Φφφφφ φ φ φφφφ φφφ φ · ··· φφφ φφφ φφφφ · · φφ φ φ* ·· ·· ·· φφφφ • φ

Other abnormal developmental states are known in each cell type, by analysis of Nothernov··”'

Φ · fr transfer prove that they have BAS-1 mRNA. See Berkow (ed.) The Merck Manual Diagnosis an3··* í

Φ

Therapy, Merck & Co., Rahway N.J. and Thorne et al. Harrison's Principles of Internal Medicine^····.

Φ

McGraw-Hill, N.Y. For example, therapeutic immunosuppression can be achieved by blocking T·***·

Φ Φ « of the lymphocyte and the interaction of the B lymphocyte with this molecule. It represents an important therapy for the control*' ” of autoimmune diseases and graft rejection during transplantation. Alternatively, anti-B cell tumor φφφφ therapy may be achieved by blocking the growth and residual effects of BAS-1 on B cells. Blokacf ·”;

ΦΦΦΦ can be affected by the composition of blocking bonds, eg, neutralizing antibodies or molecules that interfere with the interaction of BAS-1 with the counterreceptor. Soluble BAS-1 can block anti-receptor binding.

Recombinant BAS-1 or BAS-1 antibodies can be purified and then introduced into the patient. These substances can be combined for therapeutic purposes with other active ingredients, e.g., conventional pharmaceutically acceptable carriers or solvents, e.g., an immunogenic adjuvant together with physiologically harmless stabilizers and ointment bases. These combinations can be sterilized by filtration and dispensed into dosing vials by lyophilization or stored in the form of a stabilized aqueous solution. This invention also describes the use of antibodies or binding fragments thereof that are not supplemented with binding.

Drug screening using BAS-1 or fragments thereof can be performed to identify compounds with binding affinity for BAS-1 including isolation of associated components. Further biological assays can then be used to determine whether the substance has true stimulatory activity and thus causes blockade and is not antagonistic, thus blocking the activity of the ligand. Likewise, a substance with intrinsic stimulatory activity can activate an antigen and is therefore an agonist by simulating BAS-1 activity. This invention further describes the therapeutic use of BAS-1 antibodies as antagonists. In particular, this finding could be used with other BAS-1 species.

The amount of agent necessary for effective therapy will depend on many different factors, such as administration, target site, physiological state of the patient, and other drugs being administered. Thus, treatment doses should be titrated for optimal safety and efficacy. Typical doses used in vitro can provide useful guidance on the amount used for in vitro administration of these agents. Testing the effective dose in animals to treat specific conditions provides further preliminary indications of the human dose. A number of cases are described, for example, by Gilman et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8 ^th Ed., Pergamon Press and Remington's Pharmaceutical Sciences, 17* ed. (1990), Mack Publishing Co., Easton, Penn. Methods of administration are discussed herein and below, e.g., for oral, intravenous, intraperitoneal or intramuscular, transdermal diffusion, and others. Pharmaceutically suitable carriers are water, saline solutions, buffers and others, which he mentions

9 »99 9

9 9 9 9 » · 9999 9 « »99 9

9 «

9 9 9 9

999,999

9

99 Ι·*| e.g., Merck Index, Merck & Co., Rahway, New Jersey. The dose range is commonly assumed*·»* · 9 to be in an amount of concentration less than 1 mM, typically less than 10 μΜ, typically less than·** Σ 100 nM, preferably less than ΙΟρΜ (picomolar) and preferably less than lfM (femtomolar) vg«···.

• 9 to a suitable carrier. Slow-release substances or devices for slow-release substances are often·”*.

9 9 used for continuous administration. **

Human BAS-1, its fragments and antibodies against it or its fragments, antagonists 5 ;'*· • 999 agonists can be introduced directly into the host to treat it or depending on the size ·”· • 99 9 compounds are required to conjugated to carrier proteins such as ovalbumin, serum albumin, before their administration. Therapeutic agents may be administered in a number of conventional doses. While it is possible to introduce the active ingredient alone, it is preferable to administer it as a pharmaceutical substance. Rather, the compositions contain at least one active ingredient as defined above together with one or more acceptable carriers. Each carrier should be both pharmaceutically and physiologically acceptable in terms of compatibility with other ingredients and should not be harmful to the patient. The agents are administered topically, orally, rectally, nasally, or parenterally (including subcutaneously, intramuscularly, intravenously, and intradermally). The agents may conveniently be administered in unit dosage form and may be prepared by methods well known in the art of pharmacy. See, for example, Gilman et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8 ^th Ed., Pergamon Press and Remington's Pharmaceutical Sciences, 17 ^th ed. (1990), Mack Publishing Co., Easton, Penn. The therapy of this invention may be combined or used in conjunction with other chemotherapeutic or chemopreventive agents.

Both forms of BAS-1 antigens, native and recombinant form, are used in the present invention particularly in kits and assay methods capable of examining the protein binding activity of the compounds. In recent years, some methods of automatic determinations have been developed, which make it possible to examine tens of thousands of substances in a short time.

See, for example, Fodor et al. (1991) Science 251: 767-773, where binding affinity testing of a number of defined polymers synthesized on a solid substrate is described. The development of suitable assays can be greatly facilitated by the availability of large quantities of purified, soluble BAS-1 as provided by the present invention.

For example, antagonists can usually be discovered once BAS-1 is structurally defined. Testing potential ligand antagonists is now possible thanks to the development of highly automated assay methods using purified BAS-1. In particular, novel agonists and antagonists may be discovered using the screening techniques available herein. Especially important • fc «fcfc* ♦ fc fc fcfcfc fcfc · fcfcfcfc • fcfcfc fcfc · fcfcfcfc • fc fcfcfcfc fcfcfc fcfc fcfcfc fcfcfc • fcfc fcfcfcfc · · fcfc · fcfc fcfc ·· fcfc fcfcfcfc • · are those detected substances that have a combined binding activity to numerous BAS-1 proteins'·*·»· ^{9 99} eg compounds that can serve as antagonists for BAS-1 allelic variants. *··* ί

In addition, since signaling BAS-l.BAS-l binding partner can work in combination §····.

• · other signals, e.g. CD28/CTLA-4:B7/B70 and or CD40:CD40 ligand pathway, combination^'**.

• · · components or combination therapy with such pathways may also be considered. Thus, antagonism of multiple signaling pathways or stimulation with multiple pathways may be used. ; ·**· fcfcfcfc

The present invention is particularly directed to the screening of compounds using recombinant ·**· fcfcfcfc antigens by any type of drug screening technique. The advantages of using recombinant proteins in the investigation of specific ligands are: a) an improvement in the renewable source of BAS-1 from a specific source, b) theoretically a larger number of antigen molecules per cell with a better signal-to-noise ratio in the assay, and c) a type of variant specificity (theoretically with greater biological specificity and specific disease).

One method of drug discovery uses eukaryotic and prokaryotic host cells that are stably transformed with recombinant DNA molecules expressing BAS-1. Cells can be isolated, expressing BAS-1 during isolation from others. Such cells can be used, either live or in fixed form, for standard antigen/ligand binding assays. See also Parce et al. (1989) Science 246: 243-247 and Owicki et al. (1990) Why. Naľl Acad. Sci. USA 87: 4007-4011, where sensitive methods for detecting cellular responses are described. In particular, competitive assays are used when cells (the source of BAS-1) are bound and incubated with a labeled ligand of known antigen binding affinity, such as ¹²⁵ I ligand, and a test compound whose binding affinity to BAS-1 is measured. Bound and free ligand are then separated to determine the degree of ligand binding. The amount of test compound bound is inversely proportional to the amount of labeled ligand binding measured. Any of a number of techniques can be used to separate the bound from the free ligand to determine the degree of ligand binding. This separation step could require a procedure such as adhesion to filters, subsequent washing, adhesion to plastic, subsequent washing, or centrifugation of cell membranes. Live cells could also be used to investigate the effects of drugs on BAS-1 functions, eg the second mediator level, i.e. Ca ²⁺ , cell proliferation, changes in inositol phosphate stores and others. Some detection methods allow the elimination of the separation step, e.g. the proximity of a sensitive detection system. A calcium-sensitive dye can be used to detect Ca ²⁺ levels using a fluorimeter or a fluorescence cell-splitter.

Other methods use the membranes of transformed eukaryotic or prokaryotic host cells as a source of human BAS-1. These cells are stably transformed with DNA vectors that direct the expression of human BAS-1 antigens. Essentially, the membranes would be prepared from ·* ··<·· ··· · · · * ·· · • · · · ·· ♦ · · · · • ···»· · · · · · ··· · ·· • · » · · · · · t

0· · ·· ·· ·· ♦· other cells and used in any receptor/ligand binding assays such as the competition assay described above. j * * * * j • · · ·

Still another approach is to use a soluble, unpurified or soluble, · ·*!

• · · punctate BAS-1 from transformed eukaryotic and prokaryotic host cells. · • ·

This enables the determination of "molecular" binding with the advantages of increased specificity, the ability to automate and the high throughput of the drug being tested. ·..·.:

Another drug screening technique involves an approach that provides high throughput screening • · · for compounds with suitable binding affinity to BAS-1 and is described in more detail by Geysen, European Patent·····* • · ·

Application 84/03564, published September 13, 1984. First, it is on a solid substrate, eg *·!··* plastic pins or some other suitable surface, see Fodor et al. (1991), synthesized a large number of different small peptide test compounds. All pins are then reacted with soluble, unpurified or soluble, purified BAS-1 and washed. The next step is to detect bound BAS-1.

A rational drug design can also be based on structural studies of the molecular shape of BAS-1 and other effectors or ligands. Effectors may be other proteins that mediate other functions in response to ligand binding or other proteins that normally interact with the antigen.

One way to determine the site of interaction with specific other proteins is to determine the physical structure, eg structural crystallography or two-dimensional NMR techniques. These will provide information on which amino acid residues form the molecular bond. For a more detailed description of protein structure determination, see, for example, Blundell and Johnson (1976) Protein Crystallography, Academie Press, New York.

Spotted BAS-1 can be poured directly onto dishes for use in the above drug screening technique. However, antibodies that are not neutralizing against these antigens can be used as antibody carriers when immobilizing the respective BAS-1 on a solid phase.

IX Kits

The present invention also includes the use of BAS-1 proteins, fragments thereof and fusion products thereof in a variety of diagnostic kits and methods for detecting the presence of BAS-1 or ligand. Typically, the kit has a portion containing either a defined BAS-1 peptide or a gene segment, or an agent that recognizes one or the other.

A kit for detecting the binding affinity of a test compound to BAS-1 typically includes a test substance, a labeled substance, e.g., a ligand or antibody with known binding affinity to BAS-1, a source of BAS-1 (naturally occurring or recombinant), and a means for cleaving the binding from loosely

44

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4 4 4

4 »44,444

4 4

44 • 4- 4444

4« 4

4 4 4 4

4 4 4 4 4 >444444 4

4 4 4 4 4 · 44 labeled substances such as the solid phase for immobilized BAS-1. Once the compounds are screened, those with suitable binding affinity to BAS-1 can be;”**;

4 4 4 evaluated by appropriate biological assays well known in the art to determine whether

4 4 are agonists or antagonists. The applicability of recombinant BAS-1 polypeptides also

44 4 provides well-defined standards for the calibration of such determinations. *....* ·

A more suitable kit to determine the concentration of, for example, BAS-1 in a sample should normally contain ί . í ^J 4 4 4 4 labeled substance, e.g., ligand or antibody, with well-known binding affinity to BAS-1, source

4 4

BAS-1 (naturally occurring or recombinant) and an agent for unbinding from free*·!··*

V 4 4 4 labeled substances, e.g. solid phase for immobilized BAS-1. A section containing reagents and instructions*.:..* will usually be provided.

A method to determine the concentration of BAS-1 in a sample should typically include the following steps: 1) preparing membranes from a sample containing a source of BAS-1, 2) washing the membrane and suspending it in buffer, 3) dissolving BAS-1 by incubating the membrane in culture medium, to to which a suitable detergent is added, 4) adjusting the concentration of the detergent dissolved in BAS-1, 5) connecting and incubating the aforementioned solution with the radiolabeled ligand in complexes, 6) recovering the complexes by filtering through a polyethylenimine-treated filter and 7) measuring the radioactivity of the recovered complexes.

Antibodies, including antigen-binding fragments, specific for human BAS-1 or BAS-1 fragments are used in diagnostics, e.g. to detect the presence of elevated levels of BAS-1 and/or its fragments. Lysates, live cells, fixed cells, immunofluorescence, cell cultures, body fluids, and others that can detect BAS-1-related antigens in serum or the like can be used in such diagnostic assays. Diagnostic assays can be homogeneous (without a separation step between the free substance and the antigen-ligand complex) or heterogeneous (including a separation step). There are many commercial assays such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (EIA), enzyme-linked immunosorbent assay (EMIT), substrate-labeled fluorescence immunoassay (SLFIA), and the like. For example, unlabeled antibodies can be used with a second antibody that is labeled and that recognizes an antibody against BAS-1 or a particular fragment thereof. These determinations have been widely debated in the literature. See, for example, Harlow and Lané (1988) Antibodies: A Laboratory Manual, CSH.

Anti-idiotype antibodies can similarly be used to determine the presence of antibodies against human BAS-1, as such they can be diagnostic for various abnormal conditions. For example, overproduction of BAS-1 can result from the production of various immunological reactions, • · · · • · · · »«» ··» • · ·· ·· *· r»»t «« « s * ♦ · · · « · · · · · * • 9999999 · ·

9 9 9 9 9 9

9 9· 99 which may be diagnostic of abnormal physiological conditions, particularly in the proliferation of cellular forms such as cancer or abnormal differentiation. j

Often reagents for diagnostic assays are supplied in kits to optimize assay sensitivity. Subject matter of the invention depending on the nature of the assay, the protocol and the labeled substance, either labeled or unlabeled antibody or labeled BAS-1 is provided. It is usually combined with other ingredients, such as buffers, stabilizers, materials necessary for signaling and production, such as enzyme substrates, etc. It is convenient if the kit contains •

instructions for proper use and disposal of content after use. The kit normally has parts for each reagent used. Reagents are conveniently supplied in the form of a dry lyophilized powder,* whereby they can be dissolved in an aqueous medium providing suitable reagent concentrations for functional assays.

Some of the basic components of drug testing and diagnostic assays mentioned above may be used without modification or may be modified in various ways. For example, labeling can be achieved by the attachment of a covalent or non-covalent group that provides a directly or indirectly detectable signal. In some of these assays, the ligand, test substance, BAS-1, or antibodies may be labeled either directly or indirectly. Direct labeling options include labeled groups: a radiolabeled substance such as ¹²⁵ I, enzymes (US Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescently labeled substances (US Pat. No. 3,940,475) capable of recording a change in fluorescence intensity , wavelength shift or fluorescence polarization. Both patents are cited here. Options for indirect labeling include biotinylation of one component, followed by binding to an avidin pair on one of the labeled groups.

There are also a number of methods for separating binding from free ligands or otherwise binding from a free tested substance. BAS-1 can be immobilized on different matrices and subsequently washed. Suitable matrices can be plastic, such as an ELISA plate, filters or beads. Methods for immobilizing BAS-1 on a matrix include, without limitation, direct adhesion to plastic, use of captured antibody, chemical conjugation, and biotin-avidin. The final step in the latter case requires precipitation of the antigen-ligand complex by any method including this use, eg an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate. Other suitable separation techniques include, but are not limited to, the particle-attracting fluorescent antibody method described in Rattle et al. (1984) Cl. Chem. 30: 1457-1461 and dual magnetic particle antibody separation as described in U.S. Pat. Pat. No. 4,659,678.

The method of binding proteins or their fragments to various labeled substances has been extensively described in the literature. Many techniques involve the use of activated carboxyl groups either by using ·· · ·

99 9 ♦ · ♦· • · * · • · · · ··· ··· • « ·· *· ·· «

• ♦ • · · • ···· • · • · · · · • · * · · • · · · ·· ·· carbodiimide or active ester to form peptide bonds, formation of thioethers by reaction of mercapte group with activated halogen, such as chloracetyl or with activated;”**;

• ·· · an olefin such as maleimide by chemical bonding or the like. Protein fusion also finds application in this ΙΊ ·· · application. · • 0· ·

Another diagnostic aspect of the present invention involves the use of polynucleotide or·.,.·• 0 oligonucleotide sequences from the BAS-1 sequence. These sequences can be used as probes for the detection of antigen levels in samples from patients suspected of having an abnormal condition.

* · · eg cancer or developmental problem. The preparation of both RNA and DNA nucleotide sequences, sequence labeling, and preferred sequence sizes are abundantly described and discussed in the literature. Typically, an oligonucleotide probe should be at least 14 nucleotides, commonly at least 18 nucleotides, and polynucleotide probes can be at most a few kilobases. A number of labeled substances can be used, the most common being radionucleotides, particularly ³² P. However, other techniques can also be used, such as biotin-modified nucleotides for incorporation into the polynucleotide.

Biotin is then administered as a binding site for avidin or antibodies, which can be labeled with various types of labels such as radionucleotides, fluorescent labels, enzymes, and the like. Alternatively, antibodies that recognize specific double helixes including DNA double helix, RNA double helix, hybrid DNA-RNA double helix, or DNA-protein double helix can be used. The antibodies can be labeled sequentially and the assay performed while the double helix is bound to the surface such that after the double helix is formed on the surface and the presence of the antibody bound to the double helix can be detected. Application of the probe to the novel anti-mediator RNA can be performed by any conventional technique, such as nucleic acid hybridization, with and without probing, recombination, hybrid release translation (HRT), and hybrid retention translation (HART). Amplification techniques such as polymerase chain reaction (PCR) may also be included.

Also described are diagnostic kits that also test the quantity and quality of other patterns. Diagnosis or prognosis is dependent on a combination of multiple indications used as patterns. Thus, kits can test combinations of patterns. See, for example, Viallet et al. (1989) Progress in Growth Factor Res. 1: 89-97.

X. Ligand or counterreceptor.

The description of the BAS-1 protein herein provides the means to identify a counterreceptor or ligand. Such a ligand or counterreceptor would specifically bind to BAS-1 with sufficiently high affinity. A number of constructs that provide additional labeling of BAS-1 to detect its partner are being made available. For example, direct labeling of BAS-1, fusions to markers for secondary labeling, eg FLAG or other tumor epitopes, Ig domain fusions, etc., provide • ·· · detection of binding partners. It can be a histological or an affinity method biochemical· ·'!

• · · purification, labeling or selection in expression cloning. A two-hybrid selection system · generating suitable constructs with available BAS-1 sequences can also be applied. See ·....· Fields and Song (1989) Nature 340: 245 - 246.

The broad concept of this invention is best understood by reference to the following examples, which are not intended to limit the invention to specific instances.

EXAMPLES OF IMPLEMENTATION OF THE INVENTION

Example 1: General methods.

Some standard methods are described or presented, for example, 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, ( ^2nd ed.) vols. 1-3, CSH Press, NY, Ausubel et al., Biology, Greene Publishing Associates, Brooklyn, NY or Ausubel et al. (1987 and Supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York. Protein purification methods include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, for example, Ausubel et al. (1987 and periodic Supplements), Deutscher (1990) Guide to Protein Purification in Methods in Enzymology, vol. 182 and other volumes in this series and literature on the use of protein purified products, e.g., Pharmacia, Piscataway NJ or Bio-Rad, Richmond, ca. Combination with other recombinant techniques allows fusion with suitable segments, eg the sFLAG sequence or equivalent, which can be fused through a protease-cleavable sequence. See e.g. Hochuli (1989) Chemische Industrie 12: 69 - 70, Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic Engineering, Principle and Methods 12: 87 - 98, Plenům Press, NY and Crowe et al. (1992) QIAexpress: The High Level Expression & Protein Purification System QIAGEN, lne., Chatsworth, CA.

Computerized sequence analysis was performed, eg, using available software programs contained in the GCG (U. Wisconsin) and GenBank resources. Public sequence databases such as GenBank and others were also used.

·· 44 • · · · ···· · · · 4 · · · • · · · 4 4 · 4 · · · 4 ·4 · · ·

4 4 4 4·· · · ·· · · · · · ·· ··

Example 2: Amplification of human BAS-1 fragment by PCR method.

9 · » · • · • · • · · ·

Two primers were designed according to mouse RP105 sequences. See Miyake et al. (1995) J. Immunol. í i*:

154: 3333-3340. The nucleotide sequence was calculated from the CTT sequence. 5'-primer A (TTG...) matches · • ··· 24 nucleotides from 162 to 185 and 3'-primer F (...TTC) matches 24 nucleotides from 1462 ·,...· ·

after 1485. No PCR products were detected from human tonsillar B cells.

Three 5'-primers were created: primer B (ACA...) includes nucleotides in position 321 to 344, • · 4 primer C (AAG...) contains nucleotides in positions 568 to 591 and primer D (TCT... ) includes'····* • 4 · nucleotides at position 859 to 882. Three other 3'-primers were also created: primer G (,..ΤΤΑ)'·ϊ··* containing nucleotides 1765 to 1788, primer H (...GAA) containing nucleotides at position 2024 to 2047 and primer E (.. .TGG) containing nucleotides 1164 to 1187.

PCR reactions of one 5'-primer in combination with four 3'-primers were used to increase the probability of obtaining PCR products from human tonsillar B cells. From the four groups of PCR reactions, only one pure 300 bp product was obtained that joined the stretch from 5'-primer D to 3'-primer E. This product was purified, subcloned into the pCR™ vector (Invitrogen, San Diego CA) and then sequenced. See SEQ ID NO: 1.

According to the 300 bp human sequence, 3 primers were designed: 5'-primer K, 3'-primers I and L. Using a similar PCR method, additional 300 bp human fragments were obtained by combining primers C and I (see SEQ ID NO: 1).

Example 3: Tissue expansion of human BAS-1.

A PCR product amplified by a pair of DE primers (see SEQ ID NO: 1) was used as a probe (DE probe) to study the tissue distribution of BAS-1 in human tissues. A 2.6 kb mRNA was detected in the human spleen and at a higher level in the human heart by the Nothern transfer method. Which was not so readily detected in human brain, thymus, lung, liver, skeletal muscle, kidney, prostate, testis, ovary, small (small) intestine, large intestine and peripheral blood leukocytes. In addition, a 2.6 kb mRNA was detected by the Nothern transfer method in the sIgD ⁺ lymphoma cell line B104 and at a higher level in the Burkitt lymphoma cell line BL2. However, it was not detected in the renal epithelial carcinoma cell line CHA, the lung epithelial carcinoma cell line MRC-5, the EBV cell line JY, or the monocyte cell line U937.

By PCR, human BAS-1 mRNA was found in vsIgD ⁺ CD38' simple B cells, s!gD ⁺ CD38 ⁺ germinal center B cells, s!gD'CD38 ⁺ germinal center B cells, in slgD' • · · · · · • · • · • · · · ·

CD38' memory B cells, in sCD38 ⁺⁺ CD20 ^marrow plasma cells, dendritic cells generated from CD14 + monocytes cultured with GM-CSF and IL-4, and dendritic cells; cells generated from CD34+ cord blood cells cultured with GM-CSF and TNFα. This was not detectable in the human T cell line MT9 by PCR.

• ·· · ··· • ·

Example 4: Screening of a cDNA plasmid library derived from the human sIgD+ lymphoma cell line B104.

• · • · * ···· • ·

9 9

9

From the results of the Nothern transfer analysis, BAS-1 expression was detected in 6 pg poly-A mRNA of B104* cell line. Accordingly, a B104 cDNA library was constructed in plasmid pSPORT1 (Gibco Life Technologies, Cergy Pontoise, France). After completion of screening of 150,000 bacterial colonies, no positive clones were found using the D-E probe. This suggests that the expression of BAS-1 in human cells may be very low.

Example 5: Human Spleen Bacteriophage XgtlO Library Screening.

A human spleen bacteriophage XgtlO library (Clontech) was used to screen a larger number of clones. After screening 2.4 x 10 ⁶ clones, 8 positive clones were identified. These were isolated, purified, and subcloned into plasmid pME18S (DNAX, Palo Alto, CA). After sequencing, two clones, S2 and LI, were found to represent the full length of human BAS-1 (see SEQ ID NO: 1). The full length of the isolated human BAS-1 cDNA is about 2635 bp and encodes a protein of about 661 amino acids. This protein product contains about 11 potential glycosylation sites and about 22 leucine-rich repeats. The coding region of this human BAS-1 cDNA shares about 72% homology with the coding region of the mouse RP105 sequence, and this complete BAS1 cDNA has 67% homology with the complete mouse cDNA sequence. The amino acid homology between human BAS-1 and mouse RP105 is about 73.5%.

Example 6: Expression of human BAS-1 protein.

All constructs were made by removing the 5' and 3' non-coding regions and adding the Marylin Kozák (Ref: J. Biol. Chem. 266: 19867, 1991) consensus sequence (ACCATGG, SEQ ID NO: 3) to increase the level of translation.

Soluble BAS-1-FLAG protein was transiently expressed in Cos 7 cells. A recombinant form of BAS-1 with a FLAG peptide at the carboxyl terminus (Hoppe et al. (1988) Biotechnology 6:

• ♦ 9 • · ···· · · · 9 9 999 999

9 9 9 9 9 9 9 9 ·· · ·· ·· ·· ··

1205 - 1210) was inserted into the expression vector pME18S and subsequently transfected into Cos-7 cells by electroporation. Electroporated cells were grown in DMEM medium supplemented with either:**”:

«

1% Nutridoma HU (Boehringer Mannheim, Mannheim, Germany) or in DMEM alone; ·'· • ♦ · medium for 5 days. · ··♦» • · • 9,999 9

9

Example 7: Purification of soluble BAS-1 Flag protein.

• · • · ·

Typically, 280 ml of supernatant containing soluble BAS-1 FLAG was applied to a 20 ml column with Cu ²⁺ *····* · 9 ions attached to a Chelating Sepharose Fast Flow matrix (Pharmacia, Upsalla, Sweden). After washing with 16 volumes of binding buffer (His-Bind Buffer Kit, Novagen, Madison, WI), proteins captured on metal ions were eluted with a gradient of 20 to 100 mM imidazole. The human BAS-1 FLAG content of the veluate fraction was determined by dot blotting using anti-FLAG monoclonal antibody M2 (Eastman Kodak, New Haven, CT) and Coomassie blue and silver-stained SDS-PAGE. Fractions of BAS-1 FLAG protein were then pooled together and dialyzed against PBS. As previously estimated from amino acid sequences by Western blotting, the enriched human BAS-1 corresponds to proteins of around 95 and 97 kD.

Example 8: Stable expression of membrane BAS-1 in NIH-3T3 cells.

The native membrane form was subcloned into the expression vector pMAMneo (Clontech,

Palo Alto, California), which contains the RSV-LTR enhancer linked to the dexamethasone-inducible MMTV-LTR promoter. This construct was then transfected into NIH3T3 cells by electroporation. Transfected NIH-3T3 cells were plated on selective 0.5 mg/ml Geneticin (G418) (Boehringer-Mannheim, Mannheim, Germany) DMEM medium supplemented with 10% Fetal Calf serum.

Biochemical characterization of the membrane BAS-1 protein in these stably transfected NIH-3T3 cells is performed by metabolic labeling. Cells were then cultured for 24 h in DMEM medium supplemented with 10% Fetal Calf serum and ΙμΜ dexamethasone (Sigma, Saint Quentin Fallavier, France). To label cellular proteins, cells were then incubated with ³⁵ S-Met and ³⁵ S-Cys for the last 12 hours. Protein analyzes under reducing conditions on SDS-PAGE demonstrated the presence of the predicted 110 kD protein in the lysate of transfected NIH-3T3 cells, but not in the lysate of non-transfected NIH-3T3 cells.

·· · • · · · · · ···· • · · · ·· · · · « · • ····· · · · · · ··· ··· • · · ···· · · ·· · ·· · · ♦· · ·

Example 9: Preparation of Antibodies Specific to BAS-1. .*·*'.

• ·· · • ··· • · · • · ·

Inbred Balb/c mice were immunized intraperitoneally with recombinant forms of human .

··♦· protein. First, purified soluble BAS-1 FLAG was obtained and then stable 5· transfected NIH-3T3 cells. Animals were stimulated at appropriate times with or without protein; . ;

·· ·· other auxiliary substances for further stimulation of antibody formation. Serum is obtained or hybridomas · • · · are produced by obtained spleens. *. í.. ’ • *

Alternatively, Balb/c mice are immunized with gene-transformed cells or fragments of either endogenous or exogenous cells, or are immunized with isolated membranes enriched for antigen expression. The serum is conveniently obtained normally after a series of further applications. Various gene therapy techniques can be used to induce an immune response, eg in the production of a protein in situ.

Monoclonal antibodies can be produced. For example, splenocytes are fused to an appropriate fusion partner and hybridomas are selected in growth medium by standard methods. Hybridoma supernatants are examined for the presence of antibodies that bind to human BAS-1, eg, by ELIS A or other assays. Antibodies that specifically recognize human BAS-1, rather than species variants, can also be selected or prepared.

Another method is represented by synthesized peptides or purified protein of the immune system, inducing monoclonal or polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene and Harlow and Lané (1989) Antibodies: A Laboratory Manual, Cold Spring Harbor Press. In appropriate circumstances, the binding agent is either labeled as above, eg fluorescently or otherwise, or is immobilized on the substrate by a panning method. Nucleic acids can be introduced into animal cells to produce an antigen that serves to elicit an immune response. See, for example, Wang et al. (1993) Why. Nachl.

Acad. Sci. 90: 4156-4160, Barry et al. (1994) BioTechniques 16: 616-619 and Xiang et al.

(1995) Immunity 2: 129–135.

Example 10: Large-scale production of BAS-1 in NSO cells.

Large amounts of soluble BAS-1 and soluble BAS-1 FLAG can be produced from stable transformants of NSO cells prepared, for example, according to the methodology developed by Celltech (Slough, Berkshire, UK, International Patent Applications WQ86/05807, WO87/04462, • · · · · · φφφ · · · · · · · φφφφ ·· · · φ φ · • · ···· · · · · · ··· φφφ φφφ · · · · · · ·· · · · « φ φφ φφ

WO89/01036 and WO89/10404). Both BAS-1 and BAS-1 FLAG were subcloned into pEE12 and subsequently transfected by electroporation into NSO cells. Transfected NSO cells werej*”*·

ΦΦΦ · inoculated into selective DMEM without glutamine supplemented with 10% Fetal Calf serum, as described in the Celltech protocol. Supernatants from the best producing line were used *.

ΦΦΦΦ in biological assays and purification of soluble BAS-1 and soluble BAS-1 -FLAG.

Other expression systems can also be used for greater production of recombinant proteins. J. ί

Example 11: Production of fusion proteins with BAS-1.

<

A number of fusion constructs with the BAS-1 extracellular domain have been generated. This part of the gene is attached to another protein, eg FLAG tumor epitope, Ig domain or to a two-hybrid system construct. See, for example, Fields and Song (1989) Nature 340: 245-246.

The tumor epitope can be used in expression cloning with detection of a binding partner with an antiFLAG antibody, eg a ligand for BAS-1. Alternatively, the Ig domains can be used for purification using an antigen-like substance with ligand affinity. Two hybrid systems can also be used to isolate proteins that specifically bind to BAS-1.

Example 12: Mapping of human BAS-1 by in situ hybridization.

A chromosome was prepared. In situ hybridization was performed on a chromosome preparation obtained from phytohemagglutinin-stimulated human lymphocytes cultured for 72 hours. To guarantee good quality of the post-hybridization chromosome bands, 5-bromodeoxyuridine (60 ug/ml medium) was added for the last 7 hours of culture.

A 655 bp PCR fragment amplified with primers eg corresponding to nucleotides 395 to 411 and 1027 to 1050 of the total B cell cDNA template was cloned into the appropriate vector. The vector was labeled with a ³ H shift of the single-stranded break. The radiolabeled probe hybridized to metaphase in a final concentration of 200 ng/ml hybridization solution as described by Mattei et al. (1985) Hum. Genet. 69: 327–331.

After coating with nuclear tracer emulsion (KODAK NTB ₂ ), the slides were exposed for 18 days at 4 °C. To avoid any streaks of silver beads during the binding procedure, the chromosome was washed with buffered Giemsa solution and the metaphase was photographed. R-bands were then visualized by fluorochrome photolysis-Giemsa (FPG) and metaphase rephotographed before analysis.

0··· • 00 · · · 0000

0000 00 0 0000

0 0000 0 0 0 0 0 000 000

000 0000 0 ·

0 00 00 00 0·

In 100 metaphase cells tested after in situ hybridization, 20% of the silver grains were located on chromosome 5. Grain expansion was not random, 67% of the harvesters mapped in the region;****;

0000 ql 1.2-ql3.1 of chromosome 5 long arm. This maps to human BAS-1 in the 5q 11.2-q 13.15 ΓΪ ^Π 0 0 0 region of the human genome. .

0000 0 0 • 0

0000

Example 13: Isolation of receptors for human BAS-1. ;* , J

0 0 0

0« • 0 0

Human BAS-1 can be used as a specific binding agent to identify its binding*····’

0

0 0 partner, for its advantageous binding specificity, antibodies should be used instead. The binding agent*,;..* is either labeled as described above, eg fluorescently or otherwise, or immobilized on a substrate for panning methods.

The binding mixture is used to screen an expression library that has been generated from a cell line expressing a binding partner, eg a receptor. Standard staining techniques are used to detect or include an intracellular or surface-expressed receptor, or surface-expressing transformed cells are monitored by panning. Intracellular expression monitoring is performed by a variety of staining or immunofluorescence procedures. See also McMahan et al. (1991) EMBO J. 10: 2821-2832.

For example, on day 0, pre-coat a two-chamber permanox slide with fibronectin 1 ml per chamber, 10 ng/ml in PBS for 30 min at room temperature. Rinse once with PBS. Then inoculate with COS cells at 2 - 3 x 10 ⁵ cells per chamber in 1.5 ml of growth medium. Incubate overnight at 37 °C.

On day 1, for each sample, prepare 1.5 ml of a solution of 66 pg/ml DEAE-dextran, 66 μΜ chlorquine and 44 pg DNA in serum without DME. A positive control, e.g., human BAS-1-FLAG cDNA at 1 and 1/200 dilution and a negative control, were prepared for each set. Rinse cells with DME-free serum. Add the DNA solution and incubate for 5 hours at 37 °C. Remove the medium and add 0.5 ml of 10% DMSO in DME for 2.5 min. Remove and wash once in DME. Add 1.5 ml growth medium and incubate overnight.

On day 2, replace the medium. On day 3 or day 4, cells are fixed and stained. Rinse cells twice with HBSS (Hank's Buffered Saline Solution) and fix for 5 minutes in 4% paraformaldehyde (PFA)/glucose solution. Wash three times with HBSS solution. After all liquid was removed, the slides were stored at -80°C. In each chamber, 0.5 ml was incubated as follows. Add HBSS/saponin solution (0.1%) with 32 μΐ/ml 1M NaN ₃ for 20 minutes. Cells were then washed once with HBSS/saponin solution. Add human BAS-1 or BAS-1/antibody complex to the cells and incubate for 30 min. Wash cells twice with solution ··· · ·· · • · · · · · 99·· ···· ·· · · · · · « · ···· · · · · · ··· ··« · · · · 9 · · • 9 · ·· ·· ·· ··

HBSS/saponin. If appropriate, add the first antibody for 30 minutes. Add a second antibody, e.g.

anti-mouse antibody vector at a dilution of 1/200 and incubate for 30 minutes. Prepare ELISA solution,;****;

···· e.g. vector elite ABC horseradish peroxidase solution and pre-incubate for 30 minutes. Use; !*!

• · · for example, 1 drop of solution A (avidin) and 1 drop of solution B (biotin) per 2.5 ml of HBSS/saponin. · ····

Wash cells twice with HBSS/saponin solution. Add ABC HRP solution and incubate for 30 minutes *....* • ·

Wash the cells twice with HBSS solution, wash the second time for 2 min to seal the cells!...J. Then add diamine benzoic acid (DAB) vector for 5 to 10 minutes. Use 2 drops of *., buffer with 4 drops of DAB and 2 drops of H ₂ O ₂ per 5 ml of distilled water. Carefully remove the chamber and ····* wash the slide with water. Air dry for a few minutes, then add 1 drop of Crystal Mount and*··* cover with a coverslip. Dry for 5 minutes at 85 to 90 °C.

Evaluate positive staining and gradually subclone to isolate individual genes responsible for binding.

Alternatively, BAS-1 reagents are used to affinity purify or select cells expressing the receptor. See, for example, Sambrook et al. or Ausubel et al.

Another method is tracking the membrane-bound receptor by the panning method. The receptor cDNA is constructed as described above. The ligand can be immobilized and used to immobilize expressing cells. Immobilization can be achieved by using suitable antibodies that recognize e.g. the FLAG sequence of the BAS-1 fusion construct or by using antibodies selected against the first antibodies. Reusable cycles of selection and amplification lead to enrichment of suitable clones and ultimate isolation of receptor expressing clones.

Phage expression libraries can be screened by human BAS-1. Suitable labeling techniques, e.g. anti-FLAG antibodies, allow specific labeling of suitable clones.

All cited citations are incorporated by reference to the same extent as if the individual publications or patent applications were specifically and individually indicated to be incorporated by reference.

Many modifications and variations of this invention may be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific examples described herein are presented by way of example only, and the invention may be limited by the terms appended to the claims along with the full range of equivalents to which those claims point.

9999 • 9 · · · · · 4

999,999

LIST OF SEQUENCES (1) GENERAL INFORMATION:

(i) APPLICANT: Schering Corporation (ii) TITLE OF THE INVENTION: Isolated and Recombinant Nucleic Acid, BAS-1 Protein or Peptide and Agent, Antibody, Expression Vector, Host Cell and Methods of Use Thereof (iii) NUMBER OF SEQUENCES: 3 (iv) CONTACT ADDRESS:

(A) ADDRESS: Schering-Plough Corporation (B) STREET: 2000 Galloping Hill Road (C) CITY: Kenilworth (D) STATE: New Jersey (E) COUNTRY: USA (F) ZIP CODE: 07033 (v) TYPE OF COMPUTER PROCESSING:

(A) MEDIA TYPE: Floppy disk (B) COMPUTER: Macintosh (C) OPERATING SYSTEM: 7.5.3 (vi) CURRENT APPLICATION DATE:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(vii) PREVIOUS APPLICATION DATE:

(A) APPLICATION NUMBER (B) SUBMISSION DATE: 17.5. 1996 (viii) LEGAL REPRESENTATIVE AND AGENT INFORMATION:

9999 • 9«·

9 9 • 9 ·

9 his *

9999 9 9

9

9999

9

9

9 9

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9 9 9 (A) NAME: Cynthia L. Foulke, Esq.

(B) REGISTRATION NUMBER: 32,364 (C) CERTIFICATE NUMBER: DX0580PCT (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 908-298-2987 (B) FAX: 908-298-5388 (2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2635 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTER:

(A) NAME/KEY: CDS (B) POSITION: 97..2082 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATTCCGGCGG CTGCCAAATT GCCAGGGCCT TCACAGTTTG ATTCCATTTC TCAGCTCCAA • 4

4444

44

4 4 4 4 4 4

4 4 4 4 4 4 « · 4 4 444 444

4 4 4

44 44 44

4444

GCATTAGGTA AACCCACCAA GCAATCCTAG CCTGTG ATG GCG TTT GAC GTC AGC 114

Met 1 Alas Phew Asp Wall 5 Ser TGC 162 TTC TTT TGG GTG GTG CTG TTT TCT GCC GGC TGT AAA GTC ATC acc Cys Phew Phew Suffering 10 Wall Wall Leo Phew Ser 15 Alas Glee Cys Fox Wall 20 How many Thr TCC 210 TGG GAT CAG ATG TGC ATT GAG AAA GAA GCC AAC AAA ACA MELT AAC Ser Suffering Asp 25 Gin Met Cys How many Gosh 30 Fox Gosh Alas Association Fox 35 Thr Tire Association TGT 258 GAA AAT TTA GGT CTC AGT GAA ATC CCT GAC ACT CTA Approx AAC ACA Cys Gosh 40 Association Leo Glee Leo Ser 45 Gosh How many For Asp Thr 50 Leo For Association Thr ACA 306 GAA TTT TTG GAA TTC AGC TTT AAT TTT TTG CCT ACA ATT CAC AAT Thr 55 Gosh Phew Leo Gosh Phew 60 Ser Phew Association Phew Leo 65 For Thr How many His Association 70 AGA 354 acc TTC AGC AGA CTC ATG AAT CTT acc TTT TTG GAT TTA ACT AGG Arg Thr Phew Ser Arg 75 Leo Met Association Leo Thr 80 Phew Leo Asp Leo Thr 85 Arg TGC 402 CAG ATT AAC TGG ATA CAT GAA GAC ACT TTT CAA AGC CAT CAT CAA Cys Gin How many Association 90 Suffering How many His Gosh Asp 95 Thr Phew Gin Ser His 100 His Gin TTA 450 AGC ACA CTT GTG TTA ACT GGA AAT CCC CTG ATA TTC ATG GCA GAA Leo Ser Thr 105 Leo Wall Leo Thr Glee 110 Association For Leo How many Phew 115 Met Alas Gosh ACA 498 TCG CTT AAT GGG CCC AAG TCA CTG AAG CAT CTT TTC TTA ATC CAA Thr Ser 120 Leo Association Glee For Fox 125 Ser Leo Fox His Leo 130 Phew Leo How many Gin ACG 546 GGA ATA TCC AAT CTC GAG TTT ATT Approx GTG CAC AAT CTG GAA AAC Thr 135 Glee How many Ser Association Leo 140 Gosh Phew How many For Wall 145 His Association Leo Gosh Association 150 TTG 594 GAA AGC TTG MELT CTT GGA AGC AAC CAT ATT TCC TCC ATT AAG TTC Leo Gosh Ser Leo Tire 155 Leo Glee Ser Association His 160 How many Ser Ser How many Fox 165 Phew CCC 642 AAA GAC TTC Approx GCA CGG AAT CTG AAA GTA CTG GAT TTT CAG AAT For Fox Asp Phew 170 For Alas Arg Association Leo 175 Fox Wall Leo Asp Phew 180 Gin Association AAT 690 GCT ATA CAC TRAY ATC TCT AGA GAA GAC ATG AGG TCT CTG GAG CAG Association Alas How many 185 His Tire How many Ser Arg 190 Gosh Asp Met Arg Ser 195 Leo Gosh Gin

44 4

4 4 4

4 4

4 4

4

4444

4 4

4 4

4444

4

4·

4494

4444 * 9 4

4 4 4

44444

4 4

4 ·♦ • 4 9·· • 4 4 4

4,444,444

4 4

99

GCC 738 Alas ATC AAC CTA AGC Ser CTG AAC TTC AAT GGC AAT AAT GTT AAA GGT ATT 9 4 9 4 4 44 4 4 44« 4 4 9 9 9 · 4 9 9 How many Association Leo Leo Asn Phe Association Glee Association Association Wall Fox Glee How many 5 200 - 205 210 4 4444 GAG CTT GGG GCT TTT GAT TCA ACG GTC TTC CAA AGT TTG AAC TTT GGA 4 4 9 4 786 4 4 Gosh Leo Glee Alas Phew Asp Ser Thr Wall Phew Gin Ser Leo Association Phew Glee 4 4 4 *4^4 10 215 220 225 230 4 GGA ACT Approx AAT TTG TCT GTT ATA TTC AAT GGT CTG CAG AAC TCT ACT 44 4 4 4 4 4 4 834 4 · 4 · 4 4 Glee Thr For Association Leo Ser Val Ile Phew Association Glee Leo Gin Association Ser Thr 9 4 4 4 4 9 15 235 240 245 9 49 4 ACT CAG TCT CTC TGG CTG GGA ACA TTT GAG GAC ATT GAT GAC GAA GAT 882 Thr Gin Ser Leo Suffering Leo Gly Thr Phew Gosh Asp How many Asp Asp Gosh Asp 20 250 255 260 ATT AGT TCA GCC ATG CTC AAG GGA CTC TGT GAA ATG TCT GTT GAG AGC 930 How many Ser Ser Alas Met Leo Lys Gly Leo Cys Gosh Met Ser Wall Gosh Ser 25 265 270 275 CTC AAC CTG CAG GAA CAC CGC TTC TCT GAC ATC TCA TCC acc ACA TTT 978 Leo Association Leo Gin Gosh His Arg Phe Ser Asp How many Ser Ser Thr Thr Phew 30 280 285 290 CAG TGC TTC acc CAA CTC CAA GAA TTG GAT CTG ACA GCA ACT CAC TTG

1026

35 Gin Cys 295 Phew Thr Gin Leo 300 Gin Gosh Leo Asp Leo 305 Thr Alas Thr His Leo 310 AAA GGG 1074 TTA CCC TCT GGG ATG AAG GGT CTG AAC TTG CTC AAG AAA TTA 40 Lys Gly Leo For Ser 315 Glee Met Fox Glee Leo 320 Association Leo Leo Fox Fox 325 Leo GTT CTC 1122 AGT GTA AAT CAT TTC GAT CAA TTG TGT CAA ATC AGT GCT GCC 45 Val Leu Ser Wall 330 Association His Phew Asp Gin 335 Leo Cys Gin How many Ser 340 Alas Alas AAT TTC CCC TCC CTT ACA CAC CTC TRAY ATC AGA GGC AAC GTG AAG AAA

1170

50 Asn Phe For 345 Ser Leo Thr His Leo 350 Tire How many Arg Glee Association 355 Wall Fox Fox CTT CAC 1218 CTT GGT GTT GGC TGC TTG GAG AAA CTA GGA AAC CTT CAG ACA 55 Leu His 360 Leo Glee Wall Glee Cys 365 Leo Gosh Fox Leo Glee 370 Association Leo Gin Thr CTT GAT 1266 TTA AGC CAT AAT GAC ATA GAG GCT TCT GAC TGC TGC AGT CTG 60 Leu Asp 375 Leo Ser His Association 380 Asp How many Gosh Alas Ser 385 Asp Cys Cys Ser Leo 390 CAA CTC 1314 AAA AAC CTG TCC CAC TTG CAA acc TTA AAC CTG AGC CAC AAT Gin Leu Fox Association Leo Ser His Leo Gin Thr Leo Association Leo Ser His Association

»» ··»»

• «4 9 · ) 9 * » · ♦ · • * * < «· > « ·· 595 400 405 999 9 • 9 GAG CCT 1362 CTT GGT and CTC CAG AGT CAG GCA TTC AAA GAA TGT CCT CAG CTA 9 9 • 99 9 • 99« • 9 9 5 Glu Pro Leo Glee Leo Gin Ser Gin Alas Phew Fox Gosh Cys For Gin Leo • 9 9 99 9 410 415 420 - 9 • 999 9 9 GAA CTC CTC GAT TTG GCA TTT acc CGC TTA CAC ATT AAT GCT Approx CAA • 9 • 999 1410 • 9 9 9 10 Go Lea Leo Asp Leo Alas Phew Thr Arg Leo His How many Association Alas For Gin 9 9 9 99 99 425 430 435 • 99 AGT CCC TTC CAA AAC CTC CAT TTC CTT CAG GTT CTG AAT CTC ACT TRAY 9 9 9 9 9 9 • 9 99 1458 • 9 9 9 9 15 Sir Pro Phew Gin Association Leo His Phew Leo Gin Wall Leo Association Leo Thr Tire 9 9 9 9,999 440 445 450 TGC TTC 1506 CTT GAT acc AGC AAT CAG CAT CTT CTA GCA GGC CTA Approx GTT 20 Cys Phe Leo Asp Thr Ser Association Gin His Leo Leo Alas Glee Leo For Wall 455 460 465 470 CTC CGG 1554 CAT CTC AAC TTA AAA GGG AAT CAC TTT CAA GAT GGG ACT ATC 25 Leu Arg His Leo Association Leo Fox Glee Association His Phew Gin Asp Glee Thr How many 475 480 485 ACG AAG 1602 acc AAC CTA CTT CAG acc GTG GGC AGC TTG GAG GTT CTG ATT 30 Thr Lys Thr Association Leo Leo Gin Thr Wall Glee Ser Leo Gosh Wall Leo How many 490 495 500 TTG TCC 1650 TCT TGT GGT CTC CTC TCT ATA GAC CAG CAA GCA TTC CAC AGC 35 Leu Ser Ser Cys Glee Leo Leo Ser How many Asp Gin Gin Alas Phew His Ser 505 510 515 TTG GGA 1698 AAA ATG AGC CAT GTA GAC TTA AGC CAC AAC AGC CTG ACA TGC 40 Leu Gly Fox Met Ser His Wall Asp Leo Ser His Association Ser Leo Thr Cys 520 525 530 GAC AGC 1746 ATT GAT TCT CTT AGC CAT CTT AAG GGA ATC TRAY CTC AAT CTG 45 Asp Ser How many Asp Ser Leo Ser His Leo Fox Glee How many Tire Leo Association Leo 535 540 545 550 GCT GCC 1794 AAC AGC ATT AAC ATC ATC TCA CCC CGT CTC CTC CCT ATC TTG 50 Ala Ala Association Ser How many Association How many How many Ser For Arg Leo Leo For How many Leo 555 560 565 TCC CAG CAG AGC acc ATT AAT TTA AGT CAT AAC CCC CTG GAC TGC ACT

1842

55 Sir Gin Gin Ser Thr How many Association Leo Ser His Association For Leo Asp Cys Thr 570 575 580 TGC TCG AAT ATT CAT TTC TTA ACA TGG TRAY AAA GAA AAC CTG CAC AAA 1890 60 Cys Ser Association How many His Phew Leo Thr Suffering Tire Fox Gosh Association Leo His Fox 585 590 595

CTTGAA GGC TCG GAG GAG ACA CGT GTG CAA AAC CCG CCA TCT CTA AGG 1938 ·· · ·· ···· •···» * • · · · ··· ··· • «· · · · ·· · · »♦ ♦·

Leu Glu 600 Glee Ser Glu Glu Thr Arg Val Gin Asn Pro Pro Ser Leu Arg • · • · 9 99 9 605 610 GGA GTT AAG CTA TCT GAT GTC AAG CTT TCC TGT GGG ATT ACA GCC ATA 9,999 9 9 9 9 9 9 5 1986 99 9 Gly Val Fox Leu Ser Asp Val Lys Leu Ser Cys Gly Ile Thr Ala How many 9 615 620 625 630 9 9 9· 9999 GGC ATT TTC TTT CTC ATA GTA TTT CTA TTA TTG TTG GCT ATT CTG CTA 9 9 9· 10 2034 99 99 Gly Ile Phew Phe Leu Ile Val Phe Leu Leu Leu Leu Ala Ile Leu Leo • 635 640 645 * * 9 9 9 9 9 9 9999 TTT TTT GCA GTT AAA TAC CTT CTC AGG TGG AAA TAC CAA CAC TTT TAG 9 · » · » 15 2082 • 99 9 Phew Phew Alas Val Lys Tyr Leu Leu Arg Trp Lys Tyr Gin His Phe 650 655,660

20 TGCTGAAGGT 2142 TTCCAGAGAA AGCAAATAAG TGTGCTTAGC AAAATTGCTC TAAGTGAAAG AACTGTCATC 2202 TGCTGGTGAC CAGACCAGAC TTTTCAGATT GCTTCCTGGA ACTGGGCAGG 25 GACTCACTGT 2262 GCTTTTCTGA GCTTCTTACT CCTGTGAGTC CCAGAGCTAA AGAACCTTCT 30 AGGCAAGTAC 2322 ACCGAATGAC TCAGTCCAGA GGGTCAGATG CTGCTGTGAG AGGCACAGAG CCCTTTCCGC 2382 ATGTGGAAGA GTGGGAGGAA GCAGAGGGAG GGACTGGGCA GGGACTGCCG 35 GCCCCGGAGT 2442 CTCCCACAGG GAGGCCATTC CCCTTCTACT CACCGACATC CCTCCCAGCA CCACACACCC 2502 CGCCCCTGAA AGGAGATCAT CAGCCCCCAC AATTTGTCAG AGCTGAAGCC 40 AGCCACTAC 2562 CCACCCCCAC TACAGCATTG TGCTTGGGTC TGGGTTCTCA GTAATGTAGC CATTTGAGAA ACTTACTTTGG GGACAAAGTC TCAATCCTTA TTTTAAATGA AAAAAAAA

2622

GAAAAGCATA ΑΤΑ 2635 (2) INFORMATION FOR SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 661 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO.2.

Met Ala Phe Asp Val Ser Cys Phe Phe Trp Val Val Leu Phe Ser Ala 1.5 10 15

Gly Cys Lys Val Ile Thr Ser Trp Asp Gin Met Cys Ile Glu Lvs »4· 4 4 ·

····

4 »4 » 4* a » 4 4 a

444 44a a

44

20 25 30 4444 4 4 4 4 Alas Association Fox 35 Thr Tire Association Cys Gosh 40 Association Leo Glee Leo Ser 45 Gosh How many For 4444 4 44« 4 4 4 4 4 4 44 4 Asp Thr 50 Leo For Association Thr Thr 55 Gosh Phew Leo Gosh Phew 60 Ser Phew Association Phew 4 444* 4 4 4 4 4 4 44 , Leo 65 For Thr How many His Association 70 Arg Thr Phew Ser Arg 75 Leo Met Association Leo Thr 80 4 4 4 4 4 4 4 4 4 44 4 Phew Leo Asp Leo Thr 85 Arg Cys Gin How many Association 90 Suffering How many His Gosh Asp 95 Thr 4« 4 4 4 4 4 » 4444 • 4 4 4 4 Phew Gin Ser His 100 His Gin Leo Ser Thr 105 Leo Wall Leo Thr Glee 110 Association For 4 4 4 444« Leo How many Phew 115 Met Alas Gosh Thr Ser 120 Leo Association Glee For Fox 125 Ser Leo Fox His Leo 130 Phew Leo How many Gin Thr 135 Glee How many Ser Association Leo 140 Gosh Phew How many For Wall 145 His Association Leo Gosh Association 150 Leo Gosh Ser Leo Tire 155 Leo Glee Ser Association His 160 How many Ser Ser How many Fox 165 Phew For Fox Asp Phew 170 For Alas Arg Association Leo 175 Fox Wall Leo Asp Phew 180 Gin Association Association Alas How many 185 His Tire How many Ser Arg 190 Gosh Asp Met Arg Ser 195 Leo Gosh Gin Alas How many 200 Association Leo Ser Leo Association 205 Phew Association Glee Association Association 210 Wall Fox Glee How many Gosh 215 Leo Glee Alas Phew Asp 220 Ser Thr Wall Phew Gin 225 Ser Leo Association Phew Glee 230 Glee Thr For Association Leo 235 Ser Wall How many Phew Association 240 Glee Leo Gin Association Ser 245 Thr Thr Gin Ser Leo 250 Suffering Leo Glee Thr Phew 255 Gosh Asp How many Asp Asp 260 Gosh Asp How many Ser Ser 265 Alas Met Leo Fox Glee 270 Leo Cys Gosh Met Ser 275 Wall Gosh Ser Leo Association 280 Leo Gin Gosh His Arg 285 Phew Ser Asp How many Ser 290 Ser Thr Thr Phew Gin 295 Cys Phew Thr Gin Leo 300 Gin Gosh Leo Asp J Leo 305 Thr Alas Thr His Leo 310 Fox Glee Leo For Ser 315 Glee Met Fox Glee Leo 320 Association Leo Leo Fox Fox 325 Leo Wall Leo Ser Wall 330 Association His Phew Asp Gin 335 Leo Cys Gin How many Ser 340 Alas Alas Association Phew For 345 Ser Leo Thr His Leo 350 Tire How many Arg Glee Association 355 Wall Fox Fox Leo His 360 Leo Glee Wall Glee Cys 365 Leo Gosh Fox

370

385

390

395

400 • ·· · • · ···« • » • ·

Leu Asn. Leu Ser His Asn Glu Pro Leu Gly Leu Gin Ser Gin Ala Phe 405,410,415

Lys Glu Cys Pro Gin Leu Glu Leu Leu Asp Leu Ala Phe Thr Arg Leu 420 425 430

His Ile Asn Ala Pro Gin Ser Pro Phe Gin Asn Leu His Phe Leu Gin 435 440 445 • 4 • · · • · * ···♦ • « • * · • ·· ·

Val Leu Asn Leu Thr Tyr Cys Phe Leu Asp Thr Ser Asn Gin His Leu 450 455 460

Leu Ala Gly Leu Pro Val Leu Arg His Leu Asn Leu Lys Gly Asn His 465 470 475 480

Phe Gin Asp Gly Thr Ile Thr Lys Thr Asn Leu Leu Gin Thr Val Gly 485 490 495

Ser Leu Glu Val Leu Ile Leu Ser Ser Cys Gly Leu Leu Ser Ile Asp 500 505 510

Gin Gin Ala Phe His Ser Leu Gly Lys Met Ser His Val Asp Leu Ser

515 ,, 520 525

His Asn Ser Leu Thr Cys Asp Ser Ile Asp Ser Leu Ser His Leu Lys

530 535 540

Gly Ile Tyr Leu Asn Leu Ala Ala Asn Ser Ile Asn Ile Ile Ser Pro 545 550 555 560

Arg Leu Leu Pro Ile Leu Ser Gin Gin Ser Thr Ile Asn Leu Ser His 565 570 575

Asn Pro Leu Asp Cys Thr Cys Ser Asn Ile His Phe Leu Thr Trp Tyr 580 585 590

Lys Glu Asn Leu His Lys Leu Glu Gly Ser Glu Glu Thr Arg Val Gin 595 600 605

Asn Pro Pro Ser Leu Arg Gly Val Lys Leu Ser Asp Val Lys Leu Ser 610 615 620

Cys Gly Ile Thr Ala Ile Gly Ile Phe Phe Leu Ile Val Phe Leu Leu 625 630 635 640

Leu Leu Ala Ile Leu Leu Phe Phe Ala Val Lys Tyr Leu Leu Arg Trp ⁶⁴⁵ 650 655

Lys Tyr Gin His Phe

660 (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS· (A) LENGTH: 7 base pairs (B) TYPE: nucleic acid (C) STRAND: single • 9 9 ♦ * · • · · · • · · · · · • · · ·

•9 «949 • · 9 · ·

9 * 9 9

9 9 9 «9

99 « 9 9 9 » 9 9 9

999,999

9

99 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3

ACCATGG 7

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999

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9 •

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9

9999

9

9

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99

9 9

9 9

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Ρΐ/ΜΓ»- Ή • · · · « fc fcfc • · · · « • fc fcfc » fcfc I » ·· a ··· ·· a • a ·· ·· »· · ·· «fcfcfc • fcfc · · · • · · · · · « • · ···· fc · · · · fc · · ···· • fc · fcfc ·· • fc fcfc ύ t · · fc · · · •fcfc ··· « · ·« ·· /fcgrs-

Claims (26)

PATENT CLAIMS An isolated or recombinant nucleic acid encoding a human BAS-1 protein or fragment thereof. The isolated or recombinant nucleic acid of claim 1, wherein said nucleic acid comprises the sequence defined in SEQ ID NO: 1. The nucleic acid of claim 2, wherein said nucleic acid has at least 80% identity to the natural cDNA encoding said segment. The isolated or recombinant nucleic acid of claim 2, which encodes at least 8 consecutive residues of SEQ ID NO: 2. The nucleic acid of claim 4, which encodes at least 12 consecutive residues. The nucleic acid of claim 4, wherein said nucleic acid has at least 80% identity to the natural cDNA encoding said segment. A substantially pure BAS-1 protein or peptide thereof. The protein or peptide of claim 7 selected from the group consisting of: (a) protein or peptide from monkeys, including human; b) a protein or peptide comprising at least one polypeptide segment as defined in SEQ ID NO: 2, c) a protein or peptide having a post-translational modification profile different from the native BAS-1 protein; and d) a protein that lacks the intracellular domain of BAS-1. The protein or peptide of claim 8 comprising a segment that exhibits sequence homology to the corresponding portion of human BAS-1: (a) said homology has at least 90% identity and said portion is at least 9 amino acids; (b) said homology has at least 80% identity and said part is at least 17 amino acids; or • said homology has at least 17 amino acids; or · · · · · · · · ·. ·. ·. · · · · · ~ ·· · ♦ · ···· ·· ·· 49 ··· · · · · · · · • · · · '· • ·' · '' · · · '· · · · ·· ·· ·· ·· c) said homology has at least 70% identity and said portion is at least 25 amino acids. The fusion protein of claim 7 comprising a BAS-1 peptide. The composition of claim 7 comprising a protein and a pharmaceutically acceptable carrier. An antibody or antibody-bound fragment that specifically binds a recombinant or purified monkey BAS-1 protein or fragment thereof. The antibody of claim 12, wherein said BAS-1 protein is a monkey protein including one human. The antibody of claim 12, wherein said antibody is obtained against the peptide sequence defined in SEQ ID NO: 2. The antibody of claim 12, wherein said antibody has a K d of at least 10 µΜ. The antibody of claim 12, wherein said antibody is a monoclonal antibody. The antibody of claim 12, wherein said antibody is labeled. The antibody of claim 12, wherein: (a) induces strong B cell proliferation; or b) protect B cells from radiation-induced apoptosis. An expression vector according to claim 1 comprising a nucleic acid. The host cell of claim 19 comprising a vector. 21. A method of examining a sample for a BAS-1 binding partner comprising the steps of producing purified or recombinant BAS-1 protein and monitoring in said sample for specifically binding said binding partner to said BAS-1. · · · ΦΦΦ · * · φ · φ · φ · φ · φ · φ · φ · φ · φ · • · • · φ · Φ φ • • φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ 22. The method of claim 21, wherein said sample comprises proteins derived from T cells, dendritic cells including follicular dendritic cells or stromal cells including fibroblast cells, endothelial and epithelial cells. 23. The method of claim 21, wherein said binding partner is an antibody and said example is a hybridoma supernatant. 24. A method of modulating the physiology and development of cells comprising contacting said cells with an agonist or antagonist of a BAS-1 protein. 25. The method of claim 24, wherein said antagonist is an antibody against murine BAS-1 protein. 26. The method of claim 24, wherein said linkage is in combination with a signal mediator via an antigen receptor, a CD40: CD40 ligand pathway, or a CD28 / CTLA- pathway.