AU2004231176B2 - Human CTLA-8 and uses of CTLA-8-related proteins - Google Patents

Description

AUSTRALIA

Patents Act 1990 GENETICS INSTITUTE, LLC COMPLETE SPECIFICATION STANDARD PATENT Invention Title.

Human CTLA-8 and uses of CTLA-8-related proteins The following statement is a full description of this invention including the best method of performing it known to us:- 1 HUMAN CTLA-8 AND USES OF CTLA-8-RELATED PROTEINS 0 SThis is a divisional of AU 776563, the entire contents of which are incorporated herein by reference.

Field of the Invention The present invention relates to human CTLA-8 proteins, nucleic acids encoding such proteins, methods of treatment using such proteins. The invention also relates to C1 the use of rat CTLA-8 proteins and herpesvirus Saimiri ORF13 proteins in methods of 0 10 treatment.

Background of the Invention Cytokines are secreted proteins which act on specific hematopoietic target cells to cause a differentiation event or on other target cells to induce a particular physiological response, such as secretion of proteins characteristic of inflammation.

Cytokines, also variously known as lymphokines, hematopoietins, interleukins, colony stimulating factors, and the like, can be important therapeutic agents, especially for diseases or conditions in which a specific cell population is depleted. For example, erythropoietin, G-CSF, and GM-CSF, have all become important for treatment of anemia and leukopenia, respectively. Other cytokines such as interleukin-3, interleukin-6, interleukin-11 and interleukin-12 show promise in treatment of conditions such as thrombocytopenia and modulation of immune response.

For these reasons a significant research effort has been expended in searching for novel cytokines and cloning the DNAs which encode them. In the past, novel cytokines were identified by assaying a particular cell such as a bone marrow cell, for a measurable response, such as proliferation. The search for novel cytokines has thus been limited by the assays available, and if a novel cytokine has an activity which is unmeasurable by a known assay, the cytokine remains undetectable. In a newer approach, cDNAs encoding cytokines have been detected using the polymerase chain 1 Sreaction (PCR) and oligonucleotide primers having homology to share motifs of known cytokines or their receptors. The PCR approach is also limited by the z necessity for knowledge of previously cloned cytokines in the same protein family. Cytokines have also been cloned using subtractive hybridization to 5 construct and screen cDNA libraries, or they can potentially be cloned using PCR followed by gene electrophoresis to detect differentially expressed genes.

The subtractive hybridization methods are based on the assumption that cytokine mRNAs are those that are differentially expressed, and these methods S do not require any prior knowledge of the sequence of interest. However, many 10 cytokines may be encoded by mRNAs which are not differentially expressed, 0and thus are undetectable using these methods.

It would be desirable to develop new methods for identifying novel cytokines and other secreted factors and to isolate polynucleotides encoding them.

Summary of the Invention In developing the present invention, methods were employed which selectively identify polynucleotides which encode secreted proteins. One such polynucleotide was isolated which encodes "human CTLA-8". In accordance with the present invention, polynucleotides encoding human CTLA-8 and active fragments thereof are disclosed. "CTLA-8" is used throughout the present specification to refer to both proteins and polynucleotides encoding those proteins and to refer to proteins and polynucleotides from all mammalian species.

In a first aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: the nucleotide sequence of SEQ ID NO:1 from nucleotide 55 to nucleotide 544; the nucleotide sequence of SEQ ID NO:1 from nucleotide 85 to nucleotide 544; the nucleotide sequence of SEQ ID NO:1 from nucleotide 139 to nucleotide 544; the nucleotide sequence of SEQ ID NO:1 from nucleotide 146 to nucleotide 544; the nucleotide sequence of the full length protein coding sequence of clone B18 deposited under accession number ATCC 69868;

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C a nucleotide encoding the full length protein encoded by the cDNA insert of clone B18 deposited under accession number ATCC 69868; z a nucleotide sequence that specifically hybridizes to a nucleic acid sequence specified in to a nucleotide sequence varying from the sequence of the nucleotide sequence specified in to as a result of degeneracy of the genetic code; and S(i) an allelic variant of the nucleotide specified in to In a second aspect, the present invention provides an isolated human C CTLA-8 protein comprising an amino acid sequence selected from the group consisting of: S(a) the amino acid sequence of SEQ ID NO:2; the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163; a fragment of SEQ ID NO:2, the fragment comprising amino acids 11 to 163 of SEQ ID NO:2; a fragment of SEQ ID NO:2, the fragment comprising amino acids 29 to 163 of SEQ ID NO:2; a fragment of SEQ ID NO:2, the fragment comprising amino acids 31 to 163 of SEQ ID NO:2; and fragments of or having a CTLA-8 activity selected from the group consisting of: biological activity in a factor-dependent cell proliferation assay; induction of expression or secretion of y-IFN; chemoattractant or chemotactic activity in a chemoattraction or chemotaxis assay; and induction or expression of secretion of IL-3 or GM-CSF.

In a third aspect, the present invention provides a human CTLA-8 protein produced according to a process which comprises the steps of: growing a culture of a cell in a suitable culture medium, wherein the cell is transformed with a polynucleotide operably linked to an expression control sequence, wherein the polynucleotide is selected from the group consisting of: (aa) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide 55 to nucleotide 544; 1 i T c'i (ab) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide 146 to nucleotide 544; Z (ac) a polynucleotide comprising the nucleotide sequence of the full-length coding sequence of clone B18 deposited under accession number ATCC 69868; (ad) a polynucleotide encoding the full-length protein encoded by N- the cDNA insert of clone B18 deposited under accession number ATCC 69868; and C (ae) a polynucleotide comprising a nucleotide sequence varying from the sequence of the nucleotide sequence specified in (aa) to (ad) as a result 0of degeneracy of the genetic code; and purifying the human CTLA-8 protein encoded by said polynucleotide from the culture.

In certain embodiments, the present invention provides an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: the nucleotide sequence of SEQ ID NO:1 from nucleotide 146 to nucleotide 544; a nucleotide sequence capable of hybridizing to a nucleic acid sequence specified in a nucleotide sequence varying from the sequence of the nucleotide sequence specified in as a result of degeneracy of the genetic code; and an allelic variant of the nucleotide sequence specified in Preferably, the polynucleotide of the invention encodes a protein having CTLA-8 activity. In other embodiments the polynucleotide is operably linked to an expression control sequence. In other preferred embodiments, the polynucleotide is contained in a vector suitable for in vivo expression in a mammalian subject. Polynucleotides comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide 55 to nucleotide 544, the nucleotide sequence of SEQ ID NO:1 from nucleotide 139 to nucleotide 544 or the nucleotide sequence of SEQ ID NO:1 from nucleotide 86 to nucleotide 544 are particularly preferred.

Host cells transformed with the polynucleotides of the invention are also provided, comprising mammalian cells.

Processes are also provided for producing a human CTLA-8 protein, said processes comprising:

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c growing a culture of the host cell of the invention in a suitable culture medium; and z purifying the human CTLA-8 protein from the culture.

Isolated human CTLA-8 protein is also provided comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO:2; N the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163; M the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 10 163; the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163; and fragments of or having CTLA-8 activity.

Proteins comprising the amino acid sequence of SEQ ID NO:2 and comprising the sequence from amino acids 29 to 163, from amino acid 31 to 163, or from amino acids 11 to 163 of SEQ ID NO:2 are particularly preferred.

Preferably, the protein has CTLA-8 activity. Pharmaceutical compositions comprising a human CTLA-8 protein of the invention and a pharmaceutically acceptable carrier are also provided.

Compositions are also disclosed which comprise an antibody which specifically reacts with a human CTLA-8 protein of the invention.

Methods of treating a mammalian subject are also provided which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a human CTLA-8 protein.

Rat CTLA-8 and active having CTLA-8 activity) fragments thereof may also be used in such methods of treatment. Preferably the rat protein is administered as a composition comprising a pharmaceutically acceptable carrier and a protein comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO:4; the amino acid sequence of SEQ ID NO:4 from amino acids 18 to 150; and fragments of or having CTLA-8 activity.

Herpesvirus Saimiri ORF13, referred to herein as "herpes CTLA-8", and active having CTLA-8 activity) fragments thereof and active fragments thereof may also be used in such methods of treatment. Preferably the herpes 10/04 '08 16:13 FAX 613 8618 4199 FB RICE CO. @1013 00 0 0 CTLA-8 protein is administered as a composition comprising a pharmaceutically acceptable carrier and a protein comprising an amino acid sequence selected o from the group consisting of: the amino acid sequence of SEQ ID NO:6; the amino acid sequence of SEQ ID NO:6 from amino acids 19 to O 151; and fragments of or having CTLA-8 activity.

Cc The invention also provides a method of treating a mammalian subject comprising administering a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and IL-17 or an active Sfragment thereof.

In methods of treatment provided by the present invention, preferably the subject is treated to produce an effect selected from the group consisting of inhibition of angiogenesis, inhibition of growth or proliferation of vascular endothelial cells, inhibition of tumor growth, inhibition of angiogenesis-dependent tissue growth, proliferation of myeloid cells or progenitors, proliferation of erythroid cells or progenitors, proliferation of lymphoid cells or progenitors, induction of IFNy production, induction of IL-3 production and induction of GM-CSF production.

The present invention further provides an antibody that specifically reacts with a protein comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO:2; the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163; and a fragment of the amino acid sequence of SEQ ID NO:2 having at least one CTLA-8 activity selected from the group consisting of induction of expression or secretion of y-IFN, induction of expression or secretion of IL-6, induction of expression or secretion of IL-8, and inhibition of angiogenesis.

In an embodiment, the antibody specifically reacts with; i) a protein comprising the amino acid sequence of SEQ ID NO:2, ii) a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163, iii) a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163, 4B COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10 10/04 '08 16:13 FAX 613 8618 4199 FB RICE CO. 1014 00 0 0 iv) a protein comprising the amino acid sequence of SEQ ID NO:2 from amino <acids 31 to 163, o v) a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having at least one CTLA-8 activity selected from the group consisting of induction of expression or secretion of y-IFN, induction of expression or secretion of IL-6, induction ID of expression or secretion of IL-8, and inhibition of angiogenesis, and/or vi) a protein comprising a fragment of the amino acid sequence of SEQ ID CC) NO:2 having at least one CTLA-8 activity, wherein the at least one CTLA-8 activity is C induction of expression or secretion of y-IFN.

o 10 The present invention further provides an antibody that specifically reacts with a 0. protein comprising an amino acid sequence selected from the group consisting of: an amino acid sequence encoded by the cDNA insert of clone B18 having ATCC Accession No. 69868; an amino acid sequence encoded by SEQ ID NO:1; an amino acid sequence encoded by SEQ ID NO:1 from nucleotide 56 to 544; an amino acid sequence encoded by SEQ ID NO:I from nucleotide 86 to 544; an amino acid sequence encoded by SEQ ID NO:1 from nucleotide 140 to 544;and an amino acid sequence encoded by SEQ ID NO:1 from nucleotide 146 to 544.

In an embodiment, the amino acid sequence is encoded by the cDNA insert of clone B18 having ATCC Accession No. 69868.

In a further embodiment, the amino acid sequence is encoded by SEQ ID NO:1, SEQ ID NO:1 from nucleotide 56 to 544, SEQ ID NO:1 from nucleotide 86 to 544, SEQ ID NO:1 from nucleotide 140 to 544, or SEQ ID NO:1 from nucleotide 146 to 544.

The present invention further provides an antibody that specifically reacts with a protein comprising an amino acid sequence encoded by the complement of a first nucleotide sequence capable of hybridizing under highly stringent conditions to a second nucleotide sequence selected from the group consisting of: the cDNA insert of clone B18 having ATCC Accession No, 69868; SEQ ID NO:1; SEQ ID NO:1 from nucleotide 56 to 544; SEQ ID NO:1 from nucleotide 86 to 544; 4C COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10 10/04 '08 16:14 FAI 613 8618 4199 FB RICE CO. 015 00 0 0 e, SEQ ID NO:1 from nucleotide 140 to 544; and SEQ ID NO:1 from nucleotide 146 to 544, O wherein each encode a protein having at least one CTLA-8 activity selected from the group consisting of induction of expression or secretion of y-IFN, induction of expression or secretion of IL-6, induction of expression or secretion of IL-8, and

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inhibition of angiogenesis.

In an embodiment, the at least one CTLA-8 activity is induction of expression or C) secretion of y-IFN.

The present invention further provides an antibody obtained by a process of o 10 immunizing an animal with an immunogen selected from the group consisting of: 0. a protein comprising the amino acid sequence of SEQ ID NO:2; a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163; a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163; and a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163, wherein the antibody specifically reacts with the selected immunogen.

In an embodiment, the immunogen is a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163.

In another embodiment, the immunogen has a cysteine residue at the carboxyl terminus and is conjugated to keyhole limpet hemocyanin, and/or the immunogen has at least one tyrosine residue replaced with a sulfated tyrosine residue.

The present invention further provides an antibody obtained by a process of immunizing an animal with an immunogen selected from the group consisting of: a fragment of a protein comprising the amino acid sequence of SEQ ID NO:2 having a cysteine residue at the carboxyl terminus and being conjugated to keyhole limpet hemocyanin; and a fragment of a protein comprising the amino acid sequence of SEQ ID NO:2 having at least one tyrosine residue replaced with a sulfated tyrosine residue, wherein the antibody specifically reacts with the selected immunogen comprising a fragment of a protein comprising the amino acid sequence of SEQ ID NO:2.

The present invention further provides an antibody obtained by a process of immunizing an animal with an immunogen selected from the group consisting of: 4D COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10 10/04 '08 16:14 FAX 613 8618 4199 FR RTCE CO.

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a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163 having a cysteine residue at the carboxyl terminus and being conjugated O to keyhole limpet hemocyanin; and a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163 having at least one tyrosine residue replaced with a sulfated tyrosine I residue, wherein the antibody specifically reacts with the selected immunogen comprising the c protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 31 to S163.

In an embodiment, the antibody is a polyclonal antibody or a monoclonal 0 antibody.

In another embodiment, the antibody is a neutralizing antibody.

The invention further provides a monoclonal antibody that specifically reacts with a protein comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO:2; the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163; and a fragment of the amino acid sequence of SEQ ID NO:2 having at least one CTLA-8 activity selected from the group consisting of induction of expression or secretion of y-IFN, induction of expression or secretion of IL-6, induction of expression or secretion of IL-8, and inhibition of angiogenesis.

The invention further provides a monoclonal antibody that specifically reacts with a protein comprising an amino acid sequence selected from the group consisting of: an amino acid sequence encoded by the cDNA insert of clone B18 having ATCC Accession No. 69868; an amino acid sequence encoded by SEQ ID NO:1; an amino acid sequence encoded by SEQ ID NO:1 from nucleotide 56 to 544; an amino acid sequence encoded by SEQ ID NO:1 from nucleotide 86 to 544; an amino acid sequence encoded by SEQ ID NO:1 from nucleotide 140 to 544; and 4E COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10 10/04 '08 16:15 FAX 613 8618 4199 FB RICE CO. @]017 00

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0 S(f) an amino acid sequence encoded by SEQ ID NO:1 from nucleotide 146 to 544.

SThe present invention further provides a monoclonal antibody that specifically reacts with a protein comprising an amino acid sequence encoded by the complement of a first nucleotide sequence capable of hybridizing under highly stringent conditions IN to a second nucleotide sequence selected from the group consisting of: the eDNA insert of clone B 18 having ATCC Accession No. 69868; S(b) SEQ ID NO:1; c1 SEQ ID NO:1 from nucleotide 56 to 544; 0 10 SEQ ID NO:1 from nucleotide 86 to 544; 0 SEQ ID NO:1 from nucleotide 140 to 544; and SEQ ID NO: 1 from nucleotide 146 to 544, wherein each encode a protein having at least one CTLA-8 activity selected from the group consisting of induction of expression or secretion of y-IFN, induction of expression or secretion of IL-6, induction of expression or secretion of IL-8, and inhibition of angiogenesis.

In an embodiment, the at least one CTLA-8 activity is induction of expression or secretion of y-FN.

Also provided is a pharmaceutical composition comprising an antibody as in the invention and a pharmaceutically acceptable carrier.

The present invention further provides a method of treating a subject with a condition selected from the group consisting of an autoimmune disorder, asthma and related respiratory conditions, the method comprising administering to the subject an antibody that specifically reacts with a protein comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO:2; the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163; and a fragment of the amino acid sequence of SEQ ID NO:2 having at least one CTLA-8 activity selected from the group consisting of induction of expression or secretion of y-IFN, induction of expression or secretion of IL-6, induction of expression or secretion of IL-8, and inhibition of angiogenesis.

In an embodiment, the autoimmune disorder is rheumatoid arthritis or inflammatory bowel disease.

4F COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10 10/04 '08 16:15 FAX 613 8618 4199 FB RICE CO, [a 018 00 0 ci -Also provided is method of treating a subject with a condition selected from the group consisting of an autoir-mune disorder, asthma and related respiratory conditions, o the method comprising administering to the subject an antibody of the invention.

The present invention further provides the use of an antibody that specifically reacts with a protein comprising an amino acid sequence selected from the group INO consisting of: the amino acid sequence of SEQ ID NO:2; the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163; ci(c) the amino acid sequence ofSEQ ID NO:2 from amino acids 29 to 163; 10(d) the amino acid sequence of SEQ ID 10:2 from amino acids 9 to 163;a the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163; and o(e) a fragment of the amino acid sequence of SEQ ID NO:2 having at least one ci CTLA-8 activity selected from the group consisting of induction of expression or secretion of 7-WFN, induction of expression or secretion of IL-6, induction of expression or secretion of IL-8, and inhibition of angiogenesis, for the manufacture of a medicament for treating a subject with a condition selected from the group consisting of an autoimmune disorder, asthma and related respiratory conditions.

Also provided is the use of an antibody of the invention for the manufacture of a medicament for treating a subject with a condition selected from the group consisting of an autoimmune disorder, asthma and related respiratory conditions, Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

4G COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10 Brief Description of the Figures Fig. 1 is a comparison of homologous regions of the amino acid sequences of human CTLA-8 (indicated as "B 18_Fl"), rat CTLA-8 (indicated as "Musctla8") and Z herpes CTLA-8 (indicated as "Hsvie_2").

S 5 Fig. 2 depicts autoradiographs demonstrating expression of human CTLA-8 in COS cells.

SFig. 3 presents data relating to the ability of human CTLA-8 to inhibit angiogenesis.

c Figs. 4 and 5 present data relating to the ability of human CTLA-8 to produce or induce hematopoietic activity.

Figs. 6 and 7 present data demonstrating the ability of human CTLA-8 to induce production of IL-6 and IL8.

Detailed Description of Preferred Embodiments The inventors of the present application have identified and provided a polynucleotide encoding a human CTLA-8 protein. SEQ ID NO:1 provides the nucleotide sequence of a cDNA encoding the human CTLA-8 protein. SEQ ID NO:2 provides the amino acid sequence of the human CTLA-8 protein. Alternatively, the initiating methionine may be at amino acid 11 of SEQ ID NO:2. On the basis of amino terminal sequencing, the mature protein sequence is believed to begin at amino acid 31 of SEQ ID NO:2 (encoded by the sequence beginning with nucleotide 146 of SEQ ID NO:1).

The region from amino acid 29 to amino acid 163 of human CTLA-8 (SEQ ID NO:2) shows marked homology to portions of rat CTLA-8 (amino acids 18 to 150 of SEQ ID NO:4) and herpesvirus Saimiri ORF13 ("herpes CTLA-8") (amino acids 19 to 151 of SEQ ID NO:5). A cDNA sequence encoding rat CTLA-8 is listed at SEQ ID NO:3 and its corresponding amino acid sequence is reported at SEQ ID NO:4. A cDNA sequence encoding herpes CTLA-8 is listed at SEQ ID NO:5 and its corresponding amino acid sequence is reported at SEQ ID NO:6. Homology between rat CTLA-8 and herpes CTLA-8 was reported by Rouvier et al., J. Immunol. 1993, 150, 5445-5456.

SApplicants had previously incorrectly identified the rat sequences of SEQ ID 0 NO:3 and SEQ ID NO:4 as applying to murine CTLA-8. Applicants' human CTLA-8 (B 18) does also show homolgy to the true murine CTLA-8 sequence.

z Golstein et al. (W095/18826; Fossiez et al., Microbial Evasion and Subversion of Immunity 544:3222 (Abstract)) have also reported a species they initially identified as "human CTLA-8." However, examination of the sequence of the Golstein et al.

ND species and the human CTLA-8 (B18) sequence of the present invention readily reveals that they are two different proteins, although they are homologous with each other and with the rat CTLA-8 and herpes CTLA-8 identified herein. The Golstein 10 et al. species has now been renamed as interleukin-17 (IL-17). Because of the O homology between applicants' human CTLA-8 (B18) and IL-17, these proteins are expected to share some activities.

It has also been preliminarily determined that human CTLA-8 (B18) forms homodimers when expressed. As a result, human CTLA-8 proteins may possess activity in either monomeric or dimeric forms. Human CTLA-8 proteins can also be produced as heterodimers with rat and herpes CTLA-8 proteins and with human IL-17.

These heterodimers are also expected to have activities of the proteins of which they are comprised.

Forms of human CTLA-8 protein of less than full length are encompassed within the present invention and may be produced by expressing a corresponding fragment of the polynucleotide encoding the human CTLA-8 protein (SEQ ID NO: 1).

These corresponding polynucleotide fragments are also part of the present invention.

Modified polynucleotides as described above may be made by standard molecular biology techniques, including site-directed mutagenesis methods which are known in the art or by the polymerase chain reaction using appropriate oligonucleotide primers.

For the purposes of the present invention, a protein has "CTLA-8 activity" if it either displays biological activity in a factor-dependent cell proliferation assay (preferably an assay in which full-length the corresponding species full-length CTLA- 8 is active) (including without limitation those assays described below), or induces expression or secretion of y-IFN, or displays chemoattractant of chemotactic activity in a chemoattraction or chemotaxis assay (preferably as assay in which fulllength the corresponding species full-length CTLA-8 is active) or induces expression of secretion of IL-3 or GM-CSF.

>Human CTLA-8 protein or fragments thereof having CTLA-8 activity may be Z fused to carrier molecules such as immunoglobulins. For example, human CTLA-8 5 protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin.

°The invention also encompasses allelic variations of'the nucleotide sequence as set forth in SEQ ID NO:1, that is, naturally-occurring alternative forms of the C isolated polynucleotide of SEQ ID NO:1 which also encode human CTLA-8 or CTLA-8 proteins having CTLA-8 activity. Also included in the invention are isolated polynucleotides which hybridize to the nucleotide sequence set forth in SEQ ID NO: 1 under highly stringent (0.2xSSC at 65"C), stringent 4xSSC at 65 C or formamide and 4xSSC at 42 0 or relaxed (4xSSC at 50'C or 30-40% formamideand 4xSSC at 42 0 C) conditions. Isolated polynucleotides which encode human CTLA-8 protein but which differ from the nucleotide sequence-set forth in SEQ ID NO:1 by virtue of the degeneracy of the genetic code are also encompassed by the present invention. Variations in the nucleotide sequence as set forth in SEQ ID NO: 1 which are caused by point mutations or by induced modifications which enhance CTLA-8 activity, half-life or production level are also included in the invention.

The isolated polynucleotides of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19. 4485-4490 (1991), in order to produce the CTLA-8 protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990).

As defined herein "operably linked" means enzymatically or chemically ligated to form a covalent bond between the isolated polynucleotide of the invention and the expression control sequence, in such a way that the CTLA-8 protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.

A number of types of cells may act as suitable host cells for expression of the human CTLA-8 protein. Any cell type capable of expressing functional human CTLA-8 protein may be used. Suitable mammalian host cells include, for example, Smonkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, 5 human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, SU937, HaK, Rat2, BaF3, 32D, FDCP-1, PC12 or C2C12 cells.

The human CTLA-8 protein may also be produced by operably linking the B 10 isolated polynucleotide of the invention to suitable control sequences in one or more Sinsect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. Soluble forms of the human CTLA-8 protein may also be produced in insect cells using appropriate isolated polynucleotides as described above.

Alternatively, the human CTLA-8 protein may be produced in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.

Suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins.

The human CTLA-8 protein of the invention may also be expressed as a product of transgenic animals, as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a polynucleotide sequence encoding the human CTLA-8 protein.

The human CTLA-8 protein of the invention may be prepared by growing a culture of transformed host cells under culture conditions necessary to express the desired protein. The resulting expressed protein may then be purified from the culture medium or cell extracts. Soluble forms of the human CTLA-8 protein of the invention can be purified from conditioned media. Membrane-bound forms of human CTLA-8 protein of the invention can be purified by preparing a total membrane fraction from the expressing cell and extracting the membranes with a non-ionic detergent such as Z Triton X-100.

5 The human CTLA-8 protein can be purified using methods known to those skilled in the art. For example, the human CTLA-8 protein of the invention can be 0 concentrated using a commercially available protein concentration filter, for example, San Amicon or Millipore Pelicon ultrafiltration unit. Following the concentration step, Sthe concentrate can be applied to a purification matrix such as a gel filtration medium.

Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred S-Sepharose® columns). The purification of the human CTLA-8-protein from culture supernatant may also include one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; or by hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or by immunoaffinity chromatography. Finally, one or more reversephase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the human CTLA-8 protein. Some or all of the foregoing purification steps, in various combinations or with other known methods, can also be employed to provide a substantially purified isolated recombinant protein.

Preferably, the human CTLA-8 protein is purified so that it is substantially free of other mammalian proteins.

It is believed that human CTLA-8, active fragments and variants thereof, and CTLA-8 related proteins (such as, for example, rat CTLA-8 and herpes CTLA-8) (collectively "CTLA-8 proteins") possess or induce cytokine activities. Human CTLA-8 expression correlated with y-IFN expression in induced primary cells and can Sinduce the expression of IL-3 and/or GM-CSF, which expression can in turn produce effects associated with the induced cytokine. Therefore, human CTLA-8 and CTLA-8 z related proteins may have an effect on proliferation or function of myeloid cells, r 5 erythroid cells, lymphoid cells and their progenitors. Human CTLA-8 proteins may also play a role in formation of platelets or their progenitors.

A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) C activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited Sactivity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DAIG, T10, B9, B9/11, BaF3, MC9/G, M+ (preB 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.

The activity of a protein of the invention may, among other means, be measured by the following methods: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1- 3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.

137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J.

Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994.

Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon y, Schreiber, R.D.

In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6 John 0Wiley and Sons, Toronto. 1994.

N Assays for proliferation and differentiation of hematopoietic and z lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp.

6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med.

173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., c Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6 Nordan, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds.

0 Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl.

Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11 Bennett, Giannotti, Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto.

1991; Measurement of mouse and human Interleukin 9 Ciarletta, Giannotti, J., Clark, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci.

USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.

A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), in regulating (up or down) growth and proliferation of T and/or B Slymphocytes, as well as effecting the cytolytic activity of NK cells and other cell Z populations. These immune deficiencies may be genetic or be caused by viral 5 HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, leshmania, malaria and various fungal infections such as candida. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally would be indicated, in the treatment of cancer.

Autoimmune disorders which may be treated using a protein of the present invention include, for example, multiple sclerosis, systemic -lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graftversus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, asthma and related respriatory conditions), may also be treatable using a protein of the present invention.

A protein of the present invention may also suppress chronic or acute inflammation, such as, for example, that associated with infection (such as septic shock or systemic inflammatory response syndrome (SIRS)), inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1 (such as the effect demonstrated by IL-11).

The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., SProc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.

S128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al..

Z J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; S 5 Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J.

Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992- 1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.

0 10 Assays for T-cell-dependent immunoglobulin responses and isotype switching CN (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect ThlfTh2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J.

Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others, proteins expressed by denritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimenal Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, among others, Z proteins that prevent apoptosis after superantigen induction and proteins that regulate 5 lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia IN 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of S 10 Oncology 1:639-648, 1992.

O

Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.

A protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentarily to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, Sincluding, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post Z irradiation/chemotherapy, either in-vivo or ex-vivo in conjunction with bone 5 marrow transplantation) as normal cells or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, be O measured by the following methods: Suitable assays for proliferation and differentiation of various hematopoietic Slines are cited above.

Assays for embryonic stem cell differentiation (which will identify, among Sothers, proteins that influence embyronic differentation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.L Freshney, et al. eds. Vol pp.

1-21, Wiley-Liss, Inc.., New York, NY. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, Dexter, M. and Alien, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term culture initating cell assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.

CTLA-8 proteins are useful in the treatment of various immune deficiencies O and disorders (including SCID), in regulating (up or down) growth, proliferation and/or activity of T and/or B lymphocytes, as well as the cytolytic activity of NK cells.

Z These immune deficiencies may be caused by viral HIV) as well as bacterial 5 infections, or may result from autoimmune disorders. More specifically, infectious diseases caused by viral bacterial, fungal or other infection may be treatable using IND CTLA-8 proteins, including infections by HIV, hepatitis, influenza, CMV, herpes, mycobacterium, leishmaniasis, malaria and various fungal infections (such as candida). Of course, in this regard, the CTLA-8 proteins may also be useful where a boost to the immune system generally would be indicated, in the treatment of Ci cancer or as an adjuvant to vaccines. Autoimmune disorders which may be treated using factors of the present invention include, for example, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes melitis and autoimmune inflammatory eye disease. The CTLA-8 proteins are also expected to be useful in the treatment of allergic reactions and conditions.

CTLA-8 proteins are also expected to have chemotactic activity. A protein or peptide has "chemotactic activity," as used herein, if it can stimulate, directly or indirectly, the directed orientation or movement of cells, including myeloid and lymphoid cells. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells (particularly T-cells). Whether a particular protein or peptide has chemotactic activity for cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.

CTLA-8 proteins also inhibit growth and proliferation of vascular endothelial cells. As a result, human CTLA-8 proteins are effective in inhibiting angiogenesis vascular formation). This activity will also be useful in the treatment of tumors and other conditions in which angiogenesis in involved. Inhibition of angiogenesis by human CTLA-8 proteins will also result in inhibition or prevention of the condition to which normal angiogenesis would contribute.

Isolated CTLA-8 proteins, purified from cells or recombinantly produced, may be used as a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may contain, in addition to CTLA-8 protein and carrier, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic Z material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain ND cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, IL- 1, IL-2, IL-3, II4, IL-5, IL-6, IL-7, IL-8, IL9, IL-10, IL-11, IL-12, IL-15, G-CSF, y- IFN, stem cell factor, and erythropoietin. The pharmaceutical composition may 0 10 contain thrombolytic or anti-thrombotic factors such as plasminogen activator and C- Factor VII. The pharmaceutical composition may further contain other antiinflammatory agents. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with CTLA-8 protein, or to minimize side effects caused by the CTLA-8 protein. Conversely, CTLA-8 protein may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.

The pharmaceutical composition of the invention may be in the form of a liposome in which CTLA-8 protein is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers which in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporated herein by reference.

As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, amelioration of symptoms of, healing of, or increase in rate of healing of such conditions. When applied to an C individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the z active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, a N therapeutically effective amount of CTLA-8 protein is administered to a mammal.

SCTLA-8 protein may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing 10 cytokines, lymphokines or other hematopoietic factors. When co-administered with Sone or more cytokines, lymphokines, other hematopoietic factors or vaccine components (such as antigens or other adjuvants), CTLA-8 protein may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering CTLA-8 protein in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.

Administration of CTLA-8 protein used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection. Intravenous administration to the patient is preferred.

When a therapeutically effective amount of CTLA-8 protein is administered orally, CTLA-8 protein will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% CTLA-8 protein, and preferably from about 25 to 90% CTLA-8 protein. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the C pharmaceutical composition contains from about 0.5 to 90% by weight of CTLA-8 protein, and preferably from about I to 50% CTLA-8 protein.

Z When a therapeutically effective amount of CTLA-8 protein is administered by intravenous, cutaneous or subcutaneous injection, CTLA-8 protein will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of r- such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical c, C composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to CTLA-8 protein an isotonic vehicle such as Sodium Chloride Injection, C1 Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art.

The amount of CTLA-8 protein in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.

Ultimately, the attending physician will decide the amount of CTLA-8 protein with which to treat each individual patient. Initially, the attending physician will administer low doses of CTLA-8 protein and observe the patient's response. Larger doses of CTLA-8 protein may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not generally increased further.

It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.1 pg to about 100 mg of CTLA-8 protein per kg body weight, preferably about 0.1 pg to about 10 mg of CTLA-8 protein per kg body weight, more preferably about 0.1 pg to about 100 pg of CTLA-8 protein per kg body weight, most preferably preferably about 0.1 pg to about pg of CTLA-8 protein per kg body weight.

The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the CTLA-8 protein will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy Z using the pharmaceutical composition of the present invention.

5 CTLA-8 protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the CTLA- S8 protein and which may inhibit CTLA-8 binding to its receptor. Such antibodies are Salso useful for performing diagnostics assays for CTLA-8 in accordance with known C methods. Such antibodies may be obtained using the entire CTLA-8 protein as an S 10 immunogen, or by using fragments of human CTLA-8 protein. The peptide C -immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Additional peptide immunogens may be generated by replacing tyrosine residues with sulfated tyrosine residues. Methods for synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J.Amer.Chem.Soc. 85, 2149-2154 (1963); J.L.

Krstenansky, et al., FEBS Lett. 211. 10 (1987).

Neutralizing or non-neutralizing antibodies (preferably monoclonal antibodies) binding to human CTLA-8 protein may also be useful therapeutics for certain tumors and also in the treatment of conditions described above. These neutralizing monoclonal antibodies are capable of blocking the ligand binding to the human CTLA-8 protein or mayh promote clearance of protein from the patient.

Because of their homology to human CTLA-8, rat CTLA-8 proteins, herpes CTLA-8 proteins and IL-17 proteins (the "human CTLA-8" of Golstein et al., supra) will also possess CTLA-8 activity as described above. As a result, rat and herpes CTLA-8 proteins and IL-17 proteins, as well as active fragments and variants thereof, can be used in preparation of pharmaceutical compositions and in methods of treatment as described for human CTLA-8. Rat and herpes CTLA-8 proteins, and active fragments and variants thereof, can be produced as described above using the polynucleotides (or fragments or variants thereof) described in SEQ ID NO:3 and SEQ ID NO:5, respectively. Rat and herpes CTLA-8 may also be produced as described in Rouvier et al., J. Immunol. 1993, 150, 5445-5456. CTLA-8 proteins of other species can also be used as described herein. cDNAs encoding rat CTLA-8 and herpes CTLA-8 were deposited with the American Type Culture Collection on July 6, 1995 and assigned accession numbers ATCC 69867 and ATCC 69866, respectively. IL-17 Z proteins may also be produced as described in Golstein et al., supra.

5 Because of its homology to IL-17, the human CTLA-8 (B 18) proteins of the present invention may also share some activities with IL-17.

0 For the purposes of treatment or therapy, any of the proteins discussed or Sdisclosed herein may be administered by in vivo expression of the protein in a Smammalian subject. In such instances, a polynucleotide encoding the desired protein is administered to the subject in manner allowing expression in accordance with known methods, including without limitation the adenovirus methods disclosed herein.

Example 1 Isolation of Human CTLA-8 cDNA A partial clone for human CTLA-8 was isolated from a cDNA library made from RNA isolated from stimulated human peripheral blood mononuclear cells. This partial was identified as "B 18." B18 is sometimes used herein to refer to the human CTLA-8 of the present invention. Homology searches identified this partial clone as being related to the herpes and rat CTLA-8 genes. DNA sequence of this partial clone was used to isolate the full-length clone.

In order to isolate a full-length cDNA for B 18, a directional, full-length cDNA library by standard means in the COS expression vector pMV2. The cDNA library was transformed into E. coli by electroporation. The bulk of the original transformed cDNA library was frozen in glycerol at -80 0 C. An aliquot was titered to measure the concentration of transformed E. coli. The E. coli were thawed, diluted to 76,000/0.1 ml in media containing ampicillin, and 0.1 ml was distributed into the wells of a microtiter dish in an 8 x 8 array. The microtiter dish was placed at 37"C overnight to grow the E. coli.

To prepare DNA for PCR, 20 pl aliquots of culture from each well were withdrawn and pooled separately for each row and column of eight wells, giving 16 pools of 160 pl each. The E. coli were pelleted, resuspended in 160 pi of standard 0 lysis buffer consisting of 10 mM TrisHCI pH8, I mM EDTA, 0.01% Triton X-100, and lysed by heating to 95*C for 10 minutes.

0 z To identify which of the wells contained E. coli transformed with B 18, PCR was performed first on the DNA preps corresponding to the eight columns. The PCR consisted of two sequential reactions with nested oligonucleotides using standard conditions. The oligonucleotides used for the PCR reaction were derived from the sequence of the partial B 18 clone. They were: B185: CACAGGCATACACAGGAAGATACATTCA (SEQ ID NO:7) B 183: TCTTGCTGGATGGGAACGGAATTCA (SEQ ID NO:8) B18N: ATACATTCACAGAAGAGCTTCCTGCACA (SEQ ID NO:9) The PCR conditions were 2.5 mM MgC12 and 95C x 2 min for one cycle, 95*C x 1 min plus 68C x I min for 30 cycles, and 68C x 10 for one cycle. Each reaction was pl. The first reaction contained oligonucleotides B185 and B183 and pli of the DNA preparations. The second reaction contained oligonucleotides B 183 and B 18N and 1 pl of the first reaction.

DNA preps that potentially contained a full-length B18 cDNA clone were identified by agarose gel electrophoresis on an aliquot of the second PCR reaction.

A DNA band of the correct mobility was assumed to be derived from a B18 cDNA.

SNext the same sequence of PCR reactions and gel analysis was done on the DNA preps corresponding to the eight rows. The intersection of a row and a column identified well A2 as potentially containing B18, narrowing it down to the 76,000 E.

coli originally seeded into that well.

To further purify the individual E. coli containing the putative full-length B 18 cDNA clone, the concentration of E. coli in well A2 was measured by titering and plating dilutions of the well. Then 7600 E. coli were seeded into the wells of a second microtiter plate in an 8 x 8 array. The E. coli were grown overnight; wells were pooled, and DNA was prepared as described above. To identify which of these wells contained E. coli transformed with B18, sequential PCR reactions were performed 0 essentially as described above. Agarose gel electrophoresis identified well B2 as NC potentially containing a B18 cDNA.

O The E. coli containing this cDNA was further purified by seeding wells of a Z microtiter plate with 253 E. coli per well and proceeding as for the purification of the E. coli in well A2. Well C3 was identified as containing a putative full-length B18 cDNA clone. The exact E. coli was identified by plating the contents of the well onto IN bacterial culture media and then screening the E. coli colonies following established protocols. The probe for these hybridizations was a PCR fragment generated by doing Cl a PCR reaction on the B18 clone using as primers the oligonucleotides described 0 10 above (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9). When a single colony was C identified, DNA was prepared and sequenced by standard methods. Comparison of this sequence to the sequence of the original partial clone confirmed identity and that the isolated cDNA was full-length.

The full-length clone was deposited with the American Type Culture Collection on July 6, 1995 and assigned accession number ATCC 69868.

Example 2 Expression of Human CTLA-8 The full-length B18 clone for human CTLA-8 was transfected into COS cells which were then labelled with 3 S-methionine. An aliquot of conditioned medium from the transfected cell culture was reduced, denatured and electrophoresed on polyacrylamide gels. Autoradiographs of those gels are reproduced in Fig. 2. The band indicated by the arrow demonstrates expression of human CTLA-8.

Example 3 Inhibition of Angiogenesis by Human CTLA-8 The ability of human CTLA-8 to inhibit angiogenesis was examined in an angiostatic activity assay (endothelial cell proliferation assay). The assay was done in a 96 well plate. Primary human umbilical cells (HUVECs) were seeded to 2xl0 3 cells per well in EGM medium (Clonetics)/20% FCS and incubated at 37 C for 24 hr.

The cells were then starved in M 199 medium (GIBCO BRL) containing 10% charcoal g treated serum (M199-CS) for 48 hr at 37 0 C. Conditioned media containing B18 S(human CTLA-8) was obtained from transfected COS or stably expressing CHO cells and 1:10. 1:50, 1:250, and 1:1250 dilutions prepared in M199-CS medium containing z 100 ng/ml FGF. The dilutions of B18 were added to the starved cells and incubated 5 for 72 hr at 37 0 C. The cells were then radiolabeled by 3 H]-thymidine for 6 hr.

Radiolabeled cells were washed with PBS and trypsinized for liquid scintilation O counting. Results were plotted using Kaleidograph software. The results are shown in Fig. 3. In the figure, "Med" is the mock control, "B18" and "B18-1" were S conditioned medium from two independent transfections of COS with DNA encoding 10 human CTLA-8 (B18). IFNy was used as a positive control angiostatic angiogenesis inhibition) activity. These data demonstrate that human CTLA-8 (B 18) inhibits angiogenesis.

Example 4 Hematopoietic Activity of Human CTLA-8 The hematopoietic activity of human CTLA-8 (B18) expressed in vivo was examined by construction of a recombinant adenovirus vector.

The B 18 cDNA in the expression plasmid Adori 2-12 B18 was driven by the cytomegalovirus(CMV) immediate early promoter and enhancer.

The Adori 2-12 vector was created by addition of an SV40 origin and enhancer to a known adenovirus vector (Barr et al., Gene Therapy 1:51 (1994); Davidson et al., Nature Genetics 3:219 (1993)). The Hindll/BamHI fragment encoding the origin and enhancer was isolated from the pMT2 mammalian expression vector, blunted with Klenow and cloned into the NatI site (blunted with Klenow) of the expression vector.

The vector was derived by digesting pNOT-B 18 cDNA with Sail, filling in the overhang with Klenow to generate a blunt end and digesting with EcoRI to isolate the B 18 cDNA. The blunted- EcoRI B18 fragment was inserted into the restriction sites EcoRV-EcoRI of the adenovirus vector Adori 2-12. The CMV-B 18 expression cassette was located downstream of the SV40 origin and enhancer, and 0-1 map units of the left hand end of the adenovirus type 5(Ad5). The SV40 splice donor and acceptor were located between the CMV promoter and B 18 cDNA. Following the insert was SV40 poly A site, 9-16 map units of Ad5 and the puc 19 origin.

A recombinant adenovirus was generated by homologous recombination in 293 z cells. AscI linearized Adori 2-12 B 18 and ClaI digested AdCMVlacZ were introduced 5 into the 293 cells using lipofectamine. Recombinant adenovirus virus was isolated and amplified on 293 cells. The virus was released from infected 293 cells by three cycles of freeze-thawing. The virus was further purified by two cesium chloride centrifugation gradients and dialyzed against PBS 40C. Following dialysis of the virus Sglycerol was added to a concentration of 10 and the virus was stored at 70 oC until use. The virus was characterized by expression of the transgene, plaque forming units on 293 cells, particles/ml and Southern analysis of the virus.

A single dose of 5 X 10 "0 particles of recombinant adenovirus encoding B 18 was injected into the tail vein of male C57/b16 mice, age 7-8 weeks. Control mice received an adenovirus encoding B-galactosidase. Four mice from each experimental group were killed on day 7 and 14. Blood was collected and automated hematologic analysis was performed using-a Baker 9000. Differential counts were performed on blood smears. Tissue was harvested, fixed in formalin, and stained with hematoxylin and eosin for histopathology. In the first set of experiments, serum and tissues were analyzed 7 and 14 days post injection. A slight increase in peripheral platelet counts were observed. The animals that received B18 exhibited a slight increase in spleen size. Macroscopic analysis of the spleen showed an increase in splenic extramedullary hematopoiesis on day 7 compared to the control. These results showed a hematopoietic growth activity associated with B 18.

In a second set of experiments 5 X 10 0 particles of recombinant adenovirus encoding B18 were injected into the tail vein of male C57/b16 mice, age 17-18 weeks.

Control mice received an adenovirus encoding B-galactosidase. Blood samples were collected via retro-orbital sinus on days 2, 5, 7, 10, 14, and 21. The hematologic analyses were performed on the Baker 9000 automated cell counter with murinespecific settings. Analyses included WBC, RBC, HCT and PLT counts. Blood smears were prepared and stained with Wright-Geimsa for WBC differentials based on a 100 cellcount. Reticulocytes and reticulated platelets were quantitated using flow cytometry. Four mice from each group were killed on days 7, 14, and 21. In addition 0to peripheral blood analysis, serum was collected via cardiac puncture for quantitation of systemic 1-6 using a commercial kit (Endogen). Spleen and liver were collected for z histopathology, spleen and bone marrow hematopoietic progenitors were quantitated, 5 and bone marrow smears were prepared and stained with Wright-Geimsa for cell counts.

INO Administration of adenovirus encoding B 18 resulted in a marked increase in peripheral blood neutrophils and WBC (Fig. Maximum increases in neutrophils C were observed at day 5 and day 7. The control mice showed little difference at day and day 7. Peripheral blood neutrophils were similar in the control mice and mice that received B18 at day 21. In both the B18 and control groups an increase in white blood cells was also observed. The mice that received B 18 had a greater increase in WBC between day 2 and day 7. By Day 21 a more pronounced increase was observed in the B-gal group. No other changes in cellular chemistries were observed (Table I).

Bone marrow cellularity was calculated from pooled femurs in each group (Table Il). No significant differences were observed in either group. No significant changes were observed in bone marrow hematopoietic progenitors from day 7, 14, and 21. The CFU-GM, BFU-E and CFU-MEG in the B18 mice were similar to the Bgal control (Table II).

Administration of the adenovirus encoding B18 resulted in an increase in CFU-GM (myeloid) and BFU-E (erythroid) progenitors in the spleen compared to animals that received the B-gal virus on day 7. The increase in progenitors in the B 18 mice was 11-fold in CFU-GM and a 52-fold in BFU-E (Table II). There was a 2-fold increase in CFU-MEG at day 7 for the B18 mice. By day 21 no significant differences were observed in splenic CFU-MEG or BFU-E between the groups (Table II). A 3fold decrease in CFU-GM was observed in mice that received adenovirus encoding B 18. A slight increase in spleen size at day 7 was observed in the B 18 group. This is consistent with an increase in splenic cellularity. By day 14 and day 21 spleen weights were similar to the control group (Table II). Macroscopic analysis of the spleen showed an increase in splenic extramedullary hematopoiesis of the B 18 mice on day 7 compared to the control.

The bone marrow myeloid: erythroid ratios (Table IV) suggest a granulocytic Shyperplasia with a possible erythroid hypoplasia in mice that received adenovirus B 18 on day 7. By day 21 the ratio in the B-gal group was higher. No changes were Z observed in the 1L6 serum levels.

These results show a hematopoietic activity associated with the administration of adenovirus encoding B18 (human CTLA-8). Increases in neutrophils and white I\D blood cells were observed at day 7 in animals that received B18 adenovirus. The data Sshowed that B18 resulted in increase in splenic CFU-GM and BFU-E 7 days post Sadministration compared to the control animals. Splenic extramedullary hematopoiesis on day 7 support that B18 exhibits a hematopoietic growth activity.

These data suggest that B 18 may moblize early hematopoietic precursors.

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i 10 Table nI: Bone marrow and Splenic Hematopoietic Progenitors CFU-MEG CFU-GM BFU-E Bone B-Gal B18 B-Gal B18 B-Gal B18 Marrow* Day 7 16.0 15.7 307 241±78 51 19 25 ±11 3.5 3.1 ±117 Day 14 10.7 15.3 233 373±35 30 10 60 2.3 1.2 Day 21 5.7 6.7 ±3.1 170 160±27 40 10 27 ±6 0.6 17 Spleen** Day 7 9.3 19.5 27 3 298 6 1.3 68 1.6 1.5 1.2 Day 14 9.7 12.7 267 197 ±21 33 6 10 0.6 0.6 32 Day 21 17.0± 19.3 187 6 73 a 15 23 6 23 6 Hematopoietic precursors were determined form pooled spleen and bone marrow samples from four animals in each group. For quantitation of CFU-GM and BFU-E, either 1 x 104 bone marrow cells or 1 x 105 spleen cells were added to complete alpha methylcelluose medium methylcellulose in alpha medium, 30% fetal bovine serum, 1% bovine serum albumin, 104M 2-mercaptoethanol, 2 mM L-glutamine, 2% murine spleen cell conditioned medium, and 3 U/mL erythropoietin) and aliquoted into 35 mm tissue culture dishes in a final volume of 1.0 mL. Cultures were incubated for 7 days at 37"C, and 5% CO 2 Microscopic colonies were defined as clusters of or more cells. For quantitaion of CFU-MEG, either 1 x 105 bone marrow cells or 1 x 106 spleen cells were added to complete alpha methylcellulose medium and incubated as described above. Megakaryocyte colonies were defined as a group of 3 or more cells.

*Bone marrow progenitors are represented as mean sd number of colonies per 105 cells.

**Spleen progenitors are represented as mean sd number of colonies per 106 cells.

Table m: Spleen Weights and Femur Cellularity Spleen Wt. B-Gal B 18 (Mg) Day 7 187±.19 224±29 Dayl14 175 ±13 170± 10 Day 21 174 ±21 1151 ±27 Femur B-Gal B 18 Cellularity (X 10 6 Day 7 28 23 Day 14 28 27 Day 21 28 26 Q lapn,121 tt A k, r ume o sacrifce are represented as means sd from four animals.

Table IV: Bone Marrow Myeloid:Erythoid Ratios Group Mouse Day 7 Day 14 Day 21 B-gal 1 1.43 2.12 5.78 2 0.91 2.46 5.83 3 1.62 1.03 3.66 4 5.44 4.82 AVG 1.32 2.76 5.02 SD 0.37 0.37 1.89 B-gal 1 5.59 2.01 2.02 6.51 1.25 2.13 5.49 1.58 1.81 0.50 2.51 .2.92 AVG 4.52 1.86 2.22 SD- 1.29 2.72 0.56 All entries represent the number of myeloid cells per 1 erythroid cell. Normal mouse ratios are approximately 1: 1 to 2: 1.

Example Additional Experiments Relating to Hematopoietic Activity of Human CTLA-8 B 18 (human CTLA-8) was tested for the ability to induce production of factors having hematopoietic activity in a factor-dependent cell proliferation assay using the r human erythroleukemic cell line, TF-I (Kitamura et al., J. Cell Physiol. 140:323 S(1989)). The cells were initially grown in the presence of rhGMCSF (100 U/ml). The >cells were fed three days prior to setting up the assay. The assay conditions were as 0 Z follows: S 5 cells/well 5000/200pl incubation time 3 days I pulse time 4 hours amount of tritiated thymidine counting time 1 minute replicates 2 1 B 18 alone, conditioned medium (CM) from B 18 induced HS-5 cells were assayed. Buffer alone, CM from HS-5 cells induced with buffer and CM from uninduced HS-5 cells were assayed as controls. Results are shown in Fig. 5. B18 (human CTLA-8) demonstrated an abilit to induce production of factors which induced TF-1 proliferation. This activity was substantially eliminated by the addition of anti-GMCSF antibodies. These data demonstrate that human CTLA-8 (B 18) is able to induce hematopoiesis. Particularly, without being bound by any theory, it appears that human CTLA-8 (B18) induces production of GM-CSF and/or IL-3.

Example 6 Ability of Human CTLA-8 to Induce Production of IL-6 and IL-8 cells were incubated in the presence of human CTLA-8 (B18) and production of IL-6 and IL-8 were measured. Herpes CTLA-8 (IL-17) was used as a positive control. Applicants' human CTLA-8 (B18) demonstrated titratable production of both IL-6 and IL-8 (see Figs. 6 and 7).

All patent and literature references cited herein are incorporated by reference as if fully set forth.

EDITORIAL NOTE APPLICATION NUMBER 2004231176 The claims pages 39 45 follow on from here. Sequence listing pages 32 38 are located following the Drawings pages.

Claims (4)

1. An antibody that specifically reacts with a protein comprising an amino acid sequence selected from the group consisting of: S(a) the amino acid sequence of SEQ ID N0:2; the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163; C the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163; and S(e) a fragment of the amino acid sequence of SEQ ID NO:2 having at least one CTLA-8 activity selected from the group consisting of induction of expression or secretion of y-IFN, induction of expression or secretion of IL-6, induction of expression or secretion of IL-8, and inhibition ofangiogenesis.

2. The antibody of claim I that specifically reacts with; i) a protein comprising the amino acid sequence of SEQ ID NO:2, ii) a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163, iii) a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163, iv) a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163, v) a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having at least one CTLA-8 activity selected from the group consisting of induction of expression or secretion of y-IFN, induction of expression or secretion of IL-6, induction of expression or secretion of IL-8, and inhibition of angiogenesis, and/or vi) a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having at least one CTLA-8 activity, wherein the at least one CTLA-g activity is induction of expression or secretion of y-TFN. 39 COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10 10/04 '08 16:16 FAX 613 8618 4199 FB RICE CO. 020 00 0 O ci

3. An antibody that specifically reacts with a protein comprising an amino acid O sequence selected from the group consisting of: an amino acid sequence encoded by the cDNA insert of clone B 18 having ATCC Accession No. 69868; an amino acid sequence encoded by SEQ ID NO 1; an amino acid sequence encoded by SEQ ID NO: I from nucleotide 56 to 544; S(d) an amino acid sequence encoded by SEQ ID NO:I from nucleotide 86 to 544; C an amino acid sequence encoded by SEQ ID NO: 1 from nucleotide 140 to S544; and an amino acid sequence encoded by SEQ ID NO:1 from nucleotide 146 to

544. 4. The antibody of claim 3, wherein the amino acid sequence is encoded by the cDNA insert of clone B18 having ATCC Accession No. 69868. The antibody of claim 3, wherein the amino acid sequence is encoded by SEQ ID NO:1, SEQ ID NO:1 from nucleotide 56 to 544, SEQ ID NO:1 from nucleotide 86 to 544, SEQ ID NO:1 from nucleotide 140 to 544, or SEQ ID NO:1 from nucleotide 146 to 544. 6. An antibody that specifically reacts with a protein comprising an amino acid sequence encoded by the complement of a first nucleotide sequence capable of hybridizing under highly stringent conditions to a second nucleotide sequence selected from the group consisting of: the cDNA insert of clone B18 having ATCC Accession No. 69868; SEQ ID NO:1; SEQ ID NO:1 from nucleotide 56 to 544; SEQ ID NO:1 from nucleotide 86 to 544; SEQ ID NO:1 from nucleotide 140 to 544; and SEQ TD NO:1 from nucleotide 146 to 544, COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10 10/04 '08 16:16 FAX 613 8618 4199 FB RICE @021 00 0 0 pi wherein each encode a protein having at least one CTLA-8 activity selected from the C group consisting of induction of expression or secretion of y-IFN, induction of expression or secretion of IL-6, induction of expression or secretion of TL-8, and inhibition of angiogenesis. VO 7. The antibody of claim 6, wherein the at least one CTLA-8 activity is induction of expression or secretion ofy-IFN. ci 8. An antibody obtained by a process of immunizing an animal with an immunogen o selected from the group consisting of: a protein comprising the amino acid sequence of SEQ ID NO:2; a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163; a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163; and a protein comprising the amino acid sequence of SEQ ID NO:2 fron amino acids 31 to 163, wherein the antibody specifically reacts with the selected immunogen. 9. The antibody of claim 8, wherein the immunogen is a protein comprisin the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163. The antibody of claim 8 or 9, wherein the immunogen has a cysteine residue at the carboxyl terminus and is conjugated to keyhole limpet hemocyanin, and/or the immunogen has at least one tyrosine residue replaced with a sulfated tyrosine residue. 11. An antibody obtained by a process of immunizing an animal with an immunogen selected from the group consisting of: a fragment of a protein comprising the amino acid sequence of SEQ ID NO:2 having a cysteine residue at the carboxyl terminus and being conjugated to keyhole limpet hemocyanin; and 41 COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10 10/04 '08 1i3~17 FAT Rift ARia Alan rfl I iLt 5c CU. 10/0 '0816:1 FAXOil RIQ fQQ ICE CO 022 00 a fragment of a protein comprising the amino acid sequence of SEQ ID NO:2 o having at least one tyrosine residue replaced with a sulfated tyrosine residue, wherein the antibody specifically reacts with the selected irninunogen comprising a fragment of a protein comprising the amino acid sequence of SEQ ID NO:2. 12. An antibody obtained by a process oF immunizing an animal with an immunogen Cfl selected from the group consisting of: a protein comprising the amino acid sequence of SEQ ID NO:2 from amino oacids 31 to 163 having a cysteine residue at the carboxyl terminus and being conjugated to keyhole limpet hemocyanin; and a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163 having at least one tyrosine residue replaced with a sulfated tyrosine residue, wherein the antibody specifically reacts with the selected immunogen comprising the protein comprising the amino acid sequence of SEQ ID NO.2 from amino acids 31 to 163. 13. The antibody as in any one of claims 1 to 12 that is a polyclonal antibody or a monoclonal antibody. 14. The antibody as in any one of claims 1 to 13 that is a neutralizing antibody. A monoclonal antibody that specifically reacts with a protein comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO:2; the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163; the amino acid sequence of SEQ ID N0:2 from amino acids 31 to 163; and a fragment of the amino acid sequence of SEQ ID N0:2 having at least one CTLA-8 activity selected from the group consisting of induction of expression or secretion of y-IFN, Induction of expression or secretion of IL-6, induction of expression or secretion of IL-8, and inhibition of angiogenesis. 42 COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10 10/04 '08 16:17 FAX 613 8618 4199 FB RICE CO. M023 00 0 0 o 16, A monoclonal antibody that specifically reacts with a protein comprising an amino acid sequence selected from the group consisting of: an amino acid sequence encoded by the cDNA insert of clone B1 8 having ATCC Accession No. 69868; an amino acid sequence encoded by SEQ ID NO:1; n an amino acid sequence encoded by SEQ ID NO: from nucleotide 56 to 544; c(d) an amino acid sequence encoded by SEQ ID NO:1 from nucleotide 86 to 544; S(e) an amino acid sequence encoded by SEQ ID NO: from nucleotide 140 to 544; and an amino acid sequence encoded by SEQ ID NO:1 from nucleotide 146 to 544. 17. A monoclonal antibody that specifically reacts with a protein comprising an amino acid sequence encoded by the complement of a first nucleotide sequence capable of hybridizing under highly stringent conditions to a second nucleotide sequence selected from the group consisting of: the cDNA insert of clone BI 8 having ATCC Accession No. 69868; SEQ ID NO:1; SEQ ID NO:1 from nucleotide 56 to 544; SEQ ID NO:1 from nucleotide 86 to 544; SEQ ID NO:1 from nucleotide 140 to 544; and SEQ ID NO:1 from nucleotide 146 to 544, wherein each encode a protein having at least one CTLA-8 activity selected from the group consisting of induction of expression or secretion of y-IFN, induction of expression or secretion ofIL-6, induction of expression or secretion of fL-8, and inhibition of angiogenesis. 18. The antibody of claim 17, wherein the at least one CTLA-8 activity is induction of expression or secretion of y-IFN. 43 COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10 10/04 '08 16:17 FAX 613 8618 4199 FB RICE CO, 1 024 00 0 0 19. A pharmaceutical composition comprising an antibody as in any one of claims 1 o to 18 and a pharmaceutically acceptable carrier. A method of treating a subject with a condition selected from the group consisting Sof an autoimmune disorder, asthma and related respiratory conditions, the method comprising Sadministering to the subject an antibody that specifically reacts with a protein comprising an Cr amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO:2; S(b) the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163; and a fragment of the amino acid sequence of SEQ ID NO;2 having at least one CTLA-8 activity selected from the group consisting of induction of expression or secretion of y-IFN, induction of expression or secretion of IL-6, induction of expression or secretion of IL-8, and inhibition of angiogenesis. 21. The method of claim 20, wherein the autoimmune disorder is rheumatoid arthritis or inflammatory bowel disease. 22. A method of treating a subject with a condition selected from the group consisting of an autoimmune disorder, asthma and related respiratory conditions, the method comprising administering to the subject an antibody as in any one of claims 1 to 18. 23. Use of an antibody that specifically reacts with a protein comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO:2; the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163; the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163; and 44 COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10 10/04 '08 16:18 FAX 613 8618 4199 FB RICE CO. 1 025 00 0 O S(e) a fragment of the amino acid sequence of SEQ ID NO:2 having at least one CTLA-8 activity selected from the group consisting of induction of expression or secretion of IFN, induction of expression or secretion of IL-6, induction of expression or secretion of IL-8, and inhibition ofangiogenesis, Sfor the manufacture of a medicament for treating a subject with a condition selected from the Sgroup consisting of an autoimmune disorder, asthma and related respiratory conditions. 24. Use of an antibody as in any one of claims I to 18 for the manufacture of a Smedicament for treating a subject with a condition selected from the group consisting of an autoimmune disorder, asthma and related respiratory conditions. COMS ID No: ARCS-186355 Received by IP Australia: Time 15:16 Date 2008-04-10