i ii in u mi i n iimiiiiiii
`
`US006043344A
`[ii] Patent Number:
`[45] Date of Patent:
`
`6,043,344
`Mar. 28, 2000
`
`United States Patent [w]
`Jacobs et al.
`
`[54] HUMAN CTLA-8 AND USES OF CTLA-8-
`RELATED PROTEINS
`
`[75]
`
`Inventors: Kenneth Jacobs, Newton; Kerry
`Kelleher, Marlborough; McKeough
`Carlin, Cambridge; Samuel Goldman,
`Acton, all of Mass.; Debra Pittman,
`Windham, N.H.; Sha Mi, Belmont,
`Mass.; Steven Neben; Joanne
`Giannotti, both of Acton, Mass.;
`Margaret M. Golden-Fleet, Medford,
`Mass.
`
`[73] Assignee: Genetics Institute, Inc., Cambridge,
`Mass.
`
`[21] Appl. No.: 09/034,810
`
`[22] Filed:
`
`Mar. 4, 1998
`
`Related U.S. Application Data
`
`[60] Division of application No. 08/685,239, Jul. 18, 1996,
`abandoned, which is a continuation-in-part of application
`No. 08/504,032, Jul. 19, 1995, which is a continuation-in-
`part of application No. 08/514,014, Aug. 11, 1995, Pat. No.
`5,707,829
`[60] Provisional application No. 60/035,347, Jul. 19, 1995.
`
`Int. CI.7
`[51]
`[52] U.S. CI
`[58] Field of Search
`
`C07K 14/54
`530/351; 530/350; 424/85.1
`530/351, 350;
`424/85.1
`
`[56]
`
`References Cited
`FOREIGN PATENT DOCUMENTS
`
`9518826 7/1995 WIPO .
`OTHER PUBLICATIONS
`Rouvier et al, /. Immunol, 150, 1993, p. 5445-556.
`Yao et al, /. Immunol 155 (12) 1995, p. 5483-486.
`Nicholas et al., Virology 179, 1990, p. 189-200.
`
`Primary Examiner—Garnette D. Draper
`Attorney, Agent, or Firm—Lahive & Cockfield, LLP; Amy
`E. Mandragouras, Esq.; Peter C. Lauro, Esq.
`[57]
`ABSTRACT
`
`Polynucleotides encoding human CTLA-8 and related pro(cid:173)
`teins are disclosed. Human CTLA-8 proteins and methods
`for their production are also disclosed. Methods of treatment
`using human CTLA-8 proteins, rat CTLA-8 proteins and
`herpesvirus herpes CTLA-8 proteins are also provided.
`
`13 Claims, 7 Drawing Sheets
`
`Lassen - Exhibit 1037, p. 1
`
`

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`U.S. Patent
`
`Mar. 28, 2000
`
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`Lassen - Exhibit 1037, p. 2
`
`

`

`U.S. Patent
`
`Mar. 28, 2000
`
`Sheet 2 of 7
`
`6,043,344
`
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`Lassen - Exhibit 1037, p. 3
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`

`

`U.S. Patent
`
`Mar. 28, 2000
`
`Sheet 3 of 7
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`Lassen - Exhibit 1037, p. 4
`
`

`

`U.S. Patent
`
`Mar. 28, 2000
`
`Sheet 4 of 7
`
`6,043,344
`
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`Lassen - Exhibit 1037, p. 5
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`

`

`U.S. Patent
`
`Mar. 28, 2000
`
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`6,043,344
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`Lassen - Exhibit 1037, p. 6
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`

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`U.S. Patent
`
`Mar. 28, 2000
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`

`

`U.S. Patent
`
`Mar. 28, 2000
`
`Sheet 7 of 7
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`6,043,344
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`
`

`

`6,043,344
`
`HUMAN CTLA-8 AND USES OF CTLA-8-
`RELATED PROTEINS
`
`This application is a divisional of application Ser. No.
`08/685,239, filed Jul. 18, 1996, now abandoned, which is a 5
`continuation-in-part of application of Ser. No 08/504,032
`filed Jul. 19,1995, now converted to Provisional application
`No. 60/035347 filed Jul. 19,1995, and a continuation-in-part
`of application Ser. No. 08/514,014, filed Aug. 11,1995, now
`U.S. Pat. No. 5,707,829.
`
`10
`
`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 the use of
`rat CTLA-8 proteins and herpesvirus Saimiri 0RF13 pro(cid:173)
`teins in methods of treatment.
`
`BACKGROUND OF THE INVENTION
`
`20
`
`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 25
`lymphokines, hematopoietins, interleukins, colony stimulat(cid:173)
`ing 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 30
`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 35
`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 prolifera(cid:173)
`tion. The search for novel cytokines has thus been limited by 40
`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 encod(cid:173)
`ing cytokines have been detected using the polymerase chain
`reaction (PCR) and oligonucleotide primers having homol- 45
`ogy to shared motifs of known cytokines or their receptors.
`The PCR approach is also limited by the necessity for
`knowledge of previously cloned cytokines in the same
`protein family. Cytokines have also been cloned using
`subtractive hybridization to construct and screen cDNA 50
`libraries, or they can potentially be cloned using PCR
`followed by gel 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 do not 55
`require any prior knowledge of the sequence of interest.
`However, many cytokines may be encoded by mRNAs
`which are not differentially expressed, and thus are unde(cid:173)
`tectable using these methods.
`
`It would be desirable to develop new methods for iden(cid:173)
`tifying 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
`
`60
`
`65
`
`encode secreted proteins. One such polynucleotide was
`isolated which encodes "human CTLA-8." In accordance
`with the present invention, polynucleotides encoding human
`CILA-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 mam(cid:173)
`malian species.
`In certain embodiments, the present invention provides an
`isolated polynucleotide comprising a nucleotide sequence
`selected from the group consisting of:
`(a) the nucleotide sequence of SEQ ID NO: 1 from
`nucleotide 146 to nucleotide 544;
`(b) a nucleotide sequence capable of hybridizing to a
`nucleic acid sequence specified in (a);
`(c) a nucleotide sequence varying from the sequence of
`the nucleotide sequence specified in (a) as a result of
`degeneracy of the genetic code; and
`(d) an allelic variant of the nucleotide sequence specified
`in (a). 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, including mammalian cells.
`Processes are also provided for producing a human
`CTLA-8 protein, said processes comprising:
`(a) growing a culture of the host cell of the invention in
`a suitable culture medium; and
`(b) purifying the human CTLA-8 protein from the culture.
`Isolated human CTLA-8 protein is also provided which
`comprising an amino acid sequence selected from the group
`consisting of:
`(a) the amino acid sequence of SEQ ID NO:2;
`(b) 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;
`(d) the amino acid sequence of SEQ ID NO:2 from amino
`acids 31 to 163; and
`(e) fragments of (a), (b), (c) or (d) 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. Pharmaceuticals composi(cid:173)
`tion comprising a human CTLA-8 protein of the invention
`and a pharmaceutically acceptable carrier are also provided.
`Compositions are also disclosed which comprise an anti(cid:173)
`body which specifically reacts with a human CTLA-8 pro(cid:173)
`tein of the invention.
`Methods of treating a mammalian subject are also pro(cid:173)
`vided which comprise administering a therapeutically effec(cid:173)
`tive amount of a pharmaceutical composition comprising a
`human CTLA-8 protein.
`Rat CTLA-8 and active (i.e., having CTLA-8 activity)
`fragments thereof may also be used in such methods of
`
`Lassen - Exhibit 1037, p. 9
`
`

`

`6,043,344
`
`treatment. Preferably the rat protein is administered as a
`composition comprising a pharmaceutically acceptable car(cid:173)
`rier and a protein comprising an amino acid sequence
`selected from the group consisting of:
`(a) the amino acid sequence of SEQ ID NO:4;
`(b) the amino acid sequence of SEQ ID NO:4 from amino
`acids 18 to 150; and
`(c) fragments of (a) or (b) having CTLA-8 activity.
`Herpesvirus Saimiri ORF13, referred to herein as "herpes
`CTLA-8", and active (i.e., having CTLA-8 activity) frag(cid:173)
`ments thereof and active fragments thereof may also be used
`in such methods of treatment. Preferably the herpes CTLA-8
`protein is administered as a composition comprising a
`pharmaceutically acceptable carrier and a protein compris(cid:173)
`ing an amino acid sequence selected from the group con(cid:173)
`sisting of:
`(a) the amino acid sequence of SEQ ID NO:6;
`(b) the amino acid sequence of SEQ ID NO:6 from amino
`acids 19 to 151; and
`(c) fragments of (a) or (b) having CTLA-8 activity.
`The invention also provides a method of treating a mam(cid:173)
`malian subject comprising administering a therapeutically
`effective amount of a composition comprising a pharmaceu(cid:173)
`tically acceptable carrier and IL-17 or an active fragment
`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 vas(cid:173)
`cular endothelial cells, inhibition of tumor growth, inhibi(cid:173)
`tion 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 L-3
`production and induction of GM-CSF production.
`
`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_F1"), rat CTLA-8 (indicated as "MusctlaS") and herpes
`CTLA-8 (indicated as "Hsvie 2").
`FIG. 2 depicts autoradiographs demonstrating expression
`of human CTLA-8 in COS cells.
`FIG. 3 presents data relating to the ability of human
`CTLA-8 to inhibit angiogenesis.
`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 CILA-8 to induce production of L-6 and L-8.
`
`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:l 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
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`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:6). AcDNAsequence
`encoding rat CTLA-8 is listed at SEQ ID NO:3 and its
`corresponding amino acid sequence is reported at SEQ ID
`NO:4. AcDNAsequence 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.
`Applicants had previously incorrectly identified the rat
`sequences of SEQ ID NO:3 and SEQ ID NO:4 as applying
`to murine CTLA-8. Applicants' human CTLA-8 (B 18) does
`also show homology to the true murine CTLA-8 sequence.
`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. 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 et al. species has
`now been renamed as interleukin-17 (IL-17). Because of the
`homology between applicants' human CTLA-8 (B118) 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 pro(cid:173)
`teins 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 polynucle(cid:173)
`otides as described above may be made by standard molecu(cid:173)
`lar 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 (1) 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 (2) induces expression or secre(cid:173)
`tion of y-IFN, or (3) displays chemoattractant or chemotactic
`activity
`in a chemoattraction or chemotaxis assay
`(preferably as assay in which full-length the corresponding
`species full-length CTLA-8 is active) or (4) induces expres(cid:173)
`sion or secretion of IL-3 or GM-CSF.
`Human CTLA-8 protein or fragments thereof having
`CTLA-8 activity may be fused to carrier molecules such as
`immunoglobulins. For example, human CTLA-8 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 isolated poly(cid:173)
`nucleotide of SEQ ID NO:l which also encode human
`CTLA-8 or CTLA-8 proteins having CTLA-8 activity. Also
`included in the invention are isolated polynucleotides which
`
`Lassen - Exhibit 1037, p. 10
`
`

`

`6,043,344
`
`hybridize to the nucleotide sequence set forth in SEQ ID
`NO: 1 under highly stringent (0.2xSSC at 65° C), stringent
`(e.g. 4xSSC at 65 C or 50% formamide and 4xSSC at 42°
`C), or relaxed (4xSSC at 50° C. or 30-40% formamide and
`4xSSC at 42° C.) conditions. Isolated polynucleotides which
`encode human CTLA-8 protein but which differ from the
`nucleotide sequence set forth in SEQ ID NO:l 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:l 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, 4485^1490 (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 Enzymol-
`ogy 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, monkey COS cells, Chinese Hamster Ovary
`(CHO) cells, human kidney 293 cells, 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, HL-60,
`U937, HaK, Rat2, BaF3, 32D, FDCP-1, PC12 or C2C12
`cells.
`The human CTLA-8 protein may also be produced by
`operably linking the isolated polynucleotide of the invention
`to suitable control sequences in one or more insect expres(cid:173)
`sion vectors, and employing an insect expression system
`Materials and methods for baculovirus/insect cell expression
`systems are commercially available in kit form from, e.g.,
`Invitrogen, San Diego, Calif., 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 poly(cid:173)
`nucleotides as described above.
`Alternatively, the human CTLA-8 protein may be pro(cid:173)
`duced in lower eukaryotes such as yeast or in prokaryotes
`such as bacteria. Suitable yeast strains include Saccharomy-
`ces cerevisiae, Schizosaccharomyces pombe, Kluyveromy-
`ces strains, Candida, or any yeast strain capable of express(cid:173)
`ing 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, e.g., as a
`component of the milk of transgenic cows, goats, pigs, or
`sheep which are characterized by somatic or germ cells
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`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 prepar(cid:173)
`ing a total membrane fraction from the expressing cell and
`extracting the membranes with a non-ionic detergent such as
`Triton X-100.
`The human CTLA-8 protein can be purified using meth(cid:173)
`ods known to those skilled in the art. For example, the
`human CTLA-8 protein of the invention can be concentrated
`using a commercially available protein concentration filter,
`for example, an Amicon or Millipore Pellicon ultrafiltration
`unit. Following the concentration step, the 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 com(cid:173)
`monly 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 (e.g., 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 inter(cid:173)
`action chromatography using such resins as phenyl ether,
`butyl ether, or propyl ether; or by immunoafSnity chroma(cid:173)
`tography. Finally, one or more reverse-phase high perfor(cid:173)
`mance liquid chromatography (RP-HPLC) steps employing
`hydrophobic RP-HPLC media, e.g., silica gel having pen(cid:173)
`dant 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 expres(cid:173)
`sion in induced primary cells and can induce 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 related proteins
`may have an effect on proliferation or function of myeloid
`cells, 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) activity or may
`induce production of other cytokines in certain cell popu(cid:173)
`lations. Many protein factors discovered to date, including
`all known cytokines, have exhibited activity in one or more
`factor dependent cell proliferation assays, and hence the
`assays serve as a convenient confirmation of cytokine activ-
`
`Lassen - Exhibit 1037, p. 11
`
`

`

`6,043,344
`
`ity. The activity of a protein of the present invention is
`evidenced by any one of a number of routine factor depen(cid:173)
`dent cell proliferation assays for cell lines including, without
`limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/
`G, M+ (preB M+), 2E8, RB5, DAI, 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 with(cid:173)
`out 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 Publish(cid:173)
`ing Associates and Wiley-Interscience (Chapter 3, In Vitro
`assays for Mouse Lymphocyte Function 3.1-3.19; Chapter
`7, Immnologic 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 Immunol(cid:173)
`ogy 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, with(cid:173)
`out limitation, those described in: Polyclonal T cell
`stimulation, Kruisbeek, A. M. and Shevach, E. M. In Cur(cid:173)
`rent 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. 6.8.1-6.8.8, John Wiley and Sons,
`Toronto. 1994.
`Assays for proliferation and differentiation of hematopoi(cid:173)
`etic and lymphopoietic cells include, without limitation,
`those described in: Measurement of Human and Murine
`Interleukin 2 and Interleukin 4, Bottomly, K., 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, Proc. Natl. Acad. Sci. U.S.A.
`80:2931-2938, 1983; Measurement of mouse and human
`interleukin 6 - Nordan, R. In Current Protocols in Immu(cid:173)
`nology. J. E. e.a Coligan eds. 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, E, Giannotti, J., 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 Interleu(cid:173)
`kin 9 - Ciarletta, A., 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 Publish(cid:173)
`ing Associates and Wiley-Interscience (Chapter 3, In Vitro
`assays for Mouse Lymphocyte Function; Chapter 6, Cytok(cid:173)
`ines and their cellular receptors; Chapter 7, Immunologic
`studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci.
`USA77:6091-6095, 1980; Weinbergeret al, Eur. J. Immun.
`11:405^111,1981; 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, includ-
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`8
`ing without limitation the activities for which assays are
`described herein. Aprotein may be useful in the treatment of
`various immune deficiencies and disorders (including severe
`combined immunodeficiency (SCID)), e.g., in regulating (up
`or down) growth and proliferation of T and/or B
`lymphocytes, as well as effecting the cytolytic activity of
`NK cells and other cell populations. These immune defi(cid:173)
`ciencies may be genetic or be caused by viral (e.g., HIV) as
`well as bacterial or fungal infections, or may result from
`autoirmune 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 infec(cid:173)
`tions 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, i.e., in the
`treatment of cancer.
`Autoimmune disorders which may be treated using a
`protein of the present invention include, for example, mul(cid:173)
`tiple sclerosis, systemic lupus erythematosus, rheumatoid
`arthritis, autoimmune pulmonary inflammation, Guillain-
`Barre syndrome, autoimmune thyroiditis, insulin dependent
`diabetes mellitis, myasthenia gravis, graft-versus-host dis(cid:173)
`ease 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 pro(cid:173)
`duction 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., 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; Takai et al., J. Immunol
`140:508-512, 1988; 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.