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Title:  Variant B7 co-stimulatory molecules
United States Patent: 
7,183,376
Issued: 
February 27, 2007

Inventors: 
Punnonen; Juha (Belmont, CA), Lazetic; Alexandra (San Jose, CA), Leong; Steven R. (Berkeley, CA), Chang; Chia-Chun (Los Gatos, CA)
Assignee: 
Maxygen, Inc. (Redwood City, CA)
Appl. No.: 
10/032,214
Filed: 
December 20, 2001


 

Training Courses -- Pharm/Biotech/etc.


Abstract

The invention provides polynucleotides and polypeptides encoded therefrom having advantageous properties, including an ability of the polypeptides to preferentially bind a CD28 or CTLA-4 receptor at a level greater or less than the ability of human B7-1 to bind CD28 or CTLA-4, or to induce or inhibit altered level of T cell proliferation response greater compared to that generated by human B7-1. The polypeptides and polynucleotides of the invention are useful in therapeutic and prophylactic treatment methods, gene therapy applications, and vaccines.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides novel co-stimulatory molecules (abbreviated as "NCSM") molecules, including polypeptides and proteins, related fusion polypeptide or fusion protein molecules, or functional equivalents thereof, homologues, and fragments of said polypeptide and protein molecules or equivalents, analogs, or derivatives thereof, and vectors, cells, and compositions comprising such NCSM molecules. The invention also provides nucleic acids encoding any of these polypeptides, proteins, fragments or variants thereof. In addition, the invention provides vectors, cells, and compositions comprising such nucleic acids, and uses of such NCSM polypeptides and NCSM nucleic acids; and other features are apparent upon further review.

Generally speaking, a "co-stimulatory molecule" refers to a molecule that acts in association or conjunction with, or is involved with, a second molecule or with respect to an immune response in a co-stimulatory pathway. In one aspect, a co-stimulatory molecule may be an immunomodulatory molecule that acts in association or conjunction with, or is involved with, another molecule to stimulate or enhance an immune response. In another aspect, a co-stimulatory molecule is immunomodulatory molecule that acts in association or conjunction with, or is involved with, another molecule to inhibit or suppress an immune response. A "co-stimulatory molecule" need not act simultaneously with or by the same mechanism as the second molecule. The term "NCSM" in reference to a molecule is not intended to limit the molecule to only those molecules that have positive co-stimulatory properties (e.g., that stimulate or augment T cell proliferation). In the initial recombination procedures described below, libraries of recombinant molecules were generated by recombining nucleotide sequences of parental co-stimulatory molecules (CSM) as discussed herein. As shown by the data and analyses presented herein, novel recombinant molecules having a variety of properties were identified and selected. For example, polypeptide and nucleic acid molecules that enhance an immune response, such as by inducing T cell activation or proliferation (e.g., agonists), and molecules that down-regulate or inhibit an immune response, such as by inhibiting T cell activation or proliferation (e.g., antagonists) were identified and selected. Further, molecules that preferentially bind and/or signal through either or both the CD28 and CTLA-4 receptors were identified and selected. Thus, the term "NCSM" refers to a co-stimulatory molecule and is not limited to molecules having the co-stimulatory properties of the parent sequences, but is intended to refer collectively to all polypeptides of the invention, and nucleic acids encoding them, and other embodiments as described herein, unless specifically noted otherwise.

The term "NCSM" also includes variants, mutants, derivatives, and fragments of: 1) B7-1 and B7-2 polypeptides and nucleic acids, and 2) B7-1 and B7-2 polypeptides and nucleic acids of the Artiodactyla family (including, e.g., bovine B7-1 and B7-2), including all such polypeptide variants (and nucleic acids encoding such polypeptide variants) that exhibit properties similar or equivalent to the properties of the CD28BPs or CTLA-4BPs described herein. For example, the term includes B7-1, B7-2, and Artiodactyla (e.g., bovine) B7-1 and B7-2 polypeptide variants (and nucleic acids encoding such polypeptide variants) of the invention, wherein such polypeptide variants have a CD28/CTLA-4 binding affinity ratio about equal to, equal to, or greater than the CD28/CTLA-4 binding affinity ratio of hB7-1 or hB7-2 and/or an ability to induce a T-cell proliferation, and/or a T-cell activation response about equal to, equal to, or greater than that of hB7-1. The term "NCSM" also is intended to include B7-1, B7-2, and Artiodactyla (e.g., bovine) B7-1 and B7-2 polypeptide variants (and nucleic acids encoding such polypeptide variants), wherein such polypeptide variants have a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of hB7-1 or hB7-2, and/or an ability to induce a T-cell proliferation and/or T-cell activation response about equal to or less than that of hB7-1 or hB7-2.

In one aspect, the invention includes isolated or recombinant NCSM polypeptides, variants, homologues, derivatives, analogs, and fragments thereof. The invention includes recombinant NCSM polypeptides having varied abilities to preferentially bind to and/or signal through CD28 and/or CTLA-4 receptor and provide for selected and differential manipulation of T cell responses in vitro, ex vivo, and in vivo. The invention also includes isolated or recombinant NCSM nucleic acids, variants, homologues, derivatives, analogs, and fragments thereof that encode polypeptides having varied abilities and uses described above. Such NCSM polypeptide and polynucleotides are useful in a variety of applications, including e.g., therapeutic and prophylactic treatment methods, vaccinations, and diagnostic assays described below. The invention also provides NCSM polypeptides, and polynucleotide encoding such polypeptides, that strongly or preferentially bind at least one of CD28 or CTLA-4, but do not effectuate signaling; such molecules are useful in methods as potential antagonists of endogenous molecules, such as e.g., endogenous co-stimulatory molecules. Further, the invention provides NCSM polypeptides, and polynucleotides encoding them, having improved or altered receptor/ligand binding affinities and methods of using such molecules, including in pharmaceutical, prophylactic, therapeutic, vaccine, and diagnostic applications.

In one aspect, the invention provides an isolated or recombinant polypeptide comprising an amino acid sequence of an extracellular domain (ECD) amino acid sequence having at least about 75% amino acid sequence identity to an extracellular domain amino acid sequence of, or the full-length sequence of, at least one of SEQ ID NOS:48 68, 174 221, 283 285, and 290 293, and not being a naturally-occurring extracellular domain amino acid sequence, and wherein said polypeptide has a CD28/CTLA-4 binding affinity ratio about equal to or greater than the CD28/CTLA-4 binding affinity ratio of human B7-1 and/or has an ability to induce a T-cell proliferation and/or T-cell activation response about equal to or greater than that of hB7-1. Some such polypeptides induce T-cell proliferation or T-cell activation or both T-cell proliferation and T-cell activation. In some embodiments, the T cell activation or proliferation response is at least about equal to or greater than that caused by WT hB7-1.

Some such polypeptides of the invention may comprise an amino acid sequence having 75% identity to a full-length polypeptide sequence of SEQ ID NOS:48 68, 174 221, 283 285, and 290 293. Such polypeptides may be expressed on the surface of a cell membrane (e.g., following transfection of the cell with a nucleic acid that encodes said polypeptide) or associated with or bound to a cell membrane, or form an integral membrane protein (e.g., by further comprising a transmembrane domain amino acid sequence). Through such expression, such polypeptides typically become integral membrane proteins. Preferably, such cell-expressed polypeptides, membrane-associated, or membrane-bound polypeptides, have an ability to induce a T cell proliferation response that is equal to or greater than that induced by hB7-1.

Other such polypeptides modulate T-cell activation, but do not induce proliferation of purified T-cells activated by soluble anti-CD3 mAbs.

In one embodiment, the isolated or recombinant polypeptide comprises an ECD that has at least about 90% amino acid sequence identity to an ECD or the full-length sequence of at least one of SEQ ID NOS:48 68, 174 221, 283 285, and 290 293. In another embodiment, the ECD amino acid sequence comprises an amino acid subsequence of at least one of SEQ ID NOS:48 68, 174 221, 283 285, and 290 293.

Some such isolated or recombinant polypeptides are soluble (e.g., not cell membrane-bound or membrane-associated or forming an integral part of a cell membrane). The invention includes monomeric and multimeric (or aggregated) forms of such soluble polypeptides. A soluble monomer comprises one soluble polypeptide of the invention (e.g., one soluble NCSM-ECD), while a soluble multimer or soluble aggregate comprises at least two soluble polypeptides of the invention (e.g., two soluble ECDs). The multimer or aggregate may be formed by cross-linking or other means to link polypeptide sequences. Such polypeptides may comprise a fusion protein comprising at least one additional amino acid sequence, such as an Ig polypeptide. Some such soluble polypeptides, in the presence of a population of activated T cells, have an ability to induce a T cell proliferation response, in the presence of a population of activated T cells, that is less than the T cell proliferation response induced by a soluble human B7-1 polypeptide in the presence of a population of activated T cells.

Some such isolated or recombinant polypeptides further comprise at least one of a signal peptide domain, transmembrane domain (TMD), and/or cytoplasmic domain(CD), including, e.g., wherein such domain is a WT, variant, or mutant domain of a co-stimulatory polypeptide, including, e.g., a recombinant domain derived from a mammalian B7-1 or B7-2. In one embodiment, the isolated or recombinant polypeptide comprises an amino acid subsequence, including any of a signal peptide, TMD, or CD of any of SEQ ID NOS:48 68, 174 221, 283 285, and 290 293. Nucleic acids encoding such polypeptides and domains are also provided.

In another aspect, the invention provides an isolated or recombinant polypeptide, which polypeptide comprises a non-naturally-occurring amino acid sequence encoded by a nucleic acid comprising a polynucleotide sequence selected from the group of: (a) a polynucleotide sequence selected from SEQ ID NOS:1 21 and 95 142, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NOS:48 68, 174 221, 283 285, and 290 293, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence which, but for the degeneracy of the genetic code, hybridizes under at least stringent or highly stringent conditions over substantially the entire length of polynucleotide sequence (a) or (b); (d) a polynucleotide sequence comprising all or a nucleotide fragment of (a), (b), or (c), wherein the nucleotide fragment encodes a polypeptide having a CD28/CTLA-4 binding affinity ratio about equal to or greater than the CD28/CTLA-4 binding affinity ratio of human B7-1, and/or has an ability to induce a T-cell proliferation and/or T-cell activation response about equal to or greater than that of hB7-1; (e) a polynucleotide sequence encoding a polypeptide, the polypeptide comprising an amino acid sequence which is substantially identical over at least about 150 contiguous amino acid residues of any one of SEQ ID NOS:48 68, 174 221, 283 285, and 290 293; and (f) a polynucleotide sequence encoding a polypeptide that has a CD28/CTLA-4 binding affinity ratio about equal to or greater than the CD28/CTLA-4 binding affinity ratio of human B7-1, and/or has an ability to induce T-cell proliferation or activation or both that is about equal to or greater than that induced by hB7-1, which polynucleotide sequence has at least about 70% amino acid sequence identity to at least one polynucleotide sequence of (a), (b), (c), or (d).

Also provided is an isolated or recombinant polypeptide comprising an amino acid sequence according to the formula: MGHTM-X6-W-X8-SLPPK-X14-PCL-X18-X19-X20-QLLVLT-X27-LFYFCSGITPKSVTKRVKETVM- LSCDY-X55-TSTE-X60-LTSLRIYW-X69-KDSKMVLAILPGKVQVWPEYKNRTITDMNDN-X101-RIVI-- X106-ALR-X110-SD-X113-GTYTCV-X120-QKP-X124-LKGAYKLEHL-X135-SVRLMIRADFPVP-X- 149-X150-X151-DLGNPSPNIRRLICS-X167-X168-X169-GFPRPHL-X177-WLENGEELNATNTT-X- 192-SQDP-X197-T-X199-LYMISSEL-X208-FNVTNN-X215-SI-X218-CLIKYGEL-X227-VSQIF- PWSKPKQEPPIDQLPF-X249-VIIPVSGALVL-X261-A-X263-VLY-X267-X268-ACRH-X273-ARWK- RTRRNEETVGTE RLSPIYLGSAQSSG (SEQ ID NO:284), or a subsequence thereof comprising an extracellular domain, wherein position X6 is Lys or Glu; position X8 is Arg or Gly; position X14 is Arg or Cys; position X18 is Trp or Arg; position X19 is Pro or Leu; position X20 is Ser or Pro; position X27 is Asp or Gly; position X55 is Asn or Ser; position X60 is Glu or Lys; position X69 is Gln or Arg; position X101 is Pro or Leu; position X106 is Leu or Gln; position X110 is Pro or Leu; position X113 is Lys or Ser; position X120 is Val or Ile; position X124 is Val or Asp; position X135 is Thr or Ala; position X149 is Thr, Ser, or del; position X150 is Ile or del; position X151 is Asn or Thr; position X167 is Thr or del; position X169 is Ser or del; position X169 is Gly or del; position X177 is Cys or Tyr; position X192 is Val or Leu; position X197 is Gly or Glu; position X199 is Glu or Lys; position X208 is Gly or Asp; position X215 is His or Arg; position X218 is Ala or Val; position X227 is Ser or Leu; position X249 is Trp, Leu, or Arg; position X261 is Ala or Thr; position X263 is Val, Ala, or Ile; position X267 is Arg or Cys; position X268 is Pro or Leu; and position X273 is Gly or Val. Typically, the ECD comprises at least about amino acids 35 to 244 or amino acids 35 to 255 of SEQ ID NO:284. Some such polypeptides have a CD28/CTLA-4 binding affinity ratio about equal to or greater than the CD28/CTLA-4 binding affinity ratio of human B7-1, and/or has an ability to induce T-cell proliferation or activation or both that is about equal to or greater than that induced by hB7-1.

In yet another aspect, the invention provides an isolated or recombinant polypeptide comprising a subsequence of an amino acid sequence set forth in any of SEQ ID NOS:48 68, 174 182, 184 221, 283 285, and 290 293, wherein the subsequence is the extracellular domain of said amino acid sequence.

A soluble form of an NCSM polypeptide or a human B7-1 polypeptide typically comprises a polypeptide, comprising at least one ECD, which is not expressed on the surface of a cell, is not embedded in a cell membrane, and/or is not associated with a lipid bilayer. Such soluble polypeptides may contain a TD or a fragment thereof such that the polypeptide remains soluble (e.g., is not embedded membrane as an integral membrane protein or associated with a lipid bilayer). Such soluble polypeptides may also include one or more additional amino acid sequences, such as an Fc portion of an Ig (e.g., IgG). Such soluble polypeptide may comprise an ECD monomer, ECD-Ig fusion protein monomer, ECD multimer or aggregate, or ECD-Ig fusion protein multimer or aggregate (see data and discussion below).

Also provided are isolated or recombinant polypeptides, each of which comprise an amino acid sequence of an extracellular domain, wherein said extracellular domain amino acid sequence has at least about 75% amino acid sequence identity to an extracellular domain amino acid sequence of at least one of SEQ ID NOS:48 68, 174 221, 283 285, and 290 293, and is not a naturally-occurring extracellular domain amino acid sequence, wherein such polypeptide, in the presence of activated T cells, has an ability to down-regulate or inhibit a T cell proliferation response compared to the response caused by soluble hB7-1 (e.g., hB7-1-ECD or hB7-1-ECD-Ig) in the presence of activated T cells. The polypeptide can be either a soluble ECD monomer or a soluble ECD-Ig fusion protein. Such polypeptides may comprise a fusion protein comprising at least one additional polypeptide, such as, e.g., an Ig polypeptide.

The invention also includes each nucleic acid comprising a polynucleotide sequence (including degenerate sequences) that encodes each such isolated or recombinant polypeptide comprising a soluble polypeptide as described above, and a complementary polynucleotide sequence thereof. Polynucleotide sequences that, but for the degeneracy of the genetic code, hybridizes under at least stringent conditions over substantially the entire length of each such nucleic acid are also included.

The invention further provides isolated or recombinant polypeptides comprising an amino acid sequence having at least about 95% amino acid sequence identity to a full-length sequence of at least one of SEQ ID NOS:69 92, 222 252, 286 289, or to a subsequence thereof comprising the extracellular domain, wherein said sequence (a) is a non naturally-occurring sequence, and (b) comprises at least one of: Gly at position 2; Thr at position 4; Arg at position 5; Gly at position 8; Pro at position 12; Met at position 25; Cys at position 27; Pro at position 29; Leu at position 31; Arg at position 40; Leu at position 52; His at position 65; Ser at position 78; Asp at position 80; Tyr at position 87; Lys at position 120; Asp at position 122; Lys at position 129; Met at position 135; Phe at position 150; Ile at position 160; Ala at position 164; His at position 172; Phe at position 174; Leu at position 176; Asn at position 178; Asn at position 186; Glu at position 194; Gly at position 196; Thr at position 199; Ala at position 210; His at position 212; Arg at position 219; Pro at position 234; Asn at position 241; Leu at position 244; Thr at position 250; Ala at position 254; Tyr/ at position 265; Arg at position 266; Glu at position 273; Lys at position 275; Ser at position 276; an amino acid deletion at position 276; or Thr at position 279, wherein the position number corresponds to that of the human B7-1 amino acid sequence (SEQ ID NO:278), wherein said polypeptide has a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of human B7-1, and/or an ability to induce a T-cell proliferation or T-cell activation response about equal to or less than that of hB7-1. A subsequence comprises signal peptide, EDC, TMD, or CD. Each such polypeptide may further comprise at least one additional amino acid sequence, including, e.g., a sequence corresponding to a signal peptide, TMD, ECD, or CD.

In another aspect, the invention provides isolated or recombinant polypeptides, each comprising an amino acid sequence that differs from the amino acid sequence of a primate B7-1, wherein the difference between the amino acid sequence of the polypeptide and the amino acid sequence of the primate B7-1 comprises a different amino acid at at least one amino acid residue position selected from the group consisting of 12, 25, 27, 29, 40, 52, 65, 122, 129, 135, 164, 174, 196, 199, 210, 219, 234, 241, 254, 275, 276, and 279, wherein the amino acid residue positions correspond to the amino acid residue positions in the amino acid sequence of human B7-1 of SEQ ID NO:278. For some such polypeptides, the different amino acid comprises at least one of: Pro at position 12; Met at position 25; Cys at position 27; Pro at position 29; Arg at position 40; Leu at position 52; His at position 65; Asp at position 122; Lys at position 129; Met at position 135; Ala at position 164; Phe at position 174; Gly at position 196; Thr at position 199; Ala at position 210; Arg at position 219; Pro at position 234; Asn at position 241; Ala at position 254; Lys at position 275; Ser at position 276; or Thr at position 279. Preferably, for some such polypeptides, the different amino acid is His at position 65.

In another aspect, the invention provides isolated or recombinant polypeptides comprising an amino acid sequence that differs from a primate B7-1 sequence in at least one mutation selected from: Ser 12 Pro; Leu 25 Met; Gly 27 Cys; Ser 29 Pro; Lys 40 Arg; His 52 Leu; Tyr 65 His; Glu 122 Asp; Glu 129 Lys; Thr 135 Met; Thr 164 Ala; Ser 174 Phe; Glu 196 Gly; Ala 199 Thr; Thr 210 Ala; Lys 219 Arg; Thr 234 Pro; Asp 241 Asn; Val 254 Ala; Arg 275 Lys; Arg 276 Ser; or Arg 279 Thr; the mutation being indicated relative to human B7-1 with the amino acid sequence shown in SEQ ID NO:278, wherein said sequence does not occur in nature, and wherein said polypeptide has a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of human B7-1, and/or an ability to induce a T-cell proliferation and/or T-cell activation response about equal to or less than that of hB7-1.

Also included are isolated or recombinant polypeptides comprising an amino acid sequence having at least about 75% sequence identity to at least one of SEQ ID NOS:263 272, or a subsequence thereof comprising the extracellular domain, where the amino acid sequence is not naturally-occurring, and the polypeptide has a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of human B7-1, and/or an ability to induce a T-cell proliferation and/or activation response about equal to or less than that of hB7-1.

In yet another aspect, the invention includes an isolated or recombinant polypeptides which comprises a non naturally-occurring amino acid sequence encoded by a nucleic acid comprising a polynucleotide sequence selected from: (a) a polynucleotide sequence selected from SEQ ID NOS:22 45, 143 173, 253 262, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ I) NOS:69 92, 222 247, 263 272, 286 289, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence which, but for the degeneracy of the genetic code, hybridizes under highly stringent conditions over substantially the entire length of polynucleotide sequence (a) or (b); (d) a polynucleotide sequence comprising all or a fragment of (a), (b), or (c), wherein the fragment encodes a polypeptide having a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of human B7-1, or an ability to induce a T-cell proliferation or activation response about equal to or less than that of hB7-1; (e) a polynucleotide sequence encoding a polypeptide, the polypeptide comprising an amino acid sequence which is substantially identical over at least about 150 contiguous amino acid residues of any one of SEQ ID NOS:69 92, 222 247, 263 272, 286 289, and (f) a polynucleotide sequence encoding a polypeptide that has a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of human B7-1 or an ability to induce a T-cell proliferation or activation response about equal to or less than that of hB7-1, which polynucleotide sequence has at least about 70% identity to at least one polynucleotide sequence of (a), (b), (c), or (d).

The invention also includes an isolated or recombinant polypeptide comprising an amino acid sequence according to the formula:

MGHTRRQGTSP-X12-KCPYLKFFQLLV-X25-ACL-X29-HLCSGVIHVT-X40-EVKEVATLSCGLNVSVEE- LAQTRIHWQKEKKMVLTM MSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKY-X122-KDAFKR-X129-HLAEVMLSVK- AD FPTPSITDFEIPPSNIRRIICS-X164-SGGFPEPHLFWLENGEELNAINTTVSQDPET-X196-LYTVSS- KLDFNM TANHSFMCLI-X219-YGHLRVNQTFNWNTPKQEHFP-X241-NLLPSWA ITLISANGIFVICCLTYRFAPRCRERKSNETLRRESVCPV (SEQ ID NO:287), or a subsequence thereof comprising the extracellular domain, wherein position X12 is Ser or Pro; position X25 is Leu or Met; position X29 is Ser or Pro; position X40 is Lys or Arg; position X122 is Glu or Asp; position X129 is Glu or Lys; position X164 is Thr or Ala; position X196 is Glu or Gly; position X219 is Lys or Arg; position X241 is Asp or Asn. Some such polypeptides have a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of hB7-1, and/or ability to induce T-cell proliferation or activation or both about equal to or less than that induced by hB7-1.

The invention also provides an isolated or recombinant polypeptide comprising a subsequence of an amino acid sequence set forth in any of SEQ ID NOS:69 92, 222 247, 263 272, and 286 289, wherein the subsequence is the extracellular domain or full-length sequence of such amino acid sequence. Furthermore, the invention includes the full-length polypeptide sequence and one or more subsequences thereof, e.g., signal peptide, extracellular domain (ECD), transmembrane domain (TMD), and/or cytoplasmic domain (CD) of any of SEQ ID NOS:66, 81, 85, 86, 88, 90, 91, 285, 288, 289, 291, and 294, and nucleic acid sequences encoding any of these amino acid sequences.

The invention provides isolated or recombinant nucleic acids comprising a polynucleotide sequence selected from: (a) a polynucleotide sequence selected from SEQ ID NOS:1 21 and 95 142, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NOS:48 68, 174 221, 283 285, and 290 293, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence which, but for codon degeneracy, hybridizes under at least stringent or highly stringent conditions over substantially the entire length of polynucleotide sequence (a) or (b); and (d) a polynucleotide sequence comprising all or a nucleotide fragment of (a), (b), or (c), wherein the fragment encodes a polypeptide having a CD28/CTLA-4 binding affinity ratio about equal to or greater than the CD28/CTLA-4 binding affinity ratio of human B7-1, or an ability to induce a T-cell proliferation or activation response about equal to or greater than that of hB7-1. In some such nucleic acids, the polynucleotide sequence of (d) encodes a nucleotide fragment of (a) or (b) that encodes an ECD having a CD28/CTLA-4 binding affinity ratio or an ability to induce a T-cell proliferation or activation response about equal to or greater than that of hB7-1.

The invention also includes isolated or recombinant nucleic acids comprising a polynucleotide sequence encoding a polypeptide, wherein the encoded polypeptide comprises an amino acid sequence which is (a) substantially identical over at least about 150 or 200 contiguous amino acid residues of any one of SEQ ID NOS:48 68, 174 221, 283 285, and 290 293 and (b) is a non naturally-occurring sequence.

In addition, the invention includes isolated or recombinant nucleic acids comprising a nucleotide sequence coding for a polypeptide comprising the amino acid sequence set forth in any of SEQ ID NOS:48 68, 174 221, 283 285, and 290 293, or a subsequence thereof, wherein the subsequence comprises at least one of: the signal sequence, extracellular domain, or transmembrane domain of said polypeptide, and the cytoplasmic domain of said polypeptide, and wherein the amino acid sequence or subsequence is a non naturally-occurring sequence. Similarly, fragments of the above nucleotides that encode a polypeptide that has a substantially equivalent or equivalent binding activity of a NCSM polypeptide molecule, produces a substantially equivalent or equivalent NCSM-polypeptide-mediated immune response, e.g., induction or inhibition of T cell activation or proliferation, or cytokine production are a feature.

Also provided is an isolated or recombinant nucleic acid comprising a polynucleotide sequence selected from: (a) a polynucleotide sequence selected from SEQ ID NOS:22 45, 143 173, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NOS:69 92, 222 247, 286 289, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence which, but for the degeneracy of the genetic code, hybridizes under highly stringent conditions over substantially the entire length of polynucleotide sequence (a) or (b); and (d) a polynucleotide sequence comprising all or a fragment of (a), (b), or (c); wherein (c) or (d) encodes a polypeptide having a non naturally-occurring sequence comprising at least one of:

Gly at position 2; Thr at position 4; Arg at position 5; Gly at position 8; Pro at position 12; Met at position 25; Cys at position 27; Pro at position 29; Leu at position 31; Arg at position 40; Leu at position 52; His at position 65; Ser at position 78; Asp at position 80; Tyr at position 87; Lys at position 120; Asp at position 122; Lys at position 129; Met at position 135; Phe at position 150; Ile at position 160; Ala at position 164; His at position 172; Phe at position 174; Leu at position 176; Asn at position 178; Asn at position 186; Glu at position 194; Gly at position 196; Thr at position 199; Ala at position 210; His at position 212; Arg at position 219; Pro at position 234; Asn at position 241; Leu at position 244; Thr at position 250; Ala at position 254; Tyr at position 265; Arg at position 266; Glu at position 273; Lys at position 275; Ser at position 276; an amino acid deletion at position 276; and Thr at position 279, wherein the position number corresponds to that of the human B7-1 amino acid sequence (SEQ ID NO:278), and wherein said polypeptide has a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of human B7-1.

The invention further provides an isolated or recombinant nucleic acid comprising a polynucleotide sequence selected from: (a) a polynucleotide sequence selected from SEQ ID NOS:253 262, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NOS:263 272, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence which, but for codon degeneracy, hybridizes under highly stringent conditions over substantially the entire length of polynucleotide sequence (a) or (b) and encodes a polypeptide having a non naturally-occurring sequence; and (d) a polynucleotide sequence comprising all or a fragment of (a), (b), or (c), wherein the fragment encodes a polypeptide having (i) a non naturally-occurring sequence and (ii) a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of human B7-1, or an ability to induce a T-cell proliferation or activation response about equal to or less than that of hB7-1.

The invention also includes an isolated or recombinant nucleic acid comprising a polynucleotide sequence encoding a polypeptide that comprises an amino acid sequence which is substantially identical over at least about 150 contiguous amino acid residues of any one of SEQ ID NOS:69 92, 222 247, 263 272, and 286 289.

The invention also provides an isolated or recombinant nucleic acid comprising a nucleotide sequence coding for a polypeptide comprising the amino acid sequence set forth in any of SEQ ID NOS:69 92, 222 247, 263 272, and 286 289, or a subsequence thereof, wherein the subsequence comprises at least one of the signal sequence, ECD, transmembrane domain, and cytoplasmic domain of said polypeptide, and the amino acid sequence or subsequence is a non naturally-occurring sequence.

In another aspect, the invention provides an isolated or recombinant nucleic acid encoding a polypeptide that has a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of human B7-1, or an ability to induce a T-cell proliferation and/or activation response about equal to or greater than that of hB7-1, produced by mutating or recombining at least one nucleic acids described above. Also included is an isolated or recombinant polypeptide comprising an amino acid sequence having the formula:

MGHTMKWGSLPPKRPCLWLSQLLVLTGLFYFCSGITPK-SVTKRVKETVM-X50-SCDY-X55-X56-STEELT- SLRIYWQKDSKMVL AILPGKVQVWPEYKNRTITD-MNDNPRIVILALRLSD-X113-GTYTCV-X120-QK-X123-X124-X125-- X126-G-X128-X129-X130-X131-EHL-X135-SV-X138-L-X140-IRADFPVPSITDIGHPAPNVK RIRCSASG-X170-FPEPRLAWMEDGEEL-NAVNTTV-X193-X194-X195-LDTELYSVSSELD-X209-N- -X211-TNNHSIVCLIKYGELSVSQIFPWSKPK QEPPIDQLPFWVI-X252-X253-VSGALVLTAVVLYCLACRHVAR (SEQ ID NO:290), or a subsequence thereof comprising the extracellular domain, wherein position X50 is Leu or Pro; position X55 is Asn or Ser; position X56 is Ala or Thr; position X113 is Ser or Lys; position X120 is Ile or Val; position X123 is Pro or deleted; position X124 is Val, Asn, or Asp; position X125 is Leu or Glu; position X126 is Lys or Asn; position X128 is Ala or Ser; position X129 is Tyr or Phe; position X130 is Lys or Arg; position X131 is Leu or Arg; position X135 is Ala or Thr; position X138 is Arg or Thr; position X140 is Met or Ser; position X170 is Asp or Gly; position X193 is Asp or is deleted; position X194 is Gln or is deleted; position X195 is Asp or is deleted; position X211 is Val or Ala; position X252 is Ile or Val; and position X253 is Leu or Pro. The polypeptide may comprise a sequence of any of SEQ ID NOS:59, 62, 180, 184, 188, 195, 196, 200, 201, 204, 211, 213, 219, and 291. Some such polypeptides have a CD28/CTLA-4 binding affinity ratio about equal to or greater than that of hB7-1, and/or an ability to induce T-cell proliferation and/or activation about equal to or greater than that induced by hB7-1.

Another feature of the invention is an isolated or recombinant polypeptide comprising an amino acid sequence according to the formula:

MGHTMKWG-X9-LPPKRPCLWLSQLLVLTGLFYFCSG-X35-TPKSVTKRV KETVMLSCDY-X55-TSTEELTSLRIYWQKDSKMVLAILPGKVQVW PEYKNRTITDMNDNPRIVILALR-X110-SDSGTYTCVIQKP-X124-LKGAYKLEHL-X135-SVRLMIRAD- FPVPTINDLGNPSPNIRRLICSTSGGFPRPHLYWLENG-X183-ELNATNTT-X192-SQDPETKLYMISSELD- FN-X211-TSN-X215-X216-X217-LCLVKYGDLTVSQ-X231-FYWQESKPTPSANQHLTWTIIIPVSAFG- ISVIIAVI LTCLTCRNAAIRRQRRENEV-X288-M-X290-SCSQSP (SEQ ID NO:292), or a subsequence thereof comprising the extracellular domain, wherein position X9 is Thr or Ser; position X35 is Ile or Thr; position X55 is Asn or Ser; position X110 is Leu or Pro; position X124 is Asp or Val; position X135 is Thr or Ala; position X183 is Lys or Glu; position X192 is Leu or Val; position X211 is Met or Thr; position X215 is His or is deleted; position X216 is Ser or is deleted; position X217 is Phe or is deleted; position X231 is Thr or Ser; position X288 is Lys or Glu; position X290 is Glu or Gln, and wherein said amino acid sequence is a non naturally-occurring sequence. Some such polypeptides have a CD28/CTLA-4 binding affinity ratio about equal to or greater than that of hB7-1, and/or an ability to induce T-cell proliferation and/or activation about equal to or greater than that induced by hB7-1.

The invention includes an isolated or recombinant polypeptide comprising the amino acid sequence of SEQ ID NO:93 or SEQ ID NO:94, or a subsequence thereof, wherein the subsequence comprises at least one of the signal sequence, ECD, transmembrane domain, and cytoplasmic domain of said polypeptide. Also provided is an isolated or recombinant nucleic acid comprising a polynucleotide sequence selected from: (a) a polynucleotide sequence selected from SEQ ID NO:46 or SEQ ID NO:47, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NO:93, SEQ ID NO:94, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence encoding a subsequence of a polypeptide selected from SEQ ID NO:93, SEQ ID NO:94, or a complementary polynucleotide sequence thereof, wherein the subsequence comprises at least one of: the signal sequence, extracellular domain, transmembrane domain, and cytoplasmic domain of the polypeptide.

In another aspect, the invention provides a polypeptide which is specifically bound by a polyclonal antisera raised against at least one antigen, the antigen comprising the polypeptide sequence selected from any of SEQ ID NOS:48 94, 174 252, 263 272, 283 293, or a fragment thereof, wherein the antisera is subtracted with one or more (and optionally all) polypeptides encoded by one or more of the sequences set forth at GenBank Nucleotide Accession Nos: A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950. Some such polypeptides have a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of human B7-1, and/or an ability to induce a T-cell proliferation or T-cell activation response about equal to or greater than that of hB7-1.

The invention further includes an antibody or antisera produced by administering any NCSM polypeptide described above to a mammal, which antibody specifically binds at least one antigen, the antigen comprising a polypeptide comprising at least one amino acid sequence of any of SEQ ID NOS:48 94, 174 252, 263 272, and 283 293, which antibody does not specifically bind to a polypeptide encoded by at least one (optionally all) of the sequences at GenBank Nucleotide Accession Nos: A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950.

The invention provides an antibody or antisera which specifically binds a polypeptide which comprises any sequence selected from any of SEQ ID NOS:48 94, 174 252, 263 272, and 283 293, wherein the antibody or antisera does not specifically bind to at least one (optionally all) polypeptide encoded by at least one of GenBank Nucleotide Accession Nos: A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950. The antibodies are, e.g., polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments, fragments produced by an Fab expression library, or the like.

In another aspect, the invention provides a nucleic acid which comprises a unique subsequence in a nucleic acid selected from SEQ ID NOS:1 47, 95 173, and 253 262, wherein the unique subsequence is unique as compared to at least one (optionally all) nucleic acid corresponding to any of GenBank Nucleotide Accession Nos.: A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950. The invention also includes a polypeptide which comprises a unique subsequence in a polypeptide selected from: SEQ ID NOS:48 94, 174 252, 263 272, and 283 293, wherein the unique subsequence is unique as compared to at least one (optionally all) polypeptide encoded by any of GenBank Nucleotide Accession Nos. shown above.

The invention includes a target nucleic acid which, but for nucleotide codon degeneracy, hybridizes under stringent conditions to a unique coding oligonucleotide that encodes a unique subsequence in a polypeptide selected from SEQ ID NOS:48 94, 174 252, 263 272, and 283 293, wherein the unique subsequence is unique as compared to at least one (optionally all) polypeptide encoded by any of GenBank Nucleot. Access. Nos.: A92749, A92750, AA983817, AB026121, AB030650, AB030651, AB038153, AF010465, AF065893, AF065894, AF065895, AF065896, AF079519, AF106824, AF106825, AF106828, AF106829, AF106830, AF106831, AF106832, AF106833, AF106834, AF203442, AF203443, AF216747, AF257653, AH004645, AH008762, AX000904, AX000905, D49843, L12586, L12587, M27533, M83073, M83074, M83075, M83077, NM005191, S74541, S74540, S74695, S74696, U05593, U10925, U19833, U19840, U26832, U33063, U33208, U57755, U88622, X60958, Y08823, and Y09950.

The invention also includes compositions comprising any polypeptide and/or polynucleotide described herein in an excipient, preferably a pharmaceutically acceptable excipient. In one aspect, the invention provides compositions comprising an isolated or recombinant NCSM polypeptide comprising the amino acid sequence SEQ ID NOS:48 68, 174 221, 283 285, 290 293, or a costimulatory fragment thereof, wherein said costimulatory fragment has a CD28/CTLA-4 binding affinity ratio about equal to or greater than the CD28/CTLA-4 binding affinity ratio of human B7-1, or an ability to induce a T-cell proliferation or activation response about equal to or greater than that of hB7-1, and a carrier or excipient. Compositions comprising an isolated or recombinant NCSM polypeptide comprising the amino acid sequence of SEQ ID NOS:69 92, 222 247, 263 272, 286 289, or a costimulatory fragment thereof, wherein said costimulatory fragment has a CTLA-4/CD28 binding affinity ratio about equal to or greater than the CTLA-4/CD28 binding affinity ratio of human B7-1, or an ability to induce a T-cell proliferation or activation response about equal to or less than that of hB7-1, and a carrier are also a feature of the invention.

Also included are isolated or recombinant polypeptides, each comprising an amino acid sequence corresponding to an extracellular domain, wherein said amino acid sequence has at least about 92%, 93%, 94%, 95% or more amino acid sequence identity to the amino acid sequence corresponding to the extracellular domain of SEQ ID NO:66, and wherein said polypeptide has a CD28/CTLA-4 binding affinity ratio greater than the CD28/CTLA-4 binding affinity ratio of human B7-1. Some such polypeptides may further comprise at least one further amino acid sequence corresponding to a signal peptide, transmembrane domain or a cytoplasmic domain.

The invention also includes isolated or recombinant polypeptide variants, each of which comprises an amino acid sequence that differs from the amino acid sequence of a primate B7-1, wherein the difference between the amino acid sequence of the variant and the amino acid sequence of the primate B7-1 comprises a different amino acid at position 65 other than alanine, wherein the position corresponds to the position in the amino acid sequence of human B7-1 of SEQ ID NO:278. The different amino acid may comprise His, Arg, Lys, Pro, Phe, or Trp, and the primate B7-1 may be hB7-1. Some such polypeptide variants have a CTLA-4/CD28 binding affinity ratio greater than that or hB7-1 or an ability to induce a T cell proliferation response less than that of hB7-1.

The invention also includes an isolated or recombinant nucleic acid comprising a polynucleotide sequence encoding a polypeptide, where the polypeptide comprises an amino acid sequence which is substantially identical over at least 175 contiguous amino acids of any one of those NCSM polypeptide sequences listed. In various embodiments, the encoded polypeptide comprises at least about 100, 150, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 220, 225, 230, 240, 250, 260, 265, 270, 275, or 285 or more contiguous amino acid residues or substantially identical variants of any one of the polypeptide sequences listed, or encoded by any nucleic acid listed. These polypeptides can exist separately or as components of one of more fusion proteins.

The invention also includes a cell comprising any nucleic acid described herein, or which expresses any polypeptide or nucleic acid noted herein. In one embodiment, the cell expresses a polypeptide encoded by the nucleic acids herein.

The invention also includes a vector comprising any nucleic acid of the invention. The vector can comprise a plasmid, a cosmid, a phage, or a virus or a virus-like particle (VLP) (or virus fragment); the vector can be, e.g., an expression vector, a cloning vector, a packaging vector, an integration vector, or the like. The invention also includes a cell transduced by the vector. The invention also includes compositions comprising any nucleic acid described herein, and an excipient, preferably a pharmaceutically acceptable excipient. Cells and transgenic animals that include any polypeptide or nucleic acid herein, e.g., produced by transduction of the vector, are also a feature of the invention.

The invention also includes compositions produced by digesting one or more of the nucleic acids described herein with a restriction endonuclease, an RNAse, or a DNAse; and, compositions produced by incubating one or more nucleic acids described herein in the presence of deoxyribonucleotide triphosphates and a nucleic acid polymerase, e.g., a thermostable polymerase.

The invention also includes compositions comprising two or more nucleic acids described herein. The composition may comprise a library of nucleic acids, where the library contains at least 5, 10, 20 or 50 or more nucleic acids.

In another aspect, the invention includes an isolated or recombinant polypeptide encoded by any nucleic acid described herein. In one embodiment, the polypeptide may comprise a sequence selected from any of SEQ ID NOS:48 94, 174 252, 263 272, and 283 293. These sequences and fragments thereof can be present separately or as components of larger proteins such as fusion proteins.

Any polypeptide described herein optionally can effect or alter an immune response, e.g., either induce or inhibit proliferation or activation of T cells. In other embodiments, any polypeptide described above can bind preferentially either CD28 or CTLA-4 or both CD28 and CTLA-4 as described herein. In other embodiments, any polypeptide described herein optionally can enhance or limit cytokine production as described herein. Nucleotides encoding any such polypeptides having these properties are also a feature of the invention.

In one class of embodiments, any polypeptide described herein may further include a secretion signal or localization signal sequence. e.g., a signal sequence, an organelle targeting sequence, a membrane localization sequence, and the like. Any polypeptide described herein may further include a sequence that facilitates purification, e.g., an epitope tag (such as, e.g., a FLAG.TM. epitope), a polyhistidine tag, a GST fusion, and the like. The polypeptide optionally includes a methionine at the N-terminus. Any polypeptide described herein optionally includes one or more modified amino acids, such as a glycosylated amino acid, a PEG-ylated amino acid, a farnesylated ammo acid, an acetylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, an acylated amino acid, or the like. Any polypeptide described herein further may be incorporated into a fusion protein, e.g., a fusion with an immunoglobulin (Ig) sequence.

Methods for producing the polypeptides of the invention are also included. One such method comprises introducing into a population of cells any NCSM nucleic acid described herein, which is operatively linked to a regulatory sequence effective to produce the encoded polypeptide, culturing the cells in a culture medium to produce the polypeptide, and isolating the polypeptide from the cells or from the culture medium. Another such method comprises introducing into a population of cells a recombinant expression vector comprising any NCSM nucleic acid described herein; administering the expression vector into a mammal; and isolating the polypeptide from the mammal or from a byproduct of the mammal.

The invention also includes a method of treating an autoimmune or allergic disorder in a subject in need of such treatment by administering to the subject an effective amount of any NCSM polypeptide (or polynucleotide or expression vector encoding such polypeptide) described herein. In various embodiments, the autoimmune disorder may be multiple sclerosis, rheumatoid arthritis, lupus erythematosus, type I diabetes, psoriasis and the like.

The invention also includes a method of enhancing or reducing an immune response in a subject, such as either by inducing or inhibiting T cell proliferation or activation, by administration of at least one NCSM polypeptide and/or NCSM polynucleotide described herein to a population of cells. The population of cells to which the nucleic acid or polypeptide is administered can be in vivo, ex vivo, or in vitro (e.g., cultured cells). The invention includes a method of inducing, modifying, or inhibiting T-cell proliferation, the method comprising contacting a population of T cells with a polypeptide of the invention, thereby inducing, modifying, or inhibiting, respectively, proliferation of the T cells (relative to the response generated by WT hB7-1). Polypeptides that induce such T cell proliferation include CD28BP polypeptides; polypeptides that inhibit T cell proliferation include the CTLA-4BP polypeptides and B7-1 and B7-2 polypeptide variants discussed herein.

The invention also includes, in a method of treating a disorder or medical condition treatable by administration of NCSM polypeptides (or fragments thereof) or NCSM polynucleotides (or fragments thereof) to a subject, an improvement comprising administering to the subject an effective amount of a polypeptide and/or nucleic acid (or fragments thereof) described herein. The disorder, disease, or medical condition treatable by administration of NCSM polypeptides and/or nucleic acids (or fragments thereof, including soluble NCSMs and fusion proteins and vectors encoding them) may be, but is not limited to, e.g., chronic disease, autoimmune disorder, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, type I diabetes, psoriasis, AIDS or AIDS-related complexes, allogeneic or xenogeneic grafts or transplants, a variety of cancers, viral and/or bacterial infections, or the like.

Also included is a method of therapeutic or prophylactic treatment of a disease or disorder in a subject in need of such treatment, comprising administering to the subject any NCSM polypeptide described herein and an immunogen specific for said disease or disorder, wherein the combined amount of polypeptide and immunogen is effective to prophylactically or therapeutically treat said disease or disorder.

In yet another aspect, the invention includes a method of enhancing, diminishing, modifying, or potentiating an immune response in a subject, comprising: directly administering to the subject a polynucleotide comprising any NCSM nucleic acid sequence described herein, operably linked to a promoter sequence that controls the expression of said nucleic acid sequence, said polynucleotide being present in an amount sufficient that uptake of said polynucleotide into one or more cells of the subject occurs and sufficient expression of said nucleic acid sequence results to produce an amount of a polypeptide effective to enhance, diminish, or modify an immune response.

In another aspect, the invention provides a method of modulating or altering a T-cell response specific to an antigen in a subject, the method comprising administering to the subject at least one polynucleotide sequence encoding a polypeptide comprising any of SEQ ID NOS:48 94, 174 252, 263 272 and 283 293 or fragment thereof, and a polynucleotide sequence encoding the antigen or antigenic fragment thereof, wherein each of the at least one polynucleotide sequences is expressed in the subject in an amount effective to modulate or alter a T cell response.

The invention also includes a method of modulating or altering an immune response in a subject, the method comprising introducing into cells of a tumor of the subject at least one polynucleotide sequence encoding a polypeptide comprising any of SEQ ID NOS:48 94, 174 252, 263 272 and 283 293 or fragment thereof, wherein the polypeptide or fragment thereof interacts with or binds to a T cell receptor when expressed in a subject, and wherein the at least one polynucleotide sequence is operably linked to a promoter for expression in the subject and is present in an amount sufficient that when expressed is effective to modulate or alter a T cell response.

In addition, the invention includes a vector comprising at least one polynucleotide sequence encoding a polypeptide comprising any of SEQ ID NOS:48 94, 174 252, 263 272 and 283 293 or fragment thereof, wherein the polypeptide or fragment thereof interacts with or binds to a T cell receptor when expressed in a subject, wherein the at least one polynucleotide sequence is operably linked to a promoter for expression in the subject and is present in an amount sufficient that when expressed is effective to modulate or alter a T cell response.

In another aspect, the invention provides vector comprising at least one polynucleotide sequence encoding a polypeptide comprising any of SEQ ID NOS:48 94, 174 252, 263 272 and 283 293 or fragment thereof, and a polynucleotide sequence encoding the antigen or antigenic fragment thereof, wherein the NCSM polypeptide or fragment thereof interacts with or binds to a T cell receptor when expressed in a subject, and wherein each of the at least one polynucleotide sequences is operably linked to a promoter for expression in the subject and is present in an amount sufficient that when expressed is effective to modulate or alter a T cell response.

In general, nucleic acids and proteins derived by mutation, recursive sequence recombination (RSR) or other alterations of the sequences herein are a feature of the invention. Similarly, those produced by recombination, including recursive sequence recombination, are a feature of the invention. Mutation and recombination methods using the nucleic acids described herein are a feature of the invention. For example, one method of the invention includes recombining one or more nucleic acids described herein with one or more additional nucleic acids (including, but not limited to those noted herein), the additional nucleic acid encoding a NCSM polypeptide, co-stimulatory homologue or subsequence thereof. The recombining steps are optionally performed in vivo, ex vivo, or in vitro. Also included in the invention are a recombinant nucleic acid produced by this method, a cell containing the recombinant nucleic acid, a nucleic acid library produced by this method comprising recombinant polynucleotides, and a population of cells containing the library comprising recombinant polynucleotides.

The invention also includes a method of designing or identifying agonists and antagonists of CD28 and CTLA-4 (which either enhance or inhibit signaling through CD28 or CTLA-4) based on the 3-dimensional structure of the polypeptides of the invention (e.g., SEQ ID NOS:48 94, 174 252, 263 272, and 283 293).

The invention also includes soluble polypeptides and proteins (including fusion polypeptides and proteins) and nucleic acids encoding such soluble polypeptides and proteins. The invention also includes the use of such polypeptides and proteins as therapeutics, prophylactics, and diagnostics in therapeutic treatment and/or prevention of a variety of diseases and conditions. Soluble polypeptides and proteins (and nucleic acids encoding them) include, e.g., extracellular domain (ECD) amino acid sequences of each NCSM (e.g., each CTLA-4 binding protein and CD28 binding protein) described herein (or fragments thereof) and nucleic acids encoding same, as well as constructs comprising, e.g., each of said ECD, or fragments thereof, with an Ig polypeptide sequence (or fragment or variant thereof) (and nucleotide sequences encoding same) as fusion proteins. Some such soluble polypeptides and proteins exhibit a CD28/CTLA-4 binding affinity ratio that is greater than that of hB7-1 and/or have an ability to induce a T cell proliferation response, in the presence of activated T cells, that is less than that induced by soluble hB7-1 polypeptide in the presence of activate T cells.

In another aspect, the invention provides a computer or computer readable medium comprising a database comprising a sequence record comprising one or more character strings corresponding to a nucleic acid or protein sequence selected from any of SEQ ID NOS:1 272 and 283 293. The invention further includes an integrated system comprising a computer or computer readable medium comprising a database comprising one or more sequence records, each comprising one or more character strings corresponding to a nucleic acid or protein sequence selected from any of SEQ ID NOS:1 272 and 283 293, the integrated system further comprising a user input interface allowing a user to selectively view one or more sequence records. Also provided are methods of using a computer system to present information pertaining to at least one of a plurality of sequence records stored in a database, said sequence records each comprising one or more character strings corresponding to any of SEQ ID NOS:1 272 and 283 293.
 


Claim 1 of 30 Claims

1. An isolated or recombinant polypeptide variant of an extracellular domain of a wild-type primate B7-1 comprising a polypeptide sequence that has at least 95% identity to the polypeptide sequence of the extracellular domain of the wild-type primate B7-1 and differs from the polypeptide sequence of the extracellular domain of the wild-type primate B7-1 by the substitution of an amino acid other than alanine at an amino acid residue position corresponding to position 65 of the polypeptide sequence of wild-type human B7-1 (SEQ ID NO:278), wherein said polypeptide variant has a CTLA-4/CD28 binding affinity ratio greater than the CTLA-4/CD28 binding affinity ratio of the extracellular domain of the wild-type primate B7-1.

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