EP1127124A2 - Nouvelles compositions et nouveaux procedes pour identifier par criblage des modulateurs d'activation des lymphocytes t et des lymphocytes b - Google Patents

Nouvelles compositions et nouveaux procedes pour identifier par criblage des modulateurs d'activation des lymphocytes t et des lymphocytes b

Info

Publication number
EP1127124A2
EP1127124A2 EP99958694A EP99958694A EP1127124A2 EP 1127124 A2 EP1127124 A2 EP 1127124A2 EP 99958694 A EP99958694 A EP 99958694A EP 99958694 A EP99958694 A EP 99958694A EP 1127124 A2 EP1127124 A2 EP 1127124A2
Authority
EP
European Patent Office
Prior art keywords
protein
nucleic acid
rasgrp
jest
lymphocyte activation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99958694A
Other languages
German (de)
English (en)
Inventor
David A. Ferrick
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rigel Pharmaceuticals Inc
Original Assignee
Rigel Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=22309999&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=EP1127124(A2) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Rigel Pharmaceuticals Inc filed Critical Rigel Pharmaceuticals Inc
Publication of EP1127124A2 publication Critical patent/EP1127124A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • the invention relates to proteins useful in T-cell and B-cell activation, and more particularly to a human SWAP70 homolog found in a T-cell library and a human RasGRP homolog, and their use in methods for identifying candidate agents which modulate these activities
  • Lymphocytes are the white blood cells responsible for the immune response Their characteristics account for the immune system's attributes of diversity, specificity, memory, and self/nonself recognition Lymphocytes, which constitute 20-40% of the body's white blood cells, circulate in the blood and lymph and are capable of migrating into the tissue spaces and lymphoid organs
  • the lymphocytes can be broadly subdivided on the basis of function and cell-membrane components into three populations B cells, T cells, and NK cells Of particular interest are B cells and T cells, and more particularly of interest is the activation of lymphocytes As used herein, lymphocytes refer to B cells and T cells
  • SWAP70 was originally identified as a B cell specific protein involved in B cell isotype switching (Borggrefe et al , J Biol , Chem , 17025-17035 (1998), incorporated herein in its entirety)
  • JEST also sometimes called T-SWAP
  • SWAP70 proteins involved in activation of T and B cells
  • RasGRP binds to JEST and SWAP70 (also sometimes called SWAP herein)
  • B and T cell activation signaling Also provided herein are methods for screening for modulators of B and T cell activation
  • a recombinant nucleic acid encoding a JEST protein that is at least about 85% identical to the ammo acid sequence depicted in Figure 2 is provided. Also provided is a recombinant nucleic encoding the ammo acid sequence depicted in Figure 2 Further provided herein is a recombinant nucleic acid which will hybridize under high stringency conditions to the nucleic acid sequence depicted in Figure 2 or its complement Moreover, a recombinant nucleic acid that is at least about 90% identical to the nucleic acid sequence depicted in Figure 2 is provided Also provided is a recombinant nucleic acid having the nucleic acid sequence depicted in Figure 2
  • a recombinant JEST protein is provided that is at least about 85% identical to the ammo acid sequence depicted in Figure 2
  • a JEST protein according comprising the ammo acid sequence of Figure 2
  • a JEST protein according encoded by a nucleic acid at least about 85% identical to the nucleic acid sequence depicted in Figure 2
  • a JEST protein is provided herein which is encoded by a nucleic acid that will hybridize under high stringency conditions to the nucleic acid sequence of Figure 2 or its complement
  • an isolated polypeptide which specifically binds to a JEST protein is provided
  • the polypeptide is an antibody, more preferably, a monoclonal antibody
  • a monoclonal antibody is provided that reduces or eliminates the biological function of JEST protein encoded by a nucleic acid that will hybridize under high stringency conditions to the nucleic acid of Figure 2 or its complement
  • the present invention further provides a recombinant nucleic acid encoding a human RasGRP protein that is at least about 85% identical to the am o acid sequence depicted in Figure 7B Moreover, a recombinant nucleic acid is provided which encodes the ammo acid sequence depicted in Figure 7B Also provided herein is a recombinant nucleic acid which will hybridize under high stringency conditions to the nucleic acid sequence depicted in Figure 7A or its complement Further provided is a recombinant nucleic acid that is at least about 90% identical to the nucleic acid sequence depicted in Figure 7A In another embodiment, a recombinant nucleic acid having the nucleic acid sequence depicted in Figure 7A is provided Also provided are the sequences depicted in Figures 6A, 6B, 6C and 6D, which each bind to SWAP70 In one embodiment, the sequences of Figures 6A-6D bind to JEST
  • a recombinant human RasGRP protein that is at least about 95% identical to the ammo acid sequence depicted in Figure 7B
  • a human RasGRP protein is provided which comprises the ammo acid sequence of Figure 7B
  • a human RasGRP protein is provided which is encoded by a nucleic acid at least about 85% identical to the nucleic acid sequence depicted in Figure 7A
  • a human RasGRP protein encoded by a nucleic acid that will hybridize under high stringency conditions to the nucleic acid sequence of Figure 7A or its complement
  • an isolated polypeptide which specifically binds to human RasGRP protein is provided in one embodiment, such a polypeptide is an antibody, preferably a monoclonal antibody In a preferred embodiment, a monoclonal antibody that reduces or eliminates the biological function of RasGRP protein encoded by a nucleic acid that will hybridize under high stringency conditions to the nucleic acid of Figure 7A or its complement is provided
  • expression vectors comprising the nucleic acids described herein
  • host cells comprising the nucleic acids and/or vectors described herein
  • a process for producing a human RasGRP protein or JEST comprises cultunng the host cells provided herein under conditions suitable for expression of a human RasGRP protein or JEST
  • a method for screening for a bioactive agent capable of binding to a JEST protein comprises combining a JEST protein and a candidate bioactive agent, and determining the binding of said candidate agent to said JEST protein
  • a method for screening for a bioactive agent capable of binding to a human RasGRP protein comprises combining a human RasGRP protein and a candidate bioactive agent, and determining the binding of said candidate agent to said human RasGRP protein
  • a method for screening for agents capable of interfering with the binding of a SWAP70 protein and RasGRP comprises combining a SWAP70 protein, a candidate bioactive agent and a RasGRP protein and determining the binding of said SWAP70 protein and said RasGRP protein
  • Also provided herein is a method for screening for agents capable of interfering with the binding of a
  • said method comprises combining a JEST protein, a candidate bioactive agent and a RasGRP protein and determining the binding of said JEST protein and said RasGRP protein
  • a method for screening for an bioactive agent capable of modulating the activity of JEST protein comprises adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding a JEST protein and determining the effect of the candidate bioactive agent JEST bioactivity including lymphocyte activation
  • Also provided herein is a method for screening for an bioactive agent capable of modulating the activity of human RasGRP protein comprising the steps of adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding a human RasGRP protein and determining the effect of the candidate bioactive agent on RasGRP bioactivity including T-cell and B- cell activation
  • the methods provided herein can be performed wherein a library of candidate bioactive agents are added to a plurality of cells comprising said recombinant nucleic acid
  • SWAP70, JEST or RasGRP is provided It is understood that any one of these proteins, can be used Said method comprises combining a nucleic acid encoding SWAP70, JEST or RasGRP and a nucleic acid encoding a candidate protein, wherein an identifiable marker is expressed wherein said candidate protein binds to said SWAP70, JEST or RasGRP
  • Figure 1 depicts a nucleic acid sequence (cDNA) which includes an embodiment of the coding sequence of human JEST The start codon begins at nucleotide 541 and the stop codon begins at nucleotide 2434 The start and stop codons are circled
  • Figures 2A-2B depict an embodiment of the coding sequence of human JEST wherein the ammo acid sequence translation is shown below the coding sequence
  • Figure 3 depicts a schematic of where human ESTs and human RasGRP fragments provided herein aline with rat RasGRP
  • Figures 4A-4B depict a nucleic acid sequence and ammo acid sequence, respectively, for SWAP70
  • FIGS 5A-5B depict an ammo acid alignment of SWAP70 and JEST, wherein the SWAP70 is shown above JEST such that SWAP70 is the query and JEST is the subject Specific parameters utilized for the generation of the alignment are also shown
  • Figures 6A-6D depict nucleic acid sequences of human RasGRP, wherein each of these nucleic acids encoded a product which binds with SWAP70
  • Figure 6A is shown as "swap70 14" in Figure 3
  • Figure 6B is shown as “swap70 36" in Figure 3
  • Figure 6C is shown as "swap70 52" in Figure 3
  • Figure 6D is shown as "swap70 55" in Figure 3
  • Figures 7A-7B depict a nucleic acid and ammo acid sequence, respectively, of a consensus sequence for human RasGRP which binds to SWAP70
  • the lymphocyte activation proteins are from vertebrates and more preferably from mammals, including rodents (rats, mice, hamsters, guinea pigs, etc ), primates, farm animals (including sheep, goats, pigs, cows, horses, etc) and in the most preferred embodiment, from humans
  • a lymphocyte activation protein of the present invention may be identified in several ways "Protein” in this sense includes proteins, polypeptides, and peptides
  • a lymphocyte activation protein may be initially identified by its association with a protein known to be involved in T-cell and B-cell activation
  • lymphocyte activation proteins bind to SWAP or JEST Lymphocyte activation proteins may be novel or may have been known in the art to exist, but not known to bind to SWAP or JEST or related to lymphocyte activation or lymphocyte activation proteins
  • Novel lymphocyte activation nucleic acids or lymphocyte activation proteins are initially identified by substantial nucleic acid and/or ammo acid sequence identity or similarity to the respective sequences shown in the figures Such sequence identity or similarity can be based upon the overall nucleic acid or ammo acid sequence
  • the invention provides nucleic acids encoding lymphocyte activation proteins, namely, those shown in the figures, and their homologues
  • Preferred embodiments include JEST and the human homologue of RasGRP
  • RasGRP guanyl nucleotide-releasmg protein for the small guanosme tnphosphatase Ras
  • RasGRP has been shown to activate Ras and cause transformation in fibroblasts (Ebinu et al , Science, 280 1082-1086 (1998), incorporated herein in its entirety)
  • Signaling of RasGRP was associated with its partitioning in the membrane fraction Based on its expression in neurons and ability to activate Ras, it may function to promote neuron differentiation, axonal growth and synaptic plasticity It is now appreciated as shown herein that RasGRP message is induced in activated T and B cells Moreover, the relationship between SWAP and RasGRP shown herein indicates a novel cell activation pathway having numerous homologs in different cell lineages
  • JEST has sequence homology to a human EST (AA306449) from a Jurkat T cell library The full length sequence was derived from an activated human peripheral blood T-B cell switching library and from a Jurkat cDNA library JEST has different expression pattern by Northern than SWAP, being most prominent in thymus, lymphoid organs, peripheral blood leukocytes and testis As provided herein, JEST is involved in T cell signaling as well as mediating those signaling events in part by binding to RasGRP
  • lymphocyte activation proteins include proteins which bind to SWAP or JEST, which are themselves considered lymphocyte activation proteins
  • RasGRP as a lymphocyte activation protein
  • human RasGRP is provided
  • a protein is a "lymphocyte activation protein" if the overall sequence identity of the protein sequence to any one of the ammo acid sequences shown in the figures is about or greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90% In some embodiments the sequence identity will be as high as about 93 to 95 or 98% It is understood that each sequence identification number provides an individual embodiment which can be selected individually or with any combination of members of the group Sequence identity will be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv Appl Math 2 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J Mol
  • PILEUP PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments It can also plot a tree showing the clustering relationships used to create the alignment PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J Mol Evol 35 351-360 (1987), the method is similar to that described by Higgins & Sharp CABIOS 5 151-153 (1989)
  • Useful PILEUP parameters including a default gap weight of 3 00, a default gap length weight of 0 10, and weighted end gaps
  • BLAST algorithm described in Altschul et al , J Mol Biol 215, 403-410, (1990) and Karlm et al , PNAS USA 90 5873-5787 (1993)
  • WU-BLAST-2 program was obtained from Altschul et al , Methods in Enzvmolo ⁇ v.
  • WU-BLAST-2 uses several search parameters, most of which are set to the default values
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched, however, the values may be adjusted to increase sensitivity
  • a % am o acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region
  • the "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored)
  • percent (%) nucleic acid sequence identity with respect to the coding sequence of the polypeptides identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the lymphocyte activation protein
  • a preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0 125, respectively.
  • the alignment may include the introduction of gaps in the sequences to be aligned
  • sequences which contain either more or fewer ammo acids than the protein shown in the Figures it is understood that the percentage of sequence identity will be determined based on the number of identical ammo acids in relation to the total number of ammo acids
  • sequence identity of sequences shorter than that shown in the Figures, as discussed below, will be determined using the number of ammo acids in the shorter sequence
  • Lymphocyte activation proteins of the present invention may be shorter or longer than the ammo acid sequences shown in the Figures
  • portions or fragments of the sequences provided herein are considered lymphocyte activation proteins if a) they share at least one antigenic epitope, b) have at least the indicated sequence identity, c) and preferably have lymphocyte activation biological activity, including binding to SWAP
  • the sequence is used diagnostically, that is, when the presence or absence of lymphocyte activation protein nucleic acid is determined, only the indicated sequence identity is required
  • the nucleic acids of the present invention may also be shorter or longer than the sequences in the Figures
  • the nucleic acid fragments include any portion of the nucleic acids provided herein which have a sequence not exactly previously identified, fragments having sequences with the indicated sequence identity to that portion not previously identified are provided in an embodiment herein
  • lymphocyte activation proteins can be made that are longer than those depicted in the Figures for example, by the addition of epitope or purification tags, the addition of other fusion sequences, etc
  • Lymphocyte activation proteins may also be identified as being encoded by lymphocyte activation nucleic acids
  • lymphocyte activation proteins are encoded by nucleic acids that will hybridize to any one of the complementary sequences to the sequences depicted in the figures
  • any of the nucleic acid sequences in the Figures can be utilized Hybridization conditions are further described below
  • the lymphocyte activation protein when the lymphocyte activation protein is to be used to generate antibodies, the lymphocyte activation protein must share at least one epitope or determinant with the full length protein shown in the figures
  • epitope or “determinant” herein is meant a portion of a protein which will generate and/or bind an antibody
  • the epitope is unique, that is, antibodies generated to a unique epitope show little or no cross-reactivity
  • antibody includes antibody fragments, as are known in the art, including
  • the antibodies to lymphocyte activation are capable of reducing or eliminating the biological function of lymphocyte activation proteins, as is described below That is, the addition of anti-lymphocyte activation antibodies (either polyclonal or preferably monoclonal) to lymphocyte activation (or cells containing these proteins) may reduce or eliminate the lymphocyte activation activity Generally, at least a 25% decrease in activity is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred
  • lymphocyte activation antibodies of the invention bind to lymphocyte activation proteins
  • the antibodies specifically bind to lymphocyte activation proteins
  • the nucleic acid sequence identity may be either lower or higher than that of the protein sequence
  • the sequence identity of the nucleic acid sequence as compared to the nucleic acid sequences of the figures is preferably greater than 65%, and preferably greater than 75%, more preferably greater than about 80%, particularly greater than about 85% and most preferably greater than 90%
  • the sequence identity will be as high as about 93 to 95 or 98%
  • a lymphocyte activation nucleic acid encodes a lymphocyte activation protein
  • a lymphocyte activation protein As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the lymphocyte activation proteins of the present invention Thus, having identified a particular ammo acid sequence, those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the ammo acid sequence of the lymphocyte activation protein
  • the nucleic acid is determined through hybridization studies
  • nucleic acids which hybridize under high stringency to the nucleic acid sequences which complements are shown in the figures and is considered a lymphocyte activation gene
  • High stringency conditions are known in the art, see for example Maniatis et al , Molecular Cloning A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed Ausubel, et al , both of which are hereby incorporated by reference
  • Stringent conditions are sequence-dependent and will be different in different circumstances Longer sequences hybridize specifically at higher temperatures
  • An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993)
  • stringent conditions are selected to be about 5-10°C lower than the thermal melting point (TJ for the specific sequence at a defined ionic strength pH The TJ
  • less stringent hybridization conditions are used, for example, moderate or low stringency conditions may be used, as are known in the art, see Maniatis and Ausubel, supra, and Tijssen, supra
  • nucleic acid may refer to either DNA or RNA, or molecules which contain both deoxy- and nbonucleotides
  • nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids
  • nucleic acids may also contain modifications in the ⁇ bose-phosphate backbone to increase stability and half life of such molecules in physiological environments
  • the nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence
  • the depiction of a single strand also defines the sequence of the other strand (“Crick")
  • the sequences depicted in the Figures also include the complement of the sequence
  • recombinant nucleic acid herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature
  • an isolated lymphocyte activation nucleic acid, in a linear form, or an expression vector formed in vitro by ligatmg DNA molecules that are not normally joined are both considered recombinant for the purposes of this invention It is understood that once a recombinant nucleic acid is made and remtroduced into a host cell or organism, it will replicate non-recombmantly, i e using the
  • a "recombinant protein” is a protein made using recombinant techniques, i e through the expression of a recombinant nucleic acid as depicted above
  • a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics
  • the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure
  • an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0 5%, more preferably at least about 5% by weight of the total protein in a given sample
  • a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred
  • the definition includes the production of a lymphocyte activation protein from one organism in a different organism or host cell Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a mducible
  • lymphocyte activation proteins of the present invention are ammo acid sequence variants These variants fall into one or more of three classes substitutional, msertional or deletional variants These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the lymphocyte activation protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above
  • variant lymphocyte activation protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques
  • Ammo acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or mterspecies variation of the lymphocyte activation protein ammo acid sequence
  • the variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below
  • the mutation per se need not be predetermined
  • random mutagenesis may be conducted at the target codon or region and the expressed lymphocyte activation variants screened for the optimal combination of desired activity
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis Screening of the mutants is done using assays of lymphocyte activation protein activities
  • Ammo acid substitutions are typically of single residues, insertions usually will be on the order of from about 1 to 20 ammo acids, although considerably larger insertions may be tolerated Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger
  • substitutions deletions, insertions or any combination thereof may be used to arrive at a final derivative Generally these changes are done on a few ammo acids to minimize the alteration of the molecule However, larger changes may be tolerated in certain circumstances When small alterations in the characteristics of the lymphocyte activation protein are desired, substitutions are generally made in accordance with the following chart
  • substitutions are less conservative than those shown in Chart I
  • substitutions may be made which more significantly affect the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain
  • substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e g seryl or threonyl, is substituted for (or by) a hydrophobic residue, e g leucyl, isoleucyl, phenylalanyl, valyl or alanyl, (b) a cysteme or prolme is substituted for (or by) any other residue, (c) a residue having an electropositive side chain, e g lysyl, argmyl, or histidyl, is substituted for (a) a hydrophilic residue, e g se
  • variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the lymphocyte activation proteins as needed Alternatively, the variant may be designed such that the biological activity of the lymphocyte activation protein is altered For example, glycosylation sites may be altered or removed.
  • mutations in the JEST binding domain are made so as to modulate binding characteristics
  • Covalent modifications of lymphocyte activation polypeptides are included within the scope of this invention
  • One type of covalent modification includes reacting targeted ammo acid residues of a lymphocyte activation polypeptide with an organic de ⁇ vatizing agent that is capable of reacting with selected side chains or the N-or C-termmal residues of a lymphocyte activation polypeptide
  • Denvatization with bifunctional agents is useful, for instance, for crosslinkmg lymphocyte activation to a water-insoluble support matrix or surface for use in the method for purifying anti-lymphocyte activation antibodies or screening assays, as is more fully described below
  • Commonly used cross nking agents include, e g , 1 ,1-b ⁇ s(d ⁇ azoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxy- succ imide esters, for example, esters with 4-az ⁇ dosal ⁇ cyl ⁇ c acid, homobifunctional imidoesters, including disuccinimidyl esters
  • Another type of covalent modification of a lymphocyte activation polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence lymphocyte activation polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence lymphocyte activation polypeptide
  • lymphocyte activation polypeptides may be accomplished by altering the ammo acid sequence thereof The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonme residues to the native sequence lymphocyte activation polypeptide (for 0-l ⁇ nked glycosylation sites)
  • a lymphocyte activation ammo acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding a lymphocyte activation polypeptide at preselected bases such that codons are generated that will translate into the desired ammo acids
  • Another means of increasing the number of carbohydrate moieties on a lymphocyte activation polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide Such methods are described in the art, e g , in WO 87/05330 published 11 September 1987, and in Aplin and W ⁇ ston,
  • Removal of carbohydrate moieties present on a lymphocyte activation polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for ammo acid residues that serve as targets for glycosylation
  • Chemical deglycosylation techniques are known m the art and described, for instance, by Hakimuddin, et al , Arch Biochem Biophvs . 259 52 (1987) and by Edge et al , Anal Biochem .
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al , Meth Enzvmol . 138 350 (1987)
  • lymphocyte activation polypeptide comprises linking a lymphocyte activation polypeptide to one of a variety of nonprotemaceous polymers, e g , polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U S Patent Nos 4,640,835, 4,496,689, 4,301 ,144, 4,670,417, 4,791 ,192 or 4,179,337
  • Lymphocyte activation polypeptides of the present invention may also be modified in a way to form chime ⁇ c molecules comprising a lymphocyte activation polypeptide fused to another, heterologous polypeptide or ammo acid sequence
  • a chime ⁇ c molecule comprises a fusion of a lymphocyte activation polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind
  • the epitope tag is generally placed at the amino-or carboxyl-terminus of a lymphocyte activation polypeptide
  • the presence of such epitope-tagged forms of a lymphocyte activation polypeptide can be detected using an antibody against the tag polypeptide
  • the epitope tag enables a lymphocyte activation polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag
  • the chime ⁇ c molecule may comprise a fusion of a lymphocyte activation polypeptide with an immunoglobulin or a particular region of an immunoglobulin
  • a fusion could be to the Fc region of an IgG molecule as discussed further below
  • tag polypeptides and their respective antibodies are well known in the art Examples include poly-histidme (poly-his) or poly-histidine-glycme (poly-his-gly) tags, the flu HA tag polypeptide and its antibody 12CA5 [Field et al , Mol Cell Biol . 8 2159-2165 (1988)], the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al , Molecular and Cellular Biology. 5 3610-3616
  • Tag polypeptides include the Flag-peptide [Hopp et al , BioTechnolo ⁇ v. 6 1204-1210 (1988)], the KT3 epitope peptide [Martin et al , Science. 255 192-194 (1992)], tubu n epitope peptide [Skinner et al , J Biol Chem . 266 15163-15166 (1991 )], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl Acad Sci USA. 8_Z 6393-6397 (1990)]
  • lymphocyte activation proteins of the lymphocyte activation family and lymphocyte activation proteins from other organisms are cloned and expressed as outlined below
  • probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related lymphocyte activation proteins from humans or other organisms
  • particularly useful probe and/or PCR primer sequences include the unique areas of a lymphocyte activation nucleic acid sequence
  • preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosme as needed
  • the conditions for the PCR reaction are well known in the art It is therefore also understood that provided along with the sequences listed herein are portions of those sequences, wherein unique portions of 15 nucleotides or more are particularly preferred The skilled artisan can routinely synthesize or cut a nucleotide sequence to the desired length
  • a lymphocyte activation nucleic acid Once a lymphocyte activation nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombmed to form an entire full length or mature lymphocyte activation nucleic acid Wherein the full length nucleic acid has a signal peptide and/or transmembrane reg ⁇ on(s), it can be modified to exclude one or more of these regions so as to encode a peptide in its mature soluble form
  • the recombinant lymphocyte activation nucleic acid can be further-used as a probe to identify and isolate other lymphocyte activation nucleic acids It can also be used as a "precursor" nucleic acid to make modified or variant lymphocyte activation nucleic acids and proteins
  • the overlapping port ⁇ on(s) of one of the overlapping nucleic acids can be cloned and, if necessary, its constituent parts recombmed to form an entire full length or mature lymphocyte activation nucleic acid Wherein the full length nucleic acid
  • the expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome
  • these expression vectors include transc ⁇ ptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding a lymphocyte activation protein
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ⁇ bosome binding site
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers
  • Nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as
  • transcnptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ⁇ bosomal binding sites, transcnptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences
  • the regulatory sequences include a promoter and transcnptional start and stop sequences
  • Promoter sequences encode either constitutive or mducible promoters
  • the promoters may be either naturally occurring promoters or hybrid promoters Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention
  • the expression vector may comprise additional elements
  • the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification
  • the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct
  • the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector Constructs for integrating vectors are well known in the art Preferred methods to effect homologous recombination are described in PCT US93/03868 and PCT US98/05223, hereby incorporated by reference
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells Selection genes are well known in the art and will vary
  • a preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference
  • the lymphocyte activation proteins of the present invention are produced by cultunng a host cell transformed with an expression vector containing nucleic acid encoding a lymphocyte activation protein, under the appropriate conditions to induce or cause expression of a lymphocyte activation protein
  • the conditions appropriate for lymphocyte activation protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation
  • the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction
  • the timing of the harvest is important
  • the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield
  • Appropriate host cells include yeast, bacteria, archebactena, fungi, and insect and animal cells, including mammalian cells Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E coll, Bacillus subti s, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid and lymphoid cell lines, Jurkat cells, neuronal cells, and those from the spleen, thymus, prostate, testes, uterus, colon, small intestine, PBL, lymph nodes, bone marrow and liver, and all cells involved in the immune system
  • the lymphocyte activation proteins are expressed in mammalian cells
  • Mammalian expression systems are also known in the art, and include retroviral systems
  • a mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for lymphocyte activation protein into mRNA
  • a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site
  • a mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation
  • an upstream promoter element determines the rate at which transcription is initiated and can act in either orientation
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence The 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation Examples of transcription terminator and polyadenlytion signals include those derived form SV40
  • lymphocyte activation proteins are expressed in bacterial systems
  • Bacterial expression systems are well known in the art
  • a suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of lymphocyte activation protein into mRNA
  • a bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site
  • Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan Promoters from bactenophage may also be used and are known in the art
  • synthetic promoters and hybrid promoters are also useful, for example, the tac promoter is a hybrid of the trp and lac promoter sequences
  • a bacterial promoter can include naturally occurring promoters of non- bacterial origin that have the ability
  • the nbosome binding site is called the Shine-Delgamo (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream of the initiation codon
  • the expression vector may also include a signal peptide sequence that provides for secretion of a lymphocyte activation protein in bacteria
  • the signal sequence typically encodes a signal peptide comprised of hydrophobic ammo acids which direct the secretion of the protein from the cell, as is well known in the art
  • the protein is either secreted into the growth media (gram-positive bacteria) or into the pe ⁇ plasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria)
  • the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillm, chloramphenicol, erythromycin, kanamycm, neomycin and tetracyclme Selectable markers also include biosynthetic genes, such as those in the histidme, tryptophan and leucine biosynthetic pathways
  • Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E coli, Streptococcus cremons, and
  • Streptococcus lividans among others
  • the bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others
  • lymphocyte activation proteins are produced in insect cells
  • Expression vectors for the transformation of insect cells and in particular, baculovirus-based expression vectors, are well known in the art
  • lymphocyte activation proteins are produced in yeast cells
  • Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K lactis, Pichia guille ⁇ mondii and P pastons, Schizosaccharomyces pombe, and Yarrowia lipolytica
  • Preferred promoter sequences for expression in yeast include the inducible GAL1.10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokmase, phosphofructokmase, 3-phosphoglycerate mutase, pyruvate kmase, and the acid phosphatase gene
  • Yeast selectable markers include ADE2, HIS4, LEU2, TRP1 , and ALG7, which confers resistance to tunicamycin, the neomycin phosphotransferase gene, which confers resistance to G418, and the CUP1 gene, which allows yeast to grow in the presence of copper ions
  • a lymphocyte activation protein may also be made as a fusion protein, using techniques well known in the art Thus, for example, for the creation of monoclonal antibodies, if the desired epitope is small, the lymphocyte activation protein may be fused to a carrier protein to form an immunogen
  • a lymphocyte activation protein may be made as a fusion protein to increase expression, or for other reasons
  • a lymphocyte activation protein is a lymphocyte activation peptide
  • the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes
  • lymphocyte activation proteins of the invention an be linked to protein labels, such as green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), etc
  • the lymphocyte activation nucleic acids, proteins and antibodies of the invention are labeled
  • labeled herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound
  • labels fall into three classes a) isotopic labels, which may be radioactive or heavy isotopes, b) immune labels, which may be antibodies or antigens, and c) colored or fluorescent dyes
  • isotopic labels which may be radioactive or heavy isotopes
  • immune labels which may be antibodies or antigens
  • colored or fluorescent dyes The labels may be incorporated into the compound at any position
  • a lymphocyte activation protein is purified or isolated after expression lymphocyte activation proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusmg
  • a lymphocyte activation protein may be purified using a standard anti-lymphocyte activation antibody column Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful For general guidance in suitable purification techniques, see Scopes, R , Protein Purification, Sp ⁇ nger-Verlag, NY (1982) The degree of purification necessary will vary depending on the use of the lymphocyte activation protein In some instances no purification will be necessary
  • lymphocyte activation proteins and nucleic acids are useful in a number of applications
  • the nucleotide sequences (or their complement) encoding lymphocyte activation proteins have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA lymphocyte activation protein nucleic acids will also be useful for the preparation of lymphocyte activation protein polypeptides by the recombinant techniques described herein
  • a full-length native sequence lymphocyte activation protein gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate a full-length lymphocyte activation protein gene or to isolate still other genes (for instance, those encoding naturally-occurring variants of a lymphocyte activation protein or a lymphocyte activation protein from other species) which have a desired sequence identity to a lymphocyte activation protein coding sequence
  • the length of the probes will be about 20 to about 50 bases
  • the hybridization probes may be derived from the nucleotide sequences herein or from genomic sequences including promoters, enhancer elements and introns of native sequences as provided herein
  • a screening method will comprise isolating the coding region of a lymphocyte activation protein gene using the known DNA sequence to synthesize a selected probe of about 40 bases
  • Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 32 P or 35 S, or enzymatic labels such as al
  • the probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related lymphocyte activation protein coding sequences
  • Nucleotide sequences encoding a lymphocyte activation protein can also be used to construct hybridization probes for mapping the gene which encodes that lymphocyte activation protein and for the genetic analysis of individuals with genetic disorders
  • the nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries
  • Nucleic acids which encode lymphocyte activation proteins or their modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents
  • a non-human transgenic animal e g , a mouse or rat
  • a non-human transgenic animal is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e g , an embryonic stage
  • a transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops
  • cDNA encoding a lymphocyte activation protein can be used to clone genomic DNA encoding a lymphocyte activation protein in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express the desired DNA Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U S Patent Nos
  • non-human homologues of a lymphocyte activation protein can be used to construct a lymphocyte activation protein "knock out" animal which has a defective or altered gene encoding a lymphocyte activation protein as a result of homologous recombination between the endogenous gene encoding a lymphocyte activation protein and altered genomic DNA encoding a lymphocyte activation protein introduced into an embryonic cell of the animal
  • cDNA encoding a lymphocyte activation protein can be used to clone genomic DNA encoding a lymphocyte activation protein in accordance with established techniques
  • a portion of the genomic DNA encoding a lymphocyte activation protein can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration
  • several kiiobases of unaltered flanking DNA are included in the vector [see e g , Thomas and Capecchi, Cell.
  • the vector is introduced into an embryonic stem cell line (e g , by electroporation) and cells in which the introduced DNA has homologously recombmed with the endogenous DNA are selected [see e g , Li et al , CeJJ, 69 915
  • the selected cells are then injected into a blastocyst of an animal (e g , a mouse or rat) to form aggregation chimeras [see e g , Bradley, in Teratocarcinomas and Embryonic Stem Cells A Practical Approach, E J Robertson, ed (IRL, Oxford, 1987), pp 113-152]
  • a chime ⁇ c embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal
  • Progeny harboring the homologously recombmed DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombmed DNA Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of a lymphocyte activation protein polypeptide
  • Nucleic acids encoding lymphocyte activation polypeptides, antagonists or agonists may also be used in gene therapy In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo It has already been shown that short antisense oligonucieotides can be imported into cells where they act as inhibitors, despite their low mtracellular concentrations caused by their restricted uptake by the cell membrane (Zamecnik et al . Proc Natl Acad Sci USA 83. 4143-4146 [19861) The oligonucieotides can be modified to enhance their uptake, e g by substituting their negatively charged
  • nucleic acid source there are a variety of techniques available for introducing nucleic acids into viable cells The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc
  • the currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protem- liposome mediated transfection (Dzau et al , Trends in Biotechnology 11 , 205-210 [1993])
  • an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc
  • proteins which bind to a cell surface membrane protein associated with endocytosis may
  • lymphocyte activation proteins for review of gene marking and gene therapy protocols see Anderson et al , Science 256. 808-813 (1992) in a preferred embodiment, the lymphocyte activation proteins, nucleic acids, modified proteins and cells containing the native or modified lymphocyte activation proteins are used in screening assays Identification of this important T-cell and B-cell activation protein permits the design of drug screening assays for compounds that modulate lymphocyte activation activity
  • Screens may be designed to first find candidate agents that can bind to lymphocyte activation proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate lymphocyte activation activity
  • assays there are a number of different assays which may be run, binding assays and activity assays
  • the methods comprise combining a lymphocyte activation protein and a candidate bioactive agent, and determining the binding of the candidate agent to the lymphocyte activation protein
  • Preferred embodiments utilize a human lymphocyte activation protein, although other mammalian proteins may also be used, including rodents (mice, rats, hamsters, guinea pigs, etc ), farm animals (cows, sheep, pigs, horses, etc ) and primates These latter embodiments may be preferred in the development of animal models of human disease
  • variant or derivative lymphocyte activation proteins may be used, including deletion lymphocyte activation proteins as outlined above.
  • candidate bioactive agent or “exogeneous compound” as used herein describes any molecule, e g , protein, o gopeptide, small organic molecule, polysaccha ⁇ de, polynucleotide, etc , with the capability of directly or indirectly altering the bioactivity of Lymphocyte activation protein Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations Typically, one of these concentrations serves as a negative control, i e , at zero concentration or below the level of detection
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups
  • Candidate agents are also found among biomolecules including peptides, saccha ⁇ des, fatty acids, steroids, punnes, py ⁇ midines, derivatives, structural analogs or combinations thereof Particularly preferred are peptides
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucieotides Alternatively, libraries of
  • the candidate bioactive agents are proteins
  • protein herein is meant at least two covalently attached ammo acids, which includes proteins, polypeptides, o gopeptides and peptides
  • the protein may be made up of naturally occurring ammo acids and peptide bonds, or synthetic peptidomimetic structures
  • “ammo acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic ammo acids
  • homo-phenylalanme, citrulline and noreleucme are considered ammo acids for the purposes of the invention
  • Ammo acid also includes imino acid residues such as prolme and hydroxyprolme
  • the side chains may be in either the (R) or the (S) configuration
  • the ammo acids are in the (S) or L-configuration If non-naturally occurring side chains are used, non-ammo acid substituents may be used, for example to prevent or retard in vivo degradations
  • the candidate bioactive agents are naturally occuring proteins or fragments of naturally occuring proteins
  • cellular extracts containing proteins, or random or directed digests of prote aceous cellular extracts may be used
  • libraries of procaryotic and eucaryotic proteins may be made for screening against Lymphocyte activation protein
  • Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred
  • the candidate bioactive agents are peptides of from about 5 to about 30 ammo acids, with from about 5 to about 20 ammo acids being preferred, and from about 7 to about 15 being particularly preferred
  • the peptides may be digests of naturally occuring proteins as is outlined above, random peptides, or "biased” random peptides
  • randomized or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and ammo acids, respectively Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or ammo acid at any position
  • the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents
  • the library is fully randomized, with no sequence preferences or constants at any position
  • the library is biased That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities
  • the nucleotides or ammo acid residues are randomized within a defined class, for example, of hydrophobic ammo acids, hydrophilic residues, stencally biased (either small or large) residues, towards the creation of cystemes for cross-linking, prolmes for SH-3 domains, se ⁇ nes, threon es, tyrosmes or histid es for phosphorylation sites, etc , or to purines, etc
  • the candidate bioactive agents are nucleic acids By “nucleic acid” or
  • nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al , Tetrahedron 49(10) 1925 (1993) and references therein, Letsmger, J Org Chem 35 3800 (1970), Sblul et al , Eur J
  • nucleic acid candidate bioactive agents may be naturally occuring nucleic acids, random nucleic acids, or "biased" random nucleic acids
  • digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins
  • the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature
  • lymphocyte activation proteins as defined herein
  • portions of lymphocyte activation proteins are utilized, in a preferred embodiment, portions having lymphocyte activation activity are used
  • the assays described herein may utilize either isolated lymphocyte activation proteins or cells comprising the lymphocyte activation proteins
  • the lymphocyte activation protein or the candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e g a microtiter plate, an array, etc )
  • insoluble assays can also be used, for example those which can be detected by fluorescent changes with binding, etc
  • the insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening
  • the surface of such supports may be solid or porous and of any convenient shape
  • suitable insoluble supports include microtiter plates, arrays, membranes and beads These are typically made of glass, plastic (e g , polystyrene), polysaccha ⁇ des, nylon or nitrocellulose, teflonTM, etc Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples In some cases magnetic beads and the like are included The particular manner of binding
  • BSA bovine serum albumin
  • casein casein or other innocuous protein or other moiety
  • the lymphocyte activation protein is bound to the support, and a candidate bioactive agent is added to the assay
  • the candidate agent is bound to the support and the lymphocyte activation protein is added
  • Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc Of particular interest are screening assays for agents that have a low toxicity for human cells
  • assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc ) and the like
  • the determination of the binding of the candidate bioactive agent to a lymphocyte activation protein may be done in a number of ways
  • the candidate bioactive agent is labelled, and binding determined directly
  • this may be done by attaching all or a portion of a lymphocyte activation protein to a solid support, adding a labelled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support
  • a labelled candidate agent for example a fluorescent label
  • label herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e g radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc
  • Specific binding molecules include pairs, such as biotin and streptavid , digoxm and antidigoxm etc
  • the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above
  • the label can directly or indirectly provide a detectable signal
  • the proteins may be labeled at tyrosine positions using 125 l, or with fluorophores Altematively, more than one component may be labeled with different labels, using 125 l for the proteins, for example, and a fluorophor for the candidate agents
  • the binding of the candidate bioactive agent is determined through the use of competitive binding assays
  • the competitor is a binding moiety known to bind to the target molecule (lymphocyte activation molecule), such as an antibody, peptide, binding partner, gand, etc
  • the competitor is SWAP or JEST
  • there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent This assay can be used to determine candidate agents which interfere with binding between lymphocyte activation proteins and SWAP or JEST
  • the candidate bioactive agent is labeled Either the candidate bioactive agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40°C Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening Typically between 0 1 and 1 hour will be sufficient Excess reagent is generally removed or washed away The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding
  • the competitor is added first, followed by the candidate bioactive agent Displacement of the competitor is an indication that the candidate bioactive agent is binding to the lymphocyte activation protein and thus is capable of binding to, and potentially modulating, the activity of the lymphocyte activation protein
  • either component can be labeled
  • the presence of label in the wash solution indicates displacement by the agent
  • the candidate bioactive agent is labeled
  • the presence of the label on the support indicates displacement
  • the candidate bioactive agent is added first, with incubation and washing, followed by the competitor
  • the absence of binding by the competitor may indicate that the bioactive agent is bound to the lymphocyte activation protein with a higher affinity
  • the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the lymphocyte activation proteins
  • the methods comprise combining a lymphocyte activation protein and a competitor in a first sample
  • a second sample comprises a candidate bioactive agent, a lymphocyte activation protein and a competitor
  • the binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the lymphocyte activation protein and potentially modulating its activity That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to
  • a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native lymphocyte activation protein, but cannot bind to modified lymphocyte activation proteins
  • the structure of the lymphocyte activation protein may be modeled, and used in rational drug design to synthesize agents that interact with that site
  • Drug candidates that affect lymphocyte activation bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein
  • Positive controls and negative controls may be used in the assays
  • all control and test samples are performed in at least triplicate to obtain statistically significant results
  • Incubation of all samples is for a time sufficient for the binding of the agent to the protein Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined
  • the samples may be counted in a scintillation counter to determine the amount of bound compound
  • reagents may be included in the screening assays These include reagents like salts, neutral proteins, e g albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc , may be used The mixture of components may be added in any order that provides for the requisite binding
  • kits can be based on the use of the protein and/or the nucleic acid encoding the lymphocyte activation proteins Assays regarding the use of nucleic acids are further described below Screening for agents that modulate the activity of lymphocyte activation may also be done.
  • methods for screening for a bioactive agent capable of modulating the activity of lymphocyte activation comprise the steps of adding a candidate bioactive agent to a sample of lymphocyte activation protein, as above, and determining an alteration in the biological activity of lymphocyte activation protein "Modulating the activity of Lymphocyte activation protein" includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present
  • the candidate agent should both bind to lymphocyte activation (although this may not be necessary), and alter its biological or biochemical activity as defined herein
  • the methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the
  • the methods comprise combining a lymphocyte activation sample and a candidate bioactive agent, and evaluating the effect on T-cell and B-cell activation
  • lymphocyte activation activity or grammatical equivalents herein is meant one of lymphocyte activation protein's biological activities, including, but not limited to, its ability to affect T-cell and B-cell activation
  • One activity herein is the capability to bind to SWAP or JEST
  • the activity of the lymphocyte activation protein is increased, in another preferred embodiment, the activity of the lymphocyte activation protein is decreased.
  • bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments
  • the invention provides methods for screening for bioactive agents capable of modulating the activity of a lymphocyte activation protein
  • the methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising lymphocyte activation proteins
  • Preferred cell types include almost any cell
  • the cells contain a recombinant nucleic acid that encodes a lymphocyte activation protein
  • a library of candidate agents are tested on a plurality of cells
  • the assays include exposing the cells to an T-cell and B-cell activation agent that will induce T-cell and B-cell activation in control cells, i e cells of the same type but that do not contain the exogeneous nucleic acid encoding an activation protein
  • the cells may be exposed to conditions that normally result in T-cell and B-cell activation, and changes in the normal T- cell and B-cell activation progression are determined
  • the cells into which the lymphocyte activation nucleic acids are introduced normally under T-cell and B-cell activation, and thus changes (for example, inhibition of T-cell and B-cell activation) are determined
  • the cells normally do not undergo T-cell and B-cell activation, and the introduction of a candidate agent causes T-cell and B-cell activation
  • T-cell and B-cell activation may be done as will be appreciated by those in the art
  • these agents can be used as an affinity ligand, and attached to a solid support such as a bead, a surface, etc and used to pull out cells that are undergoing T-cell and B-cell activation
  • these agents can be coupled to a fluorescent dye such as PerCP, and then used as the basis of a fluorescent-activated cell sorting (FACS) separation
  • the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described
  • the agents may be administered in a variety of ways, orally, parenterally e g , subcutaneously, intrape ⁇ toneally, intravascularly, etc Depending upon the manner of introduction, the compounds may be formulated in a variety of ways
  • the concentration of therapeutically active compound in the formulation may vary from about 0 1-100 wt %
  • compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like
  • Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds
  • Diluents known to the art include aqueous media, vegetable and animal oils and fats Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents
  • the invention provides methods for identifying cells containing variant lymphocyte activation genes comprising determining all or part of the sequence of at least one endogeneous lymphocyte activation genes in a cell As will be appreciated by those in the art, this may be done using any number of sequencing techniques
  • the invention provides methods of identifying the lymphocyte activation genotype of an individual comprising determining all or part of the sequence of at least one lymphocyte activation gene of the individual This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue The method may include comparing the sequence of the sequenced lymphocyte activation gene to a known lymphocyte activation gene, i e a wild-type gene
  • the sequence of all or part of the lymphocyte activation gene can then be compared to the sequence of a known lymphocyte activation gene to determine if any differences exist This can be done using any number of known sequence identity programs, such as Bestfit, etc and others outlined herein.
  • the presence of a difference in the sequence between the lymphocyte activation gene of the patient and the known lymphocyte activation gene is indicative of a disease state or a propensity for a disease state, as outlined herein
  • lymphocyte activation proteins and particularly lymphocyte activation fragments, are useful in the study or treatment of conditions which are mediated by T-cell and B-cell activation, i e to diagnose, treat or prevent T-cell and B-cell activation-mediated disorders
  • T-cell and B-cell activation mediated disorders or "disease state” include conditions involving both insufficient or excessive T-ceil and B-cell activation Immunological disorders are numerous and known in the art
  • the methods comprise administering to a cell an anti-lymphocyte activation antibody or other agent identified herein or by the methods provided herein, that reduces or eliminates the biological activity of the endogeneous lymphocyte activation protein
  • the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a lymphocyte activation protein or modulator including anti-sense nucleic acids
  • the activity of lymphocyte activation is increased by increasing the amount of lymphocyte activation in the cell, for example by overexpressing the endogeneous lymphocyte activation or by administering a gene encoding an Lymphocyte activation protein, using known gene-therapy techniques, for example
  • the gene therapy techniques include the incorporation of the exogeneous gene using enhanced homologous recombination (EHR), for example as
  • the invention provides methods for diagnosing an T-cell and B-cell activation related condition in an individual
  • the methods comprise measuring the activity of lymphocyte activation in a tissue from the individual or patient, which may include a measurement of the amount or specific activity of Lymphocyte activation protein This activity is compared to the activity of lymphocyte activation from either a unaffected second individual or from an unaffected tissue from the first individual When these activities are different, the first individual may be at risk for an T-cell and B-cell activation mediated disorder
  • the proteins and nucleic acids provided herein can also be used for screening purposes wherein the protein-protein interactions of the lymphocyte activation proteins can be identified Genetic systems have been described to detect protein-protein interactions The first work was done in yeast systems, namely the "yeast two-hybrid" system The basic system requires a protein-protein interaction in order to turn on transcription of a reporter gene Subsequent work was done in mammalian cells See
  • two nucleic acids are transformed into a cell, where one is a "bait” such as the gene encoding SWAP, JEST or a portion thereof, and the other encodes a test candidate Only if the two expression products bind to one another will an indicator, such as a fluorescent protein, be expressed Expression of the indicator indicates when a test candidate binds to SWAP or JEST and can be identified as a lymphocyte activation protein Using the same system and the identified lymphocyte activation proteins the reverse can be performed Namely, the lymphocyte activation proteins provided herein can be used to identify new baits, or agents which interact with lymphocyte activation proteins Additionally, the two-hybrid system can be used wherein a test candidate is added in addition to SWAP or JEST and the lymphocyte activation protein encoding nucleic acids to determine agents which interfere with the bait, such as SWAP or JEST, and the lymphocyte activation protein RasGRP can also be used
  • a mammalian two-hybrid system provides post- translational modifications of proteins which may contribute significantly to their ability to interact
  • a mammalian two-hybrid system can be used in a wide variety of mammalian cell types to mimic the regulation, induction, processing, etc of specific proteins within a particular cell type
  • proteins involved in a disease state such as those described above could be tested in the relevant disease cells
  • for testing of random proteins assaying them under the relevant cellular conditions will give the highest positive results
  • the mammalian cells can be tested under a variety of experimental conditions that may affect mtracellular protein-protein interactions, such as in the presence of hormones, drugs, growth factors and cytokmes, cellular and chemical stimuli, etc , that may contribute to conditions which can effect protein-protein interactions, particularly those involved in T-cell and B-cell activation
  • the activity assays such as having an effect on T-cell and B-cell activation can be performed to confirm the acitivity of lymphocyte activation proteins which have already been identified by their sequence identity/similarity or binding to SWAP, JEST or RasGRP as well as to further confirm the activity of lead compounds identified as modulators of T-cell and B-cell activation
  • the lymphocyte activation proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to lymphocyte activation proteins, which are useful as described herein Similarly, the lymphocyte activation proteins can be coupled, using standard technology, to affinity chromatography columns These columns may then be used to purify lymphocyte activation antibodies In a preferred embodiment, the antibodies are generated to epitopes unique to the lymphocyte activation protein, that is, the antibodies show little or no cross- reactivity to other proteins These antibodies find use in a number of applications For example, the lymphocyte activation antibodies may be coupled to standard affinity chromatography columns and used to purify lymphocyte activation proteins as further described below The antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the lymphocyte activation protein
  • the anti-lymphocyte activation protein antibodies may comprise polyclonal antibodies Methods of preparing polyclonal antibodies are known to the skilled artisan Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intrape ⁇ toneal injections
  • the immunizing agent may include the lymphocyte activation protein polypeptide or a fusion protein thereof It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanm, serum albumin, bovine thyroglobu n, and soybean trypsm inhibitor Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynom
  • the anti-lymphocyte activation protein antibodies may, alternatively, be monoclonal antibodies
  • Monoclonal antibodies may be prepared using hybndoma methods, such as those described by Kohler and Milstein, Nature. 256 495 (1975)
  • a hybndoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent
  • the lymphocytes may be immunized in vitro
  • the immunizing agent will typically include the lymphocyte activation protein polypeptide or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybndoma cell [Godmg, Monoclonal Antibodies Principles and Practice. Academic Press.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybndoma cell [Godmg, Monoclonal Antibodies Principles and Practice. Academic Press.
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin Usually, rat or mouse myeloma cell lines are employed
  • the hybndoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells
  • the parental cells lack the enzyme hypoxanthme guanme phospho ⁇ bosyl transferase (HGPRT or HPRT)
  • HGPRT or HPRT hypoxanthme guanme phospho ⁇ bosyl transferase
  • the culture medium for the hyb ⁇ domas typically will include hypoxanthme, aminopte ⁇ n, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium
  • More preferred immortalized cell lines are mu ⁇ ne myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J Immunol . 133 3001 (1984), Brodeur et al , Monoclonal Antibody Production Techniques and Applications Marcel Dekker, Inc , New York, (1987) pp 51-63]
  • the culture medium in which the hybndoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against lymphocyte activation protein
  • the binding specificity of monoclonal antibodies produced by the hybndoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme- linked immunosorbent assay (ELISA)
  • RIA radioimmunoassay
  • ELISA enzyme- linked immunosorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal Biochem . 107 220 (1980)
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Godmg, supra] Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium Alternatively, the hybndoma cells may be grown in vivo as ascites in a mammal
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U S Patent No 4,816,567
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , by using o gonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of mu ⁇ ne antibodies)
  • the hybndoma cells of the invention serve as a preferred source of such DNA
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous mu ⁇ ne sequences [U S Patent No 4,816,
  • the antibodies may be monovalent antibodies
  • Methods for preparing monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinkmg
  • the relevant cysteme residues are substituted with another ammo acid residue or are deleted so as to prevent crosslinkmg
  • the anti-lymphocyte activation protein antibodies of the invention may further comprise humanized antibodies or human antibodies Humanized forms of non-human (e g , mu ⁇ ne) antibodies are chime ⁇ c immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues
  • Humanized antibodies may also comprise residues which are found neither in the
  • a humanized antibody has one or more ammo acid residues introduced into it from a source which is non-human
  • These non-human ammo acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain Humanization can be essentially performed following the method of Winter and co-workers [Jones et al , Nature. 321 522-525 (1986), Riechmann et al , Nature, 332 323-327 (1988), Verhoeyen et al , Science.
  • humanized antibodies are chime ⁇ c antibodies (U S Patent No 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J Mol Biol . 227 381 (1991), Marks et ai , J Mol Biol . 222 581 (1991)]
  • the techniques of Cole et al and Boerner et al are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy. Alan R Liss, p 77 (1985) and Boerner et al , J Immunol .
  • human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e g , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire
  • transgenic animals e g , mice in which the endogenous immunoglobulin genes have been partially or completely inactivated
  • human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire
  • This approach is desc ⁇ bed, for example, in U S Patent Nos 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661 ,016, and in the following scientific publications Marks et al , Bio/Technology 10. 779-783 (1992), Lonberg et al . Nature 368 856-859 (1994), Morrison. Nature 368. 812
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens
  • one of the binding specificities is for the lymphocyte activation protein, the other one is for any other antigen, and preferably for a cell- surface protein or receptor or receptor subunit
  • bispecific antibodies are known in the art Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy- chain/hght-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature. 305 537-539 (1983)] Because of the random assortment of immunoglobulin heavy and light chains, these hyb ⁇ domas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure The purification of the correct molecule is usually accomplished by affinity chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al , EMBO J . 10 3655-3659 (1991)
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions It is preferred to have the first heavy-chain constant region (CH1 ) containing the site necessary for light-chain binding present in at least one of the fusions DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism
  • CH1 first heavy-chain constant region
  • Heteroconjugate antibodies are also within the scope of the present invention Heteroconjugate antibodies are composed of two covalently joined antibodies Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U S Patent No 4,676,980], and for treatment of HIV infection [WO 91/00360, WO 92/200373, EP 03089] It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinkmg agents For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond Examples of suitable reagents for this purpose include immothiolate and methyl-4-mercaptobutyr ⁇ m ⁇ date and those disclosed, for example, in U S Patent No 4,676,980
  • anti-lymphocyte activation protein antibodies of the invention have various utilities
  • anti-lymphocyte activation protein antibodies may be used in diagnostic assays for a lymphocyte activation protein, e g , detecting its expression in specific cells, tissues, or serum
  • diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies A Manual of Techniques, CRC Press, Inc (1987) pp 147-158]
  • the antibodies used in the diagnostic assays can be labeled with a detectable moiety
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal
  • the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or 125 l, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate,
  • Anti-Lymphocyte activation protein antibodies also are useful for the affinity purification of lymphocyte activation protein from recombinant cell culture or natural sources
  • the antibodies against lymphocyte activation protein are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art
  • the immobilized antibody then is contacted with a sample containing the lymphocyte activation protein to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the lymphocyte activation protein, which is bound to the immobilized antibody Finally, the support is washed with another suitable solvent that will release the lymphocyte activation protein from the antibody
  • the anti-lymphocyte activation protein antibodies may also be used in treatment
  • the genes encoding the antibodies are provided, such that the antibodies bind to and modulate the lymphocyte activation protein within the cell
  • a therapeutically effective dose of a lymphocyte activation protein, agonist or antagonist is administered to a patient
  • therapeutically effective dose herein is meant a dose that produces the effects for which it is administered
  • the exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques As is known in the art, adjustments for lymphocyte activation degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art
  • a "patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms Thus the methods are applicable to both human therapy and veterinary applications
  • the patient is a mammal, and in the most preferred embodiment the patient is human
  • lymphocyte activation protein, agonist or antagonist of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intrape ⁇ toneally, intramuscularly, mtrapulmonary, vagmally, rectally, or mtraocularly
  • the lymphocyte activation may be directly applied as a solution or spray
  • compositions of the present invention comprise a lymphocyte activation protein, agonist or antagonist in a form suitable for administration to a patient
  • the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as buffers
  • fillers such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des protéines pouvant être utilisées dans l'activation des lymphocytes T et des lymphocytes B, et plus particulièrement un homologue SWAP 70 humain découvert dans une bibliothèque de lymphocytes T, ainsi qu'un homologue RasGRP humain. Cette invention concerne également la liaison de RasGRP à SWAP70 et son homologue humain, ci-après dénommé JEST. Cette invention concerne enfin des procédés permettant d'identifier des modulateurs des protéines susmentionnées, ainsi que leurs interactions.
EP99958694A 1998-10-28 1999-10-28 Nouvelles compositions et nouveaux procedes pour identifier par criblage des modulateurs d'activation des lymphocytes t et des lymphocytes b Withdrawn EP1127124A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US10618798P 1998-10-28 1998-10-28
US106187P 1998-10-28
PCT/US1999/025333 WO2000026241A2 (fr) 1998-10-28 1999-10-28 Nouvelles compositions et nouveaux procedes pour identifier par criblage des modulateurs d'activation des lymphocytes t et des lymphocytes b

Publications (1)

Publication Number Publication Date
EP1127124A2 true EP1127124A2 (fr) 2001-08-29

Family

ID=22309999

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99958694A Withdrawn EP1127124A2 (fr) 1998-10-28 1999-10-28 Nouvelles compositions et nouveaux procedes pour identifier par criblage des modulateurs d'activation des lymphocytes t et des lymphocytes b

Country Status (5)

Country Link
EP (1) EP1127124A2 (fr)
JP (1) JP2003516712A (fr)
AU (1) AU766809B2 (fr)
CA (1) CA2348733A1 (fr)
WO (1) WO2000026241A2 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0790304A1 (fr) * 1995-12-21 1997-08-20 AMERSHAM INTERNATIONAL plc Améliorations relatives à des systèmes d'analyse
ID27813A (id) * 1998-01-28 2001-04-26 Corixa Corp Senyawa-senyawa untuk terapi dan diagnosa kanker paru-paru dan metoda untuk penggunaannya
WO2000024768A2 (fr) * 1998-10-23 2000-05-04 Massachusetts Institute Of Technology Genes integrant des trajets de transduction de signaux

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0026241A3 *

Also Published As

Publication number Publication date
WO2000026241A3 (fr) 2000-11-16
AU766809B2 (en) 2003-10-23
WO2000026241A9 (fr) 2001-08-02
CA2348733A1 (fr) 2000-05-11
AU1600400A (en) 2000-05-22
JP2003516712A (ja) 2003-05-20
WO2000026241A2 (fr) 2000-05-11

Similar Documents

Publication Publication Date Title
US6589725B1 (en) Tankyrase H, compositions involved in the cell cycle and methods of use
US6709839B1 (en) SYK-UBP proteins, compositions and methods of use
US7371537B2 (en) Modulators of B-lymphocyte activation, myosin-1F compositions and methods of use
US7420045B1 (en) IAPs associated cell cycle proteins, compositions and methods of use
AU766809B2 (en) Novel compositions and methods of screening for T-cell and B-cell activation modulators
US20060228761A1 (en) Novel RIP3 associated cell cycle proteins, compositions and methods of use
US6696263B1 (en) PCNA associated cell cycle proteins, compositions and methods of use
US6387658B1 (en) PCNA-associated cell cycle proteins, compositions and methods of use
CA2385879A1 (fr) Proteines du cycle cellulaire associees a la traf4, compositions et modalites d'utilisation
CA2385235A1 (fr) Proteines du cycle cellulaire associees a des genes points de controle, compositions et modes de mise en oeuvre

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010511

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

RIN1 Information on inventor provided before grant (corrected)

Inventor name: FERRICK, DAVID, A.

17Q First examination report despatched

Effective date: 20030908

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040119

RIN1 Information on inventor provided before grant (corrected)

Inventor name: FERRICK, DAVID, A.