WO2001030377A1 - Porcine b7-1 and antibodies thereto - Google Patents
Porcine b7-1 and antibodies thereto Download PDFInfo
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- WO2001030377A1 WO2001030377A1 PCT/US2000/029155 US0029155W WO0130377A1 WO 2001030377 A1 WO2001030377 A1 WO 2001030377A1 US 0029155 W US0029155 W US 0029155W WO 0130377 A1 WO0130377 A1 WO 0130377A1
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- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70532—B7 molecules, e.g. CD80, CD86
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- Soluble and transmembrane porcine B7-1 proteins, their amino acid sequences, DNA and cDNA, as well as antibodies reactive with the B7-1 proteins are ⁇ isclosed.
- T cells also called T lymphocytes
- T cells recognize foreign pathogens (such as bacteria, viruses, or parasites) , foreign cells tissues, and or organs, become activated and thereby initiate an immune response that results in elimination of the foreign agent from the body.
- T cell activation is not only dependent on antigen recognition, but also on engagement of costimulatory molecules found on antigen presenting cells (APCs) .
- APCs antigen presenting cells
- costimulatory signal that determines whether antigen recognition leads to T cell activation is that generated by the interaction of CD28 on the T cells with the B7 molecules on the APCs. It is known that both B7-1 (CD80) and B7-2 (CD86) molecules on APCs provide critical costimulatory signals in T cell activation through their binding with the CD28 molecule on the T cell, and, moreover, that antigens presented in the absence of such costimulatory signals results in T cell anergy.
- the genomic organization of botn human and mouse B7-1 molecules are known m the art.
- the B7-1 molecule is a 60 KD trans-membrane glycoprotem usually present on the surface of APCs, and having two ligands, CD28 (discussed a ⁇ ove) and CTLA-4. Interaction of B7 molecules with CTLA-4 is known to down-regulate T cell activation.
- PCT application number WO 92/00092 to Linsley et al . describes the B7 antigen as a ligand for the CD28 receptor on T cells.
- the application states that "the B7 antigen, or its fragments or derivatives are reacted with CD28 positive T cells to regulate T cell interactions with other cells . . . B7 antigen or CD28 receptor may be used to inhibit interaction of cells associated with these molecules, thereby regulating T cell responses.”
- U.S. Patent No. 5,869,050 to de Boer et al . describes the use of ant ⁇ -B7 and ant ⁇ -CD40 antibodies to treat allograft transplant rejection, graft versus host disease and rheumatoid arthritis, stating that " . . . ant ⁇ -B7 and ant ⁇ -CD40 antibodies . . . can be used to prevent or treat an antibody-mediated or immune system disease in a patient.”
- Porcine B7-1 DNA in the form of, for example, cDNA including transmembrane porcine coding sequences, nucleic acid molecules coding for a soluble form of the porcine B7-1 protein, as well as, both transmembrane and soluble porcine B7-1 proteins lac ing a transmembrane and cytoplasm c domain, have surprisingly been created.
- an isolated nucleic ac d molecule including a DNA encoding for a polypepticie having at least 80% identity (preferably at least 90% anct mere preferable at least 95%) sequence identity with porcine B7-1 protein is provi ⁇ ed.
- ant porcine 37-1 antibodies are provided.
- the term "antibodies” refers to 1) immunoglobulms produced in vivo; 2) those produced in vitro by a hybriaoma; 3) antigen binding fragments (e.g., Fab' preparations) of such immunoglobulms; and 4) recombinantly expressed antigen binding proteins (including chimeric immunoglobulms, bispecific immunoglobulms , heterocon ugate immunoglobulms, "humanized” immuncglobulins , single chain antibodies, antigen binding fragments thereof, and other recomb ant proteins containing antigen binding domains derived from immunoglobulms) .
- Antibodies that bind to porcine B7-1 molecules, but not to human B7-1 molecules are also provided.
- therapeutic agents and methods for their use in the prevention and/or treatment of porcine xenograft rejection are described. These agents contain the porcine proteins and/or the anti-porcine antibodies discussed hereinabove.
- therapeutic agents and methods for their use in the treatment of inflammatory diseases such as autoimmune diseases are described.
- These agents contain the soluble porcine proteins discussed herein.
- porcine cells, tissues and whole organs lacking the B7-1 molecule are provided.
- FIG. 1 Sequence comparisons between porcine and human B7-1.
- sB7-l Amino acid sequences of soluble porcine B7-1 (sB7-l), the transmembrane form of porcine B ⁇ -l (tmB7-l) and human 37-1 (hB7-i) were aligned based on ammo acid identity and structural similarity. Identical amino acids are denoted by asterisks, and gaps in the sequences are indicated by dashes.
- FIG. 1 Porcine B7-1 binding to the human receptors CD28 and CTLA-4.
- Human Jurkat cells that are known to express CD28 but net CTLA-4 were incubated with sB7-l tagged with histidine residues (sB7-l-His) followed by incubation with fluorescein isothior- cyanate (FITC) -labelled polyclonal IgG recognizing the histidine tag. Binding was determined by Flow cytometry (described in the Examples herein below) .
- Panel A depicts the binding of sB ⁇ -l-His to Jurkat cells that have been previously incubated with buffer alone (no Ab (antibodies) ) , with a functionally blocking anti-CD28 mAb cr with an irrelevant isotype matched mAb (Anti-CD59) .
- Panel B shows the binding of sB7-l following preincubaticn with buffer alone (no Ab) human CTLA-4Ig or an irrelevant isotype matched mAb (Anti-CD3) . The binding of the anti-histidine IgG in the absence of s37-l-His is shown in both panels (control) .
- sB7-l blocks human T cell activation by xenogeneic or allcgeneic cells. Mitomcycin-C treated porcine cr human stimulator cells were incubated with human responder cells in the presence or absence of sB7-l and levels of T cell activation were determined. Curves show thymidine incorporation by human T cells in response to either porcine (panel A) or human (panel 3) P5L in the presence of increasing concentrations of s ⁇ ⁇ -l (closed circles) .
- Bars represent absorbances in O.D. units cf CTLA-4-Ig binding to 37-1 in the presence of the various hybridoma supernatants.
- Other bars represent hybridoma supernatants that either inhibited cr augmented CTLA-4-Ig/B7-l binding.
- Anti-porcine B7-1 hybridoma supernatants modify interactions between porcine B7-1 and human CD28.
- the binding of sE7-l to human CD28 on the surface of Jurkat cells was assayed in the presence of anti-B7-l hybridoma supernatants.
- Bolded peaks i: each panel represent the binding of sB7-l to CD28 in the absence cf hybridoma supernatants. Peaks shown by solid lines represent background binding in the absence of sB7-l. Peaks indicated by dotted lines show the binding between sB7-l and CD28 in the presence of the various supernatants.
- FIG. 6 mAbs generated to porcine B7-1 and 37-2 inhibit human T cell activation by porcine stimulator cells .
- Mitomycin C-treated porcine stimulator cells (porcine peripheral blood lymphocytes; PBL) were incubated with human responder cells in the presence or absence of anti-porcine B7 mAbs and levels of T cell activation were determined.
- Curves show thymidine incorporation by human T cells in response to porcine PBL in the presence of increasing concentrations of anti-B7-l (closed circles) , anti-B7-2 (open cirles) or a combination of both antibodies (open triangles) .
- Single points indicate levels of T cell activation in the absence of anti-B7 mAbs (open square) , in the presence of human CTLA-4-
- Ig at 100 ⁇ g/ml (closed square) or in the presence of an isotype matched murine mAb at 100 ⁇ g/ml (closed triangle) .
- Figure 7 cDNA encoding soluble porcine 37-1 (s 37-1) .
- Capital letters represent the coding sequence while lower letters show 5 or 3' untranslated regions.
- Figure 8 cDNA encoding transmembrane porcine B7-1 1 (tmB7-l)
- Capital letters represent the coding sequence while lower letter; show 5' or 3' untranslated regions.
- Isolated nucleic acid molecules including sequences that are unique to the porcine genome are provided.
- the isolated nucleic acid molecules include sense sequences of contiguous nucleotides of the porcine sequences disclosed herein, for example in Figures 7 & 8. These sense sequences are unique to the porcine genome, and can be used as PCR primers or hybridization probes for the identi ication and/or isolation of the homologous porcine genes from porcine genomic DNA, and for the quantification of 37-1 message in porcine RNA samples. Antisense sequences of contiguous nucleotides complementary to such sense sequences are also required in order to practice PCR, and may also be used as hybridization probes. In order to be used for such purposes, the sequences of contiguous nucleotides must span a sufficient length.
- oligonucleotide length required for specific hybridization i.e., hybridization under conditions requiring an essentially perfect match of complementary nucleotides wherein the sequence of the probe can be expected to occur only once in the genome of the organism being probed
- this span is at least 14 nucleotides, and, preferably, at least 18 nucleotides. Because at least 2 PCR primers are generally required to carry out a PCR reaction, the specificity of the PCR reaction is greater than that of each cf the oligonucleotide primers used to drive the reaction.
- a cloned porcine cDNA molecule having a sequence of nucleotides unique to the porcine genome is provided.
- This cloned molecule is characterized by hybridizing specifically to an isolated nucleic acid molecule as described in the preceding paragraph. Specific hybridization is used to clone this DNA molecule.
- This cloning can be accomplished by several methods well known in the art, such as by PCR using porcine reverse-transcribed DNA templates, or by conventional screening cf phage or other libraries of porcine cDNA or genomic DNA, (e.g., by- plaque lift filter hybridization) .
- recombmant expression vectors which include synthetic or cDNA-derived DNA fragments encoding porcine B7-1 proteins are provided.
- the nuclecti ⁇ e sequences coding for porcine B7-1 proteins can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements fcr the transcription and translation of tne inserted protein-coding sequence.
- the necessary transcripticr.al and translational signals can also be supplied by the native gene and/or its flanking regions.
- Suitable host vector systems include, but are not limited to: mammalian cell systems infected with viral vectors (e . g .
- vaccinia virus adenovirus, retroviruses, etc.
- mammalian cell systems transfected with plasmids include mammalian cell systems transfected with plasmids; insect cell systems infected with viral vectors (e.g., baculovirus) ; microorganisms such as yeast containing yeast expression vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.
- yeast containing yeast expression vectors or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.
- Suitable bacterial expression vectors include a selectable marker and bacterial origin of replication derived from commercially available plasmids having genetic elements of the well-known cloning vector pBR322 (American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America; ATCC Accession No. 37017) . These pBR322 "backbone sections,” or functionally equivalent sequences, are combined with an appropriate promoter and the structural gene to be expressed.
- Suitable promoters include, but are not limited to, the lactose promoter system (Chang, et al . , Nature 275,, pp. 615. 1978) the tryptophan (trp) promoter; see Goeddel, et al . , Nucl Acids Res 8, pp. 4057 1980, and the tac promoter, cr a fusion between the tac and trp promoters referred to as the trc promoter; (see Maniatis, Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp.412; 1982).
- Preferred bacterial expression vectors include, but are not limited to, vector pSE420 (comimercialy available from Invitrogen Corporation, San Diego, California) . This vector harbors the trc promoter, the lacO operon, an anti-terminator sequence, the glO ribosome binding sequence, a translation terminator sequence, the laclq repressor, the ColEl origin of replication, and the ampicillin resistance gene.
- Recombinant porcine B7-1 proteins may also be expressed in suitable fungal hosts, such as yeast of the Saccharomyces genus such as S. cerevisiae. Fungi of other genera such as Asner ⁇ illus , Pichia or Kluyveromyces may also be employed.
- Fungal vectors will generally contain an origin cf replication from the 2 ⁇ m yeast piasmid or another autonomously replicating sequence (ARS) , a promoter, DNA encoding a porcine B7-1 molecule, sequences directing pclyadenylation and transcription termination, and a selectable marker gene.
- fungal vectors will include an origin of replication and selectable markers permitting transformation and selection in both ⁇ . coli and fungi.
- Suitable promoter systems in fungi include the promoters for metallothionein, 3-phosphoglycerate kinase, or other glycolytic enzymes such as enolase, hexokinase, pyruvate kinase, glucokinase, the giucose-repressible alcohol dehydrogenase promoter (ADH2), the constitutive promoter from the alcohol dehydrogenase gene, ADHl, and others (see, for example, Schena, et al . Meth Enzymol 194, pp.
- Secretion signals such as those directing the secretion of yeast ⁇ -factor or yeast invertase, can be incorporated into the fungal vector to promote secretion of soluble porcine B7-1 proteins into the fungal growth medium. (See Moir, et al . , Meth Enzymol 194, pp.491; 1991.)
- Preferred fungal expression vectors can be assembled using DNA sequences from pBR322 for selection and replication in bacteria, and fungal DNA sequences, including the ADHl promoter and the alcohol dehydrogenase ADHl termination sequence, as found in vector pAAH5 (Ammerer, 1983) .
- the ADHl promoter is effective in yeast in that ADHl mRNA is estimated to be 1 - 2% of total poly (A) RNA.
- Mammalian or insect cell culture systems can be employed to express recombinant porcine B7-1 proteins.
- Suitable baculovirus systems for production of heterolcgous proteins in insect cells are reviewed by Luckow, et al . , 3io/Technology 6, pp. 47. 1988
- Mammalian expression vectors may have non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the porcine B7-1 protein gene to be expressed, and other 5' or 3 ' flanking sequences such as a ribosome binding site, a polyadenylation sequence, splice donor and acceptor sites, and transcriptional termination sequences.
- the transcriptional and translational control sequences in mammalian expression vector systems to be used in transforming vertebrate cells may be provided by viral sources.
- viral sources for example, commonly used promoters and enhancers are derived from polyoma virus, adenovirus, Simian virus 40 (SV40) , and human cytomegalovirus . (CMV) , including the cytomegalcvirus immediate- early gene 1 promoter and enhancer.
- Suitable eukaryotic vectors for the expression of porcine B7-1 proteins include but are not limited to pAPEX-3(see Evans et al . Gene 84, pp. 135; 1989), and pcDNAI/Amp (commercially available from Invitrogen Corporation, San Diego, California) .
- a particularly preferred host cell for the expression of inserts in the pAPEX-3 vector is the human 293 EBNA cell line (commercially available from Invitrogen, San Diego, CA) .
- the pcDNAI/Amp expression vector contains the human CMV immediate-early gene I promoter and enhancer elements, the SV40 consensus intron donor and acceptor splice sequences, and the SV40 consensus polyadenylation signal.
- This vector also contains an SV40 origin of replication that permits episomal amplification in cells (e.g., COS cells, MOP8 cells, etc.) transformed with the SV40 large T antigen, and an ampicillin resistance gene for propagation and selection in bacterial hosts.
- cells e.g., COS cells, MOP8 cells, etc.
- an ampicillin resistance gene for propagation and selection in bacterial hosts.
- EF-1 alpha promoters or inducible promoter systems (using e.g., the tetracycline-dependent derepression mechanism or an ecdvsone-inducible expression system) are also suitable for use herein .
- inducible promoter systems using e.g., the tetracycline-dependent derepression mechanism or an ecdvsone-inducible expression system
- purified porcine B7-1 proteins are prepared by culturing suitable host/vector systems to express the recombinant translation products of the nucleic acid molecules of the present invention, which are then purified from the culture media or cell extracts of the host system (e.g., the bacterial, insect, fungal, or mammalian cells) .
- the host system e.g., the bacterial, insect, fungal, or mammalian cells
- Fermentation of fungi cr mammalian cells that express soluble porcine B7-1 proteins containing a histidine tag sequence comprising a string of at least 5 histidine residues in a row
- Such histidine tagged sequence enables binding, under specific conditions, to metals such as nickel, thus permitting efficient purification by passage over nickel Columbus.
- the purification of recombinant porcine B7-1 proteins is performed using a suitable set of concentration, fractionation, and chromatcgraphy steps well known in the art (see, for example, Deutscher, Guide to Protein Purifications, Vol. 182; Academic Press, Inc., San Diego Calif. 1990; and Harris and Angal, Protein Purification Methods: A Practical Approach. IRL Press Oxford University Press. Oxford, 1989). Denaturation of the purified protein, followed by refolding under reducing conditions, is then performed to enable proper disulfide bond formation.
- Porcine 37-1 proteins purified from bodily fluids of transgenic animals engineered to produce such proteins, as well as porcine B7-1 proteins that are produced in part cr entirely by in vitro methods are provided.
- porcine B7-1 proteins include, but are net limited to, 1) reagents for blocking T cell activation; 2) immunogens for the purpose of raising antibodies to porcine 57-1 proteins; and, 3) antigens for use in immunoassays to detect soluble porcine B7-1 proteins as markers of inflammation in primate recipients of porcine xenografts, discussed hereinbelow.
- antibodies reactive with porcine 37-1 proteins, but not human B7-1 proteins are provided.
- Suitable antibodies include, but are not limited to, polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.
- Such antibodies can be prepared by applying methods known in the art. See for example; Reichmann, et al . , Nature 332, pp. 323, 1988. Winter and Milstein, 1991; Clackson, et al., Nature 352, pp.624. 1991; Morrison, Annu Rev Immunol 10, pp. 239; 1992; Haber, Immunol Rev 130, pp. 189; 1992; and Rodrigues, et al . , J Immune1 151, pp. 6954; 1993.
- Polyclonal anti B7-1 antibodies are provided. Methods cf preparing such antibodies are known to one skilled in the art and the immunization protocol may be selected without undue experimentation. Suitable methods for raising polyclonal anti 37-1 antibodies in a mammal include injecting the mammal with an immunizing agent and optionally in the presence in the presence orabsence of an adjuvant. The regimen includes multiple subcutaneous or interperitoneal injections with the immunizing agent, such as the B7-1 protein or fragments thereof. It may be useful to conjugate the immunizing agent to a carrier known to be immunogenic in the mammal being immunized.
- Monoclonal anti 37-1 antibodies are provided. Monoclonal antibodies may be prepared by using methods to generate hvbridcmas such as those described in Kohler et al, Nature , 256:495 (1975; . Briefly, a mouse, hamster, cr other suitable host is immunized with an immunizing agent to elicit lymphocytes that produce cr are capable of producing antibodies that will bind to the lmmunizmq agent. The lymphocytes may also be activated to produce antibodies immunized in vitro. The lymphocytes are then fused to myeloma cells in vitro to immortalize the antibody-producing cells .
- Suitable hybridomas for producing such antibodies can be obtained using purified porcine B7-1 proteins as the immunizing agent followed by screening to identify hybridomas producing antibodies with the desired properties. Such screening can be carried out using suitable immunoassays such the ELISA described below and in copending U.S. patent application serial no.: 08/252,493, filed June 1, 1994, which is incorporated herein by- reference. A simple modification of this ELISA (i.e., substituting soluble human 37-1 proteins for soluble porcine 37-1 proteins) can be used to identify those of the hybridomas producing antibodies that bind to porcine B7-1 proteins but do net bind to human B7-1 proteins.
- Humanized anti B7-1 antibodies are provided.
- Humanized forms cf non-human (e.g., munne) antibodies are chimeric immunoglobulms, immunoglobulin chains or fragments thereof (such as Fv, scFv, Fab, Fab',F(ab'); cr other antigen- binding subsequences cf antibodies) which contain minimal secuence derived from non-human immunoglobulin.
- Humanized antibodies include human immunoglobulms (recipient antibody) in which residues from complementary determining regions (CDRs) of the recipient are replaced by residues from CDRs cf a non-human species (donor antibody) such as mouse, rat cr rabbit having the desired specificity, affinity and binding capacity.
- CDRs complementary determining regions
- donor antibody such as mouse, rat cr rabbit having the desired specificity, affinity and binding capacity.
- specific Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
- Humanized antibodies may also comprise residue
- a humanized antibody contains one or more amino acid residues that are introduced from a non-human antibody source. These non-human amino 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, 332:323-32 '7 (1988); Verhoeyen et al .
- 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 al. J. Mol. Biol., 222:581 (1991)].
- the techiniques 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. 147 (1) : 86-95 (1991) 1.
- human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, (e.g., mice) in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge with antigens, only human antibodies are produced in a manner similar to that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. See 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 .
- Polyspecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at leas: two different antigens. One cf the binding specificities may be for the B7-1 protein, while the other may be for any other antigen, cell-surface protein, 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/light-chain pairs, where the two heavy chains and/or the two light chains have different specificities [See Milstein and Cuello, Nature, 305:537-539 (1983)]. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in Traunecker et al . , EMBO J. 10:3655-3659 (1991).
- Heteroconjugate antibodies are provided. Heteroconjugate antibodies are composed of two covalently joined antibodies. Sue: antibodies have, for example, been proposed to link immune system cells to unwanted target cells to enable their rapid elimination [U.S. Patent No. 4, 676,980], and to treat HIV infection [WO 91/00360; WO 92/200373; EP 03089] . It is contemplated that the antibodies may be prepared in vi tro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructec using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
- Antibody glycosylation Porcine B7-1 proteins and anti-porcme 37-1 antibodies with or without associated native patterns of glycosylation are also provided. For example, expressing proteins recombinantly in bacteria such as E. coli provides non-glycosylated molecules, while expressing porcine 37-1 proteins or anti porcine B7-1 protein antibodies in mammalian cells can provide glycosylated molecules .
- Anti-porcine 37-1 antibodies or soluble B7-1 proteins can be used for the prevention or treatment of xenotrasplant rejection in patients suffering from that have received porcine cells or tissues.
- a combination of anti-porcine B7-1 and anti-porcine B7-2 antibodies can be used to inhibit host T cell activation.
- the therapeutic porcine B7-1 agents can be administered in a variety of unit dosage forms.
- the dose will vary according to the particular agent.
- antibodies prepared as Fab' cr F(ab')2 fragments are of considerably smaller mass than the equivalent intact immunoglobulins, and thus require lower dosages to reach the same molar levels in the patient's blood.
- Antibodies with different affirmatives will also differ in their regarded dosages.
- the dose will also vary depending on the manner cf administration, the particular symptoms of the patient being treated, the overall health, condition, size, and age of the patient, and the judgment of the prescribing physician.
- Dosage levels of the therapeutic porcine 37-1 agents for human subjects range between about 1 mg per kg and about 100 mg per kg per patient per treatment.
- the therapeutic porcine 37-1 agent concentrations are preferably in the range from about 25 ⁇ g/ml to about 500 ⁇ g/ml.
- a typical therapeutic treatment includes a series of doses, which are usually administered concurrent with the monitoring of clinical endpoints. These may include xenctransplant biopsies cr measures of organ function (e.g. for xenotransplanted kidneys, BUN creatines and, proteinuria levels, etc.), with treatment dosage levels adjusted as needed to achieve the desired clinical outcome.
- Therapeutic porcine B7-1 agents can be administered by- methods well known in the art, such as by bolus injection, intravenous delivery, continuous infusion, sustained release from implants, etc.
- the therapeutic porcine 37-1 agents may also be entrapped microcapsules (such as hydroxymethylcellulose cr gelatin-micrccapsules) ; liposomes; and other sustained-release matrices such as polyesters, hydrogels (for example, polyhydroxyethylmethacrylate or polyvinylalcohol) or mjectable microspheres cf biodegradeable materials, such as polymers and copolymers cf glycolide, lactide, and/or ethylene glycol.
- microcapsules such as hydroxymethylcellulose cr gelatin-micrccapsules
- liposomes such as hydroxymethylcellulose cr gelatin-micrccapsules
- other sustained-release matrices such as polyesters, hydrogels (for example, polyhydroxyethylmethacrylate or polyvinylalcohol) or mjectable microspheres cf biodegradeable materials, such as polymers and copolymers cf glycolide, lactide, and/or
- the therapeutic porcine B7-1 agents can be used in compositions to treat episodes of xenograft rejection, either alone cr in combination with known immunosuppressive agents such as cyclosporin A, FK506, rapamycin, corticosteroids, etc.
- Formulations suitable for injection are found in Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985) .
- Such formulations must be sterile and non- pyrogenic, and generally will include purified therapeutic porcine B7-1 agents in conjunction with a pharmaceutically effective carrier, such as saline, buffered (e.g., phosphate buffered) saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions, and the like.
- a pharmaceutically effective carrier such as saline, buffered (e.g., phosphate buffered) saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions, and the like.
- the formulations may contain pharmaceutically acceptable auxiliary substances as required, such as, tonicity adjusting agents, wetting agents, bactericidal agents, preservatives, stabilizers, and the like.
- the therapeutic porcine B7-1 agent is formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose, albumin) as diluents.
- excipient solutions e.g., sucrose, albumin
- the amount and frequency of administration will depend, of course, on such factors as the nature and severity of the rejection episode being treated, the desired response, the condition of the patient, and so forth.
- the formulations can be distributed in sterile form as articles of manufacture comprising packaging material and the therapeutic porcine B7-1 agents.
- the packaging material will include a label which indicates that the formulation s for use in the prevention or treatment of porcine xenctransplant rejection.
- Certain of the isolated nucleic acid molecules of the invention are also useful as to direct and/cr modulate the expression cf porcine E7-1 molecules in porcine cells, e.g., by altering the expression cf porcine B ⁇ -1 genes. Such modulation may be accomplished by the use of techniques well known in the art .
- a suitable method by which the expression of porcine B ⁇ -l proteins on transgenic pig cells can be inhibited (to thereby reduce a potential human immune response against transplanted transgenic cells or tissues) is by genetic manipulations referred to in the art as "gene disruption” or “gene knockout.”
- Gene knockout uses specially designed DNA molecules (gene knockout constructions) to achieve targeted inactivaticn (knockout) cf a particular gene, via homologous recombination,- upon introduction of the construct into a cell. See for example; Thomas, et al.,Cell 44(3), pp. 419; 1986; Thomas, et al.,Cell 51(3), pp. 503;1987; Jasin and Berg, Genes & Development 2, pp.
- Gene knockouts and gene replacements can be achieved in mammalian zygotes through techniques well known in the art, including, but not limited to, microinjection, of nuclei or pronuclei, electroporation of ova or zygotes, nuclear transplantation, and/or the stable transfection or transducticn of embryonic stem cells (see for example Brinster, et al . , Proc Natl Acad Sci 86. pp. 7087; 1989, U.S. Patent No. 5,032,407 to Wagner et al., PCT Publication WO90/08832, and preferably, PCT Publication WO 99/07829.)
- swine lacking B7-1 mclecules can be created by the genetic manipulation of embryonic stem cells (ES cells) as described in PCT Patent Publication No. WO 93/02188 and Robertson, 198 " , Teratocarcinomas and Embryonic Stem Cells a Practical Approach. IRL Press Eynsham, Oxford, England.
- ES cells are grown in vitro as described in, for example, Robertson, in Robertson, ed. "Teratocarcinomas and Embryonic Stem Cells a Practical Approach” IRL Press, Eynsham, Oxford, England 1987, and in U.S. Patent No. 5,166,065.
- Genetic material having a defective or altere ⁇ gene encoding porcine B7-1 is introduced into the embryonic stem cells by elec t roporation according, for example, to the method cf McMahon, et al . , Cell 62, pp. 1073; 1990. cr by transducticn with a retroviral vector according, for example, to the method cf Robertson, et al . , Nature 323, pp. 445; 1986 cr by any cf the various t echniques described by Lovell-Badge , Teratocarcinomas anc Embryonic Stem Cells a Practical Approach. IRL Press Eynsham, Oxford, England 1987.
- the selected cells then may be injected mt a blastocysts of an animal (e.g. a pig) to form aggregation chimeras (see for example, Teratocarcinomas and Embrionic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, pp 113 ; 1987) . a suitable pseu opregnant female foster animal and the embryo brought to term to create the "knockout" progency.
- an animal e.g. a pig
- aggregation chimeras see for example, Teratocarcinomas and Embrionic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, pp 113 ; 1987.
- mutant porcine B7-1 nucleotide sequences including nucleotide deletions, insertions, subs t itutions, and additions to the exon and/or intron regions cf the porcine B7-1 such that the resultant mutant does not encode a functional B7-1 protein.
- These nucleotide sequences may be utilized in homologous recombination techniques. In such techniques, mutant sequences are used in homologous recombination techniques. In such techniques, mutant sequences are recombined with wild type genomic sequences in stem cells, ova, or newly fertilized cells of about 1 to about 500 cells. Nucleotide sequences used in this homologous recombination may be in the form of isolated nucleic acid sequences or in the context cf vectors. Upon recombination, destruction of the functional gene takes place.
- Diagnostic use of the anti-porcine B7-1 antibodies include but are not limited to assaying the patient's blood for levels of one cr more porcine B7-1 proteins. Measurement cf porcine B7-1 protein levels may be accomplished by RIA, ELISA, or other suitable immunoassay using the anti-porcine E7-1 antibodies as detection reagents. General methods for performing such assays are set forth in Coligan, et al., Current Protocols in Immunol, John Wiley & Sons, New York 1992. Blood porcine B7-1 protein levels xeno intanspla t recipients must be monitored at regular intervals, e.g., daily cr weekly, and changes in such levels recorded.
- a significant increase in the levels of porcine B7-1 protein levels in the patient's bleed is an indication that the porcine tissue is becoming inflammed, and may indicate the onset of a rejection episode.
- An alternative test for rejection (cr a test confirming tha: rejection is occurring, as indicated by measurement cf soluble B7- 1 protein levels) may be obtained by monitoring porcine cell, tissue, or organ function or by biopsy and histopathological examination of the porcine cell, tissue or organ. Such examination will be carried out in order to detect the typical manifestations of transplant rejection such as, cellular infiltration, inflammation, and necrosis.
- An immunohistopathological examination of the biopsied materials will also be performed, using certain of the anti-porcine B7-1 antibodies to detect the levels of expression cf one or mere porcine B7-1 proteins on the surface cf the xenotransplanted cells. High levels of such expression (compared to levels on non- transplanted control tissue samples) are indicative of xenotransplant rejection.
- RNA was prepared from freshly isolated porcine peripheral blood leukocytes (PBL) using the acid/guanidinium thiocyanate technique as described in Chomczynski and Sacchi, 1987. Ten micrograms of total RNA were heated at 65°C for 3 minutes, quenched on ice, and subjected to first strand cDNA synthesis for (1 hour at 37°C) in the following 100 ⁇ l reaction mixture: 10 mM Tris-HCl (pH 8.3), 50 mM KCL 1.5 mM MgCl 2; 10 mM dithiothreotol, 0.20 mM of each dNTP, 0.5 ⁇ g oligo dT 16 and 20 Units of avian mveloblastosis virus reverse transcriptase, commercially available from Seikagaku Inc., Rockville, MD.
- PBL porcine peripheral blood leukocytes
- oligonucleotide primers were generated from regions of high homology between human and mouse B7-1 sequence: 1) 5' TGGCCCGAGTATAAGAACCGGAC3' and 2) 5' TCAGTTTCAGGATCTTGGGAAA 3'.
- PCR amplification was performed for 30 cycles (94°C for 1 min, 50°C for 1 min, 72°C for 1 min) followed by 1 cycle at 72°C for 10 min.
- the resulting 338 bp fragment was cloned into the pCR2.1 vector using the T/A cloning system (commercially available from Invitrogen, San Diego, CA) and identified by DNA sequence analysis as a porcine B7-1 homologue.
- Two gene-specific oligonucleotides were derived from the porcine B7-1 sequence and a 250 bp fragment was generated by PCR. This DNA fragment was used to screen a ⁇ gtlO porcine macrophage library (provided by Dr. Michael Murtaugh, Department of Veterinary PathoBiology, University of Minnesota).
- Membranes were hybridized at 60°C overnight and subsequently washed using the following conditions: two-30 min washes with 2x SSC/0.1% SDS at room temperature, one-30 min wash with 0.5x SSC/0.1% SDS at 50°C and one-30 min wash with 0.2x SSC/0.1% SDS at 60°C. Positive plaques present on duplicate filters were purified and the B7-1 DNA was recovered by PCR using primers that flanked the insertion site of the ⁇ gtlO vector. After cloning the PCR fragment into pCR2.1-TOPO. both strands of the putative full-length clone were sequenced using the chain termination method. Clones derived from different PCR reactions were also sequenced to rule out potential errors introduced during amplification. The DNA templates were primed with vector sequence primers flanking the multiple cloning site, or primers constructed from internal cDNA sequence.
- Clones containing the B7-1 related sequence were identified at a frequency of approximately one clone per 1 x 10 5 phage.
- DNA sequence analysis performed as described above revealed a full-length cDNA that lacked both the transmembrane and cytoplasmic domains normally found in B7-1. Therefore the clones represented a soluble form of B7-1 (sB7-l) and containing 1620 bp comprised of a 718 bp 5' untranslated region (UTR), a 215 bp 3'UTR and an open reading frame that encoded 229 amino acids.
- sB7-l soluble form of B7-1
- 1620 bp comprised of a 718 bp 5' untranslated region (UTR), a 215 bp 3'UTR and an open reading frame that encoded 229 amino acids.
- the transmembrane form of porcine B7-1 (tmB7-l) was isolated by RT-PCR of freshly isolated porcine lung RNA using an oligonucleotide from the 3' end of the sB7-l coding region as the 5' primer (GCTACCAACACGATGCTTTCC) and oligo dT ]6 as the 3 ' primer.
- RNA isolation and RT-PCR were otherwise identical to those described in Example I.
- the two major products resulting from the RT-PCR were cloned into pCR2.1-TOPO and inserts were sequenced for identification.
- tmB7-l One of the clones obtained through PCR (tmB7-l) contained the complete transmembrane domain coding region and most of the cytoplasmic domain coding region (based on comparison with B7- 1 from other species), but the translational stop site and 3' UTR were not present.
- the truncation of tmB7-l, and the lack of detection of tmB7-l in the oligo dT primed porcine macrophage library suggested strong 3' UTR secondary structure in these transcripts.
- porcine B7-1 and human B7-1 shared 65% sequence identity and an overall conservation of the Ig V-like and Ig C-like structural domains characteristic of other B7 molecules (Judge et al., 1995).
- the signal peptide for sB7-l was 29 amino acids in length as determined by amino terminal sequencing of purified protein. Amino acids that have been shown to be critical for the binding of B7- 1 to both CD28 and CTLA-4 (Guo et al., 1995) were highly conserved. A clone containing the complete coding region for tmB7-l was not found, but based on sequence comparison with various other species, the terminal amino acid is expexted to be very close to the translational stop site.
- sB7-l-His expression of sB7-l protein tagged with a carboxy-terminal histidine hexapeptide (sB7-l-His) was generated in the mammalian expression vector Apex3P (Evans et al., 1995) by PCR amplification of B7-1 cDNA in pCR2.1-TOPO.
- the 5' primer (CCGGGGATCCCTTCTGTTTTCATCCTCATCAAGC) was derived from the 5'UTR of B7-1 and ' contained a BamHI site for subcloning.
- the 3' primer (GGCCTGCAGGTCATCAATGGTGATGGTGATGGTGGCATTTTTGCCAGTTGAA GGTCTGTGAC) inserted the histidine tag just upstream of the stop codon and an Sse83371 subcloning site.
- 293-EBNA embryonic kidney cells (commercially available from Invitrogen, Carlsbad, CA) were transfected with sB7-l-His in Apex3P according to methods described by Evans et al., 1995.
- Cells were grown in D10 medium (DMEM containing 5% FCS, 2 mM glutamine, 100 IU/ml penicillin and 100 ⁇ /ml streptomycin) supplemented with puromycin at a final concentration of 1 ⁇ g/ml.
- D10 medium DMEM containing 5% FCS, 2 mM glutamine, 100 IU/ml penicillin and 100 ⁇ /ml streptomycin
- puromycin at a final concentration of 1 ⁇ g/ml.
- Cells that expressed sB7-l-His were cloned by limiting dilution, and those producing high levels of protein were chosen by standard Western immuno-blot analysis of cell supernatants using rabbit anti-histidine polyclonal IgG (coomercially available from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
- the sB7-l-His protein was purified by affinity chromatography using a nickel charged nitrilotriacetic acid (NT A) resin commercially available from Qiagen, Chatsworth, CA as previously described (see Mueller et al., 1995).
- NT A nickel charged nitrilotriacetic acid
- mAbs Monoclonal antibodies
- polyclonal antisera directed against porcine B7-1 were generated by repeated immunization of mice and rabbits, respectively, with sB7-l-His according to methods routinely performed at Cocalico, Inc., Reamstown, Pennsylvania.
- MAbs were produced by immune eymphoetes and immortal myeloma cells according to methods discribed by Kohler et al.. Nature 1975. Select clones were injected into mice in order to produce ascites using protocols already established and commercially available at Cocalico.
- MAb supernatants were collected from hybridomas that had been cloned from the original fusion.
- Anti-B7-1 specific IgG was purified from selected hybridoma super natants by passage over a Protein A-Sepharose column (commercially available from Pharmacia, Piscataway, NJ). This purified IgG was utilized to assess the effect of anti-porcine B7-1 mAbs on human xenogeneic MLRs. Total IgG preimmune rabbit serum, or from serum from B 7-1 -immunized rabbits was also purified by passage over a protein A-sepharose column. This polyclonal IgG was utilized to assess the cell surface expression of B 7-1 on porcine PBL.
- Freshly isolated or activated cell populations were pre-incubated in PBS containing 5% goat serum. Cells were then incubated with anti-porcine-B7-l or preimmune rabbit IgG, in combination with murine antibodies directed against porcine CD3, IgM, class II and B7-2 cell surface antigens, in PBS containing 2% goat serum. Cells were washed in the same and then reacted with fluorescein isothyocyanate (FITC)-labeled goat anti-mouse Ig and with phycoerythrin (PE)-labeled goat anti-rabbit Ig.
- FITC fluorescein isothyocyanate
- PE phycoerythrin
- Cell-surface B7-1 was detected on subpopulations of activated lymphocytes, defined by the expression of CD3 (T cells), IgM (B cells) and Class II and B7-2 (antigen presenting cells), by two-color immunofluorescence and flow cytometry as described in Materials and Methods.
- Example VII sB7-l binds human CD28 and CTLA4
- porcine adhesion and costimulatory molecules have been shown to be conserved across species, including humans. These include the ligands for porcine E-selectin, porcine VCAM-1 and porcine B7-2 (Matis et al, 1995; Maher et al., 1996). The fact that all amino acids shown to be critical for the binding of B7-1 to both CD28 and CTLA-4 are conserved in porcine B7-1 ( Figure 1, panel A), suggests that this molecule will interact with its human counterpart ligands.
- sB7-l-His was incubated with human Jurkat T cells (available from the American Type Culture Collection, Rockville Maryland, hereinafter "ATCC"; clone TIB 152) and the interaction was assayed by indirect immunoflourescence and flow cytometry as described in Example VI, using a polyclonal anti-porcine B7-1 IgG followed by an FITC conjugated goat anti rabbit IgG.
- Jurkat cells have been shown to express CD28 but not the alternative B7-1 ligand, CTLA-4 (Freeman et al., 1992).
- Example VIII sB7-l Inhibits Human T Cell IL-2 Production in Response to Costimulation by Porcine or Human Cells
- Efficient T cell activation requires both a primary signal through the T cell receptor and a secondary signal mediated through binding of B7-1 and B7-2 costimulatory molecules on APCs, with CD28 on the surface of T cells.
- Jurkat cells do not constitutively elaborate IL-2, even in the presence of PHA, which can provide an obligate primary signal through the TCR.
- PHA signaling in the presence of a source of APCs that provide a second signal, or in the presence of stimulatory anti-CD28 antibodies results in significant IL-2 production (Williams et al., 1992).
- porcine aortic endothelial cells PAEC porcine aortic endothelial cells PAEC
- Jurkat T cells (1 x 10 6 cells/well) were then added to the wells in the presence or absence of 10 ⁇ g/ml phytohemagglutinin (PHA; commercially available from Sigma) and increaseing concentrations of sB7-l, or humans CTLA-4Ig or porcine P-selectin-His.
- PHA phytohemagglutinin
- Raji cells (1 x 10 6 cells/well) were substituted for PAEC, but were added to the Jurkat cells at the initiation of the experiment.
- the cultures were maintained at 37°C in 5% CO 2 for 24 h, at which time the culture supernatants were harvested for IL-2 detection.
- Assays to detect and quantitate human IL-2 were performed using an ELISA kit (Quantikine Human IL-2 Immunoassay; commercially available from R&D Systems), according to the manufacturer's protocol. Briefly, serial dilutions of a recombinant human IL-2 standard (commercially available from R&D Systems) or culture supernatants were added, in duplicate, to ELISA wells that had been previously coated with a "capture" monoclonal antibody specific for human IL-2, and incubated overnight at 4°C.
- Bound IL-2 was revealed by incubation with a second IL-2-specific antibody conjugated with horseradish peroxidase (HRP), followed by extensive washing and addition of substrate (hydrogen peroxide and chromogen) to produce a colored product. The reaction was stopped by the addition of 2 N sulfuric acid. The optical density of each well was determined using a microtiter plate reader (commercially available from BioRad. Model 3550. Hercules, CA) set to 450 nm. with values corrected by subtraction of readings taken at 595 nm. IL-2 concentrations were calculated using Microplate Manager software (commercially available from BioRad).
- sB7-l can bind to human T cells and block their activation by costimulatory signals delivered by either allogeneic human cells (Raji) or xenogeneic porcine cells (PAEC). Since Jurkat cells do not express CTLA-4, it can be concluded that sB7-l inhibits activation by binding to CD28 and blocking signaling through that molecule.
- MLRs were performed in the presence of increasing amounts of sB7-l, an irrelevant histidine tagged protein or CTLA-4Ig.
- Cells were maintained for 4-5 days at 37°C in 5% C0 2 in air.
- [ ⁇ jthymidine (1.0 ⁇ Ci/well; commercially available from NEN Dupont, Boston. MA) was added to the cell cultures during the last 16-18 h of the incubation.
- the cells were harvested onto glass fiber filters with an automated sample harvester (Wallac; Turku, Finland) and the filters counted in a beta liquid scintillation counter. Results were expressed as mean counts per minute (cpm) of triplicate cultures.
- Hybridoma supernatants (generated at Cocalico Inc., Reamstown, PA) were diluted 1 :2 with 4 ⁇ g/ml human CTLA-4-Ig-biotin (commercially available from Ancell, Bayport, MN), at 4 ⁇ g/ml and added to the wells for 1 h at 37°C. Plates were washed 3x before the addition of streptavidin-horseradish peroxidase (commercially available from Pharmingen, San Diego, CA) at a 1 :1000 dilution. Finally, plates were washed 3x, developed with O-phenylenediamine dihydrochloride (commercially available from Sigma, St.
- a blocking assay was performed. Human Jurkat cells that are known to express CD28 but not CTLA-4 were incubated with sB7-l in the presence of hybridoma supernatants. First. sB7-l (2.5 ⁇ g/ml) was preincubated in HBSS containing either human CTLA-4Ig (20 ⁇ g/ml) or various anti-B7-l hybridoma supernatants (neat) or buffer alone before addition to Jurkat cells (2.5 x 10 D cells/reaction) for an additional incubation.
- Short-term primary human xenogeneic MLRs (as described in Example IX) were performed to assess the ability of anti-porcine-B7-l and B7-2 mAbs to block human T cell activation to response to by porcine stimulator cells.
- Human PBL were incubated with mitomycin-C treated porcine PBL and human T cell activation was determined.
- MLRs were performed in the presence of increasing amounts of anti-B7-l, anti-B7-2 or a combination of the two antibodies and responder cell proliferation was assessed on day 4- 5 of culture. The effect of an isotype matched murine mAb or CTLA-4-Ig on T cell activation was also measured.
- CD28-mediated signaling co-stimultes murine T cells and prevents induction of anergy in T cell clones. Nature 356:607.
- CTLA-4 ligation blocks CD28-dependent T cell activation. J. Exp. Med. 183:2541.
- VCAM Porcine vascular cell adhesion molecule
- Porcine aortic endothelial cells activate human T cells: direct presentation of MHC antigens and costimulation by ligands for human CD2 and CD28. Immunity 1 :57.
- CTLA-4 and CD28 mRNA are coexpressed in most T cells after activation: expression of CTLA-4 and CD28 mRNA does not correlate with the pattern of lymphokine production. J. Immunol. 149:3795.
- CTLA-4 is a second receptor for the B cell activation antigen B7. J. Exp. Med. 174:561.
- nucleotide sequences cf the porcine B7-1 protein-encoding nucleic acid molecules may be modified by creating nucleic acid mutations which include third nucleotide changes in degenerate codcns (and other "silent" mutations that do not change the encoded ami ⁇ c acid sequence) .
- nucleotide and amino acid sequences encoding naturally occurring allelic variants of the porcine B7-1 protein genes; sequences which have been truncated so as to only encode the mature porcine B7-1 protein pcIypeptides, (i.e., a porcine B7-1 polypeptide without the amino terminal leader sequence that directs the protein to its typical transmembrane orientation in the cell); sequences in which the B7-1 protein amino terminal leader sequences have been altered (e.g., substituted with a different leader sequence); sequences in which a peptide "tag" sequence has been inserted or added on to enable the ready identification and/or purification of recombinant proteins (such as the FLAG epitope (which enables specific binding to anti-FLAG antibodies) or a histidine tag sequence) ; and sequences that have been altered to produce a soluble porcine B7-1 protein by, for example, truncation.
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US5747034A (en) * | 1992-07-09 | 1998-05-05 | Chiron Corporation | Methods and materials for the induction of T cell anergy |
US5759550A (en) * | 1993-05-06 | 1998-06-02 | Pharmagenesis, Inc. | Method for suppressing xenograft rejection |
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US5759550A (en) * | 1993-05-06 | 1998-06-02 | Pharmagenesis, Inc. | Method for suppressing xenograft rejection |
Non-Patent Citations (5)
Title |
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AUCHINCLOSS ET AL.: "Xenogeneic Transplantation", ANNU. REV. IMMUNOL., vol. 16, 1998, pages 433 - 470, XP002938442 * |
BASTA ET AL.: "Modulation of Monocytic Cell Activity and Virus Susceptibility During Differentiation into Macrophages", J. IMMUNOL, vol. 162, 1 April 1999 (1999-04-01), pages 3961 - 3969, XP002938440 * |
COZZI ET AL.: "The generation of transgenic pigs as potential organ donors for humans", NATURE MEDICINE, vol. 1, no. 9, September 1995 (1995-09-01), pages 964 - 966, XP002938443 * |
See also references of EP1140146A4 * |
TADAKI ET AL.: "Costimulatory molecules are active in the human xenoreactive T-cell response but not in natural killer-mediated cytotoxicity", TRANSPLANTATION, vol. 70, no. 1, 15 July 2000 (2000-07-15), pages 162 - 167, XP002938441 * |
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