# Patent

## Title
Use of PD-L3 proteins and PD-L3 specific antibodies or antibody fragments to regulate CD4+ and CD8+ T cell immunity

## Abstract

The present invention relates to novel regulatory T cell proteins. One protein, designated PD-L3, resembles members of the PD-L1 family, and co-stimulates αCD3 proliferation of T cells in vitro. A second, TNF-like, protein has also been identified as being upregulated upon αCD3/αGITR stimulation. This protein has been designated Treg-sTNF. Proteins, antibodies, activated T cells and methods for using the same are disclosed.

## Background
INTRODUCTION
This application claims benefit of U.S. Provisional Patent Application Ser. No. 60/674,567, filed Apr. 25, 2005, the content of which is incorporated herein by reference in its entirety.


BACKGROUND OF THE INVENTION
Induction of an immune response requires T cell expansion, differentiation, contraction and establishment of T cell memory. T cells must encounter antigen presenting cells (APCs) and communicate via T cell receptor (TCR)/major histocompatibility complex (MHC) interactions on APCs. Once the TCR/MHC interaction is established, other sets of receptor-ligand contacts between the T cell and the APC are required, i.e. co-stimulation via CD154/CD40 and CD28/B7.1-B7.2. The synergy between these contacts is suggested to result, in vivo, in a productive immune response capable of clearing pathogens and tumors, and in some cases capable of inducing autoimmunity.
Another level of control has been identified, namely regulatory T cells (Treg). This specific subset of T cells is generated in the thymus, delivered into the periphery, and is capable of constant and inducible control of T cells responses in vitro and in vivo (Sakaguchi (2000) Cell 101(5):455-8; Shevach (2000) Annu. Rev. Immunol. 18:423-49; Bluestone and Abbas (2003) Nat. Rev. Immunol. 3(3):253-7). Treg are represented by a CD4+CD25+ phenotype and also express high levels of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), OX-40, 4-1BB and the glucocorticoid inducible TNF receptor-associated protein (GITR) (McHugh, et al. (2002) Immunity 16(2):311-23; Shimizu, et al. (2002) Nat. Immun. 3(2):135-42). Elimination of Treg cells by 5 day neonatal thymectomy or antibody depletion using anti-CD25, results in the induction of autoimmune pathology and exacerbation of T cells responses to foreign and self-antigens, including heightened anti-tumor responses (Sakaguchi, et al. (1985) J. Exp. Med. 161(1):72-87; Sakaguchi, et al. (1995) J. Immunol. 155(3):1151-64; Jones, et al. (2002) Cancer Immun. 2:1). In addition, Treg have also been involved in the induction and maintenance of transplantation tolerance (Hara, et al. (2001) J. Immunol. 166(6):3789-3796; Wood and Sakaguchi (2003) Nat. Rev. Immunol. 3:199-210), since depletion of Treg with anti-CD25 monoclonal antibodies results in ablation of transplantation tolerance and rapid graft rejection (Jarvinen, et al. (2003) Transplantation 76:1375-9). Among the receptors expressed by Treg, GITR seems to be an important component since in vitro or in vivo ligation of GITR on the surface of Treg with an agonistic monoclonal antibody results in rapid termination of Treg activity (McHugh, et al. (2002) supra; Shimizu, et al. (2002) supra), also resulting in autoimmune pathology (Shimizu, et al. (2002) supra) and ablation of transplantation tolerance.
DNA microarray analysis has been conducted with a population of Treg to identify genes differentially expressed by Treg (Gavin, et al. (2002) Nat. Immunol. 3(1):33-41; McHugh, et al. (2002) supra). The expression pattern of genes of CD4+CD25− and CD4+CD25+ T cells was compared (Gavin, et al. (2002) supra) as was the expression pattern of these two populations of cells after activation by anti-CD3 antibody and IL-2 for 12 and 48 hours (McHugh, et al. (2002) supra). However, gene regulation by GITR signaling was not assessed.
T cell activation is dependent upon signs transferred through antigen-specific T cells receptor recognition and accessory receptors on the T cell. As the maintenance of immunologic peripheral homeostatis is regulated by co-stimulatory molecules, which play a critical role in suppressing autoreactive lymphocytes, identification of these co-stimulatory molecules is needed.
A novel T cell co-stimulatory molecule has now been identified and will be useful in modulating immune responses in autoimmunity, cancer, infectious disease and transplantation.
SUMMARY OF THE INVENTION
The present invention is a composition containing an isolated PD-L3 protein comprising the amino acid sequence set forth in SEQ ID NO:5 and a pharmaceutically acceptable carrier. In one embodiment, the PD-L3 protein is operably linked to a heterologous protein.
The present invention is also an expression vector harboring an isolated nucleic acid encoding PD-L3 protein comprising the amino acid sequence set forth in SEQ ID NO:5; and host cells containing said vector.
The present invention is also an isolated binding agent which specifically binds to a PD-L3 protein comprising the amino acid sequence set forth in SEQ ID NO:5.
The present invention is further a method for modulating an immune cell response by contacting an immune cell with a PD-L3 protein, or binding agent thereof, in the presence of a primary signal so that a response of the immune cell is modulated.


DETAILED DESCRIPTION OF THE INVENTION
A novel member of the PD-L1 family has now been identified in Treg cells. This novel protein has been designated PD-L3. Like other members of the PD-L1 family, PD-L3 co-stimulates αCD3 proliferation of T cells in vitro. In addition, the expression of PD-L3 is increased in αCD3 activated Treg and reduced in the presence of αGITR. A second, TNF-like, protein has also been identified as being upregulated upon αCD3/αGITR stimulation. This protein has been designated Treg-sTNF. These proteins may be involved in contact-dependent and paracrine suppression of immunity and would therefore be useful for modulating (e.g., inhibiting or stimulating) an immune response and in the treatment of diseases and conditions involving Treg signaling.
PD-L3 and Treg-sTNF were identified by global transcriptional profiling of resting Treg, Treg activated with αCD3, and Treg activated with αCD3/αGITR. αGITR was selected for this analysis as triggering of GITR on Treg has been shown to extinguish their contact-dependent suppressive activity (Shimizu, et al. (2002) supra). PD-L3 and Treg-sTNF were identified on AFFIMETRIX® DNA arrays based on their unique expression patterns (Table 1). PD-L3 exhibited an increase in expression in αCD3 activated Treg and reduced expression in the presence of αGITR; and Treg-sTNF exhibited a αCD3/αGITR-dependent increase in expression.









TABLE 1









Relative Expression












mRNA
None
αCD3
αCD3/αGITR
















PD-L3
6
10
7



Treg-sTNF
0.2
0.3
1.5







Purified CD4+CD25+ T cells were stimulated in culture overnight with none, αCD3, or αCD3/αGITR, and RNA isolated for real-time PCR analysis. Expression listed is relative for actin.






PD-L3 was cloned and sequenced and, as indicated, is a member of the PD-L1 family. This Ig family of co-stimulatory molecules is composed of positive regulatory co-receptors such as CD28 and ICOS, and also negative regulatory signals such as those mediated by CTLA-4, PD-1, and BTLA molecules. Knockout studies of negative co-stimulatory receptors have demonstrated the necessity of these receptors in controlling autoimmunity and establishing peripheral tolerance (Chen (2004) Nat. Rev. Immunol. 4:336-47). The receptors of the PD-L1 family are type I transmembrane proteins containing a single IgV domain while the ligands are type I transmembrane proteins having both an IgV and an IgC extracellular domain.
Sequence analysis revealed that PD-L3 corresponded to mouse locus Ricken ID 4632428N05 with an mRNA coding sequence given as GENBANK accession number NM—028732 and protein sequence give as NP—080401. The nucleic acid sequence encoding mouse PD-L3 is set forth herein as SEQ ID NO:1 and the mouse PD-L3 protein sequence is set forth as SEQ ID NO:2. PD-L3 has an Ig domain which shares 26.5% homology with that of PD-L1 (B7-H1). The mouse PD-L3 gene is located on chromosome 10 (62.2 Mb) and is composed of 6 exons, creating a transcript of 4799 bases in length and coding for a 309-residue type I transmembrane protein. Pfam and Interpro (Integrated resource of Protein Families, Domains and Sites) predict a signal sequence (positions 1-32) and an Ig-like domain (positions 47-147). The human homolog of PD-L3 is located on chromosome 10 (72.9 Mb) and composed of 6 exons thereby generating a transcript of 4689 bases in length coding for a 311 residue protein. The human homolog mRNA coding sequence is provided in GENBANK accession number NM—022153 and protein sequence give as NP—071436. The nucleic acid sequence encoding human PD-L3 is set forth herein as SEQ ID NO:3 and the human PD-L3 protein sequence is set forth as SEQ ID NO:4. Mouse and human genes share 74% homology and are 68% identical at the protein level. Homologs were also identified in Rattus norvegicus on chromosome 20 (27.7 Mb; GENBANK accession number BC098723), as well as Fugu rubripes and Danio rerio. In particular embodiments, PD-L3 proteins of the present share the common amino acid sequence set forth in SEQ ID NO:5.
A PD-L3-Ig fusion protein was produced according to standard methods, purified and titered into cultures of purified CD4+ T cells, APC and αCD3. On day 3, all wells were pulsed with tritiated thymidine (3H-TdR) and proliferation was determined. Like other PD-L1 proteins, PD-L3 was shown to co-stimulate αCD3 proliferation of T cells in vitro (Table 2).










TABLE 2







Treatment
Proliferation (cpm/culture)









αCD3 (0.1 μg/mL)
900 ± 600



hIgG1 (10 μg/mL)
600 ± 400



PD-L3-Fc (0.5 μg/mL)
2300 ± 1150



PD-L3-Fc (1.0 μg/mL)
3900 ± 900 



PD-L3-Fc (10 μg/mL)
4200 ± 650 










Using a rabbit anti-PD-L3 antibody, PD-L3 protein was localized to lymphoid organs and prominently found in brain tissue. Further, four transgenic mice were produced which expressed full-length PD-L3 under the control of the human elongation factor 1 promoter. These mice were generated using lentiviral vector pWPT. Similar to other PD-L1 family members (Appay, et al. (2002) J. Immunol. 168:5954-8), it is contemplated that PD-L3 will function as a negative regulator in vivo while functioning to co-stimulate αCD3 T cell proliferation in vitro.
The second co-stimulatory molecule identified, Treg-sTNF, contains a TNF-like domain similar to those found in C1q family of proteins. Sequence analysis revealed that Treg-sTNF corresponded with mouse locus Ricken ID 1110035L05 with an mRNA coding sequence given as GENBANK accession number NM—026125 and protein sequence give as NP—080401. The nucleic acid sequence encoding mouse Treg-sTNF is set forth herein as SEQ ID NO:6 and the mouse Treg-sTNF protein sequence is set forth herein as SEQ ID NO:7. This TNF-like molecule is located on chromosome 4 (154.1 Mb), near OX40 and GITR, and composed of 8 exons, creating a transcript 1301 bases in length coding for a 308 residue soluble protein. Pfam and Interpro protein predict a signal sequence (positions 1-19), a proline rich collagen triple helix-like motif (positions 99-111), and a TNF-like motif (positions 176-306). Collectively, these motifs are similar to those of the C1q family of proteins, although this TNF-like protein does not contain the characteristic C1q-like motif that identifies this family. The human homolog of Treg-sTNF is located on chromosome 1 (1.1 Mb) and is composed of 7 exons thereby generating a transcript of 1014 bases in length coding for a 337 residue protein. The human coding sequence for the human homolog of Treg-sTNF is provided as GENBANK accession number BC089443 and protein sequence give as AAH89443.1. The nucleic acid sequence encoding human Treg-sTNF is set forth herein as SEQ ID NO:8 and the human Treg-sTNF protein sequence is set forth as SEQ ID NO:9. Mouse and human genes share 65.3% homology and 66% identify at the protein level. Homologs were also identified in Rattus norvegicus on chromosome 5 (172.8 Mb; GENBANK accession number XM—233720.2), as well as Fugu rubripes and Danio rerio. In particular embodiments, Treg-sTNF proteins of the present share the common amino acid sequence set forth in SEQ ID NO:10.
Having identified a novel immune cell regulatory molecule produced by Treg cells, the present invention relates to a PD-L3 protein, agents which bind PD-L3, nucleic acids encoding PD-L3 and methods of using PD-L3 and PD-L3 binding agents to modulate immune cell responses.
As used herein, a PD-L3 protein is intended to include a protein that has a sequence which is substantially similar to that of mouse PD-L3 (i.e., SEQ ID NO:2) or human PD-L3 (i.e., SEQ ID NO:4) and in particular embodiments has the consensus amino acid sequence set forth in SEQ ID NO:5. The term substantially similar refers to sequences having sequence variation (e.g., conservative substitutions and/or variations) that do not materially affect the nature of the protein (i.e., the structure, stability characteristics, substrate specificity and/or biological activity of the protein). In general, a protein having an amino acid sequence that is substantially similar to SEQ ID NO:2 or SEQ ID NO:4 has at least 70% identity to that of SEQ ID NO:2 or SEQ ID NO:4, over its entire length and exhibits at least one biological activity of PD-L3. The present invention further provides for a protein which has an amino acid sequence which shares at least 80% identity, at least 90% identity, at least 95% identity, or more desirably at least 97-99% identity, to that of SEQ ID NO:2 or SEQ ID NO:4 over the entire length of SEQ ID NO:2 or SEQ ID NO:4.
Percent identical and percent similar are used herein in comparisons among amino acid and nucleic acid sequences. When referring to amino acid sequences, identity or percent identical refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical amino acids in the compared amino acid sequence by a sequence analysis program. Percent similar refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical or conserved amino acids. Conserved amino acids are those which differ in structure but are similar in physical properties such that the exchange of one for another would not appreciably change the tertiary structure of the resulting protein. Conservative substitutions are well-known in the art (see, e.g., Taylor (1986) J. Theor. Biol. H 9:205). When referring to nucleic acid molecules, percent identical refers to the percent of the nucleotides of the subject nucleic acid sequence that have been matched to identical nucleotides by a sequence analysis program.
Identity and similarity can be readily calculated by known methods. Nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences of the nucleic or amino acids thus define the differences. Such methods include the BLAST programs (NCBI) and the DNAstar system (Madison, Wis.). However, equivalent alignments and similarity/identity assessments can be obtained through the use of any standard alignment software. For instance, the GCG Wisconsin Package, available from the Genetics Computer Group in Madison, Wis., can also be used to compare sequence identity and similarity.
A PD-L3 protein can be in the form of a mature protein (i.e. lacking a signal sequence, residues 1-32 of SEQ ID NO:1 or SEQ ID NO:2) or can be a part of a larger protein such as a fusion protein (e.g., fused to Fc). It is often advantageous to also include amino acid sequences which contain secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production. Accordingly, one embodiment of the present invention is a mature PD-L3 protein lacking N-terminal signal sequences. Another embodiment of the present invention is a fusion protein composed of PD-L3, or a fragment thereof, operably linked to a heterologous peptide or polypeptide (e.g., GST, Ig, His6, and the like) such that the fused proteins are translated in-frame. As used herein, a heterologous peptide or protein is one which is not naturally found to be operably linked to PD-L3.
A particular suitable heterologous peptide is an immunoglobulin constant region, for example, a human Cγ1 domain or Cγ4 domain (e.g., the hinge, CH2 and CH3 regions of human IgCγ1, or human IgCγ4; see e.g., Capon, et al. U.S. Pat. Nos. 5,116,964; 5,580,756; 5,844,095 and the like). Such constant regions may retain regions which mediate effector function (e.g., Fc receptor binding) or may be altered to reduce effector function.
Fragments of a PD-L3 protein are also included in the invention. A fragment is a protein having an amino acid sequence that is entirely the same as part, but not all, of the amino acid sequence of the aforementioned PD-L3 protein. Fragments include, for example, truncation polypeptides having the amino acid sequence of a PD-L3 protein, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Other fragments are biologically active fragments. Biologically active fragments are those that mediate PD-L3 activity (e.g., co-stimulation of T cells or modulation of an immune response), including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal.
A PD-L3 protein of the invention can be prepared in any suitable manner. If produced in situ, the protein can be purified from appropriate sources, e.g., appropriate vertebrate cells e.g., mammalian cells for instance Treg cells from human, mouse, bovine or rat.
Alternatively, the availability of nucleic acid molecules encoding the PD-L3 protein enables production of PD-L3 using in vitro expression methods known in the art. For example, a cDNA or gene can be cloned into an appropriate in vitro transcription vector, for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system. In vitro transcription and translation systems are commercially available, e.g., from PROMEGA® Biotech, Madison, Wis., or GIBCO-BRL®, Rockville, Md. In vitro transcription and translation is suitable for preparing small amounts of native or mutant proteins for research purposes, particularly since it allows the incorporation of radioactive nucleotides.
Larger quantities of PD-L3 protein can be produced by expression in a suitable prokaryotic or eukaryotic system. For example, part or all of a DNA molecule, such as the coding portion of SEQ ID NO:1 or SEQ ID NO:3 can be inserted into a plasmid vector adapted for expression in a bacterial cell (such as Escherichia coli) or a yeast cell (such as Saccharomyces cerevisiae), or into a baculovirus vector for expression in an insect cell. Such vectors contain the regulatory elements necessary for expression of the DNA in the host cell, positioned in such a manner as to permit expression of the DNA into mRNA and mRNA into protein in the host cell. Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences. Suitable vectors for recombinant protein expression in mammalian, yeast, or prokaryotic systems are commercially available from such sources as STRATAGENE®, INVITROGENTM, Pharmacia and the like.
Host-specific secretion signals can be used to facilitate purification of the resulting protein. The coding sequence for the secretion peptide is operably linked to the 5′ end of the coding sequence for the protein, and this hybrid nucleic acid molecule is inserted into a plasmid adapted to express the protein in the host cell of choice. Plasmids specifically designed to express and secrete foreign proteins are available from commercial sources. For example, if expression and secretion is desired in E. Coli, commonly used plasmids include pTrcPPA (Pharmacia); pPROK-C and pKK233-2 (CLONTECH T); and pNH8a, pNH16a, pcDNAII and pAX (STRATAGENE®), among others.
A PD-L3 protein produced by in vitro transcription and translation or by gene expression in a recombinant prokaryotic or eukaryotic system can be purified according to methods known in the art (e.g., fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; or gel filtration using, for example, SEPHADEX® G-75).
Alternatively, a synthetic PD-L3 protein can be prepared using various synthetic methods of peptide synthesis via condensation of one or more amino acid residues, in accordance with conventional peptide synthesis methods. For example, peptides are synthesized according to standard solid-phase methodologies, such as may be performed on an APPLIED BIOSYSTEMSTM Model 430A peptide synthesizer (APPLIED BIOSYSTEMSTM, Foster City, Calif.), according to manufacturer's instructions. Other methods of synthesizing peptides or peptidomimetics, either by solid phase methodologies or in liquid phase, are well-known to those skilled in the art.
PD-L3 peptidomimetics (Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber and Freidinger (1985) TINS p. 392; and Evans et al. (1987) J. Med. Chem. 30:1229) are also contemplated. Peptidomimetics are usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), such as human PD-L3, but have one or more peptide linkages optionally replaced by a linkage such as —CH2NH—, —CH2S—, —CH2—CH2—, —CH═CH—(cis and trans), —COCH2—, —CH(OH)CH2—, and —CH2SO—, by methods known in the art (see, e.g., Spatola (1983) In: Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, Weinstein, B., ed., Marcel Dekker, New York, p. 267; Morley (1980) Trends Pharm. Sci. pp. 463-468; Hudson, et al. (1979) Int. J. Pept. Prot. Res. 14:177-185); Spatola, et al. (1986) Life Sci. 38:1243-1249; Hann (1982) J. Chem. Soc. Perkin Trans. I. 307-314; Jennings-White, et al. (1982) Tetrahedron Lett. 23:2533; Holladay, et al. (1983) Tetrahedron Lett. (1983) 24:4401-4404; Hruby (1982) Life Sci. (1982) 31:189-199).
Whether recombinantly-produced or chemically-synthesized, PD-L3 (including PD-L3 fusion proteins or biologically active PD-L3 fragments) can be formulated into a pharmaceutically acceptable composition for use in accordance with the methods disclosed herein. A PD-L3 protein is generally formulated with a pharmaceutically acceptable carrier, such as buffered saline; a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol and the like); carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; preservatives or suitable mixtures thereof. In addition, a pharmaceutically acceptable carrier can include any solvent, dispersion medium, and the like which may be appropriate for a desired route of administration of the composition. The use of such carriers for pharmaceutically active substances is known in the art. Suitable carriers and their formulation are described, for example, in Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippingcott Williams & Wilkins: Philadelphia, Pa., 2000.
A PD-L3 protein of the present invention can be used to identify binding partners of PD-L3, i.e., binding agents and receptors. In these assays, PD-L3 is allowed to form a physical interaction with the unknown binding partner(s), often in a heterologous solution of molecules. The binding complex is then isolated, and the identity of the binding partner is determined (e.g., via mass spec or sequence analysis). Alternatively, a panel of rational binding partners (e.g., CTLA-4, PD-1, or BTLA) can be screened with a PD-L3 protein. These procedures are greatly facilitated by simple methods for isolating PD-L3 protein, e.g., precipitation using immunologically-specific antibodies to the PD-L3 protein, or purification with PD-L3 protein bound to a solid support. In one embodiment, a PD-L3 protein is attached to a solid support via a covalent linkage. In other embodiments, attachment is via a non-covalent linkage, for example, between members of a high affinity binding pair (e.g., ligand/receptor or antigen/antibody pairs). Suitable solid supports include beads, e.g., magnetized beads or beads which are dense enough to be separated form non-associated protein by centrifugation. Alternatively, the PD-L3 protein can be used in a yeast two hybrid system such as the Ga14/LacZ system (see Clark, et al. (1998) Proc. Natl. Acad. Sci. USA 95:5401-5406) to identify binding partners.
A PD-L3 protein of the present invention can also be used as a regulatory signal in a method for modulating, i.e., stimulating or inhibiting, an immune cell response. The prototypic immune response described herein is stimulation of T cells (CD4+), but one of ordinary skill in the art will readily appreciate that the method can be applied to modulation of other T cell-mediated and/or B cell-mediated immune responses that are influenced by modulation of T cell co-stimulation. By way of example, immune responses of tumor-reactive lymphocytes (CD8+; Hellström, et al. (2001) Proc. Natl. Acad. Sci. USA 98:6783-6788), CD43+ T cells (Wang, et al. (2004) J. Immunol 173(10):6294-302), and natural killer cells can be modulated. In addition, immune responses that are indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages are also contemplated.
In one embodiment, a PD-L3 protein is used as a co-stimulatory signal for stimulating or enhancing immune cell activation. A co-stimulatory signal, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to immune cell activation. The co-stimulatory signal can be used simultaneously with or subsequent to the primary signal to achieve the desired result. The term activation, within the context of T cells, refers to the induction of cellular proliferation. Activation of a T cell may also induce cytokine production and performance of regulatory or cytolytic effector functions. T cell activation preferably results in at least a 2.5- to 4.5-fold increase in the cell population as compared to cells which have not been co-stimulated (i.e., no stimulation or stimulated with a primary signal only). T cell activation can be quantitated as exemplified herein via tritiated thymidine incorporation or by analyzing cytokine production. Cytokines can be measured according to biological activity or protein accumulation (e.g., as determined by various immuno-based, activity, or other assays). Alternatively, mRNA production can be measured to establish levels of stimulation of transcription. In particular embodiments, the primary and co-stimulatory signals are further used in combination with other agents, such as cytokines (IL-2, IL-4, IL-7, IL-10, IL-12, etc.) or antigen presenting cells, for optimal activation.
In another embodiment, a PD-L3 protein is used as an inhibitory signal for inhibiting or decreasing immune cell activation. In this embodiment, the inhibitory signal binds to an inhibitory receptor (e.g., CTLA4 or PD-1) on an immune cell thereby antagonizing the primary signal which binds to an activating receptor (e.g., via a TCR, CD3, BCR, or Fc polypeptide). Inhibition includes, e.g., inhibition of second messenger generation; an inhibition of proliferation; an inhibition of effector function in the immune cell, e.g., reduced phagocytosis, reduced antibody production, reduced cellular cytotoxicity, the failure of the immune cell to produce mediators, (such as cytokines (e.g., IL-2) and/or mediators of allergic responses); or the development of anergy.
In particular embodiments, the primary signal is a ligand (e.g., CD3 or anti-CD3) that binds TCR and initiates a primary stimulation signal. Such TCR ligands are readily available from commercial sources and specific examples include anti-CD3 antibody OKT3, prepared from hybridoma cells obtained from the American Type Culture Collection, and anti-CD3 monoclonal antibody G19-4. In an alternative embodiment, a primary signal is delivered to a T cell through other mechanisms including a protein kinase C activator, such as a phorbol ester (e.g., phorbol myristate acetate), and a calcium ionophore (e.g., ionomycin, which raises cytoplasmic calcium concentrations), or the like. The use of such agents bypasses the TCR/CD3 complex but delivers a stimulatory signal to T cells. Other agents acting as primary signals can include natural and synthetic ligands. A natural ligand can include MHC with or without a peptide presented. Other ligands can include, but are not limited to, a peptide, polypeptide, growth factor, cytokine, chemokine, glycopeptide, soluble receptor, steroid, hormone, mitogen, such as PHA, or other superantigens, peptide-MHC tetramers (Altman, et al. (1996) Science 274(5284):94-6) and soluble MHC dimers (Dal Porto, et al (1993) Proc. Natl. Acad. Sci. USA 90: 6671-5).
Immune cells activated in accordance with the method of the instant invention can subsequently be expanded ex vivo and used in the treatment and prevention of a variety of diseases; e.g., human T cells which have been cloned and expanded in vitro maintain their regulatory activity (Groux, et al. (1997) Nature 389(6652):737-42). Prior to expansion, a source of T cells is obtained from a subject (e.g., a mammals such as a human, dog, cat, mouse, rat, or transgenic species thereof). T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, spleen tissue, tumors or T cell lines. T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLLTM separation.
Alternatively, T cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis are washed to remove the plasma fraction and to place the cells in an appropriate buffer (e.g., phosphate buffered saline (PBS) or wash solution lacking calcium or other divalent cations) or media for subsequent processing steps. After washing, the cells are resuspended in a variety of biocompatible buffers, such as, calcium-free, magnesium-free PBS.
Isolation of T cells from peripheral blood lymphocytes can be carried out by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient. A specific subpopulation of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques well-known to the skilled. Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
T cells for stimulation can also be frozen after isolation. Many freezing solutions and parameters are known in the art and will be useful in this context. One method involves suspending the cells in PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media, and subsequently freezing the cells at −80° C. at a rate of 1° C. per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing can be used as well as uncontrolled freezing immediately at −20° C. or in liquid nitrogen.
Those of ordinary skill in the art will readily appreciate that stimulation and expansion of T cells described herein can be carried out in a variety of environments (i.e., containers). For example, such containers can be culture flasks, culture bags, or any container capable of holding cells (e.g., a bioreactor), preferably in a sterile environment. For example, several manufacturers currently make devices that can be used to grow cells and be used in combination with the methods of the present invention. See for example, Celdyne Corp. (Houston, Tex.), Unisyn Technologies (Hopkinton, Mass.), Synthecon, Inc. (Houston, Tex.), Aastrom Biosciences, Inc. (Ann Arbor, Mich.), Wave Biotech LLC (Bedminster, N.J.). Further, patents covering bioreactors include U.S. Pat. Nos. 6,096,532; 5,985,653; 5,888,807; and 5,190,878.
The present invention also relates to a vector, in particular an expression vector, containing nucleic acids encoding a PD-L3 protein for therapeutic use and production of recombinant PD-L3 protein. A PD-L3 nucleic acid of the present invention includes nucleic acids encoding the PD-L3 protein containing the amino acid sequence set forth in SEQ ID NO:5 and fragments, and nucleic acids substantially similar thereto. More specifically, PD-L3 nucleic acids of the invention include nucleic acids encoding mouse and human PD-L3 protein, wherein such nucleic acids are set forth in SEQ ID NO:1 and SEQ ID NO:3, respectively. A nucleic acid that is substantially similar to a PD-L3 nucleic acid shares at least 70% identity over its entire length with a nucleotide sequence encoding a PD-L3 protein of SEQ ID NO:2 or SEQ ID NO:4, and a nucleic acid having a nucleotide sequence that is at least 70% identical to that of SEQ ID NO:1 or SEQ ID NO:3, over its entire length. In particular embodiments, a nucleic acid that is substantially similar to a PD-L3 nucleic acid shares at least 80% identity, at least 90% identity, at least 95% identity, or more desirably at least 97-99% identity, to that of SEQ ID NO:1 or SEQ ID NO:3 over the entire length of SEQ ID NO:1 or SEQ ID NO:3. Also encompassed within the scope of a PD-L3 nucleic acid is a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO:1 or SEQ ID NO:3, to hybridize under conditions useable for amplification, as a probe or marker, or antisense or siRNA.
In accordance with the present invention, nucleic acids having the appropriate level sequence homology (i.e., 70% identity or greater) with part or all the coding regions of SEQ ID NO:1 or SEQ ID NO:3 can be identified by using hybridization and washing conditions of appropriate stringency. For example, hybridizations can be performed, according to the method of Sambrook, et al. ((1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories, New York) using a hybridization solution containing 1.0% SDS, up to 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 0.05% sodium pyrophosphate (pH 7.6), 5×Denhardt's solution, and 100 microgram/mL denatured, sheared salmon sperm DNA. Hybridization is carried out at 37-42° C. for at least six hours. Following hybridization, filters are washed as follows: 5 minutes at room temperature in 2×SSC and 1% SDS; 15 minutes at room temperature in 2×SSC and 0.1% SDS; 30 minutes to 1 hour at 37° C. in 2×SSC and 0.1% SDS; and 2 hours at 45-55° C. in 2×SSC and 0.1% SDS, changing the solution every 30 minutes.
One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified percent identity is set forth by: Tm=81.5° C.+16.6 log 10([Na+]/(1.0+0.7[Na+]))+0.7% GC−500/size (Wetmur (1991) Crit. Rev. Biochem. Mol. Biol. 26:227-259)
The stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25° C. below the calculated Tm of the of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In regards to the nucleic acids of the present invention, a moderate stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/mL denatured salmon sperm DNA at 42° C., and wash in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. A high stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/mL denatured salmon sperm DNA at 42° C., and wash in 1×SSC and 0.5% SDS at 6-5° C. for 15 minutes. Very high stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/mL denatured salmon sperm DNA at 42° C., and wash in 0.1×. SSC and 0.5% SDS at 65° C. for 15 minutes.
Oligonucleotides (sense or antisense strands of DNA, cDNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule encoding the PD-L3 protein are useful as probes for detecting PD-L3 genes or transcripts and can also be useful in the treatment of various diseases or conditions, when delivered by an appropriate vehicle to the affected cells. Oligonucleotides for use as probes, primers, antisense, or siRNA are based on rationally-selected nucleic acid sequences chosen from SEQ ID NO:1 or SEQ ID NO:3. Such oligonucleotides can be used for the detection and isolation of nucleic acids encoding PD-L3 or inhibition of PD-L3 expression. Further, amino acid sequences of SEQ ID NO:2 and SEQ ID NO:4 can be used to design degenerate oligonucleotide primers, as is commonly done by those skilled in the art, for screening cDNA libraries from, e.g., bovine, canine, and feline to obtain PD-L3 homologs from bovine, canine, and feline, respectively.
The nucleotide sequences encoding the PD-L3 protein of SEQ ID NO:2 or SEQ ID NO:4 can be identical to the protein encoding sequence contained in SEQ ID NO:1 or SEQ ID NO:3, or can be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the protein of SEQ ID NO:2 or SEQ ID NO:4.
When the nucleic acids of the invention are used for the recombinant production of a PD-L3 protein, the nucleic acid can include the coding sequence for the mature protein or a fragment thereof, by itself; the coding sequence for the mature protein or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, or other fusion peptide or protein as discussed supra. For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded. The PD-L3 nucleic acid can also contain non-coding 5′ and 3′ sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
Nucleic acids of the present invention can be maintained as DNA in any convenient cloning vector, e.g., in plasmid cloning/expression vector, such as pBLUESCRIPT® (STRATAGENE®), that is propagated in a suitable E. coli host cell. As described above, PD-L3 nucleic acids may be used to produce large quantities of substantially pure PD-L3 proteins, or selected fragments thereof.
Hence, the present invention also relates to vectors, in particular expression vectors, that contain a PD-L3 nucleic acid, and isolated host cells that are genetically engineered with said vectors. Expression vectors harboring PD-L3 nucleic acids are discussed supra and generally contain all the necessary regulatory sequences, for example, promoter and terminator sequences, operably linked to the PD-L3 nucleic acids such that the PD-L3 coding sequence is transcribed into RNA and subsequently translated into protein or in the case of antisense, transcribed into RNA. Large numbers of suitable vectors and regulatory sequences are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example, bacterial vectors pQE70, pQE60, pQE-9 (QIAGEN®), pBS, pD10, pBLUESCRIPT® SK, pBSKS, pNH8A, pNHI8A, pNH46A (STRATAGENE®) and pRIT5 (Pharmacia); and eukaryotic vectors PWLNEO, pSV2CAT, pOG44, pXTI, pSG (STRATAGENE®) pSVK3, pBPV, pMSG, pSVL (Pharmacia). As further examples, a PD-L3 cDNA of can be inserted in the pEF/myc/cyto vector (INVITROGENTM) or the pCMV-Tag3b vector (STRATAGENE®) and transformed (e.g., calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid-mediated transfection, electroporation) into Hela thereby facilitating purification and use of PD-L3.
However, any other plasmid or vector can be used as long as they are replicable and viable in the host. In addition, a complete mammalian transcription unit and a selectable marker can be inserted into a prokaryotic plasmid for use in in vivo procedures. The resulting vector is then amplified in bacteria before being transfected into cultured mammalian cells or delivered directly to the subject with an acceptable carrier. Examples of vectors of this type include pTK2, pHyg and pRSVneo. Hence, these plasmids, constructs and vectors can be used in both in vivo and ex vivo procedures. Ex vivo procedures involve the removal of a host cell (e.g., a Treg cell) from a subject, recombinant manipulation of the cell (i.e., transformation, transduction or transfection with a suitable PD-L3 expression vector), and the re-delivery of the cell back into its host environment.
Representative examples of appropriate hosts for in vitro procedures include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, and HEK 293 cells, and plant cells. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
Genetic material, such as the nucleic acids of the present invention, can be delivered to cells, in vivo, using various different plasmid-based delivery platforms, including but not limited to recombinant ADV (such as that described in U.S. Pat. No. 6,069,134), AAV (such as those described by U.S. Pat. No. 5,139,941), MMLV, Herpes Simplex Virus (U.S. Pat. No. 5,288,641), cytomegalovirus, lentiviral, and overall, retroviral gene delivery systems, well-known and practiced with in the art.
Techniques for preparing replication defective, infective viruses are well-known in the art (see, e.g., Gluzman et al. (1982) Virology 123(1):78-92). These systems typically include a plasmid vector including a promoter sequence (e.g., CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-1) operably linked to the nucleotide coding the gene of interest (inserted into an appropriate gene insertion site, i.e., an IRES site), as well as a terminating signal (such as a poly-A tail, i.e., BGH), and the appropriate mutations so as to make the delivery vehicle replication defective (e.g., Psi sequence deletions) and safe for therapeutic uses. The construction of the appropriate elements in a vector system containing the nucleotides of the present invention is well within the skills of one versed in the recombinant arts.
Therapeutic nucleic acids can be delivered to target cells via basic transfection methods such as permeabilizing the cell membrane physically or chemically. Liposomes or protein conjugates formed with certain lipids and amphophilic peptides can also be used for transfection (Stewart, et al. (1992) Hum. Gene Ther. 3(3):267-75; Zhu, et al. (1993) Science 261(5118):209-11). This approach is particularly effective in ex vivo procedures involving leukocytes, which can be temporarily removed from the body and can tolerate the cytotoxicity of the treatment.
A second, transduction approach, capitalizes on the natural ability of viruses to enter cells, bringing their own genetic material with them. For example, retroviruses can integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell-lines (Miller (1992) Curr. Top. Microbiol. Immunol. 158:1-24).
A third method uses other viruses, such as adenovirus, herpes simplex viruses (HSV), cytomegalovirus (CMV), and adeno-associated virus (AAV), which are engineered to serve as vectors for gene transfer. For example, in an adenovirus gene transfer systems recombinant engineered adenovirus is rendered replication-incompetent by deletion of a portion of its genome, such as El, and yet still retains its competency for infection. Relatively large foreign proteins can be expressed when additional deletions are made in the adenovirus genome. For example, adenoviruses deleted in both E1 and E3 regions are capable of carrying up to 10 Kb of foreign DNA and can be grown to high titers in 293 cells with persistent expression of transgenes following adenoviral infection in vivo.
In addition to therapeutic uses and recombinant protein production, vectors and host cells disclosed herein are useful for producing transgenic animals which constitutively overexpress PD-L3 or are deficient in PD-L3 protein production (i.e., knock out animals).
The present invention further relates to an isolated binding agent which specifically recognizes and binds to a PD-L3 protein. Binding agents are intended to include antibodies as well as peptide aptamers.
Peptide aptamers which specifically bind to a PD-L3 protein can be rationally designed or screened for in a library of aptamers (e.g., provided by Aptanomics SA, Lyon, France). In general, peptide aptamers are synthetic recognition molecules whose design is based on the structure of antibodies. Peptide aptamers consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to that of an antibody (nanomolar range).
An antibody to a PD-L3 protein can be generated using methods that are well-known in the art. An anti-PD-L3 antibody is intended to include a polyclonal and monoclonal antibody; humanized antibody; murine antibody; mouse-human antibody; mouse-primate antibody; and chimeric antibody; wherein the antibody can be an intact molecule, a fragment thereof (such as scFv, Fv, Fd, Fab, Fab′ or F(ab)′2 fragment), or a multimer or aggregate of intact molecules and/or fragments; and can occur in nature or be produced, e.g., by immunization, synthesis or genetic engineering. An antibody fragment, as used herein, refers to fragments, derived from or related to an antibody, which bind antigen and which can be derivatized to exhibit structural features that facilitate clearance and uptake, e.g., by the incorporation of galactose residues. This includes, e.g., F(ab), F(ab)′2, scFv, light chain variable region (VL), heavy chain variable region (VH), and combinations thereof.
Monoclonal antibodies to PD-L3 protein of the invention can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, et al. (1975) Nature 256:495-497; Kozbor, et al. (1985) J. Immunol. Methods 81:31-42; Cote, et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, et al. (1984) Mol. Cell. Biol. 62:109-120).
In addition, techniques developed for the production of humanized and chimeric antibodies, the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, et al. (1984) Proc. Natl. Acad. Sci. 81, 6851-6855; Neuberger, et al. (1984) Nature 312:604-608; Takeda, et al. (1985) Nature 314:452-454). Alternatively, techniques described for the production of single-chain antibodies can be adapted, using methods known in the art, to produce specific, single-chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton (1991) Proc. Natl. Acad. Sci. 88, 11120-11123).
Antibodies can also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as is well-known in the art (Orlandi, et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, et al. (1991) Nature 349:293-299).
Diabodies are also contemplated. A diabody refers to an engineered antibody construct prepared by isolating the binding domains (both heavy and light chain) of a binding antibody, and supplying a linking moiety which joins or operably links the heavy and light chains on the same polypeptide chain thereby preserving the binding function (see, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444; Poljak (1994) Structure 2:1121-1123). This forms, in essence, a radically abbreviated antibody, having only the variable domain necessary for binding the antigen. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. These dimeric antibody fragments, or diabodies, are bivalent and bispecific. It should be clear that any method to generate diabodies, as for example described by Holliger, et al. (1993) supra, Poljak (1994) supra, Zhu, et al. (1996) Biotechnology 14:192-196, and U.S. Pat. No. 6,492,123, herein incorporated by reference, can be used. In one embodiment, an antibody or diabody of the present invention is a bispecific agonistic antibody which specifically agonizes CD3 and PD-L3 receptors and co-stimulates T cell activation.
Various immunoassays can be used for screening to identify antibodies, or fragments thereof, having the desired specificity for PD-L3 protein. Numerous protocols for competitive binding (e.g, ELISA), latex agglutination assays, immunoradiometric assays, and kinetics (e.g. BIACORETM analysis) using either polyclonal or monoclonal antibodies, or fragments thereof, and are well-known in the art. Such immunoassays typically involve the measurement of complex formation between a specific antibody and its cognate antigen. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes is suitable, but a competitive binding assay can also be employed.
An antibody of the instant invention is useful producing a corresponding anti-idiotypic antibody. Anti-idiotypic antibodies, or anti-idiotypes are antibodies directed against the antigen-combining region or variable region (idiotype) of another antibody. Based on Jerne's network model of idiotypic relationships (Jerne (1974) Ann. Immunol. 125:373; Jerne, et al. (1982) EMBO J. 1:234), immunization with an antibody molecule expressing a paratope (antigen-combining site) for a given antigen produces a group of anti-antibodies, some of which share with the antigen a complementary structure to the paratope. Such anti-idiotypic antibodies would be useful for antagonizing or agonizing the PD-L3 receptor.
In one embodiment, an anti-PD-L3 antibody, or peptide aptamer, of the instant invention is agonistic which, like PD-L3 protein, binds a PD-L3 receptor and activates the receptor. In another embodiment, the anti-PD-L3 antibody, or peptide aptamer, is antagonistic and blocks the binding of PD-L3 protein to its cognate receptor on an immune cell thereby blocking activation of the receptor. Like a PD-L3 protein, such PD-L3 binding agents are useful in methods for modulating an immune cell response.
Also encompassed by the present invention are small molecules which can modulate (either enhance or inhibit) interactions between PD-L3 and its cognate receptor(s). Such small molecules can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the one-bead one-compound library method; and synthetic library methods using affinity chromatography selection. (Lam (1997) Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in, DeWitt, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb, et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann, et al. (1994) J. Med. Chem. 37:2678; Cho, et al. (1993) Science 261:1303; Carrell, et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; and Gallop, et al. (1994) J. Med. Chem. 37:1233.
Libraries of compounds can be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria or spores (U.S. Pat. No. 5,223,409), plasmids (Cull, et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla, et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310). Compounds can be screened in cell-based or non-cell-based assays. Compounds can be screened in pools (e.g., multiple compounds in each testing sample) or as individual compounds.
A small molecule for modulating interactions between PD-L3 and its cognate receptor can be identified, for example, in a cell-based assay. Such an assay involves contacting a cell expressing a PD-L3 receptor (e.g., a T cell), with a test molecule and determining the ability of the test molecule to modulate (e.g., stimulate or inhibit) the binding of PD-L3 to its binding partner. Determining the ability of the PD-L3 to bind to, or interact with, its binding partner can be accomplished, e.g., by measuring direct binding or by measuring a parameter of immune cell activation (e.g., cell proliferation or cytokine production). In a direct binding assay, the PD-L3 protein can be coupled with a radioisotope (e.g., 125I, 35S, 14C, or 3H) such that binding of PD-L3 can be determined by detecting the labeled protein in a complex (e.g., direct counting of radioemmission or by scintillation counting). Alternatively, PD-L3 can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
In one embodiment, a small molecule binds to antagonizes the interaction between PD-L3 and at least one cognate receptor. In another embodiment, the small molecule agonizes the interaction between PD-L3 and at least one cognate receptor.
PD-L3 protein, PD-L3 binding agents (antagnostic or agonistic), small molecule agonists or antagonists, vectors, isolated host cells or T cells activated by the methods described herein can be used for modulating immune responses in subjects for treating and preventing cancer, infectious disease, autoimmune disease, immune dysfunction related to aging, transplantation or any other disease state where such agents are desired for treatment. Such modulatory agents (i.e., PD-L3 protein, PD-L3 binding agents, small molecule agonists or antagonists, vectors, isolated host cells or activated T cells) can be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as cytokines or other cell populations (e.g., APCs). In general, a pharmaceutical composition containing a modulatory agent is formulated with one or more pharmaceutically acceptable carriers such as those described herein.
A modulatory agent described herein can be used in methods of prevention or treatment (e.g., by up- or down-modulating the immune response). Such methods involve administration to a subject, at risk of having or having a disease or condition associated with an unwanted or less than desirable immune response, a prophylactic or therapeutic agent prior to or after the manifestation of symptoms associated with an unwanted or less than desirable immune response. Subjects at risk or having a disease that would benefit from treatment with such agents or methods can be identified, for example, by any or a combination of diagnostic or prognostic assays known in the art. The appropriate agent used for treatment (e.g. antibodies, peptides, fusion proteins or small molecules) can be determined based on clinical indications and can be identified, e.g., using screening assays described herein. Modulatory agents can be administered in vitro (e.g., by contacting the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention relates to methods of treating an individual afflicted with a disease or disorder that would benefit from modulation of an immune response, e.g., by modulation of the interaction between a PD-L3 ligand its cognate receptor(s). As PD-L3 expression was predominantly localized to the brain, one embodiment of the present invention is the prevention or treatment of a disease or condition of the brain.
To illustrate the efficacy of using a PD-L3 protein, co-stimulation (i.e., CD3/CD30-mediated) has been successfully used in in vivo tumor-specific activation of the T cell cytolytic machinery (see, Bauer, et al. (1999) Cancer Res. 59:1961-5). Likewise, CD3/CD28-activated T cells and interleukin-2 administration has achieved tumor regression in bone metastases in Phase I clinical trials of metastatic renal cell carcinoma (Thompson, et al. (Clin. Cancer Res. 2003 Sep. 1; 9(10 Pt 1):3562-7). Accordingly, it is contemplated that co-stimulation with PD-L3 in combination with a primary signal (e.g., anti-CD3) will be useful for modulating cellular immune responses mediated by cytotoxic T cells, capable of killing tumor and infected cells, and helper T cell responses.
The invention is described in greater detail by the following non-limiting examples.
Example 1
Expression Profiling
To facilitate comparisons with established expression profiles of Treg cells, standard growth and activation conditions were employed (McHugh, et al. (2002) supra). Briefly, fresh isolated Treg cells ( ̃96% positive) were inoculated at 106/mL into complete RPMI medium supplemented with 10% fetal bovine serum and 100 units IL-2 in a 24-well plate precoated with anti-CD3 with or without anti-GITR (DTA-1) (Shimizu, et al. (2002) supra). The cells were cultured at 37° C. for 0 and 12 hours, RNA was purified and subsequently analyzed using an AFFYMETRIX® mouse genome A430 oligonucleotide array.
By comparing the data from resting or activated CD4+CD25+ T cell groups, gene expression patterns were found to be similar to those established in the art (Gavin, et al. (2002) supra; McHugh, et al. (2002) supra). It identify genes regulated by GIRT signaling, gene expression profiles were compared between the different cell populations with or without anti-GITR treatment. A list of known as well as unknown genes were compiled including the previously uncharacterized PD-L3 and Treg-sTNF.
Example 2
Inhibitory Activity of PD-L3
The inhibitory activity of PD-L1 was revealed by using antigen presenting cells over-expressing PD-L1 in vitro with CD4+ and CD8+ T cell antigen receptor transgenic T cells and antigen stimulation (Carter, et al. (2002) Eur. J. Immunol. 32:634-43). Similarly, the lentivector disclosed herein, which expresses the full-length PD-L3, is transduced into cell lines expressing class II major histocompatibility complex (MHC) and class I MHC. The response of TEa Tg or the 2C transgenic T cells to antigen presented by empty vector-transduced or PD-L3-transduced antigen presenting cells is determined according to established methods.
Example 3
Protein Expression
Expression patterns in lymphoid, monocyte and dendritic cell subsets, as well as non-hemoatopoietic tissues, is determined by RT-PCR and western blot analysis using standard protocols in combination with the rabbit αPD-L3 antibody disclosed herein.
Example 4
PD-L3 Transgenic Mice
Using Lentiviral infection of embryos, four transgenic mice ubiquitously expressing PD-L3 have been produced. These mice are expected to spontaneously develop autoimmunity and in vivo immune responses in the PD-L3 transgenic mice (i.e., humoral immune responses, T cell priming, etc.) are evaluated to assess systemic autoimmune disease development.
Example 5
PD-L3 Knock-Out Mice
PD-L3 is inactivated by homologous recombination. A BAC clone containing full-length PD-L3 sequence was purchased from INVITROGENTM (Carlsbad, Calif.). A PD-L3 targeting vector was generated by inserting a 1.6 kb fragment located at the 5′ side of the second exon of PD-L3 gene upstream the neomycin gene and the 5 kb fragment located at the 3′ side of the third exon of PD-L3 gene downstream the neomycin gene. B6-derived embryonic stem (ES) cells are electroporated with PD-L3 targeting vector and recombined clones are selected. Selected clones are then injected into C57BL/6 blastocytes and the resulting chimeric male offspring are mated to FLP-deleter mice to remove the neomycin cassette. Transmission of the targeted allele in the offspring is determined by PCR from genomic DNA. The second and the third exon contain the PD-L3 domain, therefore, the resulting mice have only the inactivated form of the PD-L3 molecule.
The overall immune capacity of PD-L3 deficient mice is determined as with other PD-L−/− mice, including assessment of T cell responses to antigen, humoral immune responses, overt autoimmunity (Systemic Lupus Erythematosus, inflammatory bowel disease), and increased susceptibility to induced autoimmune disease (experimental autoimmune encephalomyelitis) (Chen (2004) supra).

## Claims

1. A method for enhancing CD4+ or CD8+ T cell mediated immune cell responses in a subject in need thereof comprising administering an effective amount of an antibody or antibody fragment that specifically binds to the human PD-L3 protein in SEQ ID NO:4 and which antibody or antibody fragment antagonizes the immune inhibitory effect of PD-L3 protein in vivo resulting in said antibody or antibody fragment enhancing antigen-specific CD4+ or CD8+ T cell responses upon administration in a subject in need thereof.

2. The method of claim 1 where said antibody or antibody fragment is administered to a subject in need thereof which specifically binds a PD-L3 protein which is identical to the amino acid sequence in SEQ ID NO:4.

3. The method of claim 2 wherein the antibody is administered to a subject with a cancer.

4. The method of claim 3 wherein said administration enhances the proliferation of said CD4+ or CD8+ T cells.