Patent Grant
8273864
This invention relates to materials and methods for enhancing T cell activation.
Antigen-specific activation and proliferation of lymphocytes are regulated by both positive and negative signals from costimulatory molecules. The most extensively characterized T cell costimulatory pathway is B7-CD28, in which B7-1 (CD80) and B7-2 (CD86) each can engage the stimulatory CD28 receptor and the inhibitory CTLA-4 (CD152) receptor. In conjunction with signaling through the T cell receptor, CD28 ligation increases antigen-specific proliferation of T cells, enhances production of cytokines, stimulates differentiation and effector function, and promotes survival of T cells (Lenshow et al. (1996) Annu. Rev. Immunol. 14:233-258; Chambers and Allison (1997) Curr. Opin. Immunol. 9:396-404; and Rathmell and Thompson (1999) Annu. Rev. Immunol. 17:781-828). In contrast, signaling through CTLA-4 is thought to deliver a negative signal that inhibits T cell proliferation, IL-2 production, and cell cycle progression (Krummel and Allison (1996) J. Exp. Med. 183:2533-2540; and Walunas et al. (1996) J. Exp. Med. 183:2541-2550). Other members of the B7 family include B7-H1 (Dong et al. (1999) Nature Med. 5:1365-1369; and Freeman et al. (2000) J. Exp. Med. 192:1-9), B7-DC (Tseng et al. (2001) J. Exp. Med. 193:839-846; and Latchman et al. (2001) Nature Immunol. 2:261-268), B7-H2 (Wang et al. (2000)Blood 96:2808-2813; Swallow et al. (1999) Immunity 11:423-432; and Yoshinaga et al. (1999) Nature 402:827-832), and B7-H3 (Chapoval et al. (2001) Nature Immunol. 2:269-274). B7-H1 and B7-DC are ligands for PD-1, B7-H2 is a ligand for ICOS, and B7-H3 remains at this time an orphan ligand (Dong et al. (2003) Immunol. Res. 28:39-48).
B7 family molecules are expressed on the cell surface as homodimers with a membrane proximal constant IgC domain and a membrane distal IgV domain. Receptors for these ligands share a common extracellular IgV-like domain. Interactions of receptor-ligand pairs are mediated predominantly through residues in the IgV domains of the ligands and receptors (Schwartz et al. (2002) Nature Immunol. 3:427-434). In general, IgV domains are described as having two sheets that each contain a layer of β-strands (Williams and Barclay (1988) Annu. Rev. Immunol. 6:381-405). The front and back sheets of CTLA-4 contain strands A′GFCC′ and ABEDC,″ respectively (Ostrov et al. (2000) Science 290:816-819), whereas the front and back sheets of the B7 IgV domains are composed of strands AGFCC′C″ and BED, respectively (Schwartz et al. (2001) Nature 410:604-608; Stamper et al (2001) Nature 410:608-611; and Ikemizu et al. (2000) Immunity 12:51-60). Crystallographic analysis revealed that the CTLA-4/B7 binding interface is dominated by the interaction of the CDR3-analogous loop from CTLA-4, composed of a MYPPPY (SEQ ID NO:1) motif, with a surface on B7 formed predominately by the G, F, C, C′ and C″ strands (Schwartz et al. (2001) supra; and Stamper et al. supra). Data from amino acid homologies, mutation, and computer modeling provide support for the concept that this motif also is a major B7-binding site for CD28 (Bajorath et al. (1997) J. Mol. Graph. Model. 15:135-139). Although the MYPPPY motif is not conserved in ICOS, studies have indicated that a related motif having the sequence FDPPPF (SEQ ID NO:2) and located at the analogous position is a major determinant for binding of ICOS to B7-H2 (Wand et al. (2002) J. Exp. Med. 195:1033-1041).
B7-H1 (also called PD-L1) and B7-DC (also called PD-L2) are relatively new members of the B7 family, and have amino acid sequences that are about 34% identical to each other. Human and mouse orthologues of these molecules share about 70% amino acid identity (i.e., the human and mouse B7-H1 amino acid sequences are about 70% identical, and the human and mouse B7-DC amino acid sequences are about 70% identical). While B7-H1 and B7-DC transcripts are found in various tissues (Dong et al. (1999) supra; Latchman et al. supra; and Tamura (2001) Blood 97:1809-1816), the expression profiles of the proteins are quite distinct. Expression of B7-H1 protein, although essentially not found in normal tissues other than macrophage-like cells, can be induced in a variety of tissues and cell types (Dong et al. (1999) supra; Tamura et al. supra; and Ishida et al. (2000) Immunol. Lett. 84:57-62). In contrast, B7-DC is expressed only in dendritic cells and monocytes (Tseng et al. supra; and Ishida et al. supra).
The invention provides materials and methods for enhancing a costimulatory response, enhancing T cell activation, and inhibiting interaction between a costimulatory molecule and PD-1. For example, the invention provides purified variant costimulatory polypeptides that have altered binding affinity for PD-1, but that retain substantial costimulatory activity. Since it is likely that the interaction of costimulatory molecules such as B7-H1 and B7-DC with PD-1 suppresses an immune response, variant B7-H1 and variant B7-DC polypeptides with decreased binding affinity for PD-1 can be useful to enhance an immune response.
Methods of the invention can be used to enhance an immune response, enhance T cell activation, or inhibit binding of costimulatory polypeptides to PD-1, for example. Such methods can involve contacting a T cell with any of the purified variant costimulatory polypeptides or fragments provided herein. The T cell can be contacted in vitro or in vivo (e.g., in a mammal such as a mouse or a human). In some embodiments, the T cell can be contacted with a host cell [e.g., an antigen presenting cell (APC)] transfected or transduced with a nucleic acid encoding a polypeptide of the invention. If the T cell is contacted in a mammal, the host cell can be from that mammal or from another mammal. For example, the host cell can be from another mammal of the same species (e.g., another mammal that is histocompatible with the mammal to which the cells are being administered).
In one aspect, the invention provides a purified variant costimulatory polypeptide, wherein the variant polypeptide is a variant of a wild-type costimulatory polypeptide that binds to PD-1 and has reduced binding affinity for PD-1 compared to the wild-type costimulatory polypeptide, wherein the binding affinity is reduced by at least 50 percent as compared to the binding affinity of the wild-type costimulatory polypeptide, and wherein the costimulatory polypeptide retains substantial costimulatory activity. The variant costimulatory polypeptide can contain a substitution of one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more than ten) amino acids of the wild-type polypeptide.
The wild-type polypeptide can be a B7-H1 polypeptide [e.g., murine B7-H1 (SEQ ID NO:4)]. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 67 of SEQ ID NO:4. The substitution can involve replacing the amino acid at position 67 of SEQ ID NO:4 with an alanine residue. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 126 of SEQ ID NO:4. The substitution can involve replacing the amino acid at position 126 of SEQ ID NO:4 with an alanine residue. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 37 of SEQ ID NO:4. The substitution can involve replacing the amino acid at position 37 of SEQ ID NO:4 with a tyrosine residue. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 115 of SEQ ID NO:4. The substitution can involve replacing the amino acid at position 115 of SEQ ID NO:4 with an alanine residue. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 124 of SEQ ID NO:4. The substitution can involve replacing the amino acid at position 124 of SEQ ID NO:4 with a serine residue.
The wild-type polypeptide can be a B7-DC polypeptide [e.g., murine B7-DC (SEQ ID NO:6)]. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 111 of SEQ ID NO:6. The substitution can involve replacing the amino acid at position 111 of SEQ ID NO:6 with a serine residue. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 113 of SEQ ID NO:6. The substitution can involve replacing the amino acid at position 113 of SEQ ID NO:6 with a serine residue. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 56 of SEQ ID NO:6. The substitution can involve replacing the amino acid at position 56 of SEQ ID NO:6 with a serine residue. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 67 of SEQ ID NO:6. The substitution can involve replacing the amino acid at position 67 of SEQ ID NO:6 with a tyrosine residue. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 71 of SEQ ID NO:6. The substitution can involve replacing the amino acid at position 71 of SEQ ID NO:6 with a serine residue. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 101 of SEQ ID NO:6. The substitution can involve replacing the amino acid at position 101 of SEQ ID NO:6 with a serine residue. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 105 of SEQ ID NO:6. The substitution can involve replacing the amino acid at position 105 of SEQ ID NO:6 with an alanine residue.
The invention also provides a purified variant costimulatory polypeptide, wherein the variant polypeptide is a variant of a wild-type costimulatory polypeptide that binds to PD-1 and has increased binding affinity for PD-1 compared to the wild-type costimulatory polypeptide, wherein the affinity is increased by at least 50 percent as compared to the affinity of the wild-type costimulatory polypeptide, and wherein the costimulatory polypeptide retains substantial costimulatory activity. The variant costimulatory polypeptide can contain a substitution of one or more amino acids of the wild-type polypeptide. The wild-type polypeptide can be a B7-H1 polypeptide [e.g., murine B7-H1 (SEQ ID NO:4)]. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 58 of SEQ ID NO:4. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 69 of SEQ ID NO:4. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 113 of SEQ ID NO:4. The wild-type polypeptide can be a B7-DC polypeptide [e.g., murine B7-DC (SEQ ID NO:6)]. The purified variant costimulatory polypeptide can contain a substitution of the amino acid at position 58 of SEQ ID NO:6.
In another aspect, the invention features an isolated nucleic acid molecule containing a nucleic acid sequence that encodes any of the variant costimulatory polypeptides provided herein. The invention also features a vector containing the nucleic acid. The nucleic acid sequence can be operably linked to an expression control sequence in the vector. In addition, the invention features a host cell containing the vector of the invention.
In another aspect, the invention features a method for enhancing T cell activation. The method can include contacting a T cell with any of the purified variant costimulatory polypeptides provided by the invention. The contacting can be in vitro. The T cell can be in a mammal. The method can include administering the variant polypeptide to the mammal. Alternatively, the method can involve administering a nucleic acid encoding the variant polypeptide to the mammal. The method can include administering a cell transfected or transduced with the nucleic acid to the mammal, wherein the cell is a cell, or a progeny of a cell, that prior to the transfection or the transduction, was obtained from the mammal. The mammal can be a human.
In yet another aspect, the invention features a polypeptide fragment of murine B7-H1 (SEQ ID NO:4), wherein the polypeptide fragment inhibits binding of murine B7-H1 to murine PD-1. The polypeptide fragment can contain, for example, amino acids 67-69, amino acids 113-115, or amino acids 124-126 of SEQ ID NO:4. The polypeptide fragment can also contain amino acids 62-69 of SEQ ID NO:4.
The invention also features a method of inhibiting the interaction between PD-1 and B7-H1. The method can include contacting a PD-1 polypeptide with a B7-H1 polypeptide fragment provided herein. The PD-1 can be in vitro or in a mammal. The method can involve administering the polypeptide fragment to the mammal, or the method can involve administering a nucleic acid encoding the polypeptide fragment to the mammal. The method can involve administering a cell transfected or transduced with the nucleic acid to the mammal, wherein the cell is a cell, or a progeny of a cell, that prior to the transfection or the transduction, was obtained from the mammal. The mammal can be a human.
In another aspect, the invention features a polypeptide fragment of murine B7-DC (SEQ ID NO:6), wherein the polypeptide inhibits binding of murine B7-DC to murine PD-1. The polypeptide fragment can contain, for example, amino acids 67-71, amino acids 101-105, or amino acids 111-113 of SEQ ID NO:6. The polypeptide fragment can also contain amino acids 101-113 of SEQ ID NO:6.
In another aspect, the invention features a method of inhibiting the interaction between PD-1 and B7-DC. The method can include contacting a PD-1 polypeptide with a B7-DC polypeptide fragment provided herein. The PD-1 can be in vitro or in a mammal. The method can involve administering the polypeptide fragment to the mammal. The method can involve administering a nucleic acid encoding the polypeptide fragment to the mammal. The method can involve administering a cell transfected or transduced with the nucleic acid to the mammal, wherein the cell is a cell, or a progeny of a cell, that prior to the transfection or the transduction, was obtained from the mammal. The mammal can be a human.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
The invention provides materials and methods for enhancing a costimulatory response, enhancing T cell activation, and inhibiting interaction between a costimulatory molecule and PD-1. For example, the invention provides purified variant costimulatory polypeptides that have altered binding affinity for PD-1, but that retain substantial costimulatory activity. Since it is likely that the interaction of costimulatory molecules such as B7-H1 and B7-DC with PD-1 suppresses an immune response, variant B7-H1 and variant B7-DC polypeptides with decreased binding affinity for PD-1 can be useful to enhance an immune response.
1. Purified Polypeptides
Purified costimulatory polypeptides (e.g., purified B7-H1 and B7-DC polypeptides) are provided herein. As used herein, the term “polypeptide” refers to a chain of amino acids of any length, regardless of modification (e.g., phosphorylation or glycosylation). A “costimulatory polypeptide” is a polypeptide that, upon interaction with a cell-surface molecule on a T cell, enhances a T cell response (e.g., stimulates T cell proliferation and/or cytokine release). The T cell response that results from the interaction typically is greater than the response in the absence of the costimulatory polypeptide. The response of the T cell in the absence of the costimulatory polypeptide can be no response or can be a response significantly lower than in the presence of the costimulatory polypeptide. It is understood that the response of the T cell can be an effector (e.g., CTL or antibody-producing B cell) response, a helper response providing help for one or more effector (e.g., CTL or antibody-producing B cell) responses, or a suppressive response.
A costimulatory polypeptide can be a full-length costimulatory molecule, or can it be a portion (i.e., a functional fragment) of a costimulatory molecule. In some embodiments, a costimulatory polypeptide can be a variant of a full-length, immature B7-H1 polypeptide having the amino acid sequence shown in
The costimulatory polypeptides provided herein can be variant polypeptides (e.g., variant B7-H1 or B7-DC polypeptides). As used herein, a “variant” costimulatory polypeptide contains at least one amino acid sequence alteration as compared to the amino acid sequence of the corresponding wild-type costimulatory polypeptide (e.g., a polypeptide having the amino acid sequence set forth in any of SEQ ID NOs:3-6). An amino acid sequence alteration can be, for example, a substitution, a deletion, or an insertion of one or more amino acids. With respect to SEQ ID NO:4, for example, a variant B7-H1 polypeptide can contain, without limitation, a substitution at position 67 (e.g., an alanine substitution for phenylalanine at position 67), a substitution at position 126 (e.g., an alanine substitution for isoleucine as position 126), a substitution at position 37 (e.g., a tyrosine substitution for threonine at position 37), a substitution at position 115 (e.g., an alanine substitution for isoleucine at position 115), or a substitution at position 124 (e.g., a serine substitution for lysine at position 124). With respect to SEQ ID NO:6, a variant B7-DC polypeptide can contain, without limitation, a substitution at position 111 (e.g., a serine substitution for aspartic acid at position 111), a substitution at position 113 (e.g., a serine substitution for lysine at position 113), a substitution at position 56 (e.g., a serine substitution for arginine at position 56), a substitution at position 67 (e.g., a tyrosine substitution for serine at position 67), a substitution at position 71 (e.g., a serine substitution for glutamic acid at position 71), a substitution at position 101 (e.g., a serine substitution for arginine at position 101), or a substitution at position 105 (e.g., an alanine substitution for isoleucine at position 105). It is understood, however, that the recited substitutions can be made using any amino acid or amino acid analog. For example, the substitutions at the recited positions can be made with any of the naturally-occurring amino acids (e.g., alanine, aspartic acid, asparagine, arginine, cysteine, glycine, glutamic acid, glutamine, histidine, leucine, valine, isoleucine, lysine, methionine, proline, threonine, serine, phenylalanine, tryptophan, or tyrosine).
While the substitutions described herein are with respect to mouse B7-H1 and mouse B7-DC, it is noted that one of ordinary skill in the art could readily make equivalent alterations in the corresponding polypeptides from other species (e.g., rat, hamster, guinea pig, gerbil, rabbit, dog, cat, horse, pig, sheep, cow, non-human primate, or human). For example, a variant human B7-H1 polypeptide can contain a substitution at position 67, 126, 37, or 124 with respect to the amino acid sequence set forth in SEQ ID NO:3, and a variant human B7-DC polypeptide can contain a substitution at position 111, 113, 67, 71, or 105 with respect to the amino acid sequence set forth in SEQ ID NO:5. Thus, the invention features variants of B7-H1 and B7-DC from all the above mammals, nucleic acids (e.g., DNA or RNA) encoding the variant B7-H1 and variant B7-DC polypeptides, vectors containing the nucleic acids, host cells containing the vectors, and methods of using the variants, the nucleic acids, and the host cells.
A variant costimulatory polypeptide of the invention can have reduced binding affinity for PD-1 as compared to the binding affinity of the corresponding wild-type costimulatory polypeptide. The binding affinity of a variant typically is reduced by at least 50 percent (e.g., at least 50 percent, 55 percent, 60 percent, 70 percent, 75 percent, 80 percent, 90 percent, 95 percent, 99 percent, or more than 99 percent) as compared to the binding affinity of the corresponding wild-type polypeptide. In addition, a variant costimulatory polypeptide with reduced binding affinity for PD-1 can retain substantial costimulatory activity. For example, a variant costimulatory polypeptide can have at least 20 percent (e.g., at least 20 percent, 25 percent, 30 percent, 40 percent, 50 percent, 60 percent, 75 percent, 90 percent, 100 percent, or more than 100 percent) of the level of costimulatory activity exhibited by the corresponding wild-type costimulatory polypeptide. Costimulatory activity can be measured by any of a number of methods, including those disclosed herein (e.g., T cell proliferation and cytokine assays).
The invention also provides polypeptides that are fragments of full-length costimulatory molecules (e.g., costimulatory molecules having the amino acid sequences set forth in SEQ ID NOs:3-6). Such polypeptide fragments typically contain a region of a costimulatory polypeptide that is important for binding affinity for PD-1. For example, a polypeptide fragment of mouse B7-H1 (SEQ ID NO:4) can contain amino acids 67-69, 113-115, or 124-126. A polypeptide fragment of mouse B7-DC can contain, for example, amino acids 67-71, 101-105, or 111-113. Without being bound by a particular mechanism, these fragments can be useful to inhibit the binding of a costimulatory polypeptide (e.g., a full-length, native costimulatory polypeptide) to PD-1 and consequent activation of T cell suppression. The binding to PD-1 typically is inhibited by at least 50 percent (e.g., at least 50 percent, 60 percent, 70 percent, 75 percent, 80 percent, 90 percent, 95 percent, or more than 95 percent) as compared to the level of binding in the absence of the fragment. In addition, such fragments can be useful to enhance an immune response, as inhibiting interactions of B7-H1 and B7-DC with PD-1 may also inhibit the suppression of immune responses that would otherwise occur.
Isolated costimulatory polypeptides of the invention can be obtained by, for example, chemical synthesis or by recombinant production in a host cell. To recombinantly produce a costimulatory polypeptide, a nucleic acid containing a nucleotide sequence encoding the polypeptide can be used to transform, transduce, or transfect a bacterial or eukaryotic host cell (e.g., an insect, yeast, or mammalian cell). In general, nucleic acid constructs include a regulatory sequence operably linked to a nucleotide sequence encoding a costimulatory polypeptide. Regulatory sequences (also referred to herein as expression control sequences) typically do not encode a gene product, but instead affect the expression of the nucleic acid sequences to which they are operably linked.
Useful bacterial systems include, for example, Escherichia coli strains such as BL-21. An E. coli strain can be transformed with a vector such as one of the pGEX series of vectors (Amersham Biosciences Corp., Piscataway, N.J.), which produce fusion proteins containing glutathione S-transferase (GST). Transformed E. coli typically are grown exponentially, and then stimulated with isopropylthiogalactopyranoside (IPTG) to induce expression of the polypeptide of interest. The expressed polypeptide may be soluble and easily purified from lysed cells by, for example, adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the target gene product can be released from the GST moiety.
In eukaryotic host cells, a number of viral-based expression systems can be utilized to express costimulatory polypeptides (e.g., variant B7-H1 and variant B7-DC polypeptides). A nucleic acid encoding a costimulatory polypeptide of the invention can be cloned into, for example, a baculoviral vector such as pBLUEBAC™ (Invitrogen Life Technologies, Carlsbad, Calif.), which can be used to co-transfect insect cells such as Spodoptera frugiperda (Sf9) cells with wild type DNA from Autographa californica multiply enveloped nuclear polyhedrosis virus (AcMNPV). Recombinant viruses producing costimulatory polypeptides can be identified by standard methodology. Alternatively, a nucleic acid encoding a costimulatory polypeptide such as variant B7-H1 or variant B7-DC can be introduced into an SV40, retroviral, or vaccinia based viral vector for infection of suitable host cells.
Mammalian cell lines that stably express variant costimulatory polypeptides can be produced using expression vectors with appropriate control elements and a selectable marker. For example, the eukaryotic expression vectors pCR®3.1 (Invitrogen Life Technologies) and p91023(B) (see Wong et al. (1985) Science 228:810-815) are suitable for expression of variant costimulatory polypeptides in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC). Following introduction of an expression vector by electroporation, lipofection, calcium phosphate, or calcium chloride co-precipitation, DEAE dextran, or other suitable transfection method, stable cell lines can be selected (e.g., by antibiotic resistance to G418, kanamycin, or hygromycin). The transfected cells can be cultured such that the polypeptide of interest is expressed, and the polypeptide can be recovered from, for example, the cell culture supernatant or from lysed cells. Alternatively, a variant costimulatory polypeptide can be produced by (a) ligating amplified sequences into a mammalian expression vector such as pcDNA™ 3 (Invitrogen Life Technologies), and (b) transcribing and translating in vitro using wheat germ extract or rabbit reticulocyte lysate.
Variant costimulatory polypeptides can be purified using, for example, chromatographic methods such as DEAE ion exchange, gel filtration, and hydroxylapatite chromatography. For example, a costimulatory polypeptide in a cell culture supernatant or a cytoplasmic extract can be purified using a protein G column. In some embodiments, variant costimulatory polypeptides can be “engineered” to contain an amino acid sequence that allows the polypeptides to be captured onto an affinity matrix. For example, a tag such as c-myc, hemagglutinin, polyhistidine, or FLAG™ (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus. Other fusions that can be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. Immunoaffinity chromatography also can be used to purify costimulatory polypeptides.
2. Isolated Nucleic Acid Molecules
The invention provides isolated nucleic acids that include a nucleic acid sequence encoding a costimulatory polypeptide (e.g., a variant B7-H1 or B7-DC polypeptide). As used herein, “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome (e.g., nucleic acids that encode non-B7-H1 or B7-DC proteins). The term “isolated” as used herein with respect to nucleic acids also includes any non-naturally-occurring nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.
An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, a cDNA library or a genomic library, or a gel slice containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.
Nucleic acids of the invention can be in sense or antisense orientation, or can be complementary to a reference sequence encoding a costimulatory polypeptide. Reference sequences include, for example, the nucleotide sequences set forth in SEQ ID NOS:7-10 (shown in
Isolated nucleic acid molecules of the invention can be produced by standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid encoding a variant costimulatory polypeptide. PCR is a technique in which target nucleic acids are enzymatically amplified. Typically, sequence information from the ends of the region of interest or beyond can be employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptase can be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292-1293.
Isolated nucleic acids of the invention also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated DNA synthesis in the 3′ to 5′ direction). For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.
Isolated nucleic acids of the invention also can be obtained by mutagenesis. For example, a reference sequence (e.g., the nucleotide sequence set forth in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18) that encodes a costimulatory polypeptide having an amino acid sequence shown in
3. Vectors and Host Cells
The invention also provides vectors containing nucleic acids such as those described above. As used herein, a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors of the invention can be expression vectors. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
In the expression vectors of the invention, the nucleic acid is operably linked to one or more expression control sequences. As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.
Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen Life Technologies (Carlsbad, Calif.).
An expression vector can include a tag sequence designed to facilitate subsequent manipulation of the expressed nucleic acid sequence (e.g., purification or localization). Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.
The invention also provides host cells containing vectors of the invention. The term “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Suitable methods for transforming and transfecting host cells are found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, New York (1989), and reagents for transformation and/or transfection are commercially available [e.g., Lipofectin (Invitrogen Life Technologies); Fugene (Roche, Indianapolis, Ind.); and SuperFect (Qiagen, Valencia, Calif.)]. A host cell (e.g., a prokaryotic cell or a eukaryotic cell such as a COS cell) can be used to, for example, produce the costimulatory polypeptides provided herein. In some embodiments, a host cell (e.g., an APC) can be used to express the costimulatory polypeptides of the invention for presentation to a T cell.
4. Methods
The invention provides methods that include using variant costimulatory polypeptides to enhance T cell responses. The costimulatory molecules B7-H1 and B7-DC can bind to PD-1 (programmed cell death-1), a CD28 homolog with an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic domain (Ishida et al. (1992) EMBO J. 11:3887-3895). PD-1 is expressed on a subset of thymocytes and is up-regulated on T cells, B cells, and myeloid cells after their activation (Agata et al. (1996) Int. Immunol. 8:765-772).
PD-1 appears to be a negative regulator of immune responses in vivo. For example, PD-1−/− mice in the C57BL/6 background slowly developed a lupus-like glomerulonephritis and progressive arthritis (Nishimura et al. (1999) Immunity 11:141-151). Additionally, PD-1−/− mice in the BALB/c background rapidly developed a fatal autoimmune dilated cardiomyopathy (Nishimura et al. (2001) Science 291:319-322). Evidence also indicates, however, that both B7-H1 and B7-DC can function to costimulate a T cell response. In the presence of suboptimal TCR signals, B7-H1 or B7-DC can stimulate increased proliferation and production of cytokines in vitro. In addition, infusion of a B7-H1Ig fusion polypeptide can increase CD4 T cell responses and Th-dependent humoral immunity. Thus, B7-H1 and B7-DC may also bind to T cell receptors other than PD-1. Indeed, it has been shown that B7-H1 expressed on tumor cells can actively inhibit immune responses by promoting the apoptosis of effector CTLs, and the apoptotic effect of B7-H1 is mediated largely by receptors other than PD-1 (Dong et al. (2002) Nature Med. 8:793-800).
The experiments described in the Examples below indicate that the costimulatory activity of B7-H1 and B7-DC, and the described variants of each, is not mediated by the PD-1 receptor. Thus, the invention provides methods for using a variant costimulatory polypeptide with reduced affinity for PD-1 to stimulate a T cell response. The methods can include contacting a T cell with a purified variant costimulatory polypeptide. The contacting can be in vitro, ex vivo, or in vivo (e.g., in a mammal such as a mouse, rat, rabbit, dog, cow, pig, non-human primate, or a human).
The contacting can occur before, during, or after activation of the T cell. Typically, contacting of the T cell with variant costimulatory polypeptide can be at substantially the same time as activation. Activation can be, for example, by exposing the T cell to an antibody that binds to the T cell receptor (TCR) or one of the polypeptides of the CD3 complex that is physically associated with the TCR. Alternatively, a T cell can be exposed to either an alloantigen (e.g., a MHC alloantigen) on, for example, an APC [e.g., an interdigitating dendritic cell (referred to herein as a dendritic cell), a macrophage, a monocyte, or a B cell] or an antigenic peptide produced by processing of a protein antigen by any of the above APC and presented to the T cell by MHC molecules on the surface of the APC. The T cell can be a CD4+ T cell or a CD8+ T cell.
In some embodiments, a purified variant costimulatory polypeptide can be administered directly to a T cell. Alternatively, an APC such as a macrophage, monocyte, interdigitating dendritic cell (referred to herein as a dendritic cell), or B cell can be transformed, transduced, or transfected with a nucleic acid containing a nucleotide sequence that encodes a variant costimulatory polypeptide, and the T cell can be contacted by the transformed, transduced, or transfected APC. The transformed, transduced, or transfected cell can be a cell, or a progeny of a cell that, prior to being transformed, transduced, or transfected, was obtained from the subject to which it is administered, or from another subject (e.g., another subject of the same species).
The variant costimulatory polypeptide can be any of those described herein. For example, a B7-H1 polypeptide can contain a substitution at position 67, position 126, position 37, position 115, or position 124 (e.g., an alanine substitution for phenylalanine at position 67, an alanine substitution for isoleucine as position 126, a tyrosine substitution for threonine at position 37, an alanine substitution for isoleucine at position 115, or a serine substitution for lysine at position 124), each with respect to SEQ ID NO:4. Alternatively, the variant costimulatory polypeptide can be, for example, a B7-DC polypeptide containing a substitution at position 111, 113, 56, 67, 71, 101, or 105 (e.g., a serine substitution for aspartic acid at position 111, a serine substitution for lysine at position 113, a serine substitution for arginine at position 56, a tyrosine substitution for serine at position 67, a serine substitution for glutamic acid at position 71, a serine substitution for arginine at position 101, or an alanine substitution for isoleucine at position 105), each with respect to SEQ ID NO:6.
If the activation is in vitro, the variant B7-H1 molecule or variant B7-DC molecule can be bound to the floor of a the relevant culture vessel, e.g. a well of a plastic microtiter plate.
In vitro application of the purified variant costimulatory polypeptides can be useful, for example, in basic scientific studies of immune mechanisms or for production of activated T cells for use in studies of T cell function or, for example, passive immunotherapy. Furthermore, variant B7-H1 and variant B7-DC polypeptides can be added to in vitro assays (e.g., T cell proliferation assays) designed to test for immunity to an antigen of interest in a subject from which the T cells were obtained. Addition of variant costimulatory polypeptides to such assays would be expected to result in a more potent, and therefore more readily detectable, in vitro response. Moreover, a variant B7-H1 or B7-DC polypeptide, or an APC transformed, transfected, or transduced with a nucleic acid encoding such a polypeptide, can be used: (a) as a positive control in an assay to test for co-stimulatory activity in other molecules; or (b) in screening assays for compounds useful in inhibiting T costimulation (e.g., compounds potentially useful for treating autoimmune diseases or organ graft rejection).
The variant B7-H1 and variant B7-DC polypeptides provided herein are generally useful as in vivo and ex vivo as immune response-stimulating therapeutics. For example, the polypeptides of the invention can be used for treatment of disease conditions characterized by immunosuppression, such as, without limitation, cancer, AIDS or AIDS-related complex, other virally or environmentally-induced conditions, and certain congenital immune deficiencies. The polypeptides also can be employed to increase immune function that has been impaired by the use of radiotherapy of immunosuppressive drugs (e.g., certain chemotherapeutic agents), and therefore can be particularly useful when given in conjunction with such drugs or radiotherapy. In addition, variant B7-H1 molecules can be used to treat conditions involving cellular immune responses, including, for example, inflammatory conditions (e.g., conditions induced by infectious agents such as Mycobacterium tuberculosis or M. leprae), or other pathologic cell-mediated responses such as those involved in autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, or insulin-dependent diabetes mellitus).
Methods of the invention can be applied to a wide range of species, e.g., humans, non-human primates, horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice.
In some in vivo approaches, a variant B7-H1 or variant B7-DC polypeptide (or a functional fragment thereof) itself is administered to a subject in a therapeutically effective amount. Typically, polypeptides of the invention can be suspended in a pharmaceutically-acceptable carrier. Pharmaceutically acceptable carriers are biologically compatible vehicles (e.g., physiological saline) that are suitable for administration to a human. A therapeutically effective amount is an amount of a variant costimulatory polypeptide that is capable of producing a medically desirable result (e.g., an enhanced T cell response) in a treated animal. Variant costimulatory polypeptides can be administered orally or by intravenous infusion, or injected subcutaneously, intramuscularly, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily. The variant costimulatory polypeptides can be delivered directly to an appropriate lymphoid tissue (e.g., spleen, lymph node, or mucosal-associated lymphoid tissue). The dosage required typically depends on the choice of the route of administration, the nature of the formulation, the nature of the subject's illness, the subject's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending physician. Suitable dosages typically are in the range of 0.01-100.0 μg/kg. Wide variations in the needed dosage are to be expected in view of the variety of polypeptides and fragments available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Encapsulation of a purified variant costimulatory polypeptide in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.
Alternatively, a nucleic acid containing a nucleotide sequence encoding a variant B7-H1 or B7-DC polypeptide or functional fragment thereof can be delivered to an appropriate cell of an animal. Expression of the coding sequence typically is directed to lymphoid tissue of the subject by, for example, delivery of the nucleic acid to the lymphoid tissue. This can be achieved by, for example, using a polymeric, biodegradable microparticle or microcapsule delivery vehicle, sized to optimize phagocytosis by phagocytic cells such as macrophages. For example, PLGA (poly-lacto-co-glycolide) microparticles approximately 1-10 μm in diameter can be used. The nucleic acid can be encapsulated in these microparticles, which are taken up by macrophages and gradually biodegraded within the cell, thereby releasing the nucleic acid. Once released, the nucleic acid can be expressed within the cell. A second type of microparticle that also can be useful is intended not to be taken up directly by cells, but rather to serve primarily as a slow-release reservoir of nucleic acid. The nucleic acid is taken up by cells only upon release from the microparticle through biodegradation. Such polymeric particles typically are large enough to preclude phagocytosis (i.e., larger than 5 μm, and typically larger than 20 μm).
Another way to achieve uptake of a nucleic acid is to use liposomes, which can be prepared by standard methods known in the art, for example. Nucleic acid vectors can be incorporated alone into these delivery vehicles or can be co-incorporated with tissue-specific antibodies. Alternatively, a molecular conjugate can be prepared to contain a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to a receptor on target cells (Cristiano et al. (1995) J. Mol. Med. 73:479-486). Alternatively, lymphoid tissue specific targeting can be achieved using lymphoid tissue-specific transcriptional regulatory elements (TREs) such as a B lymphocyte-, T lymphocyte-, or dendritic cell-specific TRE. Lymphoid tissue specific TREs include, for example, those known in the art [see, e.g., Thompson et al. (1992) Mol. Cell. Biol. 12:1043-1053; Todd et al. (1993) J. Exp. Med. 177:1663-1674; and Penix et al. (1993) J. Exp. Med. 178:1483-1496]. Delivery of “naked DNA” (i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site is another means to achieve in vivo expression.
For in vivo expression of the variant costimulatory polypeptides provided herein, short amino acid sequences can act as signals to direct the polypeptides to specific intracellular compartments. For example, hydrophobic signal peptides having amino acid sequences such as MAISGVPVLGFFIIAVLMSAQESWA (SEQ ID NO:19) are found at the amino terminus of proteins destined for the ER. In addition, amino acid sequences such as KFERQ (SEQ ID NO:20) and other closely related sequences can target intracellular polypeptides to lysosomes, while amino acid sequences such as, for example, MDDQRDLISNNEQLP (SEQ ID NO:21) can direct polypeptides to endosomes. In addition, the amino acid sequence KDEL (SEQ ID NO:22) can act as a retention signal for the ER. Each of these signal peptides, or a combination thereof, can be used to traffic the variant B7-H1 and variant B7-DC polypeptides of the invention to specific cellular compartments. DNAs encoding the variant B7-H1 and B7-DC polypeptides containing targeting signals can be generated by, for example, PCR or other standard genetic engineering or synthetic techniques.
Similar to polypeptides, a therapeutic amount of a nucleic acid can be administered in a pharmaceutically acceptable carrier such as, for example, physiological saline. A therapeutically effective amount of a nucleic acid is an amount of the nucleic acid that is capable of producing a medically desirable result (e.g., an enhanced T cell response) in a treated animal. As described above, dosages will vary. Typical dosages for administration of nucleic acids are from approximately 106 to 1012 copies of the nucleic acid molecule. This dose can be repeatedly administered, as needed. Routes of administration can be any of those listed above.
Ex vivo methods can include, for example, the steps of harvesting cells from a subject, culturing the cells, transducing them with an expression vector, and maintaining the cells under conditions suitable for expression of the variant costimulatory polypeptides provided herein. These methods are known in the art of molecular biology. The transduction step can be accomplished by any standard means used for ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymeric microparticles can be used. Cells that have been successfully transduced then can be selected, for example, for expression of the coding sequence or of a drug resistance gene. The cells then can be lethally irradiated (if desired) and injected or implanted into the subject.
In some ex vivo methods, peripheral blood mononuclear cells (PBMC) can be withdrawn from a patient or a suitable donor and exposed ex vivo to an activating stimulus (see above) and a variant costimulatory polypeptide (whether in soluble form or attached to a solid support by standard methodologies). The PBMC containing highly activated T cells then can be introduced into the same or a different patient.
An alternative ex vivo strategy can involve transfecting or transducing cells obtained from a subject with a nucleic acid encoding a variant B7-H1 or B7-DC polypeptide. The transfected or transduced cells then can be returned to the subject. While such cells typically would be hemopoietic cells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells, or B cells), they can also be any of a wide range of types including, without limitation, fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells in which they act as a source of the variant B7-H1 or B7-DC polypeptide for as long as they survive in the subject. The use of hemopoietic cells, including the above APC, can be particularly useful, as such cells typically are expected to home to, among others, lymphoid tissue (e.g., lymph nodes or spleen) and thus the variant B7-H1 or B7-DC polypeptide can be produced in high concentration at the site where its effect (i.e., enhancement of an immune response) is exerted. In addition, if APC are used, the APC expressing the exogenous variant costimulatory molecule can be the same APC that present an alloantigen or antigenic peptide to the relevant T cell. The variant B7-H1 or variant B7-DC can be secreted by the APC or expressed on its surface.
Methods for inhibiting interactions of B7-H1 or B7-DC with PD-1 also are provided herein. These methods can include contacting a PD-1 polypeptide with a fragment of B7-H1 or B7-DC. The fragment can contain, for example, amino acids 67-69 of B7-H1, amino acids 113-115 of B7-H1, or amino acids 124-126 of B7-H1, each with respect to SEQ ID NO:4. Alternatively, the fragment can contain, for example, amino acids 67-71 of B7-DC, amino acids 101-105 of B7-DC, or amino acids 111-113 of B7-DC, each with respect to SEQ ID NO:6.
The PD-1 polypeptide can be contacted in vitro or in vivo (e.g., in a mammal such as a mouse, rat, rabbit, dog, cow, non-human primate, or human). The polypeptide fragment can be administered directly, or the method can include administering to a mammal a nucleic acid containing a nucleotide sequence encoding the polypeptide fragment. In some embodiments, the method can include administering to a mammal a cell, or the progeny of a cell, that has been transformed, transduced, or transfected with a nucleic acid encoding the polypeptide fragment. The cell can be a cell, or a progeny of a cell that, prior to being transformed, transduced, or transfected, was obtained from the mammal to which the cell is administered.
5. Articles of Manufacture
The invention also provides articles of manufacture that can contain the variant costimulatory polypeptides, functional fragments of costimulatory polypeptides, and/or inhibitory fragments of costimulatory polypeptides provided herein. Articles of manufacture also can include the nucleic acids and host cells provided herein. For example, an article of manufacture can include a variant B7-H1 or B7-DC polypeptide, or a nucleic acid encoding a variant B7-H1 or variant B7-DC polypeptide, packaged in a container (e.g., a vial). In addition, such an article of manufacture can include a label or instructions indicating that the polypeptide or nucleic acid can be used to enhance T cell activation. Alternatively, an article of manufacture can include a fragment of a B7-H1 or B7-DC polypeptide packaged in a container, and also can include a label or instructions indicating that the fragment can be used to inhibit binding to PD-1.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Mice and Cell lines: Female C57BL/6 (B6) mice were purchased from the National Cancer Institute (Frederick, Md.). PD-1-deficient (PD-1−/−) mice were generated as described previously (Nishimura et al. (1998) Int. Immunol. 10:1563-1572). Stably transfected Chinese hamster ovary (CHO) cell clones secreting fusion proteins were maintained in CHO-SF II medium (Invitrogen Life Technologies) supplemented with 1% dialyzed fetal bovine serum (FBS; HyClone, Logan, Utah). Lymphocytes and COS cells were grown in Dulbecco's modified Eagle medium (DMEM; Invitrogen Life Technologies) supplemented with 10% FBS, 25 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% MEM nonessential amino acids, 100 U/ml penicillin G, and 100 μg/ml streptomycin sulfate.
Ig Fusion Proteins: Fusion proteins containing the extracellular domain of mouse PD-1 linked to the Fc portion of mouse IgG2a (PD-1Ig) were produced in stably transfected CHO cells and purified by protein G affinity column as described previously (Wand et al. supra). Total RNA was isolated from mouse spleen cells and B7-DC cDNA was obtained by reverse-transcription PCR. B7-H1Ig and B7-DCIg were prepared by transiently transfecting COS cells with a plasmid containing a chimeric cDNA that included the extracellular domain of mouse B7-H1 (Tamura et al. supra) or B7-DC linked in frame to the CH2-CH3 portion of human IgG1. The transfected COS cells were cultured in serum-free DMEM, and concentrated supernatants were used as sources of Ig fusion proteins for initial binding assays. The Ig proteins were further purified on a protein G column for BIAcore analysis and functional assays as described previously (Wand et al. supra).
Molecular modeling: Molecular models of the Ig V-type domains of human B7-H1 (hB7-H1), mouse B7-H1 (mB7-H1), human B7-DC (hB7-DC), and mouse B7-DC (mB7-DC) were generated by homology (or comparative) modeling based on X-ray coordinates of human CD80 and CD86, as seen in the structures of the CD80/CTLA-4 and CD86/CTLA-4 complexes. First, the V-domains of CD80 and CD86 were optimally superimposed, and sequences of B7 family members were aligned based on this superimposition. The superimposition and initial alignments were carried out using the sequence-structure alignment function of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Quebec, Canada). The alignment was then manually adjusted to match Ig consensus positions and to map other conserved hydrophobic residues in the target sequences to core positions in the X-ray structures. Corresponding residues in the aligned sequences thus were predicted to have roughly equivalent spatial positions. Taking this kind of structural information into account typically is a more reliable alignment criterion than sequence identity alone if the identity is low, as in this case. In the aligned region, the average identity of the compared B7 sequences relative to the two structural templates, CD80 and CD86, was only approximately 16%. The final version of the structure-oriented sequence alignment, which provided the basis for model building, is shown in
Site-directed Mutagenesis: All mutants of B7-H1Ig and B7-DCIg were constructed using a two-step PCR technique, in which B7-H1Ig and B7-DCIg cDNAs, respectively, were used as templates. Overlapping oligonucleotide primers were synthesized to encode the desired mutations, and two flanking 5′ and 3′ primers were designed to contain EcoR I and Bgl II restriction sites, respectively. Appropriate regions of the cDNAs initially were amplified using the corresponding overlapping and flanking primers. Using the flanking 5′ and 3′ primers, fragments with overlapping sequences were fused together and amplified. PCR products were digested with EcoR I and Bgl II and ligated into EcoR I/Bgl II-digested pHIg vectors. To verify that the desired mutations were introduced, each mutant was sequenced using an ABI Prism 310 Genetic Analyzer. Plasmids were transfected into COS cells, and serum-free supernatants were harvested and used for in vitro binding assays or purified on a protein G column for BIAcore analysis and functional assays.
ELISA: A sandwich ELISA specific for B7-H1Ig and B7-DCIg was established as described previously. Briefly, microtiter plates were coated with 2 μg/ml goat anti-human IgG (Sigma, St. Louis, Mo.) overnight at 4° C. Wells were blocked for 1 hour with blocking buffer (10% FBS in PBS) and washed with PBS containing 0.05% Tween 20 (PBS-Tween). COS cell culture supernatants were added and incubated for 2 hours at room temperature. Known concentrations of purified B7-H1Ig also were added to separate wells on each plate for generation of a standard curve. After extensive washing, horseradish peroxidase (HRP)-conjugated goat anti-human IgG (TAGO, Inc., Burlingame, Calif.) diluted 1:2000 was added and subsequently developed with TMB substrate before stopping the reaction by the addition of 0.5 M H2SO4. Absorbance was measured at 405 nm on a microtiter plate reader. Concentrations of mutant fusion proteins were determined by comparison with the linear range of a standard curve of B7-H1Ig. Data from triplicate wells were collected, and the standard deviations from the mean were <10%. Experiments were repeated at least three times.
The ability of mutant and wild type B7-H1Ig and B7-DCIg fusion polypeptides to bind PD-1 was measured using a capture ELISA assay. Recombinant PD-1Ig fusion proteins were coated on microtiter plates at 5 μg/ml overnight at 4° C. The plates were blocked and washed, and COS cell culture media was added and incubated for 2 hours at room temperature. After extensive washing, HRP-conjugated goat anti-human IgG was added, followed by TMB substrate and measurement of absorbance at 405 nm.
Flow Cytometry: Human embryonal kidney 293 cells were transfected with a PD-1GFP vector, which was constructed by fusing GFP (green fluorescent protein cDNA) in frame to the C terminal end of a full-length mouse PD-1 cDNA. The cells were harvested 24 hours after transfection and incubated in FACS (fluorescence activated cell sorting) buffer (PBS, 3% FBS, 0.02% NaN3) with equal amounts of fusion proteins, which had been titrated using wild type B7-H1Ig and B7-DCIg, in COS cell culture media on ice for 45 minutes. An unrelated fusion protein containing human Ig was used as a negative control. The cells were washed, further incubated with fluorescein isothiocyanate (PE)-conjugated goat anti-human IgG (BioSource, Camarillo, Calif.), and analyzed on a FACScaliber (Becton Dickinson, Mountain View, Calif.) with Cell Quest software (Becton Dickinson). GFP-positive cells were gated by FL1.
Surface Plasmon Resonance Analysis: The affinity of purified wild type and mutant B7-H1 and B7-DC polypeptides was analyzed on a BIACOR™ 3000 instrument (Biacore AB, Uppsala, Sweden). All reagents except fusion proteins were purchased, pre-filtered, and degassed from BIAcore. All experiments were performed at 25° C. using 0.1 M HEPES, 0.15 M NaCl (pH 7.4) as a running buffer. Briefly, PD-1Ig was first immobilized onto a CM5 sensor chip (BIAcore) by amine coupling according to the BIAcore protocol. A flow cell of the CM5 chip was derivatized through injection of a 1:1 EDC:NHS [N-ethyl-N′-(diethylaminopropyl) carbodiimide:N-hydroxysuccinimide] mixture for seven minutes, followed by injection of 20 μg/ml of PD-1Ig at 10 μl/min diluted in 10 mM sodium acetate (pH 4.5). The PD-1Ig was immobilized at 2000 RUs. This was followed by blocking the remaining activated carboxyl groups with 1 M ethanolamine (pH 8.5). A control flow cell was prepared in a similar fashion as above, substituting running buffer alone in place of PD-1Ig. The fusion proteins were diluted in running buffer in a concentration series of 3.75, 7.5, 15, 30, and 60 μg/ml. The proteins were injected at a flow rate of 20 μl/min for 3 minutes, and buffer was allowed to flow over the surface for 5 minutes for dissociation data. The flow cells were regenerated with a single 30-second pulse of 10 mM NaOH. Data analysis was performed using BIAevaluation software package 3.1 (BIAcore).
T cell proliferation and cytokine assays: T cells from wild type B6 mice or PD-1−/− mice were purified using nylon wool columns (Robbins Scientific Co, Sunnyvale, Calif.) as described previously (Wang et al. supra). The enriched T cells were cultured at 3×105 cells per well in flat-bottomed 96-well microplates that were pre-coated with anti-CD3 mAb (clone 145-2C11, Pharmingen, San Diego, Calif.) in the presence of 5 μg/ml of fusion or control polypeptides. Proliferation of T cells was determined by incorporation of 1 μCi/well 3H-TdR during the last 12 hours of the 3-day culture. 3H-TdR incorporation was counted using a MICROBETA® Trilux liquid scintillation counter (Wallac, Finland). To detect cytokine, culture supernatants were collected at various time points, and the concentration of IFN-γ was measured by sandwich ELISA following the manufacturer's instructions (Pharmingen).
The V-regions in CD80 and CD86 share only limited sequence identity (approximately 20%), but their three-dimensional structures are very similar as revealed by independent crystallographic studies. Many core or Ig superfamily consensus residue positions seen in CD80/CD86 also are conserved or conservatively replaced in other B7 family members, including B7-H1 and B7-DC (
Molecular models of mouse and human B7-H1 and B7-DC molecules were constructed. These models revealed that in the V-regions, B7-H1 and B7-DC share more sequence identity than average across the B7 family—approximately 34%. Since both B7-H1 and B7-DC bind PD-1, residue conservation could be significant for formation of the receptor binding structure. Therefore, the models were used to compare the putative distribution of conserved residues that are exposed on the protein surface. A side-by-side comparison of these molecular models revealed significant conservation of surface residues on the BED faces of B7-H1 and B7-DC, more so in the human than the mouse proteins. In contrast, the opposite A′GFCC′C″ faces did not display significant residue conservation. This result was somewhat unexpected because the corresponding A′GFCC′C″ faces of both CD80 and CD86 contain the CD28/CTLA-4 binding sites.
With the aid of the molecular models, the V-domains of B7-H1 and B7-DC were scanned for important residues. Conserved and non-conserved residues on both the BED and A′GFCC′C″ faces were selected for site-specific mutagenesis. Residues in the mouse molecules were mutated to enable subsequent functional studies of selected mutant proteins. The binding characteristics of the resulting mutant proteins were assessed by specific ELISA and FACS analysis for binding to PD-1. A total of 21 mB7-H1 and 17 mB7-DC mutants were prepared and tested. The results are summarized in Tables 1 and 2. Particular residues within mB7-H1 and mB7-DC were only considered to be important for ligand-receptor interactions if their mutation caused at least a 50% loss of binding by FACS, or at least an order of magnitude loss by ELISA.
Mutation of about half of these residues significantly abolished binding to mPD-1. In particular, mB7-H1 residues F67, I115, K124, and I126 were identified as important for binding to mPD1. For mB7-DC, the identified residues were E71, I105, D111, and K113. All of these important residues are located in the C′, C″, F, and G strands of the A′GFCC′C″ face, but not in the BED face. Mutation of three residues in mB7-H1 (E58, A69, and C113) increased binding to mPD-1 three- to four-fold as determined by ELISA. Thus, these positions must at least be proximal to the receptor-ligand interface and have not only some tolerance for substitution but also potential optimization of binding interactions.
aAmino acids are numbered from the initiation methionine.
bThe nucleotides or amino acids in front of the slash were replaced with the
cSpecific binding activities were determined for each of the indicated fusion proteins.
The concentration of a fusion protein required to give an Arbitrary A405 the same as that found for wild type B7-H1Ig was determined from the linear region of binding curves and expressed as a percentage of specific binding activity of B7-H1Ig. Values represent the average of three determinations from each binding curve, and are representative of four experiments.
aAmino acids are numbered from the initiation methionine.
bThe nucleotides or amino acids in front of the slash were replaced with the
cSpecific binding activities were determined for each of the indicated fusion proteins.
The concentration of a fusion protein required to give an arbitrary A405 the same as that found for wild type B7-DCIg was determined from the linear region of binding curves and expressed as a percentage of specific binding activity of B7-DCIg. Values represent the average of three determinations from each binding curve, and are representative of four experiments.
Residues in mB7-H1 and mB7-DC that significantly reduced or abolished PD-1 when mutated were clustered in equivalent regions of the A′GFCC′C″ face of the domains (with one exception in each case), although they often were distant in sequence. In contrast, mutation of conserved residues on the BED face did not reduce binding. Thus, residue conservation on the BED face may be important for other effects such as ligand dimerization or recognition of other proteins.
The PD-1 binding sites mapped to equivalent regions on the opposite A′GFCC′C″ face. Mapping of the binding site regions revealed that residues whose mutation negatively (or positively) affected PD-1 binding could form coherent surfaces in both ligands. The proximity of important residues and some residues not important for binding again suggested that the observed effects were specific, and were not a consequence of global structural changes. This was further supported by the ability to produce higher avidity mutants of mB7-H1. Comparison of important residue positions confirmed that the location of the putative binding sites in mB7-H1 and mB7-DC closely corresponded to the CD28/CTLA-4 binding sites in CD86 and CD80.
Surface plasmon resonance analysis of binding of wild type and mutant proteins to PD-1 was largely consistent with the results from the FACS and ELISA analyses. The B7-H1 mutant C113 had a similar or slightly higher maximal response (Rmax) value than the wild type protein (321 resonance units (RU) vs. 291 RU), while the mutant I126 (56.1 RU) showed relatively minimal binding to mPD-1 (
The costimulatory potential of selected mutants also was tested. B7-H1 mutants F67 and I126, and B7-DC mutants K113 and D111 were selected for analysis. Both F67 and I126 had minimal binding to PD-1 in both FACS and ELISA assays (Table 1). Similarly, K113 and D111 did not bind PD-1 (Table 2). As shown in
Although B7-H1 and B-DC might costimulate T cell growth through a yet unknown receptor, these findings could be interpreted as an integrated stimulatory effect of unidentified costimulatory receptor(s) and PD-1. Therefore, the costimulatory effects of these mutants were tested in PD-1 deficient T cells. Wild type and variant B7-H1 polypeptides costimulated proliferation of PD-1−/− T cells as well as or better than PD-1 cells (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a divisional of U.S. Ser. No. 11/932,471, filed Oct. 31, 2007, which is a divisional of U.S. Ser. No. 10/679,775, filed Oct. 6, 2003, now U.S. Pat. No. 7,432,351, which claims priority from U.S. Provisional Application Ser. No. 60/416,203, filed Oct. 4, 2002.
This invention was made with government support under grant no. CA97085, awarded by the National Institutes of Health. The government has certain rights in the invention.
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