KR20020001865A - Use of soluble costimulatory molecules to enhance immune responses - Google Patents

Abstract

Methods of enhancing immune response are provided in which a soluble form of a costimulatory molecule, eg, a B7 molecule, is administered to enhance an immune response against an antigen, eg, tumor cells and an infectious agent. The methods of the invention are useful for both prophylactic and therapeutic immunization of a subject.

Description

Use of soluble co-stimulatory molecules to enhance immune response {USE OF SOLUBLE COSTIMULATORY MOLECULES TO ENHANCE IMMUNE RESPONSES}

1 depicts the antigen specific proliferative response of cells from mice immunized with class II-restricted peptides with or without B7-2Ig (100 μg).

FIG. 2 shows that cells from a single B7-2Ig treated mouse at the time of primary immunization have more proliferative responses than cells from mice not receiving B7-2Ig following secondary immunization.

3 shows that co-administration of B7-2Ig augments CTL responses to immunization with class I restriction peptides.

4 depicts the CTL response of mice immunized with class I restriction peptides with or without class II restriction peptides and B7-2 Ig treatment.

FIG. 5 shows that B7-IgG provides costimulatory signals for in vitro augmentation and secretion of lymphokines in splenocytes.

6 shows that B7Ig is effective as an adjuvant in a prophylactic tumor vaccine model.

FIG. 7 shows that therapeutic vaccination of mice with irradiated P815 tumor cells mixed with B7-1- or B7-2-IgG induces tumor regression and prolongation of viability.

8 shows that vaccination of mice with B7-IgG as an adjuvant for therapeutically investigated tumor cell vaccines is effective in several different mouse tumor models.

9 shows that the antitumor effect of therapeutic administration of B7-IgG alone in mice is comparable to its effect as a vaccine adjuvant.

10 shows that T or B cells are essential for B7-IgG-mediated antitumor activity.

11 shows that CD8 + T cells but not CD4 + are essential for B7-IgG antitumor activity.

12 shows that B7-IgG treatment of established tumors is independent of host IFN-γ.

Detailed description of the invention

The present invention provides an improved method of enhancing an immune response by administering a soluble costimulatory molecule (eg, an extracellular domain of the B7 molecule or a B7 fusion protein) to enhance the immune response. Soluble costimulatory molecules are administered without crosslinkers but surprisingly stimulate T cell responses. Indeed, Applicants have discovered that the methods of the present invention result in increased levels of costimulatory molecules expressed on the cell surface, eg, costimulatory molecules on the cell surface.

Prior to further description of the invention, certain terms used in the specification, examples and claims are defined.

Justice

As used herein, “prophylactically” encompasses administering a costimulatory molecule prior to or concurrent with exposing to an antigen that causes the development, augmentation, and / or enhancement of an immune response.

As used herein, "therapeutically" includes the administration of a costimulatory molecule to treat an existing or ongoing infection or disease (eg, cancer or a viral or bacterial infection) that is efficacious by treatment with the costimulatory molecule. . For therapeutic treatment, the costimulatory molecule is administered after exposure to the antigen causing the development, augmentation, and / or augmentation of the immune response. It is understood that treatment with co-stimulatory molecules, for example, may not be specific for a particular antigen but may have other beneficial effects on the subject's immune response.

As used herein, “immune cells” include cells of hematopoietic cell origin and act in an immune response. Immune cells include lymphocytes such as B cells and T cells; Natural killer cells; Myeloid cells such as monosite, macrophages, eosinophils, mast cells, basophils, and granulocytes.

As used herein, “immune response” includes T cell and / or B cell responses, ie, cellular and / or humoral immune responses. In one embodiment, the proposed method can be used to reduce T helper cell response. In other embodiments, the proposed method can be used to reduce cytotoxic T cell responses. The proposed methods can be used to reduce primary and secondary immune responses. The subject's immune response can be measured, for example, by analyzing antibody production, immune cell augmentation, cytokine release, expression of cell surface markers, cytotoxicity, and the like.

As used herein, “co-stimulation” in the context of activated immune cells includes the ability of a co-stimulatory molecule to provide a second, non-activating receptor mediated signal (“co-stimulation signal”) that induces proliferation or effector function. do. For example, costimulatory signals can result in cytokine secretion in T cells that receive, for example, T cell-receptor-mediated signals. As used herein, a “costimulatory molecule” refers to a costimulatory receptor on T cells (eg, CD28, CTLA4, ICOS (Ref. Hutloff et al. 1999. Nature 397: 263), B7h ligand (Ref. Swallow et al) Immunity. 11: 423) and / or antigen-expressing cells that bind to related molecules (eg, B7-1, B7-2, B7RP-1 (see Yoshinaga et al. 1999. Nature 402: 827). ), B7 (Refer to Swallow et al. 1999. Immunity. 11: 423) and / or molecules present on related molecules (eg, homologues.) These molecules are also collectively referred to as "B7 molecules. Is referred to herein.

As used herein, “B7” or “B7 molecule” refers to naturally occurring B7-1 molecules, B7-2 molecules, B7RP-1 molecules (Ref. Yoshinaga et al. 1999. Nature 402: 827), B7h molecules (Ref. Swallow et al. 1999. Immunity. 11: 423), structurally related molecules, fragments of such molecules, and / or functional equivalents thereof. The term "equivalent" refers to an amino acid sequence encoding the activity of a B7 molecule, eg, a natural ligand of B7 on immune cells, such as a functionally equivalent costimulatory molecule with binding to CTLA4, ICOS, and / or CD28 on T cells. It is intended to include.

As used herein, "polypeptide" refers to any peptide or protein comprising two or more amino acids linked by peptide bonds or modified peptide bonds. "Polypeptides" include both short chains, oligopeptides and oligomers, commonly referred to as peptides, and longer chains, generally referred to as proteins. The polypeptide may comprise amino acids other than twenty gene encoded amino acids. A “polypeptide” includes modifications by natural processes such as modifications by processing and other post-translational modifications, as well as by chemical modification techniques. Such modifications are well described in the basic textbooks and are described in more detail in monographs and in multivolume research literature, which are known to those skilled in the art. The same type of modification may be present at the same or different degrees in several places in a given polypeptide. Modifications can occur anywhere in the polypeptide, including peptide backbones, amino acid side chains, and amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent bonds of flavins, covalent bonds of heme components, covalent bonds of nucleotides or nucleotide derivatives, covalent bonds of lipids or lipid derivatives, phosphatides Covalent bonds, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cysteines, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, Hydroxylation, iodide, methylation, myristoylation, oxidation, proteolysis, phosphorylation, prenylation, racemization, glycosylation, lipid binding, sulfated, gamma-carboxylation, hydroxylation, and glutamic acid residues and ADP-ribosylation, selenoylation, sulfated, t-RNA mediated addition of amino acids to proteins, including, for example, arginylation, and ubiquitination (see Protein Structure And Molecular Properties, 2nd Ed., TE Creighton, WH Freeman and Compay, New York (1993) and World, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Posttranslatonal Covalent Modifications Of Proteins, BC Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182: 626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and aging, Ann. NY Acad. Sci. 663: 48- 62 (1992) Polypeptides may be branched, branched or cyclic without branching Cyclic, branched, and branched cyclic polypeptides can be induced by post-translational natural processes and are fully synthetically prepared. Can be.

As used herein, an “isolated polypeptide” or “isolated protein” is a chemical precursor if other polypeptides, proteins, cellular materials and culture media are chemically synthesized when produced from a cell or produced by recombinant DNA techniques. A polypeptide or protein is substantially free of substances or other chemicals. A "isolated" or "purified" polypeptide or a biologically active portion thereof is a chemical precursor or other if the cellular material or other contaminating polypeptide from the tissue member from which the B7 polypeptide is derived is substantially free or chemically synthesized. It means that it is virtually free of chemicals. “Substantially free of cellular material” refers to the preparation of a B7 polypeptide, where the polypeptide is separated from the cellular component of the cell from which it is isolated or recombinantly produced. In one embodiment, the term “substantially free of cellular material” refers to non-B7-polypeptides (herein referred to as “polluting polypeptides”) less than about 30% (dry weight basis), even more preferably non- B7-polypeptides include preparations of B7 polypeptides having less than about 20%, even more preferably less than about 10% non-B7-polypeptide, and most preferably less than about 5% non-B7-polypeptide. When recombinantly produced a B7 polypeptide or biologically active portion thereof, the culture medium is substantially free, that is, the culture medium is less than about 20%, even more preferably less than about 10% by weight of the polypeptide, and Most preferably less than about 5%.

In one embodiment, the term “substantially free of chemical precursors or other chemicals” includes preparations of B7 polypeptides in which the polypeptide is isolated from chemical precursors or other chemicals involved in the synthesis of the polypeptide. In one embodiment, the term "substantially free of chemical precursors or other chemicals" means less than about 30% (by dry weight) of chemical precursors or non-B7 chemicals, even more preferably chemical precursors or non- Preparation of B7 polypeptide having less than about 20% of B7 chemical, even more preferably less than about 10% of chemical precursor or non-B7 chemical, most preferably less than about 5% of chemical precursor or non-B7 chemical It includes.

Preferably, the B7 nucleic acid molecule and polypeptide are "naturally occurring". As used herein, “naturally” molecule means a B7 molecule having a naturally occurring nucleotide sequence (eg, encodes a native B7 polypeptide). It also refers to naturally occurring or non-naturally occurring variants of these polypeptides and nucleic acid molecules that possess the same functional activity in microorganisms, such as the ability to modulate adaptability to stress and / or virulence. Such variants can be prepared, for example, by using mutation techniques known in the art. In addition, variants may be synthesized chemically.

As used herein, “variant” includes nucleic acid molecules or polypeptides that retain essential properties but differ from sequences from a reference nucleic acid molecule or polypeptide. Changing the nucleotide sequence of the variant does not or does not change the amino acid sequence of the polypeptide encoded by the reference nucleic acid molecule. Changes in nucleotides can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as described below. Typical polypeptide variants differ in amino acid sequence from the reference polypeptide. In general, these differences are limited to the extent that the reference polypeptide and the variant are very similar throughout and the same in many regions. Variants and reference polypeptides may be different by any combination of one or more substitutions, additions and / or deletions. Variants of nucleic acid molecules or polypeptides may be variants that are known to occur naturally or naturally, such as, for example, allelic variants. Non-naturally occurring variants of nucleic acid molecules and polypeptides can be prepared by mutagenesis techniques, direct synthesis, and other recombinant methods known to those skilled in the art.

For example, the B7 polypeptides described herein also include equivalents thereof. Such variants can be prepared, for example, by using techniques known in the art. In addition, variants may be synthesized chemically. For example, mutant forms of functionally equivalent B7 polypeptides (eg, having the ability to bind CTLA4 and / or CD28) can be prepared using techniques well known in the art. Mutations can include, for example, isolated point mutations that can result in one substitution, or by one or more deletions or insertions. For example, random mutagenesis can be used. Mutations can also be generated by random mutagenesis or utilize cassette mutagenesis. In the former case, the entire coding region of the molecule is mutagenesis by any of several methods (chemical, PCR, doped oligonucleotide synthesis), and randomly mutated molecules are subjected to selection and screening processes. In the latter case, an isolated region of the polypeptide corresponding to one of the defined structural or functional determinants is subjected to saturation or semi-random mutations and such mutated cassettes are reintroduced into the context of an allele that is otherwise wild-type. . In one embodiment, PCR mutagenesis can be used. For example, megaprimer PCR can be used (O.H. Landt, Gene 96: 125-128).

As used herein, "enhancing an immune response" includes increasing T and / or B cell responses, ie cellular and / or humoral immune responses, by treatment using the proposed method. In one embodiment, the proposed method can be used to enhance the T helper cell response. In other embodiments, the proposed methods can be used to enhance cytotoxic T cell responses. The proposed method can be used to enhance both primary and secondary immune responses. Preferably, the proposed method increased the immune response by the subject when compared to the immune response by the untreated subject or the subject not treated. An increase in the immune response can be presented, for example, by an increased response of immune cells to the antigen from the subject upon treatment with the proposed method. The subject's immune response can be measured by measuring immune cell activation in vivo or in vitro, for example, by analyzing antibody production, increase in immune cells, release of cytokines, expression of cell surface markers, cytotoxicity, and the like. have.

As used herein, "soluble" includes molecules such as costimulatory molecules not associated with a cell. Soluble costimulatory molecules retain the function of the cell-related molecules from which they are derived. That is, they can bind their cognate ligands on T cells and can modulate signal induction via CD28 and / or CTLA4 molecules on T cells, but they are in soluble form, ie not bound to the membrane. Preferably, the soluble composition comprises the extracellular domain of the B7 molecule.

As used herein, "extradomain domain of a costimulatory molecule" includes a portion of a costimulatory molecule that is extracellular as a cell-related form of a costimulatory molecule. Preferably, the extracellular domain of the costimulatory molecule comprises the extracellular domain of the B7 molecule. The B7 extracellular domain comprises a portion of a costimulatory molecule that regulates binding to CD28 and / or CTLA4. For example, the human B7-1 extracellular domain comprises about 1 to about 208 amino acids of the mature form of B7-1 (SEQ ID NO: 1), and the human B7-2 extracellular domain is B7-2 (SEQ ID NO: About 24 to about 245 amino acids of the mature form of 2). In one embodiment, the soluble costimulatory molecule comprises an extracellular domain of the B7 molecule and additionally a signal sequence.

As used herein, “class I restriction antigen” includes antigens that bind to major histocompatibility complex (MHC) class I grooves and are delivered to T cells within the context of MHC class I molecules. Class I restriction antigens primarily stimulate CD8 + T cells. As used herein, "class II restriction antigen" includes antigens that bind to major histocompatibility complex (MHC) class II grooves and are delivered to T cells within the context of MHC class II molecules. Class II restriction antigens primarily stimulate CD4 + T cells.

As used herein, "adjuvant" includes antigens that enhance an immune response to an antigen. Adjuvants can be administered in combination with costimulatory molecules to further enhance antigen response.

As used herein, “monospecificity” includes soluble costimulatory molecules having only one specificity, which specifically binds to their cognate ligands such as CD28 or CTLA4 on T cells. Such monospecific agents have not been engineered to include additional specificity and therefore do not bind with other cell surface molecules in a targeted manner. As used herein, “oligospecificity” includes costimulatory molecules having one or more specificities, eg, specificity for molecules other than B7 ligands, eg, cell surface molecules such as tumor cell antigens or specificity for T cell receptors. . As used herein, “bivalent” includes soluble costimulatory molecules having two binding sites for interacting with their cognate ligands, eg, CD28 and / or CTLA4, for one soluble costimulatory molecule. As used herein, "dimer" includes soluble forms that exist as homodimers, ie, one unit comprising two identical subunits joined together by, for example, disulfide bonds. As used herein, "multimer" includes soluble forms having two or more subunits.

II. Soluble co-stimulatory molecules

B7 antigens are B lymphocytes that are professional antigen expressing cells (e.g., monosite, dendritic cells, Langerhans cells) and cells providing antigens to immune cells (e.g. keratinocytes, endothelial cells, astrocytes, fibroblasts, Is a family of costimulatory molecules found on the surface of histoliocytes. These costimulatory molecules bind CTLA4, CD28, and / or ICOS on T cells or other known or not yet identified receptors on immune cells. Members of this class of costimulatory molecules induce T cell expansion and / or cytokine secretion by providing costimulation to activated T cells.

Purification techniques for B7 molecules have been established and also the B7 gene (cDNA) has been cloned from a number of species including humans and mice (see, eg, Freeman, GJ et al. (1993) Science 262: 909-911; Azuma, M. et al. (1993) Nature 366: 76-79; Freeman, GJ et al. (1993) J. Exp. Med. 178: 2185-2192).

The nucleotides of the costimulatory molecules are known in the art and can be found in the literature or in a database such as GenBank. For example, B7-2 (Ref. Freeman et al. 1993 Science. 262: 9090 or GeneBank Accession numbers P42081 or A48754); B7-1 (Ref. Freeman et al. J. Exp. Med. 1991. 174: 625 or GeneBank Accession numbers P33681 or A45803); CTLA4 (see, eg, Ginsberg et al. 1985. Science. 228: 1401; or GenBank Accession numbers P16410 or 291929); And CD28 (Aruffo and Seed. Proc Natl. Acad. Sci. 84: 8573or Genbank Accession number 180091), ICOS (Hultoff et al. 1991. Nature. 397: 263; WO 98/38216), and Related sequences can be found.

In addition to the naturally occurring form of the costimulatory molecule, the term “costimulatory molecule” also refers to a non-naturally occurring form of a costimulatory molecule that retains the function of the costimulatory molecule, eg, the ability to bind homologous counter receptors Variants or mutant forms. For example, conditions comparable to 65 ° C. in 5 × SSC (1 × SSC = 150 mM NaCl / 0.15 M Na citrate) under conditions that avoid hybridization with non-operating polar genes. Below), DNA sequences capable of hybridizing with DNA encoding B7 molecules can be used to prepare anti-B7 antibodies. In addition, a DNA sequence that retains sequence identity to a region of a nucleic acid molecule encoding the function of a costimulatory molecule, e.g., a protein domain important for binding to another costimulatory molecule, may be used as an immunogen. Can be used to produce. Preferably, the non-naturally occurring costimulatory molecule has significant amino acid identity with the amino acid sequence of the naturally occurring costimulatory molecule extracellular domain (eg, greater than 70%, preferably greater than 80% and more More preferably 90-95%).

To determine the amino acid residues of a costimulatory molecule that appear to be important for binding the costimulatory molecule and its counter receptor, the amino acid sequence comprising the extracellular domains of the co-stimulatory molecules of other species, eg, mouse and human, is aligned. And conserved (eg, identical) residues can be noted. This can be done, for example, by using any standard alignment program such as Meg-Align (DNA STAR). Such an alignment program is described in more detail below. Such conserved or identical residues appear to be essential for the costimulatory molecules to bind to their receptors and therefore do not appear to be altered.

For example, regions of the B7-1 molecule important for regulating functional interactions with CD28 and CTLA4 have been identified by mutations. Two hydrophobic residues in the V-type domain of B7-1, including Y87 residues and conserved among all B7-1 and B7-2 molecules, have been found to be very important (Refer to Fargeas et al. 1995. J. Exp. Ed. 182: 667). Using these or similar techniques, amino acid residues in the extracellular domain of the costimulatory molecule that are very important and therefore cannot be altered can be determined.

Using B7 cDNA molecules, peptides with B7 activity can be generated using standard techniques. The host cell transfected to express the peptide can be any prokaryotic or eukaryotic cell. For example, peptides with B7 activity are expressed in bacterial cells such as E. coli , insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO) and NS0 cells. Can be. Other suitable host cells and expression vectors can be found in this reference (Goeddel, 1990), which is known to those skilled in the art. Examples of the expression vector in Saccharomyces as MY process serenity non - (S. cerivisae) are pYepSec1 (reference:.. Baldari et al, ( 1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz, (1982 ) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Baculovirus vectors (SF 9Cells) that can be used for protein expression in cultured insect cells are pAc family (Ref .: Smith et al., (1983) Mol. Cell Biol. 3: 2156-2165) and pVL family (Lucklow, VA, and Summers, MD, (1989) Virology 170: 31-39). In general, COS cells (Gluzman, Y., (1981) Cell 23: 175-182) are combined with a vector such as pCDM8 (Seed, B., (1987) Nature 329: 840) for transient amplification / expression. While used, CHO cells (dhfr-Chinese Hamster Overy) are vectors such as pMT2PC for stable amplification / expression in mammalian cells (Kaufman et al. (1987), EMBO J. 6: 187-195). It can be used with. Preferred cell lines suitable for the production of recombinant proteins are NS0 myeloma cell lines available from ECACC (List # 85110503) and described in Galfre, G. and Milstein, C. ((1981) Methods in Enzymology 73 (13): 3- 46; and Preparation of Monoclonal Antibodies: Strategies and Procedures, Academic Press, NY, NY.Vector DNA is known techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated trans It can be introduced into mammalian cells via speculation, lipofectin, or electroporation Methods suitable for transformation of host cells can be found in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989), and in other experimental texts, when mammalian cells are used, the regulatory function of the expression vector is often provided by viral material. Rio e, Adenovirus 2, is derived from cytomegalovirus and is derived from the most common is the monkey virus 40 (SV40).

Peptides with B7 activity are expressed in mammalian cells, or ammonium sulfate precipitation, fractional column chromatography (eg, ion exchange, gel filtration, electrophoresis, affinity chromatography, etc.) and final crystallization (reference: " Enzyme Purification and Related Techniques ", Methods in Enzymology, 22: 233-577 (1971).

B7 molecules for preparing soluble B7 molecules for use in the methods of the invention can be derived from any mammalian species and preferably humans. The nucleotide sequence of B7 molecules from several sources is known in the art. The complete DNA sequence of human B7-1 (CD80) has GenBank No. M27533 and is disclosed in Freeman et al. In 1989 in J. Immunol. 143: 2714. The complete cDNA sequence of human B7-2 has GenBank No. L25259 and is disclosed in Freeman et al. In Science in 1993. 262; 9090 or Azuma et al. Nature. 1993. 366; 76. See WO 96/40915 for the sequences of both B7-1 and B7-2. The nucleotide sequences of human B7-1 and human B7-2 are also described in SEQ ID NOs: 1 and 2 (B7-1) and SEQ ID NOs: 3 and 4 (B7-2). In addition, such sequences can be measured by isolating B7 nucleic acid molecules from known origin based on the ability of the sequence to be known, for example, to human B7 sequences. For example, B7 molecules can be determined by their ability to hybridize with known nucleic acid molecules that encode peptides having B7 activity under high or low stringent conditions. Suitable stringent conditions that promote DNA hybridization, for example, 6.0 x sodium chloride / sodium citrate (SSC) at about 40 ° C. and subsequently 2.0 × SSC at 50 ° C. are known to those skilled in the art and are described in Current Protocols in Molecular Biology, John. Wiley & Sons, NY (1989), 6.3.1-6.3.6). For example, the salt concentration in the washing step can be selected from low stringent conditions of about 2.0 x SSC at 50 ° C to high stringent conditions of about 0.2 x SSC at 50 ° C. It may also be increased from room temperature at about 22 ° C. at high or low stringent conditions to about 65 ° C. at high stringent conditions in the washing step. In a preferred embodiment, the B7 molecule is a human B7 molecule.

In one embodiment, the soluble costimulatory molecule is derived from naturally occurring B7-1 or B7-2 molecules. As described herein, polypeptides having B7 molecular activity and having a sequence different from naturally occurring B7 molecules due to denaturation of the genetic code can be expressed as soluble forms, which are also within the scope of the present invention. Such nucleic acids encode polypeptides that are functionally equivalent to B7 (eg, polypeptides having B7 activity) or that differ from the sequences of B7-1 or B7-2 known in the art. For example, several amino acids are designed by one or more triplets. Codons that characterize the same amino acid, or synonyms (eg, CAU and CAC are synonyms of histidine) can be caused by denaturation in the genetic code. As an example, DNA sequence polymorphism (particularly within the third base of the codon) in the nucleotide sequence of the B7 molecule can result in a "silent" mutation that does not affect the encoded amino acid. However, DNA sequence polymorphisms that induce changes in the amino acid sequence of the B7 antigen are expected to be present in the population. Those skilled in the art will predict that these variants (up to about 3-4% of nucleotides) in one or more nucleotides of a nucleic acid encoding peptides having novel B lymphocyte antigen activity may be present in individuals within the population due to natural allelic variations. Can be. Such nucleotide variations and resulting amino acid polymorphisms are also within the scope of the present invention. In addition, they may be one or more isoforms or related cross-reactive B7 molecules.

In addition to naturally occurring allelic variants, B7 molecules within the scope of the present invention can be prepared using techniques known in the art. In another embodiment, the soluble costimulatory molecule is a modified form of B7-1 or B7-2 that retains the function of the B7 costimulatory molecule, ie, is functionally identical. The DNA sequence of the B lymphocyte antigen can be modified to produce a protein or polypeptide having an amino acid sequence altered by genetic techniques. Such sequences are considered to be within the scope of the present invention, wherein the expressed polypeptides can bind CTLA4 and / or CD28 and modulate T cell mediated immune responses and immune functions.

For example, in one embodiment, B lymphocytes by any one of several methods, wherein the mutation comprises a simple deletion or insertion, a global deletion, an insertion or substitution of a base cluster or a substitution of a single base. It can be introduced into the DNA molecule to produce variants or modified equivalents of the antigenic DNA. For example, changes in B7-1 or B7-2 cDNA sequences, such as amino acid substitutions or deletions, can preferably be obtained by site-directed mutagenesis. Site directed mutagenesis systems are known in the art. Protocols and reagents are commercially available from Amersham International PLC, Amersham, U.K.

B7 activity, ie binding to the natural ligand of the B7 molecule, is responsible for the T cell mediated immune response, as evidenced by its ability and augmentation of T cells by, for example, T cells that receive cytokine production and / or primary activation signals. Modified polypeptides with the ability to modulate are considered to be within the scope of the present invention.

Another example of modification of a peptide having the activity of a B7 molecule is to replace the cysteine residues with preferably alanine, serine, threonine, leucine or glutamic acid residues to minimize dimerization via disulfide bonds. In addition, amino acid side chains having B7 activity may be chemically modified. Another modification is the cyclization of the peptide.

To increase stability and / or reactivity, polypeptides with B7 activity can be modified to incorporate one or more polymorphisms in the amino acid sequence of an antigen resulting from any natural allelic variation. In addition, D-amino acids, non-natural amino acids, or non-amino acid analogs may be substituted or added to produce modified proteins within the scope of the present invention. Peptides can also be modified using polyethylene glycol (PEG) to produce peptides conjugated with PEG according to the methods of A. Sehon and co-workers (Weet et al., Supra). PEG can also be added during chemical synthesis of the peptide. Other modifications of the peptide include reduction / alkylation (Tarr in: Methods of Protein Microcharacterization, J. E. Silver ed., Humana Press, Clifton NJ 155-194 (1986)); Acylation (Tarr); Chemical bonds with a suitable carrier (Mishell and Shiigi, eds, Selected Methods in Cellular Immunology, WH Freeman, San Francisco, CA (1980), US Patent 4,939,239; or mild formalin treatment (Marsh (1971), Int.Arch. Of Allergy and Appl.Imunmun. 41: 199-215).

Preferred B7 polypeptides have at least about 60%, preferably at least about 70%, even more preferably at least about 80% identity with B7 activity and naturally occurring B7 amino acids. Polypeptides having at least about 90, preferably at least about 95%, and even more preferably at least about 98-99% identity to B7 activity and naturally occurring B7 amino acids are also within the scope of the present invention. Amino acid “identity” at a given position refers to two peptides having the same amino acid at the corresponding position when the amino acid sequence of the peptide is aligned. If any position in the compared sequences is occupied by the same amino acid, the molecules are identical at that position. The degree (or percentage) of identity of a sequence is a function of the number of positions matched or identical by the sequences.

One skilled in the art can easily align two amino acids to provide a biologically significant alignment. Comparison of sequences and determination of percent identity between two sequences can be achieved by using mathematical algorithms. In one embodiment, the percent identity between the two amino acid sequences is a needle incorporated into the GAP program in the GCG software package (available from http://www.gcg.com) using the Blowsome 62 matrix or the PAM250 matrix. Measured by using Needleman and Bunsch algorithms, and gap weights of 16, 14, 12, 10, 8, 6, 5 or 4 and length weights of 1, 2, 3, 4, 5 or 6 Can be. In another embodiment, the percent identity between two amino acids or nucleotides is incorporated in the ALIGN program (version 2.0). E. Meyers and W. By using the algorithm of W. Miller (CABIOS, 4: 11-17 (1989)), it can be measured using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

Nucleic acid and protein sequences of the invention can be used, for example, as "search sequences" to perform searches against known databases to identify members or related sequences of other families. This search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altscul, et al. (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleic acid sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the B7 protein molecules of the invention. To obtain a gapped alignment for comparison purposes, the gap BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using the BLAST and GAP BLAST programs, default variables of each program (eg XBLAST and NBLAST) can be used (reference website address: http://www.ncbi.nlm.nih.gov).

“At least a portion of the extracellular domain of the B7 molecule” refers to the entirety or activity of the extracellular domain sequence of B7 (ie binding to a native ligand of B7 on immune cells, eg binding to CTLA4 and / or CD28 on T cells). It is defined to include a portion of it that encodes a polypeptide having. Peptides with B7 activity bind CTLA4 and / or CD28 and bind T cell mediated immune responses to, eg, cytokines by T cells that bind their ligands or receive primary activation signals as shown in the Examples. Adjust as evidenced by inducing production and / or augmentation. In a preferred embodiment, “at least a portion of the extracellular domain of the B7 molecule” is the entirety of the extracellular portion of the human B7 antigen that can be used to bind CTLA4 and / or CD28 (eg, approximately of the sequence of B7-1 Polypeptides comprising amino acid residues 1-208 or amino acid residues 24-245 of the sequence of approximately B7-2.

In addition to B7 polypeptides comprising only naturally occurring amino acids, B7 peptidomimetics are also provided. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the template peptide. This type of non-peptide compound is termed "peptide mimetics" or "peptidomimetics" (reference: incorporated herein by reference, Fauchere, J. (1986) Adv. Drug Res. 15:29 Veber and Fridinger (1985) TINS p. 392; and Evans et al. (1987) J. Med. Chem 30: 1229), usually developed with the aid of computer-based molecular modeling.

Peptide mimetics, which are structures similar to therapeutically useful peptides, can be used to provide an equivalent therapeutic or prophylactic effect. In general, peptidomimetics are paradigm polypeptides (i.e., polypeptides having biological or pharmacological activity), for example, structurally similar to B7, but by methods known in the art CH2NH-, CH2S-, -CH2-CH2-, And one or more peptide bonds optionally substituted by a bond selected from the group consisting of -CH = CH- (cis and trans), -COCH2-, -CH (OH) CH2-, and -CH2SO-, each of which Incorporated by reference (Spatola, AF in "Chemistry and Biochemistry od Amino Acids, Peptides, and Proteins," B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, AF, Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review); Moley, JS, Trends Pharm Sci (1980) pp. 463-468 (general review); Hudson, D. et al., Int J Pept Prt Res (1979) 14: 177-185 (-CH2NH-, CH2CH2-); Spotola, AF et al., Life Sci (1986) 38: 1243-1249 (-CH2-S-); Hann, MM, J Chem Soc Perkin Trans I (1982) 30 7-314 (-CH-CH-, cis and trans); Almquist, RG et al., J Med Chem (1980) 23: 1392-1398 (-COCH2-); Jennings-White, C. et al., Tetrahedron Lett (1982) 23: 2533 (-COCH2-): Szelke, M. et al., European Appln. EP 45665 (1982) CA: 97: 39405 (1982) (-CH (OH) CH2-); Holladay, M.W. et al., Tetrahedron Lett (1983) 24: 4401-4404 (-C (OH) CH 2-); And Hurby, V. J., Life Sci (1982) 31: 189-199 (-CH2-S-). Particularly preferred non-peptide bonds are -CH2NH-.

Such peptide mimetics can, for example, be economically more productive, better chemical stability, enhanced pharmacological properties (half-life, absorbency, efficacy, effect, etc.), altered specificity (e.g., , Light spectrum of biological activity), reduced antigenicity and the like. Labeling of peptidomimetics is generally one or more labels directly or via spacers (eg, amide groups) to uninterrupted sites on the peptidomimetics predicted by quantitative structure-activity data and / or molecular modeling. Covalent bonds Generally, such uninterrupted sites are those that do not form direct contact with the macromolecules to which the peptidomimetics bind to produce a therapeutic effect. Derivatization (eg, labeling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of peptidomimetics.

Intended substitutions of the same type of D-amino acid with one or more amino acids of the B7 amino acid sequence (eg, D-lysine in place of L-lysine) can be used to produce more stable peptides. In addition, constrained peptides comprising a B7 amino acid sequence or substantially identical sequence variation can be prepared by methods known in the art (see, eg, Rizo and Gierasch (1992) Ann, Rev. Biochem. 61: 387). It may be provided by adding an internal cysteine residue that can form intermolecular disulfide bonds to cyclize.

One skilled in the art can produce polypeptides corresponding to B7 peptide sequences and sequence variants thereof without undue experimentation. Such polypeptides can often be produced as part of larger polypeptides in prokaryotic or eukaryotic cells by expression of polynucleotides encoding the B7 peptide sequence. Such peptides can also be synthesized by chemical methods. Methods of expressing heterologous polypeptides in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are incorporated herein by reference (Maniatis et al., Molecular Cloning: A Laboratory Manual (1989). ), 2nd Ed., Cold Spring Harbor, NY; Berger and Kimmel, methods in Enzymology, Volume 152, Guide to Molecular Cloning Technology (1987), Academic Press, Inc., San Die, Calf .: Merrifield, J. ( 1969) j.aM.cHEM.sOC.91: 501; cHAIKEN I. m. (1981) crc cRIT.rEV.bIOCHEM. 11: 255; kAISER ET AL. (1989) sCIENCE 243: 187; mERIFIELD, b (1986) ) sCIENCE 232: 342; kENT, SBH (1988) Ann. Rev. Biochem. 57: 957; and Offord, RE (1980) Semisynthetic Proteins, Wieley Publishing).

Peptides can be produced, for example, by direct chemical synthesis. Peptides may be produced as modified peptides having non-peptide moieties covalently attached to the N-terminus and / or C-terminus. In certain preferred embodiments, either or both of the carboxy terminus or the amino terminus can be chemically synthesized. The most common modifications of terminal amino and carboxy groups are acetylation and amidation, respectively. Amino terminus modifications such as acylation (eg, acetylation) or alkylation (eg, methylation) and carboxy terminus modifications, such as amidation, and cyclization, are various embodiments of the present invention. Can be integrated. Peptide elongation to certain amino terminus and / or carboxy terminus modifications and / or central sequences may result in physical, chemical, biological and pharmacological properties such as increased stability, increased potency and / or effect, resistance to serum proteases, and desirable. Such as pharmacokinetic properties.

The invention can also provide a B7 chimeric or fusion polypeptide. A B7 “chimeric polypeptide” or “fusion polypeptide” herein includes a B7 polypeptide that is operably linked to a non-B7 polypeptide. "B7 polypeptide" refers to a polypeptide having an amino acid sequence that corresponds to a B7 polypeptide, while a "non-B7 polypeptide" refers to a polypeptide that is not substantially homologous to a B7 polypeptide (eg, a polypeptide different from and identical to or different from a B7 polypeptide). Polypeptides derived from an organism). Within a B7 fusion polypeptide, the B7 polypeptide may correspond to all or part of the B7 polypeptide. In a preferred embodiment, the B7 fusion polypeptide comprises one or more biologically active moieties of the B7 polypeptide. Within a fusion polypeptide, the term "operably linked" indicates that the B7 polypeptide and the non-B7 polypeptide are fused in frame with each other. Non-B7 polypeptides may be fused with the N-terminus or C-terminus of the B7 polypeptide.

Preference is given to nucleic acid fragments capable of encoding a B7 polypeptide of at least about 40 amino acid residues in length, preferably at least about 80 amino acid residues in length, even more preferably at least about 120 amino acid residues in length. Particularly preferred are fragments that are at least about 200 amino acid residues in length, for example fragments comprising the entire extracellular domain of a B7 molecule. Several methods can be used to generate fragments of an isolated DNA sequence. Small subregions or fragments of nucleic acids encoding B7-1 or B7-2 proteins, eg, 1-30 base lengths, can be prepared by standard organic chemical synthesis methods. This method is also useful for the preparation of primers for use in the generation of larger synthetic fragments of B7 DNA.

DNA fragments associated with larger subregions or fragments of genes encoding B lymphocyte antigens are identified by polymerase chain reaction (PCR) (Sambrook, Fritsch and Maniatis, 2 Molecular Cloning; A Laboratory Manual, Cold Spring Harbor, NY). (1989)), and the DNA thus obtained can be expressed as a peptide by combining with a suitable expression vector. While using PCR, specific sequences of cloned double-stranded DNA are generated and cloned into expression vectors, which are then analyzed for CTLA4 / CD28 binding activity. For example, to express secreted (soluble) forms of human B7-1 or B7-2 protein using PCR, DNA that does not encode the membrane proteins and the cytoplasmic regions of the protein can be synthesized. Such DNA molecules can be ligated with a suitable expression vector and introduced into a host cell such as CHO, where the B7 protein fragment is synthesized and secreted. Next, the B7 protein fragment can be easily obtained from the culture medium.

In one embodiment, at least a portion of the B7 molecule, such as an extracellular domain portion deficient in the membrane protein portion of the molecule, is placed in an expression vector and expressed by the host cell such that the B7 molecule is not expressed at the surface of the cell. For example, cDNA encoding the extracellular domain of the B7 molecule can be synthesized by PCR (US Pat. No. 4,683,202) using primers derived from known sequences of B7-1 or B7-2 (Ref. Freeman) et al., J. Immunol. 1989. 143: 2714 or Science. 1993. 262: 9090). The resulting cDNA sequence can be collected as a prokaryotic or eukaryotic expression vector, which vector can be used for the synthesis of the extracellular domain of B7 by a suitable host such as COS or CHO cells.

In another embodiment, the expression vector comprises a DNA encoding a peptide having B7 antigen activity and a DNA encoding a second polypeptide. It is preferred that the second polypeptide is not derived from a costimulatory molecule. Preferably, the B7 chimeric or fusion protein of the invention is produced by standard recombinant DNA methods. For example, DNA fragments for encoding other polypeptide sequences may be prepared by known methods such as blunt ends or stagger ends for ligation, restriction enzyme digestion to provide suitable ends, cohesive ends as suitable. It can be ligated in-frame by using peel-in, alkaline phosphatase treatment to avoid undesired binding, and enzymatic ligation. In other embodiments, the fusion gene can be synthesized by known techniques, including automated DNA synthesizers. In addition, PCR amplification of gene fragments can be performed using anchor primers that produce complementary entanglements between two consecutive gene fragments that can subsequently be reannealed and reamplified to generate chimeric gene sequences (see Current Protocols in Molecular Biology, eds.Ausuel ey al. John Wiley & Sons: 1992). In addition, many expression vectors encoding the fusion moiety are commercially available (eg, GST polypeptides). As the B7-encoding nucleic acid molecule is encoded with this expression vector, the fusion moiety binds to the B7 protein in frame. For example, hexa-histidine can be added to the peptide for purification by immobilized metal ion affinity chromatography (Hochuli, E. et al., (1988 (Bio / Technology 6: 1321-1325)). In addition, to isolate B lymphocyte antigens that have no extraneous sequence, specific endoprotease cleavage sites can be introduced between the fusion moiety and the sequence of the peptide Peptides by adding functional groups to the peptide or removing the hydrophobic sites of the peptide It may be necessary to increase the availability.

In one embodiment, the DNA encoding the B7 molecule or portion thereof is bound in frame with the DNA encoding an immune response, eg, a viral antigen or a tumor cell antigen.

In one embodiment, the DNA encoding the amino acid sequence corresponding to the extracellular domain of the B7 antigen is combined with the DNA encoding the amino acid sequence corresponding to the constant region of an immunoglobulin molecule (see US Pat. No. 5,580,756 or WO). 97/28267). The second peptide may comprise immunoglobulin constant regions such as human Cγ1 domains or Cγ4 domains or Cμ or portions thereof (eg, CH2 and CH3 regions of hinges, human Cγ1 domains or Cγ4 domains, herein See US Pat. No. 5,116,964 to Capon et al., Incorporated by reference). The resulting B7Ig fusion protein may have altered solubility, binding affinity, stability and / or value (ie, the number of possible binding sites per molecule) and may increase the efficiency of protein purification.

In a preferred embodiment, the cysteine residues in the immunoglobulin constant region can be conserved to form disulfide bonds and soluble dimer B7Ig proteins. In one embodiment, a portion of the B7 molecule can be fused to the constant region of an IgM antibody or portion thereof to form a soluble multimeric form of B7Ig protein.

Particularly preferred B7Ig fusion proteins comprise an extracellular domain portion or variable region sequence domain of human B7-1 or B7-2 coupled with an immunoglobulin constant region. Immunoglobulin constant regions used in soluble B7 molecules may contain genetic modifications that reduce or eliminate effector activity determined by immunoglobulin structure. For example, DNA encoding the extracellular portion of B7-1 or B7-2, and DNA encoding the variable region family domain of B7-1 or B7-2 or the constant region family domain of B7-1 or B7-2 The encoding DNA can be joined with DNA encoding the CH2 and CH3 regions of the hinge, human Cγ1 domain or Cγ4 domain by site directed mutation.

If a non-human immunoglobulin constant region is used, the constant region is preferably humanized. Methods for preparing chimeric or humanized antibodies are known in the art (Ref. Morrison et al., Proc. Natl. Acad. Sci. U, SA 81: 6851 (1985); Takeda et al., Nature 314 : 452 (1985), Cabilly et al., US Patent No. 4,816,567; Boss et al., US Patent No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom Patent GB 2177096B, Teng et al., Proc. Natl. Acad. Sci. USA, 80: 7308-7312 (1983); Kozbor et al., Immunology Today, 4: 7279 (1983); Olsson et al., Meth.Enzymol., 92: 3-16 (1982); PCT publication WO92 / 06193 and EP 0239400).

Methods for Assembling and Expressing DNA Encoding Amino Acid Sequences Corresponding to B7 Antigen and Soluble B7Ig Molecules For example, synthesis of oligonucleotides, PCR, cell transformation, vector construction, expression systems, and the like are known in the art. to be.

B7 fusion proteins and polypeptides produced by recombinant techniques can be secreted and isolated from a mixture of cells and media containing the protein or peptide. In addition, proteins or peptides are retained in the cytoplasm and cells can be harvested and lysed to isolate proteins. Cell culture generally includes host cells, media and other components. Suitable media for cell culture are known in the art. Proteins and polypeptides can be isolated from cell culture media, host cells, or both using protein and peptide purification techniques known in the art. Techniques for transfecting host cells and purifying proteins and peptides are further described herein.

III. Screening of Soluble Costimulatory Molecules Structurally Related to Activity

Screening of B7 molecules that are structurally related to retaining the characteristic B lymphocyte antigen activity described herein can be accomplished by using one or more of several different assays. For example, peptides can be screened to suggest that they maintain reactivity with anti-B7 monoclonal antibodies that bind to naturally occurring B7 molecules. In particular, suitable cells, such as COS cells, can be transfected using DNA encoding the polypeptide to be tested. Production of the secreted form of B7 can be assessed using anti-B7 monoclonal antibodies or CTLAIg or CD28 fusion proteins in immunoprecipitation assays. The ability of cells to express peptides of interest to bind to CTLA4 or CD28 on platelets by panning can be tested. In addition, antagonism of the test peptide with naturally occurring B7 molecules for binding to CD28 or CTLA4 can be tested.

Even more preferably, other assays test the functional properties of the B7 antigen. As noted above, the ability of T cells to synthesize cytokines may be attributed to the occupancy or crosslinking of T cell receptors for antigen (eg, anti-CD3 or “activated T cells”). as well as the "primary activation signal" provided by the ester), as herein provided by the interaction with B lymphocyte antigens, for example by the interaction of B7-1 or B7-2 with CD28 and / or CTLA4 on T cells. do. The binding of B7 molecules to their natural ligands on T cells that receive signals via T cell receptors has the effect of signal transduction to T cells leading to an increase in the levels of cytokines, particularly interleukin-2, which is also T Induces an increase in lymphocytes. Other assays for B7 fusions include assays for the synthesis of cytokines such as interleukin-2, interleukin-4 or other cytokines, and / or assays for T cell augmentation by CD28 + T cells that have received a primary activation signal. .

In vitro, T cells can be provided with a first or primary activation signal by contacting them with an anti-T3 monoclonal antibody (eg anti-CD3) or phorbol ester. T cells that receive the primary activation signal are referred to herein as activated T cells. B7 function adds the origin of B7 (eg, a cell expressing a peptide having B7 activity or a secreted form of B7) and a primary activation signal such as an antigen associated with MHC class I or II to the T cell culture, Analyzing Functional Results For example, it is analyzed by analyzing supernatants for interleukin-2, gamma interferon, or other known or unknown cytokines. For example, any one of several conventional assays for interleukin-2, such as described in Proc. Natl. Acd. Sci. USA, 86: 1333 (1989), the relevant part of which is incorporated herein by reference. Assays can be used. Analytical kits for interferon production are available from Genzyme Corporation (Cambridge, Mass.). Enhancement of T cells can be measured as described in the Examples below. Peptides possessing the properties of the B7 antigen as described herein can result in increased cytokine production, eg, IL-2, per cell by T cells, compared to negative controls lacking costimulatory signals. Can lead to an increase in T cells.

Ⅳ. Method of Administration of Soluble Costimulatory Molecules

Compositions and / or formulations of the subject matter described herein are administered to a subject in need thereof to enhance an immune response. In one embodiment, the soluble B7 molecule is prophylactically administered with the antigen, eg, prior to infection of the toxin or to a subject without cancer. In another embodiment, the soluble B7 molecules of the subject are therapeutically administered to a subject with an already developed disease, eg, a subject having cancer or infected with a toxin, that is to obtain a beneficial effect from, for example, enhanced costimulation. Can be.

In one embodiment, the subject's costimulatory molecules are administered co-administered with the antigen formulation. The antigen can be a protein, polysaccharide, lipoprotein, or any combination thereof. For example, the antigen comprises a natural protein or protein fragment, synthetic protein or protein fragment, or peptide; Glycoproteins, glycopeptides, lipoproteins, nucleoproteins, nucleopeptides; Peptide-peptide conjugation; Recombinant nucleic acid expression products.

In one embodiment, the antigenic preparation may comprise a mixture of antigens. For example, the antigen can be administered in the form of irradiated cells (eg, tumor cells or live infected cells), viral particles, or preparation homogenates. In another embodiment, a purified formulation of an antigenic peptide or a recombinant form of an antigenic peptide, such as a viral peptide or tumor associated antigen, is administered to a subject. In one embodiment, the antigenic preparation comprises an MHC class I restriction peptide. In another embodiment, the antigen preparation comprises an MHC class II restriction peptide. In another embodiment, the antigen preparation comprises a class I restriction peptide and a class II restriction peptide for administration to a subject.

In one embodiment, the antigen is administered by "genetic immunization." In one embodiment, a DNA expression vector encoding a peptide of interest is injected into a host animal, eg, into the subject's skin or muscle. The gene product is correctly synthesized, glycosylated, folded and expressed by the subject. Using this method, antigens that are difficult to obtain in sufficient quantity or purity can be administered. In one embodiment, DNA is injected intramuscularly or coated onto gold particles by a particle bombardment device, or "gene gun," and delivered into the skin. Genetic immunization has been shown to induce specific humoral and cellular immune responses (Mor et al., 1995. J. Immunol. 155: 2039; Xu and Liew. 1995. Immunology. 84: 173; Davis et al. 1994. Vaccine. 12: 1503).

The proper route of administration of the soluble B7 molecule depends on the subject to be treated. For example, the soluble B7 molecule may be administered alone with the antigen and / or prior to administration of the antigen, or alone for several days after administration of the antigen. In one embodiment, the soluble B7 molecule may be administered “genetically” by administering a nucleic acid molecule that encodes a soluble B7 molecule or portion thereof. In other embodiments, soluble B7 molecules and antigens can be administered in a conjugated form.

Dosage regimens of soluble B7 molecules can be adjusted without undue testing to provide the optimum therapeutic response for each subject. For example, antibody titers to antigens or cellular immune responses to antigens (e.g., DTH responses (Ref. Puccetti et al. 1994. Eur. J. Immunol. 24: 1446) may cause the subject to It may be measured to determine whether it is developing or exhibits an enhanced immune response to the antigen, and the dosage regimen may be adjusted accordingly.

The active agents and compositions can be administered parenterally or intraperitoneally. The formulation can be administered, for example, intranasally, orally, intravenously, intramuscularly, subcutaneously or mucosally. Dispersions can be prepared in glycols, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these formulations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for infusion include sterile aqueous solutions (water soluble) or dispersions or sterile powders for the temporary preparation of sterile infusion solutions or dispersions. The pharmaceutical composition of the present invention may be formulated to be suitable for a particular route of administration. For example, in many embodiments, the pharmaceutical compositions of the invention may be infused, inhaled, or insufflation (through the mouth or nose), or intranasally, mucosa, oral cavity, buccal, parenteral, rectal, intramuscular, intravenous And intraperitoneal, and subcutaneous delivery.

The formulation or composition is sterile. In addition, they must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion containing water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycols, and the like), and appropriate mixtures thereof. Appropriate fluid properties can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, assorbic acid, thimerosal and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the infusion composition can be achieved by including an agent in the composition that prolongs absorption, such as aluminum monostearate and gelatin.

Sterile infusion solutions can be prepared by integrating the active composition or formulation with the required amount in an appropriate solvent with one or a combination of ingredients enumerated above as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound (eg, a costimulatory molecule and / or antigen and any additives) into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. . In the case of sterile powders for the preparation of sterile infusion solutions, the preferred method of preparation is to add to the powder of the active ingredient (e.g., a formulation or composition) to yield any additional desired components from its presterile filtered solution. Vacuum drying and lyophilization. Such formulations or compositions are described, for example, in Mol Med 1988. 4: 109) in a form suitable for use with a needleless infusion device (as known in the art, eg, US Pat. Nos. 5,383,851; 5,581,198; 5,846,233).

If the active agent or composition is properly protected as described above, the protein can be administered orally, eg with an inert diluent or an absorbent edible carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active agents is known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Ancillary active compounds can also be incorporated into the compositions.

It is particularly advantageous to formulate parenteral compositions in dosage unit form to facilitate administration and to unify dosages. Dosage unit form herein refers to physically discrete units suited as one dose for the stray animal subject to be treated; Each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect with the required pharmaceutical carrier. Detailed descriptions of the dosage unit forms of the present invention provide endogenous limitations in the field of compound preparation such as (a) the unique properties of the active compound and the particular therapeutic effect to be achieved, and (b) the active agent or composition for the treatment of the individual. It is regulated and directly depends on.

As used herein, “pharmaceutically acceptable carrier” includes, for example, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorbing smokers, and the like. The use of such media and agents for pharmaceutically active substances is known in the art. Additional agents may be incorporated.

The formulations of the present invention may be administered to a subject in a biologically compatible form suitable for in vivo administration to enhance an immune response. "Biologically compatible form suitable for in vivo administration" means the form of protein to be administered when any toxic effect is eliminated by the therapeutic effect of the agent. Subjects are intended to include living organisms, for example mammals, in which an immune response can be initiated. Examples of subjects include humans, primates other than humans, dogs, cats, mice, mice, and genetically transcribed species thereof. Administration of a peptide with activity of a B7 molecule as described herein can be in any pharmacological form, including soluble B7 peptide alone, soluble B7 peptide in combination with an antigen, and soluble B7 peptide in combination with a pharmaceutically acceptable carrier. Can be.

A therapeutically or prophylactically active amount of a composition of the present invention is defined as an effective amount at a dose and for a period necessary to achieve the desired result. Preferably, administration of soluble B7 molecules (with or without antigen) results in an enhanced immune response to the antigen (eg, viral or tumor cell antigen).

Immune responses to antigens (eg, cellular and / or humoral immune responses) by the subject can be measured using techniques known in the art.

Measurement of the immune response to the antigen can be made in the body or analyzed in vitro. For example, the responsiveness of immune cells to tumor cells can be measured by running a biopsy and examining cell infiltrates. In situ cytokine staining can be performed at the site of infection, near the tumor site, or in draining lymph nodes. In vitro culture of cells can be performed to test for cellular responsiveness to the antigen (eg, proliferation and / or cytokine production in the presence of the antigen and / or cytotoxic activity against the antigen). For example, the therapeutic or prophylactically active amount of a polypeptide having B7 activity may vary depending on factors such as the disease state, age, gender and weight of the individual, and the ability to elicit the desired response in the individual. Dosages may be adjusted to provide the optimum therapeutic or prophylactic response. For example, the dosage may be administered separately several times a day, or the dosage may be proportionally reduced as the therapeutic situation requires.

The active compound can be administered in a conventional manner by infusion (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application or rectal administration. Depending on the route of administration, the active compound may be coated with a substance that protects the compound from enzymes, acid activity, and other natural conditions that may inactivate the compound.

The present invention relates to an active compound in the form of a medicament for use in therapy as described herein. The active compound may also be used in the manufacture of a medicament for use in therapy.

V. Administration of Additional Agents

The subject can be treated by administering additional agents that further enhance the immune response. For example, an adjuvant and / or cytokine can be administered to the subject.

In one embodiment, cytokines such as granulocyte-macrophage colony stimulating factor, macrophage colony stimulating factor, granulocyte colony stimulating factor, interleukin 1, interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 7, interleukin 10, and / or interleukin 12 may be administered. Interferons such as α, β, and / or gamma interferon may also be administered. Preferably, cytokines such as IL-12 may be administered which support the development of T1 type T helper responses and the development of cellular immunity. In other embodiments, additional agents that modulate costimulatory signals, such as anti-CD28 antibodies, can be administered to the subject.

In other embodiments, agents that are known auxiliaries may be administered. At this time, the only adjuvant widely used in humans is allum (aluminum phosphate or aluminum hydroxide). Other adjuvants such as saponins and their purified components Quill A, Freund's complete adjuvant and other adjuvants used in research and veterinary applications have potential uses in human vaccines. However, phospholipid conjugates, polys as described in Muramil Dipeptide, Monophosphoryl Lipid A, Goodman-Snitkoff et al., J. Immunol. 147: 410-415 (1991) Resorcinol) Nonionic surfactants such as oxyethylene oleyl ether and n-hexadecyl polyethylene ether, enzyme inhibitors including pancreatic trypsin inhibitors, such as diisopropylfluorophosphate (DEP) and trasylol New chemically defined formulations may be used. In embodiments in which the antigen is administered, the antigen is contained in a proteoliposome or Novasome TM, eg, as described in Miller et al., J. Exp. Med. 176: 1739-1744 (1992). Encapsulated in lipid vesicles such as lipid vesicles (Micro Vescular Systems, Inc., Nashua, NH) can further enhance immune response.

The present invention will use the prior art of cell biopsy, cell culture, molecular biopsy, microbiology, recombinant DNA, and immunology unless otherwise noted, these techniques are known to those skilled in the art. These techniques are explained fully in the literature. See, eg, Genetics; Molecular Cloning A Laboratory Manual, 2nd Ed., Ed. by Sambrook, J. et al. Cold Spring Harbor Laboratory Press (1989); Short Protocols in Molecular Biology, 3rd Ed., Ed. by Ausubel, F. et al. Wiley, NY (1995); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed. (1984)); Mullis et al. U.S.Patent No: 4,683,195; Nucleic Acid Hybridization (B.D. Hames & S. J. Higgins eds. (1984)); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London (1987)); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C. C. Blackwell, eds. (1986)); and Miller, J. Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1972)).

The contents of all references, pending patent applications, and published patents cited herein are expressly incorporated herein by reference. Each reference described herein is incorporated by reference in its entirety. Any patent application to which this application claims priority is hereby fully incorporated by reference.

The invention is further illustrated by the following examples and should not be construed as further limitations.

Example

Example 1 Enhancement of CTL Response of Soluble Costimulatory Molecules

Optimal T cell activity requires signaling and costimulation through T cell receptors. B7-1 and B7-2 are two potent auxiliary stimulatory molecules on the surface of APCs. The effect of soluble forms of B7-2 on T cell responses in vivo was investigated. Soluble molecules are chimeric proteins containing the extracellular domain of B7-2 fused to the Fc region of mouse IgG2a. Administration of the B7-2Ig fusion protein for class II restricted peptide immunity significantly enhanced antigen-specific T cell proliferation and cytokine response. B7-2Ig administration enhanced the CTL response to immunity with class I-restricted peptides. Enhancement of the CTl response by B7-2Ig was significantly increased in the presence of T helper cell responses to class II-restricted peptides. These findings illustrate the immune stimulatory activity of soluble protein forms of co-stimulatory molecules, such as B7-2, against multiple T cell immune response variables, and have demonstrated that these molecules have clinical utility in infectious diseases and vaccine applications.

Materials and Methods Used in Example 1

mouse

Female BALB / cJ mice (Jackson Labs, Bar Harbor, ME) between 7 and 10 weeks of age were used for the study.

Preparation of Peptide Immunogen

All peptides used are influenza virus A / PR18 / 34 from the nucleic acid protein (NP) H-2 ^d - was restricted immune dominant epitopes. Class I-limiting peptides, amino acids 147-155, TYQRTRALV, (Ref .: Taylor, PM, et al. 1987, Immunogenetics 26: 267) and amino acids 206-229, FWRGENGRKTRIAYERMCNILKGK, (Ref .: Brett, SJ et al. 1991 Journal Immunology 147: 1647) was synthesized in a PE biosystem 430A peptide synthesizer using standard Fmoc / NMP chemistry with HOBT / DCC amino acid activity. These were analyzed and purified on Beckman HPLC, and the mass was confirmed using a Bruker Maldi (MALDI) mass spectrometer.

Preparation of AIPR18 / 34 Virus Stock

AIPR / 8/34 influenza stocks were prepared and titrated by injecting seed virus into 10-day hatched eggs at dilution of 104. After 42 hours of incubation, the ureteral fluids of the infected eggs were individually collected and aseptically confirmed on blood agar plates. Sterile body fluids were collected, aliquots were prepared, stored at -70 ° C and frozen quickly. Plaque titration (Bucher, D. J. et al. 1991, J Clin Microbiol 29: 2484) was performed and then thawed rapidly and virus dilution (in ureter body fluids) was performed.

B7-2Ig Fusion Protein

The B7-2Ig fusion protein was expressed and purified as previously disclosed (Fields, P.E., et al. 1998. J Immunol 161: 5268). In summary, the expression plasmid pED consisted of the extracellular domain of murine B7-2 coupled with cDNA and hinge encoding the signal, and genome DNA encoding CH2 and CH3 of mouse IgG2a.

Expression vectors were transfected into CHO cell lines and amplified by the techniques described above (Kaufman, RJ, et al. 1988. Journal of Biological Chemistry 263: 6352 ). The concentrated cell culture supernatant was passed through a rProtein A Sepharose Fast Flow column (Pharmacia Biotech). B7-2Ig was eluted using 20 mM citrate pH 3.0, neutralized using 1M Tris pH 8.0 and formulated at PBS pH 7.2 by buffer exchange. Protein concentration was calculated using absorbance at 280 nm and the absorbance coefficient was 1.33 cm / mg / ml. Endotoxin levels were less than 0.25 EU / mg as determined by gel coagulation assay (Cape Cod Associates). The percentage of protein as multimer was less than 1% as determined by analysis using TSK 3000 SWXL column (Toso Haas USA, Mongomeryville, Pa.). In vitro costimulatory activity of B7-2Ig has been demonstrated (Ref. Fields, PE, et al. 1998. J Immunol 161: 5268 ). This experiment was performed three more times and similar results were obtained. One or more replicate experiments for each experiment were performed using purified mouse monoclonal IgG2a against unrelated human protein GDF-9 (Genetics Institute, Cambridge MA) as a control for B7-2Ig treatment.

Peptide Immunization

100 μg of the indicated peptide or peptide mixture was mixed with IFA (Sigma) in volume ratio 1: 1 and emulsified. Mice were immunized subcutaneously at the beginning of the tail using an antigen / IFA emulsion in 100 μl. B7-2Ig in 100 μl was administered subcutaneously to the site adjacent to the peptide immunization site. In experiments harvesting lymph node cells, peptides and B7-2Ig were administered at the beginning of the tail and behind the neck as described above. The treatment group consisted of 5 mice each. B7-2Ig was administered in 0.1% aluminum hydroxide (Rehydragel, Rehies, Dublin, Ireland).

Live virus immunization and test administration

Metapan (Mallinckrodt Veterinary, Mundelein, IL) was administered as a nose cone until the mice lost reflux. Live virus diluted in PBS was administered intranasally in a final volume of 40 μl. Lethal dose of virus (4,000 PFU / mouse) resulted in 100% mortality in unimmunized mice. The immunization dose of 40 PFU / mouse did not cause any morbidity and the mice were completely protected from lethal challenge.

Proliferation assay

Lymph nodes (exclude mesenteric lymph nodes) and spleens are harvested on the indicated dates, and a single cell suspension is prepared to give 5 x 10 ^-5 M 2ME, 2 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin (gibco). In 200 μl in RPMI 1640 (Gibco / Biel) supplemented with 10% FCS, incubated in flat bottom 96 well plates at 4 × 10 ⁵ cells / well. Class II-restricted peptides were added at the indicated concentrations at the start of the culture. Proliferation was measured by 18 hour pulses (1 μC / well) using 3H thymidine incorporation followed by 1450 microbeta plate reader (Wallac). Comparison of proliferative responses from lymph node cells was performed 3, 5, 7 and 9 days after primary immunization. Secondary responses were evaluated from the spleen collected three days after the second immunization.

Cytokine black

Cells were cultured as described above for the proliferation assay. Supernatants were collected on day 3 and levels of IFN-γ, IL-5 and IL-13 were measured in commercial kits (Genzyme, Cambridge MA for IFN-γ, Endogen, Woburn MA for IL-5, and R & D Systems, Minneapolis , MN for IL-3) was determined by ELISA.

CTL Assay for Mouse Blood Cell Samples Not Separately Fractionated

Two to three weeks after the first immunization, blood samples (150 μl to 200 μl each) were obtained by orbital posterior bleeding from mice anesthetized with Airan (Ohmeda Caribe, Inc, Guayama, PR). Samples were diluted using 100 μl PBS containing 50 U heparin. 40 μl samples were removed for leukocyte counting. Differential counts on samples were not usually performed. The percentage of lymphocytes in the leukocyte fraction was 69% ± 8% by the differential count of 100 samples taken at various times during the study. The cells were washed to remove heparin. 8 × 10 ⁵ WBC / mouse as a whole were supplemented as described above for the proliferation assay, further supplements for the production of primary in vitro CTL responses (4 μg / ml anti-CD28 (PV1.17) and 10 U / ml recombinant murine IL-12 (rmIL-12, Genetics Institute, Cambridge, MA), 10 U / ml IL-2 and 200 U / ml IL-6, (Genzyme, Cambridge, MA), 0.1 pM Class I Treated with restriction peptides (TYQRTRALV, amino acids 147-155), and 1 x 10 ⁶ / ml irradiated (2,000 rads) syngeneic splenocytes (0.17 M NH4CL buffered with 0.017 M Tris for Iyse RBC) (Gajewski, TF 1996. J Immunol 156: 465 ))) and resuspended in 10 ml of medium consisting of RPMI 1640.

Cells were plated in 96 well round bottom Costa plates at a volume of 100 μl for all 8 × 10 ³ leukocytes per well. All 80 wells were plated for each blood sample. Sixteen wells were added with medium only with splenocytes irradiated on each plate. On day 7 of culture, the supernatant was removed by inverting the plate and the cell pellet was suspended by plate agitation. Antigen-positive (Ag-positive) targets were prepared by overnight pulses of P815 cells with 10 ug / ml of class I-restricted peptide. 1 × 10 ³ Ag-positive, ⁵¹ Cr labeled, P815 target was added to 50% wells in 100 μl culture medium and 1 × 10 ³ Ag-negative, ⁵¹ Cr labeled, control P815 target was added to the remaining 50%. . Of the 16 wells per plate without effector cells, eight were marked as spontaneous release and eight wells as maximum release measurements. Total release was achieved by the addition of 20 μl of 10% Triton X100. After 4 hours of incubation, 50 μl supernatant / well was collected into Wallac 96 well plate (1450-401), 125 μl scintillant (OptiPhase SuperMix, Wallac, Turku, Finland) was added, and the plate was transferred to Wallac microbeta 1450. Counted.

Calculation of Mean Specific Dissolution

The percent dissolution for each well was calculated according to the standard formula:

Experimental Release-Medium Release X 100

Total Release-Medium Release

In the above formula, total release was the mean cpm of all wells to which target and Triton-X-100 was added, and media release was SD of all wells to which only average cpm plus target and medium were added. The average specific percent dissolution per well was calculated according to the following formula:

(Ag ⁺ Dissolution ∑% of 40 Wells with Target)-(Dissolution ∑% of 40 Wells with Control Target)

40

Data is expressed as mean specific dissolution ± S.D calculated by the average of the mean specific dissolution values per well obtained from each of 5 mice in the group. Statistical significance was determined using the two tailed Stuent's test.

result:

B7-2Ig Enhances Primary CD4 + T Cell Proliferative Response to Immunization with Class II-Restricted Peptides

To determine whether B7-2Ig enhances or inhibits primary T cells for peptide immunization, mice were immunized with peptides in the presence or absence of B7-2Ig administration. Class II-restricted peptides amino acids 55-77 and amino acids 206-229 from NP of A / PR / 8/34 influenza virus were delivered subcutaneously as a mixture in IFA. These peptides were found to be immunodominant CD4 + Th cell epitopes (Brett, SJ, et al. 1991. Journal Immunology 147: 1647, Brett, SJ, and JP Tite. 1996). H-2-linked and H-2-unbound genes affect influenza nucleoprotein epitope recognition by CD4 + T cells ( Immunology 87:42 ). B7-2Ig (in 0.1% alum) was administered to adjacent sites. In preliminary studies, peptide-specific proliferative responses were measured from lymph node cells 3, 5, 7 and 9 days after immunization. Response kinetics were not affected by administration of B7-2Ig. Optimal proliferative responses were observed in both B7-2Ig and control mice 7 and 9 days after immunization. As shown in FIG. 1, treatment with B7-2Ig upon peptide immunization significantly increased antigen-specific proliferative responses from lymph node cells collected 9 days after immunization (test with 50 μg / ml peptide). P = 0.014) in case of intubation restimulation.

In FIG. 1, five mice per group were immunized at two subcutaneous sites with an IFA emulsion containing 100 μg per injection of two Class II restriction peptides or PBS. Only 100 μg of B7-2IgG2a or 0.1% alum in 0.1% alum were administered to the site adjacent to the immunization site. Lymph node cells (-O-) from mice immunized with peptide alone, lymph node cells from mice treated with B7-2Ig only (-Δ-), or lymph node cells from mice treated with B7-2Ig (-■-) Nine days after the immunizations were taken and re-stimulated in vitro with two peptides of the indicated concentrations used for immunization. Peptide immunized mice not injected with B7-2Ig were allowed to receive alum as a control. Proliferation was assessed by incorporation of ³ H-thymidine. Data is expressed as mean cpm / well ± SD of 5 mice per group. Similar results were obtained when mouse IgG2a mAb was used as a control. The data is from one of three representative experiments. Administration of B7-2Ig performed without immunity showed no proliferative response, demonstrating that antigen dependence of B7-2Ig occurs after primary immunity. These results demonstrate that in vivo administration of B7-2Ig enhances CD4 ⁺ Th cell response to peptide immunity.

B7-2Ig Increases Recall

To test whether B7-2Ig dependent enhancement of the primary antigen specific proliferative response triggers an enhanced memory response, mice are immunized with peptide with or without B7-2Ig and additional B7-2Ig is administered. Additional administration was 45 days later without. Splenocytes were taken 3 days after the second immunization and peptide specific proliferative responses were measured. As shown in FIG. 2, mice that received a single B7-2Ig treatment at the first immunization were secondary immunized than mice that received no B7-2Ig at all (p = 0.0006, in vitro restimulation with 100 pg / ml peptide). Greater proliferative response after the test. Five mice per group were immunized with peptide only and treated with (-O-) B7-2Ig only (-Δ-), immunized with peptide and treated with B7-2Ig (-■-). At 46 days after primary immunization, both groups of mice immunized with peptides were reimmunized with peptides without B7-2Ig coadministration. Non-immunized mice received only IFA. Three days after re-immunization, splenocytes were restimulated in vitro, respectively, with two peptides at the indicated concentrations used for immunization. Proliferation was assessed by incorporation of ³ H-thymidine. Data is expressed as mean cpm / well ± SD of 5 mice per group. Data is data from one of two replicate experiments. These data show that B7-2Ig augments recall responses when used as an adjuvant to primary T cell responses.

B7-2Ig augments Th1 and Th2 dependent primary cytokine responses.

To determine if B7-2Ig as an immunoadjuvant differentially promotes the development of a Th1 or Th2 response, the mice immunized peptide specific cytokine responses after 3, 5, 7 and 9 post immunization following primary immunization. Analysis from lymph node cells. As in the case of the proliferative response, an optimal cytokine response was observed on day 9. As shown in Table 2, administration of B7-2Ig for immunization with peptides resulted in Th1-bound cytokine IFN-γ (p = 0.017) and Th2-bound cytokine IL-5 (p = 0.011), and IL- The production level of 13 (p = 0.002) was significantly increased. Antigen specific cytokine production was not observed in cultures from immunized mice treated with B7-2Ig. These results show that B7-2Ig augments the primary T cell response of both the Th1 and Th2 phenotypes.

B7-2Ig enhances the response to class I restriction peptides.

The interaction of CD7 with membrane bound B7 has been demonstrated to induce a CTL response in vitro in the absence of CD4 + cells (Harding, FA et al., 1993, J Exp Med 177: 1791) . To test the effect of B7-2Ig on the primary CTL response, mice were immunized with immunodominant Class I restriction peptides of IFA with or without B7-2Ig concurrently. Peptide specific CTL responses were measured from undifferentiated peripheral blood cells using small blood samples, such as the CTL assay as described in Materials and Methods. This assay is a single experiment that analyzes multiple mice, assesses the statistical significance of differences between groups, repeated CTL assays from individual mice during the course of the immune response, and investigates the correlation between CTL responses and in vivo results. Made possible.

Data was expressed as one value per mouse (average specific lysis) (FIG. 3). Peptides (100 μg / mouse) served as IFA. B7-2Ig (100 μg / mouse in 0.1% alum) was subcutaneously co-administered. CTLs were determined from unsorted blood collected three weeks after immunization. Data is expressed as mean specific lysis ± SD. The values for the live virus immunized group were from a single pool of two mice, and the average specific lysis of this group from 18 mice tested in nine separate experiments was 52 ± 12. All other groups had 5 mice per group. Data presented is one of three replicates. The response to class I restricted peptides in IFA was not significant above the reference level. This finding was consistent with results from Fayolle et al. 1991 Journal Immunology 147: 406, which showed that the CTL response to class I restricted peptides in IFA in the absence of T cell help. When B7-2Ig was administered for immunization with class I restriction peptides of IFA, a small but statistically significant increase in response (p = 0.013) was observed (FIG. 3). However, the magnitude of peptide specific CTL response in mice immunized with peptide and co-administered B7-2Ig was smaller than that of mice immunized with live virus. Thus, coadministration of 100 μg doses of B7-2Ig did not elevate the response to immunity by class I restriction peptides to the maximum level achieved by the live viral immunity method.

Optimal B7-2Ig augmentation in the CTL response requires T cell help.

Since the optimal CTL response depends on Th cells (Ref .: Fayolle, C. et al. 1991, Journal Immunology 147: 4069; Keene, JA et al. 1982, J Exp Med 155: 768; Von Herrath, MG et al 1996 J Virol 70: 1072 and Ossendorp, F. et al. 1998, Journal Experimental Medicine 187: 693), the effect of B7-2Ig on the CTL response was induced by peptides in which mice are known to induce Th cell responses. Tested under concurrent immunized conditions. Mice were immunized with IFA emulsions containing only class I restriction peptides or a mixture of two class II restriction peptides and class I restriction peptides shown in FIGS. 1 and 2 and Table 1 above. CTL response was measured from unfractionated peripheral blood cells (FIG. 4). Peptide immunization was performed as a single emulsion in IFA. Mice were immunized and tested as indicated. After 3 weeks, the CTL response was measured from peripheral blood. Data are expressed as mean specific lysis ± SD. The values for the live virus immunized groups were from a single pool of two mice and the average specific lysis of this group from 18 mice tested in nine separate experiments was 52 ± 12. All other groups had 5 mice per group. Mean specific lysis of the group that received the regulatory Ig and the class II restriction peptides in the IFA in the alum were subtracted from the mice that received the regulatory Ig. Mean specific lysis of the group containing B7-2Ig and class II restriction peptides in IFA in the alum were subtracted from the group containing B7-2Ig. Data presented are data from one of four replicate experiments.

Mean specific lysis of cells from mice immunized with a mixture of class I and class II restriction peptides was significantly higher than the lysis of cells from mice immunized with class I restriction peptides alone (p = 0.046 and repetition in the experiments presented). 0.004 in the experiment). Immunization with a mixture of class I restriction peptides and class II restriction peptides and treatment with 100 μg of B7-2Ig resulted in significantly greater mean specificity than average specific lysis from peptide immunized mice that did not receive B7-2Ig. Dissolution was shown (p = 0.003, p = 0.0001, p = 0.045 and p = 0.0012 in 4 replicate experiments). This response did not increase when the dose of B7-2Ig was doubled to 200 μg / mouse, indicating that the 100 μg dose caused a maximum increase in B7-2Ig dependency.

The combination of immunizations using a mixture of these peptides with B7-2Ig treatment resulted in a peptide-specific CTL response to a similar extent as induced after immunization with live virus. The mean specific lysis value, 52 ± 12, was obtained by averaging the values obtained from 18 live virus immunized mice in 9 separate experiments, which is a reproducibility of a small amount of peripheral blood sample CTL assay and the T cell help (help ) And the conclusion that the peptide immunization in the presence of B7-2Ig elicits a response comparable to that induced by efficient live viral immunization. CTL responses detected from peripheral blood cells were highest at 2-3 weeks in both mice immunized with live virus and mice treated with B7-2Ig and weakened after 5 weeks. This again suggests the similarity of these reactions. However, as discussed below, mice immunized with peptide mixtures and treated with B7-2Ig were not protected from lethal viral challenge.

The data in FIGS. 3 and 4 show B7-2Ig formulated in 0.1% alum, which is itself a clinically recognized immune adjuvant (April, MA et al., 1966, Can J Public Health 57: 343 ). Will appear. Administration of alum alone or control Ig in alum did not affect the anti-peptide response, and formulation in alum was not required for B7-2Ig dependent immune boosting. When comparing the effects of B7-2Ig in alum directly with the effect of B7-2Ig in PBS, a large enhancement of the CTL response was observed when B7-2Ig was administered in alum (compared to 38 ± 12 Mean specific dissolution value is 72 ± 13, p = 0.007).

Optimal antigen specific activation and T cell regulation require the transmission of costimulatory signals from B7 molecules on the APC surface to CD28 and CTLA-4 on the T cell surface (B Boussiotis, VA, et al., 1996, Immunol Rev 153: 5 ; Lenschow, DJ et al. 1996, Annual Review Immunology 14: 233 ; Green, JM et al. 1994. Immunity 1: 501 ; Tivol, EA et al. 1995, Immunity 3: 541 and Waterhouse, P., 1995, Sceience 270: 985). Numerous mouse model tests have shown that immune responses can be enhanced by increased cell surface expression of B7. In these studies, an increase in B7 expression was observed in tumor cell transduction (Bassker, S. et al. 1993; Proc Natl Acad Sci USA 90: 5687 ; Cavallo, F. et al. 1995, Eur J Immunol 25: 1154 ; Coughlin, CM et al. 1995, Cancer Research 55: 4980 ; Chen, L., et al. 1992, Cell 71: 1093; Chen, L. et al. 1994, Journal of Experimental Medicine 179: 523 ; Gajewski, TF et al. 1996, J Immunol 8: 2909 ; Yang, G. et al. 1995, Journal of Immunology 154: 279 and Dunussi-Joannopoulos K, et al. 1996, Blood 87: 2938 ), cDNA injection (Kim, JJ et al. 1997, Nat Biotechnol 15: 641 ; Kim, JJ et al. 1998, Vaccine Nov; 16: 1828 and Horspool, JH et al. 1998, J Immunol 160: 2706 ), or infection with a viral vector (Ref. : Chamberlain, RS et al. 1996, Cancer Res 56: 2832 ; Emtage, PC et al. 1998, J Immunol 160: 2531 ; Hodge, JW et al. 1994, Cancer Res 54: 5552 and Putzer, BM et al. 1997 , Proc Natl Acad Sci USA 94: 10889 ). Using another method to increase the level of B7 in vivo, the solubility of B7-2, B7-2Ig, consisting of the Fx of mouse IgG2a fused with the extracellular portion of B7-2 bound to each Ig chain Protein forms have been developed. In vivo administration of B8-2Ig enhances antigen specific Th cells and CTL responses. Thus, in vivo administration of B7-2Ig provides a simple alternative to the use of viral or DNA vectors to optimize B7 transduction or B7-2 mediated costimulation in vivo of tumor cells.

Since the development of infections and autoimmune diseases can be greatly influenced by the pattern of cytokines produced by reacting Th cells, the B7 pathway can be used to skew towards either a Th1 or Th2 type cytokine response. It is worth deciding whether it can be used (Kuchroo, VK et al. 1995, Cell 80:10 ; Lenschow, DJ et al. 1995, J Exp Med 181: 1145 ; Corry ,, DB et al 1994, J Immunol 153 : 4142 ; Freeman, GJ et al. 1995, Immunity 2: 523 ; Schweitzer, AN et al. 1997, J Immunol 2 158: 713 ; Rulifson, IC et al. 1997, J Immunol 158: 658 and McAdam, AJ et al 1998 Immunol Rev 165: 231) . Many experimental systems suggest that Th2 differentiation is more dependent on B7 costimulation than Th1 differentiation (Lenschow, DJ et al. 1995, J Exp Med 181: 1145 ; Corry, DB et al. 1994, J Immunol 153: 4142 ; Freeman, GJ et al. 1995, Immunity 2: 523 and Rulifson, IC, A. et al. 1997, J Immunol 158: 658) . Anti-B7 mAbs have been used successfully to manipulate T cell differentiation in vivo. Blocking of B7-1 to mAb favors Th2 differentiation, while blocking of B7-2 has been reported to favor Th1 differentiation (Kuchroo, VK et al. 1995, Cell 80: Mar 10 (5) Lenschow, DJ et al. 1995, JExp Med 181: 1145 ; Corry, DB et al. 1994, J Immunol 152: 4142 ; Freeman, GJ et al. 1995, Immunity 2: 523 and Rulifson, Ic et al. 1997 , J Immunol 158: 658) . In contrast, using APC from B7 transgenic mice, Schweitzer et al. Found that neither B7-1 nor B7-2 appears to selectively regulate Th1 or Th2 differentiation in vitro (see Schweitzer, AN et al. 1997, J. Immunol 2 158: 713) . Data from this example showed that in vitro administration of B7-2Ig augmented antigen specific cytokine responses of both Th1 and Th2 types. Therefore, therapeutically effective B7-2Ig will be maximal in situations where the goal is to raise response rather than skew.

To test the effect of B7-2Ig on the CTL response, peptide specific derived with peptide from NP of influenza virus A / PR / 8/34 for the response derived with the same peptide from live virus immunized mice CTL reactions were compared. B7 2Ig treatment resulted in a small but marked enhancement of the response to class 1 restricted peptides in IFA. These data are consistent with data demonstrating that in the absence of exogenous help, tumor cells transfected with B7 are sufficient for the generation of in vitro CTL responses (Harding, FA et al. 1993, J Exp Med 177: 1791) .

However, the B7-2Ig enhanced response was significantly lower than the CTL response to the same peptide in mice immunized with live virus. These data show that these mice can induce a much larger anti-peptide response than the response induced by class I restricted peptide immunization and B7-2Ig simultaneous administration.

In an attempt to further enhance the response to class I restricted peptides, the effect of the addition of T cells, class II restricted peptide antigens to IFA emulsions containing class I restricted CTL peptide antigens was investigated. It has already been reported that the optimal CTL response depends on Th cells (Fayolle, c. Et al. 1991, Journal Immunology 147: 4069 ; Keene, JA et al. 1982, J Exp Med 155: 768 ; von Herrath) , MG 1996, J Virol 70: 1072 and Ossendorp, F. et al. 1998 Journal Experimental Medicine 187: 693 ). CTL responses were markedly elevated in mice immunized with both class I and class II restriction peptides. These data indicate that help of CTL responses to peptide antigens can be provided by simultaneous immunization with Th cell antigens. Covalent linkages between CTL and Th peptide epitopes are not required, suggesting that any immunogenic protein can be used as a co-immunogen to provide help for the CTL response. This conclusion is further supported by the inventors' finding that the addition of KLH to IFA emulsions containing class I restricted peptides resulted in a significant enhancement of peptide specific CTL responses.

Despite enhanced, the peptide specific CTL response of mice immunized with class I and class II restriction peptides is less than that of mice immunized with live virus. The addition of B7-2Ig to the protocol increased the CTL response of mice immunized with a mixture of peptides to the level of mice immunized with live virus. The response of these two groups is comparable, but the former group was not protected from viral lethal viral challenge. This result is consistent with Lawson et al. (Lawson, cM et al. 1994, Journal Virology 68: 3505 ), Lawson et al. Used the same peptide used in this study by administering a recombinant vaccinia virus expressing the peptide. Vigorous CTL response was induced. According to the present point, a strong CTL response to this peptide alone was not sufficient to confer protective immunogenicity to BALB / c mice.

Although not required for the immune enhancing effect of B7-2Ig, the formulation in alum is significantly enhanced than that in PBS. Administration of control Ig in alum did not affect the response, suggesting that alum alone did not enhance the response.

Infection with live viruses is a natural and effective route for the activation of class I restriction responses (Zinkernagel, RM et al. 1979, Adv Immunol 27:51) . Therefore, the data presented herein showing that comparable antipeptide responses can be obtained from mice immunized with live virus and from B7-2Ig treated mice immunized with a mixed emulsion of class I and class II restriction peptides, the latter immunization Confirm the efficacy of the protocol. In recent years, induction of CTL responses to class I restriction peptides has been of great interest.

Recent evidence that human CTLs recognize class I restricted tumor-associated peptides similarly to mouse CTLs suggested that CTL responses to these peptides could be demonstrated to be effective against human tumors (see van der Bruggen, P. et al., 1991, Science 254: 1643; Wolfel, T. et al., 1993, Int. J Cancer 55: 237; Mauerer, M. J. 1996, Melanoma Res 6: 11; Cormier, JN et al., 1997, Cancer J Sci Am 3: 37; Apostolopoulos, V. et al., 1997, J. Immunology 159: 5211; Rosenberg, SA 1998, Nature Medicine 4: 321 and Nestle, FO et al., 1998, Nature Medicine 4: 328). This possibility has spurred a strong effort to devise an immunization protocol that optimizes the response to class I restricted peptide vaccines. Success has been reported in a wide variety of methods, including insertion of minigenes into recombinant viruses and bacteria, peptide oligomerization, lipid modification, cDNA immunization, use of toxins and cytokines as adjuvants, and antigen pulsed dendritic cells (see : Allsopp, CE et al., 1996, European Journal of Immunology 26: 1951; Rotzschke, O et al., 1997, Proc Natl Acad Sci USA 94: 14642; Alving, CA et al., 1995, Immunological Reviews 145: 1 ; Ciernik, IF et al., 1996, J Immunol 156: 2369; Porgador, A. et al., 1997, Journal of Immunology 158: 834; Noguchi Y. et al. 1995, Proceedings of the National Academy of Science; USA 92: 2219 and Takahashi H. et al. 1992, International Immunology 5: 849). An advantage of the methods reported herein is that they are uniformly applicable to any peptide without the need for further modifications and are effective for both class I and class I restriction reactions.

Coadministration of B7-2Ig with peptide immunization raises relatively poor class I restriction peptide responses to levels comparable to those obtained from live viral immunization, enhancing the response of soluble forms of B7-2 protein to other poor immunogens Suggests that you can. This finding indicates that soluble protein form B7-2 can enhance antigen specific immune responses in the treatment of cancer or inflammatory diseases in which the immune system responds ineffectively.

Table I. B7-2Ig dependent enhancement of Th cytokine response ^a

Expression of Cytokines During In Vitro Restimulation

In vivo treatment IFNγ (pg / ml ± SD) IL-5 (pg / ml ± SD) IL-13 (pg / ml ± SD) Peptide Immunization bd ^b 54 ± 44 136 ± 125 Peptide Immunization + B7-2Ig 1720 ± 986 2479 ± 1104 5365 ± 1795 IFA + B7-2Ig bd ^b bd ^b bd ^b

5 mice per group for ^a class II restriction 2 were immunized with each of the peptides in two subcutaneous sites with IFA emulsion contains an ㎍ 100 PBS per injection. 100 μg of B7-2IgG2a in 0.1% alum or 0.1% alum alone was administered to the site adjacent to the immunization site. After 9 days, lymph node cells were collected and restimulated upon incubation for 3 days with 5 μg / ml of each peptide. Values for each mouse were obtained by averaging the results from triplicate wells. Data are expressed as mean mouse cytokine concentrations in group ± SD. The results were similar to those obtained when the control mouse IgG2a mAb was used as a control. Data presented are from one of three replicates. ^b Below detection limit of the test.

Example 2 B7-IgG Fusion Proteins Enhance Anti-tumor Immune Responses and Induce Recovery of Induced Tumors

Fusion proteins between the extracellular region of B7-1 or B7-2 and IgG.2a were evaluated for their ability to enhance antitumor response in four different murine tumor models, including poor immunogenic melanoma B16 / F10. . Single immunization with irradiated tumor cells in combination with B7-1 or B7-2-IgG protected mice against active tumor attack. More significantly, the 7-day induced tumors recovered after vaccination with irradiated tumor cells mixed with B7-IgG. Even therapeutic administration of B7-IgG alone achieved similar relief of tumors. Animals that rejected established tumors were resistant to recurrence, suggesting that the antitumor effect of B7-IgG is mediated by tumor specific immune mechanisms. In addition, single vaccination with irradiated tumor cells mixed with B7-1 or B7-2-IgG resulted in a CD8 response to tumor associated antigens. Thus, B7-IgG showed potent adjuvant activity in combination with exogenously delivered antigens and endogenous antigens. This finding suggests clinical value for B7-IgG that enhances antitumor and antiviral responses.

Materials and Methods Used in Example 2

Expression plasmids for the murine B7-1-IgGIgG and B7-2-IgG2a fusion proteins are hinges derived from murine IgG2a antibodies for DNA encoding signals and extracellular domains of murine B7-1 or B7-2. It was constructed by binding the CH2-CH3 domain with DNA encoding. Cysteine residues remaining in the antibody hinge region were preserved such that the prepared B7-1-IgGIgG2a or mB7-2-IgG2a is dimer and divalent. Fusion proteins with mutated IgG2a regions were generated (designated B7-IgG2mut) to prevent binding and complement activation by high affinity Fc receptors. The following amino acid residues in the CH2 domain were substituted by alanine: leucine at position # 235, glutamic acid at position # 318, lysine at position # 320, lysine at position # 322. For the preparation of B7-IgG protein, the plasmid was expressed in CHO cell line and the protein was isolated.

P815 is a tumor cell line derived from mastocytoma growing into a solid tumor after intradermal (id) injection of ⁵ × 10 ⁴ cells into a DBA / 2 mouse (Jackson Laboratories). The clones used in this experiment naturally metastasized after id injection resulting in death of mice 25 to 35 days later. Meth A, a tumor cell line derived from sarcoma of Balb / C mice, grows into solid tumors after intradermal (id) injection of ⁵ × 10 ⁵ cells (Taconic Balb / C mice). B16 / F10, a non-immunogenic tumor line derived from melanoma of C57BL / 6 mice (Taconic), grows into solid tumors after intradermal (id) injection of 5X10 ⁵ cells and dies due to metastasis 25 to 35 days later Cause. Bladder carcinoma MB49 is also derived from C57BL / 6 mice and induced solid tumors in 100% of mice 5-7 days after intradermal injection of 5 × 10 ⁵ cells.

In vitro co-stimulation test

2 × 10 ⁵ naive murine spleen cells were plated in triplicate in 96-well plates coated with low concentrations of anti-CD3 mAb (0-500 ng-ml −2C11, Pharmingen). Plates were co-coated with anti-CD28 mAb (0-1 μg / ml) as a positive control to provide the signals needed for costimulation, or B7-1-IgG or B7-2-IgG (0-9 μg / ml). Co-coated). In addition, B7-IgG was added in soluble form or crosslinked by secondary anti-mouse IgG2a Ab. Splenocytes were stimulated for 72 hours. Aliquots of supernatants were removed for IFN- [gamma] analysis and cells were pulsed with H ³ -thymidine for 8 hours. IFN- [gamma] analysis was performed by standard ELISA using capture Ab R46A4 and biotinylated XGM1.2 as detection Abs.

Vaccination Protocol:

In a prophylactic tumor vaccine model, mice were vaccinated on day 0 and simultaneously challenged with tumor cells. Mice were immunized with irradiated tumor cells (1 × 10 ⁷ cells) in PBS, either mixed with 75-100 μg mB7-1-IgG, B7-2-IgG, murine IgG, or nothing mixed. Injected into the plantar (i.fp.) of both hind legs. Injection of B7-IgG was repeated once on day 3 or day 5. On day 7, animals were challenged by injecting 50 μl intradermal into defined tumor cells of the right flank. Primary tumor growth and animal survival were monitored over 40-60 days.

In therapeutic tumor vaccine models, primary tumors were induced by id inoculation with a defined number of tumor cells. On days 5-7 (when tumor is clearly promoted), mice are vaccinated plantarized with irradiated 1 × 10 ⁷ tumor cells mixed with or without 100 μg B7-1-IgG, B7-2-IgG I was. After 3 days 100 μg of B7-IgG was reinjected into the plantar. This vaccination regimen was repeated for 2 or 3 weeks. Primary tumor growth and mouse survival were monitored from 7 to 40 to 70 days.

In the method of treatment, tumor-bearing mouse B7-IgG was treated only with fusion proteins. Only 50-100 μg of B7-1-IgG or B7-2-IgG was injected intraplantarly (i.fp.) twice a week for 3 weeks. Tumor growth and survival were monitored for 40 days.

Measurement of PIA-specific CTL Response:

Spleens were harvested from DBA / 2 mice 10-14 days after single immunization with irradiated P815 cells with or without B7-1-IgG or B7-2-IgG. After lysing 20 × 10 ⁶ , the splenocytes were restimulated for 6 days in a T25 flask with 0.1 ng / ml of P1A peptide and 5 U / ml of IL-2 (paminegen: pharmingen). Next, a standard 5h Cr ⁵¹ -release assay was performed with A20 cells pulsed with P1A peptide as a target or not pulsed with peptide. The ratio of P1A-peptide specific dissolution was expressed as the difference between the dissolution rate of the peptide pulsed target and the dissolution rate of the unpulsed target.

result:

Immobilized or crosslinked B7-IgG provides costimulatory signals for suboptimally stimulated murine spleen cells in vitro.

FACS® or Biocore® analysis determined that B7-1-IgG and B7-2-IgG fusion proteins bind to murine CD28 and CTLA-4. To determine whether the B7-IgG fusion protein increases or inhibits T cell activity, mouse splenocytes are cultured in vitro with anti-CD3 mAb bound to the plate in combination with B7-IgG immobilized on the plate. Stimulated. Interstimulation with anti-CD28 mAb bound to plates provided as positive controls B7-1-IgG and B7-2-IgG induced a dose dependent increase in proliferation and IFN-γ secretion action (FIG. 5). 2 × 10 ⁵ untreated splenocytes were stimulated three times for 72 hours with 50 ng / ml of anti-CD3 monoclonal antibody and increased amount of immobilized anti-CD28 antibody, or B7-1 or B7-2 IgG It was. The proliferative response was measured after 6 h pulses incorporating 3H-thymidine. Panels B and C show the respective amounts of IFN-γ or IL2 released after stimulation for 72 hours with 50 ng / ml of anti-CD3 antibody and the indicated amount of immobilized B7-2-IgG. Cytokines were measured by standard ELISA. In the absence of anti-CD3 stimulation, B7-IgG did not induce proliferation. Since soluble B7-IgG proteins do not enhance proliferation or IFN- [gamma] production, immobilization and crosslinking of B7-IgG molecules by secondary anti-murine IgG mAbs is essential for efficient costimulation. B7-IgG proteins (B7-1-IgG and B7-2-IgGmut) mutated in the Fc binding region are effective in costimulatory splenocytes when immobilized on the plate.

B7-1-IgG or B7-2-IgG enhances the protective effect of the investigated tumor cell vaccines.

The B7-1-IgG fusion protein was evaluated as an adjuvant within the prophylactic tumor vaccine model. Inoculation of 5 × 10 ⁴ live P815 tumor cells developed solid tumors after 5-7 days in 100% untreated DBA / 2 mice. Ten DBA / 2 mice per group were immunized intraperitoneally with 1 × 10 ⁷ irradiated P815 tumor cells at one time. Cell vaccines were mixed or given alone with 100 μg of B7-1-IgG, B7-2-IgG, or B7-1-IgG, B7-1-IgG mutant proteins. For the control group, 100 μg of B7-1-IgG or B7-2-IgG alone was administered intraperitoneally on days 0 and 5. Mice were challenged with 5 × 10 ⁴ live P815 tumor cells on day 7. Protection against vaccination was determined 24 days later with no apparent tumor. 6 shows the results obtained from one of five representative tests. During the week prior to vaccination of live tumors, vaccination of mice with the investigated P815 tumor cells did not provide protection against tumor growth. However, single challenge with irradiated tumor cells mixed with B7-1-IgG or B7-2-IgG induced 60-70% protection (FIG. 6, Table 2). In contrast, immunization with B7-1-IgG or B7-2-IgG alone provided no protection at all (FIG. 6). Protection was determined by the absence of solid tumors after 14-24 days and / or survival of at least 60 days. The latter indicated the absence of metastatic disease.

To assess the role of the IgG domain on the function of the fusion protein, immunized with irradiated P815 cells mixed with B7-1-IgGmut, a mutated protein that did not bind Fc-receptor and did not activate complement. The mutated molecule is less effective than the wild type, which provides a role for Fc binding of the B7-1-IgG molecule (FIG. 6, Table 2).

The effect of irradiated tumor cells mixed with B7-1-IgG 'and the effect of tumor cells transfected with B7-1 or B7-2 were compared. Vaccination with B7-transfectant induced significantly lower anti-tumor immunity and protected only 30% of mice. This compares to 60% after immunization with irradiated wild type P815 cells mixed with B7-IgG (Table 2). This finding indicated that B7-1-IgG 'is an effective adjuvant for generating anti-tumor protection and is more effective than B7-transfected tumor cells.

Vaccination with irradiated P815 tumor cells mixed with B7-1-IgG or B7-2-IgG treated mice in which P815 tumors were established.

To test the adjuvant activity of B7-1-IgG in a therapeutic tumor vaccine model, DB2 / 2 mice were injected with P815 tumor cells 5 days after administration at doses that would produce palpable solid tumors. . Solid P815 tumors were introduced on day 0 by intradermal injection of 5 × 10 ⁴ P815 cells. Vaccination was performed on day 7 after palpable tumors developed. Figure 7 shows injection of PBS into the plantar as control (A, E), P815 tumor cells alone (B) or mixed with irrelevant mouse IgG2a Ab (F), or irradiated P815 cells B7-1- Results of mice injected mixed with IgG (C) or B7-2-IgG (G) are shown. Immunization was repeated on days 7, 14 and 21. PBS, unrelated Ab, or B7-IgG were each administered again after 3 days. Tumor growth was monitored for 40 days. After 25-30 days, animals in the control group died of spontaneous metastasis. Panels D and H show the survival time of different treatment groups. Data is representative of four independent experiments. The kinetics of tumor growth and survival after vaccination are shown in FIG. 7 and are representative of four independent experiments. Starting from about a week after the first immunization, reduced tumor growth was observed with tumor regression in the population of mice immunized with tumor cells mixed with B7-IgG. Tumor growth did not decrease in the group of irradiated tumor cells alone or in combination with unrelated mouse B7-IgG2a Ab. After three cycles of immunization with irradiated P815 tumor cells mixed with B7-IgG, primary tumors disappeared in 60-80% of mice. Regression of primary tumors is associated with long-term survival of mice. Immunization with irradiated P815 cells mixed with B7-1-IgG or B7-2-IgG increased long-term survival by about 80% (FIG. 7, D, H). The large number of mice in untreated or irradiated tumor vaccine treated controls apparently died between 25 and 35 days due to spontaneous modifications in the liver, spleen and lymph nodes.

B7-IgGs as Therapeutic Tumor Vaccine Adjuvant in Other Tumor Models

To determine if the results of B7-IgG were unique to the P815 tumor or DBA / 2 mouse system, the effect of B7-IgG as an adjuvant was tested on three additional tumor models in two different mouse systems. Similar positive effects were obtained for all models.

Seven-day-old Balb / c mice with MethA carcinoma identified were immunized with PBS, irradiated MethA alone, or irradiated MethA cells mixed with B7-1-IgG or B7-2-IgG. Faithful MethA or B16 / F10 tumors were established in Balb / c or C57BL / 6 mice, respectively. FIG. 8 shows 10 mice per group, irradiated tumor cells alone (B, E), or 25 μg (C, D) or 100 μg (F, G) of B7-1-IgG or B7-2 It is shown to be immunized into plantar, respectively mixed with -IgG. PBS, or B7-1-IgG or B7-2-IgG, respectively, was injected after 3-4 days. One group was treated with PBS alone (not shown for A, B16 / F10). Tumor growth was monitored for 35 days. Mice were euthanized once MethA tumors reached 300 mm ² in size. Mice with B16 / F10 tumors were euthanized once the tumors reached a size of 400 mm ² or died of spontaneous metastasis. The percentage of surviving animals is shown in panel H. The experiment was repeated three more times. Two immunizations with irradiated MethA cells mixed with B7-IgG treated 100% of the mice, compared to 10-15% of the spontaneous treatment of the control group (FIG. 8). Similar results were observed in C57BL / 6 mice with bladder carcinoma MB49. In C57BL / 6 mice, the response of melanoma B16 / F10, which is highly metastatic and poorly immunogenic, was studied. Three immunizations with irradiated B16 tumor cells mixed with either of the B7-IgG proteins reduced tumor growth and improved long term survival compared to the control (FIG. 8). All of the animals in the control group died within 35-40 days, but at least 40-80% of mice treated with the B7-IgG vaccine survived at least 60 days (FIG. 8). These results support the observations in the P815 tumor model and demonstrate the effective activity of B7-IgG as adjuvant for therapeutic tumor vaccines.

Therapeutic administration of B7-IgG alone to induce anti-tumor response:

As noted above, prophylactic immunization of untreated mice with B7-IgG in the absence of irradiated tumor cells failed to defend mice against tumor challenge. However, for all four therapeutic tumor models, treatment of tumor containing mice with B7-1-IgG or B7-2-IgG alone reduced tumor growth and increased survival (FIG. 9). Mice were inoculated with live P815 (A), MethA (B), MB49 (C) or C57BL / 6 (D) tumor cells on day 0. Immunization occurred on days 6-8 as described. Mice were treated with PBS (□), irradiated tumor cells alone (◇), irradiated tumor cells mixed with B7-1-IgG (Δ), or B7-2-IgG (O), or B7-1-IgG (* ) Or B7-2-IgG alone (+). The mean value of tumor size for a group of 7 to 10 mice was plotted. Mice were euthanized when tumors reached 400 mm 2 or assigned to this value when they died of metastatic disease. For all models tested, therapeutic treatment of tumor containing mice with B7-IgG alone slowed tumor growth, induced tumor regression, and increased survival. However, data from the B16 / F10 tumor model suggests that vaccination with irradiated tumor cells + B7-IgG is a more potent anti-tumor therapy than treatment with B7-IgG alone for tumors that are at least poorly immunogenic.

These results associated with soluble B7-IgG are surprising given the results obtained using costimulatory molecules (solid costimulatory) provided on the surface of the cell. Therapeutic vaccination with B7 transfected tumor cells in all three of these models was evaluated and did not show a modest effect on tumor growth and survival. About 10 ¹⁶ B7 molecules are provided by mixing 100 μg of B7-IgG with the vaccine compared to the total about 10 ¹⁰ to 10 ¹¹ B7 molecules on the surface of 10 ⁷ transfected tumor cells. This quantitative difference may explain why vaccination with investigated B7-transfectants is much less efficient than with live B7-transfectants, where live cells can proliferate and increase the number of available B7 molecules. . Other explanations may include the expanded presence of B7-IgG molecules as compared to B7 molecules expressed on the surface of the tumor cells investigated. In addition, soluble molecules can be distributed differently in the body and reach more suitable sites of immune stimulation.

B7-IgG Mediated Tumor Therapy That is CD8 T Cell Dependent but Not IFN-γ Independent

To further characterize the involvement of the adaptive immune response to B7-IgG mediated immune stimulation, B7-IgG in SCID mice lacking mature T and B cells was evaluated. After solid tumors were established in SCID mice, these tumors were treated with B7-IgG alone or irradiated cell vaccine + B7-IgG. Both treatments did not affect tumor growth, demonstrating that B7-IgG mediated tumor response is dependent on T or B cells (FIG. 10). Tumor-containing mice were also treated after removal of CD8 or CD4 T cells. Removal of CD8 or CD4 T cells by antibody injection was initiated one day before starting B7-IgG treatment. Successful clearance was confirmed by FACS analysis of PBL on day 28. For CD4-removed mice, B7-IgG induced tumor regression and treatment was indistinguishable from normal mice (FIG. 11), but CD8 clearance lost B7-IgG mediated anti-tumor activity. Tumors grew more slowly than untreated mice removed with CD8, but no tumor treatment was observed (FIG. 11).

Despite the fact that IFN-γ plays an important role in anti-tumor immune surveillance and anti-tumor response, it has been determined that B7-IgG can treat tumors established independently of IFN-γ. Solid tumors were established in IFN-γ knockout mice and treated with irradiated tumor cell vaccines mixed with B7-IgG or B7-IgG. Both treatments induced tumor regression and treatment at about 28 days was comparable to wild type mice, demonstrating the IFN-γ independence of B7-IgG tumor treatment (FIG. 12). In addition, tumors that were not reactive to IFN-γ due to mutations in IFN-γ were still treated with B7-IgG.

It has also been determined that B7-IgG in anti-tumor treatment or as a vaccine adjuvant is more potent than blocking antibody to CTLA4. For three different tumor models, B7-IgG treatment provides protection against tumor challenge where the anti-CTLA4 antibody has no effect despite past reports of its much greater affinity for CTLA4 and its blocking activity or Tumors were treated. These data indicate that the mechanism by which B7-IgG enhances the immune response is dependent on, but not limited to, blocking of negative signals mediated by CTLA4.

Table 2. B7-IgG2a Mixed with Irradiated Tumor Cells Providing Protective Immunity

Immunization Protection rate (mean ± SD) Animal Count (Protected / Vaccination Animals) Number of experiments Do not process 8% (11) 3/42 5 Investigated P815 2% (4) 1/43 5 Irradiated P815-B7-1 Transfectant 23% (4) 3/13 2 Investigated P815 + B7-1 IgG 65% (18) 29/45 4 Investigated P815 + B7-2 IgG 60% (24) 23/37 4 Investigated P815 + B7-1 IgG (mutated) 28% (4) 5/18 2 Investigated P815 + B7-2 IgG (mutated) 20% 2/10 One Investigated P815 + Anti-CD28 0% 0/10 One Investigated P815 + Anti-CTLA-4 40% 4/10 One Investigated L1210-P1A 0% (0) 0/19 2 Investigated L1210-P1A + B7-1-IgGIgG 10% (14) 2/20 2

Equivalent

Those skilled in the art will recognize, or be able to ascertain, many equivalents to the specific embodiments of the invention described herein using only routine experimentation. Such equivalents are intended to be included within the scope of the following claims.

Background

For T cell response to foreign proteins, two signals must be provided to T lymphocytes by antigen expressing cells (APCs) (Ref. Jenkins, M. and Schwartz, R. (1987) J. Exp. Med. 165, 302-319; Mueller, DL, et al. (1990) J. Immunol. 144, 3701-3709). The first signal imparts specificity to the immune response and is induced through the T cell receptor (TCR) following the recognition of the foreign antigenic peptide expressed in the context of the major histocompatibility complex (MHC). The second signal, termed co-stimulation, is intended to induce T cells to proliferate and be functional (Ref. Lenschow et al. 1996. Annu. Rev. Immunol. 14: 233). Co-stimulation is not antigen specific or MHC limited and is considered to be provided by one or more unique cell surface proteins expressed by APCs (Ref. Jenkins, MK, et al. 1988 J. Immunol. 140, 3324 -3330; Linsley, PS, et al. 1991 J. Exp. Med. 173, 721-730; Gimmi, CD, et al., 1991 Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Young, JW, et al. 1992 J. Clin. Invest. 90, 229-237; Koulova, L., et al. 1991 J. Exp. Med. 173, 759-762; Reiser, H., et al. 1992 Proc. Natl.Acad. Sci. USA.89, 271-275; van-Seventer, GA, et al. (1990) J. Immunol. 144, 4579-4586; LaSalle, JM, et al., 1991 J. Immunol.147 , 774-80; Dustin, MI, et al., 1989 J. Exp. Med. 169, 503; Armitage, RJ et al. 1992 Nature 357, 80-82; Liu, Y., et al. 1992 J. Exp Med. 175, 437-445). If T cells are stimulated only through T cell receptors without additional costimulatory signals, they will downregulate the immune response by being nonreactive, lethargic, or killed.

CD80 (B7-1) and CD86 (B7-2) proteins expressed on APCs are very important costimulatory molecules (Ref. Freeman et al. 1991. J. Exp. Med. 174: 625; Freeman et al. 1989 J. Immunol. 143: 2714; Azuma et al, 1993 Nature 366: 76; Freeman et al. 1993. Science 262: 909. B7-2 plays a dominant role in the primary immune response, whereas B7-1, upregulated later in the course of the immune response, appears to be important for prolonging primary T cell responses or costimulating secondary T cell responses. (Reference: Bluestone. 1995. Immunity. 2: 555).

CD28, a ligand that binds B7-1 and B7-2, is constitutively expressed on resting T cells and increases expression after activation. After signaling through the T cell receptor, the binding of CD28 and costimulatory signals, or induces T cells to proliferate and secrete IL-2 (Ref. Linsley, PS, et al. 1991 J. Exp. Med 173, 721-730; Gimmi, CD, et al. 1991 Proc. Natl. Acad. Sci. USA. 88, 6575-6579; June, CH, et al. 1990 Immunol.Today 11, 211-6; Harding , FA, et al. 1992 Nature. 356, 607-609). The second ligand, named CTLA4 (CD152), is homologous to CD28 but is not expressed on resting T cells and appears to follow T cell activation (Ref. Brunet, JF, et al., 1987 Nature 328, 267-270 ). In contrast to CD28, CTLA4 appears to be very important for the negative regulation of T cell responses (Waterhouse et al. 1995. Science 270: 985). Blockade of CTLA4 was found to eliminate the inhibitory signal, whereas assembly of CTLA4 was found to provide an inhibitory signal that downregulates T cell responses (Allison and Krummel. 1995. Science 270: 932).

There has been a long need for the development of methods to enhance immune responses. For example, to date, it has been difficult to enhance the immune response to specific viruses and tumor cells using methods according to the prior art. In general, methods for enhancing the immune response, and in particular for enhancing the response to tumor antigens and infectious antigens (eg viral, bacterial, and / or parasitic antigens) will be very useful.

Summary of the Invention

The present invention provides a method for enhancing an immune response by manipulating costimulatory pathways. The method of the invention is particularly effective in increasing the response to tumor antigens and antigens from infectious agents. The present invention is based, at least in part, on the discovery that soluble forms of costimulatory molecules can enhance immune responses prophylactically and therapeutically. This enhancement is observed despite the fact that the soluble costimulatory molecules of the present invention are not administered to the solid phase (eg, not to the cells) and in the absence of crosslinking agents. This finding, in particular in view of the teaching that soluble forms of B7-1 and B7-2 molecules do not cause co-stimulatory reactions (see Hayden et al. 1996. Tissue Antigens, 48: 242; US patent 5,580,756) It is very amazing.

Thus, in one aspect, the present invention provides a method for prophylactically enhancing the subject's immune response to an antigen by administering a soluble composition comprising the extracellular domain of the costimulatory molecule, such that the subject's immune response to the antigen is enhanced. To provide.

In another aspect, the invention provides a method of therapeutically enhancing an immune response by a subject to an antigen by administering a soluble composition comprising an extracellular domain of a costimulatory molecule, such that the immune response to the antigen of the subject is enhanced. To provide.

In one embodiment, the costimulatory molecule is selected from the group consisting of B7-1 and B7-2.

In another aspect, administering a first agent comprising a class I restriction antigen or fragment thereof and an extracellular domain of a B7 molecule enhances CD8 + T cell response to a class I restricted antigen in a subject, thereby administering the class to a subject when administered to the subject. Provided are methods for allowing CD8 + T cell responses to I restriction antigens to be enhanced.

In one embodiment, the methods of the present invention further comprise administering a class II restriction antigen to the subject.

In another embodiment, the methods of the present invention further comprise administering an adjuvant to the subject.

In one embodiment, the B7 molecule is a B7-1 molecule. In another embodiment, the B7 molecule is a B7-2 molecule.

In one embodiment, the costimulatory molecule is monospecific. In other embodiments, the costimulatory molecules are dimers and divalents. In one embodiment, the soluble costimulatory molecules are monospecific and dimers and divalents.

In another embodiment of the invention, the extracellular portion of the B7 molecule is fused with a second protein or polypeptide comprising a portion of an immunoglobulin molecule. In one embodiment, the portion of the immunoglobulin molecule comprises a cysteine residue. In one embodiment, the portion of the immunoglobulin molecule comprises the hinge, CH2 and CH3 regions of the human immunoglobulin molecule. In other embodiments, the portion of the immunoglobulin molecule comprises the hinge, CH1, CH2 and CH3 regions of the human immunoglobulin molecule. In one embodiment, the immunoglobulin molecule is modified to reduce complement fixation and / or Fc receptor binding.

In one embodiment, the antigen is a tumor cell antigen.

In other embodiments, the subject comprises a cancer of a type selected from the group consisting of colon cancer, breast cancer, prostate cancer, renal cell cancer, leukemia, lymphoma, melanoma, mastocytoma, sarcoma, and bladder carcinoma.

In one embodiment, the immune response is a cellular immune response. In another embodiment, the immune response is a humoral immune response.

Claims (21)

A method of prophylactically enhancing an immune response by a subject to an antigen comprising administering a soluble composition comprising an extracellular domain of a costimulatory molecule to enhance the immune response to the subject's antigen. A method of therapeutically enhancing an immune response by a subject to an antigen comprising administering a soluble composition comprising an extracellular domain of the costimulatory molecule to enhance the immune response to the subject's antigen. The method of claim 1 or 2, wherein the costimulatory molecule is selected from the group consisting of B7-1 and B7-2. Enhancing a CD8 + T cell response to a Class I restricted antigen upon administration to a subject, including a soluble composition comprising a Class I restricted antigen or fragment thereof and an extracellular domain of a B7 molecule A method of enhancing CD8 + T cell response to an antigen. 5. The method of claim 4, further comprising administering a class II restriction antigen to the subject. 5. The method of claim 1, 2 or 4, further comprising administering an adjuvant to the subject. The method of claim 1, 2 or 4, wherein the B7 molecule is a B7-1 molecule. 5. The method of claim 1, 2 or 4 wherein the B7 molecule is a B7-2 molecule. The method of claim 1, 2 or 4, wherein the costimulatory molecule is monospecific. 5. The method of claim 1, 2 or 4, wherein the costimulatory molecules are dimers and bivalent. 6. The method of claim 1, 2 or 4, wherein the soluble costimulatory molecule is monospecific, dimeric and divalent. The method of claim 11, wherein the extracellular portion of the B7 molecule is fused with a polypeptide comprising a second protein or a portion of an immunoglobulin molecule. 13. The method of claim 12, wherein the portion of the immunoglobulin molecule comprises a cysteine residue. 13. The method of claim 12, wherein the portion of the immunoglobulin molecule comprises the hinge, CH2 and CH3 regions of the human immunoglobulin molecule. 13. The method of claim 12, wherein the portion of the immunoglobulin molecule comprises the hinge, CH1, CH2 and CH3 regions of the human immunoglobulin molecule. 13. The method of claim 12, wherein the immunoglobulin molecule is modified to reduce complement immobilization and / or Fc receptor binding. The method of claim 1, 2 or 4, wherein the antigen is a tumor cell antigen. The method of claim 2, wherein the subject has a type of cancer selected from the group consisting of colon cancer, breast cancer, prostate cancer, renal cell cancer, leukemia, lymphoma, melanoma, mastocytoma, sarcoma, and bladder carcinoma. 5. The method of claim 1, 2 or 4, wherein the antigen is an antigen selected from the group consisting of bacterial antigens, viral antigens and parasitic antigens. 5. The method of claim 1, 2 or 4, wherein the immune response is a cellular immune response. 5. The method of claim 1, 2 or 4, wherein the immune response is a humoral immune response.