JP2002507392A - Optimization of immunomodulatory properties of gene vaccines - Google Patents

Abstract

  (57) [Summary] The present invention provides a method for obtaining a molecule capable of modulating an immune response, and an immunomodulatory molecule obtained using the method. This molecule finds use, for example, in tailoring the immune response induced by a genetic vaccine for the desired purpose.

Description

DETAILED DESCRIPTION OF THE INVENTION

(Statement Regarding Government Assistance) The present invention, together with government assistance, is
Grant No. N65236-98-1-54 awarded by tAgency
Made under No. 01. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to immune responses (eg, immune responses induced by genetic vaccines).
Related to the field of regulation.

BACKGROUND [0003] Antigen processing and antigen presentation are the only factors that determine the efficacy of vaccination (whether performed with genetic vaccines or with more classical methods). Other molecules involved in determining vaccine efficacy include cytokines (interleukins, interferons, chemokines,
Hematopoietic growth factor, tumor necrosis factor, and transforming growth factor). It is a low molecular weight protein that regulates the maturation, activation, proliferation and differentiation of cells of the immune system. The properties of cytokines are pleiotropic and degenerate; that is, one cytokine often has several functions, and a given function is often mediated by more than one cytokine. In addition, some cytokines have additive or synergistic effects on other cytokines, and many cytokines also share a receptor component.

[0004] Although studies on the physiological significance of certain cytokines have been challenging due to the complexity of the cytokine network, recent studies using cytokine gene deficient mice have shown that the present invention relates to the function of cytokines in vivo. Their understanding improved significantly. In addition to soluble proteins, some membrane-bound costimulatory molecules play a fundamental role in regulating the immune response. These molecules include CD40, CD40 ligand, CD27, CD80, CD86 and CD15
0 (SLAM) and these are typically expressed in lymphoid cells after activation through antigen recognition or through cell-cell interactions.

[0005] T helper (T_H) Cells (key regulators of the immune system) can produce many different cytokines and, based on these cytokine synthesis patterns,_HCells are divided into two subsets (Paul and Seder (1994) C
ell 76: 241-251). T_HOne cell produces high levels of IL-2 and IFN-γ and minimal levels of IL-4, IL-5 and IL-1
3 or do not produce IL-4, IL-5 and IL-13. versus
In contrast, T_HTwo cells produce high levels of IL-4, IL-5 and IL-13, and minimal or no IL-2 and IFN-γ production. T _H One cell activates macrophages, dendritic cells, and⁺Cell injury
Increases the cytolytic activity of sex T lymphocytes and NK cells (ibid). On the contrary
, T_HTwo cells provide efficient help for B cells, and they also_HMediates allergic responses through the ability of two cells to induce IgE isotype switching and differentiation of B cells into IgE-secreting cells (De Vries and
And Punnonen (1996) In Cytokine Regulatio
no of humoral immunity: basic and clin
ical aspects. Snapper, C.I. M. Hen, John Wile
y & Sons, Ltd. , West Sussex, UK, 195-215).
. The exact mechanism that regulates T helper cell differentiation is not well understood, but
Tocaine is thought to play a major role. IL-4 is T_HIt has been shown to direct the differentiation of two cells. In contrast, IL-12 has a T_HInduces the development of one cell (Paul and Seder, supra). Furthermore, CD80, CD8
6 and a membrane-associated costimulatory molecule such as CD150_H1 cell and / or T_H2 can indicate development and T_HThe same molecules that regulate cell differentiation are also
Has been suggested to affect the activation, proliferation and differentiation of vesicles into Ig-secreting plasma cells
(Cocks et al. (1995) Nature 376: 260-263; Le)
(1996) Immunity 5: 285-293; Punn.
onen et al. (1993) Proc. Nat'l. Acad. Sci. USA. 9
0: 3730-3734; Punnonen et al. (1997) J. Am. Exp. Med
. 185: 993-1004).

[0006] Both human and mouse studies have shown that the cytokine synthesis profile of T helper ( _TH ) cells plays a crucial role in determining the prognosis of some viral, bacterial and parasitic infections. . T _H 1 cells high frequency, typically to prevent infection lethal. Major T _H 2 phenotype on the other hand, often results in infection of chronic in disseminated. For example, T _H 1 phenotype is observed Te s tuberculosis (resistant) form odor leprosy, and T _H 2 phenotype of lepromatous resistance, observed in (susceptible) disease nest multiple bacilli (Yamamura et al., (1991) Science 2
54: 277-279). Similarly, late HIV patients have T _H 2-like cytokine synthesis profiles, and T _H 1 phenotype have advocated that protects AIDS (Maggi et al., (1994) J. Exp. Med. 180: 489.
-495). In addition, survival from meningococcal sepsis was due to TNF-α and IL
Determined genetically based on the ability of peripheral blood leukocytes to produce -10. Individuals of a family with high production of IL-10 are at increased risk of fatal meningococcal disease. In contrast, members of the family with high TNF-α production were more likely to survive infection (Westendorp et al. (1997) Lancet.
349: 170-173).

[0007] Cytokines treatment, T _H 1 / T _H 2 differentiation and macrophage cell activation,
And it can dramatically affect the prognosis of infectious diseases. For example, Leishm
BALB / c mice infected with ania major generally but develop disseminated fatal disease with T _H 2 phenotype, anti IL-4 mAb or IL
When treated at -12, the frequency of T _H 1 cells in mice increased, and it we may weaken the effect of invasion of pathogens (Chatelain et al (1992) J.I
mmunol. 148: 1182-1187). Similarly, INF-γ protects mice from lethal herpes simplex virus (HSV) infection, and MCP-1 inhibits Pseudomonas aeruginosa or Salmonella.
Typhimurium protects against lethal infection. In addition, cytokine treatment (eg, recombinant IL-2) has shown beneficial effects on human unclassifiable immunodeficiency (Cunningham-Rundles et al. (1994) N. Engl.
. J. Med. 331: 918-921).

[0008] The administration of cytokines and other molecules that modulate the immune response in a manner most appropriate for treating a particular disease can provide a significant means for treating the disease. However, currently available immunomodulator treatments may have some disadvantages, such as insufficient specific activity, induction of an immune response to the administered immunomodulator, and other potential problems . Accordingly, there is a need for immunomodulators that exhibit improved properties as compared to currently available immunomodulators. The present invention fulfills this and other needs.

SUMMARY OF THE INVENTION The present invention provides for either directly (ie, as an immunomodulatory polynucleotide) or indirectly (ie, during translation of a polynucleotide to produce an immunomodulatory polypeptide). Thus, the present invention provides a method for obtaining a polynucleotide having a regulatory effect on an immune response induced by a genetic vaccine. The method of the invention comprises the steps of: producing a library of recombinant polynucleotides;
Screening libraries and demonstrating the ability to modulate an enhanced immune response, either by the recombinant polynucleotide itself or through the encoded polypeptide, even by the form of the nucleic acid that generated the library. Identifying at least one optimized recombinant polynucleotide. Examples include: CpG-rich polynucleotide sequences, costimulators (eg, B7-1, B7-2, CD1, CD40, CD154 (ligand for CD40), CD150 (SLAM)) or cytokines. The polynucleotide sequence to be expressed. The screening step used in these methods comprises:
For example, introducing a genetic vaccine vector comprising a library of recombinant nucleic acids into cells, and identifying cells that exhibit an increased ability to modulate the immune response of interest or to express an immunomodulatory molecule May be included. For example,
Libraries of recombinant nucleic acids encoding cytokines can be screened by testing the ability of the cytokine encoded by the nucleic acid molecule to activate cells containing receptors for the cytokine. Receptors for cytokines can be naturally occurring in the cell or can be expressed from a heterologous nucleic acid encoding a cytokine receptor. For example, a costimulatory factor which is optimized test cell or culture medium can be identified mainly T _H 2 immune response, or predominantly a costimulatory factor which is capable of inducing a T _H 1 immune response.

[0010] In some embodiments, the polynucleotide having a modulating effect on the immune response is obtained by: (1) a molecule involved in modulating an immune response or a molecule involved in modulating an immune response; Recombinantly encode at least the first and second forms of the nucleic acid (where the first and second forms are different from each other in two or more nucleotides) to obtain a library of recombinant polynucleotides. Making; and (2) screening the library, either by the recombinant polynucleotide itself or through the encoded polypeptide, to enhance the form of the nucleic acid from which the library was made. At least one optimized recombinant polynucleotide exhibiting the ability to modulate an immune response. Set. If further optimization is desired, the method may further comprise: (3) at least one optimized recombinant polynucleotide and a further form of nucleic acid, comprising a first form and a second form; The same or different form) to produce an additional library of recombinant polynucleotides;
(4) screening an additional library to identify at least one further optimized recombinant polynucleotide that exhibits the ability to modulate an enhanced immune response over the form of the nucleic acid from which the library was made; and (5) If necessary, until further optimized recombinant polynucleotides show the ability to modulate an enhanced immune response over the form of the nucleic acid from which the library was made, (3)
Repeating (4) and (4).

[0011] In some embodiments of the present invention, a library of recombinant polynucleotides is screened by: the encoded peptides or proteins whose polypeptides are displayed on the surface of a replicable genetic package Expressing a recombinant polynucleotide so as to be produced as a fusion with; contacting a replicable genetic package with a plurality of cells presenting a receptor; and cells exhibiting the modulation of a receptor-mediated immune response Is identified.

[0012] The invention also provides a method for obtaining a polynucleotide encoding an accessory molecule that improves the transport or presentation of an antigen by a cell. These methods are
Producing a library of recombinant polynucleotides by providing a recombinant nucleic acid encoding all or a portion of the accessory molecule; and screening this library for comparison with accessory molecules encoded by the non-recombinant nucleic acid And identifying an optimized recombinant polynucleotide that encodes a recombinant accessory molecule that confers on the cell an enhanced or reduced ability to transport or present the antigen on the surface of the cell. In some embodiments,
The screening step includes the steps of: introducing a library of recombinant polynucleotides into a gene vaccine vector encoding the antigen to form a library of vectors; introducing the library of vectors into mammalian cells ;
And identifying mammalian cells that exhibit increased or decreased immunogenicity to the antigen.

[0013] In some embodiments of the invention, the cytokine to be optimized is interleukin-12, and the screening comprises growing a mammalian cell containing the genetic vaccine vector in culture medium; It is performed by detecting whether cell proliferation or T cell differentiation is induced by contact with the culture medium. In another embodiment, the cytokine is interferon-α,
The screen then expresses the recombinant vector module as a fusion protein displayed on the surface of the bacteriophage, forms a phage display library, and identifies phage library members that can inhibit the growth of B cell lines It is done by doing. Another embodiment provides B7 as a costimulator.
Utilizing -1 (CD80) or B7-2 (CD86), and cells or culture media are tested for their ability to modulate an immune response.

The present invention provides optimized recombinant vectors encoding cytokines and other costimulators that exhibit reduced immunogenicity as compared to the corresponding polypeptide encoded by a non-optimized vector module. DN to get the module
A method for using A shuffling is provided. This reduced immunogenicity can be detected by introducing a cytokine or costimulator encoded by the recombinant vector module into the mammal and determining whether an immune response has been induced against the cytokine. .

[0015] The present invention also provides a method for obtaining an optimized immunomodulatory sequence encoding a cytokine antagonist. For example, suitable cytokine agonists include soluble and transmembrane cytokine receptors with incomplete signal sequences. Examples include ΔIL-10R and ΔIL
-4R and the like.

DETAILED DESCRIPTION Definitions The term “cytokine” includes, for example, interleukins, interferons, chemokines, hematopoietic growth factors, tumor necrosis factors, and transforming growth factors. Generally, these are low molecular weight proteins that regulate the maturation, activation, proliferation and differentiation of cells of the immune system.

The term “screening” generally describes the process of identifying optimal immunomodulatory molecules. Several properties of each molecule can be used in selection and screening, including, for example, the ability to induce a desired immune response in a test system. Selection is a form of screening in which identification and physical separation are achieved simultaneously by expression of a selectable marker. This allows cells expressing the marker to survive, but kills other cells in some genetic settings, or vice versa. Screening markers include, for example, luciferase, β-galactosidase, and green fluorescent protein. Selectable markers include drug resistance and toxin resistance genes and the like. Due to limitations in studying the primary immune response in vitro, in vivo studies are a particularly useful screening method. In these studies, a genetic vaccine containing an immunomodulatory molecule is first introduced into a test animal, and then the immune response is determined by analyzing a protective immune response or by using lymphoid cells from the immunized animal. By studying the quality or strength of the induced immune response. Although spontaneous selection can occur during the course of natural evolution, and spontaneous selection occurs, in this method selection is made by a person.

As used herein, “exogenous DNA segment”, “heterologous sequence”
Alternatively, a "heterologous nucleic acid" is one that is derived from a source that is foreign to the particular host cell or, if the same source, has been modified from its original form. Thus, a heterologous gene in a host cell is endogenous to a particular host cell,
Includes modified genes. The heterologous sequence modifications in the present application, described herein, typically result from the use of DNA shuffling. Thus, the term refers to a DNA segment that is foreign or heterologous to the cell, or a DNA segment that is homologous to the cell, but in a position in the host cell nucleic acid where the element is not naturally found. The exogenous DNA segment is expressed to yield an exogenous polypeptide.

The term “gene” is used extensively to refer to any segment of DNA that is involved in a biological function. Thus, genes contain the coding and / or regulatory sequences required for their expression. Genes also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest, or synthesizing from known or putative sequence information, and the gene can be a sequence designed to have the desired parameters. May be included.

The term “isolated” when applied to a nucleic acid or protein, refers to a nucleic acid or protein that is substantially free of other cellular components of interest in its natural state. It can be either dry or aqueous, but preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein, the major species present in the preparation, is substantially purified. Specifically, an isolated gene is separated from open reading frames that flank the gene and encode proteins other than the gene of interest. The term "purified" indicates that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In particular, it means that the nucleic acid or protein is at least about 50% pure,
More preferably at least about 85% pure, and most preferably at least about 9
9% pure.

The term “naturally occurring” is used to describe an object that can be found in nature, as distinct from being artificially created by man. For example, a polypeptide that can be isolated from a natural source and that is present in an organism (including a virus, bacterium, protozoan, insect, plant or mammalian tissue) that has not been intentionally modified by a human in the laboratory Alternatively, the polynucleotide sequence is naturally occurring.

The term “nucleic acid” refers to deoxyribonucleotides, ribonucleotides and polymers thereof, either in single-stranded or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids that have similar binding properties to a reference nucleic acid and include known analogs of naturally occurring nucleotides that are metabolized in a manner similar to the naturally occurring nucleotides. Unless otherwise indicated, particular nucleic acid sequences also implicitly include those conservatively modified variants (eg, degenerate codon substitutions) as well as the complementary and explicitly indicated sequences. In particular, substitution of degenerate codons is accomplished by creating a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. Can be achieved (B
atzer et al., (1991) Nucleic Acid Res. 19: 508
1; Ohtsuka et al. (1985) J. Am. Biol. Chem. 260: 2605
-2608; Cassol et al. (1992); Rossolini et al. (1994).
Mol. Cell. Probes 8: 91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by the gene.

The term “gene-derived nucleic acid” refers to a nucleic acid whose gene or a partial sequence thereof ultimately served as a template for its synthesis. Thus, mRNA,
cDNA reverse transcribed from mRNA, RNA transcribed from this cDNA, cD
DNA amplified from NA, RNA transcribed from amplified DNA, etc., are all derived from this gene, and the detection of the product so derived is dependent on the detection of the original gene and / or gene transcript in the sample. Indicates presence and / or amount.

A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if they increase the transcription of the coding sequence. Operably linked means typically that the DNA sequences being linked are contiguous and, where necessary to join the two protein coding regions, contiguous and in reading frame. means. However, generally, enhancers are several kilobases away from the promoter and function when the intron sequence can be of variable length, so that some polynucleotide elements are operably linked but contiguous. Not.

Specific binding affinity between two molecules (eg, between a ligand and a receptor) refers to the preferential binding of one molecule to another in a mixture of molecules.
Binding of a molecule can be considered specific if the binding affinity is from about 1 × 10 ⁴ M ⁻¹ to about 1 × 10 ⁶ M ⁻¹ or more.

The term “recombinant” when used in reference to a cell indicates that the cell replicates the heterologous nucleic acid or expresses a peptide or protein encoded by the heterologous nucleic acid. Recombinant cells can contain genes that are not found in the natural (non-recombinant) form of the cell. A recombinant cell can also contain a gene found in the cell in its native form, where the gene is modified and reintroduced into the cell by artificial means. The term also includes cells that contain nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such modifications include gene replacement, site-specific mutations, and related Includes cells obtained by technology.

“Recombinant expression cassette” or simply “expression cassette” refers to a recombinant or synthetic expression of a nucleic acid element capable of effecting the expression of a structural gene in a host compatible with such sequences. This is the nucleic acid construct prepared in (1). The expression cassette contains at least a promoter, and optionally a transcription termination signal. Typically, a recombinant expression cassette includes a nucleic acid to be transcribed (eg, a nucleic acid encoding a desired polypeptide), and a promoter. Additional elements necessary or helpful in effecting expression may also be used as described herein. For example, an expression cassette can also include a nucleotide sequence that encodes a signal sequence that directs secretion of the expressed protein from a host cell. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in the expression cassette.

“Recombinant polynucleotide” or “recombinant polypeptide” refers to 1
A nucleic acid sequence derived from more than one source nucleic acid or polypeptide, or a non-naturally occurring polynucleotide or polypeptide comprising an amino acid sequence. Here, the source nucleic acid or polypeptide may be a naturally occurring nucleic acid or polypeptide, or may itself be subject to mutagenesis or other types of modification. Source polynucleotides or polypeptides from which different nucleic acid or amino acid sequences are derived are sometimes homologous (ie, have or encode polypeptides that encode identical or similar structures and / or functions), and Frequently, for example, from different isolates, serotypes, strains, species of the organism, or from different disease states.

In two or more nucleic acid or polypeptide sequences, the term “identity” or percentage “identity” may be determined using one of the following sequence comparison algorithms or by visual inspection: Refers to two or more sequences or subsequences that have the same or a specified percentage of amino acid residues or nucleotides that are the same or the same when compared or aligned for the greatest match.

In two nucleic acids or polypeptides, the phrase “substantially identical” refers to the comparison of the greatest match using one of the following sequence comparison algorithms or as determined by visual inspection: At least 6 when done or aligned
Refers to two or more sequences or subsequences that have 0%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity. Preferably, substantial identity exists over a region of the sequence that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequence is at least about 150 residues. Are substantially the same throughout. In some embodiments, the sequence is substantially identical over the entire length of the coding region.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, the arrangement of subsequences is specified, and, if necessary, parameters of the sequence algorithm program. The sequence comparison algorithm then calculates the percent sequence identity of the test compound relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be found, for example, in Smith and Waterma.
n, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needlman and Wunsch, J. Am. Mol.
Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sc
i. USA 85: 2444 (1988), or by computerized implementation of these algorithms (Wisconsin Genetic).
s Software Package, Genetics Computer
Group, 575 Science Dr. , Madison, WI GA
P, BESTFIT, FASTA, TFASTA) or by visual inspection (generally Ausubel et al., See below).

One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm. This is described in Altschul et al. M
ol. Biol. 215: 403-410 (1990). BL
Software for performing AST analysis is available from National Center f
or Biotechnology Information (http: //
www. ncbi. nlm. nih. gov. Available publicly through /). This algorithm uses a short word of length W in the query sequence.
Identifying rd) involves first identifying a high-scoring sequence pair (HSP). The query sequence either matches or satisfies some positive value threshold score T when aligned with words of the same length in the database-base sequence. Let T be the adjacent word score threshold (neighborhood wor
d score threshold (Altschul et al., supra).
These initial neighboring word hits serve as sources for initiating the search and find longer HSPs containing them. This word hit can then be extended in both directions along each sequence, as long as the cumulative alignment score can be increased. Cumulative scores are calculated for nucleotide sequences using the parameters M (reward score for a pair of matching residues; always> 0) and N (penalty score for a mismatching residue); always <
0). For the amino acid sequence, a cumulative score is calculated using a score matrix. Extension of word hits in each direction is stopped if: the cumulative alignment score falls below the value that reached its maximum in amount X; by accumulating one or more negative-score residue alignments, Cumulative score goes below zero; or any sequence reaches end. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as default: word length (W) of 11
Expected value (E) of 10, cutoff of 100, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses the following as initial settings: word length (W) of 3, expected value (E) of 10, and BLOSUM62.
Scoring matrices (Henikoff and Henikoff (1989) Pr
oc. Natl. Acad. Sci. USA 89: 10915)
.

In addition to calculating the percent sequence identity, the BLAST algorithm also
Perform a statistical analysis of the similarity between the two sequences (eg, Karlin and A
ltschul (1993) Proc. Nat'l. Acad. Sci. USA
90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (smallest sum p
(P (N)). This provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example,
If the minimum sum probability is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001 in the comparison of the reference nucleic acid and the test nucleic acid,
The nucleic acid is considered similar to the reference sequence.

Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase “hybridizes specifically to” means that the sequence is a complex mixture (eg,
(Total cell) When present in DNA or RNA, refers to the binding, duplex formation, or hybridization of molecules to only specific nucleotide sequences under stringent conditions. "Substantially bind" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid, and by reducing the stringency of the hybridization medium to achieve the desired detection of the target polynucleotide sequence. Accept minor mismatches that can be adapted.

In nucleic acid hybridization experiments, such as Southern and Northern hybridizations, “stringent hybridization conditions” and “stringent hybridization wash conditions” are sequence dependent and under different environmental parameters. different. Longer sequences hybridize specifically at higher temperatures. An extensive guide to nucleic acid hybridization is available in Tijssen (1993) Laboratory.
Techniques in Biochemistry and Molec
ullar Biology--Hybridization with Nuc
leic Acid Probes part I Chapter 2 "Overview
of principals of hybridization and
the strategy of nucleic acid probe a
ssays ", Elsevier, New York. Generally, highly stringent hybridization and washing conditions are selected to be about 5 ° C. below the thermal melting temperature (T _m ) for a particular sequence at a given ionic strength and pH. Typically, under "stringent conditions" a probe will hybridize to its target subsequence, but not to other sequences.

The T _m is the temperature (under a predetermined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are chosen to be equivalent to the _Tm for a particular probe. Examples of stringent hybridization conditions for hybridization of complementary nucleic acids having more than 100 complementary residues on a filter in a Southern blot or Northern blot include 50% formamide containing 1 mg of heparin, 42 ° C. and this hybridization is performed overnight. Examples of highly stringent wash conditions are 0.15 M NaCl, 72 ° C.
For about 15 minutes. An example of stringent wash conditions is a 0.2 × SSC wash at 65 ° C. for 15 minutes (for description of SSC buffer, see Sambrook
, Supra). Often, high stringency washes are performed before low stringency washes to remove background probe signals. For example, an example of a moderately stringent wash for a duplex of more than 100 nucleotides is 1 × SSC at 45 ° C. for 15 minutes. For example, an example of a low stringency wash for a duplex of more than 100 nucleotides is 4-6 × SSC at 40 ° C. for 15 minutes. Short probes (for example,
For about 10 to 50 nucleotides), stringent conditions typically include a salt concentration of Na ⁺ ion of less than about 1.0 M, typically about 0.01 to 1. The pH is 7.0-8.3 at 0 M Na ⁺ ion concentration (or other salt), and the temperature is typically at least about 30 ° C. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Generally, a signal-to-noise ratio that is twice (or higher) than that observed for an irrelevant probe in a particular hybridization assay indicates detection of specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This occurs, for example, when a copy of the nucleic acid is made using the maximum codon degeneracy permitted by the genetic code.

[0038] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-linked to the polypeptide encoded by the second nucleic acid. Is reactive or specifically binds. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.

The phrase “specifically (or selectively) binds to an antibody” or “specifically (or selectively) immunoreacts” when referring to proteins or peptides refers to proteins and other organisms. A binding reaction that is determinant of the presence of this protein or an epitope derived from this protein in the presence of a heterogeneous population of the body. Thus, under designated immunoassay conditions, a specific antibody binds to a particular protein,
And does not bind in significant amounts to other proteins present in the sample. Antibodies raised against a multivalent antigenic polypeptide generally bind to the protein from which one or more epitopes were obtained. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. Various immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays,
Western blot, or immunohistochemistry, is routinely used to select monoclonal antibodies specifically immunoreactive with the protein. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see
Harlow and Lane (1988) Antibodies, A Labo.
rattro Manual, Cold Spring Harbor Pu
publications, New York "Harlow and Lane"). Typically, a specific or selective reaction will be at least twice background signal or noise, and more typically more than 10 to 100 times background.

A “conservatively modified mutation” of a particular polynucleotide sequence refers to those polynucleotides that encode the same or essentially the same amino acid sequence, or that the polynucleotide , Refer to essentially identical sequences. Due to the degeneracy of the genetic code, many functionally identical nucleic acids
Encodes any given polypeptide. For example, codons, CGU, CGC, C
GA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid mutations are “silent mutations”, which are a type of “conservatively modified mutations”. Every polynucleotide sequence described herein that encodes a polypeptide also describes every possible silent variation, unless otherwise stated. One skilled in the art will recognize that each codon (except for AUG, which is usually the only codon for methionine) in the nucleic acid can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each "silent variant" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

In addition, one skilled in the art will recognize that a single amino acid or a low percentage of amino acids (typically less than 5%, more typically less than 1%) in the encoded sequence will be altered, added, or A "conservatively modified mutation" is one in which the individual substitution, deletion or addition that is deleted results in a substitution of the amino acid with a chemically similar amino acid.
Recognize that Tables of conservative substitutions of functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another: Aliphatics: Glycine (G), Alanine (A), Valine (V), Leucine (L),
Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: aspartic acid (D), glutamic acid (E), asparagine (A), glutamine (Q). For further groupings of amino acids, see Creighton (1984) P
proteins, W.M. H. See also Freeman and Company. In addition, individual substitutions, deletions or additions that modify, add or delete a single amino acid or a low percentage of amino acids in the encoded sequence are also referred to as "conservatively modified mutations". is there.

A “subsequence” refers to a nucleic acid sequence or amino acid sequence that includes a portion of a longer nucleic acid sequence or amino acid sequence (eg, a polypeptide), respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to polynucleotides that, when present on a gene vaccine vector, are capable of modulating an immune response either directly or indirectly (ie, by encoding a polypeptide). Provides a method for obtaining a sequence. In another embodiment, the invention provides a method for optimizing antigen transport and presentation. The optimized immunomodulatory polynucleotide obtained using the method of the present invention is particularly suitable for use in combination with vaccines (including genetic vaccines). One of the advantages of genetic vaccines is that genes encoding immunomodulatory molecules such as cytokines, costimulatory molecules, and molecules that improve antigen transport and presentation can be incorporated into genetic vaccine vectors. This provides an opportunity to modulate the immune response elicited against antigens expressed by the genetic vaccine.

A. Production of Recombinant Libraries The present invention involves producing a recombinant library of polynucleotides, and then screening this library to identify library members that exhibit desired properties. Identify. Any of a variety of methods can be used to make this recombinant library.

[0045] The substrate nucleic acid used for recombination can vary depending on the particular application. For example,
Polynucleotides that encode cytokines, chemokines, or other accessory molecules should be optimized, and different forms of nucleic acid molecules that encode all or a portion of a cytokine, chemokine, or other accessory molecule may be subject to recombination. Is done. This method requires at least two variant forms as starting substrates. Variant forms of the candidate substrate may exhibit substantial sequences, ie, secondary structures similar to each other, but they should also differ in at least two positions. The initial diversity between forms may be the result of natural variation, for example, different variant forms (homologs) may be derived from different individuals or strains of organisms (including geographic variants). Obtained or constitute related sequences from the same organism (eg, allelic variants). Alternatively, early diversity can be induced, for example, the second variant form may be due to error-prone transcription (eg, error-prone PCR) of the first variant form, or the use of a polymerase lacking proofreading activity (Liao (1990) Gene 88: 107-111.
) Or by replication of a first form in a mutagenized strain (mutagenized host cells are discussed in further detail below). The initial diversity between substrates is greatly increased in subsequent steps of recursive sequence recombination.

Often, improvements are achieved after one round of recombination and screening. However, using recursive sequence recombination, the desired properties can still be improved. Sequence recombination can be accomplished in many different or permuted formats, as described in further detail below. These forms share some common principles. Recursive sequence recombination involves successive cycles of recombination to generate molecular diversity. That is, a family of nucleic acid molecules that exhibit some degree of sequence identity to one another, but differ in the presence of mutations. In any given cycle, recombination can occur in vivo or in vitro, intracellularly or extracellularly. Furthermore, the diversity arising from recombination can be increased in any cycle by applying prior mutagenesis methods (eg, error-prone PCR or cassette mutagenesis) to either the substrate or the product for recombination. . In some cases, the new or improved property or characteristic may be, for example, a sequence of different variant forms (eg, homologs from different individuals or strains of organisms), or related sequences from the same organism. When using (eg, allelic variation), it can be achieved after only one cycle of in vivo or in vitro recombination.

In a currently preferred embodiment, the recombinant library is prepared using DNA shuffling. Use shuffling and screening or selection to "evolve" individual genes, whole plasmids or viruses, multiple gene clusters, or whole genomes (Stemmer (1995) Bi)
o / Technology 13: 549-553). Repeat cycles of recombination and screening / sorting can be performed to further evolve the nucleic acid of interest. Such techniques do not require extensive analysis and computer manipulation required by conventional methods for polypeptide engineering. Shuffling allows for the recombination of a large number of mutations in a minimal number of selection cycles, in contrast to conventional pairwise recombination events. Thus, the sequence recombination techniques described herein provide for recombination between mutations in any or all of the mutations, thereby exploring the manner in which various combinations of mutations can affect a desired result, It offers certain advantages in providing a very fast method. However, in some cases, the structural and / or functional information available provides an opportunity (although not necessary for sequence recombination) to modify the technology.

[0048] Sequence recombination (often DNA shuffling, evolution)
Illustrative formats and examples are described below by the present inventors and co-workers: co-pending application February 17, 1994.
U.S. patent application Ser. No. 08 / 198,431, filed on Feb. 17, 1995 PCT / US95 / 02126 filed on Feb. 17, 1995, and Ser. No. 08 / 425,684 filed on Apr. 18, 1995. No. 08 / filed on Oct. 30, 1995
No. 537,874, filed on Nov. 30, 1995, 08 / 564,955.
No. 5, No. 08 / 621,859, filed on March 25, 1996, 1996.
08 / 621,430 filed on March 25, 1996, PCT / US96 / 05480 filed on April 18, 1996, and 08/650 filed on May 20, 1996. No. 08/0, filed on Jul. 3, 1996.
No. 675,502, filed Sep. 27, 1996, Ser. No. 08 / 721,824.
No. PCT / US97 / 17300 filed on Sep. 26, 1997 and PCT / US97 / 24239 filed on Dec. 17, 1997;
Stemmer, Science 270: 1510 (1995);
er et al., Gene 164: 49-53 (1995): Stemmer, Bio.
/ Technology 13: 549-553 (1995); Stemmer.
Proc. Natl. Acad. Sci. U. S. A. 91: 10747-1
0751 (1994); Stemmer, Nature 370: 389-39.
1 (1994); Crameri et al., Nature Medicine 2 (1
): 1-3 (1996); Crameri et al., Nature Biotechn.
14: 315-319 (1996), each of which is incorporated by reference in its entirety for all purposes.

Other methods for obtaining diversity of recombinant polynucleotides and / or nucleic acids used as substrates for shuffling include, for example, homologous recombination (PCT / US98 / 05223; WO 98/42727). ); Oligonucleotide-directed mutagenesis (for review, see, eg, Smith, Ann. R.)
ev. Genet. 19: 423-426 (1985); Botstein and Shortle, Science 229: 1193-1201 (1985).
Carter, Biochem. J. 237: 1-7 (1986); Kunk
el, "The efficiency of oligocleotide.
e directed mutagenesis ", Nucleic acid
s & Molecular Biology, Eckstein and Lil
ed., Springer Verlag, Berlin (1987)). These methods include: Oligonucleotide-directed mutagenesis (Zoller and Smith, Nucl. Acids Res 10: 6).
487-6500 (1982), Methods in Enzymol. 10
0: 468-500 (1983), and Methods in Enzymo.
l. 154: 329-350 (1987)), phosphothioate modified DNA mutagenesis (Taylor et al., Nucl. Acids Res. 13: 8749-87).
64 (1985); Taylor et al., Nucl. Acids Res. 13: 8
765-8787 (1985); Nakamaye and Eckstein, N.
ucl. Acids Res. 14: 9679-9698 (1986); Say
ers et al., Nucl. Acids Res. 16: 791-802 (1988)
Sayers et al., Nucl. Acids Res. 16: 803-814 (1
988)), mutagenesis using a template containing uracil (Kunkel, Proc.
Nat'l. Acad. Sci. USA 82: 488-492 (1985) and Kunkel et al., Methods in Enzymol. 154: 367
-382)); Mutagenesis using gapped double-stranded DNA (Kramer et al., N.
ucl. Acids Res. 12: 9441-9456 (1984); Kra.
mer and Fritz, Methods in Enzymol. 154: 3
50-367 (1987); Kramer et al., Nucl. Acids Res.
16: 7207 (1988); and Fritz et al., Nucl. Acids R
es. 16: 6987-6999 (1988)). Further suitable methods include point mismatch repair (Kramer et al., Cell 38: 879-887).
(1984)), mutagenesis using repair-deficient host strains (Carter et al., Nucl.
. Acids Res. 13: 4431-4433 (1985); Carter.
, Methods in Enzymol. 154: 382-403 (1987)
)), Deletion mutagenesis (Eghteddarzadeh and Henikoff, N
ucl. Acids Res. 14: 5115 (1986)), restriction selection and restriction purification (Wells et al., Phil. Trans. R. Soc. London. A
317: 415-423 (1986)), Mutagenesis by whole gene synthesis (Nam
biar et al., Science 223: 1299-1301 (1984); Sa.
kamar and Khorana, Nucl. Acids Res. 14:63
61-6372 (1988); Wells et al., Gene 34: 315-323.
(1985); and Grundstrom et al., Nucl. Acids Res
. 13: 3305-3316 (1985)). Kits for mutagenesis are commercially available (e.g., Bio-Rad, Amersham International).
onal, Anglian Biotechnology).

B. Screening Methods A recombination cycle usually involves at least one cycle of screening or selecting for molecules having the desired properties or characteristics. If the recombination cycle is performed in vitro, the recombinant product (ie, the recombinant segment) is sometimes introduced into the cells prior to the screening step. The recombination segment also
Prior to screening, they may be ligated to a suitable vector or other regulatory sequence. Alternatively, recombinant products produced in vitro are sometimes packaged as viruses prior to screening. If the recombination is performed in vivo, the recombination products can sometimes be screened in the cells where the recombination occurs.
In other applications, the recombinant segments are extracted from the cells and optionally packaged as viruses prior to screening.

The nature of the screening or selection depends on what property or feature is obtained or on the property or feature sought for improvement, and many examples are discussed below. Generally, it is not necessary to understand the molecular basis on which a particular recombinant product (recombinant segment) has acquired new or improved properties or characteristics relative to the starting substrate. For example, a gene vaccine vector contains a number of component sequences (eg, coding sequences, regulatory sequences, target sequences, sequences that provide stability, immunoregulatory sequences, sequences that affect antigen presentation, each having various intended roles). , And sequences that affect insertion). Each of these component sequences can be changed and recombined simultaneously. Screening / selection can then be performed, for example, on recombinant segments that increase episomal maintenance in the target cell, with the need to attribute such improvement to any of the individual component sequences of the vector. Absent.

Depending on the particular screening protocol used for the desired property,
The initial round of screening can often be performed on bacterial cells due to high transfection efficiency and ease of culture. The latter round (and other screening types that are unacceptable for screening in bacterial cells) involves the use of mammalian cells to optimize recombinant segments for use in environments similar to those of their intended use. Done. The final round of screening can be performed on the correct cell type for the intended use (eg, human antigen presenting cells). In some cases, the cells may be obtained, for example, from a patient to be treated with the intent of minimizing immunogenicity issues in the patient.

The screening or selection process identifies a subpopulation of recombinant segments that have evolved to obtain new or improved desired properties useful for genetic vaccination. Depending on the screen, the recombinant segment may be identified as a component of a cell, a component of a virus or a free form. More than one screening or selection round may be performed after each round of recombination.

If further improvements in properties are desired, at least one of the recombinant segments, and usually the population, that survives in the first round of screening / selection,
Provided for further recombination rounds. These recombinant segments can be recombined with each other or with an exogenous segment presenting an initial substrate or a further variant thereof. Furthermore, recombination can be continued in vivo or in vitro. If the previous screening step identifies the desired recombinant segment as a component of a cell, the component can be subjected to further recombination in vivo, or to further recombination in vitro, or a round of in vitro recombination. Can be isolated prior to performing. Conversely, if the previous screening step identified the desired recombinant segments, either as naked forms or as viral components, these segments could be introduced into cells to perform a round of in vivo recombination. The second round of recombination, regardless of how it is performed, produces additional recombination segments that include more diversity than exists in the recombination segments obtained from the previous round.

A second round of recombination may be followed by a further round of screening / selection according to the principles discussed above for the first round. Screening / selection stringency may be enhanced between rounds. Also, if improvement of one or more properties is desired, or if it is desired to require one or more new properties, the properties of the screened and screened properties may be changed between rounds. Further rounds of recombination and screening may then be performed until the recombination segment has evolved sufficiently to obtain the desired new or improved property or function.

Various screening methods for particular applications are described herein.
In some cases, the screening step involves expressing the recombinant peptide or polypeptide encoded by the recombinant polynucleotide in the library as a fusion with the protein displayed on the surface of a replicable genetic package. I do. For example, phage display can be used. For example, Cwirl
la et al., Proc. Natl. Acad. Sci. USA 87: 6378-6
482 (1990); Devlin et al., Science 249: 404-40.
6 (1990), Scott and Smith, Science 249: 38.
6-388 (1990); Ladner et al., U.S. Patent No. 5,571,698. Other replicable genetic packages include, for example, bacteria, eukaryotic viruses, yeast, and spores.

For display libraries, the most frequently used genetic packages are bacteriophages, especially filamentous phage, and especially phage M13, F
b and F1. Most work involved inserting a library encoding the polypeptide to be displayed into either the gIII or gVIII of these phage to form a fusion protein. For example, Dower
Devlin, WO 91/18989; MacCaf.
ferty, WO92 / 01047 (gene III); Huse, WO92 / 0
6204; Kang, WO 92/18619 (gene VIII).
Such fusion proteins usually, but need not, include a signal sequence from the phage coat protein, the polypeptide to be displayed, and either the gene III protein or the gene VIII protein or fragments thereof. Exogenous coding sequences are often, even if other insertion sites are possible.
Inserted at or near the N-terminus of gene III or gene VIII.

Eukaryotic viruses can be used to present polypeptides in a similar manner. For example, the presentation of human heregulin fused to the Moloney murine leukemia virus gp70 is described in Han et al., Proc. Natl. Aca
d. Sci. USA 92: 9747-9751 (1995). Spores can also be used as replicable genetic packages. In this case, the polypeptide is presented from the outer surface of the spore. For example, B. subtili
Spores from s are reported to be suitable. The sequence of the coat protein of these spores is described by Donovan et al. Mol. Biol. 196, 1-10 (1
987). Cells can also be used as replicable genetic packages. The polypeptide to be displayed is inserted into a gene encoding a cellular protein expressed on the cell surface. Preferred bacterial cells include Sal
monella typhimurium, Bacillus subtili
s, Pseudomonas aeruginosa, Vibrio chol
erae, Klebsiella pneumonia, Neisseria
gonorrhoeae, Neisseria meningitidis, B
acteroides nodosus, Moraxella bovis, and especially Escherichia coli. Details of outer surface proteins are discussed by Ladner et al., US Pat. No. 5,571,698,
And is incorporated herein by reference. For example, E. The lamB protein of E. coli is suitable.

The basic concept of a display method using a phage or other replicable genetic package is the establishment of a physical association of the polypeptide encoding the DNA to be screened with the polypeptide. This physical association is provided by a replicable genetic package. This presents the polypeptide as part of a capsid enclosing the genome of a phage or other package, where the polypeptide is encoded by the genome. Establishing the physical association of polypeptides with these genetic materials allows for the simultaneous mass screening of a large number of phage with different polypeptides. Phage displaying polypeptides that have an affinity for the target (eg, receptor) bind to the target, and these phages are enhanced by affinity screening for the target. The identity of the polypeptides displayed from these phages can be determined from their respective genomes. The polypeptide identified using these methods as having binding affinity for the desired target can then be synthesized in large amounts by conventional means, or the polynucleotide encoding the peptide or polypeptide can be It can be used as part of a genetic vaccine.

C. Evolution of Improved Immune Regulatory Sequences Cytokines can dramatically affect macrophage activation and T _H 1 / T _H 2 cell differentiation, resulting in infectious disease. Furthermore, recent research has shown that DN
A strongly suggests that A itself can act as an adjuvant by activating cells of the immune system. In particular, CpG-rich DNA sequence that is not methylated, enhance the differentiation of T _H 1 cells, activates cytokine synthesis by monocytes, and to induce the proliferation of B lymphocytes was shown. Accordingly, the present invention provides immunomodulation of genetic vaccines by (a) evolving the stimulatory properties of the DNA itself and (b) evolving genes encoding cytokines and related molecules involved in the regulation of the immune system. Methods for enhancing properties are provided. These genes are then used in a gene vaccine vector.

Of particular interest are INF-α and IL-12, which distort the immune response to the phenotype of T helper 1 (T _H 1) cells, thus neutralizing pathogen invasion Improve the host's ability to Also provided are methods of obtaining improved immunomodulatory nucleic acids that can inhibit or enhance antigen-specific T cell activation, differentiation, or anergy. Because of the limited information on the structures and mechanisms that regulate these events, the hybridization techniques of the present invention provide a much faster solution than rational design.

[0062] The methods of the invention typically involve the use of DNA shuffling or other methods to generate libraries of recombinant polynucleotides. This library is then screened to identify the recombinant polynucleotides in the library, and when included in or administered with a genetic vaccine, can enhance the immune response induced by the vector? Otherwise it can change. In some embodiments, the screening step comprises introducing a genetic vaccine vector comprising the recombinant polynucleotide into a mammalian cell, and wherein the cell, or the culture medium obtained by growing the cell, comprises Determining whether a response can be modulated can be included.

The optimized recombinant vector module obtained by polynucleotide recombination is useful not only as a component of a gene vaccine vector, but also for the production of polypeptides (eg, modified Satkine and the like). Can be administered to a mammal to enhance or shift the immune response. The polynucleotide sequence obtained using the DNA shuffling method of the present invention can be used as a component of a gene vaccine, or as such, for a cytokine or other immunomodulatory polypeptide used as a therapeutic or prophylactic agent. It can be used for production. If desired, the sequence of the polynucleotide encoding the optimized immunomodulatory polypeptide can be determined, and the deduced amino acid sequence is used to produce the polypeptide using methods known to those of skill in the art. obtain.

(1. Immunostimulatory DNA Sequence) The present invention provides a method for obtaining an immunostimulatory polynucleotide when introduced into a mammal. Two 5 'purines (GpA or ApA) and two 3'
Oligonucleotides containing a hexamer with a central CpG flanked by 'pyrimidines (TpC or TpT) induce cytokine synthesis and B cell proliferation in vitro (Krieg et al. (1995) Nature 374: 546; Klinm
(1996) Proc. Natl. Acad. Sci. USA 93: 2
879; Pisetsky (1996) Immunity 5: 303-10).
And acts as an adjuvant in vivo. Gene vaccine vectors into which oligos containing immunostimulatory sequences (ISS) have been inserted increase the ability to enhance antigen-specific antibody responses after DNA vaccination. The minimum length of an ISS oligonucleotide for functional activity in vitro is eight (Kli
nman et al. (supra)). The 20-mer with three CG motifs was found to induce cytokine synthesis significantly and more efficiently than the 15-mer with two CG motifs (supra). Four molecules of GGGG bind DNA to the cell surface (macrophages are receptors that bind DNA, such as scavengers).
scavenger receptor).
isetsky et al. (supra)).

In accordance with the present invention, the library comprises recombinant random DNA (eg, human,
Mouse, or other fragments of genomic DNA), known ISS, poly A, C
, G or T sequences, or a combination thereof. Recombining DNA containing at least the first and second forms that differ from each other by two or more nucleotides to produce a library of recombinant polynucleotides.

[0066] The library is then screened to identify recombinant polynucleotides that exhibit immunostimulatory properties. For example, the library can be screened for induction of cytokine production in vitro based on the introduction of the library into the appropriate cell type. A schematic diagram of this procedure is shown in FIG. Some cytokines that can be used as indicators of immunostimulatory activity include, for example, IL-2, I
L-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-1
5, and IFN-γ. IL-4 / IFN-γ, IL-4 / IL-2, I
L-5 / IFN-γ, IL-5 / IL-2, IL-13 / IFN-γ, IL-1
Changes in the 3 / IL-2 ratio can also be tested. Another screening method is
Determination of the ability to induce proliferation of cells involved in the immune response (eg, B cells, T cells, monocytes / macrophages, whole PBL, etc.). Other screens include surface antigens (eg, B7-1 (CD80), B7-2 (CD86), MHC class I
And II, and detecting the induction of APC activation based on a change in the expression level of CD14).

Another useful screen involves identifying a recombinant polynucleotide that induces T cell proliferation. The ISS sequence is used to induce B cell activation.
And, due to some homology between surface antigens expressed by T cells and surface antigens expressed by B cells, polynucleotides having stimulatory activity on T cells can be obtained.

Libraries of recombinant polypeptides can also be screened for improved CTL and antibody responses in vivo and for improved protection from infection, cancer, allergy or autoimmunity. Recombinant polynucleotides displaying the desired properties can be recovered from the cells, and if further improvement is desired, the shuffling and screening can be repeated. The optimized ISS sequence can be used as an adjuvant separately from the actual vaccine, or
The NA sequence can be fused to a gene vaccine vector.

2. The Cytokines, Chemokines, and Accessory Molecules The present invention also provides optimized cytokines, cytokine antagonists, chemokines, and other accessory molecules that direct, inhibit or enhance an immune response. Provide a way to: For example, the methods of the present invention can be used to obtain genetic vaccines and other reagents that improve or alter the immune response when administered to a mammal, such as optimized cytokines. These optimized immunomodulators are useful for treating infectious diseases, as well as other conditions, such as inflammatory diseases, in an antigen-nonspecific manner.

For example, the methods of the present invention can be used to develop optimized immunomodulatory molecules for targeted allergy. The optimized immunomodulatory molecule can be used alone or with an antigen-specific genetic vaccine to prevent or treat allergy. Four basic mechanisms are available that can achieve specific immunotherapy of allergy. First, it may be administered reagents to provoked a decrease in allergen-specific T _H 2 cells. Second, it may be administered reagents to provoked an increase in allergen-specific T _H 1 cells. Third, it may direct the expansion of inhibitory CD8 ⁺ T cells. Finally, allergies can be treated by inducing allergen-specific T cell anergy. In this example, the cytokine is determined using the method of the invention,
It can be optimized to obtain reagents effective to achieve one or more of these immunotherapy goals. The method of the present invention may include one or more properties (eg, improved specific activity,
It is used to obtain anti-allergic cytokines with improved secretion after introduction into target cells). These cytokines are effective at lower doses than the natural cytokines and have fewer side effects. Specific targets of interest include interferon α / γ, IL-10, IL-12, and IL-
4 and IL-13 antagonists.

[0071] The optimized immunomodulator, or the optimized recombinant polynucleotide encoding the immunomodulator, can be administered alone or in combination with other accessory molecules. The inclusion of optimal concentrations of appropriate molecules can enhance the desired immune response and / or indicate the induction or suppression of a particular type of immune response. A polynucleotide encoding the optimized molecule can be included in a genetic vaccine vector, or in an optimized molecule encoded by a gene that can be administered as a polypeptide.

In the methods of the invention, a library of recombinant polynucleotides encoding an immunomodulator is made by subjecting the substrate nucleic acid to a recombination protocol (eg, DNA shuffling or other methods known to those of skill in the art). The substrate nucleic acid is typically more than one form of the nucleic acid encoding the immunomodulator of interest.

[0073] Cytokines are immunomodulators that can be improved using the methods of the present invention.
Cytokine synthesis profiles play an important role in the host's ability to prevent viral, bacterial and parasitic infections, and cytokines can dramatically affect the efficacy of genetic vaccines and the consequences of infectious diseases. Some cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, I
L-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-1
3, IL-14, IL-15, IL-16, IL-17, IL-18, G-CS
F, GM-CSF, IFN-α, IFN-γ, TGF-β, TNF-α, TNF
-Β, IL-20 (MDA-7), and flt-3 ligand have been shown to stimulate an immune response in vivo or in vitro. Immune functions that can be enhanced using appropriate cytokines include, for example, B cell proliferation, Ig synthesis, Ig
Isoform transformed, T cell proliferation and cytokine synthesis, T _H 1 and T _H 2 cell differentiation, activation and proliferation of CTL, monocyte / macrophage / dendritic cells by activation and cytokine production, as well as monocytes / macrophages Differentiation of dendritic cells.

[0074] In some embodiments, the present invention provides a method for obtaining an optimized immune modulator may indicate an immune response against T _H 1 or T _H 2 response. The ability to influence the direction of the immune response in this manner is very important for the development of genetic vaccines. Changing the type of _TH response can fundamentally change the outcome of an infectious disease. T _H 1 cells frequently are generally whereas that protects from lethal infection with intracellular pathogens, T _H 2 phenotype dominant often results in chronic infection with pandemic. For example, in humans, T _H 1 phenotype is present in kind tuberculosis (resistant) form of leprosy, whereas, T _H 2 phenotype, lepromatous consists many kinds of bacilli found Te (sensitive) lesion smell (Yamamura et al., (1991) Science 254: 2)
77). Late AIDS patients have a T _H 2 phenotype. Studies on family members have shown that survival from meningococcal sepsis depends on the cytokine synthesis profile of PBL, and high IL-10 synthesis is associated with a high risk of lethal consequences.
And high TNF-α indicates that it is associated with low risk. A similar example is found in mice. For example, BALB / c mice are Leishmania
It is susceptible to major infection; these mice develop disseminated fetal diseases have a T _H 2 phenotype. Anti-IL-4 monoclonal antibody or IL-
Treatment with 12 induces a T _H 1-type response, resulting in healing. Anti-interferon gamma monoclonal antibodies exacerbate the disease. For some applications, the immune response, it is preferred to orient the direction of the T _H 2 response. For example, if increased mucosal immunity (including protective immunity) is desired, enhancement of T _H 2 responses increased antibody production, in particular to induce IgA.

T helper (T _H ) cells are probably the most important regulator of the immune system. T _H cells are classified into two subsets based on their cytokine synthesis pattern (Mosmann and Coffman (1989) Adv. Im.
munol. 46: 111). T _H 1 cells, cytokines IL-2 high-level and INF-gamma, as well as non-existent or minimum level IL-4, IL-5
And IL-13. In contrast, T _H 2 cells produce high levels of IL-4, IL-5 and IL-13, and production of IL-2 and IFN-gamma is or are missing is minimal. T _H 1 cells, macrophages, dendritic cells activated and CD8 ⁺ cytotoxic T lymphocytes and natural killer (N K) enhances the cytolytic activity of cells (Paul and Seder (1994) C
ell 76: 241) whereas, T _H 2 cells, subjected Hisage efficient assistance to B cells and induces differentiation into IgE-secreting cells isotype switching, and B cell IgE, T _H 2 cells (Pun nonen et al. (1993) Proc. Nat'l. Acad. Sci. USA
90: 3730).

[0076] Improved screening methods for cytokines, chemokines, and other accessory molecules generally require improved specific activity for target cells that are sensitive to the respective cytokine, chemokine, or other accessory molecule. Based on the identity of the modified molecule shown. Libraries of nucleic acids of recombinant cytokines, chemokines, or accessory molecules can be expressed on phage or as purified proteins and tested using, for example, in vitro cell culture assays. Importantly, when analyzing recombinant nucleic acids as components of a DNA vaccine, the optimal DNA sequence (for the function of the protein product) in terms of their immunostimulatory properties, transfection efficiency, and the ability to improve vector stability. In addition) can be identified. The identified optimized recombinant nucleic acid can then be subjected to a new round of shuffling and selection.

In one embodiment of the invention, the cytokine is evolved to direct the differentiation of T _H 1 cells. For their ability to distort the immune response to the T _H 1 phenotype, interferon α (IFN-α) and interleukin-12 (IL-12
) Is a preferred substrate for recombination and selection to obtain maximum specific activity and ability to act as an adjuvant in genetic vaccination. IFN-α is a particularly preferred target for optimization using the method of the present invention. IFN-α affects the immune system, tumor cell proliferation and virus replication. Due to these activities, IFN-α
It was the first cytokine to be used in clinical practice. Today, INF-α
Is used in a wide variety of applications, including some types of cancer and viral diseases. IFN-alpha also that instructs the differentiation of T _H 1 phenotype of human T cells efficiently (Parronchi et al (1992) J.Immunol.149: 2977
). However, IFN-α has not been carefully studied in vaccination models. This is because, in contrast to the human system, IFN-alpha is because no effect on the T _H 1 differentiation in mice. This kind of difference, like IL-12 recently,
IFN-alpha is induced STAT4 activation in human cells but not mouse cells, and STAT4 has been described by indicate to the data that is required for T _H 1 differentiation of IL-12-mediated (Thierfelder et al. (1996) Natu
re 382: 171).

[0078] Family DNA shuffling is a preferred method for optimizing IFN-α using a mammalian INF-α gene with 85% to 97% homology as a substrate. More than 10 ²⁶ different recombinants can be generated in these genes from natural diversity. High-throughput methods for their expression and biological assays can be used as fusion proteins on bacteriophage to enable rapid parallel analysis of recombinant interferons. Recombinants with improved efficacy and selectivity profiles are selectively propagated for improved activity. Variants presenting improved binding to IFN-alpha receptors, using the screening for mutants having an optimal ability to direct T _H 1 differentiation, it may be selected for further analysis. More specifically, the ability of IFN-α mutants to induce the production of IL-2 and IFN-γ in human T lymphocyte cultures in vitro was demonstrated by cytokine-specific ELISA and intracellular cytokine staining and flow cytometry. Can be studied by

[0079] IL-12 is probably find it most potent cytokine der instructing T _H 1 responses, and also acts as an adjuvant, it is shown to enhance T _H 1 response following a genetic vaccination (Kim et al. (1997) J. Immunol.
158: 816). IL-12 is a unique cytokine, both structurally and functionally. IL-2 is the only heterodimeric cytokine known to date and is composed of a 35 kD light chain (p35) and a 40 kD heavy chain (p40) (Kobatashi et al. (1989) J. Exp. Med. .1
70: 827; Stern et al. (1990) Proc. Nat'l. Acad. S
ci. USA 87: 6808). Recently, Lieshke et al. ((1997) N
atature Biotech. 15:35) showed that a fusion between the p35 and p40 genes yielded a single gene with activity comparable to the two genes expressed separately. These data indicate that the IL-2 gene can be shuffled as one entity. This is useful for designing shuffling protocols. Due to its T cell proliferation promoting activity, normal human peripheral blood T cells can be used in the selection of most active IL-12 genes, and IL-12 mutations with the most potent activity on human T cells Allows direct selection of the body. The IL-12 variant can be expressed in CHO and, for example, determine the ability of the supernatant to induce T cell proliferation (FIG. 6). The IL-2 concentration in the supernatant can be normalized based on a specific ELISA that detects the tag fused to the shuffled IL-2 molecule.

Incorporation of the evolved IFN-α gene and / or IL-12 gene into a gene vaccine vector is expected to be safe. The safety of IFN-α has been proven in vast clinical studies and in daily hospital practice. Phase II trials of IL-12 in the treatment of patients with renal cell carcinoma have produced some unexpected adverse effects (Tahara et al. (1995) Human
Gene Therapy 6: 1607). However, while IL-12 as a component of a genetic vaccine targets high local expression levels, its levels found in the circulation are lower than those seen after systemic bolus administration. Was minimal. In addition, some adverse effects of systemic treatment of IL-12 appear to be associated with its abnormally long half-life (up to 48 hours in monkeys). DNA shuffling may allow for shorter half-life selections, resulting in reduced toxicity even after high bolus doses.

[0081] In other cases, especially if the antibody production with improved are desired, genetic vaccine capable of inducing T _H 2 response are preferred. For example, IL-4, the T _H 2 cells (this cell, high levels of IL-4, IL-5 and IL-13 produced, and mediates the allergic immune response) to direct the differentiation of It has been shown. Gene vaccine, when used to prevent immune to autoimmune disease, the immune response is skewed toward the T _H 2 phenotype are preferred. T _H 1 response also, the vaccine is preferred when used to treat and modulate the autoimmune response that is present. This is because, autoreactive T cells, since it is generally T _H 1 phenotype (Liblau et al. (1995) Immuno.Today 16: 34-38) .
IL-4 is also the most potent cytokine in inducing IgE synthesis;
L-4 deficient mice cannot produce IgE. Asthma and allergy are IL-4
Associated with an increased frequency of producer cells and on chromosome 5 (IL-3, IL-5
, IL-9, IL-13 and GM-CSF (generally adjacent to the gene encoding IL-4). IL-4 produced by activated T cells, basophils and mast cells is 153
Amino acid and two potential N-glycosylation sites. Human IL-4 is only 50% identical to mouse IL-4, and IL-4 activity is species-specific. In humans, IL-13 has activity similar to that of IL-4, but is less potent than IL-4 at inducing IgE synthesis. IL-4 is the only cytokine is known to direct the T _H 2 differentiation.

[0082] The improved IL-2 agonists also_HWhile useful for indicating the differentiation of two cells, improved IL-4 antagonists_HIt can direct the differentiation of one cell. Improved IL-4 agonists and antagonists include IL-4 receptors.
May be produced by shuffling of the putter or soluble IL-4 receptor.
You. The IL-4 receptor is an IL-4R α chain (140 kD high affinity binding unit).
And IL-2R γ chains (these cytokine receptors share a common γ
Chains). IL-4R α-chain comprises IL-4 receptor and IL
It is shared by the -13 receptor complex. Both IL-4 and IL-13
Induces phosphorylation of the IL-4R α chain, but induces IL-4R α in transformants.
Expression of the chain alone does not adequately provide a functional IL-4R. Soluble IL-4
Scepters are currently in clinical trials for the treatment of allergies. D of the present invention
Soluble with improved affinity for IL-4 using the NA shuffling method
Can evolve the IL-4 receptor. Such receptors include asthma and other T _H Useful for treating two-cell mediated diseases (eg, severe allergies). This shuffling reaction involves c from activated T cells from humans and other primates.
The natural diversity present in DNA libraries can be exploited. In an exemplary embodiment
In addition, the shuffled IL-4R α chain library is
Identify variants that bind to IL-4 that are expressed and have improved affinity
You. The biological activity of the selected variants is then determined using a cell-based assay.
Assay.

[0083] IL-2 and IL-15 are also of particular interest for use in genetic vaccines. IL-2 acts as a growth factor for activated B cells and activated T cells, and it also regulates NK-cell function. IL-2 is preferentially produced by T _H 1-like T cell clone. Thus, IL-2 appears to function primarily in delayed-type hypersensitivity reactions. However, IL-2 also has strong and direct effects on proliferation and Ig synthesis by B cells. The combined immunomodulatory properties of IL-2 are phenotypic in IL-2 deficient mice, which have high mortality at young age and multiple deficiencies in immune function, including spontaneous development of inflammatory bowel disease. Is reflected in IL-15 is a cytokine that is most recently identified and is produced by multiple cell types. IL-15 shares some, but not all, activity with IL-2. Both IL-2 and Il-15 induce B cell proliferation and differentiation. However, assuming normal production of IL-15 in IL-2 deficient mice, these mice have multiple immune deficiencies, and therefore, IL-1
It is clear that 5 cannot replace the function of IL-2 in vivo. IL-2 has been shown to synergistically enhance IL-10-induced human Ig production in the presence of anti-CD40 mAb, which antagonized the effects of IL-4. IL-
2 also enhances IL-4 dependent IgE synthesis by purified B cells. On the other hand, IL-2 has been shown to inhibit IL-4-dependent mouse IgG1 and IgE synthesis both in vitro and in vivo. Similarly, IL-2 inhibited IL-4 dependent human IgE synthesis by unfractionated human PBMC, but this effect was significantly lower than that of IFN-α or IFN-γ. B
Due to their ability to activate both cells and T cells, IL-2 and IL-15 are useful in vaccination. In fact, IL-2 has been shown to improve the efficacy of vaccination as a protein and as a component of a genetic vaccine. Improving the specific activity and / or the expression level / kinetics of IL-2 and IL-15 by using the DNA shuffling method of the present invention,
Increases beneficial effects compared to wild-type IL-2 and IL-15.

Another cytokine of particular interest for optimization and use in genetic vaccines according to the methods of the present invention is interleukin-6. IL-6 is a mononuclear cell-derived cytokine originally described as a B cell differentiation factor or B cell stimulating factor-2 due to its ability to enhance the level of Ig secreted by activated B cells. IL-6 has also been shown to enhance IL-4 induced IgE synthesis. The neutralizing anti-IL-6 mAb completely blocked IL-4 induced IgE synthesis, thus also suggesting that IL-6 is an essential factor for human IgE synthesis. IL-6 deficient mice have an impaired ability to produce IgA. Due to its strong activity in B cell differentiation, IL-6 can enhance the levels of specific antibodies produced following vaccination. This is particularly useful as a component of a DNA vaccine because a high local concentration can be achieved, thereby providing the most potent effect on cells adjacent to cells expressing the transfected immunogenic antigen. is there. IL-6 with improved specific activity and / or improved expression level, obtained by DNA shuffling,
It has a more beneficial effect than -6.

[0085] Interleukin-8 is another example of a cytokine useful in a genetic vaccine when modified according to the methods of the present invention. IL-8 was originally identified as a neutrophil chemoattractant and activator from monocytes. Then, IL
-8 is chemotactic for T cells and activates basophil cells,
It has also been shown to result in enhanced release of histamine and leukotriene from these cells. In addition, IL-8 inhibits neutrophil adhesion to cytokine-activated endothelial cell monolayers and protects these cells from neutrophil-mediated damage.
Thus, it was suggested that endothelial cell-induced IL-8 attenuates inflammatory events occurring near the vessel wall. IL-8 also regulates immunoglobulin production and inhibits IL-4 induced IgG4 and IgE synthesis by both unfractionated human PBMC and purified B cells in vitro. The inhibitory effect of IFN
-Α, IFN-γ or prostaglandin E2. Furthermore, I
L-8 inhibited spontaneous IgE synthesis by PBMC from atopic patients.
Due to its ability to attract inflammatory cells, IL-8, like other chemotactic agents,
Useful in enhancing the functional properties of vaccines, including DNA vaccines (acting as adjuvants). The beneficial effects of IL-8 can be improved by using the DNA shuffling methods of the invention to obtain IL-8 with improved specific activity and / or improved expression in target cells.

[0086] Interleukin-5 and its antagonists can also be optimized for use in genetic vaccines using the methods of the invention. IL-5, because of its ability to be mainly produced by T _H 2 type T cells, and induces eosinophil,
It is thought to play an important role in the pathogenesis of allergic diseases. IL-5 acts as an eosinophil differentiation factor and survival factor in both mice and humans. Blocking the activity of IL-5 by using a neutralizing monoclonal antibody strongly inhibits pulmonary eosinophilia and hypersensitivity in a mouse model.
And IL-5 deficient mice do not develop eosinophilia. These data also suggest that IL-5 antagonists may have therapeutic potential in treating allergic eosinophilia.

[0087] IL-5 has also been shown to enhance both activated mouse and human B cell proliferation and Ig synthesis by activated mouse and human B cells. However, other studies suggested that IL-5 had no effect on human B cell proliferation, but activated eosinophils. Clearly, IL-5 is not critical for normal B cell maturation or differentiation, since the antibody response in IL-5 deficient mice was normal. However, these mice have a developmental defect in their CD5 ⁺ B cells. This indicates that IL-5 is required for normal differentiation of this B cell subset in mice. Suboptimal IL-4
At a concentration of IL-5, IL-5 was shown to enhance IgE synthesis by human B cells in vitro. Furthermore, recent studies have suggested that the effect of IL-5 on human B cells depends on the mode of B cell stimulation. IL-5 is Moraxe
Significantly enhanced IgM synthesis by B cells stimulated with lla catarrhalis. Furthermore, IL-5 synergized with suboptimal concentrations of IL-2,
It had no effect on Ig synthesis by SAC-activated B cells. Activated human B cells also expressed IL-5 mRNA. This suggests that IL-5 can also regulate B cell functions (including IgE synthesis) by the autocrine mechanism.

The present invention provides a method for evolving IL-5 antagonists that efficiently bind to and neutralize IL-5 or its receptor. These antagonists are useful as components of vaccines used for prevention and treatment of allergy. For example, nucleic acids encoding IL-5 from humans and other mammalian species can be shuffled and immobilized I for initial screening.
Screen for binding to L-5R. The polypeptides that exhibit the desired effect in the initial screening assay are then tested for inhibition of growth of an IL-5 dependent cell line cultured in the presence of recombinant wild type IL-5 for highest biological activity. Such assays can be used to screen. Alternatively, the shuffled IL-5R α-chain is screened for improved binding to Il-5.

[0089] Tumor necrosis factors (α and β) and their receptors are also suitable targets for modification and use in genetic vaccines. TNF-α (originally described as cachectin due to its ability to cause tumor necrosis) is 17 kDa
It is a protein that is produced in low quantities after activation by almost all cells of the human body. TNF-α acts as an endogenous pyrogen and induces the synthesis of some proinflammatory cytokines, stimulates the production of acute phase proteins, and induces fibroblast proliferation. TNF-
α plays a major role in the pathogenesis of endotoxin shock. TN in membrane-bound form
F-α (mTNF-α), which is involved in the interaction between B cells and T cells, is rapidly upregulated within 4 hours of T cell activation. mTNF-α is H
Plays a role in polyclonal B cell activation observed in patients infected with IV. Monoclonal antibodies specific for the mTNF-α or p55 TNF-α receptors potently inhibit IgE synthesis induced on activated CD4 ⁺ T cell clones or their membranes. Mice deficient in p55 TNF-αR are resistant to endotoxin shock, and soluble TNF-αR prevents autoimmune diabetes mellitus in NOD mice. Phase II
After the encouraging results obtained in Trail, a Phase III trail using TNF-αR in the treatment of rheumatoid arthritis
Is in progress.

Using the methods of the present invention, for example, having improved affinity,
Soluble TNF-αR that can act as an antagonist to F activity can be evolved. Nucleic acids encoding TNF-αR and exhibiting sequence diversity (eg, the natural diversity observed in cDNA libraries from activated T cells of human and other primates) are shuffled. Shuffled nucleic acid,
After selecting a mutant that binds to TNF-α with improved affinity, it is expressed, for example, on a phage. If desired, the improved variants can be subjected to further assays using biological activity, and the shuffled genes can be
More than one round of shuffling and screening.

Another target of interest for application to the method of the invention is interferon-γ.
And the evolution of antagonists of this cytokine. For IFN-γ
The receptor is a 90 kD binding component glycoprotein, the extracellular portion of 228 amino acids.
(Transmembrane region), and an intracellular region of 222 amino acids. Glycosylation is
Not required for functional activity. Single chains have high affinity binding (10^-9-10^{-1 0} M), but not sufficient for signal transduction. The receptor component dimerizes by ligand binding. Mouse IFN-γ receptor is a mouse IFN-γ receptor.
-53% identical at the amino acid level to the gamma receptor. Human receptor and
Mouse receptor binds only to human IFN-γ and mouse IFN-γ, respectively
I do. Vaccinia virus, cowpox virus and camelpox virus are sIFN
-ΓR homologs, which have relatively low amino acid sequence similarity (about 20%
), But can effectively neutralize IFN-γ in vitro. These homologs
Shows human IFN-γ, bovine IFN-γ, and rat (but not mouse) IFN-γ
Can bind and have in vivo activity as an IFN-γ antagonist. Eight
All cysteines are present in humans, mice, mucus species viruses, and
Ls (six in vaccinia virus) IFN-γR polypeptide
Be preserved. This indicates a similar 3-D structure. 100-300 pM kD
The extracellular portion of mIFN-γR with is expressed in insect cells. m
NZB / W mouse by sIFN-γ receptor (mouse model of human SLE)
Treatment (three times a week, 100 mg per intraperitoneal (ip)),
Inhibits the onset of flame. Total treated with sIFN-γ or anti-IFN-γ mAb
All mice survived 4 weeks after discontinuation of treatment and 50% died in placebo population
In contrast, 78% of IFN-γ treated mice died.

The methods of the present invention can be used to evolve soluble IFN-γR receptor polypeptides with improved affinity and improved specific activity and improved ability to activate cellular immune responses. IFN-γ can be evolved. In each case, nucleic acids encoding the respective polypeptides and exhibiting sequence diversity (eg, as observed in cDNA libraries derived from activated T cells from humans and other primates) are recombinantly transformed. Provided and screened to identify recombinant nucleic acids encoding polypeptides having improved activity. In the case of shuffled IFN-γR, a library of shuffled nucleic acids can be expressed on phage and screened to identify variants that bind to IFN-γ with improved affinity. In the case of IFN-γ, the shuffled library is analyzed for improved specific activity and improved activation of the immune system, for example, using monocyte / macrophage activation as an assay. Evolved IFN-γ molecules may improve the efficiency of vaccination (eg, when used as an adjuvant).

Diseases that can be treated using the high affinity sIFN-γR polypeptides obtained using the methods of the invention include, for example, multiple sclerosis, systemic lupus erythematosus (S
LE), post-treatment tissue rejection, and graft versus host disease. Multiple sclerosis is, for example, characterized by increased IFN-γ expression in the patient's brain and increased IFN-γ production by the patient's T cells in vitro. IFN-γ treatment has been shown to significantly worsen the disease (as opposed to EAE in mice).

[0094] Transforming growth factor (TGF) -β is another cytokine that can be optimized for use in genetic vaccines using the methods of the invention. TG
F-β has growth regulatory activity on essentially all cell types and has also been shown to have complex regulatory effects on cells of the immune system. TGF-β inhibits the proliferation of both B cells and T cells, and also inhibits cytotoxic T cells and N cells.
Inhibits the development and differentiation of K cells. TGF-β is expressed in mouse B cells and human B cells.
It has been shown to direct IgA conversion in both cells. TGF-β
Have also been shown to induce germline transcription in mouse and human B cells, supporting the conclusion that TGF-β can specifically induce IgA conversion. .

Because of its ability to direct IgA conversion, TGF-β is useful as a component of DNA vaccines (eg, vaccines against diarrhea) intended to induce strong mucosal immunity. Also, due to its strong antiproliferative effect, TGF-β is useful as a component of therapeutic cancer vaccines. TGF-β with improved specific activity and / or improved expression levels / kinetics has an enhanced beneficial effect compared to wild-type TGF-β.

The cytokines that can be optimized using the methods of the present invention include granulocyte colony stimulating factor (G-CSF) and granulocyte / macrophage colony stimulating factor (GM-C
SF) may also be mentioned. These cytokines are derived from bone marrow stem cells
Induces differentiation into macrophages. Administration of G-CSF and GM-CSF significantly improves recovery from bone marrow (BM) transplantation and radiation therapy, reducing infections and the time patients must spend in hospital. GM-CSF is
Enhances antibody production after DNA vaccination. G-CSF is a protein of 175 amino acids, whereas GM-CSF has 127 amino acids. Human G-CS
F is 73% identical at the amino acid level to mouse G-CSF. And the two proteins show cross-reactivity. G-CSF is a homodimer receptor (a dimer with a kD of about 200 pM, a monomer with a kD of about 2 to 4 nM)
And the receptor for GM-CSF is a three subunit complex. The cell line transfected with the cDNA encoding G-CSFR
-Proliferate in response to CSF. Cell lines that are dependent on GM-CSF are available (eg, TF-1). G-CSF is non-toxic and currently functions very well as a drug. However, this procedure is expensive. A stronger G-CSF may reduce the cost of medical care for the patient. Treatment with these cytokines is typically for a short period of time, and the patient will not need the same treatment again to reduce the likelihood of immunogenic problems.

The method of the present invention provides a G-CSF and / or GM-
CSF), and other polypeptides having G-CSF and / or GM-CSF activity. G-CSF and / or GM-CSF nucleic acids with sequence diversity (e.g., they are obtained from cDNA libraries from various species) are shuffled into G-CSF and / or GM-CSF.
Shuffle to create a library of genes. These libraries can be screened, for example, by selecting colonies, transfecting the plasmid into appropriate host cells (eg, CHO cells), and assaying the supernatant using a receptor positive cell line. Alternatively, phage display or related techniques may be used, again using the receptor positive cell line. Yet another screening method is to use the shuffled genes as G-CSF / GM-C
Transfecting into an SF-dependent cell line. The cells are grown at very low density, one cell per well and / or in a large flask, and the fastest growing cells are selected. Isolate the shuffled genes from these cells; if desired, these genes can be used for further rounds of shuffling and selection.

Ciliary neurotrophic factor (CNTF) is another suitable target for application of the method of the invention. CNTF has 200 amino acids, which shows 80% sequence identity between rat CNTF polypeptide and rabbit CNTF polypeptide. C
NTF has an IL-6-like inflammatory effect and induces the synthesis of acute phase proteins. CNTF is a cytosolic protein that contains IL-6 / IL-
Belongs to the 11 / LIF oncostain M family and become biologically active only after being made available either by cellular damage or by an unknown release mechanism. CNTF is expressed by myelinating Schwann cells, astrocytes, and sciatic nerve. Structurally, CNTF is a dimeric protein and has a novel antiparallel arrangement of subunits. Each subunit employs a four-helix bundle fold with double crossings, and these two helices contribute to the dimer interface.
The Lys-155 mutant lacks activity, and some Glu-153 mutants have 5-10 fold higher biological activity. The receptor for CNTF is a specific C
Consists of the NTF receptor chain, gp130, and LIF-β receptor. CN
The TFRα chain lacks a transmembrane domain instead of having a GPI anchor. At high concentrations, CNTF can mediate a CNTFR-independent response. Soluble CNTFR can bind CNTF and then LIFR, and gp130
Can induce signal transduction. CNTF enhances the survival of some types of neurons, and in animal models of Huntington's disease (NGF, neurotrophic factor, and neurotrophilin-3).
Protect neurons (as opposed to 3). CNTF receptor knockout mice have severe motor neuron deficits at birth, and CNTF knockout mice show such deficits postnatally. CNTF also reduces obesity in a mouse model. Decreased expression of CNTF is sometimes observed in psychotic patients. ALS (Annual incidence rate is about 1/100000, 5% is familial,
Phase I study in patients suffering from 90% death within 6 years)
Significant side effects after an administration of more than mg / kg / day (anorexia, weight loss,
Reactivation of herpes simplex virus (HSV1), including coughing and increased oral secretions)
Was found. Antibodies to CNTF were detected in almost all patients. Thus, this illustrates the need for alternative CNTFs with different immunological properties.

The recombinant and screening methods of the present invention can be used to obtain modified CNTF polypeptides that exhibit reduced immunogenicity in vivo; higher specific activities also use these methods. Available. Shuffling is CNTF
Using a nucleic acid that encodes In a preferred embodiment, IL-6 /
LIF / (CNTF) hybrids are obtained by shuffling with an excess of oligonucleotides encoding for the receptor binding site of CNTF. Phage display can then be used to test for loss of binding to the IL-6 / LIF receptor. Following this initial screen, we will test for high affinity binding to the CNTF receptor and, if desired, for functional assays to be used on CNTF-responsive cell lines. Shuffled CNTF polypeptides can be tested to identify CNTF polypeptides that exhibit reduced immunogenicity upon administration to a mammal.

Using another method in the recombinant and screening methods of the present invention,
CNTF can be optimized, thereby improving secretion of the polypeptide. CNTF
When the cDNA is operably linked to the leader sequence of hNGF, only 35-40% of the total CNTF produced is secreted.

The shuffled genes in an expression vector (eg, a DNA vaccine)
Alternatively, targeted diseases for treatment with optimized CNTF using any of the purified proteins include obesity, amyotrophic lateral sclerosis (ALS, Lu
Gehrig's disease), diabetic neuropathy, seizures, and brain surgery.

Using the methods of the present invention, polynucleotides encoding chemokines can also be optimized and incorporated into genetic vaccine vectors. Based on structure, at least three classes of chemokines are known: C chemokines (eg, lymphotoactin), CC chemokines (eg, MC)
P-1, MCP-2, MCP-3, MCP-4, MIP-1a, MIP-1b,
RANTES), CXC chemokines (eg, IL-8, SDF-1, EL
R, Mig, IP10) (Premac and Schall (1996) Na
cure Med. 2: 1174). Chemokines can attract other cells that mediate immune and inflammatory functions, thereby enhancing the immune response. Cells attracted by various types of chemokines include, for example, lymphocytes, monocytes and neutrophils. In general, C—X—C chemokines are chemoattractants for neutrophils, but not monocytes, C—C chemokines attract monocytes and lymphocytes, but not neutrophils, and C chemokines are lymphoid. Attract the ball.

[0103] Genetic vaccine vectors can also include optimized recombinant polynucleotides that encode surface-associated accessory molecules, such as molecules involved in regulating and enhancing the immune response. These molecules (eg, B7-1 (CD80), B7-2
(CD86), CD40, ligand for CD40, CTLA-4, CD28
And CD150 (including SLAM)) can be subjected to DNA shuffling to obtain variants with altered and / or improved activity.

[0104] Optimized recombinant polynucleotides encoding the CD1 molecule are also useful in genetic vaccine vectors for certain applications. CD1 is a non-polymorphic molecule that is structurally and functionally related to MHC molecules. Importantly, CD1 has MHC-like activity and can function as an antigen presenting molecule (Porcel
li (1995) Adv. Immunol. 59: 1). CD1 is highly expressed on dendritic cells, which are very efficient antigen presenting cells. Co-transfection of target cells with a DNA vaccine vector encoding CD1 and the antigen of interest appears to boost the immune response. Because CD1 cells, in contrast to MHC molecules, display limited allelic diversity in outbred populations (P
orcelli, supra), because a large population of individuals with different genetic backgrounds can be vaccinated with one CD1 allele. The functional properties of the CD1 molecule can be improved by the DNA shuffling method of the present invention.

[0105] The optimized recombinant TAP gene and / or gene product may also be included in a genetic vaccine vector. The TAP genes and their optimization for various purposes are discussed in detail below. Furthermore, heat shock proteins (HSP
) (Eg, HSP70) can also be deployed for improved presentation and processing of antigens. HSP70 has been shown to act as an adjuvant to induce CD8 ⁺ T cell activation and enhances the immunogenicity of certain antigenic peptides (Blachere et al. (1997) J. Exp. Med. 186:
1315-22). When HSP70 is encoded by a genetic vaccine vector, it appears to enhance presentation and processing of the antigenic peptide, thereby improving the efficacy of the genetic vaccine. DNA shuffling can be used to further improve the properties of heat shock proteins (eg, HSP70), including adjuvant activity.

Recombinantly produced cytokines, chemokines, and accessory molecule polypeptides, and antagonists of these molecules, can be used to affect the type of immune response to a given stimulus. However, administration of polypeptides sometimes has drawbacks. Disadvantages include short half-life, high cost, difficulty in storage (must be stored at 4 ° C.), and the need for large quantities.
Also, bolus injections can occasionally cause side effects. Administration of a polynucleotide encoding a recombinant cytokine or other molecule overcomes most or all of these problems. For example, DNA that can be prepared with high purity is stable,
It is temperature resistant, non-infectious and easy to manufacture. In addition, polynucleotide-mediated administration of cytokines may provide for long-lasting, consistent expression, and administration of the polynucleotide, which is generally considered safe.

Since the functions of cytokines, chemokines and accessory molecules are redundant and pleiotropic, which cytokine combination is most potent in inducing and enhancing an antigen-specific immune response after vaccination Can be difficult to determine. In addition, the most useful combinations of cytokines and accessory molecules typically vary depending on the type of immune response desired after vaccination.
For example, IL-4, the T _H 2 cells (this cells produce high levels of IL-4, IL-5 and IL-13, mediate the allergic immune response) is directed differentiation, INF-gamma and IL-12 is T _H 1 cells (the cells to produce IL-2 and INF-gamma high level) directed differentiation, and to mediate the immune response of delayed shows Have been. In addition, the most useful combinations of cytokines and accessory molecules also appear to depend on the antigen used for vaccination. The present invention provides a solution to this problem of obtaining an optimized genetic vaccine cocktail. Various combinations of cytokines, chemokines and accessory molecules are incorporated into vectors using the methods described herein. These vectors are then screened for their ability to induce an immune response in vivo and in vitro.

[0,108] were generated by gene shuffling and combinations molecular biology, if a library of wide variety of vectors, is desired, for example, the maximum ability to direct the immune response to T _H 1 phenotype or T _H 2 phenotype Screen for Libraries of different vectors are constructed with different promoters, (evolved) cytokines, (evolved) cytokine antagonists, (evolved) chemokines, (evolved) accessory molecules and immunostimulatory sequences. Can be generated. Each of these can be prepared using the methods described herein. DNA sequences and compounds that facilitate transfection and expression can be included. If the pathogen is known, specific DNA sequences encoding immunogenic antigens from the pathogen can be incorporated into these vectors and provide protective immunity to the pathogen (as in a genetic vaccine).

The initial screening is preferably performed in vitro. For example, the library can be introduced into cells that will be tested for their ability to induce T cell differentiation, which can produce cytokines that are indicative of the type of immune response desired. The T _H 1 responses, for example, by screening the library to identify a recombinant polynucleotide capable of inducing T cells producing IL-2 and IFN-gamma. On the other hand, IL-4, IL-5 , and IL-13 screening Introduction of T cell production is performed in order to identify recombinant polynucleotide which supports the T _H 2 response.

[0110] Screening is also performed in vivo using animal models. For example,
Vectors produced using the methods of the invention can be tested for their ability to protect against lethal infection. Another screening method is Leishmania ma.
Injection of the Jor parasite into the footpad of BALB / c mice (nonhealer). The pool of plasmids is injected intravenously, intraperitoneally or in the footpad of these mice, and the size of the footpad ridge is followed. Yet another method of in vivo screening involves the detection of IgE levels after infection with Nipponstrongylus brasiliensis. High level indicates T _H 2 response, while low levels of IgE indicates the T _H 1 responses.

[0111] Successful results in animal models are readily ascertainable in humans. In vitro screening can be performed to test for whether or other desired immune response human T _H 1 phenotype or T _H 2 phenotype. Vectors can also be tested in humans for their ability to induce protection against infection.

Since the principle of immune function is similar to a wide variety of infections, immunostimulating DN
If the A vaccine vector is delivered to the site of pathogen entry (eg, lung or intestine), it may be useful not only in treating a number of infectious diseases, but also in protecting against infection.

3. Agonists or Antagonists of Cell Receptors The present invention also provides optimized recombinant polynucleotides that encode peptides or polypeptides that can interact with cellular receptors involved in mediating an immune response. Provide a way to get: The optimized recombinant polynucleotide can act as an agonist or antagonist of the receptor.

[0114] Cytokine antagonists may be used as components of a genetic vaccine cocktail. Blocking immunosuppressive cytokines appears to enhance the immune response in a more general manner, rather than adding a single pro-inflammatory cytokine. Because several pathways are enhanced simultaneously. By selecting the appropriate antagonist, the immune response elicited by the genetic vaccine can be altered to obtain the most effective response to achieve the desired effect. Where appropriate, antagonists to any cytokine may be suitably used: specific cytokines for blocking purposes include, for example, IL-4, IL-13, IL-10, and the like.

The present invention provides methods for obtaining cytokine antagonists that exhibit greater efficacy in blocking the action of each cytokine. Polynucleotides encoding improved cytokine antagonists can be obtained using gene shuffling to generate a recombinant library of polynucleotides, which are then screened to obtain polynucleotides encoding improved antagonists Is identified. As a substrate for DNA shuffling,
For example, a polynucleotide encoding a receptor for each cytokine may be used. At least two forms of the substrate are present in the recombination reaction, each form being different from each other in at least one nucleotide position. In a preferred embodiment, the different forms of the polynucleotide are homologous cytokine receptor genes from different organisms. The resulting library of recombinant polynucleotides is then screened to identify polynucleotides that encode cytokine antagonists having the desired affinity and biological activity.

One example of the types of effects that can be achieved by including a cytokine antagonist in a genetic vaccine cocktail, as well as one of how this effect can be improved using the DNA shuffling methods of the present invention As an example, I
L-10 will be discussed. Similar theoretical explanations can be provided for obtaining and using antagonists of other cytokines. Interleukin 10 (IL-
10) is probably the most potent anti-inflammatory cytokine known to date. IL-10 inhibits a number of pathways that enhance the inflammatory response. Biological activities of IL-10 include inhibition of MHC class II expression on monocytes, inhibition of production of IL-1, IL-6, IL-12, TNF-α by monocytes / macrophages, and T lymphocytes Growth and inhibition of IL-2 production. The importance of IL-10 as a modulator of immune and inflammatory responses was clearly demonstrated in IL-10 deficient mice. These mice are growth inhibitory, anemic, and develop inflammatory bowel disease spontaneously (Kuhn et al. (1993) Cell).
75: 263). Furthermore, in IL-10 deficient mice, Listeria
Innate and acquired immunity to monocytogenes has been shown to be increased (Dai et al. (1997) J. Immunol. 158: 2259).
). Also, genetic differences in the level of IL-10 production indicate that meningoc
It has also been suggested that it may affect the risk of patients dying from occal infection complications. Families with high IL-10 production have a 20-fold increased risk of fatal consequences of meningococcal disease (Westendrop et al. (1997) Lancet 349: 170).

Although IL-10 has been shown to activate normal and malignant B cells in vitro, it does not appear to be the major growth promoting cytokine for normal B cells in vivo. This is because IL-10 deficient mice have normal levels of B cell lymphocytes and Igs in their circulation. In fact, IL
There is evidence that -10 can indirectly down regulate B cell function through inhibition of monocyte accessory cell function. However, IL-10 appears to play a role in the proliferation and expansion of malignant B cells. Anti-IL-10 monoclonal antibodies and IL-10 antisense oligonucleotides have been shown to inhibit EBV transformation of B cells in vitro. In addition, B-cell lymphomas are associated with EBV, and most EBV ⁺ lymphomas produce high levels of IL-10. These are human genes and IL-1 encoded by EBV.
It is derived from both 0 homologs. B-cell lymphomas associated with AIDS also secrete high levels of IL-10. Furthermore, the short-lived life of patients with detectable serum IL-10 at the time of diagnosis of medium / high grade non-Hodgkin's lymphoma further suggests a role for IL-10 in the pathogenesis of B-cell malignancies. .

[0118] Antagonizing IL-10 in vivo may have advantages in some infectious and malignant diseases, and in vaccination. IL-1
A blocking effect of 0 is an enhancement of the immune response independent of the specificity of this response. This is useful in vaccination and in the treatment of severe infectious diseases. In addition, antagonists of IL-10 include IL-10 and viral IL
Useful in the treatment of B cell malignancies displaying -10 overexpression. This may also be useful in boosting a general anti-tumor immune response in cancer patients. Combining an IL-10 antagonist with a gene therapy vector may be useful in gene therapy of tumor cells to obtain a maximal immune response against the tumor cells. If shuffling of IL-10 results in IL-10 with improved inactivity, the IL-10 molecule has potential in the treatment of autoimmune and inflammatory bowel diseases. IL-10 with improved inactivity
Can also be useful as components of gene therapy vectors that reduce the immune response to vectors recognized by memory cells, and they can also reduce the immunogenicity of these vectors.

[0119] Antagonists of IL-10 are made by producing a soluble form of the IL-10 receptor (sIL-10R; Tan et al. (1995) J. Bio.
l. Chem. 270: 12906). However, sIL-10R has 560 pM
Binds to IL-10 with a Kd of 35-20
It has an affinity of 0 pM. Consequently, a half-maximal inhibition of IL-10 biological function requires a 150 molar excess of sIL-10R. In addition, virus I
The affinity of L-10 (a homolog of IL-10 encoded by the Epstein-Barr virus) for sIL-10R is 1000 times greater than that of hIL-10.
More than twice as low. In some aspects, such as when treating EBV-related B-cell malignancies, it may be advantageous if the function of viral IL-10 can also be blocked. Taken together, this soluble form of IL-10R does not appear to be effective in antagonizing IL-10 in vivo.

IL-10 with sufficient affinity to function in vivo, and antagonistic activity
DNA shuffling can be performed using a polynucleotide encoding the IL-10 receptor to obtain an antagonist of IL-10 receptors with higher affinity than normal affinity function as antagonists of IL-10. Because it strongly reduces the amount of IL-10 available for binding to functional wild-type IL-10R. In a preferred embodiment, IL-10R is shuffled using homologous cDNAs encoding IL-10R from humans and other mammalian species. Human and mouse IL-10
An alignment of the receptor sequences is shown in FIG. 14 to illustrate the potential of family DNA shuffling when evolving IL-10 receptors with improved affinity. A phage library of IL-10 receptor recombinants is shuffled with IL-10R, human or viral IL-10.
Can be screened for improved binding. Wild-type IL-10 and / or
Alternatively, viral IL-10 is added at increasing concentrations as required for high affinity. Phage bound to IL-10 can be recovered using an anti-IL-10 monoclonal antibody. If desired, repeat this shuffling one or more times. The evolved soluble IL-10R is then analyzed in a functional assay for its ability to neutralize the biological activity of IL-10 / viral IL-10. More specifically, the evolved soluble IL-10R was examined for its ability to block the inhibitory effect of IL-10 on cytokine synthesis and MHC class II expression by monocytes, proliferation by T cells, and by anti-CD40 monoclonal antibody The ability of IL-10 to inhibit the promoting effect of activated B cells on proliferation is examined.

[0121] Variants that produce IL-10 antagonists by evolving IL-10 to bind to IL-10R with higher affinity than wild-type affinity but do not activate the receptor Can be obtained. The advantage of this approach is that IL-10 molecules with improved specific activity can be evolved using the same method. In a preferred embodiment, IL-10 is shuffled using homologous cDNAs encoding IL-10 from humans and other mammalian species. further,
The gene encoding viral IL-10 may be included in shuffling. IL-
A library of 10 recombinants is screened for improved binding to the human IL-10 receptor. Library members that bind to IL-10R can be recovered by anti-IL-10R monoclonal antibodies. This screening protocol appears to yield IL-10 molecules that have both antagonistic and agonistic activity. Because the initial screen requires higher affinity, the ratio of agonists appears to have improved specific activity when compared to wild-type human IL-10. The functional characteristics of the mutant IL-10 molecules are similar to those described above for the ultra-high affinity IL-10 receptor (cytokine synthesis and MHC class II expression by monocytes, B cell and T cell proliferation). Determined in a biological assay. Antagonistic IL-4 variants were previously generated, indicating the general potential of this approach (Kru
(1992) EMBO J. et al. 11 3237-3244). Mutation of one amino acid in IL-4 resulted in a molecule that efficiently binds to the IL-4Rα chain, but this molecule has minimal IL-4 like agonistic activity.

Another example of an IL-10 antagonist is IL-20 / mda-7, which is a 206 amino acid secreted protein. This protein was originally mda-
7, which is a negative regulator of melanoma cell-derived tumor cell growth (Jiang et al. (1995) Oncogene 11: 2577).
(1996) Proc. Nat'l. Acad. Sci. USA 93:91
60). IL-20 / mda-7 is structurally related to IL-10 and antagonizes several functions of IL-10 (13th European Im
Munology Meeting, Amsterdam, 22-25 199
Summary of June 2007). In contrast to IL-10, IL-20 / mda-7 enhances expression of CD80 (B7-1) and CD86 (B7-2) on human monocytes, and TNF-α and IL-6 Is up-regulated. IL-20 /
mda-7 also increases IFN-γ production by PHA-activated PBMC.
The present invention provides a method for improving a gene vaccine by incorporating the IL-20 / mda-7 gene into a gene vaccine vector. The methods of the present invention can be used to obtain IL-20 / mda-7 variants that exhibit improved ability to antagonize IL-10 activity.

When a cytokine antagonist is used as a component of a DNA vaccine or gene therapy vector, maximum local effect is desired. Thus, in addition to the soluble form of the cytokine antagonist, transmembrane forms of the antagonist can result. This soluble form may be provided to the patient in a purified polypeptide form, for example, by intravenous injection. Alternatively, a polynucleotide encoding a cytokine antagonist can be used as a component of a gene vaccine or a component of a gene therapy vector. In this case, either the soluble form or the transmembrane form, or both, may be used. When the soluble and transmembrane forms of the antagonist are encoded by the same vector, the target cell expresses both forms and results in maximal inhibition of cytokine function on and in the immediate vicinity of the target cells.

The peptides or polypeptides obtained using these methods may be ligands for natural receptors (eg, cytokines or other co-stimulated molecules in their ability to exert an effect on the receptor-mediated immune system). ). A potential disadvantage of the administration of cytokines or other co-stimulated molecules themselves is due to disruption of tolerance (when natural cytokines or other molecules are used) or is clearly related If different molecules are used, an autoimmune response can be induced against the native molecule, either by inducing cross-reactive immunity (humoral or cellular). Through the use of the method of the invention, agonists or antagonists that avoid these potential disadvantages can be obtained. For example, relatively small peptides can be used as agonists that can mimic or antagonize the activity of natural immunomodulators without inducing cross-reactive immunity to natural molecules. In a currently preferred embodiment, the optimized agonist or antagonist obtained using the method of the invention is
It is about 50 amino acids or less in length, more preferably about 30 amino acids or less, and most preferably about 20 amino acids or less. An agonist or antagonist peptide is preferably at least about 4 amino acids in length, and more preferably at least about 8 amino acids in length. Polynucleotides flanking the coding sequence of a mimetic peptide can also be optimized using the methods of the invention to optimize the expression, conformation, or activity of the mimetic peptide.

The optimized agonist peptide or agonist polypeptide, or antagonist peptide or antagonist polypeptide, generates a library of recombinant polynucleotides and screens the library to enhance the immune response. Obtained by identifying an agonist peptide or an agonist polypeptide, or an antagonist peptide or an antagonist polypeptide that encodes a peptide or polypeptide that exhibits the same ability. The library can be made using methods such as DNA shuffling, or other methods described herein or otherwise known to those of skill in the art. Screening is successful by expressing the peptides encoded by the library members on the surface of a population of replicable genetic packages and identifying members of the library that bind to a target of interest (eg, a receptor). Done.

The optimized recombinant polynucleotide obtained using the method of the present invention can be used in several ways. For example, the polynucleotide can be placed in a genetic vaccine vector under the control of appropriate expression control sequences so that the mimetic peptide is expressed upon introduction of the vector into a mammal. If desired, the polynucleotide can be placed in a vector that incorporates a coding sequence for a surface protein (eg, geneIII or geneVIII) to preserve the conformation of the mimetic. Alternatively, the polynucleotide encoding the mimetic can be inserted directly into the antigen coding sequence of the genetic vaccine, forming a coding sequence for the "mimotope on antigen" structure. Polynucleotides encoding mimotope structures on antigen can be used in genetic vaccines or can be used to express proteins that are themselves administered as vaccines. As an example of this type of application, the coding sequence of a mimetic peptide can be expressed as a hepatitis B surface antigen (HBs
Ag) Introduction into the polynucleotide encoding the "M-loop" of the protein.
The M-loop is a peptide sequence of six amino acids linked by cysteine residues, which is found at amino acids 139-147 (number within the S protein sequence). The M-loop in the native HBsAg protein is recognized by the monoclonal antibody RFHB7 (Chen et al., Proc. Nat'l).
. Acad. Sci. USA, 93: 1997-2001 (1996)). Ch
According to En et al., the M-loop is non-overlapping and has at least four other HBs.
It forms an epitope of HBsAg that is separated from the sAg epitope.

Amino acids 139-147 are likely to be cyclic structures due to the likely Cys-Cys disulfide bond in this hydrophilic portion of the protein. Thus, this structure is similar to the filamentous phage protein pIII in which the mimotope sequence is located.
And structures similar to those found in the region of pVIII. Thus, mimotopes obtained using the method of the invention can be inserted into this region of the HBsAg amino acid sequence.

The chemokine receptor CCR6 is an example of a suitable target for peptidomimetics obtained using this method. The CCR6 receptor is found in immature dendritic cells found in the blood and migrating to the site of antigen uptake (Dieu et al., J. Exp. M.
ed. 188: 373-386 (1998)), a 7-transmembrane domain protein involved in chemoattraction (Dieu et al., Biochem. Biophys. Res.
. Comm. 236: 212-217 (1997) and J. Am. Biol. Che
m. 272: 14893-14898 (1997)). CCR6 is a mimetic peptide that binds the chemokine MIP-3α, and thus can activate CCR6, further provides a chemoattractant function to a given antigen, thus using an antigen mimetic fusion of the antigen or a DNA vector expressing that antigen. Promotes uptake by dendritic cells after immunization.

[0129] Another application of this method of the invention is the use of macrophage scavenger receptors (
MSR; Wloch et al., Hum. Gene. Ther. 9: 1434-147
(See (1998)). MSRs are involved in mediating the action of various immune modulators. Among these are bacterial DNA, which includes plasmids used for DNA vaccination, and oligonucleotides, which are often potent immunostimulators. Oligonucleotides with a particular chemical structure (e.g., phosphothio-oligonucleotides) are particularly potent, whereas bacterial or plasmid DNA must be used in relatively large amounts to produce an effect. . The ability of oligonucleotides that contain dG residues to stimulate B cells and increase the activity of immunostimulatory CpG motifs, as well as lipopolysaccharides to activate macrophages, is also mediated by MSRs. Some of these activities are toxic. Each of these immunomodulators, along with various polyanionic ligands, bind to the MSR. MS with high affinity using the method of the invention
Mimics of one or more of these immunomodulators that bind R but lack toxicity can be obtained. Such mimetic peptides are useful as immunostimulators or adjuvants.

MSRs are trimeric integral membrane glycoproteins. The three extracellular C-terminal cysteine-rich regions are linked to the transmembrane domain by a fibrous region composed of an α-helical coil and a collagen-like triple helix (Kodama).
Et al., Nature 343: 531-535 (1990)). Thus, screening of a library of recombinant polynucleotides can be accomplished by expressing the extracellular receptor structure and artificially attaching it to a plastic surface. The library can be expressed, for example, by phage display, and screened to identify libraries that bind to receptors with high affinity. Optimized recombinant polynucleotides identified by this method can be incorporated into antigen coding sequences and enhance their regulatory effect on the immune response.

(4. Co-stimulatory molecules that can inhibit or increase the activity, differentiation, or anergy of antigen-specific T cells) When expressed, inhibit the activity, differentiation, or anergy of antigen-specific T cells or Methods for obtaining optimized recombinant polynucleotides that can be increased
Provided. T cell activation involves T cell activation by MHC on the plasma membrane of antigen presenting cells (APC) (eg, monocytes, dendritic cells (DC), Langerhans cells or B cells).
Triggered when it recognizes those specific antigenic peptides in the context of the molecule. Activation of CD4 ⁺ T cells requires recognition of antigenic peptides by the T cell receptor (TCR) in the context of MHC class II molecules, while CD8 ⁺ T cells recognize peptides in the context of MHC class I molecules. recognize. Importantly, however, recognition of antigenic peptides is not sufficient for T cell proliferation and induction of cytokine synthesis. An additional costimulatory signal "second signal" is needed. The costimulatory signal is mediated via CD28 and binds its ligand, B7-1 (CD80) or B7-2 (CD86). Typically,
It is expressed in antigen presenting cells. In the absence of a costimulatory signal, T
No cell activation occurs or the T cells become anergic. In addition to CD28, CTLA-4 (CD152) also functions as a ligand for B7-1 and B7-2. However, in contrast to CD28, CTLA-4 is
Mediates negative regulatory signals on T cells and / or induces anergy and tolerance (Walunas et al. (1994) Immunity 1:
405; Karandikar et al., (1996) J. Am. Exp. Med. 184:
783).

B7-1 and B7-2 have been shown to be able to modulate some immunological responses and to be involved in vaccination, allergy, autoimmunity and important in immunomodulation in cancer Have been. Gene therapy and gene vaccine vectors expressing B7-1 and / or B7-2 have also been shown to have therapeutic potential in treating the above diseases and in improving the efficacy of gene vaccines.

FIG. 10 illustrates the interaction between APC and CD4 ⁺ T cells, except that T cells recognize antigenic peptides in the context of MHC class I molecules.
The same principle applies to the 8 ⁺ T cells is correct. Both B7-1 and B7-2 bind to CD28 and CTLA-4 (even if the sequence similarity between these four molecules is very limited (20-30%)) . Mutations in B7-1 and B7-2 that affect only binding to one ligand, do not affect binding to other ligands, or improve activity through one ligand but decrease activity through other ligands It is desired to have a body. Furthermore, because the affinity of the B7 molecule for its ligand appears to be relatively low, it is also desirable to find mutations that improve / change the activity of this molecule. However, rational design cannot predict useful variants due to the complexity of this molecule.

The present invention overcomes these difficulties and produces functionally distinct B7 molecules with altered relative ability to induce T cell activation, differentiation, cytokine production, anergy and / or tolerance. And methods that can be identified. Through the use of the method of the present invention, B7- which affects only binding to one ligand, does not affect binding to another ligand, or improves activity through one ligand but decreases activity through another ligand One and mutations in B7-2 can be found. DNA
Shuffling appears to be the most powerful method in discovering new B7 variants with altered relative binding ability to CD28 and CTLA-4.
B7 variants that act through CD28 with improved activity (and have reduced activity through CTLA-4) are expected to have improved ability to induce T cell activation. In contrast, B7 binds and acts through CTLA-4 with improved activity (and has reduced activity through CD28)
Variants are potent negative regulators of T cell function and are expected to induce tolerance and anergy.

B7 that alters its relative ability to act through CD28 and CTLA-4 (eg, B7-1 / CD80 and B7-2 /
CD86) variants are generated using DNA shuffling methods or other recombinant methods. In a preferred embodiment, the different forms of the substrate used in the recombination reaction are B7 cDNAs from different species. Such cDNA can be obtained by methods known to those skilled in the art (including RT-PCR).

Typically, the genes encoding these variant B7 molecules are incorporated into a genetic vaccine vector encoding the antigen, so that the vector can be used to modify an antigen-specific T cell response. Vectors carrying a B7 gene that acts efficiently through CD28 are useful, for example, in inducing a protective immune response, while carrying a gene encoding a B7 gene that acts efficiently through CTLA-4 Vectors that generate, for example, allergenic specific T cells or autoantigen specific T cells
Useful in inducing cellular tolerance and anergy. In some situations, such as in tumor cells or cells that induce an autoimmune response, the antigen may already be present on the surface of the target cell, and the variant B7 molecule may be present in the absence of an additional exogenous antigen gene. Can be transfected. FIG. 11 shows a screening protocol that can be used to identify B7-1 (CD80) and / or B7-2 (CD86) variants with increased ability to induce T cell activation or T cell anergy. Is illustrated. The application of this strategy is described in more detail in Example 1.

[0137] Several approaches to screening for variants can be taken. For example, a selection system based on flow cytometry may be used. B7-1 molecule and B
A library of 7-2 molecules is generally constructed of these molecules (eg, COS-7 cells, or any other species from a different species that has limited or no cross-reactivity with humans for B7 ligand binding. Cell line). An internal marker gene can be incorporated to analyze copy number per cell. Soluble CTLA-4 and CD-28 molecules can be generated and used in flow cytometry experiments. Typically, they are fused to the Fc portion of an IgG molecule, to improve the stability of these molecules, and to van der Merwe et al. (J. Exp. Med. 185: 393,199).
Easy staining with labeled anti-IgG mAb is possible, as described by 7). The cells transfected with the library of B7 molecules are then stained with soluble CTLA-4 and CD28 molecules. CTLA-4
Cells that show an increase or decrease in the / CD28 binding ratio are classified. The plasmid is then recovered and the shuffled B7 variant coding sequence is identified. These selected B7 variants may then be subjected to a new round of shuffling and selection and / or they may be further analyzed using the functional assays described below.

[0138] B7 variants can also be selected directly based on their functional properties. For in vivo studies, the B7 molecule may also evolve function in mouse cells. Pick a bacterial colony with the plasmid containing the mutant B7 molecule and isolate the plasmid. These plasmids are then transfected into antigen presenting cells (eg, dendritic cells) and the ability of these variants to activate T cells is analyzed. One of the advantages of this approach is that no assumptions are made in binding affinity, or in specificity for known ligands, and possibly new activities may be found through ligands that are further identified. In addition to dendritic cells,
If the "T cell signal of" is induced by, for example, an anti-CD3 monoclonal antibody, other cells that are relatively easy to transfect (e.g., U937 or COS-7) can be used in screening. T cell activation can be determined by methods known to those skilled in the art (eg, measuring proliferation, cytokine production, CTL activity or I
(Including expression of activating antigens such as the L-2 receptor, CD69 or HLA-DR molecule). The use of antigen-specific T cell clones (eg, T cells specific for the house dust mite antigen Der p I) allows analysis of antigen-specific T cell activation (Yssel et al., (1992)). J. Immuno
l. 148: 738-745). Variants that can increase or inhibit T cell differentiation or increase or inhibit CTL responses are identified. Similarly, for example,
Variants that have altered ability to induce cytokine production or expression of antigenic activity as measured by cytokine-specific ELISA or flow cytometry can be identified.

[0139] B7 variants are useful in modulating the immune response in autoimmune diseases, allergies, cancer, infectious diseases and vaccinations. B acting through CD28 with improved activity (and with reduced activity through CTLA-4)
Seven variants have improved their ability to induce T cell activation. In contrast, CTLA-4 with improved activity (and with reduced activity through CD28)
B7 variants that bind and act through are potent negative regulators of T cell function and induce tolerance and anergy. Therefore, these variants B7
By incorporating the gene encoding the molecule into a genetic vaccine vector encoding the antigen, it is possible to modify the antigen-specific T cell response. Vectors carrying a B7 gene that acts efficiently through CD28 are useful, for example, in inducing a protective immune response, while vectors carrying a gene encoding a B7 gene that acts efficiently through CTLA-4 Is useful, for example, in inducing tolerance and anergy in allergenic or autoantigenic specific T cells. In some situations (eg, in tumor cells or cells that induce an autoimmune response), the antigen may already be present on the surface of the target cell, and the variant B7 molecule may be transfected in the absence of additional foreign antigen genes. Can be effected.

[0140] The method of the present invention are also useful in order to obtain a B7 variants that increase the efficacy of oriented T _H 1 cells or T _H 2 cells or differentiation. Differential roles was observed with the B7-1 molecule and B7-2 molecules in the regulation of the differentiation of T-helper (T _H) cells (Freeman et al. (1995) Immunity 2: 523;
Kuchro et al. (1995) Cell 80: 707). _TH cell differentiation can be measured by analyzing the cytokine production profile induced by each particular variant. High levels of IL-4, IL-5 and / or IL-
13 is an index of differentiation of valid T _H 2 cells, whereas, IFN-gamma or IL-2 production of high levels can be used as a marker for the differentiation of T _H 1 cells. T _H 1
Alternatively, B7 variants having altered ability to induce T _H 2 cell differentiation are useful, for example, in the treatment and vaccination of allergic, malignant, autoimmune and infectious diseases.

The present invention also provides a method of obtaining a B7 variant with enhanced ability to induce the production of IL-10 by antigen-specific T cells. Elevated IL-10 production is characteristic of regulatory T cells, which can suppress the proliferation of antigen-specific CD4 ⁺ T cells (Groux et al. (1997) Nature 389: 737). DNA shuffling was performed as described above, after which recombinant nucleic acids encoding B7 variants with increased ability to induce IL-10 were identified, for example, by ELISA using cytoplasmic cytokine staining or by flow cytometry. obtain. Variants that induce high levels of IL-10 production are useful for treating allergic and autoimmune diseases.

D. Optimization of Antigen Transport and Presentation The present invention also provides gene vaccines and methods of obtaining accessory molecules that can improve the transport and presentation of antigenic peptides. A library of recombinant polynucleotides is generated and screened to identify polynucleotides that encode molecules having improved properties as compared to a wild-type control. The polynucleotide itself can be used in a genetic vaccine, or the gene product of the polynucleotide can be used for therapeutic or prophylactic applications.

1. Proteasome Class I peptides presented in the major histocompatibility complex molecules are produced by the cellular proteasome. Interferon-γ can stimulate antigen presentation, and part of the mechanism of action of interferon can be due to the induction of proteasome β-subunits LMP2 and LMP7, which is homologous β-subunit Y (delta) And X (epsilon). Such substitutions can alter the peptide cleavage specificity of the proteasome and increase the immunogenicity of class I epitopes. The Y (delta) and X (epsilon) subunits, as well as other recently discovered proteasome subunits (eg, MECL-1 homolog MC14), are characteristic of cells that are not specified in antigen presentation. Thus, incorporation of LMP2, LMP7, MECL-1 and / or other epitope-presenting specific subunits and potential interferon-inducible subunits into cells by DNA transfer can increase epitope presentation. Peptides produced by the proteasome containing the interferon-inducible subunit are identified as TAP
It seems to be transported to the endoplasmic reticulum by molecules.

The present invention provides a method for obtaining a proteasome that exhibits increased or decreased ability to specifically process an MHC class I epitope. According to this method, DNA shuffling can be used to have novel specificities that can enhance the immunogenicity of some proteins, and / or once their subunits bind to the proteasome and Obtain any evolved protein that can increase activity. The transition from non-specific proteasomes to class I epitope-specific proteasomes can be many different, as some states (some but not all interferon-inducible subunits may be associated with the proteasome) Proteolytic specificity can potentially be achieved. Therefore,
The evolution of specific LMP-like subunits can generate novel proteasome compositions with enhanced functions for epitope presentation.

The method includes performing DNA shuffling using two or more forms of a polynucleotide encoding a proteasome component (the form of the polynucleotide differs in at least one nucleotide) as a substrate. Shuffling is performed by using one or more of various proteasome components (eg, LMP2, LMP7, M
(Including ECL-1 and other individual proteasome components that are specifically involved in class I epitope presentation) using polynucleotides encoding as described herein. Examples of suitable substrates include, for example, Stohwasser
(1997) Eur. J. Immunol. 27: 1182-1187 and Gaczynska et al. (1996) J. Am. Biol. Chem. 271: 1727
5-17280. In a preferred embodiment, family shuffling is used, wherein the different substrates are polynucleotides encoding proteasome components from different species.

After the recombination reaction is completed, the resulting library of recombinant polynucleotides is screened to identify polynucleotides that encode proteasome components that have the desired effect on class I epitope production. For example, the recombinant polynucleotide can be introduced into a genetic vaccine vector that also encodes a particular antigen of interest. The vectors of the library can then be introduced into mammalian cells that have been subsequently screened to identify those cells that exhibit increased antigen-specific immunogenicity. Methods for analyzing proteasome activity are described, for example, in Groettrup et al. (1997)
) Proc. Nat'l. Acad. Sci. USA 94: 8970-897
5 and Groetrup et al. (1997) Eur. J. Immunol. 26
: 863-869.

Alternatively, the methods of the invention can be used to evolve proteins that bind strongly to the proteasome but have reduced or no activity. Thus, it antagonizes proteasome activity and reduces the ability of cells to display class I molecules. Such molecules desire the lower immunogenicity of exogenous proteins expressed in cells as a result of gene therapy, and have been processed for Class I presentation that would otherwise allow the cells to be recognized by the immune system. To gene therapy protocols. Such high-affinity, low-activity LMP-like subunits demonstrate an immunosuppressive effect that is also useful in other therapeutic protocols where cells expressing non-self proteins need to be protected from the immune response.

The specificity of the proteasome and the TAP molecule (discussed below) may have evolved naturally. Thus, it may be important that the two pathways of a class I processing system be functionally identical. A further aspect of the present invention is to perform simultaneous DNA shuffling in two gene families, followed by performing two random combinations to find the appropriate matched proteolytic and transport specificities including.

2. Antigen Transport The present invention provides methods for improving the transport of antigenic peptides from the cytosolic compartment to the endoplasmic reticulum, thereby transporting MHC class I molecules to the cell surface. Enhanced expression of an antigenic peptide results in an enhanced immune response, particularly
Improves the activation of D8 ⁺ cytotoxic lymphocytes. This is useful in DNA vaccine development and gene therapy.

In one embodiment, the present invention provides a method for obtaining a gene that exhibits improved antigen presentation.
TAP gene (transporter related to antigen processing)
No. The TAP gene is a member of the ATP binding cassette family of membrane translocators.
Be a member. These proteins convert antigenic peptides into MHC class I molecules.
And is involved in the expression and stability of MHC class I molecules on the cell surface
You. Two TAP genes, TAP1 and TAP2, have been cloned to date.
(Powis et al. (1996) Proc. Nat'l. Acad. Sci.
. USA 89: 1463-1467; Koopman et al. (1997) Curr.
. Opin. Immunol. 9: 80-88; Monaco (1995) J. Mol.
Leukocyte Biol. 57: 543-57). TAP1 and TAP
2 form heterodimers, and these genes transfer peptides to the endoplasmic reticulum.
Required for delivery, where those genes bind to MHC class I molecules
. The basic role of the TAP gene product in the presentation of antigenic peptides has been disrupted
Demonstrated in mice with the TAP gene. TAP1-deficient mice have M
Dramatically reduces surface expression levels of HC class I and reduces CD8 in the thymus ⁺ Positive selection of T cells was strongly reduced. Thus, CD8 in the periphery of TAP-deficient mice⁺The number of T lymphocytes is extremely low. When the TAP gene is transfected back into these cells, the levels of MHC class I expression are restored.

[0151] The TAP gene is a good target for gene shuffling. Because of natural polymorphisms and in some mammalian species (human (Beck et al. (1
992) J. Mol. Biol. 228: 433-441; Genbank accession number Y13582; Powis et al., Supra), gorilla TAP1 (Lau
d et al. (1996) Human Immunol. 50: 91-102), mouse (Reiser et al. (1988) Proc. Nat'l. Acad. Sci. US).
A 85: 2255-2259; Marusina et al. (1997) J. Am. Immu
nol. 158: 5251-5256, TAP1: Genbank accession numbers U60018, U60019, U60020, U60021, U6002
2 and L76468-L67470; TAP2: Genbank accession numbers U60087, U60088, U6089, U60090, U60091
And U60092), because these genes of hamsters (including TAP1, Genbank accession numbers AF001154 and AF001157; TAP2, Genbank accession numbers AF001156 and AF001155) have been cloned and sequenced. Furthermore, it has been shown that point mutations in the TAP gene can lead to changes in peptide specificity and peptide presentation. Also, functional differences in TAP genes from different species were observed. For example, human TAP and rat TAP containing the rTAP2a allele are rather irregular, while mouse TAP is selected for restricted and C-terminal small polar / hydrophobic amino acids or peptides with positively charged amino acids. Is done. The basis for this selectivity is unknown.

The method of the present invention provides that at least two nucleotides differ by at least one nucleotide position.
Performing DNA shuffling of the TAP1 and TAP2 genes using the two forms of TAP1 and / or TAP2 polynucleotide sequences as substrates. In a preferred embodiment, TA from several mammalian species
The P sequence is used as a substrate for shuffling. Natural polymorphisms of this gene may provide additional substrate diversity. If desired, the optimized TAP gene obtained from one round of shuffling and screening may be subjected to further shuffling / screening rounds to further obtain a polynucleotide encoding the optimized TAP.

Polypeptide encoding TAP optimized from a library of recombinant TAP genes
To identify nucleotides, place this gene on the same plasmid
It can be expressed as an antigen. If this step limits the extent of antigen presentation, CD8 ⁺ Produces increased presentation for CTL. Variants of TAP can act selectively to regulate the expression of certain antigenic peptide fragments whose expression levels would otherwise limit
Increases or is usually never transferred to the RER, and with MHC class I
Causes the transport of peptides that are not used for binding.

When used in the context of gene therapy vectors in cancer therapy, the evolved TAP gene is a means of increasing the expression of MHC class I molecules on tumor cells, and the efficient use of antigenic tumor-specific peptides Provides a means to obtain a presentation. Therefore, the vector containing the evolved TAP gene can induce a strong immune response against malignant cells. The shuffled TAP gene can be transfected into a malignant cell line that expresses low levels of MHC class I molecules using retroviral vectors or electroporation. Transfection efficiency is determined by TAP
It can be monitored using a marker gene (eg, green fluorescent protein) encoded by the same vector as the gene. Cells that express equal levels of green fluorescent protein but express the highest levels of MHC class I molecules as efficient TAP gene markers are then sorted using flow cytometry, and The evolved TAP gene is then recovered from these cells, for example, by PCR or by recovering the entire vector. If further optimization is desired, these sequences can then be subjected to a new round of shuffling, selection and recovery.

The evolution of the molecule of the TAP gene can be combined with the co-evolution of the desired antigen. Co-evolution of the desired antigen may further improve the efficiency of presentation of the antigenic peptide after DNA vaccination. This antigen can be evolved using gene shuffling to include a structure that allows for optimal presentation of the desired antigenic peptide when the optimal TAP gene is expressed. The optimal TAP gene for the presentation of an antigenic peptide of one given antigen may be different from the optimal TAP gene for the presentation of an antigenic peptide of another antigen. Gene shuffling techniques are ideal and are probably the only way to solve this type of problem. The efficiency of presentation of the desired antigenic peptide can be determined using specific cytotoxic T lymphocytes, for example, by measuring cytokine production or T lymphocyte CTL activity using methods known to those of skill in the art. Can be analyzed.

3. Cytotoxic T Cell Inducing Sequences and Immunogenic Agonist Sequences Certain proteins are more capable of possessing MHC class I epitopes than other proteins. Because they are more easily used by cellular machinery involved in the processing required for the presentation of class I epitopes. The present invention provides a method for identifying expressed polypeptides that is particularly efficient in passing through various biosynthetic and degradation steps that lead to the presentation of class I epitopes, as well as methods for identifying CTL epitopes from other proteins. Provided is the use of these polypeptides to increase presentation.

[0157] In one embodiment, the present invention provides a cytotoxic T cell-inducing sequence (CTIS).
Which can be used to carry a heterologous class I epitope for the purpose of vaccination against the pathogen from which it is derived. One example of CTIS is obtained from hepatitis B surface antigen (HBsAg). This indicates that it is an effective carrier for its own CTL epitope when delivered as a protein under certain conditions. DNA immunization with a plasmid expressing HBsAg also induces high levels of CTL activity. The present invention provides shorter truncated fragments of the HBsAg polypeptide,
It functions very efficiently at inducing L activity and achieves higher levels of CTL induction than with HBsAg protein or with a plasmid encoding the full length HBsAg polypeptide. The synthesis of CTIS from HBsAg is described in Example 3; and a diagram of CTIS is shown in FIG.

ER localization of the cleaved polypeptide may be important in achieving proper proteolytic release of the peptide containing the CTL epitope (Cressw
ell and Hughes (1997) Curr. Biol. 7: R552-R
555; Craiu et al. (1997) Proc. Nat'l. Acad. Sci.
ScL USA 94: 10850-10855). The pre-S2 and transmembrane regions provide T helper epitopes that may be important for inducing a strong cytotoxic immune response. The truncated CTIS polypeptide has a simple structure (see FIG. 1), which eliminates the need to maintain any particular protein conformation and allows one or more heterologous class I epitope sequences to be added to the polypeptide. Can be attached to the C-terminus. Such sequences are then available to the processing mechanism for class I epitopes. The size of the polypeptide is not an issue for the usual constraints of the native HBsAg structure. Therefore, the length of the heterologous sequence,
Thus, the number of included CTL epitopes varies. This is shown schematically in FIG. The ability to include long sequences containing either multiple and different Class I sequences, or alternatively different variations of a single CTL sequence, allows one to apply DNA shuffling methodology.

[0159] The present invention also relates to a C-cell that can specifically lyse cells expressing a native epitope sequence.
A method for obtaining an immunogenic agonist sequence (IAS) that induces TL is provided. In some cases, the reactivity is greater than when the CTL response is induced by the natural epitope (see Example 3 and FIG. 3). Such IAS derivatized CTLs can be drawn from a different T cell repertoire than T cells induced by the native sequence. Thus, poor responsiveness to a given epitope can be overcome by recruiting T cells from a larger pool. To discover such an IAS, the amino acid at each position of the CTL-derived peptide (
Perhaps except for the so-called anchor residue positions) can vary over a range of 19 amino acids that are not present at normal positions. DNA shuffling methodology can be used to scan a large range of sequence possibilities.

Synthetic gene segments containing multiple copies of the native epitope sequence can be prepared such that each copy carries a small number of nucleotide changes. This gene segment can be shuffled to create a diverse range of CTL epitope sequences,
Some of them should function as IAS. This process is illustrated in FIG.

In practice, oligonucleotides are typically constructed according to the above design and polymerized enzymatically to form a synthetic gene segment of the linked epitope. Restriction sites can be incorporated into a fraction of the oligonucleotide to allow for cleavage and selection of a given size range of the linked epitope. Most of these have different sequences and are therefore potential IAS. This epitope-containing gene segment can be linked to CTIS like a segment of HBsAg by a suitable cloning method. The resulting plasmid construct can be used for DNA-based immunization and CTL induction.

E. Pharmaceutical Compositions of Gene Vaccines and Methods of Administration The improved immunomodulatory polynucleotides and polypeptides of the present invention treat and / or prevent various diseases and conditions involving the respective antigens. Useful to For example, genetic vaccines using reagents obtained according to the methods of the invention are useful for both prevention and treatment of infectious diseases, including diseases caused by any bacteria, fungi, viruses or other pathogens of mammals. It is. The reagents obtained using the present invention may also be useful for treating autoimmune diseases, including, for example, rheumatoid arthritis, SLE, diabetes mellitus, myasthenia gravis, reactive arthritis, ankylosing spondylitis, and multiple sclerosis. Can be used for treatment. These and other inflammatory conditions, including IBD, psoriasis, pancreatitis, and various immunodeficiencies, can be treated using genetic vaccines containing vectors and other components obtained using the methods of the present invention. Genetic vaccine vectors and other reagents obtained using the methods of the invention can be used to treat allergies and asthma. Furthermore, the use of genetic vaccines has great promise for treating cancer and preventing metastasis. Induction of an immune response to cancerous cells can result in the body's immune system reducing or eliminating cancer.

[0163] In a presently preferred embodiment, the reagents obtained by use of the present invention are used with a gene vaccine vector. The choice of vector and components may also be optimized for the particular purpose of treating allergies or other conditions. For example,
Antigens associated with treatment of a particular condition can be optimized using recombination and selection methods similar to those described herein. Suitable antigens for such methods, and for various conditions, are described in "antigen library immunization" (TTC Attorne).
y Docket No. 18097-0228710US, February 1, 1999
Concurrently assigned U.S. Patent Application No.
It is described in. A polynucleotide encoding a recombinant antigenic polypeptide can be placed under the control of a promoter, such as a highly active promoter or a tissue-specific promoter. The promoter used to express the antigenic polypeptide can be prepared using recombination and selection methods similar to those described herein, International Application No. PCT / US97 / 17300 (International Publication No. 98/1
3487), which can itself be optimized. . Reagents obtained using the methods of the invention can also be used with multi-component genetic vaccines, which can modulate the immune response to be most suitable to achieve the desired effect (eg,
See co-pending and commonly assigned U.S. Patent Application No. _, entitled "Gene Vaccine Vector Technology" (filed February 10, 1999 as TTC Attorney Docket No. 18097-030100US). . It is sometimes advantageous to use genetic vaccines that target a particular target cell type (eg, antigen presenting or processing cells);
"Targeting Gene Vaccine Vectors" (TTC Attorney Docke)
No. _______________________________________Bounded as co-pending and commonly assigned U.S. Pat.

A genetic vaccine vector comprising an optimized recombinant polynucleotide obtained as described herein can be used in mammals (including humans) to induce a therapeutic or prophylactic immune response. Can be delivered. Vaccine delivery vehicles are intended for administration to individual patients, typically systemically (eg, intravenous, intraperitoneal, intramuscular, subcutaneous, intracranial, intraanal, intravaginal, buccal, buccal or by inhalation). Can be delivered in vivo, or can be administered by topical application. Alternatively, the vector can be delivered ex vivo to cells (eg, cells transplanted from individual patients (eg, lymphocytes, bone marrow aspirates, tissue biopsies)) or to common donor hematopoietic stem cells, Thus, usually after selecting for cells that have incorporated the vector, the cells are re-implanted into the patient.

[0165] Many delivery methods are well known to those of skill in the art. Such methods include, for example, liposome-based gene delivery (Debs and Zhu (1993) WO 93 /
24640; Mannino and Gould-Fogerite (1988)
BioTechniques 6 (7): 682-691; Rose U.S. Patent No. 5,279,833; Brightham (1991) WO 91/06309.
And Felgner et al. (1987) Proc. Natl. Acad. Sc
i. USA 84: 7413-7414), as well as the use of viral vectors (
For example, adenoviral vectors (eg, as reviewed in Berns et al. (199)
5) Ann. NY Acad. Sci. 772: 95-104; Ali et al., (1.
994) Gene Ther. 1: 367-384; and Haddada et al. (
1995) Curr. Top. Microbiol. Immunol. 199 (
Pt3): 297-306), papillomavirus vectors, retroviral vectors (see, eg, Buchscher et al. (1992) J. Viro.
l. 66 (5) 2731-2739; Johan et al. (1992) J. Am. Viro
l. 66 (5) 1635-1640 (1992); Sommerfeld et al. (1
990) Virol. 176: 58-59; Wilson et al. (1989) J. Am. V
irol. 63: 2374-2378; Miller et al. Virol. 65
: 2220-2224 (1991); Wong-Stal et al., PCT / US9.
4/05700, and Rosenburg and Fauci (1993) F
unmetal Immunology, 3rd edition, Paul edition, Raven
Press, Ltd., New York and references herein, and Yu et al., Gene Therapy (1994) supra), and adeno-associated virus vectors (West et al. (1987) Virology 1).
60: 38-47; Carter et al. (1989) U.S. Pat. No. 4,797,368.
No .; Carter et al., WO 93/24641 (1993); Kotin (199).
4) Human Gene Therapy 5: 793-801; Muzyc
zka (1994) J. Mol. Clin. Invest. 94: 1351 and AAV
See Samulski (supra) for an overview of vectors;
kowski, U.S. Pat. No. 5,173,414; Tratschin et al. (19
85) Mol. Cell. Biol. 5 (11): 3251-3260; Tra
tschin et al. (1984) Mol. Cell. Biol. 4: 2072-20
81; Hermonat and Muzyczka (1984) Proc. Nat
l. Acad. Sci. USA, 81: 6466-6470; McLaughl.
(1988) and Samulski et al. (1989) J. Am. Virol.
, 63: 03822-2828).

“Naked” DNA and / or RNA, including genetic vaccines, can be introduced directly into tissues (eg, muscle). See, for example, USPN 5,580,859. Other methods (eg, “bombardment” or particle-mediated transformation (eg, Sa)
nford et al., USPN 4,945,050; USPN 5,036,006.
)) Are also suitable for introducing genetic vaccines into mammalian cells according to the invention. These methods are useful not only for the introduction of DNA into mammals in vivo, but also for ex vivo modification of cells for reintroduction into mammals. For other methods of delivering genetic vaccines, repeat the vaccine administration, if necessary, to maintain the desired level of immunomodulation.

Gene vaccine vectors (eg, adenovirus, liposomes, papillomavirus, retrovirus, etc.) can be directly administered to mammals for transduction of cells in vivo. The genetic vaccine obtained using the method of the present invention comprises:
For prophylactic and / or therapeutic treatments, any suitable mode (parenteral (eg, subcutaneous, intramuscular, intradermal, or intravenous), topical, oral, rectal, intrathecal, buccal (eg, (Sublingual), or in topical administration (including, for example, aerosol or transdermal), and may be formulated as pharmaceutical compositions for administration. For example, pre-treatment of the skin with the use of a hair removal agent may be useful in transdermal delivery. Suitable methods for administering such packaged nucleic acids are available and are well known to those skilled in the art. And although more than one route may be used to administer a particular composition, certain routes often provide a more immediate and more effective response than another route.

Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide range of suitable formulations of the pharmaceutical compositions of the present invention. A variety of water-soluble carriers can be used, such as, for example, buffered saline. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well-known sterilization techniques. Since the composition is required for approximate physiological conditions, pharmaceutically acceptable auxiliary substances such as pH and buffering agents, toxicity modifiers and the like (eg, sodium acetate, sodium chloride,
Potassium chloride, calcium chloride, sodium lactate, etc.)). The concentration of the gene vaccine vector in these formulations can vary widely, and is chosen primarily based on fluid volume, viscosity, body weight, etc., according to the particular mode of administration chosen and the needs of the patient. .

Formulations suitable for oral administration include: (a) a liquid solution (eg, an effective amount of a packaged nucleic acid suspended in a dilute solution (eg, water, saline or PEG 400)); (b) Capsules, sachets, tablets each containing a predetermined amount of active ingredient as a liquid, solid, granule or gelatin; (c) a suspension in a suitable liquid; and (d) a suitable emulsion, Can consist of Tablet forms include lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia,
Gelatin, colloidal silicon dioxide, croscarmellose
llose) sodium, talc, magnesium stearate, stearic acid,
And one or more of other excipients, colorants, fillers, binders, diluents, buffers, swelling agents, preservatives, flavoring agents, pigments, disintegrants, and pharmaceutically compatible carriers. May be included. A lozenge form as well as a tablet comprising the active ingredient in an inert substrate (e.g., gelatin and glycerin or sucrose and acacia emulsions, gels, and the like), in addition to the active ingredient, are known in the art. Active ingredients in fragrances,
Usually it can include sucrose and acacia or tragacanth. It is recognized that when administered orally, a genetic vaccine must be protected from digestion. This is typically by conjugation of the vaccine vector with a composition that renders the vector resistant to acid and enzymatic hydrolysis, or packaging the vector in a suitable resistant carrier such as a liposome. Achieved by either Means of protecting vectors from digestion are well-known in the art. The pharmaceutical composition can be encapsulated, for example, in liposomes or in a formulation to provide sustained release of the active ingredient.

The packaged nucleic acids, alone or with other suitable components, can be made into aerosol formulations to be administered via inhalation (eg, they can be “nebulized”). Aerosol formulations can be placed into an acceptable pressurized propellant (eg, dichlorodifluoromethane, propane, nitrogen, etc.).

Formulations suitable for rectal administration include, for example, suppositories, which consist of the nucleic acid packaged with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules consisting of a combination of the packaged nucleic acids and a substrate, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.

Parenteral administration (eg, intraarticular (i.
Formulations suitable for (the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes) include antioxidants, buffers, bacteriostats, and blood of the intended recipient, and the like. Aqueous and non-aqueous isotonic sterile injectable solutions that may contain solutes that make up the formulation that is tonic, and water-soluble and water-insoluble that may contain suspending agents, dissolving agents, thickening agents, stabilizers and preservatives. Sterile suspensions are included. In the practice of the present invention, the compositions may be administered, for example, by intravenous infusion, oral, topical, intraperitoneal, intracapsular or intrathecal. Parenteral administration and intravenous administration are preferred methods of administration. The packaged nucleic acid formulation may be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. Cells transduced by the packaged nucleic acid can also be administered intravenously or parenterally.

The dose administered to a patient, in the context of the present invention, should be sufficient to result in a beneficial therapeutic response in the patient over time. The dose will be determined by the effectiveness of the particular vector used and the condition of the patient, as well as the weight or vascular surface area of the patient to be treated. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of the particular vector or transformed cell type in the particular patient.

In determining the effective amount of a vector to be administered in the treatment or prevention of infections and other conditions, a physician will evaluate the toxicity of the vector, the progression of the disease, and the production of anti-vector antibodies, if any. Generally, an equivalent dose of naked nucleic acid from a vector is typically about 1 μg to 1 mg for a 70 kilogram patient, and the dose of vector used to deliver the nucleic acid is an equivalent Calculated to yield therapeutic nucleic acid. Administration can be accomplished in single or divided doses.

In therapeutic applications, the compositions will cure or at least partially arrest the disease and its complications in a patient suffering from the disease (eg, an infectious disease or an autoimmune disease). Is administered in a sufficient amount. An amount sufficient to accomplish this is defined as "therapeutically effective dose." The effective amount for this use will depend on the severity of the disease and the general health of the patient. Single or multiple administrations of the composition can be administered depending on the dosage and frequency as required and tolerated by the patient. In any situation, the composition should provide a sufficient amount of the protein of the invention to effectively treat the patient.

In prophylactic applications, the compositions are administered to humans or other mammals to induce an immune response that can help protect against the establishment of an infectious disease or other condition.

The toxicity and therapeutic efficacy of the gene vaccine vectors provided by the present invention are determined using standard pharmaceutical procedures in cell culture or experimental animals. LD ₅₀ (the dose lethal to 50% of the population) and ED ₅₀ (therapeutic in 50% of the population) using the methods presented herein and those otherwise known to those skilled in the art. Effective dose) can be determined.

A typical pharmaceutical composition for intravenous injection is about 0.1 to 10 per patient per day
mg. Especially when the drug is administered to an isolated site and not to the bloodstream (e.g., a body cavity or lumen of an organ), from 0.1 to about 100 per patient per day.
A dose of mg may be used. Subsequently, higher dosages are possible with topical administration. Actual methods of preparing parenterally administrable compositions are known or apparent to those skilled in the art, and are provided by Remington's Pharmaceut.
ical Science, 15th Edition, Mack Publishing Co.
mpany, Easton, Pennsylvania (1980).

A multivalent antigenic polypeptide of the invention and a gene vaccine expressing the polypeptide can be packaged in a bag, a dispenser device, and a kit for administering the gene vaccine to a mammal. For example, a bag or dispenser device containing one or more unit dosage forms is provided. Typically, instructions for administration of the compound are provided with the packaging, along with appropriate instructions in labels that are appropriate for treatment of the indicated condition, where the compound is indicated. For example, the label may be useful if the active ingredient in the packaging is useful for the treatment of certain infectious diseases, autoimmune disorders, tumors, or is mediated by a mammalian immune response, or is strongly sensitive May be useful for the prevention or treatment of other diseases or conditions that are:

EXAMPLES The following examples are provided to illustrate, but not to limit the invention.

Example 1 B7-1 (CD80) and / or B7-2 by DNA Shuffling
This example describes the use of the DNA shuffling method of the present invention to obtain B7-1 and B7-2 polypeptides having altered biological activity. I do.

DNA Shuffling Using DNA shuffling, B7 (B7-1) with altered relative ability to act through CD28 and CTLA-4 when compared to wild-type B7 molecules.
/ CD80 and B7-2 / CD86) variants of the variants are generated. Typically, B7 cDNAs from various species are generated by RT-PCR and these sequences are shuffled using family DNA shuffling.
An alignment of the human, rhesus monkey and rabbit B7-1 nucleotide sequences is shown in FIG. This demonstrates that family DNA shuffling is a viable approach when evolving B7 molecules.

(Screening of B7 variant) Next, this library was screened for autoimmune diseases, allergies,
Identify those variants that are useful for modulating the immune response in cancer, infectious diseases and vaccination. Any of several approaches to screening for this variant may be used.

A. Selection Systems Based on Flow Cytometry A library of B7-1 and B7-2 molecules is used to identify cells that do not normally express these molecules (eg, COS-7 cells, or humans for B7 ligand binding). (Any cell line from a different species with limited or no cross-reactivity). An internal marker gene can be incorporated to analyze copy number per cell.

To facilitate flow cytometry experiments, soluble CTLA-4 and CD28 molecules are produced. Typically, these soluble polypeptides are converted to IgG
Fused with the Fc region of a molecule to improve the stability of the molecule and to provide labeled anti-IgG
Staining with monoclonal antibodies may be facilitated (as described in van der Merwe et al. (1997) J. Exp. Med. 185: 393). B
Cells transfected with the library of seven molecules are then stained with soluble CTLA-4 and CD28 molecules. Cells that show an increase or decrease in CTLA-4 / CD28 binding ratio are classified. The plasmid was then recovered,
Then, the shuffled sequence is identified. These selected B7 variants may then be subjected to a new round of shuffling and selection and further analyzed using the functional assays described below.

B. Selection Based on Functional Properties Pick a bacterial colony containing a plasmid containing a variant of the B7 molecule and isolate the plasmid. These plasmids are then transferred to antigen presenting cells (eg,
Dendritic cells) and analyze the ability of these mutants to activate T cells. One of the advantages of this approach is that it makes no assumptions about the binding affinity or specificity for a known ligand, and perhaps new activities can be found through the ligand to be identified.

T cell activation can be analyzed by measuring proliferation, cytokine production, CTL activity or expression of an activating antigen, such as an IL-2 receptor, CD69, or HLA-DR molecule. The use of antigen-specific T cell clones (eg, specific T cells for the house dust mite antigen Der pI) allows analysis of antigen-specific T cell activation. Mutants that can promote or inhibit T cell proliferation or increase or inhibit CTL responses are identified. Similarly, variants with altered ability to induce the expression of cytokine production or antigen activation as measured, for example, by a cytokine-specific ELISA or flow cytometry may be selected. The results obtained using the proliferation-based assay are shown in FIG.
Shown in

[0189] A Differential roles of B7-1 molecules, and B7-2 molecules in the regulation of differentiation (C.T _H 1 ability to direct either cell differentiation or T _H 2 cell differentiation) T helper cell identification (Freeman et al. (1995) Immunity
2: 523; Kuchroo et al (1995) Cell 80: 707) , can be screened for the most effective B7 variants when directed to any of the T _H 1 cell differentiation or T _H 2 cell differentiation. _TH cell differentiation can be measured by analyzing the profile of cytokine production induced by each particular variant.
The high levels of IL-4, IL-5 and / or IL-13, an indicator of efficient T _H 2 cell differentiation, whereas a high level of production of IFN-gamma or IL-2 is, T _H 1 It can be used as a marker of cell differentiation. B7 variants with varying ability to induce partial reduction of T _H 1 or T _H 2 cells appears to be useful in the treatment and vaccination of allergic diseases, malignancies, autoimmune diseases and infectious diseases .

D. Enhanced IL-10 Production Increased production of IL-10 is characteristic of regulatory T cells. This may suppress the proliferation of antigen-specific CD4 ⁺ T cells (Groux et al. (1997) Nature 389: 737). Accordingly, B7 variants can be screened to identify variants with increased ability to induce IL-10 production by antigen-specific T cells. Production of IL-10 can be determined by ELISA using, for example, cytoplasmic cytokine staining.
It can be measured by SA or flow cytometry. High levels of IL-10
Variants that induce the production of are useful for treating allergic and autoimmune diseases.

Example 2 Evolution of Cytokines for Improved Specific Activity and / or Improved Expression Levels This example demonstrates improved specific activity when transfecting a genetic vaccine into mammalian cells. Methods for evolving cytokines for improved expression levels are described. IL-12 is the most potent cytokine that directs T _H 1 responses, which improves the efficacy of genetic vaccination. Advanced IL-12
The molecule is useful as a component of a genetic vaccine. IL-12 is a heterodimeric cytokine consisting of a 35 kD light chain (p35) and a 40 kD heavy chain (p40) (Kobayashi et al. (1989) J. Exp. Med. 170:
827; Stern et al. (1990) Proc. Nat'l. Acad. Sci.
USA 87: 6808). Recently, Lieshke et al. (Nature Bio
technology. (1997) 15:35) demonstrated that a fusion between the p35 and p40 genes yielded a single gene with activity comparable to that of two separately expressed genes. Thus, the IL-12 gene is shuffled as a whole encoding both subunits. This is useful when designing shuffling protocols. Also, the subunits of IL-12 can be expressed separately in the same expression vector, or the subunits can be expressed separately and screened using co-transfection of the two vectors. This provides an additional shuffling strategy.

[0192] IL-12 plays several roles in regulating allergic responses. For example, IL-12 induces the differentiation of T _H 1 cells, and down-regulate responses of T _H 2. IL-12 inhibits IgE synthesis both in vivo and in vitro, and also induces IFN-γ production. Accordingly, it is desirable to have optimized IL-12 that can better perform these functions in mammalian administration.

[0193] Cytokine genes from human and non-human primates, including the IL-12 gene, are generally 93-99% homologous (Villinger et al. (1995) J. Am.
Immunol. 155: 3946-3954), which provides a good starting point for family shuffling. A library of shuffled IL-12 genes was obtained by shuffling the p35 and p40 subunits from humans, rhesus monkeys, cats, dogs, cows, pigs, and goats, and incorporated this into a vector. . The supernatants of these transductants are then analyzed for biological activity as shown in FIG. Due to its T cell proliferation-promoting activity, it is possible to use normal human peripheral blood T cells in the selection of the most active IL-12 gene, whereby the IL having the most potent activity in human T cells It is possible to select -12 mutants directly.

As shown in FIG. 7, a functional screening assay has been successfully accomplished. In this assay, COS-7 cells were first transfected with a vector encoding the IL-12 subunit. 48 hours after transfection, the supernatants of these cultures were studied for their ability to induce the proliferation of activated human peripheral blood T cells. FIG. 8 shows the consistency of T cell proliferation levels induced in this assay. This indicates that this assay can be used to distinguish activity between supernatants with different abilities to induce T cell activation. In other words, this assay provides a means of screening for improved IL-12-like activity in culture supernatants of transfected cells. Vectors with optimized IL-12 encoding polynucleotides were tested for their ability to induce human T cell activation. The results (shown in FIG. 9) show that shuffled IL-12 significantly increases the ability to induce T cell activation compared to wild-type IL-12.

FIG. 6 illustrates a general strategy for screening evolved cytokine genes. Although a specific example is given for IL-12, a similar approach applies to all cytokines when using cell types that are sensitive to each cytokine. For example, GM-CSF can be evolved by the same approach by using it in screening GM-CSF sensitive cell line TF-1. Furthermore, although the vector is transfected into CHO cells in this example, any mammalian cell that can be transfected in vitro can be used as a host cell. In addition to CHO cells, other good host cells include cell line WI-26,
COS-1, COS-7, 293, U937 and freshly isolated human antigen presenting cells (eg, monocytes, B cells and dendritic cells).

Example 3 Cytotoxic T Cell Inducing Sequences from Hepatitis B Surface Antigen and Potent Immunogenic Agonist T Cell Epitopes This example can efficiently present T cell epitopes Preparation of polynucleotide sequences and D to identify potent immunogenic agonist T cell epitopes
Describes the strategy for applying NA shuffling.

The HBsAg polypeptide (PreS2 and S region) was modified at amino acid position 103 (
The cysteine codon TGT was converted to the stop codon TGA by introduction of a stop codon (counting from the PreS2 initiator methionine). The amino acid sequence of the cleaved protein is thus:

[0198]

Embedded imageMet. Here, the standard single letter code for amino acids is used. P
The first methionine residue in the reS2 and S regions is underlined. And mouse L ^d Restricted CTL epitopes are double underlined; asterisk (^*) Artificially
Represents the introduced stop codon.

A structure resembling this cleaved polypeptide is shown in FIG. During protein biosynthesis, the N-terminal region of the HBsAg polypeptide is transported across the membrane of the endoplasmic reticulum (ER). The first part of the S region is a transmembrane structure that confines the polypeptide to the ER. The remaining C-terminal region of the polypeptide is located in the cytoplasmic compartment, where this region can be accessed by the epitope processing machinery of the cell. This structure forms what is referred to as a cytotoxic T cell guidance sequence (CTIS).

[0200] CTIs are preferably used with immunogenic agonist sequences (IAS). Examples of IAS, was replaced with a codon encoding alanine in the DNA sequence of an epitope of L ^d restricted class I epitopes of the 12 amino acid positions from HBsAg. This result demonstrates that in some cases this reactivity is more active than when the CTL response is induced by the natural epitope.

Example 4 Treatment of Obesity, Anorexia and Cachexia Using Optimized Immunomodulatory Molecules Optimized immunomodulatory molecules obtained using the methods of the present invention Find use in a wide range of applications in addition to its use in vaccination. For example, there is increasing evidence that certain forms of obesity are associated with dysfunction of the immune system, and that molecules that modulate the immune response (eg, cytokines) can induce or inhibit obesity. The present invention provides methods for optimizing immunomodulatory molecules for the treatment of obesity, anorexia and cachexia.

Leptin and ciliary neurotrophic factor (CNTF) are examples of cytokines that have been shown to play a role in the development of obesity. Congenital leptin deficiency is severe early obesity in humans.
y-onset obesity) (Montague et al. (1997) N
atature 387: 903-908), and has shown that CNTF repairs obesity and diabetes associated with leptin deficiency and leptin resistance (G
loaguen et al. (1997) Proc. Nat'l. Acad. Sci. US
A 94: 6456-6461). Antagonists of CNTF and / or leptin may be effective in treating anorexia and / or cachexia.

[0203] The method of the present invention may be applied to leptin and / or CN with improved specific activity.
Used to generate TF molecules. This method is also useful for obtaining improved cytokines that exhibit reduced immunogenicity in vivo; immunogenicity is particularly associated with CNTF. Because wild-type CNTF is highly antigenic, it results in high levels of production of anti-CNTF antibodies when administered to humans. Administering an improved cytokine molecule prepared as a polypeptide using the method of the invention as a polypeptide or improved leptin and / or CNTF
The shuffled nucleic acid encoding the polypeptide is used in a gene vaccine vector. The present invention also provides for increased levels of leptin and / or C
Methods for generating a vector that induces the production of NTF are provided.

The methods of the present invention may also be used to obtain an agent that is useful in treating anorexia, cachexia, and related disorders. In this embodiment, leptin and / or CNTF antagonists are evolved using DNA shuffling methods. For example, the leptin receptor can be evolved to obtain a soluble form with increased affinity for leptin. The receptor for leptin in mice is the hypothalamus (Mercer et al. (1996) FEBS Lett. 38
7: 113), in areas known to be involved in maintaining energy balance, and in the choroid plexus and leptomeninges, where they form part of the blood / brain barrier.

The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in light thereof will be suggested to those skilled in the art,
It is understood that the spirit and scope of the present application fall within the scope of the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference for all purposes.

[Brief description of the drawings]

FIG. 1 shows an example of a cytotoxic T cell induction sequence (CTIS) obtained from an HBsAg polypeptide (PreS2 and S region).

FIG. 2 shows a CTIS with a heterologous epitope attached to the cytoplasmic part.

FIG. 3 shows the induction of an immunogenic agonist sequence (IAS) as described in Example 3. Specific killing (percent) is based on 5 effectors: target (E: T)
Shown for the ratio.

FIG. 4 shows a method for preparing an immunogenic agonist sequence (IAS). Wild type (WT) and mutated forms of the nucleic acid encoding the polypeptide of interest are constructed and subjected to DNA shuffling to obtain a nucleic acid encoding a polyepitope region containing a potential agonist sequence.

FIG. 5 shows a schematic diagram for improving immunostimulatory sequences by DNA shuffling.

FIG. 6 shows that a recombinant library of human IL-12 gene was screened,
FIG. 4 is an illustration of a procedure by which a shuffled IL-12 gene encoding a recombinant IL-12 with increased ability to induce T cell proliferation can be identified.

FIG. 7 shows the results of a high-throughput functional assay for a vector encoding a variant of IL-12 obtained using the method of the present invention.

FIG. 8 shows the induction of T cell proliferation upon transfection of T cells with individual vectors encoding IL-12 variants.

FIG. 9 shows the results of experiments demonstrating that shuffled IL-12 chimeras obtained using the methods of the present invention exhibit improved ability to activate human T cells.

FIG. 10 shows how T cell activation or anergy differs in B7-1
1 shows a model of whether a gene vaccine vector encoding (CD80) and / or B7-2 (CD86) variants can be induced.

FIG. 11 shows a method using DNA shuffling to obtain CD80 / CD86 variants with improved ability to induce T cell activation or anergy.

FIG. 12 shows the results obtained in a screening assay for altered function of B7.

FIG. 13 provides experimental results demonstrating that shuffled B7 chimeras provide potent T cell activation.

FIG. 14A depicts an alignment of nucleotide sequences for human and mouse IL-10 receptor sequences.

FIG. 14B depicts an alignment of nucleotide sequences for human and mouse IL-10 receptor sequences.

FIG. 14C depicts an alignment of the nucleotide sequences for the human and mouse IL-10 receptor sequences.

FIG. 14D depicts an alignment of the nucleotide sequences for human and mouse IL-10 receptor sequences.

FIG. 14E shows an alignment of nucleotide sequences for human and mouse IL-10 receptor sequences.

FIG. 14F depicts an alignment of nucleotide sequences for human and mouse IL-10 receptor sequences.

FIG. 14G depicts an alignment of nucleotide sequences for human and mouse IL-10 receptor sequences.

FIG. 14H depicts an alignment of nucleotide sequences for human and mouse IL-10 receptor sequences.

FIG. 14I depicts an alignment of nucleotide sequences for human and mouse IL-10 receptor sequences.

FIG. 15A shows an alignment of the nucleotide sequence of the B7-1 (CD80) gene from human, rhesus monkey, and rabbit.

FIG. 15B shows an alignment of the nucleotide sequence of the B7-1 (CD80) gene from humans, rhesus monkeys, and rabbits.

FIG. 15C shows an alignment of the nucleotide sequence of the B7-1 (CD80) gene from humans, rhesus monkeys, and rabbits.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/50 G01N 33/566 33/566 A61K 45/00 // A61K 45/00 C07K 14/02 C07K 14 / 02 14/705 14/705 C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC , NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (71) Applicant 3410 Central Expressway, Santa Clara, California 95051 U.S.A. S. A. (72) Inventor Stemmer, Willem P. C. United States California 95030, Los Gatos, Cathy Court 108 (72) Inventor Warren, Robert Gerald France F-75015 Paris, L'Ercourb, 332 (72) Inventor Howard, Russell United States California, Los Altos Hills, Biscaino Road 12700

Claims (20)

[Claims] 1. A method for obtaining a polynucleotide having a regulatory effect on an immune response or a polynucleotide encoding a polypeptide having a regulatory effect on an immune response, wherein the immune response comprises a gene Induced by a vaccine vector, the method comprises: creating a library of recombinant polynucleotides; and screening the library to optimize recombination with a modulating effect on the immune response induced by the genetic vaccine vector Identifying a polynucleotide or an optimized recombinant polynucleotide encoding a polypeptide having a regulatory effect, wherein the optimized recombinant polynucleotide or encoded by the recombinant polynucleotide. The polypeptide is the library Compared to non-recombinant polynucleotides to produce a show an increased ability to modulate the immune response, methods. 2. The method of claim 1, wherein said optimized recombinant polynucleotide is incorporated into a gene vaccine vector. 3. The method of claim 1, wherein said optimized recombinant polynucleotide, or a polypeptide encoded by said optimized recombinant polynucleotide, is administered in combination with a gene vaccine vector. . 4. The library of recombinant polynucleotides is selected from the group consisting of DNA shuffling, error-prone PCR, oligonucleotide-directed mutagenesis, uracil-mediated mutagenesis, and repair-deficient host mutagenesis. The method of claim 1, wherein the method is made by a process performed. 5. The method of claim 1, wherein said polynucleotide having a modulating effect on an immune response comprises: (1) at least a first and a second, involved in modulating an immune response. Creating a library of recombinant polynucleotides that recombines at least the first and second forms of nucleic acids, which encodes the nucleic acids of the form, or molecules involved in the regulation of an immune response, wherein the library comprises The first and second forms differ from each other in two or more nucleotides; and (2) the library is screened and the library is enhanced to modulate an immune response more than the form of the nucleic acid from which it was made At least one optimized recombination, which demonstrates the ability, either by the recombinant polynucleotide itself or through the encoded molecule; Identifying a polynucleotide obtained by the method. 6. The method of claim 5, wherein the method further comprises: (3) recombining a further form of the nucleic acid with at least one optimized recombinant polynucleotide. Creating a further library of recombinant polynucleotides, wherein said nucleic acid is the same or different from said first and second forms; (4) screening said further library and Identifying at least one further optimized recombinant polynucleotide that exhibits an enhanced ability to modulate an immune response over the form of the nucleic acid that generated the rally; and (5) if necessary, the further optimization Until the modified recombinant polynucleotide exhibits a further enhanced ability to modulate an immune response than the nucleic acid form from which the library was made. Repeating steps (3) and (4). 7. The optimized recombinant polynucleotide encodes a peptide or polypeptide capable of interacting with a cellular receptor involved in mediating an immune response, wherein the peptide or polypeptide is 2. The method of claim 1, which acts as an agonist or antagonist of the receptor. 8. The method of claim 7, wherein said cell receptor is a macrophage scavenger receptor. 9. The method of claim 7, wherein said cellular receptor is selected from the group consisting of a cytokine receptor and a chemokine receptor. 10. The method of claim 9, wherein said chemokine receptor is CCR6.
The method described in. 11. The method of claim 7, wherein said peptide or polypeptide mimics the activity of a natural ligand for said receptor, but does not induce immunoreactivity to said natural ligand. 12. The method of claim 7, wherein the library comprises: fusion of the encoded peptide or polypeptide with a protein displayed on the surface of a replicable genetic package. Expressing the recombinant polynucleotide so as to be produced as a product; contacting the replicable gene package with a plurality of cells presenting the receptor; and modulating an immune response mediated by the receptor Identifying cells showing
Screening method. 13. The method of claim 12, wherein said replicable genetic package is selected from the group consisting of bacteriophages, cells, spores, and viruses. 14. The method of claim 13, wherein said replicable genetic package is an M13 bacteriophage, and wherein said protein is encoded by gene III or gene VII. 15. The method further comprising introducing the optimized recombinant polynucleotide into a gene vaccine vector, and administering the vector to a mammal, wherein the peptide or polypeptide is expressed. And acting as an agonist or antagonist of the receptor.
The method described in. 16. The method, wherein the method produces the peptide or polypeptide encoded by the optimized recombinant polynucleotide, and wherein the peptide or polypeptide is introduced into a mammal in combination with a genetic vaccine vector. The method of claim 7, further comprising the step of: 17. The method of claim 7, wherein said optimized recombinant polynucleotide is inserted into a nucleotide sequence encoding an antigen of a genetic vaccine vector. 18. The method of claim 1, wherein said optimized recombinant polynucleotide encodes a costimulator. 19. The method of claim 18, wherein said costimulator is a cytokine. 20. The method of claim 1, wherein said optimized recombinant polynucleotide encodes a cytokine antagonist.