HK1068924B - Chimeric immunomodulatory compounds and methods of using the same - Google Patents

Description

Chimeric immunomodulatory compounds and methods of use thereof

Cross reference to related patent applications

The present application claims the benefit of provisional patent application No. 60/299,833 (filed on day 21/6/2001) and provisional patent application No. 60/375,253 (filed on day 23/4/2002), the entire contents of which are incorporated herein by reference for all purposes.

Technical Field

The present invention relates to chimeric immunomodulatory compounds ("CICs") comprising nucleic acid and non-nucleic acid moieties, and to the use of such compounds to modulate immune responses. The invention can be applied in the fields of biomedicine and immunology.

Background

The citation of a document in this section should not be construed as an admission that such document is prior art to the present invention.

The type of immune response elicited by infection or other antigen challenge can generally be distinguished by the subtype of T helper (Th) cells involved in the response. The Th1 subtype is responsible for classical cell-mediated functions such as delayed-type hypersensitivity and activation of Cytotoxic T Lymphocytes (CTLs), while the Th2 subtype is more potent as a helper for B-cell activation. The type of immune response to an antigen is generally influenced by the cytokines produced by the cells responding to the antigen. It is believed that the difference between the cytokines secreted by Th1 and Th2 cells may reflect the different biological functions of the two subtypes. See, for example, Romagnani (2000) Ann. 9-18.

Since the Th1 subtype secretes IL-2 and IFN-gamma which activate CTL, it is particularly suitable in response to viral infections, intracellular pathogens and tumor cells. The Th2 subtype may be more suitable for responding to free living bacterial and helminth parasites and may modulate allergic responses, since IL-4 and IL-5 are known to induce IgE synthesis and eosinophil activation, respectively. In general, Th1 and Th2 cells can secrete different patterns of cytokines, whereby one response can modulate the activity of the other response. For example, a change in Th1/Th2 balance may result in an allergic response, or, alternatively, may result in an enhanced CTL response.

It has been recognized for some time that Th 1-type immune responses can be induced in mammals by administration of certain immunomodulatory polynucleotides. Immunomodulatory polynucleotides include sequences known as immunostimulatory sequences ("ISS"), which typically comprise CG dinucleotides. See, for example, PCT publications WO98/55495, WO97/28259, U.S. Pat. Nos.6,194,388 and 6,207,646; and Krieg et al (1995) Nature 374: 546-49. For many infectious diseases, such as tuberculosis and malaria, a Th2 type response has little protective value against infection. Protein vaccines generally induce a Th2 type immune response characterized by high titers of neutralizing antibodies but lack significant cell-mediated immunity. Moreover, some types of antibody responses are not suitable for certain indications, most notably in allergy, the IgE antibody response can lead to anaphylactic shock.

In view of the need for improved immunotherapeutic approaches, there is a need to identify compounds that can be used to modulate immune responses.

DISCLOSURE OF THE INVENTION

In one aspect, the invention relates to chimeric compounds having immunomodulatory activity. The present chimeric immunomodulatory compounds ("CICs") generally contain one or more nucleic acid moieties (nucleic acid moieties) and one or more non-nucleic acid moieties (non-nucleic acid moieties). The nucleic acid moieties in a CIC comprising more than one nucleic acid moiety may be the same or different. The non-nucleic acid moieties in a CIC comprising more than one non-nucleic acid moiety may be the same or different. Thus, in one embodiment a CIC comprises two or more nucleic acid moieties and one or more non-nucleic acid spacer (spacer) moieties, wherein at least one non-nucleic acid spacer moiety is covalently linked to both nucleic acid moieties. In one embodiment, at least one nucleic acid moiety comprises the sequence 5 '-CG-3'. In one embodiment, at least one nucleic acid moiety contains the sequence 5 '-TCG-3'.

In one aspect, the invention provides a chimeric immunomodulatory compound having the formula "N₁-S₁-N₂"wherein N is₁And N₂Is part of a nucleic acid structure and S₁Is a non-nucleic acid spacer moiety, and the CIC exhibits immunomodulatory activity. In one embodiment, the core structure is "N₁-S₁-N₂-S₂-N₃", where N₃Is part of a nucleic acid structure and S₂Is part of a non-nucleic acid spacer. In one embodiment, a CIC has an "N₁-S₁-N₂-S₂-[N_v-S_v]_A"wherein A is an integer of 1 to 100, [ N ]_v-S_v]_ARepresents A additional repeats of a nucleic acid moiety conjugated to a non-nucleic acid spacer moiety, wherein S and N are at each [ N_v-S_v]All of the repetitions are independently selected. In one embodiment, a is at least 2 and at least 4 nucleic acid moieties in the CIC have different sequences.

In one aspect, CICContaining the formula N₁-S₁-N₂Or N₁-S₁-N₂-S₂-N₃Core structure of (wherein N is₁，N₂And N₃Is a nucleic acid moiety, S₁And S₂Is a non-nucleic acid spacer moiety, and S₁And S₂Covalently bound to exactly two nucleic acid moieties). Exemplary of such CIC's are those containing the formula (5' -N)₁-3’)-S₁-N₂The core structure of (c). In one embodiment, N is₁Has the sequence 5 '-TCGAX-3', wherein X is 0-20 nucleotide bases (SEQ ID NO: 1). In one embodiment, X is 0-3 nucleotide bases. In one embodiment, X is CGT. In another embodiment N ₁Has the sequence 5 '-TCGTCGA-3'. In one embodiment, the CIC has the structure N₁-S₁-N₂-S₂-[N_v-S_v]_A(wherein A is an integer of 1 to 100, [ N ]_v-S_v]_ARepresents A additional repeats of a nucleic acid moiety conjugated to a non-nucleic acid spacer moiety, wherein S and N are at each [ N_v-S_v]All of the repetitions are independently selected). In one embodiment, a is 1 to 3.

In another aspect, the invention provides a compound having the formula [ N ]_v]_A---S_pIn the core structure of (1), wherein S_pIs a moiety of a nucleic acid independently selected from the group consisting of "A", i.e., N_vA covalently bound multivalent spacer, wherein a is at least 3, and the CIC exhibits immunomodulatory activity. In one embodiment, the CIC has a core structure [ S ]_v-N_v]_A---S_pIn which S is_pIs an element independently selected from "A", i.e. [ S ]_v-N_v]Covalently bound multivalent spacer, and independently selected element [ S ]_v-N_v]Included are spacer moieties covalently bound to the nucleic acid moiety, and wherein A is at least 3. In one embodiment, a is 3 to 50. In another embodiment, a is greater than 50. In one embodiment, at least 2, 3, or all of the CICs are present in the compositionThe 4 nucleic acid moieties have different sequences.

In one aspect, the CIC contains the formula [ N ]_v]_A-S_pOr [ S ]_v-N_V]_A-S_pCore structure of (wherein S) _pIs a nucleic acid moiety N independently selected from the group consisting of_vOr an independently selected element [ S ]_v-N_v]Covalently bound multivalent spacer, each independently selected element [ S ]_v-N_v]Both contain a spacer moiety covalently bound to a nucleic acid moiety, and wherein a is at least 3). In embodiments, a is from 3 to about 50 or from about 50 to about 500. In one embodiment, S_pComprises a dendrimer (dendromer). In one embodiment, the nucleic acid moiety of the CIC has a sequence selected from the group consisting of: TCGXXXXXXX, TCGAXXX, XTCGXXX, XTCGAXX, TCGTCGA, TCGACGT, TCGAACG, TCGAGAGAT, TCGACTC, TCGAGCG, TCGATTT, TCGCTTT, TCGGTTT, TCGTTTT, TCGTCGT, ATCGTT, TTCGTTT, TTCGATT, ACGTTCG, AACGTTC, TGCGTT, TGTCGTT, TCGXXX, TCGAXXX, TCGTCG, AACGTT, ATCGAT, GTCGTT, GACGTT, TCGXX, TCGAX, TCGAT, TCGTT, TCGTC, TCGA, TCGT, TCGX, and TCG (where "X" is any nucleotide).

In another aspect, the invention provides a compound having the formula "N₁-S₁"CIC of core Structure, wherein N₁Is part of a nucleic acid structure and S₁Is a non-nucleic acid spacer moiety, and the CIC exhibits immunomodulatory activity.

CIC may comprise non-nucleotide spacer moieties comprising moieties such as triethylene glycol (triethylene glycol), hexaethylene glycol (hexaethylene glycol), polymers comprising phosphodiester and/or phosphorothioate linked oligoethylene glycol moieties, C ₂-C₁₀Alkyl (e.g., propyl, butyl, hexyl), glycerol or modified glycerols (e.g., glycerol derivatized at the 1, 2 or 3 hydroxyl positions; e.g., 3-levulinyl-glycerol), pentaerythritol or modified pentaerythritol (pentaerythritol modified at any hydroxyl position; e.g., "trebler"), glycerol derivatives, and mixtures thereof,2- (hydroxymethyl) ethyl, 1, 3-diamino-2-propanol, abasic nucleotides, polysaccharides (e.g., cross-linked polysaccharides), dendrimers, and/or other spacer moiety components disclosed herein.

In various embodiments, a CIC as described above has one or more of the following characteristics: (i) a CIC comprises at least one nucleic acid moiety that is less than 8 nucleotides (or base pairs) in length or less than 7 nucleotides in length; (ii) the length of all nucleic acid moieties in a CIC is less than 8 nucleotides, or less than 7 nucleotides; (iii) CIC includes at least one nucleic acid moiety comprising the sequence 5 '-CG-3' (e.g., 5 '-TCG-3'); (iv) the CIC comprises at least two nucleic acid moieties having different sequences; (v) all nucleic acid moieties in a CIC have the same sequence; (vi) CIC includes at least one non-nucleic acid spacer moiety that is or contains a tripethylene glycol, hexapolyethylene glycol, propyl, butyl, hexyl, glycerol or modified glycerol (e.g., glycerol derivatized at the 1, 2 or 3 hydroxyl positions; e.g., 3-levulinyl-glycerol), pentaerythritol or modified pentaerythritol (pentaerythritol modified at any hydroxyl position; e.g., "triplex"), 2- (hydroxymethyl) ethyl, 1, 3-diamino-2-propanol, abasic nucleotides, polysaccharides (e.g., cross-linked polysaccharides), or dendrimers. In some embodiments, the spacer moiety is not a polypeptide.

In various embodiments, the CIC described herein has one or more of the following characteristics: (vii) the CIC comprises at least one CIC nucleic acid moiety that does not have "independent immunomodulatory activity"; (viii) CIC does not include nucleic acid moieties with "independent immunomodulatory activity"; (ix) CIC comprises at least one CIC nucleic acid moiety having "sub-independent immunological activity". "independent immunomodulating activity" and "sub-independent immunological activity" are described herein. In various embodiments herein the CIC comprises at least one nucleic acid moiety that is double-stranded or partially double-stranded. CICs having self-complementary nucleic acid moieties can be designed so as to form a double helix. See, for example, C-84, C-85, and C-87.

Thus, in various aspects, the present invention provides a CIC comprising two or more nucleic acid moieties and one or more non-nucleic acid spacer moieties, wherein at least one spacer moiety is covalently linked to both nucleic acid moieties and at least one nucleic acid moiety comprises the sequence 5 '-CG-3', and wherein the CIC has immunomodulatory activity. The CIC may comprise at least three nucleic acid moieties, wherein each nucleic acid moiety is covalently linked to at least one non-nucleic acid spacer moiety. The CIC may have at least one immunomodulatory activity, such as (a) the ability to stimulate IFN- γ production by human peripheral blood mononuclear cells; (b) the ability to stimulate IFN- α production by human peripheral blood mononuclear cells; and/or (c) the ability to stimulate human B cell proliferation.

One or more nucleic acid moieties in a CIC may comprise a sequence such as 5 '-TCGA-3', 5 '-TCGACGT-3', 5 '-TCGTCGA-3' and 5 '-ACGTTCG-3'. In one embodiment, one or more nucleic acid moieties in a CIC may have the sequence 5' -X₁X₂CGX₃X₄-3' (where X is₁Is 0 to 10 nucleotides; x₂Is empty or is A, T or U; x₃Is a vacancy or A; x₄Is 0 to 10 nucleotides; and wherein the nucleic acid moiety is conjugated to the spacer moiety, such as at the 3' terminus). In one embodiment, X₁、X₂、X₃And X₄The total number of nucleotides in (a) may be less than 8, less than 7, less than 6, less than 5, or less than 4. In some embodiments, one or more nucleic acid moieties in a CIC may have a nucleic acid sequence such as the following: TCGXXXXXXX, TCGAXXX, XTCGXXX, XTCGAXX, TCGTCGA, TCGACGT, TCGAACG, TCGAGAGAT, TCGACTC, TCGAGCG, TCGATTT, TCGCTTT, TCGGTTT, TCGTTTT, TCGTCGT, ATCGTT, TTCGTTT, TTCGATT, ACGTTCG, AACGTTC, TGCGTT, TGTCGTT, TCGXXX, TCGAXXX, TCGTCG, AACGTT, ATCGAT, GTCGTT, GACGTT, TCGXX, TCGAX, TCGAT, TCGTT, TCGTC, TCGA, TCGT, TCGX, or TCG (where "X" is any nucleotide).

At one endIn one embodiment, one or more nucleic acid moieties comprises 3 to 7 bases. In one embodiment, the nucleic acid moiety comprises 3 to 7 bases and has the sequence 5' - [ (X)_0-2]TCG[(X)_2-4]-3 ', or 5' -TCG [ (X)_2-4]-3 ', or 5' -TCG (A/T) [ (X)_1-3]-3 ', or 5 ' -TCG (A/T) CG (A/T) -3 ', or 5 ' -TCGACGT-3 ' or 5 ' -TCGTCGA-3 ', wherein each X is an independently selected nucleotide. In some embodiments, a CIC comprises at least 3, at least 10, at least 30, or at least 100 nucleic acid moieties having a sequence as described above.

The CIC may comprise at least one nucleic acid moiety that is less than 8 nucleotides in length. Optionally all nucleic acid moieties in a CIC are less than 8 nucleotides long. In some embodiments, all nucleic acid moieties in the CIC comprising the sequence 5 '-CG-3' are less than 8 nucleotides long. The CIC may comprise at least two nucleic acid moieties having different sequences. The CIC may comprise at least one nucleic acid moiety not comprising the sequence 5 '-CG-3'. The CIC may comprise at least one nucleic acid moiety that has no independent immunological activity or has a sub-independent immunological activity. Optionally, none of the nucleic acid moieties in the CIC has independent immunomodulatory activity. The linkage between nucleotides in a nucleic acid moiety can include phosphodiester bonds, phosphorothioate bonds, phosphorodithioate bonds, other covalent bonds, and mixtures thereof. Likewise, the linkage between the nucleic acid moiety and the spacer moiety or between the constituents of the spacer moiety may include phosphodiester linkages, phosphorothioate linkages, phosphorodithioate linkages, other linkages, and mixtures thereof.

In one embodiment, the CIC includes a reactive linking group (e.g., a reactive sulfur-containing group). CIC may be bound or non-covalently bound to a polypeptide, such as a polypeptide antigen.

The invention also provides a composition comprising CIC and a pharmaceutically acceptable excipient and/or an antigen and/or a cationic microcarrier (such as a polymer of lactic acid and glycolic acid). The composition may be substantially free of endotoxins.

In one aspect, the invention provides a composition comprising a CIC as described herein and a pharmaceutically acceptable excipient, an antigen (e.g., an antigen against which an immune response is desired), or both. In one embodiment, the composition is formulated according to GMP standards. In one embodiment, the composition is prepared by a method comprising detecting the presence of endotoxin in the composition. In one embodiment, the composition is substantially free of endotoxin. In one embodiment, the composition is free of liposomes.

In one aspect, the invention provides the use of a CIC as described herein in the manufacture of a medicament.

In one aspect, the invention provides a method of modulating an immune response in an individual by administering a chimeric immunomodulatory compound or composition described herein, wherein the amount administered is an amount sufficient to modulate the immune response in the individual. In one embodiment, the individual has a disorder associated with a Th2 type immune response, such as allergy or allergy-induced asthma. In one embodiment, the subject has an infectious disease.

In one aspect, the invention provides a method of increasing interferon gamma (IFN- γ) in an individual by administering a CIC or composition described herein, wherein the amount administered is an amount sufficient to increase IFN- γ in the individual. In one embodiment, the subject has inflammation. In one embodiment, the individual has idiopathic pulmonary fibrosis.

In one aspect, the invention provides a method of increasing interferon alpha (IFN- α) in an individual by administering a CIC or composition described herein in an amount sufficient to increase IFN- α in the individual. In one embodiment, the subject has a viral infection.

In one aspect, the invention provides a method of ameliorating a symptom of an infectious disease in a subject by administering to the subject an effective amount of a CIC or composition described herein, wherein the effective amount is an amount sufficient to ameliorate the symptom of the infectious disease.

In one aspect, the invention provides methods of ameliorating an IgE-related disorder in a subject by administering to the subject having the IgE-related disorder an effective amount of a CIC or composition described herein, wherein the effective amount is an amount sufficient to ameliorate symptoms of the IgE-related disorder. In one embodiment, the IgE-associated disorder is an allergy or allergy-associated disorder.

The invention also provides a method of modulating an immune response in an individual by administering to the individual a CIC, wherein the amount administered is an amount sufficient to modulate the immune response in the individual. In embodiments, the individual has cancer and/or has a Th 2-type immune response-related disorder (e.g., allergy or allergy-induced asthma) and/or has an infectious disease.

Brief Description of Drawings

FIG. 1 shows the structure of certain agents that may be used to synthesize non-nucleic acid spacer moieties of CIC. Shown are dimethoxytrityl protected phosphoramidite spacer moiety precursors for HEG, propyl, TEG, HME, butyl and abasic spacer moieties.

FIG. 2 shows the structure of certain agents that may be used to synthesize a symmetrical or asymmetrical CIC non-nucleic acid spacer moiety. Shown are dimethoxytrityl-protected phosphoramidite spacer moiety precursors for glycerol [2] ("symmetric branching"), levulinyl-glycerol [3] ("asymmetric branching"), "triplex" [9], and "symmetric doublet" (doubler) "[ 10] spacer moieties.

FIGS. 3A and 3B illustrate the synthesis of branched CIC.

FIG. 4 shows the synthesis scheme for C-105.

FIG. 5 shows the induction of immune-related genes in mouse lungs following intranasal treatment with CIC.

FIGS. 6A-C show the effect of CIC on IL-12 p40 (FIG. 6A), IL-6 (FIG. 6B), and TNF-. alpha.levels (FIG. 6C).

FIGS. 7A-B show the structures of C-8 (FIG. 7A) and C-101 (FIG. 7B).

EMBODIMENTS FOR CARRYING OUT THE INVENTION

I. General procedure

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are within the skill of the art. These techniques are well described in the literature, such as molecular cloning: a Laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989) and Molecular Cloning: a laboratory manual, third edition (Sambrook and Russel, 2001), (referred to herein collectively and individually as Sambrook "); oligonucleotide Synthesis (Oligonucleotide Synthesis) (m.j. gate, ed., 1984); animal cell Culture (animal cell Culture) (r.i. freshney, ed., 1987); a Handbook of experimental Immunology (d.m. well & c.c. blackwell, eds); gene transfer Vectors for Mammalian Cells (Gene Transer Vectors for Mammalian Cells) (J.M.Miller & M.P.Calos, eds., 1987); modern Molecular Biology methods (CurrentProtocols in Molecular Biology) (f.m. ausubel et al, eds., 1987, including suppl-ups up to 2001); and (3) PCR: polymerase chain reaction (PCR: The Polymerase chain reaction), (Mullis et al, eds., 1994); modern immunological methods (Cnrrent Protocols in immunology) (j.e. colligan et al, eds., 1991); a manual of immunization experiments (the Immunoassay Handbook) (D.wild, ed., Stockton Press NY, 1994); bioconjugate Techniques (Bioconjugate Techniques) (Greg t. hermanson, ed., academic press, 1996); immunoassay method (Methods of Immunological Analysis) (R.Masseyeff, W.H.Albert, and N.A.Staines, eds., Weinheim: VCH Verlagsgesellschaft mbH, 1993); harlow and Lane (1988) Antibodies, Laboratory manuals (Antibodies, A Laboratory Manual), Cold Spring Harbor Publications, New York, and Harlow and Lane (1999) utilize Antibodies, Laboratory manuals (Usingantibodies: A Laboratory Manual), Cold Spring Harbor Press, Cold Spring Harbor, NY (referred to herein collectively and individually as "Harlow and Lane"); beaucage et al eds, modern Nucleic acid chemistry (Current Protocols in Nucleic acid chemistry) John Wiley & SohS, inc., New York, 2000); and Agrawal, ed., methods for oligonucleotides and Analogs, Synthesis and Properties (Protocols for oligonucleotides and Analogs, Synthesis and Properties) Humana Press inc., New Jersey, 1993).

Definition of

As used herein, the singular forms "a", "an" and "the" include the plural forms unless otherwise indicated herein or otherwise evident from the context. For example, it will be apparent from the context that a "chimeric immunomodulatory compound (" CIC ") may comprise one or more CIC. Likewise, a CIC constituent element (i.e., a nucleic acid moiety or a non-nucleic acid spacer moiety) referred to in the singular may comprise a plurality of elements. For example, the expression "nucleic acid moiety" in a CIC may also denote two or more "nucleic acid moieties" in a CIC.

The terms "polynucleotide", "oligonucleotide" and "nucleic acid" used interchangeably herein include single-stranded dna (ssdna), double-stranded dna (dsdna), single-stranded rna (ssrna) and double-stranded rna (dsrna), modified oligonucleotides and oligonucleotides or combinations thereof. The nucleic acid may be in a linear or circular configuration, or the oligonucleotide may comprise both linear and circular segments. Nucleic acids are polymers of nucleosides, wherein the nucleosides are linked by, for example, phosphodiester bonds or other linkages, such as phosphorothioate linkages. Nucleosides consist of a purine (adenine (a) or guanine (G) or derivatives thereof) or pyrimidine (thymine (T), cytosine (C) or uracil (U) or derivatives thereof) base bonded to a sugar. These four nucleoside units (or bases) are called deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine in DNA. Nucleotides are phosphate esters of nucleosides.

The term "3" generally refers to a region or position in a polynucleotide or oligonucleotide that is 3' (downstream) of another region or position in the same polynucleotide or oligonucleotide.

The term "5" generally refers to a region or position in a polynucleotide or oligonucleotide that is 5' (upstream) of another region or position in the same polynucleotide or oligonucleotide.

A region, portion (part), non-nucleic acid spacer moiety, nucleic acid moiety or sequence is "adjacent" when one element (e.g., the region, portion (part), non-nucleic acid spacer moiety, nucleic acid moiety or sequence) is directly adjacent to another element (e.g., the region, portion (part), non-nucleic acid spacer moiety, nucleic acid moiety or sequence).

The term "CIC-antigen conjugate" refers to a complex in which CIC and antigen are linked. The linkage of the conjugates includes covalent and/or non-covalent linkages.

The term "antigen" refers to a substance that is specifically recognized and bound by an antibody or T cell antigen receptor. Antigens may include peptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids, and phospholipids; portions and combinations thereof. Antigens may be natural or synthetic. Antigens suitable for administration with CIC include any molecule capable of eliciting a B cell or T cell antigen-specific response. Preferably, the antigen is capable of eliciting an antibody response specific for the antigen. Haptens are included within the scope of "antigens". A hapten is a low molecular weight compound that is not immunogenic by itself, but which is immunogenic when conjugated to an immunogenic molecule containing an antigenic determinant. Haptenation of small molecules may be required in order to confer antigenicity. Preferably, the antigens of the invention include peptides, lipids (such as sterols, fatty acids and phospholipids), polysaccharides (such as those used in haemophilus influenzae (Hemophilus influenza) vaccines), gangliosides and glycoproteins.

"adjuvant" refers to a substance that, when added to an immunogenic agent, such as an antigen, nonspecifically enhances or potentiates the immune response to the immunogenic agent in a recipient host following exposure to the mixture.

The term "peptide" is a polypeptide of sufficient length and composition to effect a biological response, such as antibody production or cytokine activity, regardless of whether the peptide is a hapten or not. Typically, peptides are at least 6 amino acid residues in length. The term "peptide" also includes modified amino acids (naturally or non-naturally occurring), such modifications including, but not limited to, phosphorylation, glycosylation, pegylation, lipidation, and methylation.

"antigenic peptides" may include purified natural peptides, synthetic peptides, recombinant peptides, crude peptide extracts, or peptides in a partially purified or unpurified active state (e.g., peptides that are part of an attenuated or inactivated virus, cell, microorganism), or fragments of such peptides. An "antigenic peptide" or "antigenic polypeptide" thus refers to all or part of a polypeptide exhibiting one or more antigenic properties. Thus, for example, an "Amb a1 antigen polypeptide" or "Amb a1 polypeptide antigen" is an amino acid sequence from Amb a1 that exhibits antigenic properties (i.e., specifically binds to an antibody or T cell receptor), whether it is a full-length sequence, a portion of a sequence, and/or a modification of a sequence.

A "delivery molecule" or "delivery vehicle" is a chemical moiety that facilitates, allows, and/or increases the delivery of a CIC, CIC-antigen mixture, or CIC-antigen conjugate to a specific site and/or at a specific time. The delivery vehicle may or may not additionally stimulate an immune response.

"allergy to antigen" refers to an immune response generally characterized by the production of eosinophils (often in the lung) and/or antigen-specific IgE and the effects that they cause. As is well known in the art, IgE binds to IgE receptors on mast cells and basophils. When subsequently exposed to an antigen recognized by IgE, the antigen will crosslink with IgE on mast cells and basophils, causing these cells to degranulate, including but not limited to the release of histamine. It is to be understood and appreciated that in the application of some methods of the present invention, the terms "allergy to antigen", "allergy" and "allergic disease" apply equally. Moreover, it is to be understood and appreciated that the methods of the present invention include those that are equally applicable to the prevention of allergies and the treatment of existing allergic diseases.

As used herein, the term "allergen" refers to an antigenic portion of an antigen or molecule, typically a protein, that can cause an allergic response upon exposure to a subject. Typically the subject is allergic to an allergen, which may be indicated, for example, by the blister and flush test or any method known in the art. A molecule is also referred to as an allergen even if only a small population of subjects exhibit an allergic (e.g. IgE) immune response upon exposure to the molecule. Many isolated allergens are well known in the art. These include, but are not limited to, the allergens provided in table 1 herein.

The term "desensitization" refers to a method of administering increasing doses of an allergen to a subject exhibiting sensitivity to the allergen. Examples of allergen doses for desensitization are well known in the art, see e.g. Fornadley (1998) otolaryngol. 111-127.

"antigen-specific immunotherapy" refers to any form of immunotherapy involving an antigen and producing an antigen-specific modulation of the immune response. In allergy, antigen-specific immunotherapy includes, but is not limited to, desensitization therapy.

The term "microcarrier" refers to a water-insoluble particulate composition having a size of less than about 150, 120 or 100 μm, more typically less than about 50-60 μm, and may be less than about 10 μm or even less than about 5 μm. Microcarriers include "nanocarriers," which are microcarriers that are less than about 1 μm in size, preferably less than about 500 nm. Microcarriers include solid phase particles such as particles formed from biocompatible natural polymers, synthetic polymers or synthetic copolymers, but microcarriers as defined herein and other biodegradable materials known in the art may or may not include microcarriers formed from agarose or cross-linked agarose. The solid phase microcarriers are formed from polymers or other materials that are not erodible and/or degradable under mammalian physiological conditions, such as polystyrene, polypropylene, silica, ceramics, polyacrylamide, gold, latex, hydroxyapatite, and ferromagnetic and paramagnetic materials. Biodegradable solid phase microcarriers can be formed from polymers that are degradable under mammalian physiological conditions (e.g., poly (lactic acid), poly (glycolic acid), and copolymers thereof, such as poly (D, L-lactide-co-glycolide)) or erodible polymers (e.g., poly (orthoesters such as 3, 9-diethylene-2, 4, 8, 10-tetraoxaspiro [5.5] undecane (DETOSU)), or poly (anhydrides), such as poly (anhydrides) of sebacic acid). Microcarriers can also be liquid phases (e.g., oil or ester based), such as liposomes, antigen-free iscom (an immunostimulatory complex that is a stable complex of cholesterol, phospholipids and saponins with adjuvant activity), or microdroplets or micelles in oil-in-water or water-in-oil emulsions. Biodegradable liquid phase microcarriers are typically incorporated into biodegradable oils, some of which are well known in the art, including squalene and vegetable oils. Microcarriers are generally spherical, but microcarriers that deviate from spherical are also acceptable (e.g., oval, rod-like, etc.). In view of the insolubility of the microcarrier, the solid phase microcarrier can be filtered from water and water-based (aqueous) solutions (e.g., using a 0.2 micron filter).

The term "non-biodegradable" as used herein refers to a microcarrier that is not degraded or eroded under normal mammalian physiological conditions. In general, a microcarrier is considered to be non-biodegradable if it is not degraded (i.e. its loss of mass or average polymer length is less than 5%) after incubation at 37 ℃ for 72 hours in normal human serum.

A microcarrier is considered "biodegradable" if it is degradable or erodible under normal mammalian physiological conditions. In general, a microcarrier is considered biodegradable if it is degraded (i.e. at least 5% of its mass or average polymer length is lost) after incubation at 37 ℃ for 72 hours in normal human serum.

The term "CIC/microcarrier complex" or "CIC/MC complex" refers to a complex of CIC and microcarriers. The components of the complex may be covalently or non-covalently linked. Non-covalent attachment may be mediated by any non-covalent binding force, including hydrophobic interactions, ionic (electrostatic) bonds, hydrogen bonds, and/or van der waals attraction. When hydrophobic, the attachment is typically achieved by a hydrophobic moiety (e.g., cholesterol) covalently attached to the CIC.

An "individual" or "subject" is a vertebrate, such as a bird, and preferably a mammal, such as a human. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, laboratory animals, rodents (e.g., rats and mice), and pets.

An "effective amount" or "sufficient amount" of a substance is an amount sufficient to achieve a desired biological effect, such as a beneficial result (including a clinical result), and thus an "effective amount" depends on the context in which it is used. In the case of administration of a composition that modulates an immune response to a co-administered antigen, the effective amounts of the CIC and antigen are amounts sufficient to achieve a modulation that is comparable to the immune response achieved when the antigen is administered alone. An effective amount may be administered in one or more administrations.

The term "co-administration" as used herein refers to the administration of at least two different substances in sufficiently close time proximity to modulate an immune response. Preferably, co-administration refers to the simultaneous administration of at least two different substances.

By "stimulating" an immune response, such as a Th1 response, is meant enhancing the response, which may result from eliciting the response and/or boosting the response. Similarly, "stimulating" a cytokine or cell type (e.g., CTL) refers to increasing the amount or level of the cytokine or cell type.

An "IgE-related disorder" is a physiological disease characterized in part by elevated IgE levels, which may or may not be persistent. IgE-related disorders include, but are not limited to, allergies and allergies, allergy-related disorders (as described below), asthma, rhinitis, conjunctivitis, rubella, shock, allergies and drug allergies caused by stings from hymenoptera insects, and parasitic infections. The term also includes the associated manifestations of these disorders. Typically, IgE is antigen-specific in such disorders.

"allergy-related disorder" refers to a disease resulting from the immune response to antigen-specific IgE. Such results may include, but are not limited to, hypotension and shock. Allergy is an example of an allergy-related condition, during which histamine is released into the circulation, causing vasodilation and increased capillary permeability, resulting in a significant loss of plasma in the circulation. Allergic reactions can occur systemically, where the entire body experiences the associated effects, or locally, where the reaction is confined to a specific target tissue or organ.

The term "viral disease" as used herein refers to a disease in which a virus acts as a causative agent. Examples of viral diseases include hepatitis b, hepatitis c, influenza, acquired immunodeficiency syndrome (AIDS), and herpes zoster.

As used herein and well known in the art, "treatment" is a process for obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilization (i.e., not aggravating) of the state of the disease, prevention of spread of the disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total) of the disease, whether detectable or undetectable. "treatment" may also refer to an extended survival compared to the expected survival in the absence of such treatment.

"alleviating" a disease or disorder refers to a reduction in the extent and/or unwanted clinical manifestations of the disease or condition and/or a slowing or lengthening of the time course of disease progression as compared to not treating the disease. Especially in allergy, as is well known to the person skilled in the art, the reduction may occur by modulating the immune response against the allergen. Moreover, the relief does not necessarily occur after administration of one dose, it often occurs after administration of a series of doses. Thus, an amount sufficient to alleviate a response or condition may be administered one or more times.

The "antibody titer" or "antibody amount" that is "elicited" by CIC and antigen refers to the amount of the specified antibody measured at a certain point in time after administration of CIC and antigen.

A "Th 1-associated antibody" is an antibody whose production and/or increase is associated with a Th1 immune response. For example, IgG2a is a mouse Th1 related antibody. For the purposes of the present invention, an assay for a Th 1-related antibody can be an assay for one or more such antibodies. As in humans, the determination of Th 1-related antibodies may require the determination of IgG1 and/or IgG 3.

A "Th 2-associated antibody" is an antibody whose production and/or increase is associated with a Th2 immune response. For example, IgG1 is a mouse Th 2-related antibody. For the purposes of the present invention, an assay for a Th 2-related antibody can be an assay for one or more such antibodies. As in humans, the determination of Th 2-related antibodies may require the determination of IgG2 and/or IgG 4.

By "inhibiting" or "suppressing" a function or activity, such as cytokine production, antibody production, or histamine release, is meant a decrease in the function or activity when compared to the same condition or alternatively to another condition other than the condition or parameter of interest. For example, a composition comprising CIC and an antigen that inhibits histamine release may reduce histamine release compared to histamine release as induced by the antigen alone. As another example, a composition comprising CIC and antigen that inhibits antibody production may reduce the extent and/or level of antibody compared to the extent and/or level of antibody produced as with antigen alone.

As used herein, "prepared or formulated in accordance with GMP standards" when referring to pharmaceutical compositions means that the compositions are formulated to be sterile, isotonic, and to fully comply with all manufacturing quality management (GMP) regulations of the U.S. food and drug administration.

The term "immunogenic" as used herein has its ordinary meaning in the art and refers to an agent (e.g., a polypeptide) that elicits an adaptive immune response when injected into the human or animal body. The immune response may be B-cell (humoral) and/or T-cell (cellular).

All ranges are intended to include terminal values. Thus, a polymer having "2 to 7 nucleotides" or having "between 2 and 7 nucleotides" includes a polymer having 2 nucleotides and a polymer having 7 nucleotides. When referring to a lower limit and an independently selected upper limit, it is understood that the upper limit is higher than the lower limit.

Chimeric immunomodulatory compounds

The present invention provides chimeric immunomodulatory compounds ("CICs") that are useful, inter alia, for modulating immune responses in individuals, such as mammals including humans. The present invention is based, in part, on the discovery that certain chimeric molecules comprising a nucleic acid moiety covalently bound to a non-nucleic acid spacer moiety have immunomodulatory activity, particularly in human cells. Surprisingly, this activity is evident even when the nucleic acid moiety has a sequence that does not exhibit significant immunomodulatory activity when provided as a separate polynucleotide.

Accordingly, the present invention provides novel agents and methods for modulating immune responses, including the treatment and prevention of diseases in humans and other animals.

The following sections describe the structure and properties of the CIC of the invention, as well as the structure and properties of its components, the nucleic acid moieties and the non-nucleic acid spacer moieties.

Core Structure of CIC

The CIC of the invention comprises one or more nucleic acid moieties and one or more non-nucleic acid spacer moieties. CICs having various structures are contemplated. For example, exemplary CICs have core structures represented by formulas I-VII below. Formulas I-III show the core sequence for "Linear CIC". Formulas IV-VI show the core sequences for "branched CIC". Formula VII shows the core structure for "single spacer CIC".

In each of the formulae below, "N" represents a nucleic acid structural moiety (oriented 5'→ 3 ' or 3 ' → 5 ' direction) and "S" represents a non-nucleic acid spacer moiety. A single dash ("-") indicates a covalent bond formed between one nucleic acid moiety and one non-nucleic acid spacer moiety. A double-dashed line ("-") indicates a covalent bond formed between one non-nucleic acid spacer moiety and at least two nucleic acid moieties. A triple line ("- - -") indicates a covalent bond formed between one non-nucleic acid spacer moiety and a plurality (i.e., at least 3) of nucleic acid moieties. Subscripts are used to indicate nucleic acid moieties or non-nucleic acid spacer moieties at different positions. However, the different nucleic acid moieties distinguished by subscripts do not indicate that the nucleic acid moieties necessarily have different structures or sequences. Likewise, different spacer moieties distinguished by subscripts does not indicate that these spacer moieties necessarily have different structures. As in formula II below, N₁And N₂The nucleic acid moieties represented may have the same or different sequences, S₁And S₂The spacer structure portions represented may have the same or different structures.

A. Linear CIC

In one embodiment, the CIC comprises a core structure

N₁-S₁-N₂ (I)。

In one embodiment, the CIC comprises a core structure

N₁-S₁-N₂-S₂-N₃ (II)。

In one embodiment, the CIC comprises a core structure

N₁-S₁-N₂-S₂-[N_v-S_v]_A (III)，

Wherein A is an integer from 1 to about 100, [ N_v-S_v]Represents A times of a nucleic acid moiety conjugated with a non-nucleic acid spacer moietyAdditional iterations are performed. The subscript "v" indicates that N and S are each "[ N ]_v-S_v]"is independently selected. "A" is sometimes 1 to about 10, sometimes 1 to 3, sometimes exactly 1, 2, 3, 4 or 5. In some embodiments, a is an integer in a range having a lower limit of 1, 2, 3, 4, or 5 and an independently selected upper limit of 10, 20, 50, or 100 (e.g., 3 to 10).

In some embodiments of the invention, the CIC has a structure as shown in formula I, II or III. However, in some embodiments according to the present invention, linear CICs comprise, but are not necessarily limited to, the structures provided by formulas I-III. That is, formulae I, II and III define a core structure in which a non-nucleic acid spacer moiety is covalently bound to no more than two nucleic acid moieties. However, in many embodiments, it is contemplated that additional chemical moieties (e.g., phosphates, mononucleotides, additional nucleic acid moieties, alkyl groups, amino groups, sulfur-or disulfide groups or linking groups, and/or spacer moieties) may be covalently attached to the end of the core structure. For example, if all of the nucleic acid moieties in a CIC are 5 '-TCGTCGA-3' and the spacer moiety is selected from the group consisting of hexapolyethylene glycol ("HEG"), phosphorothioate-linked HEG multimers, and glycerol, a CIC having a core structure as shown in formula I includes all of the following formulae:

TCGTCGA-HEG-TCGTCGA-OH (Ia)

TCGTCGA-HEG-TCGTCGA-PO₄ (Ib)

TCGTCGA-HEG-TCGTCGA-HEG (Ic)

HFG-TCGTCGA-HEG-TCGTCGA-HEG (Id)

TCGTCGA-HEG-TCGTCGA-HEG-TCGTCGA (Ie)

TCGTCGA-HEG-TCGTCGA-(HEG)₄-TCGTCGA (If)

(TCGTCGA)₂-Glycerol-TCGTCGA-HEG-TCGTCGA (Ig)

PO₄-TCGTCGA-HEG-TCGTCGA (Ih)

TCGTCGA-(HEG)₁₅-T (Ii)

(TCGTCGA-HEG)₂-Glycerol-HEG-TCGTCGA (Ij)

TCGTCGA-HEG-T-HEG-T (Ik)

It is obvious that such CIC containing a core structure as shown in formula I includes CIC containing a core structure as shown in formula II or III.

In some embodiments, one or more of the spacers comprise smaller units (e.g., HEG, TEG, glycerol, C3 alkyl, etc.) linked together. In one embodiment, the linkage is an ester linkage (e.g., phosphodiester or phosphorothioate) or other linkage, as described below.

In certain embodiments, the terminal structures of the CIC are covalently linked together (e.g., nucleic acid moiety to nucleic acid moiety; spacer moiety to spacer moiety, or nucleic acid moiety to spacer moiety) to form a cyclic conformation.

B. Branch CIC

In one embodiment, the CIC comprises a core structure

[N_v] _A---S_p (IV)，

Wherein S_pIs a nucleic acid moiety N independently selected from the group consisting of "A_vA covalently bound multivalent spacer, and a is at least 3, such as exactly 3, 4, 5, 6, or 7, or greater than 7. In various embodiments, a is an integer from 3 to 100 (including 3 and 100). In some embodiments, a is an integer in a range having a lower limit of about 3, 5, 10, 50, or 100 and an independently selected upper limit of about 5, 7, 10, 50, 100, 150, 200, 250, or 500. Also some can be considered In embodiments, a is greater than about 500.

In a related embodiment, the CIC comprises a core structure

[S_v-N_v]_A---S_P (V)，

Wherein S_pIs an element independently selected from "A", i.e. S_v-N_v(comprising a spacer moiety covalently bound to a nucleic acid moiety) a covalently bound multivalent spacer, and a is at least 3. In various embodiments, a is an integer from 3 to 100 (including 3 and 100). In some embodiments, a is an integer in a range having a lower limit of 5, 10, 50, or 100 and an independently selected upper limit of 10, 50, 100, 250, or 500. It is also contemplated that in some embodiments, a is greater than 500. In a related embodiment, the CIC comprises a core structure:

(S₁-N₁)-S_p--(N_v)_A (VI)

wherein S_pIs a nucleic acid moiety N independently selected from the group consisting of "A_vAnd at least one sum spacer moiety S₁Conjugated nucleic acid moieties N₁A covalently bound multivalent spacer, and wherein a is at least 2. In one embodiment, a is 2. In various embodiments, a is 3, 4, 5, or an integer from 3 to 100 (including 3 and 100). In some embodiments, a is an integer in a range having a lower limit of 5, 10, 50, or 100 and an independently selected upper limit of 10, 50, 100, 150, 200, 250, or 500. It is also contemplated that in some embodiments, a is greater than 500. In some embodiments of the invention, the CIC has a structure as shown in formula I, II or III. However, according to the present invention, a branched CIC may comprise, but is not limited to, the structures provided by formulas IV, V and VI. That is, formulae IV, V and VI define a core structure in which one multivalent spacer moiety (Sp) is covalently bound to at least three (3) nucleic acid moieties. It is contemplated that, in some embodiments, additional chemical moieties (e.g., phosphates, mononucleotides, additional nuclei) Acid and/or spacer moieties) are covalently bound at the ends of the core structure. For example, if all of the nucleic acid moieties in a CIC are 5 '-TCGTCGA-3' and all of the spacer moieties are glycerol or HEG, a CIC having a core structure as shown in formula IV would include:

(TCGTCGA)₂-Glycerol-TCGTCGA (IVa)

(TCGTCGA-HEG)₂-Glycerol-TCGTCGA (IVb)

(TCGTCGA-HEG-TCGTCGA)₂-Glycerol-TCGTCGA (IVc)

[(TCGTCGA)₂-Glycerol-TCGTCGA]₂-Glycerol-TCGTCGA (IVd)

It is clear that, for example, such CICs having a core structure as shown in formula IV include CICs having a core structure as shown in formula V or VI. In a preferred embodiment of the invention, the CIC comprises at least two nucleic acid moieties which are different (i.e.of different sequence).

In some embodiments, one or more of the spacers comprise smaller units (e.g., HEG, TEG, glycerol, C3 alkyl, etc.) linked together. In one embodiment, the linkage is an ester linkage (e.g., a phosphodiester or phosphorothioate).

C. CIC with single spacer

In a different aspect of the invention, a CIC contains a structure in which there is a single nucleic acid moiety covalently conjugated to a single spacer moiety, i.e.,

N₁-S₁ (VII)

In one embodiment, S₁Having a structure of multimer (multimer) comprising a plurality of smaller units (e.g., HEG, TEG, glycerol, 1 ', 2' -dideoxyribose, C2 alkyl to C12 alkyl subunits, etc.), whereinThe smaller units are typically linked by ester linkages (e.g., phosphodiesters or phosphorothioates), as described below. See formula VIIa, shown below, for example. The multimer may be a heteropolymer (heteromer) or a homopolymer (homomer). In one embodiment, the spacer is a heteropolymer of monomeric units (e.g., HEG, TEG, glycerol, 1 ', 2' -dideoxyribose, C2 alkyl to C12 alkyl linkers, etc.) joined by ester linkages (e.g., phosphodiesters or phosphorothioates). See formula VIIb, shown below, for example.

For example, if the nucleic acid moiety is 5 '-TCGTCGA-3' and the spacer moiety is a phosphorothioate-linked polymer of hexapolyethylene glycol [ "(HEG)₁₅”]Then, a CIC having a core structure as shown in formula VII includes:

TCGTCGA-(HEG)₁₅ (VIIa)

similarly, if the nucleic acid moiety is 5 '-TCGTCGA-3' and the spacer moiety is a multimer of alternating hexapolyethylene glycol and propylene subunits linked by phosphorothioate linkages, a CIC having a core structure as shown in formula VI comprises:

TCGTCGA-HEG-propyl-HEG (VIIb).

Immunomodulatory Activity of CIC

The CIC of the invention has immunomodulatory activity. The terms "immunomodulation," "immunomodulating activity," or "modulating an immune response" as used herein include immunostimulatory and immunosuppressive effects. An immune response that is immunomodulated according to the present invention will generally shift toward a "Th 1-type" immune response and away from a "Th 2-type" immune response. It is generally accepted that a Th1 type response is a cellular immune system (e.g., cytotoxic lymphocytes) response, whereas a Th2 type response is generally a "humoral" or antibody-based response. The Th 1-type immune response is generally characterized by "delayed-type hypersensitivity" reactions to antigens. Th1 type responses can be detected at a biochemical level by increased levels of Th1 associated cytokines (e.g., IFN-. gamma., IFN-. alpha., IL-2, IL-12 and TNF-. beta., and IL-6, although IL-6 may also be associated with a Th2 type response). Th 2-type immune responses are often associated with high levels of antibody production, including IgE production, no or low CTL production, and expression of Th 2-associated cytokines such as IL-4 and IL-5.

The immunomodulation of the present invention can be recognized by in vitro (in vitro), in vivo (in vivo) and/or ex vivo (ex vivo) assays (assays). Exemplary measurable immune responses capable of indicating immunomodulatory activity include, but are not limited to, production of antigen-specific antibodies, secretion of cytokines, activation or expansion of lymphocyte populations such as NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, and the like. See, for example, WO 97/28259; WO 98/16247; WO 99/11275; krieg et al (1995) Nature 374: 546-549; yamamoto et al (1992) J.Immunol.148: 4072-; ballas et al (1996) J.Immunol.157: 1840-; klinman et al (1997) J.Immunol.158: 3635-3639; sato et al (1996) Science 273: 352 and 354; pisetsky (1996) j.immunol.156: 421-; shimada et al (1986) Jpn.J. cancer Res.77: 808-; cowdery et al (1996) J.Immunol.156: 4570-4575; roman et al (1997) Nat Med.3: 849-54; lipford et al (1997) eur.j.immunol.27: 2340-; WO98/55495, WO00/61151, Pichyangkul et al (2001) J.Imm.Methods 247: 83-94. See also the examples below. For purposes of illustration and not limitation, certain useful assays are described below.

Assays are generally performed by contacting cells, tissues, animals, etc. with or administering test samples (e.g., test samples comprising CIC, polynucleotides, and/or other agents) to them and measuring responses. The test sample containing CIC or polynucleotide may be in a variety of different forms or concentrations, as deemed appropriate for the type of assay by one of ordinary skill in the art. For example, for cell-based assays, CIC or polynucleotide concentrations of 20. mu.g/ml or 10. mu.g/ml or 2. mu.g/ml are commonly used. Typically, to perform such an assay, the absorbance at 260nm is measured and 0.5OD is used₂₆₀Concentrations were determined by conversion at 20 μ g/ml. This allows for normalization of the total nucleic acid amount in the test sample and, for example, when the spacer moiety is not evident at 260nmThis is applicable at absorbance. Alternatively, the concentration or weight may be measured using other methods known in the art. If desired, the amount of nucleic acid moieties can be determined by measuring absorbance at 260nm, and CIC weight can be calculated using the formula of CIC. This method is sometimes used in cases where the ratio of the weight occupied by spacer moieties to the weight occupied by nucleic acid moieties in a CIC is high (i.e., greater than 1).

It will also be appreciated that positive and negative controls are useful in the immunomodulatory activity assay. A suitable positive control for immunomodulatory activity is an immunomodulatory phosphorothioate DNA having the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 2), but other suitable positive controls for immunomodulatory activity will be apparent to those of ordinary skill in the art. One useful negative control is no test agent (i.e., only vehicle or medium, also referred to as "cell only" for some in vitro tests). Alternatively, phosphorothioate DNA having the sequence 5'-TGACTGTGAACCTTAGAGATGA-3' (SEQ ID NO: 3) was used as a negative control in some embodiments. Other negative controls can be designed by the skilled artisan guided by the disclosure herein and the design of routine experimentation.

One useful class of assays is the "cytokine response assay". An exemplary assay for immunomodulating activity measures cytokine responses in human peripheral blood mononuclear cells ("PBMCs") (as in Bohle et al, 1999 [)]Eur.j.immunol.29: 2344-53; verthelyi et al [2001]J.immunol.166: 2372-77). In one embodiment of this assay, peripheral blood is collected and PBMCs are isolated from one or more healthy human volunteers. Generally, blood is collected by venipuncture using a heparinized syringe and then applied to FICOLL ^_(AmershamPharmacia Biotech) and centrifuged. Then from FICOLL^_PBMCs were collected and washed twice with cold Phosphate Buffered Saline (PBS). Cells were plated at 2X 10⁶cell/mL concentration in RPMI1640 (containing 10% heat-inactivated human AB serum, 50 units/mL penicillin, 50. mu.g/mL streptPlain, 300. mu.g/mL glutamine, 1mM sodium pyruvate, and 1 XMEM non-essential amino acid (NEAA)) and resuspended and incubated for 24 hours (e.g., in 48-or 96-well culture plates) in the presence or absence of test samples or controls.

Cell-free culture fluid was collected from each well and assayed for IFN-. gamma.and/or IFN-. alpha.concentration. Immunomodulatory activity is detected if the amount of IFN- γ secreted by PBMCs contacted with the test compound is significantly greater (e.g., at least about 3-fold greater, typically at least about 5-fold greater) than the amount secreted by PBMCs in the absence of the test compound, or, in some embodiments, in the presence or absence of an active control compound (e.g., 5'-TGACTGTGAACCTTAGAGATGA-3' (SEQ ID NO: 3)). Conversely, a test compound has NO immunomodulatory activity if the amount of IFN- γ secreted by PBMCs contacted with the test compound is not significantly greater (e.g., less than 2-fold greater) than the amount secreted by PBMCs in the absence of the test compound, or, alternatively, in the presence of an inactive control compound (e.g., 5'-TGACTGTGAACCTTAGAGATGA-3' (SEQ ID NO: 3)).

If IFN- α concentrations are determined, the amount of IFN- α secreted by PBMCs contacted with the test compound is often significantly greater (e.g., sometimes at least about 2-fold or at least about 3-fold greater for IFN- α) than the amount secreted by PBMCs in the absence of the test compound, or, in some embodiments, in the presence or absence of an active control compound (e.g., 5'-TGACTGTGAACCTTAGAGATGA-3' (SEQ ID NO: 3)). In some embodiments, the significantly increased level of IFN- α secretion is at least about 5-fold, at least about 10-fold, or even at least about 20-fold higher than the control. Conversely, a test compound has no immunomodulatory activity if the amount of IFN- α secreted by PBMCs contacted with the test compound is not significantly greater (e.g., less than 2 greater) than the amount secreted by PBMCs in the absence of the test compound, or, alternatively, in the presence of an inactive control compound (e.g., 5'-TGACTGTGAACCTTAGAGATGA-3' (SEQ ID NO: 3)).

As illustrated in the examples below, administration of certain CICs may result in significant secretion of both IFN- α and IFN- γ, while administration of other CICs has less effect on IFN- α secretion or, conversely, on IFN- γ secretion. See example 49 for an example.

Another useful class of assays is cell proliferation assays, such as B cell proliferation assays. The effect of an agent (e.g., CIC) on B cell proliferation can be determined using any of a variety of assays known in the art. An example of a B cell proliferation assay is provided in example 41.

Given donor differences, as in cell-based assays such as cytokine and proliferation assays, it is preferred to perform assays using cells (e.g., PBMCs) from multiple different donors. Typically, the number of donors is at least 2 (e.g., 2), preferably at least 4 (e.g., 4), and sometimes at least 10 (e.g., 10). Immunomodulatory activity is detected if the secreted amount of IFN- γ in the presence of the test compound (e.g., in at least half, preferably at least 75%, most preferably at least 85% of the healthy donors tested) is at least about 3-fold or at least about 5-fold higher than the secreted amount in the presence of the test compound, or, in some embodiments, an inactive control compound (e.g., the control compounds described above).

In vitro assays can also be performed using mouse cells as described in example 42 below or in other mammalian cells.

Exemplary in vivo assays are described in examples 43, 44 and 46 (mouse) and example 45 (non-human primate).

Unless otherwise indicated or apparent, the cytokine assays described in the examples below were performed using human PBMCs essentially following the procedures described in example 28. For example, a large number of test compounds can be detected simultaneously using multi-well plates or other multi-chamber assay materials. These tests can be performed using computer controlled robotic devices well known in the art, if desired.

3. Nucleic acid moieties

CICs of the invention contain one or more nucleic acid moieties. The term "nucleic acid moiety" as used herein refers to a nucleotide monomer (i.e., a single nucleotide) or a nucleotide polymer (i.e., containing at least 2 contiguous nucleotides). As used herein, a nucleotide includes (1) a purine or pyrimidine base linked to a sugar, wherein the sugar is linked to a phosphate group via an ester linkage, or (2) a nucleotide analog in which the base and/or sugar and/or phosphate are replaced by an analog, see below. If more than one nucleic acid moiety is contained in a CIC, these nucleic acid moieties may be the same or different.

The following three sections describe the features of the nucleic acid moiety, such as the length, presence and location of sequences or sequence motifs within the moiety, and also describe (without intending to limit the invention) the nature and structure of the nucleic acid moiety and CIC containing such moiety.

A. Length of

Typically, the nucleic acid moiety is 1 to 100 nucleotides in length, but in some embodiments the moiety may be longer. In some embodiments, one or more nucleic acid moieties in a CIC are less than 8 nucleotides in length (i.e., 1, 2, 3, 4, 5, 6, or 7 nucleotides). In various embodiments, a nucleic acid moiety (e.g., a nucleic acid moiety of less than 8 nucleotides in length) is at least 2 nucleotides in length, typically at least 3, at least 4, at least 5, at least 6, or at least 7 nucleotides in length. In other embodiments, the nucleic acid moiety is at least 10, at least 20, or at least 30 nucleotides in length.

As shown in the examples below, CIC containing only heptameric, hexameric, pentameric, tetrameric, and trimeric nucleic acid moieties were active in assays of immunomodulation activity (as in examples 36 and 37). Thus, it is contemplated that in some embodiments, a CIC comprises at least one nucleic acid moiety that is shorter than 8 nucleotides long. In some embodiments, all nucleic acid moieties in a CIC are shorter than 8 nucleotides (e.g., have a length in a range with a lower limit of 2, 3, 4, 5, or 6 nucleotides and an independently selected upper limit of 5, 6, or 7 nucleotides, wherein the upper limit is greater than the lower limit). As in one embodiment, the specified nucleic acid moieties in a CIC (including all nucleic acid moieties in the CIC) may be 6 or 7 nucleotides long. In one embodiment, a CIC contains two spacer moieties and one nucleic acid moiety inserted therein that is less than 8 bases in length (e.g., 5, 6, or 7 bases in length).

In CICs comprising multiple nucleic acid moieties, the length of the nucleic acid moieties may be considered the same or different. In one embodiment, one or more (e.g., at least about 2, at least about 4, or at least about 25%, at least about 50%, at least about 75%) or all of the nucleic acid moieties in a CIC are less than 8 nucleotides, in some embodiments less than 7 nucleotides, in some embodiments less than 6 nucleotides, in some embodiments from 2 to 7 nucleotides, in some embodiments from 3 to 7 nucleotides, in some embodiments from 4 to 7 nucleotides, in some embodiments from 5 to 7 nucleotides, in some embodiments from 6 to 7 nucleotides in length.

As discussed in more detail below, often at least one nucleic acid moiety in a CIC includes the sequence CG, such as TCG or a CG-containing motif as described herein. In one embodiment, at least one nucleic acid moiety comprises a CG-containing nucleic acid motif and is less than 8 nucleotides long (e.g., has a specified length of less than 8 nucleotides as described above). In a related embodiment, none of the nucleic acid moieties in the CIC that are longer than 8 nucleotides contain the sequence "CG" or optionally the sequence "TCG" or "ACG" (i.e., all of the nucleic acid moieties in the CIC that contain the sequence CG are less than 8 nucleotides long). In one embodiment, at least one nucleic acid moiety in the CIC does not contain a CG sequence.

B. Sequences and motifs (motifs)

As indicated above, a particular nucleic acid moiety may have a variety of lengths. In one embodiment, the nucleic acid moiety is less than 8 nucleotides long. In one embodiment, the nucleic acid moiety is 8 nucleotides long or longer. In various embodiments, at least one nucleic acid moiety in a CIC of the invention comprises a sequence as described below.

In the formulae below, all sequences are in the 5 '→ 3' orientation and the following abbreviations are used: b ═ 5-bromocytosine; 5-bromouracil; a-a ═ 2-amino-adenine; g ═ 6-thio-guanine; t ═ 4-thiothymine; h-modified cytosine containing an electron withdrawing group (e.g., halogen at the 5-position). In various embodiments, cytosine (C) in the sequences referred to below is replaced by N4-ethylcytosine or N4-methylcytosine or 5-hydroxycytosine. In various embodiments, guanosine (G) in the formula is replaced with 7-deazaguanosine.

In CIC tested to date, the presence of CG is associated with cytokine-induced activity. Thus, in one embodiment, at least one nucleic acid moiety in the CIC comprises at least one 5 '-cytosine, guanine-3' (5 '-CG-3') sequence. The cytosine is not methylated at the C-5 position, and preferably is not methylated at any position.

In one embodiment, one or more nucleic acid moieties comprises 3 to 7 bases. In one embodiment, the nucleic acid moiety comprises 3 to 7 bases and has the sequence 5' - [ (X)_0-2]TCG](X)_2-4 ]-3 ', or 5' -TCG [ (X)_2-4]-3 ', or 5' -TCG (A/T) [ (X)_1-3]-3 ', or 5 ' -TCG (A/T) CG (A/T) -3 ', or 5 ' -TCGACGT-3 ', or 5 ' -TCGTCGA-3 ', wherein each X is an independently selected nucleotide. In some embodiments, the CIC comprises at least 3, at least 10, at least 30, or at least 100 nucleic acid moieties having the aforementioned sequences.

In one embodiment, the nucleic acid moiety comprises the sequence 5 '-thymine, cytosine, guanine-3' (5 '-TCG-3'), such as, without limitation, 3-mer TCG, 4-mer TCGX (e.g., TCGA), 5-mer TCGXX (e.g., TCGTC and TCGAT), 6-mer TCGXXX, XTCGXX and TCGTCG, and 7-mer TCGXXX, XTCGXXX, XXTCGXX and TCGTCGX, wherein X is any base. Often, at least one nucleic acid moiety comprises the sequence 5 '-thymine, cytosine, guanine, adenine-3' (5 '-TCGA-3'), e.g., comprises the sequence 5 '-TCGACGT-3'.

In some embodiments, the nucleic acid moiety comprises the sequence 5 '-ACGTTCG-3';

5′-TCGTCG-3′；5′-AACGTTC-3′；5′-AACGTT-3′；5′-TCGTT-3′；

5′-CGTTCG-3′；5′-TCGTCGA-3′；5′-TCGXXX-3′；5′-XTCGXX-3′；

5′-XXTCGX-3′；5′-TCGAGA-3′；5′-TCGTTT-3′；5′-TTCGAG-3′；

5′-TTCGT-3′；5′-TTCGC-3′；5′-GTCGT-3′；

5′-ATCGT-3′；5′-ATCGAT-3′；5′-GTCGTT-3′；5′-GTCGAC-3′；

5 '-ACCGGT-3'; 5 '-AABGTT-3'; 5 '-AABGUT-3', 5 '-TCGTBG-3' wherein X is any nucleotide.

In some embodiments, the nucleic acid moiety comprises the sequence 5' -X₁X₂CGX₃X₄-3', wherein X₁Is 0 to 10 nucleotides; x₂Is empty or is A, T or U; x₃Is a vacancy or A; and X₄Is 0 to 10 nucleotides. In one embodiment, the nucleic acid moiety is conjugated to the spacer moiety at the 3' end. In some embodiments, X₁Is 0 to 5 nucleotides, alternatively 0 to 2 nucleotides; and X₄Is 0 to 5 nucleotides, or 0 to 2 nucleotides.

In some embodiments, the nucleic acid moiety comprises a sequence as shown below: 5 '-purine, C, G, pyrimidine-3'; 5 '-purine, C, G, pyrimidine, C, G-3'; or 5 '-purine, C, G, pyrimidine, C, C-3'; for example (both in the 5 '→ 3' direction), GACGCT; GACGTC; GACGTT; GACGCC; GACGCU; GACGUC; GACGUU; GACGUT; GACGTU; AGCGTT; AGCGCT; AGCGTC; AGCGCC; AGCGUU; AGCGCU; AGCGUC; AGCGUT; AGCGTU; AACGTC; AACGCC; AACGTT; AACGCT; AACGUC; AACGUU; AACGCU; AACGUT; AACGTU; GGCGTT; GGCGCT; GGCGTC; GGCGCC; GGCGUU; GGCGCU; GGCGUC; GGCGUT; GGCGTU, AACGTT, AGCGTC, GACGTT, GGCGTT, AACGTC, GACGTC, GGCGTC, AACGCC, AGCGCC, GACGCC, GGCGCC, AGCGCT, GACGCT, GGCGCT, GGCGTT, and AACGCC.

In some embodiments, the nucleic acid moiety comprises the sequence: 5 '-purine, cytosine, guanine, pyrimidine, cytosine-3' or 5 '-purine, cytosine, guanine, pyrimidine, cytosine, guanine-3'.

In some embodiments, the nucleic acid moiety comprises the sequence (both in the 5 '-3' direction) AACGTTCG; AACGTTCC; AACGUTCG; AABGTTCG; AABGUTCG and/or AABGTTBG.

In various embodiments, the nucleic acid moiety comprises the motif 5' -X₁X₂AX₃CGX₄TCG-3' (SEQ ID NO: 4), where X₁Is T, G, C or B, X₂Is T, G, A or U, X₃Is T, A or C, X₄Is T, G or U, and the sequence is not 5'-TGAACGTTCG-3' (SEQ ID NO: 5) or 5'-GGAACGTTCG-3' (SEQ ID NO: 6). Examples include (both in the 5 '→ 3' direction):

TGAACGUTCG(SEQ ID NO：7)；TGACCGTTCG(SEQ ID NO：8)；

TGATCGGTCG(SEQ ID NO：9)；TGATCGTTCG(SEQ ID NO：10)；

TGAACGGTCG(SEQ ID NO：11)；GTAACGTTCG(SEQ ID NO：12)；

GTATCGGTCG(SEQ ID NO：13)；GTACCGTTCG(SEQ ID NO：14)；

GAACCGTTCG(SEQ ID NO：15)；BGACCGTTCG(SEQ ID NO：16)；

CGAACGTTCG(SEQ ID NO：17)；CGACCGTTCG(SEQ ID NO：18)；

BGAACGTTCG(SEQ ID NO：19)；TTAACGUTCG(SEQ ID NO：20)；

TUAACGUTCG (SEQ ID NO: 21) and TTAACGTTCG (SEQ ID NO: 22).

In various embodiments, the nucleic acid moiety comprises the sequence:

5’-TCGTCGAACGTTCGTTAACGTTCG-3’(SEQ ID NO：23)；

5’-TGACTGTGAACGUTCGAGATGA-3’(SEQ ID NO：24)；

5’-TCGTCGAUCGUTCGTTAACGUTCG-3’(SEQ ID NO：25)；

5’-TCGTCGAUCGTTCGTUAACGUTCG-3’(SEQ ID NO：26)；

5’-TCGTCGUACGUTCGTTAACGUTCG-3’(SEQ ID NO：27)；

5’-TCGTCGAa-ACGUTCGTTAACGUTCG-3’(SEQ ID NO：28)；

5’-TGATCGAACGTTCGTTAACGTTCG-3(SEQ ID NO：29)；

5’-TGACTGTGAACGUTCGGTATGA-3’(SEQ ID NO：30)；

5’-TGACTGTGACCGTTCGGTATGA-3’(SEQ ID NO：31)；

5’-TGACTGTGATCGGTCGGTATGA-3’(SEQ ID NO：32)；

5’-TCGTCGAACGTTCGTT-3’(SEQ ID NO：33)；

5’-TCGTCGTGAACGTTCGAGATGA-3’(SEQ ID NO：34)；

5’-TCGTCGGTATCGGTCGGTATGA-3’(SEQ ID NO：35)；

5’-CTTCGAACGTTCGAGATG-3’(SEQ ID NO：36)；

5’-CTGTGATCGTTCGAGATG-3’(SEQ ID NO：37)；

5’-TGACTGTGAACGGTCGGTATGA-3’(SEQ ID NO：38)；

5’-TCGTCGGTACCGTTCGGTATGA-3’(SEQ ID NO：39)；

5’-TCGTCGGAACCGTTCGGAATGA-3’(SEQ ID NO：40)；

5’-TCGTCGAACGTTCGAGATG-3’ (SEQ ID NO：41)；

5’-TCGTCGTAACGTTCGAGATG-3’(SEQ ID NO：42)；

5’-TGACTGTGACCGTTCGGAATGA-3’(SEQ ID NO：43)；

5’-TCGTCGAACGTTCGAACGTTCG-3’(SEQ ID NO：44)；

5’-TBGTBGAACGTTCGAGATG-3’(SEQ ID NO：45)；

5’-TCGTBGAACGTTCGAGATG-3’(SEQ ID NO：46)；

5’-TCGTCGACCGTTCGGAATGA-3’(SEQ ID NO：47)；

5’-TBGTBGACCGTTCGGAATGA-3’(SEQ ID NO：48)；

5’-TCGTBGACCGTTCGGAATGA-3’(SEQ ID NO：49)；

5’-TTCGAACGTTCGTTAACGTTCG-3’(SEQ ID NO：50)；

5’-CTTBGAACGTTCGAGATG-3’(SEQ ID NO：51)；

5’-TGATCGTCGAACGTTCGAGATG-3’(SEQ ID NO：52).

in some embodiments, the nucleic acid moiety comprises the sequence: 5' -X₁X₂AX₃BGX₄TCG-3' (SEQ ID NO: 53), where X₁Is T, G, C or B, X₂Is T, G, A or U, X₃Is T, A or C, X₄Is T, G or U. In some embodiments, the nucleic acid moiety is not 5 '-TGAABBGTTCG-3' (SEQ ID NO: 54). Examples include (both in the 5 '-3' direction): TGAAGBUTCG (SEQ ID NO: 55)

TGACBGTTCG(SEQ ID NO：56)；TGATBGGTCG(SEQ ID NO：57)；

GTATBGGTCG(SEQ ID NO：58)；GTACBGTTCG(SEQ ID NO：59)；

GAACBGTTCG(SEQ ID NO：60)；GAAABGUTCG(SEQ ID NO：61)；

BGACBGTTCG(SEQ ID NO：62)；CGAABGTTCG(SEQ ID NO：63)；

BGAABGTTCG(SEQ ID NO：64)；BGAABGUTCG(SEQ ID NO：65)；

TTAABGUTCG (SEQ ID NO: 66); TUAABGUTCG (SEQ ID NO: 67) and

TTAABGTTCG(SEQ ID NO：68).

in some embodiments, the nucleic acid moiety comprises the sequence:

5’-TGACTGTGAABGUTCGAGATGA-3’(SEQ ID NO：69)；

5’-TCGTCGAABGTTCGTTAABGTTCG-3’(SEQ ID NO：70)；

5’-TGACTGTGAABGUTCGGTATGA-3’(SEQ ID NO：71)；

5’-TGACTGTGAABGUTCGGAATGA-3’(SEQ ID NO：72)；

5’-TCGTCGGAAABGUTCGGAATGA-3’(SEQ ID NO：73)；

5’-TCGTBGAABGUTCGGAATGA-3’(SEQ ID NO：74).

in some embodiments, the nucleic acid moiety comprises the sequence: 5' -X₁X₂AX₃CGX₄TCG-3' (SEQ ID NO: 75), where X₁Is T, C or B, X₂T, G,A or U, X₃Is T, A or C, X₄Is T, G or U. In some embodiments, this formula is not 5'-TGAACGTTCG-3' (SEQ ID NO: 5).

In other embodiments, the nucleic acid moiety comprises the sequence:

5’-TGACTGTGAABGTTCGAGATGA-3’(SEQ ID NO：76)；

5’-TGACTGTGAABGTTBGAGATGA-3’(SEQ ID NO：77)；

5’-TGACTGTGAABGTTCCAGATGA-3’(SEQ ID NO：78)；

5’-TGACTGTGAACGTUCGAGATGA-3’(SEQ ID NO：79)；

5’-TGACTGTGAACGbUTCGAGATGA-3’(SEQ ID NO：80)；

5’-TGACTGTGAABGTTCGTUATGA-3’(SEQ ID NO：81)；

5’-TGACTGTGAABGTTCGGTATGA-3’(SEQ ID NO：82)；

5’-CTGTGAACGTTCGAGATG-3’(SEQ ID NO：83)；

5’-TBGTBGTGAACGTTCGAGATGA-3’(SEQ ID NO：84)；

5’-TCGTBGTGAACGTTCGAGATGA-3’(SEQ ID NO：85)；

5’-TGACTGTGAACGtTCGAGATGA-3’(SEQ ID NO：86)；

5’-TGACTGTGAACgTTCgAGATGA-3’(SEQ ID NO：87)；

5’-TGACTGTGAACGTTCGTUATGA-3’(SEQ ID NO：88)；

5’-TGACTGTGAACGTTCGTTATGA-3’(SEQ ID NO：89)；

5’-TCGTTCAACGTTCGTTAACGTTCG-3’(SEQ ID NO：90)；

5’-TGATTCAACGTTCGTTAACGTTCG-3’(SEQ ID NO：91)；

5’-CTGTCAACGTTCGAGATG-3’(SEQ ID NO：92)；

5’-TCGTCGGAACGTTCGAGATG-3’(SEQ ID NO：93)；

5’-TCGTCGGACGTTCGAGATG-3’(SEQ ID NO：94)；

5’-TCGTCGTACGTTCGAGATG-3’(SEQ ID NO：95)；

5’-TCGTCGTTCGTTCGAGATG-3’(SEQ ID NO：96).

in some embodiments, with respect to any of the sequences disclosed above, the nucleic acid moiety further comprises one, two, three or more TCG and/or TBG and/or THG sequences, preferably located 5' to the above-described sequences. This TCG and/or TBG may or may not be directly adjacent to the indicated sequence. For example, in some embodiments, the nucleic acid moiety comprises any one of the following sequences: 5'-TCGTGAACGTTCG-3' (SEQ ID NO: 97); 5'-TCGTCGAAGTTCG-3' (SEQ ID NO: 98); 5 '-TBGTGAACGTTCG-3' (SEQ ID NO: 99); 5-TBGTBGAACGTTCG 3' (SEQ ID NO: 100); 5'-TCGTTAACGTTCG-3' (SEQ ID NO: 101).

In some embodiments, this additional TCG and/or TBG sequence is located immediately 5' to the sequence involved. In other embodiments, one or two bases are separated.

In some embodiments, the nucleic acid moiety has the sequence: 5' - (TCG)_wN_yAX₃CGX₄TCG-3' (SEQ ID NO: 102) wherein w is 1-2, y is 0-2, N is any base, X₃Is T, A or C, X₄Is T, G or U.

In some embodiments, the nucleic acid moiety comprises any one of the following sequences:

TCGAACGTTCG(SEQ ID NO：103)；

TCGTCGAACGGTTCG(SEQ ID NO：104)；TCGTGAACGTTCG(SEQ IDNO：105)；TCGGTATCGGTCG(SEQ ID NO：106)；TCGGTACGTTCG(SEQID NO：107)；TCGGAACCGTTCG(SEQ ID NO：108)；TCGGAACGGTTCG(SEQID NO：109)；TCGTCGGAACGTTCG(SEQ ID NO：110)TCGTAACGTTCG(SEQ ID NO：111)；TCGACCGTTCG(SEQ ID NO：112)；TCGTCGACCGTTCG(SEQ ID NO：113)；TCGTTAACGTTCG(SEQ ID NO：114)

in some embodiments, the nucleic acid moiety comprises any one of the following sequences: 5' - (TBG)_zN_yAX₃CGX₄TCG-3' (SEQ ID NO: 115) wherein z is 1-2, y is 0-2, B is 5-bromocytosine, N is any base, X₃Is T, A or C, X₄Is T, G or U.

In some embodiments, the nucleic acid moiety comprises:

TBGTGAACGTTCG(SEQ ID NO：116)；TBGTBGTGAACGTTCG(SEQ IDNO：117)；TBGAACGTTCG(SEQ ID NO：118)；TBGTBGAACGTTCG(SEQ IDNO：100)；TBGACCGTTCG(SEQ ID NO：119)；TBGTBGACCGTTCG(SEQ IDNO：120).

in some embodiments, the nucleic acid moiety comprises any one of the following sequences: 5' -TCGTBGN_yAX₃CGX₄TCG-3' (SEQ ID NO: 121) wherein y is 0-2, B is 5-bromocytosine, N is any base, X₃Is T, A or C, X₄Is T, G or U. In some embodiments, the nucleic acid moiety comprises any one of the following sequences:

TCGTBGTGAACGTTCG(SEQ ID NO：122)；TCGTBGAACGTTCG(SEQ IDNO：123)；TCGTBGACCGTTCG(SEQ ID NO：124).

in some embodiments, the nucleic acid moiety comprises any one of the following sequences: 5' - (TCG)_wN_yAX₃BGX₄TCG-3' (SEQ ID NO: 125) wherein w is 1-2, y is 0-2, N is any base, X₃Is T, A or C, X₄Is T, G or U. In some embodiments, the nucleic acid moiety comprises any one of the following sequences: TCGGAAABGTTCG (SEQ ID NO: 126) or TCGAABGTTCG (SEQ ID NO: 127).

In some embodiments, the nucleic acid moiety comprises any one of the following sequences: 5' - (TBG)_zN_yAX₃BGX₄TCG-3' (SEQ ID NO: 128) wherein z is 1-2, y is 0-2, B is 5-bromocytosine, N is any base, X₃Is T, A or C, X₄Is T, G or U. In some embodiments, the nucleic acid moiety comprises any one of the following sequences:

TBGAABGUTCG (SEQ ID NO: 129) or TBGAABGTTCG (SEQ ID NO: 130).

In some embodiments, the nucleic acid moiety comprises any one of the following sequences: 5' -TCGTBGN_yAX₃BGX₄TCG-3' (SEQ ID NO: 131) wherein y is 0-2, B is 5-bromocytosine, N is any base, X₃Is T, A or C, X₄Is T, G or U. In some embodiments, the nucleic acid moiety comprises any one of the following sequences:

TCGTBGAABGUTCG (SEQ ID NO: 132) or TCGTBGAABGTTCG (SEQ ID NO: 133).

In some embodiments, the nucleic acid moiety comprises the sequence: AACGTTCC, AACGTTCG, GACGTTCC, GACGTTCG.

In some embodiments, the nucleic acid moiety comprises the sequence:

GGCGTTCG；GGCGCTCG；GGCGTCCG；GGCGCCCG；GACGTTCC；

GACGCTCC；GACGTCCC；GACGCCCC；AGCGTTCC；AGCGCTCC；

AGCGTCCC；AGCGCCCC；AACGTTCC；AACGCTCC；AACGTCCC；

AACGCCCC；GGCGTTCC；GGCGCTCC；GGCGTCCC；GGCGCCCC；

GACGTTCG；GACGCTCG；GACGTCCG；GACGCCCG；AGCGTTCG；

AGCGCTCG；AGCGTCCG；AGCGCCCG；AACGTTCG；AACGCTCG；

AACGTCCG；AACGCCCG；GACGCTCC；GACGCCC； AGCGTTCC；

AGCGCTCC；AGCGTCCC；AGCGCCCC；AACGTCCC；AACGCCCC；

GGCGTTCC；GGCGCTCC；GGCGTCCC；GGCGCCCC；GACGCTCG；

GACGTCCG；GACGCCCG；AGCGTTCG；AGCGTCCG；AGCGCCCG；

AACGTCCG；AACGCCCG.

in some embodiments, the nucleic acid moiety comprises the sequence:

(5’→3’)TCGTCGA；TCGTCG；TCGTTT；TTCGTT；TTTTCG；ATCGAT；

GTCGAC；GTCGTT；TCGCGA；TCGTTTT；TCGTC； TGTT；TCGT；TCG；

ACGTTT；CCGTTT；GCGTTT；AACGTT；TCGAAAA；TCGCCCC；

TCGGGGG

in some embodiments, the nucleic acid moiety comprises an RNA having the sequence: AACGUUCC, AACGUUCG, GACGUUCC, and GACGUUCG.

In some embodiments, the nucleic acid moiety has a sequence comprising a sequence as set forth in commonly assigned co-pending U.S. patent application 09/802,685 (published as U.S. application publication No. 20020028784a1 on day 7/3/2002 and WO01/68077 on day 20/9/2001), 09/802,359 (published as WO01/68144 on day 20/9/2001), and co-pending U.S. application publication No. 10/033,243, or in PCT publication No. WO 97/28259; WO 98/16247; WO 98/55495; WO 99/11275; WO 99/62923; and the sequences or sequence motifs described in WO 01/35991. The nucleic acid moiety may also have a sequence comprising any one or several of the sequences previously reported to be associated with immunostimulatory activity if administered as a polynucleotide of length greater than (often substantially greater than) 8 nucleotides, see kandiiimalla et al, 2001, bioorg.med.chem.9: 807-13; krieg et al (1989) J.Immunol.143: 2448 and 2451; tokunaga et al (1992) Microbiol. Immunol.36: 55-66; kataoka et al (1992) Jpn. J Cancer Res.83: 244-; yamamoto et al (1992) J.Immunol.148: 4072-; mojcik et al (1993) clin. 130-136; branda et al (1993) biochem. Pharmacol.45: 2037-2043; pisetsky et al (1994) Life Sci.54 (2): 101-; yamamoto et al (1994a) Antisense Research and development.4: 119-122; yamamoto et al (1994b) Jpn.J. cancer Res.85: 775-779; raz et al (1994) proc.natl.acad.sci.usa 91: 9519-9523; kimnra et al (1994) j. biochem. (Tokyo) 116: 991-; krieg et al (1995) Nature 374: 546-549; pisetsky et al (1995) Ann.N.Y.Acad.Sci.772: 152-163; pisetsky (1996a) j.immunol.156: 421-; pisetsky (1996b) Immunity 5: 303-310; zhao et al (1996) biochem. pharmacol.51: 173-; yi et al (1996) J.Immunol.156: 558-564; krieg (1996) Trcnds Microbiol.4 (2): 73-76; krieg et al (1996) Antisense Nucleic Acid Drug Dev.6: 133-139; klinman et al (1996) Proc.Natl.Acad.Sci.USA.93: 2879 while 2883; raz et al (1996); sato et al (1996) Science 273: 352 and 354; stacey et al (1996) J.Immunol.157: 2116-2122; ballas et al (1996) J.Immunol.157: 1840-; branda et al (1996) J.Lab.Clin.Med.128: 329-338; sonehara et al (1996) J Interferon and cytokine Res.16: 799-; klinman et al (1997) J.Immunol.158: 3635-3639; sparwasse et al (1997) eur.j.immunol.27: 1671-; roman et al (1997) Nat Med.3: 849-54; carson et al (1997) j.exp.med.186: 1621-; chace et al (1997) Clin. Immunol. and Immunopathol.84: 185-193; chu et al (1997) J.Exp.Med.186: 1623-; lipford et al (1997a) eur.j.immunol.27: 2340-; lppford et al (1997b) Eur.J.Immunol.27: 3420-; weiner et al (1997) Proc.Natl.Acad.Sci.USA94: 10833-10837; macfarlane et al (1997) Immunology 91: 586-593; schwartz et al (1997) j.clin.invest.100: 68-73; stein et al (1997) Antisense Technology, Ch.11 pp.241-264, C.Lichtenstein and W.Nellen, eds., IRL Press; wooldridge et al (1997) Blod 89: 2994 as well as 2998; leclerc et al (1997) cell.immunol.179: 97 to 106; kline et al (1997) J invest.med.45 (3): 282A; yi et al (1998a) J.Immunol.160: 1240-1245; yi et al (1998b) J.Immunol.60: 4755-4761; yi et al (1998c) J.Immunol.60: 5898-; yi et al (1998d) J.Immunol.161: 4493-4497; krieg (1998) Applied antisense oligonotide Technology ch.24, pp.431-448, c.a.stein and a.m.krieg, eds., Wiley-Liss, inc; krieg et al (1998a) Trends Microbiol.6: 23-27; krieg et al (1998b) J.Immunol.161: 2428-; krieg et al (1998c) Proc. Natl. Acad. Sci. USA 95: 12631-; spiegelberg et al (1998) Allergy53 (45S): 93-97; horner et al (1998) Cell immunol.190: 77-82; jakob et al (1998) J.Immunol.161: 3042 and 3049; redford et al (1998) J.Immunol.161: 3930-; weratna et al (1998) Antisense & Nucleic Acid Drug Development 8: 351- > 356; mcclusky et al (1998) j.immunol.161 (9): 4463-4466; gramzinski et al (1998) mol. med.4: 109-118; liu et al (1998) Blood 92: 3730-3736; moldovenu et al (1998) Vaccine 16: 1216-1224; brazolot Milan et al (1998) Proc.Natl.Acad.Sci.USA95: 15553 and 15558; briode et al (1998) J.Immunol.161: 7054 and 7062; briode et al (1999) int. Arch. allergy. immunological.118: 453-456; kovarik et al (1999) J.Immunol.162: 1611-1617; spiegelberg et al (1999) Peditator, Pulmonol. Suppl.18: 118-121; Martin-Orozco et al (1999) int. immunol 11: 1111-; EP468,520; WO 96/02555; WO 97/28259; WO 98/16247; WO 98/18810; WO 98/37919; WO 98/40100; WO 98/52581; WO 98/55495; WO98/55609 and WO 99/11275. See also Elkins et al (1999) j.immunol.162: 2291-2298, WO98/52962, WO99/33488, WO99/33868, WO99/51259 and WO 99/62923. See also Zimmermann et al (1998) j.immunol.160: 3627 vs 3630; krieg (1999) Trends Microbiol.7: 64-65 and U.S. Pat. Nos. 5,663,153, 5,723,335 and 5,849,719. See also Liang et al (1996) j.clin.invest.98: 1119-1129; bohle et al (1999) Eur.J.Immunol.29: 2344-. See also WO 99/61056; WO 00/06588; WO 00/16804; WO 00/21556; WO 00/54803; WO 00/61151; WO 00/67023; WO00/67787 and U.S. Pat. No. 6,090,791. In one embodiment, at least one nucleic acid moiety in a CIC comprises a TG sequence or a pyrimidine-rich (e.g., T-rich or C-rich) sequence, as described in PCT publication WO 01/22972.

In some embodiments, the nucleic acid moiety is different from one or more of the "following" hexamers:

5′-GACGTT-3′，5′-GAGCTT-3′，5′-TCCGGA-3′，

5′-AACGTT-3′，5′-GACGTT-3′，5′-TACGTT-3′，5′-CACGTT-3′，

5′-AGCGTT-3′，5′-ATCGTT-3′，5′-ACCGTT-3′，5′AACGGT-3′，

5 '-AACGAT-3', 5 '-AACGCT-3', 5 '-AACGTG-3', 5 '-AACGTA-3' and

5′-AACGTC-3′。

in some embodiments, the CIC comprises at least 3, at least 10, at least 30, or at least 100 nucleic acid moieties having the sequences described above.

C. Sequence of the nucleic acid moiety: heterogeneity and location effects

In CICs comprising a plurality of nucleic acid moieties, the nucleic acid moieties may be the same or different.

In one embodiment, all nucleic acid moieties in a CIC have the same sequence. In one embodiment, the CIC comprises nucleic acid moieties having at least 2, at least 3, at least 4, at least 5, or at least 6 or more different sequences. In one embodiment, the CIC comprises less than 10 different nucleic acid moieties. In one embodiment, each nucleic acid moiety in a CIC has a different sequence.

In some embodiments, a single nucleic acid moiety comprises more than one repeat of the sequence motif listed in § 3(B) above, or comprises two or more different sequence motifs. Within a single nucleic acid moiety these motifs may be adjacent, overlapping, or separated by other nucleotide bases within the nucleic acid moiety. In one embodiment, the nucleic acid moiety comprises one or more palindromic sequence regions. For single stranded oligonucleotides, the term "palindromic sequence" refers to a sequence that will be in a palindromic configuration when the oligonucleotide is complexed with a complementary sequence to form a double-stranded molecule. In another embodiment, one nucleic acid moiety in a CIC has a palindromic or partially palindromic sequence relative to a second nucleic acid moiety. In one embodiment of the invention, the sequence of one or more nucleic acid moieties of a CIC does not have a palindrome. In one embodiment of the invention, the sequence of one or more nucleic acid moieties of a CIC does not comprise a palindromic sequence of more than four bases, optionally more than 6 bases.

As noted above, in various embodiments, the nucleic acid moiety of one or more (e.g., all) of the CICs comprises a 5 '-CG-3' sequence, or alternatively, comprises a 5 '-TCG-3' sequence. In one embodiment, the nucleic acid moiety is 5, 6 or 7 bases in length. In one embodiment, the nucleic acid moiety has the formula 5' -TCG [ (X)_2-4]3 'or 5' -TCG (A/T) [ (X)_1-3]Or 5 '-TCG (A/T) CG (A/T) -3' or 5 '-TCGACGT-3' (where each X is an independently selected nucleotide). In one embodiment, the aforementioned nucleic acid moiety is a 5-prime (prime) moiety.

In one embodiment, the nucleic acid moiety comprises the sequence 5 '-TCGTCGA-3'. In one embodiment, the nucleic acid moiety comprises a sequence selected from the group consisting of (both in the 5 '→ 3' direction):

TCGXXXX，TCGAXXX，XTCGXXX，XTCGAXX，TCGACGT，

TCGAACG，TCGAGAT，TCGACTC，TCGAGCG，TCGATTT，

TCGCTTT，TCGGTTT，TCGTTTT，TCGTCGT，ATCGATT， TTCGTTT，

TTCGATT，ACGTTCG，AACGTTC，TGACGTT，TGTCGTT， TCGXXX，

TCGAXX， GTCGTT， GACGTT， ATCGAT， TCGTCG； TCGTTT；

TCGAGA； TTCGAG； TTCGTT； AACGTT； AACGTTCG；AACGUTCG，

ABGUTCG, TCGXX, TCGAX, TCGAT, TCGTT, TCGTC; TCGA, TCGT, and TCGX (where X is A, T, G or C; U is 2 '-deoxyuridine, and B is 5-bromo-2' -deoxycytidine).

In one embodiment, the nucleic acid moiety is a peptide having the sequence TCGXXXXX,

TCGAXXX，XTCGXXX，XTCGAXX，TCGTCGA，TCGACGT，

TCGAACG，TCGAGAT，TCGACTC，TCGAGCG，

TCGATTT，TCGCTTT，TCGGTTT，TCGTTTT，TCGTCGT，ATCGATT，

TTCGTTT, TTCGATT, ACGTTCG, AACGTTC, TGACGTT or a 7-mer of TGTCGTT; or is a 6-mer having the sequence TCGXXX, TCGAXX, TCGTCG, AACGTT, ATCGAT, GTCGTT or GACGTT; or is a 5-mer having the sequence TCGXX, TCGAX, TCGAT, TCGTT or TCGTC; or is a 4-mer having the sequence TCGA, TCGT or TCGX; or a 3-mer having the sequence TCG; wherein X is A, T, G or C.

In one embodiment, at least about 25%, preferably at least about 50%, or at least about 75%, and sometimes all, of the nucleic acid moieties in a CIC comprise at least one of the foregoing sequences. In one embodiment, at least one nucleic acid moiety does not contain a CG motif. In other embodiments, at least about 25%, sometimes at least about 50%, sometimes at least about 75% of the nucleic acid moieties in the CIC are those that do not have a CG motif or, alternatively, a TCG motif.

The position of a sequence or sequence motif in a CIC may affect the immunomodulatory activity of this CIC, as described in the examples below. The following terminology is useful when referring to the position of a sequence motif in the nucleic acid moiety of a CIC: (1) in CICs comprising multiple nucleic acid moieties, the moiety with a free 5' -end is referred to as the "5-priming moiety". It is understood that a single CIC may have multiple 5-initiating moieties. (2) Within any particular nucleic acid moiety, when such moietyA sequence or motif in a molecule is in the "5-prime position" of the moiety when no nucleotide base is present at the 5' end of the sequence or motif. Thus, in the structural part having the sequence 5 '-TCGACGT-3', the sequences T, TC, TCG and TCGA are in the "5-priming position", whereas the sequence GAC is not. For example, a CIC containing the sequence TCG at the "5-prime position" of the nucleic acid moiety may be more active than a similar CIC except for a different localization of the TCG motif. CIC having a TCG sequence in the "5-priming moiety", e.g.in the "5-priming position" of the "5-priming moiety", may render this CIC particularly active. See example 38 for examples. The nucleic acid moiety having a free 5 '-end may be represented by the symbol "5" on the left side of the base sequence of the nucleic acid moiety in the formula' ^FIs (e.g. 5'^F-TACG-3'). As used herein, the term "free 5 'end" has its ordinary meaning in the context of a nucleic acid moiety, i.e., it means that the 5' end of the nucleic acid moiety is not conjugated to a capping group or a non-nucleotide spacer moiety.

Immunostimulatory activity may also be affected by the location of the CG motif in the nucleic acid moiety (e.g., in the 5' -moiety). For example, in one useful embodiment, a CIC contains at least one nucleic acid moiety having the sequence 5 '-X-CG-Y-3', wherein X is 0, 1, or 2 nucleotides and Y is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 nucleotides in length. In one embodiment, the 5 '-X-CG-Y-3' sequence is located in the 5 '-structural portion of the CIC, e.g., the 5' -priming position of the CIC. In one embodiment, the CIC contains 2, 3 or more nucleic acid moieties having the sequence of formula 5 '-X-CG-Y-3'. For example, in one embodiment, all of the nucleic acid moieties of the CIC have a sequence of the formula 5 '-X-CG-Y-3'.

Also, CICs containing the sequence TCGA (e.g., a sequence comprising TCGACGT) in a nucleic acid moiety have immunomodulatory activity and are effective in IFN- α induction. TCGA (e.g., a sequence comprising TCGACGT) in a 5-priming moiety (e.g., at the 5-priming position of a 5-priming moiety) makes CIC specific Has high activity. See examples 38 and 49. Thus, in one embodiment, the CIC comprises a compound having the formula (5' -N)₁-3’)-S₁-N₂(Ia) a core structure of₁Having the sequence 5 '-TCGAX-3', X is 0 to 20 nucleotide bases, often 0 to 3 bases. In one embodiment, X is CGT. The sequence TCGTCGA is also particularly effective in IFN- α induction.

In addition, the presence of a free (unconjugated) nucleic acid at the 5' end can affect immunostimulatory activity. See example 39 for examples. In various embodiments, a CIC of the invention comprises at least 1, at least 2, at least 3, at least 4, or at least 5 free 5' ends. In some embodiments, the number of free 5' ends is 1 to 10, 2 to 6, 3 to 5, or 4 to 5. In one embodiment, the number of free 5' ends is at least about 50 or at least about 100.

D. Independent immunomodulating Activity "

One property of a nucleic acid moiety is the "independent immunomodulating activity" associated with the nucleotide sequence of the nucleic acid moiety. As described above, the present inventors found that, surprisingly, CIC still shows immunomodulatory activity even when all nucleic acid moieties of the CIC do not have sequences that, if presented as separate polynucleotides, can show comparable immunomodulatory activity.

In some embodiments, as described below, the nucleic acid moiety of a CIC does not have "independent immunomodulatory activity", or it has "less independent immunomodulatory activity" (i.e., as compared to the CIC).

The "independent immunomodulatory activity" of a nucleic acid moiety can be determined by measuring the immunomodulatory activity of independent polynucleotides having the primary sequence of the nucleic acid moiety and having the same nucleic acid backbone (e.g., phosphorothioate, phosphodiester, chimeric backbone). For example, a CIC having the structure "5 ' -TCGTCG-3 ' -HEG-5 ' -ACGTTCG-3 ' -HEG-5 ' -AGATGAT-3-comprises three nucleic acid moieties. For example, to determine the independent immunomodulatory activity of the first nucleic acid moiety in a CIC, a test polynucleotide having the same sequence (i.e., 5 '-TCGTCG-3') and the same backbone structure (e.g., phosphorothioate) is first synthesized using conventional methods and then its immunomodulatory activity (if any) is determined. Immunomodulatory activity can be determined using standard assays capable of indicating different aspects of an immune response, such as those described in § 2 above. Preferably using the human PBMC assay described in § 2 above. As described above, the assay is generally performed using cells obtained from a plurality of donors, taking into account donor differences. A polynucleotide has NO immunomodulatory activity (and the corresponding nucleic acid moiety does not have "independent immunomodulatory activity") if the amount of IFN- γ secreted by PBMC contacted with the polynucleotide in the majority of donors is not significantly greater (e.g., less than about 2-fold greater) than the amount of IFN- γ secreted in the absence of a test compound or, in some embodiments, in the presence of an inactive control compound (e.g., 5'-TGACTGTGAACCTTAGAGATGA-3') (SEQ ID NO: 3)).

To compare the immunomodulatory activity of the CIC and independent polynucleotides, the immunomodulatory activity is preferably measured using the human PBMC assay as described in § 2 above. In general, the activity of two compounds is compared by performing parallel tests on them under identical conditions (e.g., using the same cell type), typically at a concentration of about 20. mu.g/ml. As described above, the concentration can be generally determined by measuring the absorbance at 260nm and using 0.5 OD₂₆₀This conversion was determined at 20. mu.g/ml. This operation allows normalization of the total amount of nucleic acid in the test sample. Alternatively, the concentration or weight may be determined using other methods known in the art. If desired, the amount of nucleic acid moieties may be determined by measuring absorbance at 260nm, and the weight of CIC may be calculated using the molecular formula of CIC. This method is sometimes used in cases where the ratio of the weight occupied by the spacer moiety to the weight occupied by the nucleic acid moiety in the CIC is high (i.e., greater than 1).

Alternatively, a 3 μ M concentration may be used, particularly when the calculated molecular weights of the two samples compared differ by more than 20%.

The nucleic acid moiety of a CIC may be characterized as having "sub-immunomodulatory activity" if the tested polynucleotide has an activity that is less than the activity of the comparable CIC. Preferably, the polynucleotides tested have an independent immunomodulatory activity of no more than about 50% of CIC activity, more preferably no more than about 20% of CIC activity, most preferably no more than about 10% of CIC activity, or in some embodiments, even less.

For CICs having multiple (e.g., multiple different) nucleic acid moieties, the immunomodulatory activity (if any) of a mixture of test polynucleotides corresponding to the multiple nucleic acid moieties can also be determined. This assay can be performed using a total amount of test polynucleotides (i.e., in a mixture) equal to the amount of CIC used. Alternatively, in this assay, the amount of each test polynucleotide or each different test polynucleotide in the mixture may be equal to the amount of CIC. As described in § 2, it is preferred to perform assays and analyses using PMBC from multiple donors, taking into account donor differences.

In one embodiment, one or more nucleic acid moieties (e.g., at least about 2, at least about 4, or at least about 25%, at least about 50%, or all of the nucleic acid moieties, measured alone or, alternatively, in combination) in a CIC do not have independent immunomodulatory activity. In one embodiment, one or more of the nucleic acid moieties (e.g., at least about 2, at least about 4, or at least about 25%, at least about 50%, or all of the nucleic acid moieties, measured alone or, alternatively, in combination) of the CIC has a sub-independent immunomodulatory activity relative to the CIC.

In a related embodiment, the CIC comprises one or more nucleic acid moieties with independent immunomodulatory activity. For example, in some embodiments, all or substantially all (e.g., at least 90%, preferably at least 95%) of the nucleic acid moieties have independent immunomodulatory activity. For example, a CIC containing multivalent spacers may comprise more than 4, often more than 10, often at least about 20, at least about 50, at least about 100, at least about 400, or at least about 1000 (e.g., at least about 2500) nucleic acid moieties having independent immunomodulatory activity (e.g., having the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 2)).

Thus, in a particular CIC, the number of nucleic acid moieties having independent immunomodulatory activity can be 0, 1, 2 or more, 3 or more, less than 3, 4 or more, less than 4, 5 or more, less than 5, at least 10, at least about 20, at least about 50, at least about 100, at least about 400 or at least about 1000, all, or less than all of the CIC nucleic acid moieties.

E. Structure of nucleic acid moiety

The nucleic acid moiety of the CIC may comprise structural modifications relative to the native nucleic acid. Such modifications include any known in the art that can be used for modification of a polynucleotide, but are not limited to modification of a 3 'OH or 5' OH group, modification of a nucleotide base, modification of a sugar component, and modification of a phosphate group. Various such modifications are described below.

The nucleic acid moiety may be DNA, RNA or mixed DNA/RNA, single-stranded, double-stranded or partially double-stranded, and may comprise other modified polynucleotides. Double-stranded nucleic acid moieties and CICs are contemplated, and unless otherwise indicated, the terms "base" or "nucleotides" are meant to include base pairs or base pair nucleotides. The nucleic acid moiety may comprise a naturally occurring or modified, non-naturally occurring base, and may comprise a modified sugar, phosphate, and/or terminus. For example, modifications of the phosphate include, but are not limited to, methylphosphonate, phosphorothioate, phosphoramidate (bridged or non-bridged), phosphotriester, and phosphorodithioate, and can be used in any combination. Other non-phosphate linkages may also be used. Preferably, the CIC and nucleic acid moieties of the invention comprise a phosphorothioate backbone. Sugar modifications well known in the art, such as 2 ' -alkoxy-RNA analogs, 2 ' -amino-RNA analogs, and 2 ' -alkoxy-or amino-RNA/DNA chimeras, as well as other sugar modifications described herein, can also be prepared and combined with any phosphate modifications. Examples of base modifications (discussed further below) include, but are not limited to, the addition of electron-withdrawing moieties to C-5 and/or C-6 of cytosine (e.g., 5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine) and the addition of electron-withdrawing moieties to C-5 and/or C-6 of uracil (e.g., 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil). See, e.g., PCT publication No. WO 99/62923.

The nucleic acid moiety may also comprise a phosphate modified nucleotide. The synthesis of nucleic acids comprising modified phosphate or non-phosphate linkages is also well known in the art. For a review see "Oligonucleotides as Therapeutic Agents" (edited by d.j.chadwick and g.cardew) John Wiley and Sons, New York, Matteucci (1997) "oligonucleotide analogues in NY: overview "(" Oligonucleotide Analogs: overview "). The phosphorus derivative (or modified phosphate group) that can be attached to the sugar or sugar analog moiety in a nucleic acid of the invention can be a monophosphate, diphosphate, triphosphate, alkylphosphate, phosphorothioate, phosphorodithioate, phosphoramidate, or the like. The preparation of the above-mentioned phosphate analogues and their incorporation into nucleotides, modified nucleotides and oligonucleotides is also known per se and need not be described in detail here. Peyrottes et al (1996) Nucleic acid research (Nucleic Acids Res.) 24: 1841-1848; chaturvedi et al (1996) Nucleic acid research (Nucleic Acids Res.) 24: 2318-2323; and Schultz et al (1996) Nucleic acid research (Nucleic Acids Res.) 24: 2966-2973. For example, Synthesis of phosphorothioate Oligonucleotides is similar to that of the natural Oligonucleotides described above, except that the oxidation step is replaced by a sulfurization step (methods, Synthesis and characterization for Oligonucleotides and Analogs (Agrawal eds.) Zon (1993) "oligonucleotide Phosphorothioates" in the Humana Press, pp. 165-190). Similarly, the synthesis of other phosphate analogues such as phosphotriester (Miller et al (1971) JACS 93: 6657-6665), non-bridged phosphoramidate (Jager et al (1988) biochemistry (Biochem.) 27: 7247-7246), N3 'to P5' phosphoraramidates (Nelson et al (1997) JOC 62: 7278-7287) and phosphorodithioate (U.S. Pat. No. 5,453,496) have been described. Other non-phosphorus based modified Nucleic Acids may also be used (Nucleic Acids Res.) 17: 6129-6141 by Stirchak et al (1989). Nucleic acids with phosphorothioate backbones appear to be more resistant to degradation upon injection into a host. Braun et al (1988) journal of immunology (j. immunoli.) 141: 2084-2089; and Latimer et al (1995) molecular immunology (mol. Immunol.) 32: 1057-1064.

The nucleic acid moieties for use in the present invention may comprise ribonucleotides (comprising ribose as the sole or primary sugar component) and/or deoxyribonucleotides (comprising deoxyribose as the primary sugar component). Modified sugars or sugar analogs may be incorporated into the nucleic acid moieties. Thus, the sugar moiety may be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, and sugar "analog" cyclopentyl group in addition to ribose and deoxyribose. The sugar may be of the pyran or furan type. The sugar moiety is preferably a furanoside of ribose, deoxyribose, arabinose, or 2' -O-alkylribose, and the sugar may be attached to each heterocyclic base in either an alpha or beta anomeric configuration. Sugar modifications include, but are not limited to, 2 ' -alkoxy-RNA analogs, 2 ' -amino-RNA analogs, and 2 ' -alkoxy-or amino-RNA/DNA chimeras. For example, modifications of sugars in CIC include, but are not limited to, 2 '-amino-2' -deoxyadenosine. The preparation of these sugars or sugar analogs, and the corresponding "nucleosides" (wherein such sugars or analogs are linked to a heterocyclic base (nucleobase)) is known per se and need not be described herein, unless such preparation may involve any particular example. Sugar modifications may also be made in the preparation of CIC in combination with any phosphate modifications.

The heterocyclic bases or nucleobases incorporated into the nucleic acid moieties can be naturally occurring primary purine and pyrimidine bases (i.e., uracil, thymine, cytosine, adenine and guanine, as described above), as well as natural and synthetic modifications of the primary bases.

One skilled in the art will recognize that a number of "synthetic" non-natural nucleosides containing various heterocyclic bases and various sugar moieties (and sugar analogs) are readily available in the art, and that the nucleic acid moiety may include one or more heterocyclic bases that are not part of this main five natural nucleobase component, so long as the other requirements of the invention are met. Preferably, however, the heterocyclic base is, but not limited to, uracil-5-yl, cytosine-5-yl, adenine-7-yl, adenine-8-yl, guanine-7-yl, guanine-8-yl, 4-aminopyrrolo [2.3-d ] pyrimidin-5-yl, 2-amino-4-oxopyrrolo [2, 3-d ] pyrimidin-5-yl or 2-amino-4-oxopyrrolo [2, 3-d ] pyrimidin-3-yl, wherein the purine is linked to the sugar moiety in the nucleic acid moiety through the 9-position, the pyrimidine through the 1-position, the pyrrolopyrimidine through the 7-position and the pyrazolopyrimidine through the 1-position.

The nucleic acid moiety may comprise at least one modified base. As used herein, the term "modified base" is synonymous with "base analog", e.g., "modified cytosine" is synonymous with "cytosine analog". Similarly, "modified" nucleoside or nucleotide is defined herein as synonymous with nucleoside or nucleotide "analog". Examples of base modifications include, but are not limited to, the addition of electron-withdrawing moieties to C-5 and/or C-6 of cytosines in a nucleic acid moiety. Preferably, the electron-withdrawing moiety is a halogen. Such modified cytosines can include, but are not limited to, azacytosine, 5-bromocytosine, 5-chlorocytosine, chlorinated cytosine, cyclic cytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, 5, 6-dihydrocytosine, 5-iodocytosine, 5-nitrocytosine, and any other pyrimidine analog or modified pyrimidine. Other examples of base modifications include, but are not limited to, the addition of electron-withdrawing moieties to C-5 and/or C-6 of uracil in a nucleic acid moiety. Preferably, the electron-withdrawing moiety is a halogen. Such modified uracils may include, but are not limited to, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil. See also kandmilia et al, 2001, bioorg.med.chem.9: 807-13.

Other examples of base modifications include the addition of one or more sulfur-containing groups to the base, including, but not limited to, 6-thioguanine, 4-thiothymine, and 4-thiouracil.

The preparation of nucleosides having modified bases and the synthesis of modified oligonucleotides using the nucleosides having modified bases as precursors has been described, for example, in U.S. Pat. nos. 4,910,300, 4,948,882 and 5,093,232. These nucleosides with modified bases are designed so that they can be incorporated into the ends or interior of the oligonucleotide by chemical synthesis. Such nucleosides with modified bases present at the end or within the oligonucleotide can serve as sites for attachment of peptides or other antigens. Nucleosides in which the sugar moiety is modified have also been described (including but not limited to, for example, U.S. patents 4,849,513, 5,015,733, 5,118,800, 5,118,802) and can be similarly used.

4. Non-nucleic acid spacer moieties

The CIC compounds of the invention comprise one or more non-nucleic acid spacer moieties covalently bound to a nucleic acid moiety. For convenience, the non-nucleic acid spacer moiety is sometimes referred to herein simply as a "spacer" or "spacer moiety".

The spacer typically has a molecular weight of from about 50 to about 500,000 (e.g., about 50 to about 50,000), sometimes from about 75 to about 5000, sometimes from about 75 to about 500, and in various embodiments is covalently bound to one, two, three, or more than three nucleic acid moieties. There are a variety of reagents suitable for attaching nucleic acid moieties. For example, a variety of compounds referred to in the scientific literature as "non-nucleic acid linkers", "non-nucleotide linkers", or "valency platform molecules" may be used as spacers in a CIC. If a spacer moiety is said to contain a particular spacer component (e.g., hexapolyethylene glycol), then the spacer comprises that component (or substituted derivative) as a subunit or part of the spacer. For example, the spacer shown in example 49 can be described as comprising a polysaccharide component, a hexapolyethylene glycol component, and a derivatized thioether linker component. As described below, in certain embodiments, the spacer comprises a plurality of subunits covalently linked together and may have a homopolymer structure or a heteropolymeric structure. Often these subunits are linked by a linker, phosphodiester linkage, and/or phosphorothioate linkage. See the examples below. A CIC non-nucleotide spacer moiety comprising or derived from a plurality of such units may be referred to as a "composite spacer". In one embodiment, for purposes of illustration and not limitation, a CIC comprises a composite spacer that contains any two or more (e.g., 3 or more than 3, 4 or more than 4, or 5 or more than 5) of the following compounds linked together via phosphodiester and/or phosphorothioate linkages: an oligo (ethylene glycol) unit (e.g., a tri (ethylene glycol) spacer; a hexa (ethylene glycol) spacer;); alkyl units (e.g., propyl spacer; butyl spacer; hexyl spacer); branched spacers (e.g., 2- (hydroxymethyl) ethyl spacers; glycerol spacers; trebler spacers; symmetric dyads (doubler) spacers).

It will be understood that single nucleotides and polynucleotides are not included within the definition of a non-nucleic acid spacer, otherwise nucleic acid moieties and adjacent non-nucleic acid spacer moieties will not be distinguishable.

Many different spacers are described herein, but are not limited thereto for purposes of illustration only. The reader will appreciate that, for ease of description, the spacer moieties (or components of the spacer moieties) are sometimes referred to by the chemical name of the compound from which the spacer moiety or component thereof is derived (e.g., hexapolyethylene glycol), and it will be understood that CIC actually comprises conjugates of these compounds with nucleic acid moieties. As will be apparent to those skilled in the art (and as described in further detail below), the non-nucleic acid spacer may be (and typically is) formed from a spacer moiety precursor that comprises a reactive group that allows one or more nucleic acids (e.g., oligonucleotides) to be coupled to the spacer moiety precursor to form a CIC and may comprise a protecting group. The reactive groups on the spacer precursors may be the same or different.

Exemplary non-nucleic acid spacers include oligo-ethylene glycol (e.g., tri-polyethylene glycol, tetra-polyethylene glycol, hexa-polyethylene glycol spacers, and other polymers containing up to about 10, about 20, about 40, about 50, about 100, or about 200 ethylene glycol units), alkyl spacers (e.g., propyl, butyl, hexyl, and other C2-C12 alkyl spacers, such as typically C2-C10 alkyl, most commonly C2-C6 alkyl), symmetrical or asymmetrical spacers derived from glycerol, pentaerythritol, 1, 3, 5-trihydroxycyclohexane, or 1, 3-diamino-2-propanol (e.g., symmetrical double and triple doubler spacer moieties described herein). Optionally these spacer components are substituted. For example, glycerol and 1, 3-diamino-2-propanol may be substituted at the 1, 2, and/or 3 positions (e.g., by replacing one or more of the carbon-attached hydrogens with one of the following groups), as would be understood by one of ordinary skill in the art. Likewise, pentaerythritol may be substituted at any or all of the methylene positions with any of the following groups. Substituents include alcohols, alkoxy groups (such as methoxy, ethoxy, and propoxy), straight or branched alkyl groups (such as C1-C12 alkyl), amines, aminoalkyl groups (such as amino C1-C12 alkyl), phosphoramidites, phosphates, phosphoramidates, dithiophosphates, thiophosphates, hydrazides, hydrazines, halogens (such as F, Cl, Br, or I), amides, alkylamides (such as C1-C12 alkylamides), carboxylic acids, carboxylic esters, carboxylic anhydrides, carboxylic acid halides, ethers, sulfonyl halides, imidoates, isocyanates, isothiocyanates, haloformates, carbodiimide adducts, aldehydes, ketones, mercapto, haloacetyl, alkyl halides, alkyl sulfonates, NR1R2 (where R1R2 is-C (═ O) CH ═ CHC (═ O)) (maleimide), thioethers, cyano groups, sugars (such as mannose, galactose, and glucose), thioethers, ketones, and ketones, Alpha, beta-unsaturated carbonyl, alkyl mercury, alpha, beta-unsaturated sulfone.

In one embodiment, a spacer may contain one or more abasic nucleotides (i.e., lacking nucleotide bases but having sugar and phosphate moieties). Exemplary abasic nucleotides include 1 '2' -dideoxyribose, 1 '-deoxyribose, 1' -deoxyarabinose, and polymers thereof.

The spacer can comprise homo-or hetero-polymeric oligomers and polymers of the non-nucleic acid components described herein (e.g., linked via phosphodiester or phosphorothioate linkages or, alternatively, amide, ester, ether, thioether, disulfide, phosphoramidate, phosphotriester, dithiophosphate, methylphosphonate, or other linkages). For example, in one embodiment, the spacer moiety contains a branched spacer component (e.g., glycerol) conjugated to an oligo-ethylene glycol, such as HEG, via a phosphodiester or phosphorothioate linkage (see, e.g., C-94). Another example is a spacer comprising a multivalent spacer component conjugated to an oligoethylene dimer, such as HEG.

Other suitable spacers include substituted alkyl groups, substituted polyglycols, optionally substituted polyamines, optionally substituted polyols, optionally substituted polyamides, optionally substituted polyethers, optionally substituted polyimines, optionally substituted polyphosphodiesters (e.g., poly (1-phospho-3-propanol)), and the like. Optional substituents include alcohols, alkoxy groups (e.g., methoxy, ethoxy, and propoxy), straight or branched alkyl groups (e.g., C1-C12 alkyl), amines, aminoalkyl groups (e.g., amino C1-C12 alkyl), phosphoramidites, phosphate esters, thiophosphate esters, hydrazides, hydrazines, halogens (e.g., F, Cl, Br, or I), amides, alkylamides (e.g., C1-C12 alkylamides), carboxylic acids, carboxylic acid esters, carboxylic acid anhydrides, carboxylic acid halides, ethers, sulfonyl halides, imidoesters, isocyanates, isothiocyanates, haloformates, carbodiimide adducts, aldehydes, ketones, mercapto groups, haloacetyl groups, alkyl halides, alkyl sulfonates, NR1R2 (where R1R2 is-C (═ O) CH ═ CHC (═ O)) (maleimide), thioethers, cyano groups, sugars (e.g., mannose, galactose, and glucose), α, β -unsaturated carbonyl groups, amino groups, and amino groups, Alkyl mercury, alpha, beta-unsaturated sulfone.

Other suitable spacers may include polycyclic molecules, such as those containing phenyl or cyclohexyl rings. The spacer may be a polyether such as polypropylene glycol, polyethylene glycol, polypropylene glycol, a bifunctional polycyclic molecule such as bifunctional pentalene, indene, naphthalene, azulene, heptaleneBiphenylene, asymmetric indacene, symmetric indacene, acenaphthene, fluorene, phenalene, phenanthrene, anthracene, fluoranthene, acenaphthylene, aceanthrylene, triphenylene, pyrene, perylene, and mixtures thereof,Tetracene, thianthrene, isobenzofuran, chromene, xanthene, benzoxathia-hexadiene, which may be substituted or modified, or may be a combination of a polyether and a polycyclic molecule. The polycyclic molecules may be substituted or polysubstituted with C1-C75 alkyl, C6 alkyl, alkenyl, hydroxyalkyl, halogen or haloalkyl groups. Nitrogen-containing polyheterocycles (e.g., indolizine) are generally not suitable spacers. The spacer may also be a polyol such as glycerol or pentaerythritol. In one embodiment, the spacer comprises (1-phosphopropane)₃-phosphoric acid esters or (1-phosphoric acid propane)₄Phosphate esters (also known as propylene glycol tetraphosphate and propylene glycol pentaphosphate). In one embodiment, the spacer comprises a derivative of 2, 2' -Ethylenedioxydiethylamine (EDDA).

Other exemplary non-nucleic acid spacers for CIC include "linkers" as described in the following references: cload and Schepartz, j.am.chem.soc. (1991), 113: 6324; richardson and Schepartz, j.am.chem.soc. (1991), 113: 5109, preparing a solution; ma et al, Nucleic acids research (1993), 21: 2585; ma et al, Biochemistry (1993), 32: 1751, preparing a mixture of water and an organic solvent; McCurdy et al, Nucleotides & Nucleotides (1991), 10: 287; jaschke et al, Tetrahedron Lett, (1993), 34: 30l of the mixture; ono et al, Biochemistry (1991), 30: 9914 of the raw materials; and Arnold et al, International publication Nos. WO89/02439 and EP0313219B1 titled "non-nucleic acid ligation reagent for nucleotide probes"; salenkhe et al, j.am.chem.soc. (1992), 1l 4: 8768; nelson et al, Biochemistry, 35: 5339-5344 (1996); bartley et al, Biochemistry 36: 14502-511 (1997); dagneeaux et al Nucleic Acids Research 24: 4506-12 (1996); durand et al, Nucleic Acids Research 18: 6353-59 (1990); reynolds et al, Nucleic Acids Research, 24: 760-65 (1996); hendry et al Biochemica Acta, 1219: 405-12 (1994); altmann et al, nucleic acids Research, 23: 4827-35(1995), and U.S. Pat. No. 6,117,657(Usman et al).

Suitable spacer moieties may provide charge and/or hydrophobicity to the CIC, provide favorable pharmacokinetic properties (e.g., increased stability, longer residence time in the blood), and/or cause the CIC to target specific cells or organs. Spacer moieties may be selected or modified to tailor the CIC to the desired pharmacokinetic properties, induction of a particular immune response, or to the desired mode of administration (e.g., oral).

In a CIC comprising more than one spacer moiety, the spacers may be the same or different. Thus, in one embodiment all non-nucleic acid spacer moieties in a CIC have the same structure. In one embodiment, the CIC comprises non-nucleic acid spacer moieties having at least 2, at least 3, at least 4, at least 5, or at least 6 or more different structures.

In some embodiments contemplated by the present invention, the spacer structure portion of the CIC is specified to not include certain structures. Thus, in some embodiments of the invention, the spacer is not an abasic nucleotide or a polymer of abasic nucleotides. In some embodiments of the invention, the spacer is not an oligo (ethylene glycol) (e.g., HEG, TEG, etc.) or a poly (ethylene glycol). In some embodiments the spacer is not a C3 alkyl spacer. In some embodiments the spacer is not an alkyl or substituted spacer. In some embodiments the spacer is not a polypeptide. Thus, in some embodiments, an immunogenic molecule, such as a protein or polypeptide, is not suitable as a component of a spacer moiety. However, as described below, it is contemplated that in certain embodiments, the CIC is a "protein-like CIC," i.e., comprises a spacer moiety comprising a polypeptide (i.e., an oligomer or polymer of amino acids). For example, as described below, in some embodiments, a polypeptide antigen can be conjugated to multiple nucleic acid moieties as a platform (multivalent spacer). However, in some embodiments, the spacer moiety is not proteinaceous and/or not antigenic (i.e. the spacer moiety, if isolated from the CIC, is not antigenic).

Suitable spacer moieties do not render CIC, which is a component thereof, insoluble in aqueous solutions (e.g., PBS, pH 7.0). Thus, the definition of spacer excludes microcarriers or nanocarriers. Furthermore, low solubility spacer moieties, such as dodecyl spacer (solubility < 5mg/ml when measured as the diol precursor 1, 12-dihydroxydodecane), are not preferred because it reduces the hydrophilicity and activity of the CIC. Preferably, the spacer moiety has a solubility, e.g., as measured as a diol precursor, that is much greater than 5mg/ml (e.g., a solubility of at least about 20mg/ml, at least about 50mg/ml, or at least about 100 mg/ml). The form of the spacer moiety used in detecting the solubility of the spacer moiety is generally the most closely related inactive and unprotected spacer precursor molecule. For example, C-19 comprises a spacer moiety comprising a dodecyl group having phosphorothioate diester linkages at the C-1 and C-12 positions, thereby linking the spacer moiety to a nucleic acid moiety. In this case, the water solubility of dodecyl spacer (1, 12-dihydroxydodecane) in the form of a diol was examined and found to be less than 5 mg/ml. Spacers with higher water solubility (when detected with their diol precursors) may result in CICs with greater immune stimulation. Such highly water soluble spacers include, without limitation, propane-1, 3-diol, glycerol, butane-1, 4-diol, pentane-1, 5-diol, hexane-1, 6-diol, tripethylene glycol, tetraethylene glycol, and HEG.

A. Charged multi-cell spacer structure moiety

The charge of the CIC may be provided by phosphate, phosphorothioate, or other groups in the nucleic acid moiety as well as groups in the non-nucleic acid spacer moiety. In some embodiments of the invention, the non-nucleic acid spacer moiety has a net charge (e.g., a net positive charge or a net negative charge as measured at pH 7). In one useful embodiment, the CIC has a net negative charge. In some embodiments, the negative charge of a CIC spacer moiety can be increased by derivatizing the spacer subunits described herein to increase their charge. For example, glycerol may be covalently bound to both nucleic acid moieties, while the remaining alcohol may be reacted with an activated phosphoramidite followed by oxidation or sulfurization to form a phosphate or phosphorothioate, respectively. In certain embodiments, the negative charge (i.e., the total amount of charge when there is more than one spacer) provided by the non-nucleic acid spacer moiety of the CIC is greater than the negative charge provided by the nucleic acid moiety of the CIC. The charge can be calculated from molecular formula or determined experimentally, e.g.by capillary Electrophoresis (Li, ed., 1992, capillary Electrophoresis, Principles, operation and Application (Electrophoresis, Principles, Practice and Application), Elsevier sciences publishers, Amsterdam, The Netherlands, pp 202-206).

As noted above, suitable spacers may be polymers of smaller non-nucleic acid (e.g., non-nucleotide) compounds, see those described herein, which themselves may serve as spacers, including compounds commonly referred to as non-nucleotide "linkers. Such polymers (i.e., "multi-unit spacers") can be hetero-or homo-polymers, and often comprise monomeric units (e.g., HEG, TEG, glycerol, 1 ', 2' -dideoxyribose, etc.) linked by ester linkages (e.g., phosphodiester or phosphorothioate). Thus, in one embodiment the spacer comprises a polymeric (e.g., heteropolymer) structure of non-nucleotide units (e.g., 2 to about 100 units, or 2 to about 50, such as 2 to about 5, or about 5 to about 50, such as about 5 to about 20 units).

For illustration, a CIC containing a multi-cell spacer includes

5′-TCGTCG-(C3)₁₅-T

5' -TCGTCG- (Glycerol)₁₅-T

5′-TCGTCG-(TEG)₈-T

5′-TCGTCG-(HEG)₄-T

Wherein (C)₃)₁₅Refers to 15 propyl linkers linked via phosphorothioate; (Glycerol)₁₅Refers to 15 glycerol linkers linked via phosphorothioate; (ii) a (TEG)₈Refers to 8 tri-polyethylene glycol linkers linked via phosphorothioate; (HEG)₄Refers to 4 hexapolyethylene glycol linkers attached via a phosphorothioate linkage. It should be appreciated that certain multi-unit spacers have a net negative charge, and the amount of negative charge can be increased by increasing the number of, e.g., ester-linked, monomer units.

B. Multivalent spacer moieties

In certain embodiments, the spacer moiety is a multivalent, non-nucleic acid spacer moiety (i.e., a "multivalent spacer"). As used herein, a CIC comprising a multivalent spacer comprises a spacer covalently bound to three (3) or more nucleic acid moieties. Multivalent spacers are sometimes referred to in the art as "platform molecules". The multivalent spacer may be polymeric or non-polymeric. Exemplary suitable molecules include glycerol or substituted glycerols (e.g., 2-hydroxymethylglycerol, levulinyl-glycerol); tetraaminobenzene, heptaamino- β -cyclodextrin, 1, 3, 5-trihydroxycyclohexane, pentaerythritol and pentaerythritol derivatives, tetraaminopentaerythritol, 1, 4, 8, 11-tetraazacyclotetradecane (Cyclam), 1, 4, 7, 10-tetraazacyclododecane (Cyclen), polyethyleneimine, 1, 3-di-amino-2-propanol and substituted derivatives (e.g., "symmetrical double doublets"), [ propyloxymethyl ] ethyl compounds (e.g., "triple doublets"), polyethylene glycol derivatives such as the so-called "StarPEG" and "bPEG" (see, for example, Gnanou et AL, 1988, Makromol. chem.189: 2885; Rein et AL, 1993, Acta Polymer 44: 225, Merrill et AL, U.S. Pat. No. 5,171,264; Shearwater Polymers Inc., Huntsville AL), dendrimers, and polysaccharides.

Dendrimers (Dendrimers) are known in the art, and are chemically defined globular molecules, generally prepared by stepwise or repeated reactions of multifunctional monomers to give a branched structure (see, for example, Tomalia et al, 1990, Angew. chem. int. Ed. Engl 29: 138-75). Many dendrimers are known, such as amine-terminated polyaminoamines, polyethyleneimine and polypropyleneimine dendrimers. Exemplary dendrimers suitable for use in the present invention include "dense star" polymers or "starburst" polymers, as described in U.S. Pat. nos. 4,587,329; 5,338,532, respectively; and 6,177,414, including the so-called "poly (aminoamine) (" PAMAM ") dendrimers". Still other multimeric spacer molecules suitable for use in the present invention include chemically defined non-polymeric valency platform molecules, as described in U.S. Pat. nos. 5,552,391; and PCT application PCT/US00/15968 (published as WO 00/75105); PCT/US96/09976 (published as WO 96/40197), PCT/US97/10075 (published as WO 97/46251); PCT/US94/10031 (published as WO 95/07073); and those disclosed in PCT/US99/29339 (published as WO 00/34231). Many other suitable multivalent spacers may also be utilized and will be known to those skilled in the art.

Conjugation of the nucleic acid moiety to the platform molecule can be achieved in a variety of ways, typically involving one or more cross-linkers and functional groups on the nucleic acid moiety and the platform molecule. The linking group can be added to the platform using standard synthetic chemistry techniques. The linking group can be added to the nucleic acid moiety using standard synthetic techniques.

Multivalent spacers having various valencies may be used in the practice of the invention, and in various embodiments the multivalent spacer of a CIC is associated with from about 3 to about 400 nucleic acid moieties, sometimes from about 100 to about 500, sometimes from about 150 to about 250, sometimes 3-200, sometimes 3 to 100, sometimes 3-50, often 3-10, and sometimes more than 400 nucleic acid moieties. In many different embodiments, a multivalent spacer is conjugated to more than 10, more than 25, more than 50, more than 100, or more than 500 (which may be the same or different) nucleic acid moieties. It will be appreciated that in certain embodiments, when CIC contains multivalent spacers, the present invention provides a group of CIC with slight differences in molecular structure. For example, when CICs are prepared using dendrimers, polysaccharides or other multivalent spacers with high valency, a slightly heterogeneous mixture of molecules may result, i.e. molecules comprising different numbers (within or predominantly within a measurable range) of nucleic acid moieties attached to such multivalent spacer moieties. If a dendrimer, polysaccharide, or the like is used as an element of a multivalent spacer, the nucleic acid moiety may be directly or indirectly attached to such element (e.g., dendrimer). For example, a CIC may comprise a nucleic acid moiety linked to a dendrimer through a low polyethylene glycol element (where dendrimer + low polyethylene glycol constitutes a spacer moiety). It will be appreciated that the nucleic acid moiety may be conjugated to more than one spacer moiety, as described in § III (1) B above.

Polysaccharides derivatized to enable attachment of nucleic acid moieties may be used as multivalent spacers in CICs. Suitable polysaccharides may be natural or synthetic. Exemplary polysaccharides include, for example, dextran, mannin, chitosan, agarose and starch. For example, mannin can be utilized, because of the mannin (mannose) receptor on immunologically relevant cell types, such as monocytes and alveolar macrophages, this polysaccharide spacer moiety can be used to target specific cell types. In one embodiment, the polysaccharide is cross-linked. One suitable compound is epichlorohydrin crosslinked sucrose (e.g., FICOLL)^_)。FICOLL^_Synthesized by crosslinking sucrose with epichlorohydrin to give a highly branched structure. For example, as shown in example 49, aminoethylcarboxymethyl-Ficoll (AECM-Ficoll) can be prepared using the following protocols imman, 1975, j.imm.114: 704 and 709. The number of nucleic acid moieties in the polysaccharide-containing CIC can be any of the ranges as described herein for CIC (e.g., multivalent CIC). For example, in one embodiment, the polysaccharide comprises from about 150 to about 250 nucleic acid moieties. Thus, AECM-Ficoll can be reacted with a heterobifunctional (heterobifunctional) crosslinker, such as 6-maleimidocaproyl N-hydroxysuccinimide ester, and conjugated to a thiol-derivatized nucleic acid moiety (see Lee et al, 1980, mol. Imm.17: 749-56). Other polysaccharides may be similarly modified.

Synthesis of CIC

It is well within the ability of those skilled in the art to prepare CIC by conventional methods in light of the present disclosure and the knowledge in the art. Techniques for preparing nucleic acid moieties (e.g., oligonucleotides and modified oligonucleotides) are known. Techniques available for synthesizing nucleic acid moieties include, but are not limited to, enzymatic and chemical methods and combinations of enzymatic and chemical methods. For example, DNA or RNA containing phosphodiester bonds can be synthesized by chemical methods by sequentially coupling the appropriate nucleoside phosphoramidite to the 5 '-hydroxyl of a growing oligonucleotide having its 3' -terminus attached to a support, followed by oxidation to convert the intermediate phosphite triester to a phosphotriester. Solid phase supports that can be used for DNA synthesis include controlled pore glass (Applied Biosystems, Foster City, Calif.), polystyrene bead matrix (Primer Support, Amersham Pharmacia, Piscataway, NJ) and TentGel (Rapp Polymer GmbH, Tubingen, Germany). Once the desired oligonucleotide sequence has been synthesized, the oligonucleotide sequence can be removed from the support, the phosphotriester group deprotected to a phosphodiester and the nucleobase deprotected using ammonia or other base.

For example, a DNA or RNA polynucleotide (nucleic acid moiety) containing phosphodiester bonds is generally synthesized by repeating the following steps repeatedly: a) removing the protecting group from the 5 '-hydroxyl group of the 3' nucleoside or nucleic acid bound to the solid support, b) coupling an activated nucleoside phosphoramidite to the 5 '-hydroxyl group, c) oxidizing the phosphotriester to a phosphotriester, and d) capping the unreacted 5' -hydroxyl group. DNA or RNA containing phosphorothioate linkages may be prepared as described above, except that the oxidation step is replaced with a sulfurization step. Once the desired oligonucleotide sequence is synthesized, the oligonucleotide sequence can be removed from the support, the phosphotriester group deprotected to a phosphodiester and the nucleobase deprotected using ammonia or other base. Examples are given in Beaucage (1993) PROTOCOLS FOROLIGONUCLEOTIEDES AND ANALOGS, SYNTHESIS ANDPROPERTIES (Agrawal, ed.) for "Synthesis of oligodeoxyribonucleotides" Humana Press, Totowa, NJ; warner et al (1984) DNA 3: 401; tang et al (2000) org. Process Res. Dev.4: 194-198; wyrzykiewica et al (1994) Bioorg. & med. chem. lett.4: 1519-; radhakrishna et al (1989) J.org chem.55: 4693-4699 and U.S. Pat. No. 4,458,066. Programmable devices capable of automatically synthesizing nucleic acid moieties having specific sequences are ubiquitous. Examples include Expedite8909 automated DNA synthesizer (Perseptive Biosystems, Framington MA), ABI 394(Applied Biosystems, Inc., Foster City, Calif.), and OligoPilot II (Amersham Pharmacia Biotech, Piscataway, NJ).

For example, polynucleotides can be assembled in the 3 'to 5' direction using nucleosides (monomers) that contain an acid labile 5 '-protecting group and a 3' -phosphoramidite and that are base protected. Exemplary such monomers include 5 ' -O- (4, 4 ' -dimethoxytrityl) -protected nucleoside-3 ' -O- (N, N-diisopropylamino) 2-cyanoethyl phosphoramidite, wherein exemplary protected nucleosides include, but are not limited to, N6-benzoyl adenosine, N4-benzoyl cytidine, N2-isobutyrylguanosine, thymidine, and uridine. In this case, the solid support used comprises a 3' -linked protected nucleoside. Alternatively, polynucleotides can be assembled in the 5 'to 3' direction using protected nucleosides containing acid labile 3 '-protecting groups and bases of 5' -phosphoramidites. Exemplary such monomers include 3 ' -O- (4, 4 ' -dimethoxytrityl) -protected nucleoside-5 ' -O- (N, N-diisopropylamino) 2-cyanoethylphosphamide, wherein exemplary protected nucleosides include, but are not limited to, N6-benzoyladenosine, N4-benzoylcytidine, N2-isobutyrylguanosine, thymidine, and uridine (Glen research, Sterling, Va.). In this case, the solid support used comprises a 5' -linked protected nucleoside. The circular nucleic acid component can be isolated or synthesized by recombinant means or chemically. Chemical synthesis can be carried out using any of the methods described in the literature, see for example Gao et al (1995) Nucleic Acids Res.23: 2025-2029 and Wang et al (1994) Nucleic Acids Res.22: 2326-2333.

Depending on the particular CIC to be prepared, the nucleic acid moiety and spacer moiety may be conjugated by a variety of methods. Adding specific spacer featuresMethods are known in the art and are described, for example, in the references cited above. See, for example, Durand et al, Nucleic Acids Research 18: 6353-59(1990). The covalent linkage between the spacer moiety and the nucleic acid moiety can be any of a variety of linkage forms including phosphodiester, phosphorothioate, amide, ester, ether, thioether, disulfide, phosphoramidite, phosphotriester, phosphorodithioate, methylphosphonate, and other types of linkages. As described above, the spacer moiety precursor may optionally be modified with a group that activates the terminus for coupling to a nucleic acid. Exemplary activated spacer moieties are shown in FIG. 1, to which a protecting group suitable for use in automated synthesis has been added. Other spacer moiety precursors include, for example, but are not limited to, (1) HOCH₂CH₂O(CH₂CH₂O)_nCH₂CH₂OH, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 or greater than 45; (2) HOCH ₂CHOHCH₂OH；(3)HO(CH₂)_nOH, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.

In one embodiment, the spacer moiety precursor utilized comprises first and second reactive groups to allow stepwise conjugation with the nucleic acid moiety, wherein the first reactive group has properties enabling it to be efficiently coupled to the end of a growing strand of nucleic acid, and the second reactive group is capable of stepwise further extending the growing strand of mixed nucleotide and non-nucleotide moieties in the CIC. It is often convenient to combine the spacer moiety and the nucleic acid moiety using the same phosphoramidite type chemistry used for the synthesis of the nucleic acid moiety. For example, CIC of the present invention can be conveniently synthesized by means of an automated DNA synthesizer (e.g., Expedite 8909; Perseptive Biosystems, Framington, Mass.) using phosphoramidite Chemistry (see Beaucage, 1993, supra; Current Protocols in Nucleic Acid Chemistry, supra). However, it will be apparent to those skilled in the art that the same (or equivalent) synthesis steps performed by an automated DNA synthesizer may also be performed manually, if desired. The resulting linkage between the nucleic acid and the spacer precursor may be a phosphorothioate linkage or a phosphodiester linkage. In such syntheses, typically, one end of the spacer (or spacer subunit of a multimeric spacer) is protected with a 4, 4' -dimethoxytrityl group, while the other end comprises a phosphoramidite group.

Many spacers with useful protecting and reactive groups are available in the market, for example:

tri-polyethylene glycol spacer or "TEG spacer"9-O- (4, 4' -dimethoxytrityl) tripethylene glycol-1-O- [ (2-cyanoethyl) N, N-diisopropylphosphoramidite](Glen Research，22825Davis Drive，Sterling，VA)；

Hexapolyethyleneglycol spacer or "HEG spacer"18-O- (4, 4' -dimethoxytrityl) hexapolyethylene glycol-1-O- [ (2-cyanoethyl) N, N-di-isopropylphosphoramidite](Glen Research，Sterling，VA)；

Propyl spacer3- (4, 4' -Dimethoxytrityloxy) propoxy-1-O- [ (2-cyanoethyl) N, N-diisopropylphosphoramidite](Glen Research，Sterling，VA)；

Butyl spacer4- (4, 4' -dimethoxytrityloxy) butoxy-1-O-](2-cyanoethyl) N, N-diisopropylphosphoramidite](Chem Genes Corporation，Ashland TechnologyCenter，200 Homer Ave，Ashland，MA)；

Hexyl spacer: 6- (4, 4' -Dimethoxytrityloxy) hexyloxy-1-O- [ (2-cyanoethyl) N, N-diisopropylphosphoramidite](Biosearch Technologies，Novoto，CA)

2- (hydroxymethyl) ethyl spacer or "HME spacer1-(4，4′-Dimethoxytrityloxy) -3- (acetylpropionyloxy) -propoxy-2-O- [ (2-cyanoethyl) N, N-diisopropylphosphoramidite](ii) a Also referred to as "asymmetric branch" spacers (see fig. 2) (Chem Genes corp., Ashland technology company, Ashland MA.);

"abasic nucleotide spacer" or "abasic spacer5-O- (4, 4' -dimethoxytrityl) -1, 2-dideoxyribose-3-O- [ (2-cyanoethyl) N, N-diisopropylphosphoramidite](GlenResearch，Sterling，VA)；

"symmetrical branched spacer" or "glycerol spacer1, 3-O, O-bis- (4, 4' -dimethoxytrityl) glycerol-2-O- [ (2-cyanoethyl) N, N-diisopropylphosphoramidite](Chem Genes, Ashland, MA) (see fig. 2);

"triple doublet spacer(see FIG. 2)2, 2, 2-O, O, O-tris [3-O- (4, 4' -dimethoxytrityloxy) propoxymethyl]Ethyl-1-O- [ (2-cyanoethyl) N, N-diisopropylphosphoramidite](Glen Research，Sterling，VA)；

"symmetrical double doubler spacer(see, FIG. 2)1, 3-O, O-bis [5-O- (4, 4' -dimethoxytrityloxy) pentanoylamino]propyl-2-O- [ (2-cyanoethyl) N, N-diisopropylphosphoramidite](Glen Research，Sterling，VA)；

Dodecyl spacer12(4, 4' -dimethoxytrityloxy) dodecyloxy-1-O- [ (2-cyanoethyl) N, N-diisopropylphosphoramidite](Glen Research，Sterling，VA)。

These and numerous other protected spacer moiety precursors (e.g., containing DMT and phosphoramidite protecting groups) are commercially available or can be synthesized using conventional methods for preparing CIC disclosed herein. The instrument can be programmed according to the manufacturer's instructions to add the nucleotide monomers and spacers in the desired order.

The preparation of CIC "in situ" on a DNA synthesizer requires a protected nucleoside and a protected spacer monomer, both of which contain reactive or activatable functional groups. The reacted and/or protected form of the spacer moiety may be referred to as a "spacer precursor component". The reactive groups in the spacer precursor form stable bonds after coupling and the protecting groups on the spacer precursor are removed in the resulting CIC spacer moiety, as will be apparent to those skilled in the art. The protecting group is typically removed during the stepwise synthesis of the CIC to allow the reaction to proceed at this site. If protecting groups are present on other reactive groups, these protecting groups may be removed after the stepwise synthesis of CIC (as shown for making levulinyl groups on the spacer precursor for C-25 in FIG. 2 Structure 3).

An exemplary spacer precursor without other reactive functional groups is 18-O- (4, 4 '-dimethoxytrityl) hexapolyethylene glycol-1-O- [ (2-cyanoethyl) N, N-diisopropylphosphoramidite ], which contains a protecting group- -4, 4' -dimethoxytrityl group and a reactive group- -the (2-cyanoethyl) N, N-diisopropylphosphoramidite group. In the chemical preparation of CIC using phosphoramidite on a DNA synthesizer, (2-cyanoethyl) N, N-diisopropylphosphoramidite groups in the spacer precursors are activated by a weak acid, such as 1H-tetrazole, and react with the free 5' -hydroxyl group of the nucleobase-protected nucleic acid moiety to form a phosphite triester. The phosphite triester groups are then oxidized or sulfurized to form stable phosphotriester or phosphorothioate triester groups, respectively. The resulting triester groups are stable in the rest of the synthesis of CIC and remain in this form until final deprotection. The 4, 4' -dimethoxytrityl group on the spacer precursor will be removed in order to couple to another spacer precursor or an activated nucleoside monomer that will become part of the next nucleic acid moiety. After coupling and oxidation or sulphurisation reactions, this group also becomes a stable phosphotriester or phosphorothioate triester group, respectively. After the protected CIC is assembled, the CIC can be cleaved from the solid support, the cyanoethyl groups on the phosphotriester or phosphorothioate triester groups are removed, and the nucleobase protection is removed. In this example, the CIC comprises a spacer moiety including a stable phosphodiester or phosphorothioate diester linkage linking the nucleic acid moieties. Both the reactive phosphoramidite group and the protected hydroxyl group of the spacer precursor are converted to stable phosphodiester or phosphorothioate linkages in the spacer moiety. Since the reaction at each end of the spacer is independent, one bond may be a phosphodiester and the other a phosphorothioate diester, or any combination thereof. CIC's with other phosphate modifications, such as phosphorodithioates, methyl phosphates and phosphoramidates, can also be prepared by this method using spacer precursors with appropriate reactive groups, appropriate auxiliaries and protocols designed for such linkages. These schemes are similar to those described for making nucleic acid moieties having modified phosphate linkages.

Although it is convenient to use phosphoramidite chemistry to prepare certain CICs, it should be understood that CICs of the present invention are not limited to compounds prepared by any particular synthesis or preparation method. For example, nucleic acid moieties containing groups incompatible with DNA synthesis and deprotection conditions, such as, but not limited to, hydrazine or maleimide, can be prepared by reacting nucleic acid moieties containing amino linkers with suitable heterobifunctional crosslinkers, such as SHNH (nicotinic acid succinimidylhydrazide) or sulfo-SMCC (4- [ N-maleimidomethyl ] -cyclohexane-l-carboxylic acid sulfosuccinimidyl ester).

Methods of conjugating proteins, peptides, oligonucleotides and small molecules in various combinations are described in the literature and these methods can be modified to achieve conjugation of a nucleic acid moiety containing a reactive linker group to a spacer moiety precursor. See, for example, bioconjugate Techniques (bioconjugate Techniques), Greg t. hermanson, Academic Press, inc., San Diego, CA, 1996. In some embodiments, the nucleic acid moieties are synthesized first, followed by the addition of reactive linking groups (e.g., amino, carboxyl, sulfur-containing groups, disulfide groups, etc.) using standard synthetic chemistry techniques. The reactive linking group (which is believed to form part of the ultimate spacer moiety) may be conjugated to other non-nucleic acid compounds (such as, without limitation, the compounds listed in § 4 above) to form part of the spacer moiety. Reactive linking groups are added to nucleic acids using standard methods of nucleic acid synthesis, which can be performed using a variety of reagents known in the art. Examples include reagents containing protected amino, carboxyl, thiol, disulfide, aldehyde, diol, dienyl, and phosphoramidite groups. Once these compounds are incorporated into nucleic acids via activated phosphoramidite groups and deprotected, they provide amino, carboxylic acid, aldehyde, diol, diene or thiol reactivity, respectively, to the nucleic acids. Exemplary reactive groups for conjugating a nucleic acid moiety comprising a reactive linking group to a spacer moiety precursor comprising a reactive group are shown below.

Nucleic acid reactive groups Spacer moiety precursor reactive group Formed stable connection

Mercaptomaleimides, haloacetylthio-ether linkages

Maleimide mercaptothioether linkages

Mercaptopyridine disulfide bond

Disulfide sulfhydryl pyridine disulfide bond

Amine NHS or other active ester amide bond

Amine carboxyamide bond

Carboxyamine amide bond

Aldehyde, ketone hydrazine, hydrazide hydrazone, hydrazide bonds

Hydrazine, hydrazide aldehyde, ketohydrazone, or hydrazide bonds

Diene dienophile aliphatic or heterocyclic rings

The reactive linking group and the spacer precursor react to form a stable bond and the entire atomic group between the two (or more) nucleic acid moieties is defined as the spacer moiety. For example, a synthesized nucleic acid moiety having a mercaptohexyl group attached thereto via a phosphorothioate group may be reacted with a spacer precursor containing one (or more) maleimide groups to form a thioether bond. The spacer moiety of this CIC includes phosphorothioate and hexyl groups from the linker on the nucleic acid moiety, newly formed thioether linkages, and the remainder of the spacer that was part of the spacer precursor.

Although linear CICs can be prepared using these conjugation strategies, these methods are most commonly used to prepare CICs of branched structure. In addition, spacer precursor molecules with several orthogonal reactive groups can be prepared to allow for the addition of more than one nucleic acid moiety (e.g., different sequence motifs).

In one embodiment, a CIC having a multivalent spacer conjugated with more than one nucleic acid moiety is prepared. For example, platforms containing two maleimide groups (which can react with sulfhydryl-containing polynucleotides) and two activated ester groups (which can react with amino-containing nucleic acids) have been described (see, for example, PCT/US94/10031, published as WO 95/07073). The two activating groups can be reacted independently of each other. This results in a CIC comprising a total of 4 nucleic acid moieties, 2 per sequence.

CICs having two different nucleic acid sequences in a multivalent spacer can also be prepared using a symmetrically branched spacer, as described above, and conventional phosphoramidite reactions (e.g., using manual or automated methods). The symmetrical branched spacer comprises one phosphoramidite group and two identical protecting groups which can be removed simultaneously. For example, in one method, the first nucleic acid is synthesized and coupled to a symmetrically branched spacer, after which the protecting group is removed from the spacer. Two more (homologous) nucleic acids are then synthesized on the spacer (twice the amount of reagent used in each step as compared to the amount used to synthesize a single nucleic acid moiety). This process is described in detail in example 15 below.

Three different nucleic acid moieties (hereinafter nucleic acids I, II, III) can be linked to a multivalent platform (e.g., asymmetric branched spacer) using similar methods. This operation is most conveniently carried out using an automated DNA analyzer. In one embodiment, the asymmetric branching spacer comprises one phosphoramidite group and two orthogonal protecting groups that can be independently removed. First, nucleic acid I is synthesized, an asymmetric branching spacer is coupled to nucleic acid I, and then nucleic acid II is added after selective removal of one of the protecting groups. Nucleic acid II is deprotected and capped, after which the other protecting group on the spacer is removed. Finally, nucleic acid III was synthesized. This process is described in detail in example 17 below.

Hydrophilic linkers of varying lengths can be used, for example, to link nucleic acid moieties to platform molecules. A variety of suitable linkers are known. Suitable linkers include, without limitation: linear oligomers or polymers of ethylene glycol. Such linkers include the formula R¹S(CH₂CH₂O)_nCH₂CH₂O(CH₂)_mCO₂R²Wherein n is 0-200, m is 1 or 2, R¹H or a protecting group such as trityl, R²H or alkyl or aryl groups such as 4-nitrophenyl ester. These linkers can be used to attach thiol-reactive group-containing molecules such as haloaceyi, maleiamide, etc. (general purpose)A peroxosulfide) is linked to another molecule containing an amino group (via an amide bond). The order of attachment can vary, i.e., the thioether bond can be formed before or after the amide bond is formed. Other useful linkers include sulfo-SMCC (4- [ N-maleimidoylmethyl)]-cyclohexane-1-carboxylic acid sulfosuccinimidyl ester) Pierce Chemical co.product 22322; sulfo-EMCS (N- [ epsilon-maleimidocaproyloxy)]Sulfosuccinimidyl ester) Pierce Chemical co.product 22307; sulfo-GMBS (N- [ gamma-maleimidobutaneacyloxy)]Sulfosuccinimidyl ester) Pierce Chemical co product22324(Pierce Chemical Company, Rockford, IL), and similar compounds of the general formula maleimidyl-R-c (o) NHS esters, where R is alkyl, cycloalkyl, ethylene glycol polymers, and the like.

Particularly useful methods for covalently linking nucleic acid moieties and multivalent spacers are described in the references and examples cited above.

Thus, in one aspect, the invention provides a method for preparing a CIC useful for modulating an immune response in a mammal by covalently linking a polynucleotide and a compound, thereby obtaining a chimeric compound comprising a nucleic acid moiety and a spacer moiety region and having immunomodulatory activity. In various embodiments, the nucleic acid region can have the structure and sequence of any of the nucleic acid moieties described herein and the spacer region can have the structure of any of the spacer moieties described herein. Often, there is more than one ligation step, e.g., ligation is repeated at least once to covalently attach two polynucleotides to one or two spacers, and typically ligation is repeated at least two or three times. The method further comprises mixing the CIC with a pharmaceutically acceptable excipient to form a composition. In one embodiment, the composition is sterile, e.g., suitable for administration to a human patient, e.g., manufactured or formulated according to GMP standards. In one embodiment, the CIC is associated with a microcarrier and/or antigen as described herein. It is also contemplated that, in some embodiments, the CIC formulation of the invention does not contain one or more of the following: (i) a colloidal dispersion system, (ii) liposomes, (iii) microcarriers, (iv) polypeptides, (v) antigens, and (vi) endotoxins.

6. Protein-like CIC

In certain embodiments, a polypeptide, such as a protein antigen or antigen fragment, is used as a multivalent spacer moiety to which multiple nucleic acid moieties may be covalently conjugated, either directly or via a linker, to form a "protein-like CIC". The polypeptide may be an antigen or immunogen against which a desired adaptive immune response may be generated, or may be a carrier (e.g., albumin). Protein-like CICs generally comprise at least one, typically several or more nucleic acid moieties that (a) are 2 to 7, more typically 4 to 7 nucleotides, or 2 to 6, 2 to 5, 4 to 6 or 4 to 5 nucleotides in length and/or (b) have sub-independent or no independent immunomodulatory activity. Methods for preparing protein-like CICs will be apparent to those skilled in the art upon review of this disclosure. For example, a nucleic acid may be covalently conjugated to a polypeptide spacer moiety, including the 3 'or 5' terminus of a nucleic acid moiety (or an appropriately modified base within a nucleic acid moiety) and an N bearing an appropriately reactive group (e.g., an N that can react with a cytosine residue) using methods known in the art ⁴N-hydroxysuccinimide ester with directly reacted amino groups). As another example, the polypeptide can be attached to the free 5' end of the nucleic acid moiety through an amine, thiol, or carboxyl group incorporated into the nucleic acid moiety. Alternatively, the polypeptide may be conjugated to a spacer moiety, as described herein. Furthermore, a linking group containing a protected amine, thiol or carboxyl group at one end and comprising a phosphoramidite may be covalently linked to a hydroxyl group of the polynucleotide, and then after deprotection, this functional group may be used for covalent attachment of the CIC to the peptide.

7. Purification of

CIC of the invention may be purified by any conventional means, such as high performance liquid chromatography, electrophoresis, nucleic acid affinity chromatography, molecular size exclusion chromatography and ion exchange chromatography. In some embodiments, the CIC is substantially pure, for example, at least about 80% pure by weight, often at least about 90% pure by weight, more typically at least about 95% pure, most typically at least about 85% pure.

8. Composition comprising a metal oxide and a metal oxide

In various embodiments, the compositions of the invention comprise one or more CIC (i.e., a single CIC or a combination of two or more CIC), wherein the CIC is optionally combined with another immunomodulator, such as a peptide, antigen (as described below), and/or additional adjuvant. The compositions of the invention may comprise CIC and a pharmaceutically acceptable excipient. By "pharmaceutically acceptable" it is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Pharmaceutically acceptable excipients are well known in the art and include sterile water, isotonic solutions such as saline and phosphate buffered saline, and other excipients known in the art. See Remington for examples: the Science and Practice of Pharmacy (19The part, 1995, Gennavo, ed.). Adjuvants (alum is an illustrative example) are known in the art. CIC preparations with other immunotherapeutic agents, such as cytokines and antibodies, can be prepared. In some embodiments, the compositions are isotonic and/or sterile, e.g., suitable for administration to a human patient, e.g., manufactured or formulated according to GMP standards.

A. CIC/MC complex

CIC may be administered in the form of a CIC/microcarrier (CIC/MC) complex. Accordingly, the present invention provides compositions comprising CIC/MC complexes.

The CIC/MC complex comprises CIC bound to the surface of microcarriers (i.e., CIC is not encapsulated in MC), and preferably multiple CIC molecules are bound per microcarrier. In certain embodiments, a mixture of different CICs may be complexed with a microcarrier, such that the microcarrier will have more than one CIC bound thereto. The bond between the CIC and the MC may be covalent or non-covalent (e.g., mediated by ionic bonds and/or hydrophobic interactions). One skilled in the art understands that CIC can be modified or derivatized and the composition of the microcarrier can be selected and/or modified to accommodate the type of binding desired for forming the CIC/MC complex.

The covalently bound CIC/MC complexes may be linked using any covalent crosslinking technique known in the art. Typically, the CIC moiety is modified to incorporate additional moieties (e.g., free amines, carboxyls, or thiols) or to incorporate modified (e.g., phosphorothioate) nucleotide bases to provide sites at which the CIC moiety can be attached to the microcarrier. The linkage between the CIC and MC moieties in the complex may occur at the 3 'or 5' end of the CIC, or at a suitably modified base within the CIC. Microcarriers are also typically modified to incorporate moieties through which covalent attachment can be formed, but functional groups normally present on microcarriers can also be utilized. CIC/MC may be formed by incubating CIC with a microcarrier under conditions that allow formation of a covalent complex, such as in the presence of a cross-linking agent or by using an activated microcarrier that comprises an activating moiety that can form a covalent bond with CIC.

A number of crosslinking techniques are known in the art and include crosslinking agents that react with amino, carboxyl, and thiol groups. It will be apparent to those skilled in the art that: the choice of cross-linking agent and method depends on the configuration of the CIC and microcarrier and the desired final configuration of the CIC/MC complex. The crosslinking agent may be a homobifunctional (homobifunctional) crosslinking agent or a heterobifunctional crosslinking agent. When a homobifunctional crosslinker is used, the crosslinker utilizes the same moiety on both the CIC and MC (e.g., an aldehyde crosslinker may be used to covalently link the CIC and MC when both comprise one or more free amines). The heterobifunctional crosslinker utilizes different moieties on the CIC and MC (e.g., maleimidoyl-N-hydroxysuccinimide ester can be used to covalently link the free thiol group on the CIC and the free amine on the MC), and is preferred in minimizing bonding between the microcarriers. In most cases, it is preferred that the crosslinking is by a first crosslinking moiety on the microcarrier and a second crosslinking moiety on the CIC, wherein the second crosslinking moiety is not present on the microcarrier. One preferred method of generating a CIC/MC complex is to activate the microcarriers by incubation with a heterobifunctional crosslinker and then incubate the CIC with the activated MC under conditions suitable for reaction to form the CIC/MC complex. The crosslinker may incorporate a "spacer arm" between the two reactive moieties, or the two reactive moieties in the crosslinker may be directly linked.

In a preferred embodiment, the CIC moiety comprises at least one free thiol group (e.g., provided by a 5' -thiol modified base or linker) for cross-linking the microcarrier, while the microcarrier comprises free amino groups. The MC may be activated using a heterobifunctional crosslinker (e.g., a crosslinker comprising a maleimide group and an NHS-ester) that reacts with both groups, such as succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, and then covalently crosslinks the CIC to form a CIC/MC complex.

The non-covalent CIC/MC complex may be linked by any non-covalent bonding or interaction, including ionic (electrostatic) bonds, hydrophobic interactions, hydrogen bonds, van der Waals attractive forces, or a combination of two or more different interactions, as is typically the case when a bonding pair is used to link the CIC and MC.

Preferred non-covalent CIC/MC complexes typically complex by hydrophobic or electrostatic (ionic) interactions or combinations thereof (e.g., by base pairing between the CIC and the polynucleotide attached to the MC). Due to the hydrophilic nature of the polynucleotide backbone, the CIC/MC complex that relies on hydrophobic interactions to form the complex typically requires modification of the CIC portion of the complex to incorporate highly hydrophobic moieties. Preferably, the hydrophobic moiety is biocompatible, non-immunogenic and naturally occurs in the body of the individual to which the composition is to be administered (e.g., the hydrophobic moiety may be found in mammals, particularly humans). Examples of preferred hydrophobic moieties include lipids, steroids, sterols such as cholesterol and terpenes. Of course, the method of attaching the hydrophobic moiety to the CIC depends on the configuration of the CIC and the nature of the hydrophobic moiety. This hydrophobic moiety may be added at any suitable site in the CIC, preferably at the 5 'or 3' end; where a cholesterol moiety is added to a CIC, it is preferably added to the 5' end of the CIC using conventional chemical reactions (see, e.g., Godard et al (1995), Eur. J. biochem., 232: 404-. Preferably, the microcarriers used in the CIC/MC complexes connected by hydrophobic bonds are made of hydrophobic materials such as oil droplets or hydrophobic polymers, but hydrophilic materials modified to incorporate hydrophobic moieties may also be used. When the microcarrier is a liposome or other liquid phase microcarrier containing a cavity, the CIC/MC complex is formed by mixing CIC and MC after MC is prepared, thereby avoiding the encapsulation of CIC during MC preparation.

Non-covalent CIC/MC complexes through electrostatic binding generally utilize the high negative charge of the polynucleotide backbone. Thus, microcarriers used in non-covalently bound CIC/MC complexes are typically positively charged at physiological pH, e.g., about pH 6.8-7.4. Microcarriers may themselves have a positive charge, but microcarriers prepared using compounds that generally do not have a positive charge may be made positively charged by derivatization or modification. For example, the polymers used to prepare microcarriers can be derivatized to add positively charged groups, such as primary amines. Alternatively, a positively charged compound can be incorporated into the microcarrier formulation during manufacture (e.g., a positively charged surfactant can be used during the manufacture of poly (lactic acid)/poly (glycolic acid) copolymers to impart a positive charge on the resulting microcarrier particles). See, for example, examples 28 and 34 below.

Non-covalent CIC/MC complexes linked by nucleotide base pairing can be prepared by conventional methods. Typically, the base-paired CIC/MC complex is prepared with a microcarrier comprising a polynucleotide ("capture polynucleotide") that is bound, preferably covalently bound, to at least part of the CIC. The complementary segment between the CIC and the capture nucleotide preferably has at least 6, 8, 10 or 15 contiguous base pairs, more preferably at least 20 contiguous base pairs. The capture nucleotide may be bound to the MC by any method known in the art, and is preferably covalently bound to the 5 'or 3' end of the CIC.

In other embodiments, a binding pair may be used to link the CIC and MC in the CIC/MC complex. The binding pair may be a receptor and ligand, an antibody and antigen (or epitope), or any other binding pair with high affinityBound binding pairs (e.g., K)_dLess than about 10^-8). One preferred class of binding pairs is biotin and streptavidin or biotin and avidin, which form a tight complex. When binding of the CIC/MC complex is mediated using a binding pair, CIC is derivatized with one member of the binding pair, typically by covalent bonding, and MC is derivatized with the other member of the binding pair. The mixing of these two derivative compounds will result in the formation of a CIC/MC complex.

Many embodiments of the CIC/MC complex do not include an antigen, while certain embodiments exclude antigens associated with a disease or condition that is the therapeutic target of the CIC/MC complex. In further embodiments, the CIC also binds to one or more antigenic molecules. The antigen may be coupled to the CIC moiety in the CIC/MC complex in a variety of ways, including covalent and/or non-covalent interactions, or the antigen may be attached to a microcarrier. In CIC/MC complexes with antigen bound to CIC, attachment of antigen to CIC can be accomplished by the techniques described herein and by techniques well known in the art.

B. Co-administered antigens

In some embodiments, the CIC is co-administered with the antigen. Any antigen may be co-administered with a CIC and/or used in the preparation of a composition comprising a CIC and an antigen.

In some embodiments, the antigen is an allergen. Examples of recombinant allergens are provided in table 1. The preparation of a number of allergens is well known in the art, including but not limited to the ragweed pollen allergen Antigen E (Amb aI) (Rafnar et al (1991) J. Biol.chem.) 266: 1229-1236), the grass allergen Lol p1 (Tambrini et al (1997) European J. Biochem. 249: 886-894), the major dust mite allergen Der pI and Der PII (Chua et al (1988) J. Experimental medicine (J.Exp.Med.) 167: 175-182; Chua et al (1990) int. Arch. allergy Appl. 91: 124-129), preparation of domestic cat allergen Fel dI (Rogers et al (1993) molecular Immunology (mol. Immunol.) 30: 559-. The preparation of protein antigens from grass pollen for in vivo administration has been reported.

In some embodiments, the allergen is a food allergen, including but not limited to peanut allergens such as Ara hI (Stanley et al (1996) adv. exp. Med. biol. 409: 213-; walnut allergens such as Jug rI (Tueber et al (1998) J. allergy Clin. Immunol.101: 807-; brazil nut allergens such as albumin (Pastorello et al (1998) J Allergy Clin. Immunol.102: 1021-1027); shrimp allergens such as Pen aI (Reese et al (1997) int. Arch. Allergy Immunol.113: 240-242); egg allergens such as ovomucoid (Crooke et al (1997) J.Immunol.) 159: 2026-2032); milk allergens such as bovine beta-lactoglobulin (Selot et al (1999) clinical and experimental allergy (Clin. exp. allergy) 29: 1055-; fish allergens such as microalbumin (Van Do et al (1999) Scand. J Immunol.50: 619; Galland et al (1998) J Chromatogr.B.biomed.Sci.appl.706: 63-71.). In some embodiments, the allergen is a latex allergen, including but not limited to Hev b7(SoWka et al (1998) European journal of biochemistry (Eur. J Biochem.) 255: 213-219). Table 1 lists a list of allergens that can be used.

TABLE 1

Recombinant allergens

Group of	Allergens	Reference to the literature
			Animals:
crustaceans
			Shrimp/lobster	Tropomyosin PansI	Leung et al (1996) j. 954-961Leung et al (1998) mol. Mar. biol. Biotechnol.7: 12-20
Insect pest
			Ant as health food	Soli2 (venom)	Schmidt et al, J Allergy Clin immunol., 1996, 98: 82-8
Bee product	Phospholipase A2(PLA) hyaluronidase (Hya)	Muuller et al, J Allergy Clin Immunol, 1995, 96: 395-: 1229-35Muller et al, clinical and laboratory Allergy (Clin Exp Allergy), 1997, 27: 915-20Soldatova et al, J Allergy Clin Immunol, 1998, 101: 691-8
			Cockroach (black beetle)	Blag Bd9OKBlag4(calycin) glutathione S-transferase Pera3	Helm et al, J Allergy Clin Immunol, 1996, 98: 172-180 valves et al, J Allergy Clin Immunol, 1998, 101: 274-280Arruda et al, J Biol Chem, 1997, 272: 20907-12Wu et al, molecular immunology (MolImmunol), 1997, 34: 1-8
Dust mite	Derp2 (major allergen) Der p2 variant Der f2Der p10Tyr p2	Lynch et al, J Allergy Clin Immunol, 1998, 101: 562-4; hakkaart et al, Clin Exp Allergy, 1998, 28: 169-74; hakkaart et al, ClinExp Allergy, 1998, 28: 45-52; hakkaart et al, Int Arch Allergy Immunol, 1998, 115 (2): 150-6Mueller et al, J Biol Chem (J Biol Chem), 1997, 272: 26893-8Snmith et al, J Allergy Clin Immunol, 1998, 101: 423-5Yasue et al, clinical and experimental immunology (Clin Exp Immunol), 1998, 113: 1-9Yasue et al, Cell immunology (Cell Immunol), 1997, 181: 30-7Asturias et al, Biochim Biophys Acta, 1998, 1397: 27-30Eriksson et al, Eur J Biochem (Eur J Biochem), 1998

Wasp	Antigen 5aka DolmV (venom)	Tomalski et al, Arch insert Biochem Physio, 1993, 22: 303-13
			Mosquito (Mosquitoes)	Aed aI (salivary adenosine triphosphate diphosphatase)	Xu et al, Int arc Allergy Immunol, 1998, 115: 245-51
(wasp)	Antigen 5, hyaluronidase and phospholipase (venom)	King et al, J Allergy Clin Immunol, 1996, 98: 588-600
			Mammal animal
Cat (cat)	Fel dI	Slunt et al, J Allergy Clin Immunol, 1995, 95: 1221-8Hoffmann et al (1997) J Allergy Clin Immunol 99: 227-32Hedlin Curr Opin Pediatr, 1995, 7: 676-82
			Dairy cow	Bos d2 (dander; lipocalin) beta-lactoglobulin (BLG, major milk allergen)	Zeiler et al J Allergy Clin Immunol, 1997, 100: 721-7 Rautiainen et al Biochem Bioph.Res Comm, 1998, 247: 746-50Chatel et al, molecular immunology (Mol Immunol), 1996, 33: 1113-8Lehrer et al Crit Rev Food Sci Nutr, 1996, 36: 553-64
Dog	Can fI and Canf2, salivary lipocalin	Konieczny et al, Immunology (Immunology), 1997, 92: 577-86Spitzauer et al, J Allergy Clin Immunol, 1994, 93: 614-27Vrtala et al, journal of immunology (J Immunol), 1998, 160: 6137-44
			Horse	Equ cl (major allergen, lipocalin)	Gregoire et al J Biol Chem, 1996, 271: 32951-9
Mouse	Mouse Urine Protein (MUP)	Konieczny et al, Immunology (Immunology), 1997, 92: 577-86
			Other mammalian allergens
Insulin		Ganz et al J Allergy Clin Immunol, 1990, 86: 45-51Grammer et al, J Lab Clin Med, 1987, 109: 141-6Gonzalo et al, Allergy (Allergy), 1998, 53: 106-7
			Interferon	Interferon alpha 2c	Detmar et al Contact Dermatis, 1989, 20: 149-50
Mollusk	topomyosin	Leung et al J Allergy Clin Immunol, 1996, 98: 954-61
			Plant allergen:
barley	Hor v9	Astwood et al Adv Exp Med Biol, 1996, 409: 269-77
			Birch (Betula platyphylla (Betula)	Pollen allergen, Bet v4rBet v1 Bet v 2: (inhibitory protein)	Twardosz et al Biochem bioph.res comm, 1997, 239: 197Pauli et al, J Allergy Clin Immunol, 1996, 97: 1100-9van Neerven et al Clin Exp Allergy, 1998, 28: 423-33Jahn-Schmid et al, immunology, 1996, 2: 103-13Breitwieser et al Biotechniques, 1996, 21: 918-25Fuchs et al J Allergy Clin Immunol, 1997, 100: 356-64
Brazil nut	Globulin protein	Bartolome et al Allergol Immunopathol, 1997, 25: 135-44

Cherry	Pru aI (major allergen)	Scheurer et al, molecular immunology (Mol Immunol), 1997, 34: 619-29
			Corn (corn)	Zml 3 (pollen)	Heiss et al FEBS Lett, 1996, 381: 217-21Lehrer et al Iht Arch Allergy Immunol, 1997, 113: 122-4
Grass (Haw)	Phlp1, Phlp2, Phlp5 (timothy grass pollen) Hol 15 chorion grass pollen poa pratensis allergen Cynd7 Cynd12 (an inhibitor protein)	Bufe et al Am J Respir Crit Care Med, 1998, 157: 1269-76Vrtala et al, J Immunol, 6.1998, 15.6.160: 6137-44Niederberger et al J Allergy Clin Immun., 1998, 101: 258-64Schramm et al, Eur J Biochem, 1998, 252: 200-6Zhang et al, J Immunol, 1993, 151: 791-9Smith et al Int Arch Allergy Immunol, 1997, 114: 265-71Asturias et al, Clin Exp Allergy, 1997, 27: 1307-13Fuchs et al, J Allergy Clin Immunol, 1997, 100: 356-64
			Cryptomeria fortunei (Thunb.) Trev	Jun a2 (Axibai Juniperus chinensis, Juniperus usashei)) Cryj1, Cryj2 (Peacock fir, Cryptomeria japoniae)	Yokoyama et al biochem. biophysis. res. commun., 2000, 275: 195-202Kingetsu et al Immunology, 2000, 99: 625-629
Juniper	Jun o2 (pollen)	Tinghino et al J Allergy Clin Immunol, 1998, 101: 772-7
			Latex	Hev，b7	Sowka et al, Eur J Biochem, 1998, 255: 213-9Fuchs et al J Allergy Clin Immunol, 1997, 100: 356-64
Indigofera tinctoria (Mercurialis)	Merai (inhibitory protein)	Vallverdu et al J Allergy Clin Immunol, 1998, 101: 363-70
			Mustard (yellow)	Sinai (seed)	Gonzalez de la Pena et al Biochem bioph. res comm, 1993, 190: 648-53
Oilseed rape	Bra rI pollen allergen	Smith et al Int Arch Allergy Immunol, 1997, 114: 265-71
			Peanut	Ara hI	Stanley et al Adv Exp Med Biol, 1996, 409: 21-6Burks et al J Clin Invest, 1995, 96: 1715-21Burks et al Int Arch Allergy Immunol, 1995, 107: 248-50
Poa pratensis	Poa p9	Parronchi et al, European journal of immunology (Eur J Immunol), 1996, 26: 697 Astwood et al Adv Exp Med Biol, 1996, 409: 269-77
			Ragweed	Amb aI	Sun et al Biotechnology Aug, 1995, 13: 779-86Hirschwehr et al J Allergy Clin Immunol, 1998, 101: 196-206Casale et al J Allergy Clin Immunol, 1997, 100: 110-21
Rye	Lol pI	Tamborini et al, Eur journal of biochemistry (E)ur J Biochem)，1997，249：886-94

Nuts	Jug rI	Teuber et al J Allergy Clin immun, 1998, 101: 807-14
			Wheat (Triticum aestivum L.)	Allergens	Fuchs et al J Allergy Clin Immunol, 1997, 100: 356-64Donovan et al, Electrophoresis (Electrophoresis), 1993, 14: 917-22
Fungi
			Aspergillus (Aspergillus)	Asp f1, Asp f2, Asp f3, Asp f4, rAsp f6 manganese superoxide dismutase (MNSOD)	Crameri et al Mycoses, 1998, 41 Suppl 1: 56-60Hemmann et al, European journal of immunology (Eur J Immunol), 1998, 28: 1155-60Banerjee et al J Allergy Clin Immunol, 1997, 99: 821-7Crameri Int arm Allergy Immunol, 1998, 115: 99-114Crameri et al Adv Exp Med Biol, 1996, 409: 111-6Moser et al J Allergy Clin Immunol, 1994, 93: 1-11Mayer et al Int Arch Allergy Immunol, 1997, 113: 213-5
Blomia	Allergens	Carabllo et al Adv Exp Med Biol, 1996, 409: 81-3
			Penicillium (Penicillium)	Allergens	Shen et al, clinical and experimental Allergy (Clin Exp Allergy), 1997, 27: 682-90
Naked cap mushroom genus (Psilocybe)	Psi c2	Horner et al Int arc Allergy Immunol, 1995, 107: 298-300

In some embodiments, the antigen is from an infectious agent, including protozoan, bacterial, fungal (including unicellular and multicellular), and viral infectious agents. Examples of suitable viral antigens are described herein and are well known in the art. Bacteria include haemophilus influenzae (Hemophilus influenza), Mycobacterium tuberculosis (Mycobacterium tuberculosis), and Bordetella pertussis (Bordetella pertussis). Protozoan infections include plasmodium, leishmania, trypanosomes, and schistosomes. Fungi include Candida albicans (Candida albicans).

In some embodiments, the antigen is a viral antigen. Viral polypeptide antigens include, but are not limited to, HIV proteins such as HIV gag protein (including, but not limited to, Membrane Anchor (MA) protein, core shell (CA) protein, and Nucleocapsid (NC) protein), HIV polymerase, influenza matrix (M) protein, and influenza Nucleocapsid (NP) protein, hepatitis b surface antigen (HBsAg), hepatitis b core protein (HBcAg), hepatitis e protein (HBeAg), hepatitis b DNA polymerase, hepatitis c antigen, and the like. Documents discussing influenza vaccination include Scherle and gerhrad (1988), proceedings of the national academy of sciences of the united states (proc. natl. acad. sci. usa) 85: 4446 and 4450; scherle and gerhrad (1986), journal of experimental medicine (j.exp.med.) 164: 1114-; granoff et al (1993), Vaccine (Vaccine) 11: s46-51; kodhalli et al (1997), journal of virology (j.virol.) 71: 3391-3396; ahmeida et al (1993), Vaccine (Vaccine) 11: 1302-; chen et al (1999), Vaccine (Vaccine) 17: 653-; govorkova and Smirnov (1997) Acta Virol (1997) 41: 251, 257; koide et al (1995), Vaccine (Vaccine) 13: 3-5; mbawuike et al (1994), Vaccine (Vaccine) 12: 1340-1348; tamura et al (1994), Vaccine (Vaccine) 12: 310-316; tamura et al (1992) european journal of immunology (eur.j Immunol.) 22: 477 ion 481; hirabayashi et al (1990), Vaccine (Vaccine) 8: 595-599. Other examples of antigenic polypeptides are group or subgroup specific antigens, and these antigenic polypeptides are known for a number of infectious agents, including, but not limited to, adenovirus (adenoviruses), herpes simplex virus (herpes simplex virus), papilloma virus (papilloma virus), respiratory syncytial virus (respiratory syncytial virus), and poxvirus (poxvirus).

Many antigenic peptides and proteins are well known and are readily available in the art; others may be identified by conventional techniques. For immunizing against neoplasia or treating an existing tumor by immunization, the immunomodulating peptide may comprise tumor cells (live or irradiated), tumor cell extracts or protein subunits of tumor antigens such as Her-2/neu, Mart1, carcinoembryonic antigen (CEA), gangliosides, Human Milk Fat Globules (HMFG), mucin (MUC1), MAGE antigens, BAGE antigens, GAGE antigens, gp100, Prostate Specific Antigen (PSA), and tyrosinase. An immunity-based contraceptive vaccine may be formed by including a sperm protein co-administered with the CIC. Lea et al (1996) Biochim.Biophys.Acta1307: 263.

attenuated and inactivated viruses are suitable for use herein as antigens. The preparation of these viruses is well known in the art and many are commercially available (see, e.g., Physicians' Desk Reference (1998) 52 th edition, Medical Economics Company, Inc.). For example, poliovirus (poliovirus) may be administered as 1POL^_(Pasteur Merieux Connaught) and ORIMUNE^_(Lederle laboratories) and hepatitis A Virus may be available as VAQTA^_(Merck) measles virus (measles virus) available as ATTENUVAX ^_(Merck) mumps Virus (mumps virus) available as MUMPSVAX^_(Merck) and rubella virus (rubella virus) may be obtained as MERUFAX^_II (Merck). In addition, attenuated and inactivated viruses such as HIV-1, HIV-2, herpes simplex virus, hepatitis B virus, rotavirus (rotavirus), human and non-human papilloma virus and lentivirus (slow vaccine virus) may provide peptide antigens.

In some embodiments, the antigen comprises a viral vector, such as vaccinia virus, adenovirus, and canarypox virus vectors.

Antigens may be isolated from their source using purification techniques well known in the art, or more conveniently, may be produced recombinantly.

Antigenic peptides may include purified natural peptides, synthetic peptides, recombinant proteins, crude protein extracts, attenuated or inactivated viruses, cells, microorganisms, or fragments of such peptides. The immunomodulating peptide may be a natural peptide, or a chemically or enzymatically synthesized peptide. Any chemical synthesis known in the art is suitable. Solution phase peptide synthesis can be used to construct medium size peptides, or chemical construction of peptides can be performed using solid phase synthesis. Atherton et al (1981) Hoppe Seylers Z.physiol.chem.362: 833-839. Peptides can also be produced using proteolytic enzymes to couple amino acids. Kullmann (1987) EnzymaticPeptide Synthesis, CRC Press, Inc. Alternatively, the peptides may be obtained by using the biochemical machinery of the cell or by isolation from a biological source. Peptides can be produced using recombinant DNA techniques. Hames et al (1987), transcription and translation: practical methods (transformation and transformation: analytical Approach), IRL Press. Peptides can also be isolated using standard techniques such as affinity chromatography.

Preferably, the antigens are peptides, lipids (such as sterols, fatty acids and phospholipids in addition to cholesterol), polysaccharides (such as those used in haemophilus influenzae vaccines), gangliosides and glycoproteins. They can be obtained by several methods well known in the art, including the use of chemical and enzymatic separations and syntheses. In some cases, such as for many sterols, fatty acids, and phospholipids, the antigenic portion of the molecule is commercially available.

Examples of viral antigens useful in the compositions and methods of using the compositions of the present invention include, but are not limited to, HIV antigens. Such antigens include, but are not limited to, those from HIV envelope glycoproteins including, but not limited to, gp160, gp120, and gp 41. The sequences of many HIV genes and antigens are well known. For example, Los Alamos National laboratory HIV Sequence Database collects, manages, and annotates HIV nucleotide and amino acid sequences. This database is accessible via the internet website http:// hiv-web. lan. gov/and published annually, see Human Retroviruses and AIDS compdendium (e.g. version 2000).

Antigens from infectious agents can be obtained by methods well known in the art, such as from natural viral or bacterial extracts, from cells infected with the infectious agent, from purified polypeptides, from recombinantly produced polypeptides, and/or in the form of synthetic peptides.

CIC may be administered with antigen in a variety of ways. In some embodiments, the CIC and the antigen are administered in spatial proximity to each other. Spatial access can be achieved in a variety of ways, including conjugation, encapsulation, addition to a platform, or adsorption to a surface, as described below. In one embodiment, the CIC and antigen are administered in a mixture (e.g., in solution). It is specifically contemplated that, in certain embodiments, the CIC and immunogen or antigen are not conjugated.

In some embodiments, the CIC is linked to a polypeptide, such as an antigen. The CIC moiety can be linked to the antigenic moiety of the conjugate in a variety of ways, including covalent and/or non-covalent interactions, through a nucleic acid moiety or a non-nucleic acid spacer moiety. In some embodiments, attachment is achieved through reactive groups such as, but not limited to, sulfur-containing groups, amines, carboxyl groups, aldehydes, hydrazines, hydrazones, disulfide groups, and the like.

The linkage between these moieties may be at the 3 'or 5' end of the nucleic acid moiety, or at an appropriately modified base at an intermediate position in the nucleic acid moiety. For example, if the antigen is a peptide and contains a suitable reactive group (e.g., N-hydroxysuccinimide ester), it may be directly reacted with the N of a cytosine residue ⁴And (4) reacting amino. Specific coupling can be achieved at one or more residues depending on the number and position of cytosine residues in the CIC.

Alternatively, modified oligonucleotides such as are known in the art may be incorporated at terminal or internal positions of the CIC. They may contain blocked functional groups that, when unblocked, can react with a variety of functional groups that may be present on or bound to the antigen of interest.

When the antigen is a peptide, the portion of the conjugate may be chemically linked to the nucleic acid moiety or the spacer moiety by a solid support. For example, the nucleic acid moiety of a CIC may be added to a portion of a polypeptide that has been pre-synthesized on a carrier. Haralambidis et al (1990) Nucleic acid research (Nucleic acids SRes.) 18: 493-499; and Haralambidis et al (1990) Nucleic acid research (Nucleic acids SRes.) 18: 501-505.

Alternatively, CIC can be synthesized such that it is attached to a solid support via a cleavable linker extending from the 3' -end of the nucleic acid moiety. When CIC is chemically cleaved from the vector, a terminal thiol or terminal amino group is left at the 3' -end of the Nucleic acid moiety (Zuckermann et al (1987) Nucleic Acids Res. 15: 5305-5321; and Corey et al (1987) Science 238: 1401-1403; Nelson et al (1989) Nucleic Acids Res. 17: 1781-1794). Conjugation of amino-modified CIC to the amino group of a peptide may be as described by Benoit et al (1987) neuromethds 6: 43-72. Conjugation of thiol-modified CIC to the carboxyl group of a peptide can be performed as described by Sinah et al (1991), oligonucleotide analogs: the Practical method (oligonucleotide assays: A Practical Approach), IRL Press. Conjugation of a nucleic acid moiety or spacer carrying an additional maleimide to the thiol side chain of a cysteine residue of a peptide has also been described. Tung et al (1991) bioconjugate Chem.2: 464-465.

The peptide portion of the conjugate can be linked to the free 5 ' -end of the nucleic acid moiety through an amine, thiol, or carboxyl group that has been incorporated (e.g., through the free 5 ' -end, 3 ' -end, through a modified base, etc.) into the nucleic acid moiety or spacer.

Conveniently, a linking group comprising a protected amine, thiol or carboxyl group at one end and comprising a phosphoramidite may be covalently bonded to a hydroxyl group of the CIC. Agrawal et al (1986) Nucleic acids research (Nucleic acids sres.) 14: 6227-6245; connolly (1985) Nucleic acid research (Nucleic Acids Res.) 13: 4485-4502; kremsky et al (1987), Nucleic Acids research (Nucleic Acids Res.) 15: 2891-; connolly (1987) Nucleic acid research (Nucleic Acids Res.) 15: 3131-; bischoff et al (1987) anal. biochem.164: 336-344; blanks et al (1988), Nucleic Acids research (Nucleic Acids Res.) 16: 10283-; and U.S. patent nos. 4,849,513, 5,015,733, 5,118,800, and 5,118,802. After deprotection, these amine, thiol and carboxyl functional groups can be used to covalently attach the CIC to the peptide. Benoit et al (1987); and Sinah et al (1991).

CIC-antigen conjugates may also be formed by non-covalent interactions such as ionic bonds, hydrophobic interactions, hydrogen bonds, and/or van der Waals attraction.

Non-covalently linked conjugates can include non-covalent interactions, such as biotin-streptavidin complexes. The biotin group can, for example, be attached to a modified base of the CIC. Roget et al (1989), Nucleic Acids research (Nucleic Acids Res.) 17: 7643-7651. Incorporating a streptavidin moiety into a peptide moiety will allow the streptavidin-conjugated peptide and biotinylated oligonucleotide to form a non-covalent binding complex.

Non-covalent attachment may also be created by ionic interaction involving residues within the CIC and antigen, such as charged amino acids, or by using a linker moiety comprising charged residues that can interact with both the oligonucleotide and the antigen. For example, non-covalent conjugation can occur between a normally negatively charged CIC and a positively charged amino acid residue in the peptide, such as polylysine, polyarginine, and polyhistidine residues.

Non-covalent conjugation between the CIC and antigen may be achieved through a DNA binding motif in a molecule that interacts with DNA, where the molecule is a natural ligand for DNA. For example, such DNA binding motifs can be found in transcription factors and anti-DNA antibodies.

Attachment of the CIC to the lipid may be formed by standard methods. These methods include, but are not limited to, the synthesis of oligonucleotide-phospholipid conjugates (Yanagawa et al (1988) Nucleic Acids Symp. Ser.19: 189-192), oligonucleotide-fatty acid conjugates (Grabarek et al (1990) Biochemical Ann. biochem.) 185: 131-135, and Staros et al (1986) Biochemical Ann. 156: 220-222), and oligonucleotide-sterol conjugates. Boujrad et al (1993), proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.usa) 90: 5728-5731.

The linkage of the oligonucleotide to the oligosaccharide may be formed by standard known methods. These methods include, but are not limited to, the synthesis of oligonucleotide-oligosaccharide conjugates, where the oligosaccharide is part of an immunoglobulin. O' Shannessy et al (1985), journal of applied biochemistry (J.applied Biochem.) 7: 347-355.

Other methods for attaching peptides and other molecules to oligonucleotides can be found in U.S. patent nos. 5,391,723; kricka (ed.), Nonisotopic DNA Probe technologies (Nonisotopic DNA probes), Kessler (1992) in Academic Press, Nonradioactive labeling methods for nucleic acids "; and Geoghegan et al (1992) bioconjugate. chem.3: 138 and 146.

CIC may be tightly associated with antigen in other ways. In some embodiments, the CIC and the antigen are tightly coupled by encapsulation. In other embodiments, the CIC and antigen are intimately associated by attachment to a platform molecule. A "platform molecule" (also referred to as a "platform") is a molecule that comprises sites that allow attachment of CIC and antigen. In other embodiments, the CIC and antigen are intimately associated by adsorption onto a surface, preferably a carrier particle.

In some embodiments, the methods of the present invention employ an encapsulating agent (or a composition comprising such an encapsulating agent), wherein the encapsulating agent maintains intimate association of the CIC and the first antigen until the complex reaches the target. Preferably, the composition comprising the CIC, antigen and encapsulating agent is in the form of an adjuvant oil-in-water emulsion, microparticles and/or liposomes. More preferably, the adjuvant oil-in-water emulsion, microparticles and/or liposomes encapsulating CIC are in the form of particles of about 0.04 μm to about 100 μm in size, preferably any of the following ranges in size: about 0.1 μm to about 20 μm; about 0.15 μm to about 10 μm; about 0.05 μm to about 1.00 μm; about 0.05 μm to about 0.5 μm.

Colloidal dispersions such as microspheres, beads, macromolecular complexes, nanocapsules and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles and liposomes can effectively encapsulate CIC-containing compositions.

The encapsulation composition may further comprise any of a variety of ingredients. These components include, but are not limited to, alum, lipids, phospholipids, Lipid Membrane Structures (LMS), polyethylene glycol (PEG), and other polymers such as polypeptides, glycopeptides, and polysaccharides.

Polypeptides suitable for use as an encapsulating component include any polypeptide known in the art and include, but are not limited to, fatty acid binding proteins. The modified polypeptide may comprise any of a variety of modifications including, but not limited to, glycosylation, phosphorylation, myristoylation, sulfation, and hydroxylation. As used herein, suitable polypeptides are those that protect CIC-containing compositions to retain their immunomodulatory activity. Examples of binding proteins include, but are not limited to, albumins such as Bovine Serum Albumin (BSA) and pea albumin.

Other suitable polymers may be any known in the pharmaceutical art including, but not limited to, naturally occurring polymers (e.g., dextran, hydroxyethyl starch, and polysaccharides) and synthetic polymers. Examples of naturally occurring polymers include proteins, glycopeptides, polysaccharides, dextrans and lipids. The other polymer may be a synthetic polymer. Examples of synthetic polymers suitable for use in the present invention include, but are not limited to, polyalkyl glycols (PAGs) such as PEG, polyoxyethylated polyols (POPs) such as polyoxyethylated glycerol (POG), polytrimethylene glycol (PTG), polypropylene glycol (PPG), hydroxyethyl methacrylate, polyvinyl alcohol (PVA), polyacrylic acid, polyethyloxazoline, polyacrylamide, polyvinylpyrrolidone (PVP), polyaminoacids, polyurethanes, and polyphosphazenes. The synthetic polymer may also be a linear or branched, substituted or unsubstituted homopolymer, a copolymer or block copolymer of two or more different synthetic monomers.

The PEG used in the encapsulation composition of the invention may be purchased from chemical suppliers or synthesized using techniques well known in the art.

The term "LMS" as used herein refers to lamellar lipid particles in which the polar head groups of the polar lipid are arranged facing the aqueous phase of the interface to form a membrane structure. Examples of LMS include liposomes, micelles, spirochetes (i.e., generally cylindrical liposomes), microemulsions, unilamellar liposomes, multilamellar liposomes, and the like.

One colloidal dispersion system that may be used to administer CIC is liposomes. Mice were immunized with liposome-encapsulated antigen, which was shown to promote a Th 1-type immune response to the antigen. Aramaki et al (1995), Vaccine (Vaccine) 13: 1809-1814. As used herein, a "liposome" or "lipid vesicle" is a vesicle surrounded by at least one and possibly more than one bilayer lipid membrane. Liposomes can be artificially prepared from phospholipids, glycolipids, lipids, steroids such as cholesterol, related molecules, or combinations thereof by any technique known in the art including, but not limited to, sonication, extrusion, or removal of detergent from a lipid-detergent complex. Liposomes may also optionally contain other components such as tissue targeting components. It is understood that the "lipid membrane" or "lipid bilayer" need not consist only of lipids, as long as the overall structure of the membrane is a sheet with two hydrophilic surfaces sandwiching a hydrophobic core, but that the "lipid membrane" or "lipid bilayer" may also contain any suitable other components, including but not limited to cholesterol and other steroids, lipid-soluble chemicals, proteins of any length, and other amphipathic molecules. For a comprehensive discussion of membrane structure see J.Kendrew (1994) Encyclopedia of molecular Biology (The Encyclopedia of molecular Biology). Suitable lipids are described, for example, in Lasic (1993) "Liposomes: from Physics to Applications "Elsevier, Amsterdam.

Methods for preparing liposomes containing compositions comprising CIC are well known in the art. Lipid vesicles may be prepared by any suitable technique known in the art. Methods include, but are not limited to, microencapsulation, microfluidization, LLC methods, alcohol injection, freon injection, "foaming" methods, detergent dialysis, hydration, sonication, and reverse phase evaporation. For review see Watwe et al (1995) Curr. Sci.68: 715-724. A variety of techniques may be combined to provide the vesicles with the most desirable properties.

The present invention includes the use of LMS containing tissue or cell targeting components. Such targeting ingredients are ingredients in LMS that promote LMS accumulation preferentially at specific tissues or cell sites and not other tissues or cell sites when the LMS is administered to a whole animal, organ or cell culture. The targeting component is typically accessed from outside the liposome and therefore it is preferably incorporated into the outer surface or inserted into an outer lipid bilayer. The targeting component may be, inter alia, a peptide, a region of a larger peptide, an antibody or antigen-binding fragment thereof specific for a cell surface molecule or marker, a nucleic acid, a carbohydrate, a region of complex carbohydrates, a specific lipid, or a small molecule such as a drug, hormone, or hapten that binds to any of the above molecules. Antibodies specific for cell type-specific cell surface markers are well known in the art and can be readily prepared by methods well known in the art.

LMS can be targeted to any cell type to which therapy is intended, such as cell types that may modulate and/or participate in an immune response. Such target cells and organs include, but are not limited to, APCs such as macrophages, dendritic cells and lymphocytes, lymphoid structures such as lymph nodes and spleen, and non-lymphoid structures, particularly those in which dendritic cells are present.

The LMS composition of the present invention may further comprise a surfactant. The surfactant may be a cationic, anionic, amphiphilic or nonionic surfactant. One preferred type of surfactant is a nonionic surfactant; and those which are water-soluble are particularly preferred.

In embodiments where the CIC and antigen are tightly linked by linkage to a platform molecule, the platform may be a protein-like or non-protein-like (e.g., synthetic) valency platform (valency platform). Examples of suitable platforms are described above with respect to the discussion of the valency platforms used as spacer moieties for CIC. The antigen and valency platform may be linked by conventional methods. For example, where the polypeptide contains amino acid side chain moieties with functional groups such as amino, carboxyl or sulfhydryl groups, these functional groups may serve as sites for conjugation of the polypeptide to the platform. If the polypeptide does not contain such groups, residues having such functional groups may be added to the polypeptide. Such residues may be incorporated by solid phase synthesis techniques or recombinant techniques, both of which are well known in the art of peptide synthesis. When the polypeptide has a carbohydrate side chain (or when the antigen is a carbohydrate), functional groups, amino, sulfhydryl and/or aldehyde groups, may be incorporated therein by conventional chemistry. For example, primary amino groups can be incorporated by reacting an oxidized saccharide with ethylenediamine in the presence of sodium cyanoborohydride; incorporation of sulfhydryl groups by reaction with cysteamine dihydrochloride followed by reduction with a standard disulfide reducing agent; while aldehyde groups may be generated after periodic acid oxidation. If the platform molecule does not have a suitable functional group, the platform molecule can also be derivatized in a similar manner to include the functional group.

In another embodiment, co-administration of the CIC and the antigen is achieved by adsorbing both to a surface, such as a nanoparticle or microcarrier surface. The CIC and/or antigen may be adsorbed to the surface by non-covalent interactions, including ionic and/or hydrophobic interactions. The adsorption of polynucleotides and polypeptides to surfaces for the delivery of the adsorbed molecules to cells is well known in the art. See, e.g., Douglas et al (1987) crit. rev. ther. drug. carrier. syst.3: 233-; hagiwara et al (1987) In Vivo 1: 241-; bousquet et al (1999) pharm. res.16: 141-147; and Kossovsky et al, U.S. patent 5,460,831. Preferably, the material comprising the adsorption surface is biodegradable.

In summary, the characteristics of nanoparticles such as surface charge, particle size and molecular weight depend on the polymerization conditions during polymerization, monomer concentration and the presence of stabilizers (Douglas et al, 1987, supra). The surface of the carrier particle may be modified, e.g. by surface coating, to allow or facilitate adsorption of the CIC and/or antigen. The CIC and/or antigen adsorbed carrier particles may be further coated with other substances. The addition of such other substances, as described herein, can extend the half-life of the particle, e.g., after administration of the particle to a subject, and/or can target the particle to a particular cell type or tissue.

Nanocrystalline surfaces that adsorb CIC and antigen have been described (see, e.g., U.S. Pat. No. 5,460,831). The nanocrystalline core particles (1 μm or less in diameter) may be coated with a surface energy modifying layer that promotes adsorption of the polypeptide, polynucleotide, and/or other agent. As described in us patent 5,460,831, for example, the core particle may be coated with a surface that promotes adsorption of the oligonucleotide, followed by coating with an antigen preparation, for example in the form of a lipid-antigen mixture. Such nanoparticles are self-assembled complexes of nanometer-sized particles, typically on the order of 0.1 μm, with an inner CIC layer and an outer antigenic layer.

The other surface is nanoparticles prepared by polymerizing alkyl cyanoacrylate. Alkyl cyanoacrylates can be polymerized by anionic polymerization in acidified aqueous media. Depending on the polymerization conditions, these small particles tend to have a size of 20 to 3000nm, and nanoparticles with specific surface characteristics and specific surface charges can be prepared (Douglas et al, 1987, supra). For example, oligonucleotides can be adsorbed onto polyisobutylcyanoacrylate and polyisohexylcyanoacrylate nanoparticles in the presence of a hydrophobic cation such as tetraphenyl chloride or a quaternary ammonium salt such as cetyltrimethylammonium bromide. The adsorption of oligonucleotides onto these nanoparticles appears to be mediated by ion pairs formed between negatively charged phosphate groups and hydrophobic cations of the nucleic acid strands. See, e.g., Lambert et al (1998), biochemistry (Biochimie) 80: 969-: 1370-1378; chavany et al (1992) pharm. Res.9: 441-449. The polypeptide may also be adsorbed onto polyalkylcyanoacrylate nanoparticles. See, e.g., Douglas et al, 1987; schroeder et al (1998) Peptides (Peptides) 19: 777-780.

The other suction surface is nanoparticles prepared by polymerization of methylene malonate. For example, as described by Bousquet et al, 1999, adsorption of a polypeptide to poly (methylene malonate 2.1.2) nanoparticles appears to be achieved initially by electrostatic forces and then stabilized by hydrophobic forces.

C. Other adjuvants

CIC may also be used in combination with an adjuvant. Administration of an antigen in combination with a CIC and an adjuvant may result in an enhanced immune response to the antigen, and thus an enhanced immune response, as compared to the immune response resulting from a composition comprising only the CIC and the antigen. Adjuvants are well known in the art and include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, alum (aluminum salts), liposomes and microparticles (including, but not limited to, polystyrene, colloidal silica, and the like),Starch, polyphosphazene and polylactide/polyglycoside). Other suitable adjuvants also include, but are not limited to, MF59, DETOX^TM(Ribi), squalene mixture (SAF-1), muramyl peptides, saponin derivatives, mycobacterial cell wall preparations, monophosphoryl ester A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunits, polyphosphazenes and derivatives, and Immune Stimulating Complexes (ISCOMs) such as Takahashi et al (1990) Nature 344: 873-875, as well as lipid-based adjuvants and other adjuvants described herein. For veterinary use and for the production of antibodies in animals, the mitogenic component of Freund's adjuvant (complete and incomplete Freund's adjuvant) can be used.

IV. the method of the invention

The present invention provides a method of modulating an immune response in an individual, preferably a mammal, more preferably a human, comprising administering to the individual a CIC as described herein. Immunomodulation may include stimulation of a Th 1-type immune response and/or suppression or reduction of a Th 2-type immune response. The CIC is administered in an amount sufficient to modulate the immune response. Modulation of the immune response may be modulation of a humoral and/or cellular immune response, as described herein, and may be determined using standard techniques in the art and as described herein.

Many individuals are adapted to receive CIC as described herein. Preferably (but not necessarily), the individual is a human.

In certain embodiments, the individual has a disorder associated with a Th 2-type immune response, such as (without limitation) allergy or allergy-induced asthma, atopic dermatitis, eosinophilic gastrointestinal inflammation, eosinophilic esophagitis, and allergic bronchopulmonary aspergillosis. Administration of CIC will result in immunomodulation, increasing the levels of one or more cytokines associated with a Th 1-type response, which may result in a reduction of Th 2-type response characteristics associated with the allergen response of the individual. Immunomodulation on of individuals with disorders associated with a Th 2-type response can result in alleviation or amelioration of one or more symptoms of the disease. If the condition is allergy or allergy-induced asthma, the amelioration of one or more symptoms comprises a reduction of one or more of the following symptoms: rhinitis, allergic conjunctivitis, circulating IgE levels, circulating histamine levels, and/or the need for 'rescue' inhalation therapy (e.g., inhaled albuterol administered by a metered dose inhaler or nebulizer).

In a further embodiment, the subject receiving the immunomodulating therapy of the invention is a subject receiving a vaccine. The vaccine may be a prophylactic vaccine or a therapeutic vaccine. A prophylactic vaccine comprises one or more epitopes associated with a condition that an individual may be at risk of developing (e.g., mycobacterium tuberculosis antigen as a vaccine for the prevention of tuberculosis). Therapeutic vaccines comprise one or more epitopes associated with a particular condition affecting an individual, such as a surface antigen of mycobacterium tuberculosis or mycobacterium bovis (m.bovis) in a tuberculosis patient, an antigen that causes an individual to become allergic in an allergic individual (i.e., allergy desensitization therapy), tumor cells from a cancer patient (e.g., as described in U.S. patent No. 5,484,596), or a tumor-associated antigen of a cancer patient. The CIC may be administered in conjunction with the vaccine (e.g., injected with the vaccine or simultaneously but separately) or the CIC may be administered separately (e.g., at least 12 hours before or after vaccine administration). In certain embodiments, the antigen of the vaccine is part of a CIC, and the antigen is covalently or non-covalently linked to the CIC. Administration of CIC treatment to an individual receiving a vaccine resulted in a shift in the immune response to the vaccine towards the Th 1-type compared to individuals receiving a vaccine that did not contain CIC. Shifts to Th 1-type responses can be recognized by the development of delayed-type hypersensitivity (DTH) responses to antigens in the vaccine, elevated IFN- γ and other Th 1-type response-associated cytokines, production of CTLs specific for antigens in the vaccine, a decrease or reduction in IgE levels specific for antigens in the vaccine, a reduction in Th 2-associated antibodies specific for antigens in the vaccine, and/or an increase in Th 1-associated antibodies specific for antigens in the vaccine. In the case of a therapeutic vaccine, administration of the CIC and vaccine may also result in an improvement in one or more symptoms of the condition to be treated by the vaccine. As will be apparent to those skilled in the art, the exact symptoms and manner of improvement will depend on the condition to be treated. For example, when the therapeutic vaccine is directed against tuberculosis, treatment with CIC and the vaccine results in a reduction in cough, pleural or chest wall pain, fever, and/or other symptoms known in the art. When the vaccine is an allergen for use in allergy desensitization therapy, the treatment results in a reduction of allergy symptoms (e.g., rhinitis, reduction of allergic conjunctivitis, reduction of circulating IgE levels, and/or circulating histamine levels).

Other embodiments of the invention relate to immunomodulating treatment of individuals already suffering from a disease or disorder, such as cancer or an infectious disease. Cancer is an attractive target for immunomodulation because most cancers express tumor-associated and/or tumor-specific antigens that are not present on other cells in the body. Stimulation of a Th 1-type response against tumor cells will result in the immune system killing the tumor cells by direct and/or bystander means, resulting in a reduction of tumor cells and/or a reduction of symptoms. Administration of CIC to an individual with cancer results in stimulation of a Th 1-type immune response against the tumor cells. This immune response kills tumor cells either by direct action of cells of the cellular immune system (e.g., CTLs) or components of the humoral immune system, or by bystander effects on cells in the vicinity of targeted cells of the immune system, including macrophages and Natural Killer (NK) cells. See, e.g., Cho et al (2000), natural biotechnology (nat. biotechnol.) 18: 509-514. In treating an existing disease or disorder, CIC may be combined with other immunotherapeutic agents, such as cytokines, adjuvants and antibodies. For example, the CIC may be administered as part of a treatment regimen that includes administration of a binding agent that binds to a tumor cell-displayed antigen. Examples of binding agents include polyclonal and monoclonal antibodies. Examples of target antigens include CD20, CD22, HER2, and other antigens known in the art or to be discovered in the future. Without wishing to be bound by theory, CIC is thought to enhance killing of binder-associated tumor cells (e.g., by enhancing antibody-dependent cytotoxicity and/or effector function). The binding agent may optionally be labeled with, for example, a radioisotope or toxin that disrupts the cells bound by the binding agent. The CIC may be administered in conjunction with (e.g., simultaneously with) or before or after (e.g., less than 24 hours before or after administration of the agent). For example, in the case of cancer, the CIC may be administered in combination with a chemotherapeutic agent known or suspected to be useful in the treatment of cancer. For another example, CIC may be administered in combination with radiation therapy or gene therapy. This CIC may be any CIC described herein.

Immunomodulating therapies according to the invention may also be used for individuals with infectious diseases, particularly those that are resistant to antibody fluid immune responses (e.g., diseases caused by mycobacterial infections and intracellular pathogens). Immunomodulating therapy is useful in the treatment of infectious diseases caused by cellular pathogens (such as bacteria or protozoa) or subcellular pathogens (such as viruses). CIC therapy can be administered to individuals with mycobacterial disease, such as tuberculosis (e.g., mycobacterium tuberculosis and/or mycobacterium bovis infection), leprosy (i.e., mycobacterium leprae (Mleprae) infection), or mycobacterium marinum (m.marinum) or mycobacterium ulcerosa (m.ulcerans) infection. CIC therapy may also be used to treat viral infections, including influenza virus, Respiratory Syncytial Virus (RSV), hepatitis B virus, hepatitis C virus, herpes virus, particularly herpes simplex virus and papilloma virus infections. Diseases caused by intracellular parasites, such as malaria (e.g., infection by Plasmodium vivax, Plasmodium ovale, Plasmodium falciparum, and/or Plasmodium malariae), leishmaniasis (e.g., Leishmania donovani, Leishmania tropicalis, Leishmania mexicana, Leishmania brasiliensis, Leishmania peru, Leishmania infantis, l.chagasi, and/or Leishmania russelliana), and Toxoplasmosis (i.e., infection by Toxoplasmosis), may also benefit from CIC treatment, as well as Toxoplasmosis (i.e., infection by Plasmodium vivax). CIC therapy can also be used to treat parasitic diseases such as schistosomiasis (i.e. schistosomiasis infection caused by schistosomiasis species such as schistosoma japonicum (s.haematbium), schistosoma mansonii (s.mansonii), schistosoma japonicum (s.japonicum) and meigongyzia (s.mekongi)) and schistosomiasis (i.e. infection caused by Clonorchis sinensis). Administration of CIC to an individual with an infectious disease can result in an improvement in the symptoms of the infectious disease. In some embodiments, the infectious disease is a non-viral disease.

The invention also provides methods of elevating or stimulating at least one Th 1-associated cytokine in an individual, wherein the Th 1-associated cytokine comprises IL-2, IL-12, TNF- β, IFN- γ, and IFN- α. In certain embodiments, the invention provides methods of elevating or stimulating IFN- γ in an individual, particularly an individual in need of elevated IFN- γ levels, by administering to the individual an effective amount of CIC to elevate IFN- γ. Those in need of increased IFN-y levels are those with a condition that is generally responsive to administration of IFN-y. Such conditions include a number of inflammatory conditions, including but not limited to ulcerative colitis. Such disorders also include a number of fibrotic disorders, including but not limited to Idiopathic Pulmonary Fibrosis (IPF), scleroderma, radiation-induced skin fibrosis, liver fibrosis (including schistosomiasis-induced liver fibrosis), kidney fibrosis, and other diseases that can be ameliorated by administration of IFN- γ. Administration of CIC of the invention will result in elevated IFN- γ levels and result in amelioration of one or more symptoms of the condition responsive to IFN- γ, stabilization of one or more symptoms, and/or prevention of development of the condition (e.g., reduction or elimination of additional damage or symptoms). The methods of the invention may be used in conjunction with other therapies that constitute the standard of care for the disease, such as in IPF in conjunction with administration of an anti-inflammatory agent such as systemic corticosteroid therapy (e.g., cortisone).

In certain embodiments, the invention provides methods of increasing IFN- α in an individual, particularly an individual in need of increased IFN- α levels, by administering to the individual an effective amount of CIC to increase IFN- α levels. Those in need of increased IFN- α are those with conditions responsive to administration of IFN- α, including recombinant IFN- α, including but not limited to viral infections and cancer.

Administration of CIC of the invention may result in elevated IFN- α levels and result in amelioration of one or more symptoms of the condition responsive to IFN- α, stabilization of one or more symptoms, or prevention of development of the condition (e.g., reduction or elimination of additional damage or symptoms). The methods of the invention may be used in combination with other therapies that constitute the standard of care for disease, such as administration of antiviral agents in the case of viral infections.

It is apparent based on the present disclosure that the spacer composition of CIC may influence the immune response elicited by CIC administration. Virtually all of the spacers tested (except dodecyl) can be used in CIC to effectively induce IFN- γ in human PBMC. However, it has been observed that the spacer composition of linear CIC has a different effect on IFN- α induction. For example, CICs containing spacers such as HEG, TEG, or C6 tend to cause higher IFN- α induction (and reduced B cell proliferation) in PBMCs compared to CICs containing C3, C4, or abasic spacers (see, e.g., example 34, below).

The invention also provides methods of reducing IgE levels, particularly serum IgE levels, in a subject having an IgE-related disorder by administering to the subject an effective amount of a CIC. In such methods, the CIC may be administered alone (e.g., without antigen) or in combination with an antigen, such as an allergen. An IgE-related disorder is a disease, disorder or a group of symptoms that can be ameliorated by lowering IgE levels. A decrease in IgE will result in an improvement in the symptoms of IgE-related disorders-such symptoms include allergic symptoms (e.g. rhinitis, conjunctivitis), reduced sensitivity to allergens, reduced allergic symptoms in allergic individuals, or reduced severity of allergies.

The methods of the invention include embodiments in which the CIC is administered in the form of a CIC/microcarrier complex.

In some embodiments, the present invention provides a method of stimulating the production of CTLs in an individual, the method comprising administering to the individual an effective amount of CIC to increase CTL production.

It will be apparent to those skilled in the art that the methods of the present invention may be used in combination with other therapies for the particular indication for which CIC therapy is administered. For example, CIC therapy may be administered to malaria patients in combination with anti-malarial drugs such as chloroquine, to leishmaniasis patients in combination with leishmaniasis-killing drugs such as pentamidine and/or allopurinol, to tuberculosis patients in combination with anti-mycobacterial drugs such as ramification, rifampin and/or ethambutol, or to atopic (allergic) patients in combination with allergen desensitization therapy.

A. Administration and assessment of immune response

As described herein, a CIC may be administered in conjunction with a pharmaceutical and/or immunogenic agent and/or other immunostimulating agent, and may be combined with a physiologically acceptable carrier therefor.

For example, a CIC or composition of the invention may be administered in combination with other immunotherapeutic agents such as cytokines, adjuvants and antibodies. The CIC may be administered in combination with (e.g., simultaneously with, or before or after (e.g., no more than 24 hours before or after) the agent.

As with all immunostimulatory compositions, the immunologically effective amounts and methods of administration of a particular CIC formulation may vary depending on the individual, the disease to be treated, and other factors apparent to those skilled in the art. Factors to be considered include the presence of the co-administered antigen, whether the CIC is administered in combination with or covalently linked to an adjuvant or delivery molecule, the route of administration and the number of immunizations to be administered. Such factors are well known in the art and their determination without undue experimentation is within the skill of one of ordinary skill in the art. Suitable dosage ranges are those that provide the desired modulation of the immune response to the antigen. Generally, the dose is determined by the amount of CIC administered to the patient and not by the total amount of CIC. Useful dosage ranges for CIC (given in the amount of CIC delivered) may be, for example, about any of the following: 1 to 500. mu.g/kg, 100 to 400. mu.g/kg, 200 to 300. mu.g/kg, 1 to 100. mu.g/kg, 100 to 200. mu.g/kg, 300 to 400. mu.g/kg, 400 to 500. mu.g/kg. The absolute amount administered to each patient depends on pharmacological characteristics such as bioavailability, clearance and route of administration.

The effective amount and method of administration of a particular CIC formulation may vary depending on the individual patient and the stage of disease and other factors apparent to those skilled in the art. The route of administration for a particular application will be apparent to those skilled in the art. Routes of administration include, but are not limited to, topical, dermal, transdermal, transmucosal, epidermal, parenteral, gastrointestinal, and nasopharyngeal and pulmonary (including bronchial and alveolar). A suitable dosage range is one that provides sufficient CIC-containing composition to achieve a tissue concentration of about 1-10 μ M by measuring blood levels. The absolute amount administered to each patient depends on pharmacological characteristics such as bioavailability, clearance and route of administration.

As described herein, APC and tissues with high concentrations of APC are preferred targets for CIC. Therefore, it is preferred to administer CIC to the skin and/or mucosa of a mammal where APCs are present in relatively high concentrations.

The present invention provides CIC formulations suitable for topical application, including but not limited to physiologically acceptable implants, ointments, creams, lotions, and gels. Topical application may be applied, for example, by a dressing or bandage in which the delivery system is dispersed, or by applying the delivery system directly into an incision or open wound, or by a transdermal applicator directed at the target site. Creams, lotions, gels or ointments having CIC dispersed therein are suitable for use as topical ointments or wound fillers.

Preferred routes of dermal administration are those that are minimally invasive. Among these, transdermal delivery, epidermal administration and subcutaneous injection are preferred. Among these, epidermal administration is preferred due to the expected presence of higher concentrations of APC in the intradermal tissue.

Transdermal administration is achieved by applying creams, lotions, gels, etc. that allow the CIC to penetrate the skin and enter the bloodstream. Compositions suitable for transdermal administration include, but are not limited to, pharmaceutically acceptable suspensions, oils, creams and ointments applied directly to the skin or incorporated into a protective vehicle such as a transdermal carrier (so-called "patch"). Examples of suitable creams, ointments and the like can be found, for example, in the Physician's Desk Reference.

For transdermal delivery, iontophoresis is a suitable method. Iontophoretic delivery can be accomplished by commercially available patches that can continue to deliver their products through unbroken skin for several days or longer. The use of this method allows for controlled delivery of the pharmaceutical composition at relatively large concentrations and allows for infusion of the combination drug and the concomitant use of an absorption enhancer.

An exemplary PATCH product for use in this method is the product sold under the trademark LECTRO PATCH by general medical Company of Los Angeles, CA. The product maintains the reservoir electrode electronically at neutral pH and is adapted to provide doses of varying concentrations and to be administered continuously and/or periodically. Preparation and use of the PATCH should be in accordance with manufacturer printed instructions with the LECTRO PATCH product; these specifications are incorporated herein by reference. Other closed patch systems are also suitable.

For transdermal delivery, low frequency ultrasound delivery is also a suitable method. Mitragoti et al (1995) Science (Science) 269: 850-853. The application of low frequency ultrasound frequencies (about 1MHz) allows for the overall controlled delivery of therapeutic compositions, including those having high molecular weights.

Epidermal administration essentially involves mechanical or chemical stimulation of the outermost epidermis, and the stimulation will be sufficient to elicit an immune response to the stimulation. Specifically, the stimulus should be sufficient to attract the APC to the stimulation site.

One exemplary mechanical stimulation regimen uses a plurality of short teeth of very narrow diameter that can be used to stimulate the skin and attract APCs to the stimulation site to ingest CIC that is transferred out of the ends of the teeth. For example, the MONO-VACC old tuberculin test, produced by Pasteur Merieux, Lyon, France, comprises a device suitable for introducing a composition comprising CIC.

The device (sold in the united states by connaughtt Laboratories, inc. of Swiftwater, PA) consists of a plastic container with a syringe pusher at one end and a toothed disc at the other end. The toothed disc has a plurality of teeth of narrow diameter fixed to it in a length such that it scrapes just the outermost cells of the epidermis. Each tooth in the MONO-VACC kit is coated with tuberculin; in the present invention, each needle is coated with a pharmaceutical composition of a CIC formulation. The use of the device is preferably in accordance with instructions written by the manufacturer included in the device product. Similar devices that may also be used in this embodiment are those currently used for conducting allergy tests.

Another suitable method of epicutaneous administration of CIC is to use chemicals that stimulate the outermost cells of the epidermis, thus provoking a sufficient immune response that may attract APCs to this area. One example is a keratolytic agent such as salicylic acid used in commercially available topical depilatory creams sold under the trademark NAIR by Noxema Corporation. The method may also be used to achieve epithelial administration of the mucosa. The chemical stimulant may also be used in combination with a mechanical stimulant (e.g., if the MONO-VACC dentition is also coated with the chemical stimulant, such a combination is present). The CIC may be suspended in a carrier that also contains a chemical irritant or co-administered with the chemical irritant.

Parenteral routes of administration include, but are not limited to, electrical injection (iontophoresis) or direct injection such as direct injection into the central vein, intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. CIC formulations suitable for parenteral administration are typically formulated in USP water or water for injection and may also contain pH buffers, salt fillers, preservatives and other pharmaceutically acceptable excipients. CIC for parenteral injection may be formulated in pharmaceutically acceptable sterile isotonic solutions such as saline and phosphate buffered saline for injection.

The gastrointestinal routes of administration include, but are not limited to, the swallow and rectal routes. The present invention includes CIC formulations suitable for gastrointestinal administration, including but not limited to pharmaceutically acceptable powders, tablets or liquids for administration by swallowing and suppositories for rectal administration. It will be apparent to those skilled in the art that: tablets or suppositories will also contain a pharmaceutically acceptable solid such as starch to fill the composition.

Nasopharyngeal and pulmonary administration can be effected by inhalation and includes routes of delivery such as intranasal, transbronchial and transalveolar routes. The present invention includes CIC formulations suitable for inhalation administration, including but not limited to liquid suspensions for forming aerosols and powders for dry powder inhalation delivery systems. For inhalation administration of CIC formulations, suitable devices include, but are not limited to, nebulizers, vaporizers, nebulizers, and dry powder inhalation delivery devices.

The choice of delivery route can be used to modulate the immune response elicited. For example, when the influenza virus vector is administered by intramuscular or epidermal (gene gun) routes, the IgG titer and CTL activity are the same; however, intramuscular vaccination produced mainly IgG2a, whereas the epidermal pathway produced mainly IgG 1. Pertmer et al (1996), journal of virology (j.virol.) 70: 6119-6125. Thus, one of skill in the art may utilize slight differences in immunogenicity elicited by different routes of administration of the immunomodulatory polynucleotides of the invention.

The above compositions and methods of administration are intended to describe, but not to limit, the method of administration of the CIC formulations of the invention. Methods of preparing the various compositions and devices are within the ability of those skilled in the art and will not be described in detail herein.

Immune responses to CIC can be assayed (both qualitatively and quantitatively) by any method known in the art, including, but not limited to, measuring antigen-specific antibody production (including measuring specific antibody + subclasses), specific lymphocyte populations such as CD4⁺Activation of T cells, NK cells or CTLs, production of cytokines such as IFN-. gamma.IFN-. alpha.IL-2, IL-4, IL-5, IL-10 or IL-12 and/or release of histamine. Methods for determining specific antibody responses include enzyme-linked immunosorbent assays (ELISAs) and are well known in the art. Specific types of lymphocytes such as CD4⁺The determination of the number of T cells can be done, for example, with Fluorescence Activated Cell Sorting (FACS). Cytotoxicity and CTL assays can be performed, for example, as reported by Raz et al (1994), Proctl.acad.sci.usa) 91: 9519-9523 and Cho et al (2000). Cytokine concentrations can be determined, for example, by ELISA. These and other assays for assessing immune responses to immunogens are well known in the art. See, e.g., selection Methods in cellular immunology (1980) edited by misshell and Shiigi, w.h.freeman and co.

Preferably, a Th 1-type response is stimulated, i.e. elicited and/or enhanced. With respect to the present invention, stimulation of a Th 1-type immune response can be determined by measuring cytokine production in vitro or ex vivo in CIC-treated cells and comparing to control cells not treated with CIC. Methods of determining cytokine production by a cell include those described herein and any method known in the art. The type of cytokine produced in response to CIC treatment indicates that the cells produce an immune response that is biased towards either the Th 1-type or the Th 2-type. As used herein, the term "Th 1-biased" cytokine production refers to a measurable increase in the production of Th 1-type immune response-associated cytokines in the presence of a stimulating agent as compared to the production of such cytokines in the absence of stimulation. Examples of such Th 1-biased cytokines include, but are not limited to, IL-2, IL-12, IFN- γ, and IFN- α. Conversely, "Th 2-biased cytokines" refer to those cytokines associated with a Th 2-type immune response, including but not limited to IL-4, IL-5, and IL-13. Cells that can be used to determine CIC activity include cells of the immune system, primary cells and/or cell lines isolated from a host, preferably APC and lymphocytes, even more preferably macrophages and T cells.

Stimulation of Th 1-type immune responses may also be measured in a host treated with CIC and may be measured by any method known in the art, including but not limited to: (1) reduced levels of IL-4 or IL-5 are measured in the CIC-treated host before and after antigen challenge as compared to an antigen-contacted or antigen-contacted and challenged, CIC-untreated control; or a lower (or even no) IL-4 or IL-5 level is detected; (2) increased levels of IL-12, IL-18, and/or IFN (α, β, or γ) in a CIC-treated host before and after antigen challenge, as compared to an antigen-contacted, or antigen-contacted and challenged, CIC-untreated control; or detecting elevated IL-12, IL-18 and/or IFN (alpha, beta or gamma) levels; (3) producing "Th 1-biased" antibodies in CIC-treated hosts compared to controls not treated with CIC; and/or (4) a decrease in the level of antigen-specific IgE is detected before and after antigen challenge in a CIC-treated host as compared to an antigen-contacted or antigen-contacted and challenged, CIC-untreated control; or a lower (or even no) level of antigen-specific IgE is detected. Many of these assays can be accomplished by measuring in vitro or ex vivo cytokines produced by APCs and/or lymphocytes, preferably macrophages and/or T cells, using the methods described herein or any method known in the art. Some of these assays can be accomplished by measuring the class and/or subclass of antigen-specific antibodies using the methods described herein or any method known in the art.

The class and/or subclass of antigen-specific antibodies produced in response to CIC treatment indicates that the cells produce an immune response that is biased toward the Th 1-type or the Th 2-type. As used herein, the term "Th 1-biased" antibody production refers to a measurable increase in production of antibodies associated with a Th 1-type immune response (i.e., a Th 1-associated antibody). One or more Th 1-related antibodies can be assayed. Examples of such Th 1-biased antibodies include, but are not limited to, human IgGI and/or IgG3 (see, e.g., Widhee et al (1998) Scand. J Immunol.47: 575-581 and de Martino et al (1999) Ann. allergy assay Immunol.83: 160-164) and murine IgG2 a. In contrast, "Th 2-biased antibodies" refer to those antibodies associated with a Th 2-type immune response, including but not limited to human IgG2, IgG4 and/or IgE (see, e.g., Widhe et al (1998) and de Martino et al (1999)) and murine IgG1 and/or IgE.

Induction of Th 1-biased cytokines due to administration of CIC can lead to enhanced cellular immune responses, such as those performed by NK cells, cytotoxic killer cells, Th1 helper cells, and memory cells. These responses are particularly beneficial for prophylactic or therapeutic vaccination against viruses, fungi, protozoan parasites, bacteria, allergic diseases, asthma and tumours.

In some embodiments, a Th2 response is inhibited. Inhibition of the Th2 response can be determined, for example, by a reduction in the levels of Th 2-related cytokines (such as IL-4 and IL-5) and a reduction in IgE response to allergens and a reduction in histamine release.

V. kit of the invention

The invention provides a kit. In certain embodiments, the kits of the invention comprise one or more containers comprising a CIC. The kit may further comprise a set of appropriate instructions, typically written instructions, regarding the intended therapeutic use of the CIC (e.g., immunomodulation, ameliorating symptoms of an infectious disease, increasing IFN- γ levels, increasing IFN- α levels, or ameliorating an IgE-related disorder).

The kit may comprise the CIC packaged in any convenient suitable package. For example, if the CIC is a dry formulation (e.g., lyophilized or dry powder), a vial with an elastomeric stopper is typically used so that the CIC can be easily resuspended by injecting a liquid through the elastomeric stopper. Ampoules with inelastic removable closures (e.g. sealing glass) or ampoules with elastomeric stoppers are most suitable for liquid formulations of CIC. It is also contemplated to use the package in combination with a specific device such as an inhaler, nasal administration device (e.g., a nebulizer) or infusion device such as a mini-pump.

Instructions for the use of CIC typically include information regarding the dosage, dosing regimen and route of administration for the intended treatment. The containers of CIC may be unit doses, bulk packs (e.g., multi-dose packs), or sub-unit doses. The instructions provided in the kits of the invention are typically written instructions written on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

In some embodiments, the kit further comprises an antigen (or comprises one or more antigens), which may or may not be packaged in the same container (formulation) as the CIC. Antigens have been described herein.

In certain embodiments, the kits of the invention comprise CIC in the form of a CIC/microcarrier complex (CIC/MC), and may further comprise a set of instructions, typically written instructions, relating to the intended therapeutic use of the CIC/MC complex (e.g., immunomodulation, ameliorating symptoms of an infectious disease, increasing IFN- γ levels, increasing IFN- α levels, or ameliorating an IgE-related disorder).

In some embodiments, the kits of the invention comprise materials for producing a CIC/MC complex, typically including containers separately containing CIC and MC, but in certain embodiments provided are materials for producing MC rather than preformed MC. The CIC and MC are preferably provided in a form that allows the CIC and MC provided to form a CIC/MC complex upon mixing. This configuration is preferred when the CIC/MC complexes are linked by non-covalent bonds. When CIC and MC are to be crosslinked by a heterobifunctional crosslinking agent; this configuration is also preferred when the CIC or MC is provided in an "activated" form (e.g., attached to a heterobifunctional crosslinker such that a moiety reactive with the CIC is available).

For a CIC/MC complex comprising a liquid phase MC, the kit preferably comprises one or more containers containing materials for producing the liquid phase MC. For example, for an oil-in-water emulsion MC, the CIC/MC kit may comprise one or more containers containing an oil phase and a water phase. The contents of the vessel are emulsified to produce the MC, which may then be mixed with the CIC, preferably with the CIC modified to incorporate a hydrophobic moiety. Such materials include oils and water used to create oil-in-water emulsions, or include a container containing freeze-dried liposomal components (e.g., a mixture of phospholipids, cholesterol, and surfactants) plus one or more containers containing an aqueous phase (e.g., a pharmaceutically acceptable aqueous buffer).

VI. examples

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

Example 1: structure of polynucleotide and chimeric compound

Table 2 shows the structures of the polynucleotides and chimeric molecules referred to in the examples. "HEG" is a hexapolyethylene glycol spacer moiety; "TEG" is a triethylene glycol; "C3" is a propyl spacer moiety; "C4" is a butyl spacer; "C6" is a hexyl spacer; "C12" is a dodecyl spacer; "HME" is 2-hydroxymethylethyl; "abasic" or "ab" is 1 ', 2' -dideoxyribose. Other spacers are described in the specification and drawings.

All nucleotide linkages and linkages between nucleic acid moieties and spacer moieties are phosphorothioate, except where specifically indicated in table 2 and the specific examples. For example, in CICs that include a composite (multi-subunit) spacer moiety having multiple HEG or C3 units (e.g., C-13, C-14, C-15, C-15, C-91, C-92, C-36, C-37, and C-38), the C3 or HEG units are linked with phosphorothioate linkers. Similarly, the branched CICs shown (e.g., C-93, C-94, C-95, C-96, C-97, C-98, C-100, C-101, C-103, C-104, C-121, C-122, C-123, C-124, C-125, C-126, C-127, C-129, C-130) include phosphorothioate linkers between the branched and linear subunits of the spacer. Other branched CICs shown (e.g., C-26, C-99, C-102, C-105, and C-137) were prepared by conjugation strategies with linking groups as described in the examples.

Table 2 also includes compounds having terminal linking groups (e.g., HS (CH)₂)₆And HO (CH)₂)₆SS(CH₂)₆) The CIC of (e.g., C-128, C-106, C-113), the end-linked gene can be used to link these CIC molecules to the branched spacer portion to construct a branched CIC. See example 18 for examples. These linking groups are attached to the CIC using phosphorothioate linkages.

TABLE 2

Test compounds and polynucleotides

Example 2: synthesis of chimeric Compounds with Linear Structure and Hexapolyethyleneglycol spacers

C-10 having the structure shown below was synthesized. The nucleic acid moiety is a DNA having phosphorothioate linkages and the spacer moiety is hexapolyethylene glycol (HEG) which is linked to the nucleic acid moiety by phosphorothioate linkages.

C-10：5′-TCGTCG-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′

The C-10 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite 8909 automated DNA synthesizer using the manufacturer's procedure for the synthesis of 1. mu. mol phosphorothioate DNA. The nucleoside monomer and spacer moiety precursor, 4' -O-dimethoxytrityl-hexapolyethylene glycol-O- (N, N-diisopropyl) 2-cyanoethylphosphonite (from Glen Research, Sterling, Va.) were dissolved in anhydrous acetonitrile to a final concentration of 0.05M. (As will be apparent to one of ordinary skill in the art, the term "nucleoside monomer" or "spacer moiety" as used herein from time to time (e.g., in the context of CIC synthesis) refers to a precursor reagent that, if deprotected and ligated to other components using synthetic methods as disclosed herein, can yield CIC nucleic acid and non-nucleic acid moieties). The HEG spacer precursor was placed in the instrument at the auxiliary monomer position. The instrument is programmed to add nucleotide monomers and HEG spacers in the desired order and synthesize nucleic acid moieties in the 3 'to 5' direction.

1. Solid phase support using 3' bound "T" to the support

2. Synthesis of 5 '-AGATGA-3' moiety

3. Adding HEG spacer

4. Synthesis of 5 '-ACGTTCG-3' moieties

5. Adding HEG spacer

6. Synthesis of 5 '-TCGTCG-3' structural moiety

The synthesis cycle consisted of a detritylation step, a coupling step (phosphoramidite monomer plus 1H-tetrazole), a capping step, a sulfurization step with 0.05M 3H-1, 2-benzodithiolan-3-one 1, 1-dioxide (Beaucage reagent), and a final capping step. After the assembly is completed, the trityl-removed compound is cut off from the controlled porous glass and reacted with concentrated ammonia water at 58 ℃ for 16 hours to deprotect the base. This compound was purified by preparative polyacrylamide electrophoresis, desalted on a Sep-pak Plus column (Waters, Milord, Mass.), and precipitated from 1M aqueous sodium chloride solution with 2.5 volumes of 95% ethanol. The molecules were dissolved in Milli Q water and the yield determined from the absorbance at 260 nm. Finally, the compound was lyophilized to form a powder. This compound was characterized by capillary gel electrophoresis, electrospray mass spectrometry, and RP-HPLC to confirm its composition and purity. In addition, an endotoxin content assay (LAL assay, Bio Whittaker) was also performed, and the results showed endotoxin levels < 5EU/mg compound (i.e., essentially endotoxin-free).

C-8, C-21, C-22, C-23, C-24, C-32 and M-1 as well as other linear HEG-CICs were synthesized similarly.

Example 3: synthesis of chimeric Compounds with Linear Structure and propyl spacer

C-11 having the structure shown below was synthesized. The nucleic acid moiety is a DNA having a phosphorothioate linkage and the spacer moiety is a propyl group (C3) which is linked to the nucleic acid moiety via a phosphorothioate linkage.

C-11：5′-TCGTCG-3′-C3-5′-ACGTTCG-3′-C3-5′-AGATGAT-3′

The C-11 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's procedure for the synthesis of 1. mu. mol phosphorothioate DNA. The nucleoside monomer and spacer moiety precursor, 4' -O-dimethoxytrityl-propyl-O- (N, N-diisopropyl) 2-cyanoethylphosphonite (from Glen Research, Sterling, Va.) were dissolved in anhydrous acetonitrile to a final concentration of 0.05M. The C3 spacer precursor was placed in the instrument at the auxiliary monomer position. The instrument is programmed to add nucleotide monomers and C3 spacers in the desired order and synthesize the nucleic acid moieties in the 3 'to 5' direction.

1. "T" solid phase support using 3' -binding support

2. Synthesis of 5 '-AGATGA-3' moiety

3. Adding C3 spacer

4. Synthesis of 5 '-ACGTTCG-3' moieties

5. Adding C3 spacer

6. Synthesis of 5 '-TCGTCG-3' structural moiety

Synthesis, deprotection, work-up, and analysis were all performed as described in example 2.

C-9 and other CICs containing C3 were synthesized similarly.

Example 4: synthesis of chimeric Compounds with Linear Structure and butyl spacer

C-17 having the structure shown below was synthesized. The nucleic acid moiety is a DNA having a phosphorothioate linkage and the spacer moiety is a butyl group (C4) which is linked to the nucleic acid moiety via a phosphorothioate linkage.

C-17：5′-TCGTCG-3′-C4-5′-ACGTTCG-3′-C4-5′-AGATGAT-3′

The C-17 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's procedure for the synthesis of 1. mu. mol phosphorothioate DNA. The nucleoside monomer and spacer moiety precursor, 4' -O-dimethoxytrityl-butyl-O- (N, N-diisopropyl) 2-cyanoethyl phosphoramidite (from Chemgenes, Ashland, Mass.) were dissolved in anhydrous acetonitrile to a final concentration of 0.05M. The C4 spacer precursor was placed in the instrument at the auxiliary monomer position. The instrument is programmed to add nucleotide monomers and C4 spacers in the desired order and synthesize the nucleic acid moieties in the 3 'to 5' direction.

1. "T" solid phase support using 3' -binding support

2. Synthesis of 5 '-AGATGA-3' moiety

3. Adding C4 spacer

4. Synthesis of 5 '-ACGTTCG-3' moieties

5. Adding C4 spacer

6. Synthesis of 5 '-TCGTCG-3' structural moiety

Synthesis, deprotection, work-up, and analysis were all performed as described in example 2.

Example 5: synthesis of chimeric Compounds with Linear Structure and TriPEG spacer

C-18 having the structure shown below was synthesized. The nucleic acid moiety is DNA having phosphorothioate linkages and the spacer moiety is a tri-polyethylene glycol (TEG) linked to the nucleic acid moiety via phosphorothioate linkages.

C-18：5′-TCGTCG-3′-TEG-5′-ACGTTCG-3′-TEG-5′-AGATGAT-3′

The C-18 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's protocol for the synthesis of 1. mu. mol phosphorothioate DNA. The nucleoside monomer and spacer moiety precursor, 4' -O-dimethoxytrityl-triethylene glycol-O- (N, N-diisopropyl) 2-cyanoethyl phosphoramidite (from Glen Research, Sterling, Va.) were dissolved in anhydrous acetonitrile to a final concentration of 0.05M. The TEG spacer precursor was placed in the instrument at the auxiliary monomer position. The instrument is programmed to add nucleotide monomers and TEG spacers in the desired order and synthesize nucleic acid moieties in the 3 'to 5' direction.

1. "T" solid phase support using 3' -binding support

2. Synthesis of 5 '-AGATGA-3' moiety

3. Adding TEG spacers

4. Synthesis of 5 '-ACGTTCG-3' moieties

5. Adding TEG spacers

6. Synthesis of 5 '-TCGTCG-3' structural moiety

Synthesis, deprotection, work-up, and analysis were all performed as described in example 2.

Example 6: synthesis of chimeric Compounds with Linear Structure and dodecyl spacer

C-19 having the structure shown below was synthesized. The nucleic acid moiety is a DNA having a phosphorothioate linkage and the spacer moiety is a dodecyl group (C12) which is linked to the nucleic acid moiety via a phosphorothioate linkage.

C-19：5′-TCGTCG-3′-C12-5′-ACGTTCG-3′-C12-5′-AGATGAT-3′

The C-19 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's procedure for the synthesis of 1. mu. mol phosphorothioate DNA. The nucleoside monomer and spacer moiety precursor, 4' -O-dimethoxytrityl-dodecyl-O- (N, N-diisopropyl) 2-cyanoethylphosphonite (from Glen Research, Sterling, Va.) were dissolved in anhydrous acetonitrile to a final concentration of 0.05M. The C12 spacer precursor was placed in the instrument at the auxiliary monomer position. The instrument is programmed to add nucleotide monomers and C12 spacers in the desired order and synthesize the nucleic acid moieties in the 3 'to 5' direction.

1. "T" solid phase support using 3' -binding support

2. Synthesis of 5 '-AGATGA-3' moiety

3. Adding C12 spacer

4. Synthesis of 5 '-ACGTTCG-3' moieties

5. Adding C12 spacer

6. Synthesis of 5 '-TCGTCG-3' structural moiety

Synthesis, deprotection, work-up, and analysis were all performed as described in example 2.

Example 7: synthesis of chimeric Compounds with Linear Structure and abasic spacers

C-20 having the structure shown below was synthesized. The nucleic acid moiety is a DNA having a phosphorothioate linkage and the spacer moiety is a 1 ', 2' -dideoxyribose (abasic) linked to the nucleic acid moiety via a phosphorothioate linkage.

C-20：

5′-TCGTCG-3′-abasic-5′-ACGTTCG-3′-abasic-5′AGATGAT-3′

The C-20 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's procedure for the synthesis of 1. mu. mol phosphorothioate DNA. The nucleoside monomer and spacer moiety precursor, 5 ' -O- (4, 4 ' -O-dimethoxytrityl) -1 ', 2 ' -dideoxyribose-3 ' -O- (N, N-diisopropyl) 2-cyanoethylphosphonite (from Glen Research, Sterling, Va.) were dissolved in anhydrous acetonitrile to a final concentration of 0.05M. The abasic spacer precursor is placed at the auxiliary monomer position of the instrument. The instrument is programmed to add nucleotide monomers and abasic spacers in the desired order and synthesize nucleic acid moieties in the 3 'to 5' direction.

1. "T" solid phase support using 3' -binding support

2. Synthesis of 5 '-AGATGA-3' moiety

3. Addition of abasic spacer

4. Synthesis of 5 '-ACGTTCG-3' moieties

5. Addition of abasic spacer

6. Synthesis of 5 '-TCGTCG-3' structural moiety

Synthesis, deprotection, work-up, and analysis were all performed as described in example 2.

Example 8: synthesis of chimeric Compounds with Linear Structure and HexaPEG and TriPEG spacers

C-29 having the structure shown below was synthesized. The nucleic acid moiety is a DNA having phosphorothioate linkages, the spacer moiety is hexapolyethylene glycol (HEG) linked to the nucleic acid moiety via phosphorothioate linkages, and the 3' -end group is tri-polyethylene glycol (TEG) linked to the nucleic acid moiety via phosphorothioate linkages.

C-29：

5′-TCGTCG-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′-TEG

The C-29 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's procedure for the synthesis of 1. mu. mol phosphorothioate DNA. The tripelenyl glycol-controlled porous glass used as solid support for the synthesis was from Glen Research (Sterling, Va.). Nucleoside monomer and spacer moiety precursor 4, 4' -O-dimethoxytrityl-hexapolyethylene glycol-O- (N, N-diisopropyl) 2-cyanoethylphosphamide (from Glen Research, Sterling, VA) was dissolved in anhydrous acetonitrile to a final concentration of 0.05M. The HEG spacer was placed at the auxiliary monomer position of the instrument. The instrument is programmed to add nucleoside monomers and HEG spacers in the desired order and synthesize the nucleic acid moieties in the 3 'to 5' direction.

1. Utilizing a solid phase carrier of tri-polyethylene glycol

2. Synthesis of 5 '-AGATGAT-3' moieties

3. Adding HEG spacer

4. Synthesis of 5 '-ACGTTCG-3' moieties

5. Adding HEG spacer

6. Synthesis of 5 '-TCGTCG-3' structural moiety

Synthesis, deprotection, work-up, and analysis were all performed as described in example 2.

Example 9: synthesis of chimeric Compounds with Linear Structure and HexaPEG and TriPEG spacers

C-30 having the structure shown below was synthesized. The nucleic acid moiety is DNA having phosphorothioate linkages, the spacer moiety and the 5 '-end group are hexapolyethylene glycol (HEG) linked to the nucleic acid moiety via phosphorothioate linkages, and the 3' -end group is tri-polyethylene glycol (TEG) linked to the nucleic acid moiety via phosphorothioate linkages.

C-30：

HEG-5′-TCGTCG-3′-HEG-5′-ACGTTCG-3′HEG-5′-AGATGAT-3′-TEG

The C-30 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's procedure for the synthesis of 1. mu. mol phosphorothioate DNA. The tripelenyl glycol-controlled porous glass used as solid support for the synthesis was from Glen Research (Sterling, Va.). Nucleoside monomer and spacer moiety precursor 4, 4' -O-dimethoxytrityl-hexapolyethylene glycol-O- (N, N-diisopropyl) 2-cyanoethylphosphamide (from Glen Research, Sterling, VA) was dissolved in anhydrous acetonitrile to a final concentration of 0.05M. The HEG spacer precursor was placed in the instrument at the auxiliary monomer position. The instrument is programmed to add nucleotide monomers and HEG spacers in the desired order and synthesize nucleic acid moieties in the 3 'to 5' direction.

1. Utilizing a solid phase carrier of tri-polyethylene glycol

2. Synthesis of 5 '-AGATGAT-3' moieties

3. Adding HEG spacer

4. Synthesis of 5 '-ACGTTCG-3' moieties

5. Adding HEG spacer

6. Synthesis of the S '-TCGTCG-3' moiety

7. Adding HEG spacer

Synthesis, deprotection, work-up, and analysis were all performed as described in example 2.

Example 10: synthesis of chimeric Compounds with Linear Structure and Hexaand TriPEG spacers and with phosphodiester linkages

C-31 having the structure shown below was synthesized. The nucleic acid moiety is DNA having phosphodiester linkages, the spacer moiety and the 5 '-end group are hexapolyethylene glycol (HEG) linked to the nucleic acid moiety via phosphodiester linkages, and the 3' -end group is tri-polyethylene glycol (TEG) linked to the nucleic acid moiety via phosphodiester linkages.

C-31：

HEG-5′-TCGTCG-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′-TEG

The C-31 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's protocol for 1. mu. mol phosphodiester DNA synthesis. The tripelenyl glycol-controlled porous glass used as solid support for the synthesis was from Glen Research (Sterling, Va.). Nucleoside monomer and spacer moiety precursor 4, 4' -O-dimethoxytrityl-hexapolyethylene glycol-O- (N, N-diisopropyl) 2-cyanoethylphosphamide (from Glen Research, Sterling, VA) was dissolved in anhydrous acetonitrile to a final concentration of 0.05M. The HEG spacer was placed at the auxiliary monomer position of the instrument. The instrument is programmed to add nucleotide monomers and HEG spacers in the desired order and synthesize nucleic acid moieties in the 3 'to 5' direction.

1. Utilizing a solid phase carrier of tri-polyethylene glycol

2. Synthesis of 5 '-AGATGAT-3' moieties

3. Adding HEG spacer

4. Synthesis of 5 '-ACGTTCG-3' moieties

5. Adding HEG spacer

6. Synthesis of 5 '-TCGTCG-3' structural moiety

7. Adding HEG spacer

The synthesis cycle consists of a detritylation step, a coupling step (phosphoramidite monomer plus 1H-tetrazole), a capping step, an oxidation step and a final capping step. After the assembly is completed, the trityl-removed compound is cut off from the controlled porous glass and reacted with concentrated ammonia water at 58 ℃ for 16 hours to deprotect the base. This compound was purified by preparative polyacrylamide electrophoresis, desalted on a Sep-pak Plus extraction column (Waters, Milford, Mass.), and precipitated from 1M aqueous sodium chloride solution with 25 volumes of 95% ethanol. This compound was dissolved in Milli Q water and the yield was determined from the absorbance at 260 nm. Finally, the compound was lyophilized to form a powder. This compound was characterized by capillary gel electrophoresis, electrospray mass spectrometry, and RP-HPLC to confirm its composition and purity. In addition, an endotoxin content assay (LAL assay, Bio Whittaker) was also performed, and the results showed endotoxin levels < 5 EU/mg compound.

Example 11: synthesis of chimeric Compounds with Linear Structure and 2- (hydroxymethyl) Ethyl spacer

C-25 having the structure shown below was synthesized. The nucleic acid moiety is a DNA having a phosphorothioate linkage, and the spacer moiety is a 2- (hydroxymethyl) ethyl (HME) linked to the nucleic acid moiety via a phosphorothioate linkage.

C-25：

5′-TCGTCG-3′-HME-5′-ACGTTCG-3′-HME-5′-AGATGAT-3′

The C-25 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's protocol for the synthesis of 1. mu. mol phosphorothioate DNA. Nucleoside monomer and spacer moiety precursor 1-O- (4, 4' -dimethoxytrityl) -3-O-levulinyl-glycerol-2-O- (N, N-diisopropyl) 2-cyanoethyl phosphoramidite (from Chemgenes, Ashland, Mass.) was dissolved in anhydrous acetonitrile to a final concentration of 0.05M. The HME spacer was placed at the auxiliary monomer position of the instrument. The instrument is programmed to add nucleotide monomers and HME spacers in the desired order and synthesize nucleic acid moieties in the 3 'to 5' direction.

1. "T" solid phase support using 3' -binding support

2. Synthesis of 5 '-AGATGA-3' moiety

3. Spacer addition of HME

4. Synthesis of 5 '-ACGTTCG-3' moieties

5. Spacer addition of HME

6. Synthesis of 5 '-TCGTCG-3' structural moiety

Synthesis, deprotection, work-up, and analysis were all performed as described in example 2. The levulinyl group is removed during the treatment with ammonia.

Example 12: synthesis of chimeric Compounds having Linear Structure and negatively charged spacer moieties

C-13 having the structure shown below was synthesized. The nucleic acid moiety is DNA having phosphorothioate linkages, and the spacer moiety is a propyl (C3) polymer linked via phosphorothioate linkages.

C-13：5′-TCGTCG-3′-(C3)₁₅-5′-T-3′

The C-13 molecule was synthesized on a Perseptive Biosystems Expedite8909 automated DNA synthesizer using the manufacturer's protocol for 1. mu. mol phosphorothioate DNA synthesis. Nucleoside monomer and spacer moiety precursor 4, 4' -O-dimethoxytrityl-propyl-O- (N, N-diisopropyl) 2-cyanoethylphosphamide (from Glen Research, Sterling, VA) was dissolved in anhydrous acetonitrile to a final concentration of 0.1M. The C3 spacer was placed at the auxiliary monomer position of the instrument. The instrument is programmed to add nucleotide monomers and C3 spacers in the desired order and synthesize the nucleic acid moieties in the 3 'to 5' direction.

1. "T" solid phase support using 3' -binding support

2. Adding 15C 3 spacers

3. Synthesis of 5 '-TCGTCG-3' structural moiety

The synthesis cycle consists of a detritylation step, a coupling step (phosphoramidite monomer plus 1H-tetrazole), a capping step, a step at 9: 1, acetonitrile: a sulfurization step with 0.02M 3-amino-1, 2, 4-dithiazole-5-thione (ADTT) in pyridine and a final capping step. After the assembly was completed, the trityl-bearing compound was cleaved from the controlled pore glass and reacted with concentrated ammonia at 58 ℃ for 16 hours to deprotect the bases. This compound was purified by HPLC on a Hamilton PRP-1 column using a gradually increasing acetonitrile gradient in 0.1M triethylammonium acetate. The purified compound was concentrated to dryness, treated with 80% aqueous acetic acid to remove the 4, 4' -dimethoxytrityl group, and then precipitated twice from 1M aqueous sodium chloride solution with 2.5 volumes of 95% ethanol. This compound was dissolved in Milli Q water and the yield was determined from the absorbance at 260 nm. Finally, the compound was lyophilized to form a powder.

This compound was characterized by capillary gel electrophoresis, electrospray mass spectrometry, and RP-HPLC to confirm its composition and purity. In addition, an endotoxin content assay (LAL assay, Bio Whittaker) was also performed, and the results showed endotoxin levels < 5EU/mg compound.

C-14, C-15 and C-16 were synthesized similarly.

Example 13: synthesis of chimeric Compounds having Linear Structure and negatively charged spacer moieties

C-38 having the structure shown below was synthesized. The nucleic acid moiety is DNA having phosphorothioate linkages and the spacer moiety is hexapolyethylene glycol (HEG) linked via phosphorothioate linkages.

C-38：5′-TCGTCGA-3′-(HEG)₄-5′-TCGTCGA-3′

The C-38 molecule was synthesized as described in example 2. Spacer moiety precursor is 4, 4' -O-dimethoxytrityl-hexapolyethylene glycol-O- (N, N-diisopropyl) 2-cyanoethylphosphamide (from Glen Research, Sterling, VA). The synthesis is accomplished by performing the following steps:

1. "A" solid phase support using 3' -binding support

2. Synthesis of 5 '-TCGTCG-3' structural moiety

3. Adding 4 HEG spacers

4. Synthesis of 5 '-TCGTCGA-3' moieties

This compound was purified by HPLC as described in example 12. Characterization and endotoxin content determination of this compound were performed as described in example 2.

Example 14: synthesis of chimeric Compounds having a Linear Structure and a negatively charged spacer moiety connecting two nucleic acid moieties by the 3' -terminus

C-37 having the structure shown below was synthesized. The nucleic acid moiety is DNA having phosphorothioate linkages and the spacer moiety is hexapolyethylene glycol (HEG) linked via phosphorothioate linkages.

C-37：5′-TCGTCGA-3′-(HEG)-3′-AGCTGCT-5′

The C-37 molecule was synthesized as described in example 2, but the synthesis utilized a 5 '-carrier bound nucleoside and 3' -O- (4, 4 '-dimethoxytrityl) -protected nucleoside-5' -O- (N, N-diisopropyl) 2-cyanoethylphosphonite (Glen Research, Sterling, Va.) to synthesize the first nucleic acid moiety. Spacer moiety precursor is 4, 4' -O-dimethoxytrityl-hexapolyethylene glycol-O- (N, N-diisopropyl) 2-cyanoethylphosphamide (from Gleh Research, Sterling, VA). The synthesis is accomplished by performing the following steps:

1. "T" solid phase support using 5' -binding support

2. Synthesis of 3 ' -AGCTGC-5 ' moiety with 3 ' -O- (4, 4 ' -dimethoxytrityl) -protected nucleoside-5 ' -O- (N, N-diisopropyl) 2-cyanoethylphosphide (5 ' to 3 ' Synthesis)

3. Adding 4 HEG spacers

4. Synthesis of 5 ' -TCGTCGA-3 ' moiety with 5 ' -O- (4, 4 ' -dimethoxytrityl) -protected nucleoside-3 ' -O- (N, N-diisopropyl) 2-cyanoethylphosphonite (3 ' to 5 ' Synthesis)

This compound was purified by HPLC as described in example 12. Characterization and endotoxin content determination of this compound were performed as described in example 2.

Example 15: synthesis of chimeric Compounds having branched structures

C-27 having the structure shown below was synthesized. The nucleic acid moiety is a DNA having a phosphorothioate linkage, and the spacer moiety is glycerol, which is linked to the nucleic acid moiety via a phosphorothioate linkage.

C-27：(5′-TCGTCGA-3′)₂-glycerol-5 '-AACGTTC-3'

The C-27 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's protocol for the synthesis of 1. mu. mol phosphorothioate DNA. Nucleoside monomer and spacer moiety precursor 1, 3-bis- (4, 4' -O-dimethoxytrityl) -glycerol-2-O- (N, N-diisopropyl) 2-cyanoethyl phosphoramidite (symmetrically branched phosphoramidite from chemces, Ashland, MA, fig. 2) was dissolved in anhydrous acetonitrile to a final concentration of 0.05M. A glycerol spacer was placed at the auxiliary monomer location on the instrument. The instrument is programmed to add nucleotide monomers and glycerol spacers in the desired order and synthesize nucleic acid moieties in the 3 'to 5' direction.

1. "C" solid phase support using 3' -binding support

2. Synthesis of 5 '-AACGTT-3' moieties

3. Addition of glycerol-based symmetrically branched phosphoramidites

4. Simultaneous synthesis of two 5 '-TCGTCGA-3' moieties

The preparation of this branched compound was carried out according to the same procedure as described in example 2, except that in step 4 the amount of each agent delivered in the synthesis cycle was doubled due to the simultaneous construction of two nucleic acid strands. The symmetrically branched phosphoramidites shown in FIG. 2 require that the nucleic acid sequence synthesized after addition of the symmetrically branched phosphoramidite must be identical, but the nucleic acid sequence synthesized prior to its addition may be identical or different from the sequence synthesized later.

The branched compounds were purified and characterized as described in example 2.

C-28 was synthesized analogously.

Example 16: a chimeric compound having a branched structure in which all nucleic acid moieties are linked via the 3' -end is synthesized.

C-95 having the structure shown below was synthesized. The nucleic acid moiety is DNA having phosphorothioate linkages, and the spacer moiety is glycerol and HEG, which are linked to the nucleic acid moiety via phosphorothioate linkages.

C-95：(5′-TCGTCGA-3′-HEG)₂-glycerol-HEG-3 '-AGCTGCT-5'

The C-95 molecule was synthesized as described in example 2, but the first nucleic acid moiety was synthesized using a 5 '-carrier-bound nucleoside and a 3' -O- (4, 4 '-dimethoxytrityl) -protected nucleoside-5' -O- (N, N-diisopropyl) 2-cyanoethylphosphonite (Glen Research, Sterling, Va.). The precursor for the branched spacer moiety was 1, 3-bis- (4, 4' -O-dimethoxytrityl) -glycerol-2-O- (N, N-diisopropyl) 2-cyanoethyl phosphoramidite (a symmetrically branched phosphoramidite from Chem Genes, Ashland, MA, FIG. 2). The synthesis is accomplished by performing the following steps:

1. "T" solid phase support using 5' -binding support

2. Synthesis of 3 ' -AGCTGC-5 ' moiety with 3 ' -O- (4, 4 ' -dimethoxytrityl) -protected nucleoside-5 ' -O- (N, N-diisopropyl) 2-cyanoethylphosphide (5 ' to 3 ' Synthesis)

3. Adding HEG spacer

4. Addition of glycerol-based symmetrically branched phosphoramidites

5. Adding two HEG spacers simultaneously

6. Simultaneous synthesis of two 5 ' -TCGTCGA-3 ' moieties using 5 ' -O- (4, 4 ' -dimethoxytrityl) -protected nucleoside-3 ' -O- (N, N-diisopropyl) 2-cyanoethylphosphamide (3 ' to 5 ' synthesis)

The preparation of this branched compound was carried out according to the same procedure as described in example 2, except that each reagent input into the synthesis cycle was doubled in steps 5 and 6 due to the simultaneous construction of two nucleic acid strands. The symmetrically branched phosphoramidites shown in FIG. 2 require that the nucleic acid sequence synthesized after the addition of the symmetrically branched phosphoramidite must be identical, but the nucleic acid sequence synthesized prior to its addition may be the same or different from the sequence synthesized later.

This compound was purified by HPLC as described in example 12. Characterization and endotoxin content determination of this compound were performed as described in example 2.

Example 17: chimeric compounds having a branched structure and containing three different nucleic acid moieties were synthesized.

C-35 having the structure shown below was synthesized. The nucleic acid moiety is a DNA having a phosphorothioate linkage, and the spacer moiety is a glycerol linked to the nucleic acid moiety via a phosphorothioate linkage.

The C-35 molecule was synthesized as described in example 2. Nucleoside monomer and spacer moiety precursor 1- (4, 4' -O-dimethoxytrityl) -3-O-levulinyl-glycerol-2-O- (N, N-diisopropyl) 2-cyanoethyl phosphoramidite (asymmetric branched phosphoramidite from Chemgenes, Ashland, Mass., FIG. 2) was dissolved in anhydrous acetonitrile to a final concentration of 0.05M. A glycerol spacer was placed on the instrument at the auxiliary monomer location. The instrument is programmed to add nucleotide monomers and glycerol spacers in the desired order and synthesize nucleic acid moieties in the 3 'to 5' direction.

1. "T" solid phase support using 3' -binding support

2. Synthesis of 5 '-AGATGA-3' moiety

3. Addition of an asymmetrically branched phosphoramidite based on glycerol

4. Synthesis of 5 '-AACGTTC-3' moiety at the dimethoxytrityl terminus

5. Detritylation and capping of the AACGTTC moiety.

6. Removal of levulinyl protecting groups

7. Synthesis of 5 '-TCGTCGA-3' moieties

The synthesis proceeds essentially as described in example 2, but after step 4, the 5 '-AACGTTC-3' moiety is detritylated and capped with acetic anhydride/N-methylimidazole to cap the nucleic acid moiety. Next, the levulinyl protecting group was removed by treatment with 0.5M hydrazine hydrate in 3: 2 pyridine: acetic acid/pH 5.1 for 5 minutes. The solid support containing the compound was washed well with anhydrous acetonitrile and the 5 '-TCGTCGA-3' moiety was added using the protocol described in example 2.

This branched compound was purified and characterized as described in example 2.

Example 18: chimeric compounds of branched structure were synthesized by conjugation strategy.

C-36 was synthesized as shown in FIG. 3. The nucleic acid moiety is DNA having a phosphorothioate linkage and the spacer moiety is based on STARBURST^_A dendrimer. The synthesis of 5' -C6-disulfide spacer (thiol modifier C6S-S, Glen Research, Sterling, VA product number 10-1926-xx) nucleic acid structure part, the disulfide spacer once reduced can provide with the tree with maleimide group reaction.

Synthesis of 5' -C6-disulfide-TCGTCGA (4):

5 '-C6-disulfide-TCGTCGA can be synthesized using a Perseptive Biosystems Expedite8909 automated DNA synthesizer using the manufacturer's protocol for the synthesis of 1. mu. mol phosphorothioate DNA. The nucleoside monomer and thiol modifier C6S-S (Glen Research, Sterling, Va.) were dissolved in anhydrous acetonitrile to a final concentration of 0.1M. The thiol modifier is placed at the auxiliary monomer position of the instrument. The instrument is programmed to add the nucleotide monomers and thiol modifiers in the desired order and synthesize the nucleic acid moieties in a 3 'to 5' direction.

1. "A" solid phase support using 3' -binding support

2. Synthesis of 5 '-TCGTCG-3' structural moiety

3. The thiol modifier precursor (S-trityl-6-mercaptohexyl- (2-cyanoethyl) - (N, N-diisopropyl) phosphoramidite) was added.

The synthesis cycle consisted of a detritylation step, a coupling step (phosphoramidite monomer plus 1H-tetrazole), a capping step, a sulfurization step with 0.02M 3-amino-1, 2, 4-dithiazole-5-thione (ADTT) in 9: 1 acetonitrile: pyridine and a final capping step. After the assembly is completed, the trityl-bearing compound is cut off from the controlled porous glass and reacted with concentrated ammonia water at 58 ℃ for 16 hours to deprotect the base. This compound was purified by HPLC on a Hamilton PRP-1 column using an increasing gradient of acetonitrile in 0.1M triethylammonium acetate. The purified compound was concentrated to dryness, treated with 80% aqueous acetic acid to remove the 4, 4' -dimethoxytrityl group, and then precipitated twice with 2.5 volumes of 95% ethanol from 1M aqueous sodium chloride. This compound was dissolved in Milli Q water and the yield was determined from the absorbance at 260 nm. Finally, the compound was lyophilized to form a powder.

This compound was characterized by capillary gel electrophoresis, electrospray mass spectrometry, and RP-HPLC to confirm its composition and purity. In addition, an endotoxin content assay (LAI assay, Bio Whittaker) was also performed, and the results showed endotoxin levels < 5EU/mg compound.

Synthesis of 5' -mercapto-C6-TCGTCGA (5):

disulfide-modified nucleic acids (4) are reduced to thiols using tris (2-carboxyethylphosphine) hydrochloride (TCEP; Pierce, Rockford, Ill.). Nucleic acid was dissolved at a concentration of 20mg/ml in a buffer containing 0.1M sodium phosphate/0.15M sodium chloride/pH 7.5. In a separate vial, TCEP was dissolved at a concentration of 0.17M in 0.1M sodium phosphate/0.15M sodium chloride/pH 7.5. 5 equivalents of TCEP were added to the nucleic acid and gently mixed. This solution was incubated at 40 ℃ for 120 min and then purified by size exclusion chromatography (Pharmacia P2 column) to yield 5' -mercapto-C6-TCGTCGA (5).

Synthesis of maleimide-modified STARBURST _ dendrimer (7):

STARBURST _ TRORIES with varying numbers (4, 8, 16, 32, 64, etc.) of amines are available from Aldrich (Milwaukee, Wis.). STARBURST dendrimer (6) with four amino groups was dissolved in Dimethylformamide (DMF) at a concentration of 0.2M. Triethylamine (10 equivalents) and sulfosuccinimidyl 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (sulfo-SMCC; Pierce, Rockford, IL, 8 equivalents) were then added and the solution was stirred for 2 hours or until the reaction was complete, as determined by thin layer chromatography (TLC; 10% methanol/dichloromethane). The reaction was quenched with water for 30 minutes before the DMF was removed under vacuum. The residue was dissolved in methylene chloride and, The reaction mixture was washed twice with saturated aqueous sodium bicarbonate solution and then with water. The organic phase was passed over MgSO₄Dried, filtered, and concentrated to dryness under vacuum. The product is purified by silica gel chromatography to produce7。

STARBURST _ TREE- (5 '-TCGTCGA-3')₄(8) The synthesis of (2):

the maleimide-modified STARBURST _ dendrimer (6) was dissolved in DMSO (5mg/ml) and to this solution purified 5' -C6-mercapto-TCGTCGA (5) (10 equiv.) was added dropwise at a concentration of 10mg/ml in 0.1M sodium phosphate/0.15M sodium chloride/pH 7.5. The resulting mixture was stirred at 40 ℃ overnight. Purification of the conjugate by molecular size exclusion chromatography (Sephadex G-25) yields the compound8。

Example 19: synthesis of chimeric Compounds having branched structures

C-94 having the structure shown below was synthesized. The nucleic acid moiety is a DNA having a phosphorothioate linkage, and the spacer moiety is a glycerol linked to the nucleic acid moiety via a phosphorothioate linkage.

C-94：(5′-TCGTCGA-3′-HEG)₂-glycerol-HEG-5 '-TCGTCGA-3'

The C-94 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's procedure for the synthesis of 1. mu. mol phosphorothioate DNA. Nucleoside monomer and spacer moiety precursor [1, 3-bis- (4, 4 '-O-dimethoxytrityl) -glycerol-2-O- (N, N-diisopropyl) 2-cyanoethyl phosphoramidite (symmetrically branched phosphoramidite from ChemGenes, Ashland, Mass., FIG. 2) and 4, 4' -O-dimethoxytrityl-hexapolyethylene glycol-O- (N, N-diisopropyl) 2-cyanoethyl phosphoramidite (from Glen Research, Sterling, Va) ] were dissolved in anhydrous acetonitrile to a final concentration of 0.05M. The glycerol and HEG spacers were placed on the instrument at the auxiliary monomer locations. The instrument is programmed to add nucleotide monomers, HEG spacers and glycerol spacers in the required order and synthesize the nucleic acid moieties in the 3 'to 5' direction.

1. "A" solid phase support using 3' -binding support

2. Synthesis of 5 '-TCGTCGA-3' moieties

3. Adding HEG spacer

4. Addition of glycerol-based symmetrically branched phosphoramidites

5. Adding two HEG spacers simultaneously

6. Simultaneous synthesis of two 5 '-TCGTCGA-3' moieties

This branched compound was prepared according to the same protocol as described in example 2, except that each reagent input into the synthesis cycle was doubled in steps 5 and 6 due to the simultaneous construction of two nucleic acid strands. The symmetrically branched phosphoramidites shown in FIG. 2 require that the nucleic acid sequence synthesized after addition of the symmetrically branched phosphoramidite must be identical, but the nucleic acid sequence synthesized prior to its addition may be identical or different from the sequence synthesized later.

This branched compound was purified by HPLC as described in example 12 and characterized as described in example 2.

C-96 and C-101 were synthesized similarly.

C-103 and C-104 were also synthesized by the same method, but with the use of a tri-polyethylene glycol or propyl spacer, respectively, instead of a hexa-polyethylene glycol spacer.

Example 20: synthesis of chimeric Compounds having branched structures

C-98 having the structure shown below was synthesized. The nucleic acid moiety is a DNA having a phosphorothioate linkage, and the spacer moiety is a glycerol linked to the nucleic acid moiety via a phosphorothioate linkage.

C-98：(5′-TCGTCGA-3′-HEG)₃-Tridymer-HEG-5 'AACGTTC-3' -HEG-5 '-TCGA-3'

The C-98 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's protocol for the synthesis of 1. mu. mol phosphorothioate DNA. The nucleoside monomer and spacer moiety [ triplex phosphoramidite (from Glen Research, Sterling, Va.) and 4, 4' -O-dimethoxytrityl-hexapolyethylene glycol-O- (N, N-diisopropyl) 2-cyanoethylphosphamide (from Glen Research, Sterling, Va) ] were dissolved in anhydrous acetonitrile to a final concentration of 0.05M. The triplex and HEG spacers were placed in the auxiliary monomer position of the instrument. The instrument is programmed to add nucleotide monomers, HEG spacers and triplex spacers in the desired order and synthesize nucleic acid moieties in the 3 'to 5' direction.

1. "A" solid phase support using 3' -binding support

2. Synthesis of 5 '-TCGA-3' moiety

3. Adding HEG spacer

4. Synthesis of 5 '-AACGTTC-3' moieties

5. Adding HEG spacer

6. Addition of Tridylic phosphoramidite (see FIG. 2)

7. Adding three HEG spacers simultaneously

8. Simultaneously synthesizing three 5 '-TCGTCGA-3' structural parts

The preparation of this branched compound was carried out according to the same protocol as described in example 2, but each reagent was input into the synthesis cycle in three times in amounts in steps 7 and 8 since 3 nucleic acid strands were constructed simultaneously. The symmetric triplex phosphoramidite shown in FIG. 2 requires that the nucleic acid sequence synthesized after addition of the symmetric triplex phosphoramidite must be identical, but the nucleic acid sequence synthesized before its addition may be identical or different from the later synthesized sequence.

This branched compound was purified by HPLC as described in example 12 and characterized as described in example 2.

Example 21: synthesis of Linear chimeric Compounds with hexapolyethylene glycol spacer and 3' -thiol linker

CIC containing a 3' -thiol linker was first synthesized and purified as its disulfide derivative. The disulfide group is then reduced to produce a reactive thiol group. For example, to synthesize C-116, C-8 was first synthesized as described in example 2, but in this synthesis the 3' -thiol modifier C3S-SCPG (Glen Research, Sterling, Va.) was used as the solid support instead of the "T" solid support.

C-116：5′-TCGTCGA-3′-HEG-5′-ACGTTCG-3′-HEG-5′-AGATGAT-3′-(CH₂)₃SS(CH₂)₃OH

It should be understood that C-116 can be represented as [ C-8] -3' -disulfide. This CIC was purified by HPLC as described in example 12. This compound was characterized as described in example 2.

C-116 was reduced to the thiol using tris (2-carboxyethylphosphine) hydrochloride (TCEP; Pierce, Rockford, Ill.). C-116 was dissolved at a concentration of 30.5mg/ml (0.8ml, 24.4 mg; 3.14. mu. mol) in 100mM sodium phosphate/150 mM sodium chloride/1 mM EDTA/pH7.4 buffer. In another flask, TCEP was dissolved at a concentration of 0.167M in 100mM sodium phosphate/150 mM sodium chloride/1 mM EDTA/pH7.4 buffer. 5 equivalents (100ul, 4.8mg, 17umol) of TCEP stock solution were added to the CIC solution. The solution was gently mixed, incubated at 40 ℃ for 120 min, and purified on a Sephadex G-25 column (5ml, Amersham Pharmacia, Piscataway, N.J.) to give C-117(13.2 mg). It is understood that C-117 may be represented as [ C-8] -3' -mercapto. This CIC was purified by HPLC as described in example 12.

C-115 was similarly synthesized from C-114.

Example 22: synthesis of Linear chimeric Compounds with propyl spacer and 5' -thiol linker

CIC containing a 5' -thiol linker was first synthesized and purified as its disulfide derivative. The disulfide group is then reduced to produce a reactive thiol group. The compound C-110 (below) can be represented as 5' -disulfide-C-11. Compound C-111 can be represented as 5' -mercapto-C-11.

C-110：HO(CH₂)₆SS(CH₂)₆-5′-TCGTCG-3′-C3-5′-ACGTTCG-3′-C3-5′-AGATGAT-3′

C-110 was synthesized as described in example 3, but the final coupling was performed using thiol modifier C6S-S (Glen Research, Sterling, Va.). This CIC was purified by HPLC as described in example 12. This compound was characterized as described in example 2. C-110 was reduced to the thiol using tris (2-carboxyethylphosphine) hydrochloride (TCEP; Pierce, Rockford, Ill.) as described in example 22.

C-107, C-113 and P-16 were synthesized analogously.

Example 23: chimeric compounds of branched structure were synthesized by conjugation strategy.

C-105 was synthesized as shown in FIG. 4. Tris (2-maleimidoethyl) amine (TMEA, Pierce, Rockford, IL) was dissolved in Dimethylformamide (DMF) at a concentration of 4.3 mg/ml. TMEA solution (12ul, 52ug, 1.0 equiv.) was added to a solution of C-117 (237ul, 4.0mg, 4.0 equiv.) in 100mM sodium phosphate/150 mM sodium chloride/1 mM EDTA/pH7.4 buffer and mixed well. This solution was purified on a Superdex200 column (24ml, Amersham Pharmacia, Piscataway, N.J.) in 10mM sodium phosphate/141 mM sodium chloride/pH 7.0 buffer after standing overnight at room temperature. The product was dried under vacuum, dissolved in 0.4ml of Milli Q water and precipitated with 1.0ml of 95% ethanol. After freezing at-20 ℃ for 1 hour, the mixture was centrifuged (2 minutes at 14 KRPM) and the supernatant carefully removed. The pellet was dissolved in 0.35ml of Milli Q water and the concentration of C105 was determined (0.4 mg isolated). This compound was analyzed as described in example 2.

C-99 was synthesized analogously.

Example 24: chimeric compounds of branched structure were synthesized by conjugation strategy.

A. Synthesis of maleimido-STARBURST dendrimer-2 generation

STARBURST dendrimer-2 generation containing 16 hydroxyl groups was purchased from Aldrich (Milwaukee, Wis.) as a 20% solution in methanol. The dendrimer (191ul, 38.2mg, 11.7umol) was vacuum dried, redissolved in 200ul of DMF and again vacuum dried to remove the last traces of methanol. To prepare the maleimido-dendrimer, N- (p-maleimidophenyl) isocyanate (PMPI, 50mg, 233.5umol) was dissolved in 200ul of DMF in a separate glass vial and then added to the dendrimer rapidly. Vortex the mixture until the dendrimer is dissolved. The solution was placed on a rotary mixer overnight at room temperature. The solution was concentrated in vacuo, dissolved in 20% methanol/dichloromethane (1ml) and purified on a 7.5g silica gel column (70-230 mesh, 60A) in 20% methanol/dichloromethane. The maleimido-dendrimer product eluted from the column in the first fraction (due to the presence of residual DMF) and was free of PMPI by-product. The product was concentrated to a tan solid (10mg, 13% yield).

STARBURST treelike^_-(5′-TGACTGTGAACGTTCGAGATGA)_X＝3-16(SEQ ID NO: 2) (C-102) Synthesis

Maleimido-dendrimer (5.7mg) was dissolved in dimethyl sulfoxide (DMSO) to form a stock solution with a concentration of 2.5 mg/ml. Maleimido-dendrimer stock solution (100ul, 0.25mg, 0.0375umol) was added to a solution (0.7ml) of C-107(9.1mg, 1.2umol) in 100mM sodium phosphate/150 mM sodium chloride/lmMEDTA/pH 7.4 buffer. The solution was placed on a rotary mixer at room temperature overnight and the product was purified on a Superdex200 column (24ml, Amersham pharmacia, Piscataway, N.J.) in 10mM sodium phosphate/141 mM sodium chloride/pH 7.0 buffer. The product eluted in the void volume (1.3mg) in 10.4 min. Analysis on a 1.2% agarose E-gel (Invitrogen, Carlsbad, Calif.) revealed that this compound was a mixture of high molecular weight species representing different loadings of polynucleotide on the dendrimer. The C-102 running electrophoresis presents a mixture of products of 1kb to more than 15kb (effective size compared to double stranded DNA marker molecules).

Example 25: synthesis of Linear chimeric Compounds with propyl spacers and Mixed phosphodiester/phosphorothioate linkages

C-84 having the structure shown below was synthesized. The nucleic acid moiety is a DNA having a phosphorothioate linkage (indicated by the lower case "s") or a phosphodiester linkage (all other linkages), and the spacer moiety is a propyl group (C3) linked to the nucleic acid moiety via a phosphodiester linkage.

C-84: 5 '-GsGs-3' -C3-5 '-TGC-3' -C3-5 '-ATCGAT-3' -C3-5 '-GCA-3' -C3-5 '-GGsGsGsGsG-3' (wherein the lower case "s" indicates a phosphorothioate bond and the other bond is a phosphodiester bond)

The C-84 molecule was synthesized by TriLink Biotechnologies (SanDiego, Calif.) on a Perseptive biosystems Expedite8909 automated DNA synthesizer using the manufacturer's protocol for the synthesis of 1. mu. mol phosphorothioate DNA. The phosphorothioate linkage procedure was arranged with capital letters as bases and the phosphodiester linkage procedure was arranged with lowercase letters as bases, the auxiliary position containing a propyl spacer phosphoramidite.

Nucleoside monomer and spacer moiety precursor 4, 4' -O-dimethoxytrityl-propyl-O- (N, N-diisopropyl) 2-cyanoethylphosphamide (from Glen Research, Sterling, VA) was dissolved in anhydrous acetonitrile to a final concentration of 0.05M. The C3 spacer was placed on the instrument at the auxiliary monomer location. The instrument is programmed to add nucleotide monomers and C3 spacers in the desired order and synthesize the nucleic acid moieties in the 3 'to 5' direction.

1. "G" solid phase support using 3' -binding support

2. Synthesis of 5 '-GGsGsGsGsG-3'

3. Adding C3 spacer

4. Synthesis of 5 '-GCA-3'

5. Adding C3 spacer

6. Synthesis of 5 '-ATCGAT-3'

7. Adding C3 spacer

8. Synthesis of 5 '-TGC-3'

9. Adding C3 spacer

10. Synthesis of 5 '-GsGs-3'

Synthesis, deprotection, work-up, and analysis were all performed as described in example 2.

C-85 and C-87 were synthesized similarly.

Example 26: synthesis of oligonucleotides containing less than 8 nucleotides

Polynucleotides containing fewer than 8 bases and phosphorothioate linkages were synthesized on a Perseptive Biosystems Expedite8909 automated DNA synthesizer. This polynucleotide was purified by RP-HPLC on a Polymer Labs PLRP-S column using an increasing gradient of acetonitrile in 0.1M triethylammonium acetate. The purified polynucleotide was concentrated to dryness, treated with 80% aqueous acetic acid to remove the 4, 4' -dimethoxytrityl group, and then the compound was precipitated twice with 3 volumes of isopropanol from 0.6M aqueous sodium acetate/pH 5.0. The polynucleotide was dissolved in MilliQ water and the yield was determined from the absorbance at 260 nm. Finally, the polynucleotide is lyophilized to a powder. This polynucleotide was characterized and endotoxin content determined as described in example 2.

Example 27: preparation of biodegradable cationic microcarriers

Cationic poly (lactic acid, glycolic acid) microcarriers (cgga) were prepared as follows. 0.875g of poly (D, L-lactide-co-glycolide) 50: 50 polymer (Boehringer Mannheim, Indianapolis, IN), which itself had a viscosity of 0.41dl/g (0.1%, chloroform, 25 ℃), was dissolved IN 7.875g of methylene chloride at a concentration of 10% w/w together with 0.3g of DOTAP. The clear organic phase was then emulsified in 500ml of aqueous PVA solution (0.35% w/v) by homogenisation with a laboratory mixer (Silverson L4R, Silverson instruments) at 4000 rpm for 30 minutes at room temperature. The system temperature was then raised to 40 ℃ with circulating hot water through the mixer sleeve. At the same time, the stirring rate was reduced to 1500 rpm and these conditions were maintained for 2 hours to extract and evaporate the dichloromethane. The microsphere suspension was cooled to room temperature with circulating cold water.

Microcarriers were separated by centrifugation (Beckman Instruments) at 8000 rpm for 10 minutes at room temperature and resuspended in deionized water by gentle in-bath sonication. This centrifugal washing was repeated two more times to remove excess PVA from the particle surface. The final pellet of particles was suspended in about 10ml of water and lyophilized overnight. Identification of the size and surface charge of the dried cationic microcarrier powder: average size (number weighed, μ) 1.4; zeta potential (mV) 32.4.

Example 28: immunomodulation of human cells by CIC

The assay was conducted to assess the immunomodulatory activity of (1) a chimeric molecule comprising a spacer moiety and (2) a polynucleotide.

Chimeric compounds and polynucleotides were synthesized as described above or by conventional phosphorothioate chemistry. Polynucleotides P-6 and P-7 were synthesized by hybrid Specialty Products (Milford MA). Immunomodulatory activity is determined by routine assays as described herein.

Peripheral blood was collected from volunteers by venipuncture with a heparinized syringe. Blood was placed on a FICOLL (Amersham Pharmacia Biotech) cushion and centrifuged. PBMCs located at FICOLL _ demarcation were collected and then washed twice with cold Phosphate Buffered Saline (PBS). At 2X 10 in 48-well plates (examples 29 to 32) or 96-well plates (examples 33 to 40)⁶Individual cells/mL in 10% heat inactivated human AB serum plus 50 units/mL penicillin, 50. mu.g/mL streptomycin, 300. mu.g/mL glutamine, 1mM sodium pyruvate and 1 XMEM nonessential amino acids (NEAA) in RPMI1640 resuspensionCells were cultured at 37 ℃.

Cells were incubated for 24 hours in the absence of test sample, in the presence of 20. mu.g/ml (0.5OD/ml) of test sample, or in the presence of 20. mu.g/ml of test sample premixed with 100. mu.g/ml of cPLGA (if utilized). Cell-free medium was then collected from each well and assayed for IFN-. gamma.and IFN-. alpha.concentrations. SAC (Pansorbin Calbiochem, 1/5000 dilution) was used as a positive control. SAC contents are staphylococcus aureus (cowan) cell material.

CYTOSCEEN, available from BioSource International, Inc^TMELISA kit, according to the manufacturer's instructions for IFN-gamma and IFN-alpha detection.

In the human PBMC assay, the background level of IFN- γ may vary, even significantly, from donor to donor. IFN-alpha levels in non-stimulated conditions generally show low background levels.

The results of such tests are given in examples 29-40 below.

In each of the experiments shown, "Medium only" and "P-7" were negative controls. "P-7" has previously been shown to have no immunomodulatory activity. SAC and "P-6" are positive controls. "P-6" has previously been shown to have significant immunomodulatory activity.

Example 29: immunomodulatory Activity of CIC

This example shows that four different CICs have significant immunomodulatory activity, as evidenced by stimulation of IFN-. gamma.and IFN-. alpha.secretion (Table 3). As expected, P-7 was not active. In addition, P-1, a 7-mer containing TCG, was inactive. Interestingly, CIC with HEG and propyl spacer moieties showed varying degrees of stimulation of IFN- α secretion. While both types of CIC stimulated IFN- α secretion, the effect of CIC with HEG was more pronounced.

TABLE 3

	IFN-γ(pg/ml)			IFN-α(pg/ml)
							Test compounds	Donor 1	Donor 2	Mean value of	Donor 1	Donor 2	Mean value of
Only culture medium	8	0	4	0	0	0
							P-7	410	51	231	0	0	0
SAC	2040	1136	1588	393	43	218
							P-6	2180	669	1425	401	39	220
							P-1	8	0	4	0	0	0
C-8	1916	696	1306	1609	44	827
							C-9	2157	171	1164	142	0	71
C-10	1595	952	1273	1662	50	856
							C-11	2308	270	1289	119	0	59

Example 30: activity of polynucleotides

This example shows that polynucleotides P-1, P-2, P-3, P-4 and P-5 have no immunomodulatory activity (Table 4). These polynucleotides have the sequences of the nucleic acid moieties of C-10 and C-11, C-10 and C-11 having immunomodulatory activity as shown in example 29.

TABLE 4

	IFN-γ(pg/ml)			IFN-α(pg/ml)
							Test compounds	Donor 3	Donor 4	Mean value of	Donor 3	Donor 4	Mean value of
Only culture medium	0	3	2	0	18	9
							P-7	3	8	5	0	31	15
SAC	1179	2000	1589	50	969	510
							P-6	99	223	161	28	106	67
							P-1	1	4	2	0	32	16
P-3	1	3	2	0	32	16
							P4	0	3	1	0	58	29
P-5	0	3	2	0	57	29
							P-2	0	4	2	0	40	20

Example 31: activity of mixtures of polynucleotides

This example shows that the mixture of polynucleotides P-1 and P-3 and the mixture of P-1, P-3, P-4 and P-5 do not have immunomodulating activity (Table 5). These polynucleotides have the sequence of the nucleic acid moieties of C-10 and C-11, while C-10 and C-11 have immunomodulatory activity. The mixture contained equal amounts of each polynucleotide at a total concentration of 20 μ g total polynucleotide/ml.

TABLE 5

Test compounds	IFN-γ(pg/ml)			IFN-α(pg/ml)
							Only culture medium	Donor 5	Donor 6	Mean value of	Donor 5	Donor 6	Mean value of
	3	52	28	20	20	20
							P-7	7	66	37	20	94	57
SAC	903	284	593	458	8215	4337
							P-6	73	1170	621	54	482	268
							(P-1)+(P-3)	3	36	19	20	40	30
(p-1)+(P-3)+(P-4)+(P-5)	1	99	50	70	65	68
							C-10	102	806	454	91	1700	896
C-11	25	792	409	76	175	126

Example 32: immunomodulatory Activity of CIC

This example demonstrates the immunomodulatory activity of C-10 and C-11 in assays using different donors than those of examples 29 and 31 (Table 6).

TABLE 6

	IFN-γ(pg/ml)			IFN-α(pg/ml)
							Test compounds	Donor 7	Donor 8	Mean value of	Donor 7	Donor 8	Mean value of
Only culture medium	1	0	1	0	0	0
							P-7	2	2	2	0	0	0
SAC	594	1100	847	22	303	163
							P-6	15	367	191	4	59	32
							C-10	23	198	111	46	539	293
C-11	5	419	212	6	56	31

Example 33: immunomodulatory Activity of CIC

This example demonstrates the immunomodulatory activity of C-8 and C-9 in an assay using a different donor than that of example 29 (Table 7). P-2, a TCG-containing 6-mer, was inactive.

TABLE 7

	IFN-γ					IFN-α
												Donor 9	Donor 10	Donor 11	Donor 12	Mean value of	Donor 9	Donor 10	Donor 11	Donor 12	Mean value of
Only culture medium	17	1	1	10	7	4	2	2	15	6
											P-7	5	2	3	2	3	0	3	1	5	2
SAC	380	688	159	73	325	2246	364	1129	1029	1192
											P-6	66	20	72	23	45	12	28	12	12	16
											P-2	2	3	1	2	2	0	2	1	4	2
C-8	312	35	31	28	102	58	30	18	49	39
											C-9	134	7	56	30	56	8	10	1	15	8

Example 34: immunomodulatory Activity of CIC

The experiments shown in table 8 demonstrate the immunomodulatory activity of several CICs of the invention, i.e. CICs characterized by various short nucleic acid moieties and various spacer moieties. Table 8 also shows that compound M-1, a compound with a mixed HEG/nucleic acid structure but lacking any 5 '-C, G-3' sequence (see Table 2), as well as certain other compounds (C-19) showed no activity. CIC formulations with cPLGA significantly enhanced the induction of IFN- α. In some cases, IFN- γ levels were also increased.

The number "28- - -" indicates the individual donor.

TABLE 8

Irritant substance	Concentration ug/ml	IFN-γ(pg/ml)					IFN-α(pg/ml)
													28065	28066	28067	28068	Mean value of	28065	28066		28067	28068	Mean value of
		Only cells P-6P-7P-2P-3P-4P-2+ P-3+ P-4C-8C-9C-10C-17C-18C-19C-20C-21C-23C-24C-25M-1C-27C-28PLGAP-6+ PLGAP-7+ PLGAP-2+ PLGAP-3+ PLGAP-4+ PLGAP-2+ P-3+ P-4+ PLGAC-8+ PLGAC-9+ PLGAC-10+ PLGAC-17+ PLGAC-18+ PLGAC-19+ PLGAC-20+ PLGAC-21+ PLGAC-23+ PLGAC-24+ PLGAC-25+ PLGAC-1 + PLGAC-27+ PLGAC-28+ PLGAC-10+ PLGAC-17+ PLGAC-18+ PLGAC-19+ PLGAC-20+ PLGAC-21+ PLGAC-23+ PLGAC-24+ PLGAC-25+ PLGAC-1 + PLGAC-27+ PLGAC-28	0202020202020 in total; 6.7 Total each 20202020202020202020202020200202020202020; 6.7 Each 20202020202020202020202020200	9643939779949399100010001000100010008435465343833754115747510825597519357134191225273346195087321000105568221623642778888601000	212112710198961028131652613511912713811428013915218410878025687416147179120588339	128811115657513227116246419402442128761040142453435575873557302703901201882893235489511	29061500104195105594596952505960238116337222641055731144343703574564203554484664884813777074995466415355	253461052031242537339440537445624222413172115226501823851650615261356533523184374084117445176072192953484448463551	014000001231311621510212275228110330388098230734182023951093204919142188599710442468789314140136216284	41783000606091524300000219457080505381303692403753753191784189208704473156		045005989622107221601318319822324291256502100004354380168635152729351314359126533721573772108283884011544												61263000035864340951621364769137677901652102000023360010646252045358627747141707920004962200034013355259520350	35031122146251423459426300532628111216787115327710590298512392380191433047797112528971138291T303207303583

As is evident from table 8, donor 28065 showed a high background in the IFN- γ assay. The value "1000" is given to indicate that the measurement is outside the sensitivity range of the assay.

Example 42: immunomodulation of mouse cells by CIC

The immunomodulatory activity of the polynucleotides and chimeric compounds was tested on murine splenocytes. Immunomodulation was assessed by measuring cytokines secreted into the culture medium. Cytokine levels in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA).

Cells are isolated and prepared using standard techniques. Spleens from 8 to 20 week old BALB/c mice were harvested and splenocytes isolated using standard sectioning methods (toasting) and treatment with ACK lysis buffer from BioWhittaker inc. Four spleens were pooled in this experiment. The isolated cells were washed in RPMI 1640 medium supplemented with 2% heat-inactivated Fetal Calf Serum (FCS), 50. mu.M 2-mercaptoethanol, 1% penicillin-streptomycin, and 2 mML-glutamine and washed at approximately 7X 10 ⁵Individual cells/ml were resuspended in 10% FCS/RPMI (RPMI 1640 medium containing 10% heat-inactivated FCS, 50. mu.M 2-mercaptoethanol, 1% penicillin-streptomycin and 2mM L-glutamine).

Cell culture was prepared by mixing approximately 7X 10 cells⁵Cells per well were established in triplicate in 100 μ l 10% FCS/RPMI in 96-well flat-bottomed microtiter plates, and cells were allowed to rest for at least 1 hour after plating. Incubate with the indicated test compounds (at the indicated concentrations) for 24 hours at 37 ℃. Cell supernatants were harvested and frozen at-80 ℃. The cytokine production by the cells was measured by ELISA and the results are shown in Table 9.

TABLE 9

Test compounds	Dosage form	IL-6	IL-12	IFNγ
					P-6	5.0μg/ml1.0μg/ml0.1μg/ml	93115760121	537445651665	25052175187
C-10	5.0μg/ml1.0μg/ml0.1μg/ml	334217619	23291738122	1991049
					C-11	5.0μg/ml1.0μg/ml0.1μg/ml	1009811814458	427949143359	33423220960
P-7	5.0μg/ml1.0μg/ml	97	177143	2330
					SAC		734	1343	18843
Only culture medium		9	124	9

Example 35: activity of CIC containing 3-nucleotide nucleic acid moieties and enhancement of Activity by cPLGA

This example shows the immunomodulatory activity of several CICs measured using human PBMC in the presence and absence of cPLGA. Interestingly, C-30(C-31) of the phosphodiester type was inactive as CIC alone, but had good activity when formulated with cPLGA. In fact, the general trend is that CICs containing only phosphodiester linkages (C-31, C-36, and C-93) are inactive as CICs alone, but their activity increases significantly when formulated with cPLGA.

C-32, a CIC containing only a trimeric nucleic acid moiety, was active alone and showed greater activity when formulated with cPLGA. See table 10.

Watch 10

	IFN-γ(pg/ml)						IFN-α(pg/ml)
												Irritant substance	28089	28090	28098	28099	Mean value of		28089	28090	28098	28099	Mean value of
Only cells P-6P-7C-10C-21C-22C-8C-9C-29C-30C-31C-32C-33C-93C-28PLGAP-6+ PLGAP-7+ PLGAC-10+ PLGAC-21+ PLGAC-22+ PLGAC-8+ PLGAC-9+ PLGAC-29+ PLGAC-30+ PLGAC-31+ PLGAC-32+ PLGAC-33+ PLGAC-93+ PLGAC-28+ PLGASAC	08403561316212630000014156061218041138772668103682597256454171427683195	0255444681552750605001521443373454244332330477233327192186628306489	0745017421811020515012011001831632779115011612127118631536447159725924917072252101	41252114012491116177202353345101605301630357506341414065796323224306	130219811862109101109013116411104755745958661842683795205647240176771866273		250017560211075134158285560081977523134761910053089094469628165899013667	796227611574612467922926312230643810385256102038668336371111691228912201738155239	331051918728697314359526246342542393403650910016049348554910282181764268114192	281053730446624736233247295930296749914713172302133916801419331361131130440007070	416821142241972053921465691063621433318344651141873710753359736099923012372352126117

Example 36: immunomodulatory Activity of CIC containing 5' TCG

This example shows the immunomodulatory effects produced by CIC comprising different nucleic acid moieties (see Table 11). In general, sequences containing 5 ' -TCG-3 ' (C-8, C-21, C-50, C-51, etc.) or 5 ' -NTCG (C-46) (where N is any nucleoside) are more active than other CG-containing CICs (C-24, C-52). In addition, although most CICs induced significant amounts of IFN- γ, the results were more variable for IFN- α induction, suggesting that IFN- α induction may place more stringent requirements on the motif than IFN- γ induction. In particular, CIC containing 5 '-TCGA-3' (C-50, C-51, C-45) produced more IFN-. alpha.than CIC containing 5 '-TCGT-3' (C-41, C-42, C-52).

In addition to C-8 and C-21 (comprising the motif 5 ' -TCGTCGA-3 '), the optimal IFN- α induction was produced by CIC with TCGA at the 5 ' position.

CIC containing only hexamer (C-22), pentamer (C-43), and tetramer (C-44) nucleic acid moieties was found to induce IFN- γ if used alone. In addition, each of these CICs, as well as C-32 containing only a trimeric nucleic acid moiety, induced significant amounts of IFN- γ and IFN- α when formulated with cPLGA. C-39, a CIC with two hexameric nucleic acid moieties, was active when used alone. C-40, a CIC with a hexamer and a tetramer nucleic acid moiety, was inactive in this experiment. Both CICs showed significant activity when formulated with cPLGA.

TABLE 11

Irritant substance	IFN-γ(pg/ml)					1FN-α(pg/ml)
											28042	28043	28044	28045	Mean value of	28042	28043	28044	28045	Mean value of
	Only the cells P-6P-7C-8C-24C-21C-42C-41C-45C-46C-47C-50C-51C-52M-1C-22C-43C-44C-32C-39C-40 PLGA-6 + PLGAP-7+ PLGAC-8+ PLGAC-24+ PLGAC-21+ PLGAC-42+ PLGAC-41+ PLGAC-45+ PLGAC-46+ PLGAC-47+ PLGAC-50+ PLGAC-51+ PLGAC-52+ PLGAC-1 + PLGAC-22+ PLGAC-43+ PLGAC-44+ PLGAC-32+ PLGAC-39+ PLGAC-40+ PLGAC	15495664681487901981745903991121324795238452061282389134326192138215213671017400015157101380220135792969201811722154000221014522211180014381618	411897693915615191067107514668145371292134921429343536359194885582153832425471380120414171940229223524000120940001726159297525944000400040004981271	39252610003121198400084198448014250911142127736566484782812055258117414901362187021901910192014321137146510001551231085135420062759227518131053	52121323426177374525363171924112834016511713723317812286523253724966344721614024631173136194276133274160123	77054566016192113265348234392028299171732133131228351312318314201661423953185013741264155716142219151118701142100204915881934227620879771016	02702005700622420361120012000310010602182029594255352408502461548183712032535898620421672758285	4485115107229312373013724530181412018726038722193312024108119874000130927128183241341118612934000114240004000110	9362232879242315226019324035136720608463103710716658862955341693257301242115434022640217687712613155657	02120501500143035364823001036283822110191691196421000327536004249192127363700											1342132725613788315100158224328182751522261132524151546879421865428714841692200445526173653223792171113

Example 37 immunomodulating Activity of CIC

This example shows immunomodulation test experiments on other linear CICs (some containing both Phosphorothioate (PS) and Phosphodiester (PO)) and branched CICs (tables 12 and 13). Comparison of C-94 (a branched CIC) and C-21 (a linear CIC containing identical nucleic acid moieties) shows that branched CIC induces four times more IFN- α than linear CIC. The amount of IFN-. gamma.and IFN-. alpha.induced was significantly increased after each CIC was formulated with cPLGA. The phosphoric acid diester type C-94 and C-93 are active only when formulated. C-87 showed significant induction of IFN- α.

TABLE 12

Irritant substance	IFN-γ(pg/ml)					IFN-α(pg/ml)
											28042	28043	28044	28045	Mean value of	28042	28043	28044	28045	Mean value of
	Only cells P-6P-7C-8C-53C-49C-84C-85C-94C-93C-21C-9PLGAP-6+ PLGAP-7+ PLGAC-8+ PLGAC-53+ PLGAC-49+ PLGAC-84+ PLGAC-85+ PLGAC-94+ PLGAC-93+ PLGAC-21+ PLGAC-9+ PLGA-49 + PLGAC-84	11324346233936729174438572691960113284211471235313241288040001451	41036197532743323131981460268535813551121219232294755316690763	052948646387676931514174140005905914744617812174000291554400086940004000	1365335604263532321348881716441306721941653113210206195611673379125125331804	6363465632480881207361716697143510933414321152188428582263782928062005	89078030003027146397115559519904177721161883778712389	234025080025239400116039604609149211640471577199	3224520100528664811911198515010130400040004000400020452572397	6410854256588222241855613496411212984322594210403641241340009881571477											194315103157571376838195295451112106615160125832362288110711358366

Watch 13

Irritant substance	IFN-g(pg/ml)					IFN-α(pg/ml)
											28218	28219	28220	28221	Mean value of	28218	28219	28220	28221	Mean value of
	Cell-only P-6P-7C-87C-94SAC	513583152552	5752439621	525538441383	51415977269647	5475281921301	323232307532483	32323232167105	323232426963332	323232265412452											3232321910311268

Example 38: positional Effect of sequence motifs in CIC

This example describes an immunomodulation test assay for various CICs, some of which were tested in different donors in the previous examples, and illustrates the positional effects of nucleic acid sequences in CICs.

The CIC to be detected comprises CIC comprising two different CG-containing nucleic acid sequences (TCGTCGA and ACGTTCG) in the nucleic acid moiety and a nucleic acid moiety without a CG sequence (AGATGAT). In a CG-containing nucleic acid sequence, a CIC containing a TCGTCGA sequence will have greater activity than a CIC containing only an ACGTTCG sequence. Of these two, CIC with TCGTCGA did have greater activity. The general structure of CIC used in this embodiment, N, can be utilized₁-S₁-N₂-S₂-N₃To illustrate the location of the motif within the CIC. Placing the most active motif TCGTCGA at N₁The location resulted in the generation of the most active CIC (C-8, C-56). Is placed in N₂The location also confers activity. For example, in N₂C-57 with TCGTCGA in position and in N₃C-58 with TCGTCGA in the position was slightly more active than TCGTCGA. In N₁CIC with ACGTTCG sequence at the position, although less active than similar CIC with TCGTCGA sequence, is still better than that in N₁CIC activity with the AGATGAT sequence at position (C-57 and C-58 compared to C-59 and C60) is large. In this experiment, C-61 contains a nucleic acid moiety containing a CG motif but no TCG motif, which when formulated with cPLGA induces IFN- γ. See table 14.

TABLE 14

Irritant substance	IFN-γ(pg/ml)					IFN-α(pg/ml)
											28156	28157	28158	28159	Mean value of	28156	28157	28158	28159	Mean value of
	Only cells P-6P-8C-8C-9C-23C-54C-55C-56C-57C-58C-60C-61PLGAP-6+ PLGAP-8+ PLGAC-8+ PLGAC-9+ PLGAC-23+ PLGAC-54+ PLGAC-55+ PLGAC-56+ PLGAC-57+ PLGAC-58+ PLGAC-60+ PLGAC-61	125113225516121162733297511122341940430492431070951083814825838104879210278046501265780	387220742729576378566543323288209179632643115186214128651150960604814106585815081184	4207313401922028855203675926355251262693071821572473211011355623883	523131197329295218186563262843863114963411299921423175123564000305610211014864659	346118472360345224532963326820914492301115671086881824974115314291250756645969677	35216102262689985135385582434000139810207525054000555179714208	348497551422359654155230220246294884877177816211265801400014769321188055	17246178598635761293470418285741 8240147788521033216	83281602016913313142231033592315734835972782112433252139506334											414414270671223120175402481383107719282710198696113992821573313683776

This experiment also compared the immunomodulatory activity of two branched CIC: c-94 has a HEG moiety between the branched glycerol component and the nucleic acid moiety, while C-28 has the nucleic acid moiety directly linked to the glycerol spacer. See table 15. Interestingly, while the induction of IFN- γ by the two branched CICs was similar, the induction of IFN- α by CIC containing HEG spacers was much higher. A branched CIC (C-99) comprising three P-6 sequences linked via the 5-terminus to a maleimido activated triethylamine spacer induces only IFN-. gamma.and not IFN-. alpha.when formulated with cPLGA. In general, maximal IFN- α production is produced using a CIC having nucleic acid moieties linked by a branched structure, and having multiple unlinked or "free" 5' -ends of the nucleic acid moieties, and including spacers that provide conformational flexibility and spacing between the nucleic acid moieties.

Watch 15

Irritant substance	IFN-γ(pg/ml)					IFN-α(pg/ml)
											110	112	119	120	Mean value of	110	112	119	120	Mean value of
	Only the cells P-6P-7C-8C-59C-63C-50C-51C-45C-41C-42C-46C-52C-39C-40C-94C-28C-99PLGAP-6+ PLGAP-7+ PLGAC-8+ PLGAC-59+ PLGAC-63+ PLGAC-50+ PLGAC-51+ PLGAC-45+ PLGAC-41+ PLGAC-42+ PLGAC-46+ PLGAC-52+ PLGAC-39+ PLGAC-40+ PLGAC-94+ PLGAC-28+ PLGAC-99+ PLGA	4415081241152256153610961528880512150812244726041805168556427632164072194868012088121240273631681612304810322024136026682104768	2434424540523762642401921002044004811612284521289681612208241580368411216302418082032201212361332124431882568672	20144161362880525252325668401084104441672960321188620234014322896305616000102123720234482285364884035725316	281044048406048403632563628322012060401202300316238418282092992924247236561908360817241244186433961320472	29525514699451336546529016945643214721554141914308658146710916859881805173040692822615839413097158432072458452323911807	20502196229471614084465825058267461608382210948146674234414837685244553235423462237264376423625658302114	20017232306388472130226222619814444226014113827381414126015447461030638208466841839280	223447426424648382200107418215620829244415226105686601298222130762796416151045474207421182372252307837948000284344	27224222286366224286364822502878262921470226502782298340261484206209420542682206265826166346198260											56132128133325656910765206211779753499013103129419941331481482495318644394189211421662016136258723645461197200

Example 39 Activity of branched CIC

This example demonstrates that branched CICs with multiple free 5' ends and conformational flexibility provided by HEG spacers induce more IFN α relative to linear CICs with HEG spacers (C-94 vs C-21 and C-96 vs C-23) or branched CICs without added (HEG) spacers (C-94 vs C-28 and C-96 vs C-27). The addition of a further HEG spacer and a 4 base nucleic acid moiety to C-96 caused a reduction in IFN α induction (C-96 compared to C-97). See table 16.

The immunomodulatory activity of two CICs containing the trimeric 5 '-TCG-3' motif was examined (C-91 and C-68). Neither CIC was active by itself, but C-91 was significantly active when formulated with cPLGA.

The hydrophilic polyamide-containing STARBURST dendrimer- (C-102) conjugated with multiple P-6 sequences has significantly greater IFN- α activity than the P-6 sequence alone for an equivalent amount of P-6 (based on the P-6 chain of each chain). This result demonstrates that multimeric delivery of 5 '-CG-3' containing nucleic acid moieties on flexible hydrophilic cores significantly enhances induction of IFN- α using compositions and synthetic methods different from those described above.

TABLE 16

Irritant substance	IFN-g(pg/ml)						IFN-α(pg/ml)
													28185	28186	28187	28188	Mean value of	×4	28185	28186	28187	28188	Mean value of	×2
	Only cells P-6P-7C-8C-94C-28C-21C-23C-27C-96C-97C-9C-86C-91C-68C-102PLGAP-6+ PLGAP-7+ PLGAC-8+ PLGAC-94+ PLGAC-28+ PLGAC-21+ PLGAC-23+ PLGAC-27+ PLGAC-96+ PLGAC-97+ PLGAC-9+ PLGAC-86+ PLGAC-91+ PLGAC-68+ PLGAC-102+ PLGAC-86	5205015418116224442491632591891301581031573193913953331992924003563841116131774195	117425612437143221624444434643127118654967170186177823101813222	1314819123384751252921195125953062101131281524228017517710295244124938411424380274	18291757331251157164392243413201514413919414715	59467816166103221998101801052778137717820616214596142218174145259417323127	20378253116442664138875392405319402073073054627712823649579382570872696579101377671293506	36120419618951444383425503072579418804710415996761843581599542750422644793388014209473	4412531239464274446711644613187411646461955314531692505855726684514494513974	32452021360159391184443725109078460139491513401104246428519343164221151	443141344554402146314444493520713642139173483544426102	194216108571612856137889648112411545322757207618642038245398928816290114231868482													38843221711421125611225157719295224823109064541014411523512840774907817857163257922847371369165

Example 40

This assay measures the activity of a series of CICs comprising the hexamer nucleic acid motif 5 ' -TCGTCG-3 ' and a plurality of spacers (C-13, C-14, C-15 and C-16) attached to the 3 ' -end of this nucleic acid moiety. See table 17. These CICs are not active if used alone, but all have significant activity when formulated on plga.

TABLE 17

Irritant substance	IFN-γ(pg/ml)					IFN-α(pg/ml)
											28057	28058	28059	28060	Mean value of	28057	28058	28059	28060	Mean value of
	Only cells P-6P-7C-13C-14C-15C-16PLGAP-6+ PLGAP-7+ PLGAC-13+ PLGAC-14+ PLGAC-15+ PLGAC-16+ PLGAAC	1831235140200040200020002000172673	2103231323220002712623595852072000	112303121004920001622173220002772000	398545316254452401682000258712000	113752622294161392663127312111821668	062103100035200005865393729911842920	39600000222200052779946871628225292000	014502490004117471615912637141382333387	01230000004031081437295317311362146											3210700007415371995302503337862017863

Example 41: role of CIC in B-cell proliferation assay

Human PBMCs were isolated from heparinized blood of two normal subjects. Some PBMCs were stored and the remainder were incubated with CD19+ MACS beads (Miltenyi Biotec). These cells were then passed through a magnet and positively selected to isolate CD19+ B cells. This population had > 98% CD19+ as determined by FACS analysis. Then B cells were plated at 1X 10 ⁵The culture was carried out at a concentration of 200. mu.l/well in 96-well round bottom plates. In some cases, PBMCs were also cultured, but at 2X 10⁵At a concentration of 200. mu.l/well. Cells were stimulated with 2. mu.g/ml polynucleotide or CIC (three replicates). The incubation period was 48 hours at 37 ℃. After the culture period is finished, use³Plates were pulsed with H-thymidine (1. mu. Ci/well) and incubated for a further 8 hours. Plates were then harvested using standard liquid scintillation techniques and data collected in counts per minute (cpm).

Experiment A: the results of experiment A (Table 18) demonstrate that polynucleotide (P-6) and CIC containing the 5 '-C, G-3' motif (C-8, C-9, C-21, C-28) cause B cell proliferation. The control compounds, P-7 and M-1, and the heptameric polynucleotide P-1, did not cause B cell proliferation at all. Branched CIC (C-28) and CIC with propyl spacers (C-9) caused greater B cell proliferation than CIC with hexapolyethylene glycol spacers (C-8 and C-21). Proliferation of PBMCs reflects proliferation of B cells.

Watch 18

		Donor 146					Donor 147				Average of both
												Cell type	Stimulation of	cpm1	cpm2	cpm3	Mean value of		cpm1	cpm2	cpm3	Mean value of
B cell	Cells only	538	481	795	605		482	360	296	379													492
											B cell	P-6	29280	33430	30056	30922		35729	18032	21166	34976	27949
B cell	P-7	4858	5810	7079	5916		4364	4066	2774	3735													4825
											B cell	P-1	761	608	721	697		569	460	687	572	634
B cell	C-8	23815	30066	22969	25617		20914	22370	23659	22314													23966
											B cell	C-9	35365	42705	45231	41100		55543	49035	44985	49854	45477
B cell	C-21	28467	16074	19258	21266		17604	18851	19887	18781													20024
											B cell	M-1	1514	2815	1173	1834		1679	1667	1436	1594	1714
B cell	C-28	50999	54630	46418	50682		65593	51040	50357	55663													53173
PBMCs	Cells only	2744	2303	2284	2444		1301	2402	2143	1949													2196
											PBMCs	P-6	22067	23740	28099	24635		26436	23830	17531	22599	23617
PBMCs	P-7	7620	8362	9686	8556		9783	9841	10476	10033													9295
											PBMCs	P-1	9724	3041	2425	5063		1706	1960	324	1330	3197
PBMCs	C-8	47202	40790	4481	44268		38845	39733	27981	35520													39894
											PBMCs	C-9	55348	24857	39953	40053		88106	65413	90665	81395	60724
PBMCs	C-21	30338	22685	22383	25135		28819	530	37088	22146													23641
											PBMCs	M-1	8753	5203	4496	6151		1034	3298	1674	2002	4076
PBMCs	C-28	94977	121595	84977	100516		103916	91439	100905	98753													99635

Experiment B: experiment B (table 19) evaluated the effect of spacer composition as well as CIC structure (linear versus branched) on B cell proliferation. Linear CIC's containing propyl, butyl, abasic, and hydroxymethyl ethyl spacers tend to induce greater B-cell proliferation than corresponding CIC's containing hexapolyethylene glycol spacers or tri-polyethylene glycol spacers (compare C-10, C-11, C-17, C-18, C-20, C-25). The dodecyl spacer rendered CIC (C-19) inactive. Notably, the B cell proliferation data does not necessarily reflect cytokine data as shown above, particularly the difference between B cell proliferation and IFN- α induction.

Watch 19

Proliferation assay

121 194

Sample (I)	Cells	Irritant substance	cpm1	cpm2	cpm3	Mean value of		cpm1	cpm2	cpm3	Mean value of	Average of both
													1234567891011121314151617	B cell	Only the cell P-6P-7C-8C-9C-10C-11C-22C-94C-28C-17C-18C-19C-20C-23C-24C-25	4511999616232604219381514230367171471141835393279758583127610628820634360	75715031182112078354001413630412140141440626954304261708510993085116221678935016	29719804290117696238771615818528684411110267809895146539262853220087279926480	5021827721151079327072151452643612668123112970922765158699613022015645593131952		20313678199293331366074801696764727361215881746710028371180828730397916060	22812732159393911671754582089855408505136911489012217403187056532340719509	1519003168676021786659431125338945349156911051810538312174819596346817384	19411804175787751608162941637353027072169901429210928362180898286361817651	3481504119369784215761072021404898596922335018529133986622415511966477524802

Example 43: induction of immune-related genes in mouse lungs following intranasal treatment with CIC

The ability of C-9, C-23 and P-6 (positive controls) to induce mRNA expression of 75 different genes in mouse lungs was investigated. Genes evaluated include genes encoding cytokines, chemokines, cell surface molecules, transcription factors, metalloproteinases, and other molecules. This study was performed in the Northview Pacific laboratory (Hercules, Calif.) using 6-8 week old female BALB/c mice from Jackson Labs (Bar Harbor, ME). Under mild isoflorine anesthesia, 5 mice per group were treated intranasally with 20ug C-9, C-23, P-6 (positive control) or P-7 (negative control) in 50uL saline. Previous experiments demonstrated that the optimal induction time for most genes was 6 hours after treatment. Thus, lungs were harvested at 6 hours and snap frozen in liquid nitrogen and stored at-80 ℃ for later use. Total RNA was isolated using RNeasy mini kit (Qiagen inc., Valencia, CA). RNA samples were treated with DNAse (Roche Diagnostics, Mannheim, Germany) and converted to cDNA using superscript II RNase H-reverse transcriptase (Invitrogen, Rockville MD), as described in scherens et al, 2001, eur.j. of lmmunoglogy31: 1465-74. The cDNA samples of each group were pooled and mRNA expression of 75 genes was determined in each pooled sample using real-time quantitative PCR (ABIPrism5700, Perkin Elmer Applied Biosystems) and sybergreen (Qiagen Inc.). In addition to the gene of interest, in each sample, mRNA expression of housekeeping genes (HPRT or ubiquitin) was also determined. To correct for the amount of RNA in each sample, all data were calculated relative to the expression of housekeeping genes. The most up-regulated genes were selected as shown in FIG. 5 and the data are expressed as fold induction over the response in control-treated (P-7) mice. This data demonstrates that C-9, C-23 and P-6 can efficiently induce the expression of a variety of genes including IL-6, IL-12P40, IFN- α, IP-10 and IL-10. However, treatment of mice with C-9 induced significantly higher expression of IFN- α mRNA compared to the C-23 or P-6 treated groups.

Example 44: in vivo Activity of CIC

In vivo studies were performed by injecting 20ug (200ul volume) of P-6 (positive control), P-7 (negative control), C-9, C-23, P-1 or P-11 subcutaneously into the back of the neck of mice (10/group). Blood samples were collected after 2 hours. For the LPS positive control group, mice were injected intraperitoneally with a volume of 200ul and blood samples were collected after 1.5 hours (i.e., at the peak of LPS-induced TNF-. alpha.activity). Blood was allowed to clot and serum was prepared and stored at-80 ℃ until the test began. Serum cytokines were assayed using the Biosource cvtoscreen kit for TNF- α detection and Pharmingen antibody for mIL-6 and mIL-12 detection. All samples were tested in duplicate.

P-6 and two CICs (C-9 and C-23) each induced IL-12P40, IL-6, and TNF-. alpha.whereas the control oligonucleotide (P-7) was inactive (FIGS. 6A-C). In this experiment, CIC C-23 was more effective than C-9 and P-6. As expected, the hexamer (P-11: 5 '-AACGTT) and heptamer (P-1: 5' -TCGTCGA) were inactive.

Example 45: immune response in primates against antigens and CIC

The baboon was tested for immune response to hepatitis b surface antigen (HBsAg) administered in the presence of CIC.

HBsAg is recombinant HBsAg produced in yeast. At the start of the study, male and female baboons were included in the baboon group (8 per group) and the body weight ranged from 8 to 31kg (group mean body weight was 13 to 16 kg).

Baboons were immunized twice by intramuscular Injection (IM) of 20. mu.g HBsAg in a volume of 1ml, with an interval of two months (months 0 and 2). As listed below, some groups also received CIC (C-8 or C-9) or positive control (P-6) plus HBsAg.

Blood was collected from all animals before and 2 weeks after immunization. anti-HBsAg IgG titers were determined as follows. Baboon serum samples were analyzed using AUSAB EIA commercial kit (Abbott Labs Cat. #9006-24 and 1459-05) using beads coated with HBsAg from human plasma. A set of HBsAg positive and negative standards from human plasma ranging from 0-150mIU/ml were tested with the test samples. Biotin-conjugated HBsAg and rabbit anti-biotin-HRP-conjugated antibody were used as secondary antibody complexes for detection. The assay was developed with o-phenylenediamine (OPD) and absorbance was measured at 492nm, with background subtraction at 600nm (Quantum II spectrophotometer, Abbott Labs). The absorbance of the sample was used to determine the corresponding anti-HBsAg concentration in milliinternational units per ml (mIU/ml) from a standard curve according to the manufacturer established parameters. For diluted samples, quantification is based on sample absorbance resulting in values of 0-150mIU/ml, which is then multiplied by a dilution factor to give the final concentration.

The log-transformed data were statistically analyzed by analysis of variance (NCSS97 statistical software program, Kaysville, UT) using a one-way analysis of variance with a prior comparison (α ═ 0.05). Significance was observed when p.ltoreq.0.05.

Groups of test animals were immunized as follows:

groups 1-20 μ g HBsAg;

groups 2-20. mu.g HBsAg + 1000. mu. g P-6;

groups 3-20. mu.g HBsAg + 1000. mu. g C-8;

groups 4-20. mu.g HBsAg + 1000. mu. g C-9

The results of the study are shown in table 20 below. CIC or positive control P-6 administered in combination with HBsAG increased the titer of anti-HBsAg antibody compared to HBsAg administered alone. In the pairwise comparison, the immune responses detected in groups 2, 3, and 4 were significantly different from the immune response detected in group 1 (p < 0.05 for group 2 and p < 0.005 for groups 3 and 4 after the second immunization). No statistical differences were found between groups 2, 3, and 4.

Watch 20

Antibody response to Baboon (AUSAB EIA)

HBsAg+CIC

#	Group # vaccine	After the first immunization against HBsAg (mIU/ml)	After the second immunization
				B339B340B341B342B343B344B345B346	1HBV(20ug)	00000000	763158055502824
	Standard deviation of mean value	00	4026
				B347B348B349B350B351B352B353B354	2HBV(20ug)P-6(1000ug)	06017001521	32912110813,569315381,4461,675
	Standard deviation of mean value	79	2200*4,637
				B379B380B381B382B383B384B385B386	3HBV(20ug)C-8(1000ug)	20012505200	1843,03841,7063,71825013,75011,62679
	Standard deviation of mean value	2245	9294**14,121
				B387B388B389B390B391B392B393B394	4HBV(20ug)C-9(1000ug)	0420040526750	5,6058,9783122,99212,6631122,36452
	Standard deviation of mean value	68139	4135**4.633

^*p＜0.05，^**p < 0.005 compared to HBV alone (group 1)

Example 46: in vivo response by CIC-antigen conjugates

This example shows that an antibody-mediated immune response is induced in mice by administration of a CIC-antigen conjugate.

At two week intervals, 10 mice/group were immunized twice with C-11/Amb a1 conjugate (synthesized as described below) (lug or 10ug), P-6/Amb a1(1ug), or Amb a1(1ug) by the intradermal (tail) route, as described below. Titers of anti-Amb a1 specific IgGI and IgG2a were determined from sera obtained two weeks after each injection. The second immunization was followed by in vitro restimulation of splenocytes at week 6 to determine Amb a1 specific IFN γ and IL-5 responses.

Mice immunized with the C-11-Amb a1 conjugate showed a characteristic immune response pattern observed when immunized with the P-6-Amb a1 reference, specifically, a shift in immune response specific from Th2 to Th 1-type Amb a 1. Mice immunized with C-11 or P-6 conjugates developed a strong IgG2a response and reduced IgGl responses. The conjugate treated group also showed a cessation of IL-5 response and an increase in IFN γ response. Furthermore, the immune response against the C-11-Amb a1 conjugate showed an increase in a dose-dependent manner, as evidenced by comparing the 1ug and 10ug dose groups. The C-11-Amb a1 conjugate elicited an immune response similar in nature to that observed with P-6-Amb a 1.

The results are shown in tables 21 to 23.

General procedure

Animal studies were performed at the Northview Pacific laboratory (Hercules, Calif.) using 8-12 week old female BAILB/c mice from Charles River laboratory (Hollister, Calif.). 10 mice/group were given two tail Intradermal (ID) injections at 2 week intervalsThe following substances are listed: C-11/Amb a1 conjugate (1ug), C-11/Amb a1 conjugate (10ug), P-6/Amb a1 conjugate (1ug) or Amb a1 antigen (1 ug). Blood samples were collected via the retroorbital route two weeks after each injection and sera were prepared for antibody assays. At 6 weeks after the second injection, spleens were harvested for in vitro restimulation assays to determine the cytokine response of IFN γ and IL-5. Spleens were tested individually. Using 25 and 5ug/ml of Amb a1 vs 5X 10⁵Cells/well were restimulated and supernatants were harvested on day 4 and stored at-80 ℃ until assayed. Controls for in vitro experiments included 0.01% SAC and 10ng/ml and 1uM PMA/IO, respectively.

Mouse anti-Amb a1 IgG1 and IgG2a assays

Mouse serum samples were analyzed by ELISA in 96-well round plates coated with 1. mu.g/ml of Amb a1 antigen at 50. mu.l/well. Goat anti-mouse IgG1 (or IgG2a) biotin conjugated antibody was used as the secondary antibody. Detection was performed using streptavidin-horseradish peroxidase conjugate.

The assay was developed with TMB and absorbance was measured at 450nm, with background subtraction at 650nm (Emax precision microplate reader, Molecular Devices, Sunnyvale, Calif.). Titers were defined as the reciprocal of the dilution of serum giving an ELISA absorbance value of 0.5OD using a 4-parameter assay (Softmax Pro97, Molecular Devices, Sunnyvale, CA). All samples were tested in duplicate in two wells on different plates, and the mean of the two values was taken as the potency value.

Mouse IL-5 and IFN-gamma assays

The supernatants were tested for IL-5 and IFN- γ levels by capture ELISA on plates coated with anti-cytokine monoclonal antibodies. Biotinylated anti-cytokine mabs were used as secondary antibodies. Detection was performed using streptavidin-horseradish peroxidase conjugate and color test with TMB. Concentrations were calculated from the standard curve measured on each plate. Absorbance at 450nm was measured and background was subtracted at 650nm (Emax accurate microplate reader, Molecular Devices, Sunnyvale, Calif.). All samples were tested in duplicate in two wells on different plates, and the average of the two values was taken as the concentration value.

The log-transformed data were statistically analyzed by the program NCSS97 (NCSS97 statistical software, Kaysville, Utah) using One-Way ANOVA with plannecroparies (α ═ 0.05) with a priori comparison. For the following studies, p < 0.05 was considered significant.

Synthesis of C-11/Amb a1 conjugates

Synthesis of activated C-11 (C-111):

5' -disulfide-C-11 (C-110) was dissolved in activation buffer (100mM sodium phosphate/150 mM sodium chloride/pH 7.5) and activated by reduction of TCEP. The activated CIC (C-111) was purified using a 5ml SephadexG25 column (Pharmacia) with the same activation buffer as the mobile phase. Fractions were collected manually at 0.5 minute intervals starting from the initial rise in baseline. After purification, the concentration of each fraction was determined using A260 and the extinction coefficient was 25.6 OD/mg.

Synthesis of activated Amb a 1:

amb a1 is activated by: first blocking its free thiol group and then adding a heterofunctional crosslinker. Excess reagent was removed by desalting using a HiTrap G-25 desalting column (Pharmacia Catalog # 17-1408-01). The resulting activated Amb a1 has an average of 9.3 sites per activated protein.

Synthesis of C-11/Amb a1 conjugates：

Activated C-11(C-111) and activated Amb a1 were combined and the resulting C-11/Amb a1 conjugate fractionated using a Superdex200 size exclusion chromatography column (pharmacia Cat. #; 17-1088-01; 1 cm. times.30 cm). Formulation buffer (10mM phosphate, 150mM NaCl, pH7.2) was used as the mobile phase. The baseline start to rise was the starting point and fractions were collected at 1 minute intervals.

Conjugate samples were analyzed by SDS-PAGE using 4-12% NuPAGE gels (Invitrogen, Catalog # NP0322) and MOPS buffer (Invitrogen, Catalog # NP0001) while molecular size exclusion chromatography (SEC-HPLC) using a BioSep SEC-S3000 column (Phenomenex, Catalog #; OOH-2146-EO). After SDS-PAGE, the proteins were visualized by Coomassie blue (GelCode, Pierce Catalog #24596) staining. The presence of CIC was confirmed by using DNA-silver staining (Pharmacia, Catalog # 17-6000-30). SDS-PAGE and SEC-HPLC were used to define the pooling criteria and to characterize the resulting pools. Protein concentration was measured by Bicinchoninic acid (Bicinchoninic acid) method (BCA, Sigma Catalog # BCA-1).

TABLE 21

Active IgG1 and IgG2a anti-Amb a1 titers of C-11/Amb a1 conjugates in mice

Group of	Animal # s	Immunization	2 weeks after first immunization		2 weeks after the second immunization
							IgG1	IgG2a	IgG1	IgG2a
			1	12345678910	C-11/Amb a1 conjugate (1ug) ID	30303030383030305630					148221943641,8949435702593030	7,90013,0379465,4853,80560010,3376002,5758,381	19,88619,73523,91810,4879,9455,24920,1568,3505,74728,971
		Standard deviation of mean value					33**8	510599	5,367**4,400	15,244*8,285
			2	11121314151617181920	C-11/Amb a1 conjugate (10ug) ID	51307730303055999930					3456674451,662674501,1371,1198,2271,613	8,982201,0086,73922,578190,8355,97129,64670,15980,0526,235	27,877612,73986,672121,77088,74517,600105,398183,152250,20663,616
		Standard deviation of mean value					53*29	1,5732,399	62,22175,298	155,778*174,925
			3	21222324252627282930	P-6/Amb a1 reference conjugate (1ug) ID	301,42248517090388333011330					373032651,1822,0272,298321558939	3,43715,65284,92737,37938,12132,4993,01124,30743,06037,116	65,3066,198177,28156,07476,572240,09824,40420,79619,5867,317
		Standard deviation of mean value					330475	662862	31,95123,568	69,36378,697
			4	31323334353637383940	Amb a1(1ug)ID	3,4057,3312,8474,0218,3331,2141,2794,3325692,696					34930353021228630803030	172,827164,673112,766100,281156,037118,407396,404187,33563,536161,039	6,2441,0037,1741,3994,9692,1256004,599600902
		Standard deviation of mean value					3,603**2,554	111*123	163,331**90,406	2,962**2,530

Value 30 for samples with a value < 30 after the first immunization

Value 600 for samples with a value < 600 after the second immunization

^*p＜0.05，^**P < 0.005 compared with P-6/Amb a1

TABLE 22

Activity of C-11/Amb a1 conjugates in mice

In vitro IFN γ response (pg/ml)

Animal(s) production	Immunization	Amb a1		PMA	Only culture medium
						25ug/ml	5ug/ml
		12345678910	C-11/Amb a1 conjugate (1ug) ID					225011629185341692523136249004383 dead 1608825067	16320100545084832211298254892716 dead 628512431	125058433583537969225215258855 dead 2772228201	454545454518545 death 4545
	Standard deviation of mean value			175367289	108896839	140119353	6147
		1112131415161718	C-11/Amb a1 conjugate (10ug) ID					1999450732547529641783356880188783949763	1346625103284227801743505512995907928468	3370236467217702460120021406043156258062	4545123130515182983211
19				102646	60332	32669	3366
		20						61939	29505	53393	756
	Standard deviation of mean value			65868**24860	3965220160	3557613331	394455
		21222324252627282930	P-6/Amb a1 reference conjugate (1ug) ID					42754307407614076435645408952553815884421563276	3083996169562364330164310271434912432460846897	3655023742154078961159151335515515116237106643680	454545118209308734545734
	Standard deviation of mean value			2755620099	1841614603	2558119548	167218
		31323334353637383940	Amb a1(1ug)ID					3016119351121301687911874492617020992209	2585277623925129726732840376511523895	10800094345975678962377808772998925370169108000103131	452104461495689282187132309
	Standard deviation of mean value			3366**2143	24651915	9152013239	212156

Value 18 for a value < 18

Values are not included in the calculation because the values for medium alone are > 3 standard deviations + the average of all values for medium alone (i.e., 2014pg/ml)^**P < 0.005 compared to the P-6/Amb a1 value for 25. mu.g/ml restimulation

TABLE 23

Activity of the C-11/Amb a1 conjugate in mice in vitro IL-5 response (pg/ml)

Animal(s) production	Immunization	Amb a1		PMA	Only culture medium
						25ug/ml	5ug/ml
		12345	C-11/Amb a1 conjugate (1ug)					67492642468	10653242445	2202240696829792851	4124242446
6	ID			104	121	2547	129
		78910						24 death 2424	24 death 24203	3935 death 13831837	24 death 2453
	Standard deviation of mean value			6886	6363	2320948	3312
		11121314151617181920	C-11/Amb a1 conjugate (10ug) ID					24411692611342537224334213	33731372212211871093523155	1655225889291865877896636561238751	248424332429242414524
	Standard deviation of mean value			153111	13080	1377940	4440
		21222324252627282930	P-6/Amb a1 reference conjugate (1ug) ID					242415796883921810324110	24602771523637217654459102	1514372517024473141412351037138221942990	24242424242424242424
	Standard deviation of mean value			8864	171151	21671174	240
		31323334353637383940	Amb a1(1ug)ID					72491930375109312062808144112401373	33157125967476349021159091228481	1332318615742506219737151727165513671683	24245624242767582468
	Standard deviation of mean value			1128**734	831588	2094808	4020

Value 24 for a value < 24

Values were not included in the calculation because the values for medium alone were > 3 standard deviations + the average of all values for medium alone (i.e., 124pg/ml)^*p＜0.05，^**P < 0.005 compared to the P-6/Amb a1 value for 25. mu.g/ml restimulation

Example 47: effect of spacer moieties on CIC Activity

This example shows the effect of different spacer moieties on IFN- α induction. A comparison of C-90(C3CIC) and C-51(HEG CIC) showed that C-51 induced IFN-. alpha.8-fold more than C-90, although each CIC induced similar amounts of IFN-. gamma.. Similarly, a comparison of branched CICs with different linkers showed that HEG (C-94) > TEG (C-103) > C3(C-104) — no linker (C-28) for IFN- α induction.

Watch 24

Irritant substance	IFN-g(pg/ml)					IFN-g(pg/ml)
											28234	28235	28236	28237	Mean value of	28234	28235	28236	28237	Mean value of
	Only cells P-6P-7C-90C-51C-71C-101C-96C-97C-100C-88C-33C-21C-28C-94C-103C-104PLGAP-6+ PL6AP-7+ PLGAC-90+ PLGAC-51+ PLGAC-71+ PLGAC-101+ PLGAC-96+ PLGAC-97+ PLGAC-100+ PLGAC-88+ PLGAC-33+ PLGAC-21+ PLGAC-28+ PLGAC-94+ PLGAC-103+ PLGAC-104+ PLGASAC	415137173014211027523410721144574139881011101436817610211815928174192401845	452411812316820523911918321861071416144431514467364479765910707371299512444411131623643731250	451749719344862735426949017247308882392508718105211831106412548791167988174211489681089100513521388641924	4116741586158016632612139698019074772076164512291116185412510377513461837483899694454715327780450553947405638684034506349305350	43217552478577865503345652130608516335381542581613508156513861513214819631780275517041369142912581546182214212342	165862161617161616169516161616161616161611753257308546794107474215208035511369164145895162374	1616164677302491201616161616161182129122694166962168224444883237421610926135661561164653148616327	1616161173525381354608140398212167316548126161571163164544800080008000666080004074240935145366184719744561281149	16741634537981665856699316193111916781636312131635344416510380008000800080008000377764126727800013480005405023744											16412813110615632546434471561093519616107894198313291628805356533262925501624026162559284040748859993061631899

Example 48: assessing independent immunomodulating activity of polynucleotides corresponding to nucleic acid moiety sequences

This example further illustrates the immunomodulatory activity of CIC containing nucleic acid moieties that do not have independent immunomodulatory activity. The activity of a polynucleotide corresponding to the sequence of a nucleic acid moiety of a CIC is detected alone or in combination with a free spacer and compared to the activity of a CIC comprising equal amounts of nucleic acid and spacer. For example, 3uM CIC C-101 was compared to 9uMP-14 or a mixture of 9uM P-14 and 9uM hexapolyethylene glycol and 3uM glycerol (since C-101 contains 3 equivalents of P-14, 3 equivalents of hexapolyethylene glycol, and 1 equivalent of glycerol). In all cases, CIC is active and short-chain polynucleotides, either alone or mixed with spacers, are inactive. See table 25. The activity of the spacers alone was tested at a concentration of 9uM, and all spacers were completely inactive.

TABLE 25

Irritant substance	IFN-g(pg/ml)					IFN-a(pg/ml)
											28250	28251	28252	28253	Mean value of	28250	28251	28252	28253	Mean value of
	Only the cell P-6P-7 propyl spacer butyl spacer tri-polyethylene glycol hexa-polyethylene glycol glycerol C-51C-101P-14P-14/HEG/glycerol C-21C-94P-1P-1/HEG/triol C-45P-13P-13/HEGC-10P-2P-3P-4P-2/P-3/P-4/HEG	181211041381512161352241014122340541510718403377327268	5181151112456334195160219261328163252111	93439117121641801110155287561995224577653724338	13711111511671461010203128118118981111	85236446781281531613133204331159142854422312927	161616161616161618154016163124516161616161616161616	16161616161616162461472161669645161610916162316291616	30161616161616169550916164132316165516162525381616	1916161616163516122626451616264119816163 82161612416311616											201616161616211643712911616101603161614016164718291616

Example 49: (5 '-TCGACGT-3' HEG)_{Average 185 ═ 185}Preparation of-Fic 0ll400(C-137)

A. Maleic diamide-Ficoll₄₀₀Preparation of

Preparation of Aminoethylcarboxymethyl (AECM) by Inman (J.Immunology, 1975, 114: 704-)₁₈₀-Ficoll₄₀₀. On average, there are 180 aminoethyl groups per mole of Ficoll (MW 400,000 Da). 27.6mg (62.6. mu. mol) of 4- (N-maleimidomethyl) -cyclohexane-1-carboxylic acid sulfosuccinimidyl ester in 300. mu.l of DMSO were added dropwise to 23.2mg (0.058. mu. mol) of AECM dissolved in 1.0ml of 0.1M sodium phosphate buffer (pH6.66)₁₈₀-Ficoll₄₀₀And continuously stirring while adding. The reaction mixture was placed on a shaker for 2 hours and then desalted on a Sephadex G-25 column to give 20mg of maleimido-Ficoll₄₀₀. There are on average about 165 maleimide groups per mole of Ficoll.

B.5′-TCGACGT-3′-HEG-(CH₂)₃Preparation of-SH (C-136)

Synthesis of 5 '-TCGACGT-3' -HEG- (CH) by a similar method to that for C-116₂)₃-SS-(CH₂)₃-OH (C-135). To a solution of 0.4mL of 0.1M sodium phosphate/150 mM chlorideTo 10mg (3.57. mu. mol) of C-135 in sodium/pH 7.5 buffer was added 5.7mg (20. mu. mol) of TCEP dissolved in 0.7ml of the same buffer. The mixture was vortexed thoroughly and then placed in a 40 ℃ water bath for 2 hours. The thiol (C-136) was purified by RP-HPLC (Polymer Labs PLRP-S column) using an increasing gradient of acetonitrile in triethylammonium acetate buffer (TEAA)/pH7.0 and used immediately in the next reaction.

C.(5′-TCGACGT-3′-HEG)x-Ficoll₄₀₀Preparation of (C-137)

To a solution of 5.5mg (0.014umol) maleimido-Ficoll in 0.7ml 0.1M sodium phosphate/pH 6.66₄₀₀6.8mg (2.5. mu. mol) of C-136 dissolved in 3.45mL of about 30% acetonitrile/TEAA/pH 7.0 buffer was added. The mixture was placed on a shaker at room temperature overnight and the product was purified on a Superdex200 column (Pharmacia). Calculation using the total weight of the isolated product and the absorbance value at 260nm showed that the product contained on average about 185 oligonucleotides per mole of Ficoll. A second fraction with a lower oligonucleotide loading was also obtained.

D.C-137 Activity

As shown in table 26, the polysaccharide-based CIC has interesting activity in cytokine response assays, especially showing significant IFN- α stimulation.

Watch 26

Stimulating compounds	IFN-g(pg/ml)						IFN-a(pg/ml)
												28313	28314	28315	28316	Mean value of	×4	28313	28314	28315	28316	Mean value of
	Cell-only P6P7C-137SAC	111187	938112277	1147121356	1130920544000	89911221055	3239543904220	3131313612346	1221301765468192	100122107624114	9813412140001172												881041093426456

***

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Accordingly, the detailed description and examples are not to be construed as limiting the scope of the invention, which is defined by the appended claims.

All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be so incorporated by reference.

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<213> Artificial sequence

<220>

<223> synthetic constructs

<400>3

tgactgtgaa ccttagagat ga 22

<210>4

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>1

<223> n ═ t, g, c, or 5-bromocytosine

<221> variants

<222>2

<223> n ═ t, g, a, or u

<221> variants

<222>4

<223> n ═ t, a, or c

<221> variants

<222>7

<223> n ═ t, g, or u

<400>4

nnancgntcg 10

<210>5

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>5

tgaacgttcg 10

<210>6

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>

ggaacgttcg 10

<210>7

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>7

tgaacgutcg 10

<210>8

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>8

tgaccgttcg 10

<210>9

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>9

tgatcggtcg 10

<210>10

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>10

tgatcgttcg 10

<210>11

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>11

tgaacggtcg 10

<210>12

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>12

gtaacgttcg 10

<210>13

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>13

gtatcggtcg 10

<210>14

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>14

gtaccgttcg 10

<210>15

<211>10

<212>DNA

<213> Artificial sequence

<220>

<222> synthetic construct

<400>15

gaaccgttcg 10

<210>16

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>1

<223> n ═ 5-bromocytosine

<400>16

ngaccgttcg 10

<210>17

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>17

cgaacgttcg 10

<210>18

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>18

cgaccgttcg 10

<210>19

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>1

<223> n ═ 5-bromocytosine

<400>19

ngaacgttcg 10

<210>20

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>20

ttaacgutcg 10

<210>21

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>21

tuaacgutcg 10

<210>22

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>22

ttaacgttcg 10

<210>23

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>23

tcgtcgaacg ttcgttaacg ttcg 24

<210>24

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>24

tgactgtgaa cgutcgagat ga 22

<210>25

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>25

tcgtcgaucg utcgttaacg utcg 24

<210>26

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>26

tcgtcgaucg ttcgtuaacg utcg 24

<210> 27

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>27

tcgtcguacg utcgttaacg utcg 24

<210>28

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>7

<223> n ═ 2-amino-adenine

<400>28

tcgtcgnacg utcgt taacg utcg 24

<210>29

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>29

tgatcgaacg ttcgttaacg ttcg 24

<210>30

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>30

tgactgtgaa cgutcggtat ga 22

<210>31

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>31

tgactgtgac cgttcggtat ga 22

<210>32

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>32

tgactgtgat cggtcggtat ga 22

<210>33

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>33

tcgtcgaacg ttcgtt 16

<210>34

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>34

tcgtcgtgaa cgttcgagatga 22

<210>35

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>35

tcgtcggtat cggtcggtat ga 22

<210>36

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>36

cttcgaacgt tcgagatg 18

<210>37

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>37

ctgtgatcgt tcgagatg 18

<210>38

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>38

tgactgtgaa cggtcggtat ga 22

<210>39

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>39

tcgtcggtac cgttcggtat ga 22

<210>40

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>40

tcgtcggaac cgttcggaat ga 22

<210>41

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>41

tcgtcgaacg ttcgagatg 19

<210>42

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>42

tcgtcgtaac gttcgagatg 20

<210>43

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>43

tgac tgtgac cgttcggaat ga 22

<210>44

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>44

tcgtcgaacg ttcgaacgtt cg 22

<210>45

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2，5

<223> n ═ 5-bromocytosine

<400>45

tngtngaacg ttcgagatg 19

<210>46

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>46

tcgtngaacg ttcgagatg 19

<210>47

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>47

tcgtcgaccg ttcggaatga 20

<210>48

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2，5

<223> n ═ 5-bromocytosine

<400>48

tngtngaccg ttcggaatga 20

<210>49

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>49

tcgtngaccg ttcggaatga 20

<210>50

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>50

ttcgaacgtt cgttaacgtt cg 22

<210>51

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>4

<223> n ═ 5-bromocytosine

<400>51

cttngaacgt tcgagatg 18

<210>52

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>52

tgatcgtcga acgttcgaga tg 22

<210>53

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>1

<223> n ═ t, g, c, or 5-bromocytosine

<221> variants

<222>2

<223> n ═ t, g, a, or u

<221> variants

<222>4

<223> n ═ t, a, or c

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<221> variants

<222>7

<223> n ═ t, g, or u

<400>53

nnanngntcg 10

<210>54

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>54

tgaangttcg 10

<210>55

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>55

tgaangutcg 10

<210>56

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>56

tgacngttcg 10

<210>57

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>57

tgatnggtcg 10

<210>58

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>58

gtatnggtcg 10

<210>59

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>59

gtacngttcg 10

<210>60

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>60

gaacngttcg 10

<210>61

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>61

gaaangutcg 10

<210>62

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>1，5

<223> n ═ 5-bromocytosine

<400>62

ngacngt tcg 10

<210>63

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>63

cgaangttcg 10

<210>64

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>1.5

<223> n ═ 5-bromocytosine

<400>64

ngaangttcg 10

<210>65

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>1.5

<223> n ═ 5-bromocytosine

<400>65

ngaangutcg 10

<210>66

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>66

ttaangutcg 10

<210>67

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>67

tuaangutcg 10

<210>68

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>68

ttaangttcg 10

<210>69

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>11

<223> n ═ 5-bromocytosine

<400>69

tgactgtgaa ngutcgagat ga 22

<210>70

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>9.19

<223> n ═ 5-bromocytosine

<400>70

tcgtcgaang ttcgttaang ttcg 24

<210>71

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>11

<223> n ═ 5-bromocytosine

<400>71

tgactgtgaa ngutcggtat ga 22

<210>72

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>1 1

<223> n ═ 5-bromocytosine

<400>72

tgact gtgaa ngutcggaat ga 22

<210>73

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>11

<223> n ═ 5-bromocytosine

<400>73

tcgtcggaaa ngutcggaat ga 22

<210>74

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5，9

<223> n ═ 5-bromocytosine

<400>74

tcgtngaang utcggaatga 20

<210>75

<211>10

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>1

<223> n ═ t, c, or 5-bromocytosine

<221> variants

<222>2

<223> n ═ t, g, a, or u

<221> variants

<222>4

<223> n ═ t, a, or c

<221> variants

<222>7

<223> n ═ t, g, or u

<400>75

nnancgntcg 10

<210>76

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>11

<223> n ═ 5-bromocytosine

<400>76

tgac tgtgaa ngt tcgagat ga 22

<210>77

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>11，15

<223> n ═ 5-bromocytosine

<400>77

tgactgtgaa ngttngagat ga 22

<210>78

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>11

<223> n ═ 5-bromocytosine

<400>78

tgactgtgaa ngttccagat ga 22

<210>79

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>79

tgac tgtgaa cgtucgagat ga 22

<210>80

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>13

<223> n ═ 5-bromouracil

<400>80

tgactgtgaa cgntcgagat ga 22

<210>81

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>11

<223> n ═ 5-bromocytosine

<400>81

tgactgtgaa ngttcgtuat ga 22

<210>82

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>11

<223> n ═ 5-bromocytosine

<400>82

tgactgtgaa ngttcggtat ga 22

<210>83

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>83

ctgtgaacgt tcgagatg 18

<210>84

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2，5

<223> n ═ 5-bromocytosine

<400>84

tngtngtgaa cgttcgagat ga 22

<210>85

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>85

tcgtngtgaa cgttcgagat ga 22

<210>86

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>13

<223> n ═ 4-thio-thymine

<400>86

tgac tgtgaa cgntcgagat ga 22

<210>87

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>12，16

<223> n ═ 6-thio-guanine

<400>87

tgactgtgaa cnttcnagatga 22

<210>88

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>88

tgactgtgaa cgttcgtuat ga 22

<210>89

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>89

tgactgtgaa cgttcgttat ga 22

<210>90

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>90

tcgttcaacg ttcgttaacg ttcg 24

<210>91

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>91

tgattcaacg ttcgttaacg ttcg 24

<210>92

<211>18

<212>DNA

<213> Artificial sequence

<220>

<222> synthetic construct

<400>92

ctgtcaacgt tcgagatg 18

<210>93

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>93

tcgtcggaac gttcgagatg 20

<210>94

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>94

tcgtcggacg ttcgagatg 19

<210>95

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>95

tcgtcgtacg ttcgagatg 19

<210>96

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>96

tcgtcgttcg ttcgagatg 19

<210>97

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>97

tcgtgaacgt tcg 13

<210>98

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>98

tcgtcgaacg ttcg 14

<210>99

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2

<223> n ═ 5-bromocytosine

<400>99

tngtgaacgt tcg 13

<210>100

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2，5

<223> n ═ 5-bromocytosine

<400>100

tngtngaacg ttcg 14

<210>101

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>101

tcgttaacgttcg 13

<210>102

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221>misc_feature

<222>(4)...(6)

<223> tcg may or may not be present

<221> variants

<222>7，8

<223> n ═ any nucleotide

<221> variants

<222>7，8

<223> n may or may not be present

<221> variants

<222>10

<223> n ═ t, a, or c

<221> variants

<222>13

<223> n ═ t, g, or u

<400>102

tcgtcgnnan cgntcg 16

<210>103

<211>11

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>103

tcgaacgttc g 11

<210>104

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>104

tcgtcgaacg ttcg 14

<210>105

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>105

tcgtgaacgt tcg 13

<210>106

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>106

tcggtatcgg tcg 13

<210>107

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>107

tcggtaccgt tcg 13

<210>108

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>108

tcggaaccgt tcg 13

<210>109

<211>12

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>109

tcggaacgtt cg 12

<210>110

<211>15

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>110

tcgtcggaac gttcg 15

<210>111

<211>12

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>111

tcgtaacgtt cg 12

<210>112

<211>11

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>112

tcgaccgttc g 11

<210>113

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>113

tcgtcgaccg ttcg 14

<210>114

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>114

tcgttaacgt tcg 13

<210>115

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2

<223> n ═ 5-bromocytosine

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<221> variants

<222>4-6

<223> tng may or may not be present

<221> variants

<222>7，8

<223> n ═ any nucleotide

<221> variants

<222>7，8

<223> n may or may not be present

<221> variants

<222>(10)...(10)

<223> n ═ t, a, or c

<221> variants

<222>(13)...(13)

<223> n ═ t, g, or u

<400>115

tngtngnnan cgntcg 16

<210>116

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2

<223> n ═ 5-bromocytosine

<400>116

tngtgaacgt tcg 13

<210>117

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2，5

<223> n ═ 5-bromocytosine

<400>117

tngtngtgaa cgttcg 16

<210>118

<211>11

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2

<223> n ═ 5-bromocytosine

<400>118

tngaacgttc g 11

<210>119

<211>11

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2

<223> n ═ 5-bromocytosine

<400>119

tngaccgttc g 11

<210>120

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2，5

<223> n ═ 5-bromocytosine

<400>120

tngtngaccg ttcg 14

<210>121

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<221> variants

<222> 7，8

<223> n ═ any nucleotide

<221> variants

<222>7，8

<223> n may or may not be present

<221> variants

<222>10

<223> n ═ t, a, or c

<221> variants

<222>13

<223> n ═ t, u, or g

<400>121

tcgtngnnan cgntcg 16

<210>122

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>122

tcgtngtgaa cgttcg 16

<210>123

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>123

tcgtngaacg t tcg 14

<210>124

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<400>124

tcgtngaccg ttcg 14

<210>125

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221>misc_feature

<222>(4)...(6)

<223> tcg may or may not be present

<221> variants

<222>7，8

<223> n ═ any nucleotide

<221> variants

<222>7，8

<223> n may or may not be present

<221> variants

<222>10

<223> n ═ t, a, or c

<221> variants

<222>11

<223> n ═ 5-bromocytosine

<221> variants

<222>(13)...(13)

<223> n ═ t, g, or u

<400>125

tcgtcgnnan ngntcg 16

<210>126

<211>13

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>8

<223> n ═ 5-bromocytosine

<400>126

tcggaaangt tcg 13

<210>127

<211>11

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>6

<223> n ═ 5-bromocytosine

<400>127

tcgaangttc g 11

<210>128

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2

<223> n ═ 5-bromocytosine

<221> variants

<222>(4)...(6)

<223> tng may or may not be present

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<221> variants

<222>7，8

<223> n ═ any nucleotide

<221> variants

<222>7，8

<223> n may or may not be present

<221> variants

<222>(10)...(10)

<223> n ═ t, a, or c

<221> variants

<222>(13)...(13)

<223> n ═ t, g, or u

<400>128

tngtngnnan ngntcg 16

<210>129

<211>11

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2，6

<223> n ═ 5-bromocytosine

<400>129

tngaangutc g 11

<210>130

<211>11

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>2，6

<223> n ═ 5-bromocytosine

<400>130

tngaangttc g 11

<210>131

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5

<223> n ═ 5-bromocytosine

<221> variants

<222>7，8

<223> n ═ any nucleotide

<221> variants

<222>7，8

<223> n may or may not be present

<221> variants

<222>10

<223>n＝t，a，c

<221> variants

<222>11

<223> n ═ 5-bromocytosine

<221> variants

<222>(13)...(13)

<223> n ═ t, g, or u

<400>131

tcgtngnnan ngntcg 16

<210>132

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5，9

<223> n ═ 5-bromocytosine

<400>132

tcgtngaang utcg 14

<210>133

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> variants

<222>5，9

<223> n ═ 5-bromocytosine

<400>133

tcgtngaang ttcg 14

<210>134

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>134

tgactgtgaa cgttcgagat ga 22

<210>135

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>135

tgactgtgaa ccttagagat ga 22

<210>136

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>136

tgactgtgaa ggttagagat ga 22

<210>137

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> modified base

<222>1

<223> n-thymine conjugated to a reactive linking group

<400>137

ngactgtgaa ccttagagat ga 22

<210>138

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> modified base

<222>1

<223> n-thymine conjugated to a reactive linking group

<400>138

ngactgtgaa cc ttagagat ga 22

<210>139

<211>66

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<400>139

tgactgtgaa cgttcgagat gatgactgtg aacgttcgag atgatgactg tgaacgttcg 60

agatga 66

<210>140

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> modified base

<222>1

<223> n-thymine conjugated to a reactive linking group

<400>140

ngactgtgaa cgttcgagat ga 22

<210>141

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> synthetic constructs

<221> modified base

<222>1

<223> n-thymine conjugated to a reactive linking group

<400>141

ngactgtgaa cgttcgagat ga 22

Claims (48)

1. A Chimeric Immunomodulatory Compound (CIC) comprising two or more nucleic acid moieties and one or more non-nucleic acid spacer moieties,

wherein at least one non-nucleic acid spacer moiety is covalently linked to two nucleic acid moieties,

wherein the spacer is not a polypeptide,

wherein at least one nucleic acid moiety comprises the sequence 5 '-CG-3', and

wherein the CIC has at least one immunomodulatory activity selected from the group consisting of:

(a) the ability to stimulate IFN- γ production by human peripheral blood mononuclear cells;

(b) the ability to stimulate IFN- α production by human peripheral blood mononuclear cells;

(c) the ability to stimulate B cell proliferation.

2. The CIC of claim 1, wherein at least one nucleic acid moiety comprises the sequence 5 '-TCG-3'.

3. The CIC of claim 1 or 2, comprising a core structure having the formula:

N₁-S₁-N₂or N₁-S₁-N₂-S₂-N₃

Wherein N is₁、N₂And N₃Is a nucleic acid moiety, S₁And S₂Is a non-nucleic acid spacer moiety, and S₁And S₂Covalently bound to exactly two nucleic acid moieties.

4. The CIC of claim 1 or 2, comprising a core structure having the formula:

[N_v]_x-S_p，

wherein S_pIs a nucleic acid moiety independently selected from X, i.e. N_vA covalently bound multivalent spacer, and wherein X is at least 3.

5. The CIC of claim 4, wherein X is 3 to about 50.

6. The CIC of claim 4, wherein X is about 50 to about 500.

7. The CIC of claim 4, wherein S_pComprising a dendrimer or a polysaccharide.

8. The CIC of claim 7, wherein S_pComprising a cross-linked polysaccharide.

9. The CIC of claim 1 or 2, comprising at least 3 nucleic acid moieties, wherein each nucleic acid moiety is covalently linked to at least one non-nucleic acid spacer moiety.

10. The CIC of claim 1 or 2, comprising a composition comprising an oligo-ethylene glycol, glycerol, C₂-C₁₀Alkyl, abasic nucleotide, pentaerythritol, 1, 3-diamino-2-propanol, 2- (hydroxymethyl) ethyl, a polysaccharide, or a non-nucleotide spacer moiety of a dendrimer.

11. The CIC of claim 1 or 2, comprising a composite non-nucleotide spacer moiety.

12. The CIC of claim 10 or 11, wherein the non-nucleotide spacer comprises a HEG subunit.

13. The CIC of claim 10 or 11, wherein the non-nucleotide spacer comprises a phosphodiester and/or phosphorothioate linked oligo-ethylene glycol moiety.

14. The CIC of claim 4, comprising a first spacer subunit comprising a dendrimer, a polysaccharide, glycerol, pentaerythritol, or 2- (hydroxymethyl) ethyl group, and further comprising at least one HEG subunit, wherein the HEG subunit is covalently bound to the first spacer subunit and to the nucleic acid moiety.

15. The CIC of claim 14, wherein the linkage between the HEG subunit and the first spacer element is a phosphodiester linkage or a phosphorothioate linkage and the linkage between the HEG subunit and the nucleic acid moiety is a phosphodiester linkage or a phosphorothioate linkage.

16. The CIC of claim 1 or 2, wherein at least one nucleic acid moiety comprises the sequence 5 '-TCGA-3'.

17. The CIC of claim 1 or 2, wherein at least one nucleic acid moiety comprises the sequence 5' - [ (X)_0-2]TCG[(X)_2-4]-3', wherein each X is an independently selected nucleotide.

18. The CIC of claim 14, wherein at least one nucleic acid moiety has the sequence 5' -TCG [ (X)_2-4]-3′；5′-TCG(A/T)[(X)_1-3]-3'; or 5' -TCG (A/T) [ (X)_1-3]-3', wherein each X is an independently selected nucleotide.

19. The CIC of claim 18, wherein at least one nucleic acid moiety has the sequence 5 '-TCGACGT-3' or 5 '-TCGTCGA-3'.

20. The CIC of claim 1 or 2, wherein all nucleic acid moieties of the CIC have the formula 5' -TCG [ (X)_2-4]-3′；5′-TCG(A/T)[(X)_1-3]-3'; or 5' -TCG (A/T) [ (X)_1-3]-3', wherein each X is an independently selected nucleotide.

21. The CIC of claim 1 or 2, wherein at least one nucleic acid moiety comprising the sequence 5 '-CG-3' is less than 8 nucleotides long.

22. The CIC of claim 1 or 2, wherein all nucleic acid moieties comprising the sequence 5 '-CG-3' are less than 8 nucleotides long.

23. The CIC of claim 1 or 2, wherein all nucleic acid moieties are identical.

24. The CIC of claim 1 or 2, wherein at least one nucleic acid moiety of the CIC is (i) not independently immunologically active or (ii) secondarily independently immunologically active.

25. The CIC of claim 24, wherein none of the nucleic acid moieties of the CIC has independent immunomodulatory activity.

26. The CIC of claim 13, having a structure selected from the group consisting of:

5’-TCGTCGA-3’-HEG-5’-ACGTTCG-3’-HEG-5’-AGATGAT-3’

5’-TCGTCG-3’-HEG-5’-ACGTTCG-3’-HEG-5’-AGATGAT-3’

5’-TCGTCGA-3’-HEG-5’-TCGTCGA-3’-HEG-5’-TCGTCGA-3’

5’-TCGTCG-3’-HEG-5’-TCGTCG-3’-HEG-5’-TCGTCG-3’

5’-TCGTCG-3’-HEG-5’-AACGTT-3’-HEG-5’-AGATGAT-3’

5’-TCGTCG-3’-HEG-5’-ACGTTCG-3’-HEG-5’-AGATGAT-3’-TEG

HEG-5’-TCGTCG-3’-HEG-5’-ACGTTCG-3’-HEG-5’-AGATGAT-3’-TEG

5’-TCGTTTT-3’-HEG-5’-TCGTTTT-3’-HEG-5’-TCGTTTT-3’

5’-TCGTCGT-3’-HEG-5’-TCGTCGT-3’-HEG-5’-TCGTCGT-3’

5’-TCGAGAT-3’-HEG-5’-TCGAGAT-3 ’-HEG-5’-TCGAGAT-3’

5’-TCGTCGT-3’-HEG-5’-TGTCGTT-3’-HEG-5’-TGTCGTT-3’

5’-TCGTCGA-3’-HEG-5’-ACGTTCG-3’-HEG-5’-TCGTCGA-3’

5’-TCGTCGA-3’-HEG-5’-ACGTTCG-3’-HEG-5’-GGGGGG-3’

5’-TCGAACG-3’-HEG-5’-TCGAACG-3’-HEG-5’-TCGAACG-3’

5’-TCGACGT-3’-HEG-5’-TCGACGT-3’-HEG-5’-TCGACGT-3’

5’-TCGTCGA-3’-HEG-5’-AACGTTC-3’-HEG-5’-AGATGAT-3’

5’-TCGTCGA-3’-HEG-5’-AACGTTC-3’-HEG-5’-TCGTCGA-3’

5’-TCGTCGA-3’-HEG-5’-AGATGAT-3’-HEG-5’-ACGTTCG-3’

5’-TCGACTC-3’-HEG-5’-TCGAGCG-3’-HEG-5’-TTCTCTT-3’

5’-TCGTCGA-3’-HEG-5’-TCGTCGA-3’-HEG-3’-AGCTGCT-5’

5’-TCGAT-3’-HEG-5’-TCGAT-3’-HEG-5’-TCGAT-3’-HEG-5’-TCGAT-3’

5’-TCGTCGA-3’-HEG-5’-TCGTCGA-3’-HEG-5’-AACGTTC-3’-HEG-5’-AGAT-3’

5’-TCGACGT-3’-HEG-5’-TCGACGT-3’-HEG-5’-TCGACGT-3’-HEG-5’-TCGACGT-3’

5’-TCGATTT-3’-HEG-5’-TCGATTT-3’-HEG-5’-TCGATTT-3’

5’-TCGCTTT-3’-HEG-5’-TCGCTTT-3’-HEG-5’-TCGCTTT-3’

5’-TCGGTTT-3’-HEG-5’-TCGGTTT-3’-HEG-5’-TCGGTTT-3’

(5 '-TCGTCGA-3' -HEG) 2-Glycerol-HEG-5 '-TCGTCGA-3'

(5 '-TCGTCGA-3' -HEG) 2-Glycerol-HEG-3 '-AGCTGCT-5'

(5 '-TCGTCGA-3' -HEG) 2-Glycerol-HEG-5 '-AACGTTC-3'

(5 '-TCGTCGA-3' -HEG) 2-Glycerol-HEG-5 '-AACGTTC-3' -HEG-5 '-TCGA-3'

(5 '-TCGTCGA-3' -HEG) 3-Triplex-HEG-5 '-AACGTTC-3' -HEG-5 '-TCGA-3'

(5 '-TCGTCGA-3' -HEG) 2-Glycerol-HEG-5 '-AACGTTC-3' -HEG-5 '-TCGACGT-3'

(5 '-TCGACGT-3' -HEG) 2-Glycerol-HEG-5 '-TCGACGT-3'

(5 '-TCGTCGA-3' -TEG) 2-Glycerol-TEG-5 '-TCGTCGA-3'

(5 '-TCGTCGA-3' -HEG-HEG) 2-Glycerol-HEG-5 '-TCGTCGA-3'

(5 '-TCGACGT-3' -HEG) 2-symmetrical double multiplier-HEG-5 '-TCGACGT-3'

(5 '-TCGACGT-3' -HEG) 3-Triplex-HEG-5 '-TCGACGT-3'

((5 '-TCGACGT-3' -HEG) 2-Glycerol-HEG-5 '-TCGACGT-3'

(5 '-TCGACGT-3' -HEG) 2-Glycerol-HEG-5 '-AACGTTC-3'

((5 '-TCGACGT-3' -HEG) 2-Glycerol-HEG-5 '-T-3'

(5 '-TCGACGT-3' -HEG) 3-Triplex-HEG-5 '-T-3'

(5’-TCGACGT-3’-HEG)x-Ficoll₄₀₀

27. The CIC of claim 1 or 2, wherein the linkage between nucleotides of the nucleic acid moiety is selected from phosphodiester bonds and phosphorothioate bonds.

28. The CIC of claim 1 or 2, wherein the linkages between nucleotides of the nucleic acid moiety, between the nucleic acid moiety and the spacer moiety, and between subunits of the spacer moiety are phosphodiesters and/or phosphorothioates.

29. The CIC of claim 1, wherein spacer structure portion comprises a dendrimer.

30. The CIC of claim 1, wherein the spacer moiety comprises a polysaccharide.

31. A composition comprising a CIC according to any preceding claim and a pharmaceutically acceptable excipient.

32. The composition of claim 31, wherein the composition is substantially free of endotoxins.

33. The composition of claim 31 or 32, further comprising an antigen.

34. The composition of claim 31 or 32, further comprising cationic microspheres.

35. The composition of claim 34 wherein the microspheres comprise a polymer of lactic acid and glycolic acid.

36. Use of a CIC according to any of claims 1-30 or a composition according to any of claims 31-35 in the manufacture of a medicament for modulating an immune response in an individual.

37. The use of claim 36, wherein the individual has a condition associated with a Th 2-type immune response.

38. The use of claim 37, wherein the disorder associated with a Th 2-type immune response is allergy or allergy-induced asthma.

39. The use of claim 36, wherein the subject has an infectious disease.

40. Use of the CIC of any one of claims 1-30 or the composition of any one of claims 31-35 in the manufacture of a medicament for increasing interferon-gamma (IFN- γ) in a subject.

41. The use of claim 40, wherein the individual has idiopathic pulmonary fibrosis.

42. Use of the CIC of any one of claims 1-30 or the composition of any one of claims 31-35 in the manufacture of a medicament for increasing interferon-alpha (IFN- α) in a subject.

43. The use of claim 42, wherein the subject has a viral infection.

44. Use of the CIC of any one of claims 1-30 or the composition of any one of claims 31-35 in the manufacture of a medicament for ameliorating a symptom of an infectious disease in a subject.

45. Use of a CIC according to any of claims 1-30 or a composition according to any of claims 31-35 in the manufacture of a medicament for ameliorating an IgE-related disorder in a subject.

46. The use of claim 45, wherein the IgE associated disorder is allergy.

47. The use of claim 45, wherein the IgE-associated disorder is an allergy-associated disorder.

48. Use of a CIC according to any of claims 1-30 or a composition according to any of claims 31-35 in the manufacture of a medicament for the treatment of cancer.