JP2014027935A - Conjugate of cd40 agonist antibody/i-type interferon synergistic adjuvant, composite comprising the same, and use thereof as treatment enhancing cellular immunity - Google Patents

Abstract

Provided is a CD40 agonist antibody / type I interferon synergistic adjuvant conjugate, a complex containing the same, and a therapeutic method such as HIV infection that enhances cellular immunity.
A synergistic adjuvant comprising a synergistically effective amount of at least one type I interferon and at least one CD40 agonist. These portions may be in the same or separate compositions. Further, fusion proteins and DNA complexes comprising a type I interferon / CD40 agonist / antigen conjugate.
[Selection] Figure 1

Description

Related Applications This application is a U.S. provisional application number 60 / 796,867,20 filed May 3, 2006.
With respect to 60 / 809,821 filed on June 1, 2006 and 60 / 842,009 filed on September 5, 2006, all of these applications are incorporated by reference in their entirety. This application is also filed in US provisional application 60 / 777,56 filed March 1, 2006.
With respect to 9, that application is also incorporated herein by reference.
FIELD OF THE INVENTION [0001] The present invention relates generally to synergistic adjuvant conjugate may be used to enhance immunity in a subject in need thereof. In particular, the invention relates to (i) type I interferons and (ii) CD40 agonists such as agonist anti-CD40 antibodies or C
It relates to a specific synergistic adjuvant conjugate further comprising a D40L polypeptide or a CD40L fragment or a complex comprising CD40L, and optionally (iii) a target antigen.

[0002] The present invention further provides a novel protein or DNA complex comprising or encoding said synergistic adjuvant conjugate, such as (i) a CD40 agonist antibody or soluble CD40L protein or CD40L fragment or CD40L complex, and ( ii) relates to proteins and DNA complexes that contain or encode type I interferons, and optionally (iii) the desired antigen.

[0003] Furthermore, the present invention provides a novel immunotherapy comprising administering such a synergistic adjuvant conjugate or DNA or protein complex to enhance antigen-specific cellular immunity, eg CD8 + immunity I will provide a. In particular, for the treatment of various chronic diseases, including cancers such as CD40 antigen expressing tumors, and for the treatment of infectious diseases such as HIV infection, autoimmune diseases, allergic and inflammatory diseases, and enhanced vaccine efficacy, The use of compositions comprising these novel adjuvant conjugates and / or polypeptide complexes and DNA complexes is also described.

[0004] The present invention also relates to such CD40 in combination with a sufficient amount of type I interferon to reduce or prevent toxicity such as hepatotoxicity that may occur when a CD40 agonist is administered alone. Novel methods are provided for reducing the toxicity of CD40 agonists, such as CD40L polypeptides and complexes or agonist CD40 antibodies, by administering agonists. This is because C at therapeutic doses that would otherwise be hampered by toxicity.
Facilitates administration of D40 agonists.

BACKGROUND OF THE INVENTION [0005] The body's defense system against microorganisms and other chronic diseases, such as those affecting cell proliferation, are mediated by early responses of the innate immune system and late responses of the adaptive immune system. Is done. Innate immunity is characteristic of, for example, microbial pathogens and involves a mechanism that recognizes structures that are not present in mammalian cells. Examples of such structures include bacterial liposaccharide (LPS), viral double stranded DNA, and unmethylated CpG DNA nucleotides. Effector cells of the innate immune response system include neutrophils, macrophages, and natural killer cells (NK cells). In addition to innate immunity, vertebrates, including mammals, have developed immunological defense systems that are stimulated by exposure to infectious agents, each increasing in size and effectiveness with continuous exposure to specific antigens. . Because of its ability to adapt to specific infections or antigenic damage, this immune defense mechanism has been termed adaptive immunity. There are two types of adaptive immune responses: humoral immunity involving antibodies produced by B lymphocytes, and cellular immunity mediated by T lymphocytes.

[0006] The two main types of T lymphocytes are called CD8 + cytotoxic lymphocytes (CTL) and CD4 helper cells (Th cells). CD8 + T cells are effector cells that recognize foreign antigens presented by class I MHC molecules on cells infected with viruses or bacteria, for example, via T cell receptors (TFR). In recognizing foreign antigens, CD8 + cells undergo an activity, maturation, and proliferation process. This differentiation process results in CTL clones that have the ability to destroy target cells that display foreign antigens. On the other hand, T helper cells are involved in both humoral and cell-mediated forms of effector immune responses. With respect to humoral or antibody immune responses, antibodies are produced by B lymphocytes through interaction with Th cells. In particular, extracellular antigens, such as circulating bacteria, are taken up by special antigen presenting cells (APC),
Processed and presented in association with class II major histocompatibility complex (MHC) molecules on CD4 + Th cells. These Th cells in turn produce antibodies by activating B lymphocytes. In contrast, a cell-mediated or cellular immune response serves to neutralize microorganisms that reside locally within the cell, for example after successful infection of the target cell. Foreign antigens, such as microbial antigens, are synthesized in infected cells and presented on the surface of such cells in association with class I MHC molecules. The presentation of such epitopes is the stimulation of CD8 + CTL as described above, followed by CD
The process is also stimulated by 4+ Th cells. Th cells are composed of at least two separate subpopulations, Th1 and Th2 cells. Th1 and Th2 subtypes represent a polarized population of Th cells that differentiate from a common precursor after exposure to antigen.

[0007] Each T helper cell subtype secretes cytokines that oppose each other and promote unique immune effects that mutually control their increase and function. Th1 cells are found in large quantities of cytokines such as interferon (IFN) γ, tumor necrosis factor alpha (TNF).
Alpha), interleukin-2 (IL-2), IL-12 and the like, as well as small amounts of IL-4. Th1-related cytokines are CD8 + cytotoxic T lymphocytes (CT
L) Promotes activity and is most frequently associated with cell-mediated immune responses against intracellular pathogens. In contrast, Th2 cells are abundant in cytokines such as IL-4, IL-13, and IL-1.
0, secretes a small amount of IFNγ and promotes antibody response. The Th2 response is particularly associated with humoral reactions such as protection from Bacillus anthracis and the elimination of parasitic infections.

[0008] Whether the resulting immune response is due to Th1 or Th2,
Depends mainly on pathogens involved and factors in the cellular environment such as cytokines. Not being able to activate the T helper response, or the correct T helper subset, may not be able to implement a sufficient response to combat a particular pathogen, but may not generate sufficient immunity against reinfection. Many infectious agents are intracellular pathogens, and cell-mediated reactions such as those exemplified by Th1 immunity are expected to play an important role in prevention and / or treatment. Furthermore, for many of these infectious agents, induction of inappropriate Th2 responses has been shown to negatively impact disease outcome. Examples include M. tuberculosis, S. It includes Manson schistosomiasis, and a Th2-like dominant immune response that produces adverse effects. Leprosy also appears to be characterized by a broad but inappropriate Th2-like reaction. HIV infection represents another example. It has been suggested that the ratio of Th1-like cells to other Th cell populations may play an important role in the progression to disease symptoms.

[0009] As a means of protecting against infectious agents, vaccination protocols aimed at protecting against some microorganisms have been developed. Vaccination protocols against infectious agents often have insufficient vaccine immunogenicity, inappropriate type of response (antibody versus cell-mediated immunity), lack of ability to derive long-term immune memory, and / or Or it is hampered by the inability to generate immunity against different serotypes of a given pathogen. Current vaccination methods are aimed at the derivation of antibodies specific to a given serotype and many common pathogens, such as viral serotypes or pathogens. Efforts to monitor which antigens are spreading throughout the world need to be repeated. An example of this is annual monitoring of the occurrence of influenza A serotypes that are expected to be the primary infectious type.

[0010] To support vaccination protocols, adjuvants have been further developed that support the generation of immune responses against specific infections. For example, aluminum salts are used as relatively safe and effective vaccine adjuvants to enhance antibody responses against specific pathogens. One drawback of such adjuvants is that they are relatively ineffective at stimulating cell-mediated immune responses and produce predominantly Th2-biased immune responses.

[0011] It is now widely recognized that the generation of protective immunity depends not only on exposure to an antigen, but also on the situation where the antigen is encountered. In non-inflammatory situations, the introduction of new antigens into host cells produces tolerance rather than long-term immunity, but inflammatory factors (
There are many examples in which immunity is induced by exposure to an antigen in the presence of an adjuvant. (M
ondino et al. , Proc. Natl. Acad. Sci. , USA 93
: 2245 (1996); Pulendran et al. , J .; Exp. Med. 1
88: 2075 (1998); Jenkins et al. , Immunity 1:
443 (1994); and Kearney et al. , Immunity 1:
327 (1994)).

[0012] A naturally occurring molecule well known to control adaptive immunity is CD40. CD40 belongs to the TNF receptor superfamily and is essential for a series of cell-mediated immune responses and is necessary for the development of T cell-dependent humoral immunity (Aruffo et a
l. Cell 72: 291 (1993); Farrington et al. , P
roc Natl Acad Sci. USA 91: 1099 (1994); Ren
shaw et al. , J Exp Med 180: 1889 (1994)). In its natural role, the CD40-ligand expressed on CD4 + T cells is
It interacts with CD40 expressed on DC or B cells to enhance the activity of APC and at the same time promote further activity of T cells (Liu et al Semin Immunol
9: 235 (1994); Bishop et al. , Cytokine Growt
h Factor Rev 14: 297 (2003)). In the case of DC, the CD40 ligation response results in responses that are classically similar to stimuli through TLRs, such as upregulation of activation markers and inflammatory cytokine production (Quezada et al. Annu Rev.
Immunol 22: 307 (2004); O'Sullivan B and Th
omas R Crit Rev Immunol 22:83 (2003)). CD8
Its importance in the response was demonstrated by studies showing that stimulation of APC through CD40 rescued the CD4-dependent CD8 + T cell response in the absence of CD4 cells (Lef
rancois et al. , J Immunol. 164: 725 (2000); B
ennett et al. , Nature 393: 478 (1998); Ridge
et al. , Nature 393: 474 (1998); Schoenberg.
r et al. , Nature 393: 474 (1998). This finding triggered the speculation that in some disease environments, CD40 agonists alone may be able to rescue unintended CD8 + T cell responses.

[0013] However, other studies have shown that CD40 stimulation alone does not sufficiently promote long-term immunity. In some model systems, anti-CD40 treatment alone did not sufficiently promote long-term immunity. In particular, anti-CD40 treatment alone does not produce enough inflammatory cytokines and deletes antigen-specific T cells (Mauri et al. Nat Med 6:67).
3 (2001); Kedl et al. Proc Natl Acad Sci, US
A 98: 10811 (2001)), and the B cell reaction may be terminated (Eri
ckson et al. , J Clin Invest 109: 613 (2002).
). Soluble trimerized CD40 ligand has been used as an agonist of the CD40 pathway in clinical practice, and it has been reported that CD40 stimulation alone provides all signals necessary for long-term CD8 + T cell immunity. Consistent with the conclusion that reconfiguration is not possible (
Vonderheide et al. , J Clin Oncol 19: 3280 (
2001)).

[0014] Various agonist antibodies have been reported by different groups. For example, mAb CD40.4 (5c3) (PharMingen, San Diego Ca)
lifornia) has been reported to increase the activity between CD40 and CD40L by about 30-40% (Schlossman et al., Leukocyte Type).
ng, 1995, 1: 547-556). Also in US Pat. No. 6,843,989, Seattle Genetics claims to provide a method for treating cancer in human antibodies using the agonist anti-human CD40. The antibody is said to have a neoplastic activity in vivo, delivering a stimulatory signal that enhances CD40-CD40L interaction by at least 45% and enhances CD40L-mediated stimulation. They have already been shown to deliver strong growth-promoting signals to B lymphocytes, S2C6, an agonist anti-human CD40
This antibody was obtained from an antibody (Pauli et al., 1989, J. Immunol.
. 142: 590-595).

[0015] Due to the role of CD40 in innate and adaptive immune responses, various CD4
CD40 agonists, including 0 agonist antibodies, are being investigated for use as vaccine adjuvants for treatments where it is desirable to enhance cellular immunity. Recently, the inventors have
Immunization with antigen in combination with some TLR agonists and anti-CD40 treatment (conjugated T
LR / CD40 agonist immunization) induced potent CD8 + T cell increase and elicited a 10-20 fold higher response than immunization with either agonist alone (Ahonen et al., J Exp Med 199: 775 (2004)
). This was the first time that a strong CD8 + T cell response could be generated in the absence of infection with viral or microbial factors. Antigen-specific CD8 + T cells derived by combined TLR / CD40 agonist immunization show enhanced secondary responses to lytic function, gamma interferon production, and antigen sensitization. As a result, synergistic activity with anti-CD40 that induces an increase in CD8 + T cells has been demonstrated with agonists of TLR1 / 6, 2/6, 3, 4, 5, 7 and 9.

[0016] There is a need to develop new effective vaccine adjuvants to enhance the effectiveness of adaptive immune responses in vaccination protocols or microbial infections. The present invention fulfills this need and provides other advantages.

[0017] It is also important to develop effective immune adjuvants that are effective at doses that do not induce side effects such as hepatotoxicity. In particular, Vanderheide et a
l. , J Clin. Oncol. 25 (7) 876-8833 (March 2007)
) Is the maximum tolerated dose of a typical agonist antibody is 0.3 mg / kg, the higher the dose,
It has been reported that side effects including venous thromboembolism, grade 3 headache, release of cytokines that cause toxic effects such as chills, and transient hepatotoxicity may be derived. Vande
rheide et al. , J Clin. Oncol. 19 (23): 4351-3
(2001) has a maximum tolerated dose of 0.1 m of the hCD40L polypeptide described therein
When g / kg / day and the polypeptide was administered at a maximum dose of 0.15 mg / kg / day, hepatotoxicity was observed in the treated subjects, characterized by elevated grade 3 or 4 liver transaminase levels. It is reported that.

SUMMARY OF THE INVENTION [0018] The present invention, in one embodiment, is a CD on dendritic cells in combination with a specific moiety.
It relates to the discovery of up-regulating 70 and eliciting a synergistic effect on immunity. For example, they promote Th1 cell immunity and CD8 T cell immune responses. In particular, the invention relates to type I interferons and CD40 agonists such as agonist CD40 antibodies or CD40.
When L polypeptide or CD40L complex, etc. are administered in combination in the same or separate compositions, or optionally in combination with further desired antigens, immunity is derived by deriving CD70 expression on CD8 + dendritic cells. Derived synergistic effect on CD8 + T
It relates to the discovery that it results in a strong increase of cells and enhancement of Th1 immunity.

[0019] Based on this discovery, the present invention provides a novel adjuvant conjugate that can be administered to a subject in need thereof as a means of enhancing immunity. The adjuvant conjugate can also be added to the vaccine or administered in combination with it to enhance its effectiveness.

[0020] In connection with the above discovery, the present invention provides (i) a type I interferon and (i
i) providing a nucleic acid construct encoding a CD40 agonist; This is further (iii
) May optionally include a nucleic acid sequence encoding a desired antigen, wherein the nucleic acid construct is synergistic to immunity, when administered in combination with the antigen, optionally to a host cell in need thereof. Is derived. Such CD40 agonists include, by way of example, CD40 agonist antibodies and CD40 agonist antibody fragments, and soluble CD40L and CD40L.
Fragments and complexes, and derivatives thereof such as oligomeric CD40L polypeptides, such as trimeric CD40L polypeptides and complexes containing the same.

[0021] The invention also provides: (i) at least one type I interferon; (ii)
At least one CD40 agonist, such as a CD40 agonist antibody or CD40L
Provided are complexes or derivatives thereof, such as a polypeptide or CD40L fragment or oligomer CD40L or a complex comprising the same, and optionally (iii) a polypeptide complex comprising an antigen. Here, these moieties may be linked directly or indirectly in any order and induce a synergistic effect on immunity when administered to a subject in need thereof.

[0022] In particular, the present invention relates to (i) an agonist anti-human CD40 antibody or human CD4
One or more genes encoding 0L polypeptides or fragments, complexes or derivatives thereof, (ii) genes encoding human type I interferons, such as human alpha or human beta interferons, and optionally (iii) Nucleic acid constructs comprising a gene encoding an antigen from which an enhanced cellular immune response is desirably derived are provided.

[0023] Also particularly the invention relates to (i) at least one agonist anti-human CD40 antibody or human CD40L polypeptide, or a fragment thereof that agonizes human CD40 / CD40L, human alpha or beta interferon, and enhanced cellular immunity Novel polypeptide structures are provided that optionally include at least one antigen from which a reaction is desirably derived.

[0024] Furthermore, the present invention provides a synergistically effective amount of (i) a type I interferon, preferably alpha or beta interferon, (ii) a CD40 agonist, preferably an agonist CD40 antibody, a monomeric or oligomeric soluble CD40L polypeptide,
Alternatively, an adjuvant polypeptide composition comprising a fragment or complex thereof and optionally (iii) one or more antigens is provided.

[0025] The present invention also provides that the CD40 agonist is type I interferon or TLR.
It relates to the discovery that toxicity of CD40 agonists can be significantly reduced when administered in combination with agonists. Thereby, since the CD40 agonist can be administered at a higher dose than described above, the present invention provides a more effective CD40 agonist treatment. For example, the MTD of a CD40L polypeptide when administered in combination with a type I interferon or TLR agonist
The (maximum tolerated dose) tide may be at least 1.5 times, more preferably at least 2 to 5 times, or 10 times or more, so that at least about 0.1 mg / kg / day. 15m
CD40L polypeptide can be administered at a maximum tolerated dose in the range of g / kg / day to 1.0 mg / kg / day or more. As a result, CD40L treatment is more effective in the treatment of CD40-related malignant tumors and other treatments disclosed herein. Furthermore, the present invention reduces the toxicity of CD40 agonist antibody treatment and facilitates administration of higher CD40 agonist antibody doses than suggested above. In particular, as described above, Vonderheide et al. , J Cli
n. Immunol. 25 (7): 876-883 (2007) reported that the MTD of the agonist CD40L antibody is 0.3 mg / kg, overdose is transient hepatotoxicity, venous thromboembolism, grade 3 headache, cytokine release , And associated toxicities and side effects such as fever and chills have been reported. Administration of a CD40 agonist antibody in combination with a type I interferon or TLR agonist may significantly increase the MTD antibody amount, for example, 1.5 to 15 times or 5 to 10 times without causing side effects. Thereby, the MTD amount of the CD40 agonist antibody is about 0.45 mg / kg to 3.0 mg / kg.
Alternatively, it may be increased further. As such, the present invention includes co-administering a sufficient amount of a Type I interferon or TLR agonist with a CD40 agonist to reduce toxic effects such as hepatotoxicity that may occur at a particular CD40 agonist dose.

[0026] The present invention further provides a novel therapy comprising administering any of the aforementioned proteins or DNA complexes, or a composition comprising a synergistic adjuvant protein. These therapies include their use as immune agonists (adjuvants), for example, synergistically enhancing the effectiveness of vaccines and conditions where enhanced immunity is desired, such as cancer, infections, autoimmune diseases, allergies Treat inflammatory diseases, gene therapy and the like.

[0027] As shown above and below, surprisingly, the aforementioned novel adjuvant conjugates or proteins or DNA complexes encoding them have a synergistic effect on immunity with single administration of CD40 agonist or type I interferon. It has been discovered that it is possible to derive and / or reduce or prevent side effects such as hepatotoxicity. Such a reduction in toxicity can be identified, for example, based on the effect of immunostimulatory conjugates on liver transaminase levels. This synergistic effect is evident because the adjuvant conjugates of the present invention induce a strong increase in CD8 + T cells in vivo by significantly inducing (upregulating) CD70 expression on CD8 + dendritic cells in vivo. can get.

[0028] Based on at least a surprising synergistic effect on such dendritic cells, CD8 + T cell immunity and Th1 immunity, a composition comprising these adjuvant conjugates, nucleic acid constructs, or polypeptide complexes is needed. May be administered as the following means.
(I) produces enhanced (exponentially superior) primary and memory CD8 + T cell responses compared to immunization with either agonist alone.
(Ii) induce an exponential increase of antigen-specific CD8 + T cells and / or (iii) generate protective immunity.

[0029] Accordingly, these adjuvant conjugates, which may comprise a protein composition, or a nucleic acid construct encoding it or a polypeptide complex comprising it, are therapeutically desirable for the enhanced cellular immune response described above. Used to treat any disease or condition, especially proliferative diseases such as infectious diseases, cancer, allergies, autoimmune diseases, inflammatory diseases, and other chronic diseases where enhanced cellular immunity is the desired therapeutic outcome Also good. Preferred applications of the present invention include the treatment of infectious diseases such as HIV infection and cancer in particular.

[0048] Detailed Description of the Invention [0049] As noted above, the present invention generally relates to synergistic adjuvant conjugates and uses thereof. Before describing the present invention in detail, the following definitions are provided. All other terms should be understood by those skilled in the art.

[0050] In the present invention, the term "agonist" refers to a receptor by modifying another compound that forms a complex with another entity that binds the receptor, or that directly binds and activates the receptor. Includes any entity that directly binds and activates or indirectly activates the receptor.

[0051] The term "CD40 agonist" specifically includes any entity that agonizes CD40 / CD40L and / or increases activity associated with one or more CD40 or CD40L. This is by way of example produced by CD40 agonist antibodies, fragments thereof, soluble CD40L, and fragments and derivatives thereof such as oligomers (eg, divalent, trimeric CD40L), and fusion proteins and recombinant or protein synthesis comprising it. Including its variants. Further, such CD40 agonists include CD40 aptamers consisting of small molecules and RNA or DNA molecules that can replace antibodies. Techniques for generating and using it as an antigen binding moiety are described, for example, in US Pat. Nos. 5,475,046, 5,720,163, 5,589,332, and 5,741,
679 can be found. These patents are incorporated herein by reference in their entirety.

[0052] In the present invention, the term "CD40L" or "CD154" refers to all mammals such as humans, rats,
CD40L, such as non-human primates, mice, and at least the corresponding mammalian CD40 polypeptides, eg, fragments, variants, oligomers, and complexes thereof that bind to human CD40. In the present invention, the CD40L to be administered may contain a CD40L polypeptide or DNA encoding the CD40L polypeptide. Such a CD
40L polypeptides and DNA are specifically described in Immunex US Patent No. 6,410,7
11, the native CD40L sequence and fragments, variants, and oligomers thereof described in US Pat. No. 6,391,637, US Pat. No. 5,981,724, US Pat. No. 5,961,974 and US Published Application No. 200400006006. including. All of these patents and publications, and the CD40L sequences disclosed therein, are incorporated herein by reference in their entirety.

[0053] In the present invention, the term 4-1BB agonist refers to the agonist 4-1B.
It includes any entity that agonizes 4-1BB receptors, such as B antibodies and 4-1MM polypeptides and complexes thereof. Such agonists may be administered in combination with type I interferons or TLR agonists to derive a synergistic effect on immunity.

[0054] In the present invention, the term "type I interferon" refers to enhanced C when administered adjacent to or in combination with a CD40 agonist.
Includes any type I interferon that elicits a D8 + immune response. This includes alpha interferons, beta interferons, and other types of interferons classified as type I interferons. In particular, this is epsilon interferon,
Zeta interferon, and tau interferon, eg tau 1, 2, 3, 4,
5, 6, 7, 8, 9, and 10. This also includes variants thereof such as fragments, consensus interferons that mimic the structure of different type I interferon molecules such as alpha interferon, PEGylated versions thereof, type I interferons altered by recombinant expression or mutation, and the like. Those skilled in the art are commercially available,
There is a good understanding of different type I interferons, including those used as treatments. Preferably, the type I interferon comprises human type I interferon, most preferably human alpha interferon.

[0055] The term "synergistic adjuvant" or "synergistic conjugate" in the context of the present invention includes a combination of two immune modulators such as receptor agonists, cytokines, adjuvant polypeptides, etc. Derives a synergistic effect on immunity compared to single administration. In particular, this application relates to at least one type I interferon and a CD40 agonist or TLR agonist and a CD40 agonist or T
Disclosed is a synergistic conjugate comprising an LR agonist or type I interferon and a 4-1BB agonist. These synergistic conjugates have a greater impact on immunity when administered concomitantly or in close proximity to each other than when administered, for example, a CD40 agonist or type I interferon in the absence of other moieties. Is derived. For example, the major effect may be evidenced by upregulation of CD70 on in vivo dendritic cells that does not occur when an immune modulator or agonist is administered alone.

[0056] In the present invention, "concurrent administration" means type I interferon and CD40.
Agonists or protein complexes or DNA complexes, or complexes encoding it, different entities such as entities, eg CD40 agonist and type I interferon, elicit synergistic effects on immunity, eg on dendritic cells It means administration under conditions that cause side effects such as upregulation of CD70 and / or hepatotoxicity. The portions may be administered in the same or different compositions, and when administered separately, will be in close proximity to each other, typically within 24 hours each, more typically about 1-8 each.
Within hours, more typically each within 1 to 4 hours, or in proximity to irritating administration. The relative amount is the dose that provides the desired synergistic effect. Furthermore, when administered in the form of a DNA complex, the agonist may be comprised on the same or different vectors, such as adenovirus or vaccinia vectors, such as the same or different vectors, plasmids or recombinant viral vectors.

[0057] "Vaccine" means a composition that has an antigen-specific effect on immunity by administration alone or in combination with an adjuvant conjugate of the invention. This includes prophylactic vaccines that provide prophylactic and therapeutic vaccines.

[0058] The term "antibody" means a normal antibody or a binding fragment thereof that competes with a specific normal antibody for binding. Binding fragments are generated by recombinant DNA techniques or by enzymatic or chemical cleavage of normal antibodies. Binding fragments are Fab, Fa
Includes b ', F (ab) 2, Fv and single chain antibodies. This includes in particular chimeric, human, humanized, bispecific and non-human antibodies. In addition, such antibodies and fragments include variants thereof that are altered to affect one or more properties such as cleavage, glycosylation, effector function, and the like.

[0059] As noted above, there is a strong need to develop and implement new vaccine adjuvants and / or adjuvant formulations that can generate strong antibody-specific T cell immunity and do not produce undesirable side effects such as hepatotoxicity.

[0060] The present invention meets this need by providing a novel adjuvant that can be administered alone or in combination with existing vaccines to enhance its effectiveness. These adjuvants are generally at least one type I interferon, preferably alpha or beta
Human interferon, at least one CD40 agonist (anti-CD40 antibody or fragment thereof) or soluble CD40L polypeptide.

[0061] The present invention relates to at least one CD40 agonist, preferably a CD40 agonist antibody or soluble CD40L, a type I interferon such as human alpha or beta interferon, and optionally a target antigen such as a tumor antigen, self antigen, allergen or viral antigen. Provides a method for deriving an enhanced cellular immune response in a subject in need thereof. These parts derive a synergistic effect on cellular immunity by inducing CD70 expression on CD8 + dendritic cells. In particular, this conjugate has (i) primary and memory CD8 + T compared to either agonist alone.
Induces an exponential increase in the generation of cellular responses, (ii) an exponential increase of CD8 + T cells, and (iii) derivation of protective immunity. As above, CD7 on CD8 + dendritic cells
Induction of 0 expression does not occur when CD40 agonist antibody or type I interferon is administered alone. Thus, surprisingly, the CD40 agonist / IFN conjugate synergizes and induces CD70 expression on CD8 + DC and a strong increase in CD8 + T cells in vivo.

[0062] In connection with this discovery, the present invention further provides a DNA construct encoding a novel synergistic agonist polypeptide complex that promotes cellular immunity. This structure is (
i) DNA encoding a CD40 agonist, preferably a CD40 agonist antibody or fragment thereof, or soluble CD40L or a fragment or derivative thereof, and (ii) DNA encoding a type I interferon such as alpha or beta interferon, Preferably (iii) further comprises DNA encoding the desired antigen.

[0063] The invention further provides a synergistic protein complex that elicits a synergistic effect on cellular immunity, comprising a CD40 agonist, preferably an agonist CD40 antibody or fragment or CD40L, type I interferon and optionally a desired target antigen. .

[0064] The present invention further provides compositions comprising these DNA constructs. When administered to a host cell, preferably a human, they can be used to generate an enhanced antigen-specific cellular immune response.

[0065] The present invention further provides expression vectors and host cells comprising a DNA construct encoding the novel synergistic agonist polypeptide conjugate. This conjugate is (
i) one or more DNA encoding a specific CD40 agonist, preferably an agonist CD40 antibody, or an antibody fragment or a fragment of CD40L;
i) one or more DNA encoding a type I interferon, preferably alpha or beta interferon, and (iii) an antigen, preferably a virus or tumor, from which an enhanced antigen-specific cellular immune response is desirably derived DNA encoding the antigen
including.

[0066] The present invention also uses the vector and the host cell to produce the novel synergistic IFN / CD40 agonist / antigen polypeptide complex, preferably agonist CD4.
A method of producing a composition comprising a 0ab / antigen / type I interferon polypeptide complex is provided.

[0067] Further, the present invention provides the above DNA structure or a composition and carrier comprising the same with a host cell from which an antigen-specific cellular immune reaction is desirably derived, for example, preferably undesirable side effects such as hepatotoxicity. Provided is a method of administration to a person with cancer or a chronic disease such as an infectious or allergic disease under reduced or eliminated conditions.

[0068] Furthermore, the present invention provides a composition comprising said novel synergistic IFN / CD40 agonist antigen polypeptide complex suitable for administration to a host cell to elicit an enhanced antigen-specific cellular immune response. provide.

[0069] The present invention also provides a composition suitable for therapeutic use, wherein the composition is administered to at least one interferon, at least one CD40 agonist, and optionally a host cell in need of such administration. If so, it includes a conjugate of the target antigen that elicits a synergistic effect on cellular immunity.

[0070] The present invention also provides the novel synergistic agonist-antigen polypeptide complex or DNA encoding the polypeptide complex or at least one type I interferon, at least one CD40 agonist, and optionally at least one Administering one or more compositions comprising one target antigen to a host cell in need of such treatment;
A novel immunotherapeutic method is provided that includes deriving an enhanced (antigen-specific) cellular immune response. In a preferred embodiment, these compositions and complexes are used in subjects with cancer, infectious diseases, particularly viruses, bacteria or parasites, or chronic diseases that result in autoimmune, inflammatory, or allergic symptoms, or Administer to subjects at risk. For example, the present invention may be used to derive an antigen-specific cellular immune response against HIV. HIV
Is a well-known example of a disease that requires a strong and long-lived cellular immune response almost certainly in protective immunity.

[0071] The present invention also relates to the effectiveness of a vaccine, particularly a vaccine intended to induce a protective cell-mediated immune response by combination or co-administration of a synergistic adjuvant conjugate of a subject that upregulates CD70 on dendritic cells. Provide a way to enhance sex. In preferred embodiments, such adjuvants include certain adjuvants disclosed herein,
In addition, TLRs such as TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,
Optionally, another adjuvant such as TLR7, TLR8, TLR9, TLR10 or TLR11 is included. Ideally, this additional adjuvant further induces CD70 expression by dendritic cells, resulting in an enhanced immune response in subjects in need thereof.

[0072] The present invention shows that immunization with antigen (bound TLR / CD40 agonist immunization) in the presence of both Toll-like receptor (TLR) and CD40 agonists leads to a significant increase in antigen-specific CD8 + T cells. This is an extension of the inventor's previous explanation. The response derived from this form of vaccination is exponentially greater than the response derived from either agonist alone and is far superior to vaccination by conventional methods. Combined TLR / CD40 agonist immunization is a strong primary and secondary CD8
It has been observed that 50-70% of antigen-specific T cells are obtained in circulating blood after generating a + T cell response and immunization only twice. However, unlike previous inventions by the inventor, this synergistic conjugate is a type I interferon and CD40 agonist or 4
-Containing conjugates of 1BB agonists. Surprisingly, both the TLR / CD40 agonist antibody conjugate and the type I interferon / CD40 agonist antibody conjugate are CD8 + D
It has been observed to induce CD70 expression on C, thereby leading to a strong increase of CD8 + T cells in vivo. As such, the CD40 pathway appears to be integrated with both TLR and type I IFN signaling pathways to provide synergistically enhanced induction of DC activity and thereby potent induction of antigen-specific cellular immunity.

[0073] To derive a synergistic effect on cellular immunity, the CD40 agonist, type I interferon and antigen (if present) are preferably in close proximity to or simultaneously with each other under conditions that produce the desired synergistic effect on immunity. It is administered as separate polypeptide moieties that may be administered in combination or separately. Whether synergy is obtained can be detected based on various means, eg, upregulation of CD70 expression on dendritic cells under the administration conditions. Alternatively, these portions may be administered as a single polypeptide fusion or a complex comprising these two or three individual entities, or a DNA complex or said two or three individual entities. It may be administered in the form of a complex that encodes.
The latter two embodiments of the present invention are advantageous in polypeptide or DNA based vaccines because they need to produce only one active agent and administer it to a subject in need of treatment, such as an HIV infected or cancer patient. It is.

[0074] The present invention meets the need to enhance its effectiveness by providing novel adjuvants that may be administered alone or in combination with existing vaccines. These adjuvants are generally at least one type I interferon, preferably alpha or beta human interferon, at least one CD40 agonist (anti-CD40 antibody or fragment thereof or soluble CD40L polypeptide) and preferably enhanced antigen-specific cells. Sexual immunity includes at least one antigen that is desirably derived as a tumor antigen or viral antigen or the like. In preferred embodiments of the invention, these polypeptide moieties are included in a polypeptide complex or encoded by a nucleic acid construct. this is,
Upon in vitro expression in a host cell, or in vivo administration to a host cell, expression of the agonist and antigen polypeptide, or expression of a complex comprising these polypeptides occurs.

[0075] Type I interferon and CD40 agonists, eg agonist CD
The dose of 40 antibody includes an amount that, in combination or co-administration, produces a synergistic effect by inducing CD70 expression on dendritic cells and an increase in antigen-specific CD8 + T cells. The ideal dose does not produce side effects such as, for example, liver toxicity that can be detected based on liver transaminase levels. For type I interferons, this amount is about 1 × 103 unit activity (U) about 1 × 1
010U, and more typically between about 104U and about 108U. The amount of agonist antibody or CD40L polypeptide may vary from about 0.00001 g to about 5 g, more typically from about 0.001 g to about 1 g. As noted above, the preferred MTD is 0.3
It may exceed mg / kg and range from about 0.45 mg / kg to about 3 mg / kg. When the therapeutic method includes administration of an antigen, about 0.0001 g to about 50 g, more typically about 0
. This may be administered in an amount ranging from 1 g to about 10 g. As mentioned above, these parts are
The same or different dosage forms may be administered. When administered separately, the portions may be administered in any order, generally within a few hours of each, and more typically at a substantially close time.

[0076] As noted above, a CD40 agonist includes any moiety that agonizes the CD40 / CD40L interaction. Generally, these portions will be CD40 agonist antibodies or agonist CD40L polypeptides. As described, these antibodies include, by way of example, human antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, scFvs, and CD40 / CD
Antibody fragments that specifically agonize 40L binding interactions are included. Most preferably, the antibody comprises a chimeric, fully human, or humanized CD40 antibody.

[0077] Human CD40L and other mammalian CD40L polypeptides are widely known and available. Its soluble forms include oligomeric CD40L polypeptides, such as the trimeric CD40L originally reported by Immunex (now Amgen). The sequences of human and mouse CD40L are also known and are commercially available.
As noted above, the CD40L dose is generally at least 0.1 mg / kg / day, more typically about 0.15-1.0 mg / kg / day. MTD so that no side effects such as hepatotoxicity and hepatic transaminase levels are elevated or minimal or negligible compared to when CD40L polypeptide is administered in the absence of type I interferon or TLR agonist Select.

[0078] As noted above, the type I interferon may be any type I interferon or variant or fragment that elicits a synergistic effect on cellular immunity when administered in the vicinity or in combination with the CD40 agonist. Such interferons are alpha interferon, beta interferon, tau 1, 2, 3, 4, 5, 6,
It may include interferon tau, interferon omega, interferon epsilon, interferon zeta, etc., particularly variants and fragments thereof, such as 7, 8, 9, or 10. This includes in particular PEGylated and consensus interferons and altered (non-natural or aglycosylated) glycosylated interferons.

[0079] The TLR agonist interacts with the anti-CD40 agonist to cause C
Although significant enhancement of D8 + T cell immunity has already been reported by the inventors, these previous studies suggest that CD40 agonists such as type I interferon and agonist antibodies also produce a synergistic effect on cellular immunity. Not. Surprisingly, the inventor has discovered that the CD40 pathway is integrated with both TLR and type I IFN signaling pathways, inducing strong cellular immunity of DC activity. Furthermore, these previous studies have not revealed the role of CD70 in this process.

[0080] Since previous studies have also involved TLR agonist / CD40 agonist conjugates, the DNA or polypeptide complex of interest may comprise an antigen, a TLR agonist, and a CLR
There is no suggestion that individual administration of D40 agonists is required. Conversely, the present invention may in some embodiments include a DNA structure and a bipartite or tripartite polypeptide comprising two or three different moieties, or a single DNA or polypeptide molecule.
Provided is a complex comprising DNA encoding one or three parts, such as a CD40 agonist antibody, alpha interferon and an antigen. This (since only one molecular entity need be generated and administered in a pharmaceutically acceptable form), its use for prophylactic or therapeutic vaccine purposes, and / or enhanced cellular immunity is desired. It should simplify its use to enhance cellular immunity in the treatment of cancer or autoimmune diseases and the like. This is particularly advantageous in the treatment of chronic diseases or conditions that may require large amounts of adjuvants for effective prophylactic or therapeutic immunity.

[0081] Bound IFN / CD40 agonist immunization using only molecular reagents inherently produces a CD8 + T cell response to the extent obtained after sensitization with an infectious agent (Ahon
en et al. , J Exp Med 199: 775 (2004)). for that reason,
The present invention relates to HIV and other chronic infections involving viruses, bacteria, fungi or parasites,
And the development of powerful vaccines against proliferative diseases such as cancer, autoimmune diseases, allergic diseases, and inflammatory diseases. Effective treatment in these diseases is bound IFN (type I)
Requires the amount and nature of cellular immunity that can only be produced by immunization / CD40 agonist immunization or other adjuvant conjugates that upregulate CD70 expression on dendritic cells.
Application of the Invention [0082] The present invention now illustrates both protein and DNA based vaccines. These are (i) at least one CD40 agonist, such as an agonist anti-CD
A conjugate of 40ab or CD40L polypeptide, (ii) optionally at least one target antigen (eg, HIV Gag) and (iii) at least one type I interferon (eg, alpha interferon). HIV Gag40 is a suitable model antigen because HIV is a chronic infection with an enhanced cellular immune response with significant therapeutic potential. However, the present invention encompasses complex structures as described above, including any antigen for which an enhanced cellular immune response is therapeutically desirable. In a preferred embodiment, the at least one target antigen is at least one type I interferon and at least one CD40.
It is included in the composition to be administered, including an agonist, or included in a polypeptide complex that includes these portions, or is encoded by a DNA complex that encodes these portions. However, in some embodiments, the complex comprising type I interferon and anti-CD40 antibody may be administered separately from the antigen, or the host cell may be naturally exposed to the antigen. Further, in some embodiments, all three parts, namely anti-CD
Forty antibodies, type I interferon and antigen may be co-administered as separate separate entities. Preferably, all these parts are administered substantially simultaneously, hepatotoxicity,
Obtain the desired synergistic enhancement in cellular immunity without side effects such as venous thromboembolism, cytokine toxicity, and / or headache. These parts, however, have enhanced CD8 + T cell proliferation and CD8 + DC by deriving a synergistic effect on cellular immunity.
They may be administered in any order that results in the induction of CD70 expression above.

[0083] Exemplary antigens include, but are not limited to, bacteria, viruses, parasites, allergens, self antigens and tumor associated antigens. When a DNA-based vaccine is used, the antigen is generally encoded by the sequence of the administered DNA construct. Alternatively, when the antigen is administered as a complex, the antigen is generally a protein contained in the administered complex. Further, when the antigen is administered separately from the CD40 agonist and the type I interferon moiety, the antigen can take any form. In particular, antigens can include protein antigens, peptides, total inactive organisms, and the like.

[0084] Specific examples of antigens that can be used in the present invention include hepatitis A, B, C or D,
Influenza virus, Listeria, Clostridium botulinum, tuberculosis, mania, smallpox (pox)
, Viral hemorrhagic fever, plague (pesticidal), HIV, herpes, antigens from papillomavirus, and other antigens associated with infectious agents. Other antigens include antigens associated with tumor cells, autoimmune diseases, allergies and asthma. Co-administration of such an antigen with a subject agonist conjugate type I interferon and an anti-CD40 antibody can be used in a therapeutic or prophylactic vaccine to immunize against such disease states.

[0085] In some embodiments, by using the methods and compositions to include an antigen from an infectious agent, an individual at risk of having an infection or an individual having an infection Can be treated. By infection is meant a disease or condition that results from the presence of foreign organisms or factors that propagate in a host cell. A subject at risk of having an infection is a subject that is prone to developing the infection. Such an individual may include, for example, a subject known or suspected of being exposed to an infectious organism or factor. A subject at risk of infection is subject to a condition associated with a deficiency in the ability to increase the immune response to an infectious agent or organism, such as a subject with congenital or acquired immunodeficiency, radiation therapy or chemotherapy. It may include a subject who is suffering, a subject who is burned, a subject who is traumatic, a subject undergoing surgery or other invasive medical or dental treatment, or an immunocompromised individual as well.

[0086] Infectious diseases to be treated or prevented using the vaccine composition of the present invention include bacterial, viral, fungal and parasitic. Other less common types of infections include rickettsia, mycoplasma, and scrapie, bovine spongiform encephalopathy (BSE), prion diseases (eg, kuru and sporadic Creutzfeldt Jacob (Creut)
zfeldt-Jacob) disease). Bacteria, viruses that infect humans,
Examples of fungi and parasites are well known. Infectious diseases may be acute, subacute, chronic, or latent, and may be local or systemic. Furthermore, the infection may be primarily intracellular or extracellular during at least one phase of the life cycle of the infecting organism or factor in the host cell.

[0087] Bacterial infections for which subject vaccines and methods can be used include both gram-negative and gram-positive bacteria. Examples of gram positive bacteria include, but are not limited to, Pasteurella species, Staphylococcus species, and Streptococcus species. Examples of gram negative bacteria include, but are not limited to, E. coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, mycobacterial species (eg, Mycobacterium tuberculosis, M. avium, M. intracellularis)
are, M.M. Kansaii, M.M. gondonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A streptococcus), Agoratie streptococci (Group B streptococcus), Streptococcus (virus group), Staphylococcus aureus, Bovine Streptococcus, Streptococcus (anaerobic species), Streptococcus pneumoniae, Pathogen Campylobacter species, Enterococcus species, Hemophilus influenza, Anthrax, Diphtheria, Corynebacterium species, Swine venom, Enterococcus enterotoxin, Tetanus, Enterobacter Includes, but is not limited to: Aerogenes, Neisseria pneumoniae, Pasteurella skin necrosis toxin, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus moniliformis, Syphilis treponema, Adenoma treponema, Leptospira, Rickettsia, and Israeli actinomycetes Not .

[0088] Examples of viruses that cause infection in humans are retroviruses (eg, human deficiency viruses such as HIV-1 also referred to as HTLV-III), HIV-II, L
Other isolates such as AC or IDLV-III / LAV or HIV-III and HIV-LP, picomavirus (eg poliovirus, hepatitis A, enterovirus, human coxsackie virus, rhinovirus, echovirus), calcivirus (Eg, strains causing gastroenteritis), togavirus (eg, equine encephalitis virus, rubella virus), flavivirus (eg, dengue virus, encephalitis virus, yellow fever virus), coronavirus (
For example, coronavirus), rhabdovirus (eg, vesicular ostomy virus, rabies virus), filovirus (eg, Ebola virus), paramyxovirus (eg, parainfluenza virus, mumps virus, measles) Viruses, respiratory syncytial viruses), orthomyxoviruses (e.g. influenza viruses), bungaviruses (e.g. Haturn virus, bungaviruses, flavoviruses and nairoviruses), arena viruses (haemorrhagic fever viruses), reoviruses ( For example, reovirus, orbivirus, rotavirus), bimavirus, hepadnavirus (hepatitis B virus), parvovirus (parvovirus), papovavirus (papillomavirus, polyomavirus), adeno Virus (adenovirus), herpes virus (for example,
Herpes simplex virus (HSV) I and II, varicella-zoster virus, varicella virus)
And iridoviruses (eg, African swine fever virus) and unclassified viruses (eg, causative factors of spongiform encephalopathy, hepatitis delta, non-A, non-B hepatitis factors (class 1)
: Intestinal infections; class 2: oral infections such as hepatitis C), norwalk and related viruses and astroviruses), but are not limited thereto.

[0089] Examples of fungi include Aspergillus spp., Coccidioides imimitis, Cryptococcus neoformans, Candida albicans and other Candida spp., Blastomisesis, Histtoplasma capsulats, Chlamydia trachomatis, Nocardia spp., And Pneumocystis carini.

[0090] Examples of parasites are blood infectious and / or tissue parasites such as Babes
ia microti, Babesi divergans, dysentery amoeba, rumble flagellate, tropical leishmania, leishmania species, brazilian leishmania, donovan leishmania, tropical malaria protozoa, plasmodium malaria parasite, oval malaria parasite, plasmodium malaria parasite, Toxoplasma gondii, Gambia trypanosome and rhodosia trypanosoma (African sleeping sickness), Crux trypanosoma (Chaga disease) and Toxoplasma gondii, worms, and roundworms are included, but are not limited thereto.

[0091] As described above, the present invention relates to a layered conjugate or protein of interest or a DNA complex containing or encoding this synergistic conjugate in the treatment of proliferative diseases such as cancer. Includes use. Cancer is a state of unrestricted growth of cells that interferes with the normal functioning of body organs and systems. A subject with cancer is a subject that can objectively measure cancer cells present in the body. A subject at risk of developing cancer is a subject who is prone to develop cancer, for example, based on family, hereditary predisposition, exposure to radiation or other factors that cause cancer. Cancers that have moved from their original location and disseminated to vital organs are affected by the deterioration of the function of the affected organs.
Eventually the subject may be killed. Hematopoietic cancers, such as leukemia, can compete with normal hematopoietic cells in a subject, resulting in hematopoietic disorders (in the form of anemia, thrombocytopenia, and neutropenia) and ultimately death.

[0092] Metastasis is a region of cancer cells different from the location of the primary tumor, and is caused by the spread of cancer cells from the primary tumor to other body parts. The subject may be monitored for the presence or absence of metastases at the time of diagnosis of the primary mass. In many cases, in addition to monitoring specific symptoms, magnetic resonance imaging (MR)
I), tomographic tomography (CT), scans, blood and platelet counts, liver function tests, chest x-rays and bone scans are used alone or in combination to detect metastases.

[0093] Risk of developing various cancers or cancers using the compositions, protein conjugates and DNA vaccines of the present invention, including tumor associated antigen (TAA) inclusion or DNA encoding CD40 expression and non-expressing cancers A subject can be treated. This is an antigen expressed in tumor cells. Examples of such cancers are breast cancer, prostate cancer, lung cancer, ovarian cancer, cervical cancer, skin cancer, melanoma, colon cancer, stomach cancer, liver cancer, esophageal cancer, kidney cancer, pharyngeal cancer,
Includes cancers of the blood such as thyroid cancer, pancreatic cancer, testicular cancer, brain cancer, osteosarcoma and leukemia, chronic lymphocytic leukemia. The vaccination method of the present invention can be used to treat a tumor by stimulating an immune response, inhibiting or slowing the growth of the tumor, or reducing the size of the tumor. A tumor-associated antigen may, but need not be, an antigen that is primarily expressed by tumor cells.

[0094] Additional cancers include basal cell cancer, bile duct cancer, bladder cancer, osteosarcoma, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, cancer of the digestive system Endometrial adenocarcinoma, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, intraepithelial neoplasia, kidney cancer, pharyngeal cancer, liver cancer,
Lymphoma, including lung cancer (small and large cells), Hodgkin lymphoma and non-Hodgkin lymphoma; melanoma; neuroblastoma; oral cancer (eg, lips, lower, mouth and pharynx); cervical cancer; pancreatic cancer; retinoblastoma Rhabdomyosarcoma; anal cancer; respiratory cancer; sarcoma; skin cancer; gastric cancer; testicular cancer; thyroid cancer; uterine cancer; urinary cancer; and other carcinomas and sarcomas; .

[0095] The compositions, protein complexes, and DNA of the present invention can also be used to treat autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, type I diabetes, psoriasis or other autoimmune diseases. . Other autoimmune diseases that may be treated using the vaccines and immune adjuvants of the invention include other inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, systemic lupus erythematosus (SLE), self Immunoencephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis, Goodpasture syndrome, pemphigus, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibody , Mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, autoimmune-related infertility, glomerulonephritis (eg, crescent-shaped glomerulonephritis, proliferative glomerulonephritis), bullous pemphigoid , Sjogren's syndrome, psoriatic arthritis, insulin resistance, autoimmune diabetes (type I diabetes; insulin-dependent diabetes), autoimmune hepatitis, autoimmune hemophilia, autoimmune lymph Proliferative syndrome (ALPS), autoimmune uveoretinitis, and Guillain-Bare syndrome. Recently, arteriosclerosis and Alzheimer's disease have been recognized as autoimmune diseases. Thus, in this embodiment of the invention, the antigen is a self antigen that elicits an undesirable immune response in which host cells contribute to tissue disruption and normal tissue destruction.

[0096] The compositions, protein conjugates and DNA vaccines of the present invention can also be used to treat asthma and allergic and inflammatory diseases. Asthma is a respiratory disease characterized by inflammation and stenosis of the airways and increased airway responsiveness to inhaled factors. Although not always so, asthma is often associated with atopic or allergic symptoms. Allergy is acquired hypersensitivity to a substance (allergen). Allergic symptoms include eczema, allergic rhinitis or nasal cold, hay fever, bronchial asthma, hives,
And food allergies and other atopic symptoms. Allergens are substances that can induce an allergic or asthmatic response in susceptible subjects. There are many allergens including pollen, insect venom, animal dander, dust, fungal spores, and drugs.

[0097] Examples of natural and plant allergens include dogs, ticks, Libyan cats,
Ambrosia, Lotium, Cedar, Alternaria, Alder, Alder, Birch, Quercus, Olive, Artemisia, Papaver, Licorice, German cockroach, Honeybee, Cypress, Juniper, Cypress, Cypress, Cypress, Cockroach, Shibamugi, Rye,
Wheat, camouflage, oxenogusa, strawberry jumpy, oat, shiragaya, hargaya,
It contains proteins specific to Ribbon Gaya, Konukagusa, Akagaeri, Kusuyoshi, Vulgaris, Sorghum, and Bromis.

[0098] It will be appreciated that the compositions, protein conjugates, and DNA vaccines of the present invention can be used in conjunction with other therapies to treat specific conditions such as, for example, infectious diseases, cancers or autoimmune diseases. For example, in the case of cancer, the method of the present invention may be used in combination with chemotherapy or radiation therapy.

[0099] Methods for producing compositions as vaccines are well known to those skilled in the art. An effective amount of protein complex or DNA can be identified empirically,
It may be based on an immunologically effective amount in animal models. Factors to consider include antigenicity, dosage form, route of administration, number of immunizing doses administered, individual physical condition, weight, age and the like. Such elements are well known to those skilled in the art and are determined by one skilled in the art (eg, Pauletti and McLnes. Eds., Vaccines, fr.
om Concept to Clinic: A Guide to the Device
lopment and Clinical Testing of Vaccines
for Human Use CRC Press (1999)). As disclosed herein, it will be appreciated that the subject DNA or protein complex can be administered alone or in combination with other adjuvants. Furthermore, the adjuvant of the subject can be added to or combined with an existing vaccine to enhance the effect. For example,
These adjuvants may be used to enhance the effectiveness of viral vaccines such as HPV vaccines recently approved for cervical cancer. They may also be combined with other adjuvants.

[00100] The DNA and protein complexes of the present invention may be any method known in the art, including intramuscular, intravenous, transdermal, subcutaneous, intraperitoneal, intranasal, oral or other mucosal routes. And can be administered locally or systemically. Additional routes include intracranial (eg, intracapsular or intraventricular), intraorbital, transocular, intracapsular, intrathecal, and topical administration. The adjuvants and vaccine compositions of the invention are administered in a suitable non-toxic drug carrier,
Alternatively, it can be formed into microcapsules or sustained release implants. The immunogenic composition of the present invention can be administered multiple times as needed to maintain the desired cellular immune response. Appropriate routes, dosage forms, and immunization schedules can be determined by one skilled in the art.

[00101] In the methods of the invention, in some examples, antigen and type I IFN / CD4
The zero agonist complex may be administered separately or may be administered in the same dosage form. In some instances it may be useful to include several antigens. These compositions may be administered individually or in combination in any order that provides the desired enhanced synergistic cellular immunity. In general, each of these compositions is administered within a short period of time. That is, administered within a few days or hours, most commonly within about half a day to 1 hour, to facilitate the treatment regimen.

[00102] In some instances, it may be beneficial to include the moiety in a complex or DNA that facilitates affinity purification. Such moieties include relatively small molecules that do not interfere with the function of the polypeptide in the complex. Alternatively, the tag may be removable by cleavage. Examples of such tags include polyhistidine tags, hemagglutinin tags, maltase binding proteins, lectins, glutathione-S transferase, avidin and the like. Other suitable affinity tags include FLAG, green fluorescent protein (GFP), myc, and the like.

[00103] The subject adjuvant conjugate and protein or DNA complex are administered using a physiologically acceptable carrier such as saline. The composition may also include other carriers or excipients such as buffers such as citrate, phosphate, acetate, and bicarbonate,
Amino acids, urea, alcohol, ascorbic acid, phospholipids, proteins, serum albumin, ethylenediaminetetraacetic acid, sodium chloride, or other salts, liposomes, mannitol, sorbitol, glycerol and the like may be included. The factors of the present invention can be formed in various ways according to the corresponding route of administration. For example, a liquid formulation can be formed for ingestion or injection gel, or a procedure can be configured for ingestion, inhalation, or topical application.
Methods for forming such formulations are well known, eg “Remington's
Pharmaceutical Sciences, “18th Ed., Mack”
It can be found in the Publishing Company, Easton Pa.

[00104] As noted above, the present invention encompasses DNA-based vaccines. These DNAs may be administered as naked DNA or included in an expression vector. Further, prior to graft implantation, the subject nucleic acid sequences may be introduced into the cells of the graft.
Preferably, the DNA is humanized to promote expression in a human subject.

[00105] The polypeptide complex of interest may further comprise a “marker” or “reporter”. Examples of marker or reporter molecules include beta-lactamase, chloramphenicol acetyltransferase, adenosine deaminase, aminoglycoside phosphotransferase, dihydrofolate reductase, hygromycin B-phosphotransferase, thymidine kinase, lacZ, and xanthine guanine phosphoribosyltransferase, etc. .

[00106] The subject nucleic acid construct can include any vector capable of inducing its expression, eg, a cell transformed with the vector. The inventor is experienced here using baculovirus vectors, and therefore this vector is illustrated here. Other vectors that may be used are T7-based vectors used in bacteria, yeast expression vectors,
Including mammalian expression vectors, viral expression vectors and the like. Viral vectors include retroviruses, adenoviruses, adeno-related vectors, herpes viruses, simian viruses 40
And bovine papillomavirus vectors.

[00107] Prokaryotic cells and eukaryotic cells that can be used to promote expression of the subject polypeptide complex include, for example, microorganisms, plant and animal cells, e.g., prokaryotic organisms such as Escherichia coli and Bacillus subtilis, and insect cells such as Sf21 cells. , Saccharomyces, Candida, Criveromyces, Schizosaccharomyces, and Pichia and other yeasts, and COS, HEK293, CHO, BH
Including mammalian cells such as K, NIH3T3, and HeLa. Those skilled in the art can readily select appropriate components for a particular expression system, including expression vectors, promoters, selectable markers, etc. appropriate for the desired cell or organism. Selection and use of various expression systems is described, for example, in Ausubel et al. , “Current Protocols
in Molecular Biology, John Wiley and Son
s, New York, N .; Y. (1993); and Pouwels et al. ,
Cloning Vectors: A Laboratory Manual ":, 19
85 Suppl. 1987). Also provided are eukaryotic cells that contain and express the DNA structure of interest.

[00108] In the case of cell transplantation, the cells can be administered using either a transplant procedure or a catheter-mediated injection procedure through the vessel wall. In some cases, cells may be administered by release into the vasculature. Cells are then distributed from this vasculature into the blood stream and / or migrate to surrounding tissue.

[00109] The subject polypeptide complex or DNA construct is CD40 and CD4.
An agonist anti-CD40 antibody that specifically binds or agonizes the binding of 0L, preferably mouse or human CD40, or CD40L or a fragment thereof,
Or code. As used herein, the term “antibody” is used in its broadest sense and includes polyclonal and monoclonal antibodies, and antigen-binding fragments thereof. This includes, for example, Fab, F (ab ′) 2, Fd and Fv fragments.

[00110] Furthermore, the term "antibody" includes naturally occurring and non-naturally occurring antibodies, such as single chain antibodies, chimeric antibodies, bifunctional and humanized antibodies. Chimeric, humanized, and fully human antibodies are suitable for use in the present invention. Chimera, humanization, CD
Methods for synthesizing R-graft, single chain and bifunctional antibodies are well known to those skilled in the art.
In addition, agonist antibodies specific for CD40 are widely known and available and are formed by immunization of suitable host cells with a CD40 antigen, preferably human CD40.

[00111] The use of anti-mouse CD40 antibody (FGK45) is demonstrated in the examples. This antibody was selected because the anti-human CD40 antibody did not specifically bind mouse CD40 and in vivo studies were rodent. For human therapy, the selected agonist CD40 antibody binds specifically to human CD40. Agonist CD40 antibodies specific for human CD40 are also known in the art and may be generated by well-known methods. As an alternative, CD4
0 agonists CD40L agonize human CD40 and CD40L interactions
Or a fusion protein containing the same.

[00112] As noted above, the synergistic conjugates of the invention comprise at least one type I interferon or fragment or variant thereof that interacts with a CD40 agonist, induces CD70 expression on CD8 + DC, and in vivo CD8 + T Derives a strong increase in cells. This includes functional variants and fragments thereof, along with, for example, alpha interferon, beta interferon, omega interferon, tao interferon, zeta interferon, and epsilon interferon.

[00113] Of course, modifications that do not significantly affect the activity of the various embodiments of the present invention are also provided within the definition of the invention provided herein.
Inventor's Theory [00114] As mentioned above, all TLR agonists tested to date interact with anti-CD40 and induce CD8 + T cell immunity. However, some TLR agonist / anti-CD40 conjugates (in the case of TLR3, 7, 9) rely heavily on type I interferon (IFNαβ) to enhance the increase in CD8 + T cells, but other TLR / CD4
It was observed that 0 agonist conjugates (in the case of TLR2 and 5) were not. Surprisingly, due to the deletion of CD4 cells, the TLR3 or 7 / CD40 agonist conjugate is CD8.
Eliminates the IFNαβ requirement for generation of + T cell responses. Collectively, these data represent IFNs in the control of CD8 + T cell responses following combined TLR / CD40 agonist immunization.
The role of αβ and CD4 cells is suggested to the inventors.

[00115] Based on these observations, the inventors have determined that TNF ligand induction on DCs is dependent or independent of IFNαβ and that this is CD8 against IFNαβ.
It was hypothesized to determine the post-dependency of the + T cell response. Since IFNαβ-dependent CD8 + T cell responses can be restored by CD4 deletion, it was hypothesized that either CD70 expression on DCs or CD8 + T cell responses were negatively affected by regulatory T cells. As a result, IFNα
β is bound TLR (3, 7, or 9) / CD40 agonist immunization followed by i) CD
Directly increases the CD8 + T cell response to 70-loaded APC (CD8 T cell center), i
i) Direct activation of DC for TNF ligand expression (DC center), iii) APC T
Proposed mechanisms that affect CD8 + T cell responses by performing one or more of the functions of inhibiting CD4 + T cell activity (Treg center) against NF ligand expression or CD8 + T cell increase. The interaction with anti-CD40 in inducing CD8 + T cell expansion is a property of all TLR agonists tested, including TLR1 / 2, 2/6, 3, 4, 5, 7, and 9 agonists. Collectively these data are combined TLR / C
FIG. 5 shows that D40 agonist immunization can reconstitute all signals necessary to elicit a strong primary CD8 + T cell response.

[00116] To identify cellular and molecular requirements for the interaction of TLR and CD40, many experiments have been performed in knockouts and / or mice in which various cell types or factors have been deleted by blocking or deletion with antibodies. went. These studies confirmed the need for normal CD40 and TRL signaling pathways (using CD40 KO and MyD88 KO mice). This interaction is independent of CD4 cells, but the observed IFNγ, IL-12, or IL-23 depends on the TLR agonist used,
It was variable dependent on the interaction with Nαβ. Ahonen, C.I. L. , C.I. L. Doxse
e, S.M. M.M. McGurran, T .; R. Ritter, W.M. F. Wade, R.W. J. et al. Ba
rth, J .; P. Vasilakos, R.A. J. et al. Noelle, and R.C. M.M. Ked
l. 2004. Combined TLR and CD40 triggering
inductors point CD8 + T cell expansion with
variable dependency on type I IFN. J Exp
Med 199: 775. It was observed that the degree of dependence on IFNαβ generally seems to be related to the amount of IFNαβ induced by a given TLR. Therefore, TLR3, 7
IFNαβ receptor knockout (IFNαβRKO) mice immunized with anti-CD40 in combination with 9 or 9 agonists failed to generate a CD8 + T cell response. vice versa,
IFNαβRKO mice immunized with anti-CD40 in combination with agonists of TLR2 or 5 produced CD8 + T cell responses. These data suggested to the inventors that IFNαβ may play a much more important role in generating adaptive immunity than already understood, as shown in the examples below.

[00117] First, it should be emphasized that it is difficult to predict and clarify the exact role of IFNαβ in generating T cell responses. Part of the reason is that many of the effects of IFNαβ on T cell function appear to be indirect. IFNa
β enhances many aspects of APC activity, including elevation of MHC molecules for major cell types. Tough, D.C. F. 2004. Type I interferon as a
link between innate and adaptive immunity
y through dendritic cell simulation. Leu
k Lymphoma 45: 257; Le Bon, A.M. , And D.D. F. Toug
h. 2002. Links between innate and adaptive
immunity via type I interferon. Curr Opi
n Immunol 14: 432. Recently, IFNαβ has been shown to advance the APC process of exogenous antigens into a process known as the class I pathway, cross-priming. L
e Bon, A.M. , N.M. Etchart, C.I. Rossmann, M.M. Ashton, S
. Hou, D .; Gewert, P.A. Borrow, and D.B. F. Tough. 200
3. Cross-priming of CD8 + T cells stimulated
d by virus-induced type I interferon. Nat
Immunol 4: 1009. This allows C after administration of exogenous protein antigen.
Allows generation of D8 + T cell responses. IFNαβ also has other effects on T cell activation and proliferation. High levels of IFNαβ are also partially activated and memory C naive
Induces proliferation of D8 T cells. Tough, D.C. F. S. Sun, X. et al. Zhang, a
nd J.M. Srent. 1999. Stimulation of naive an
d memory T cells by cytokines. Immunol Re
v 170: 39'Sprent, J.M. , X. Zhang, S.M. Sun, and D.D. T
ough. 2000. T-cell propagation in vivo a
nd the role of cytokines. Philos Trans R
Soc London B Biol Sci 355: 317; 200
3. Turnover of memory-phenotype CD8 + T cel
ls. Microbes Infect 5: 227; Zhang, X. et al. S. Sun,
I. Hwang, D.H. F. Tough, and J.M. Srent. 1998. Pote
nt and selective simulation of memory-p
henotype CD8 + T cells in vivo by IL-15. Im
community 8: 591; Tough, D .; F. , And J.M. Srent. 199
8). Bystander simulation of T cells in vi
vo by cytokines. Vet Immunol immunopathol
63: 123.
[00118] IFNαβ directly stimulates and survives naïve T cells, but the effect of IFNαβ on naive T cells may be mediated in part by APC. Marrac
k, P.I. , J .; Kappler, and T.K. Mitchell. 1999. Type
I interferons keep activated T cells ali
ve. J Exp Med 189: 521; Marrack, P .; , T. Mitche
ll, J. et al. Bender, D.C. Hildeman, R.A. Kedl, K .; Teague, a
nd J.M. Kappler. . 1998. T-cell survival. Immun
ol Rev 165: 279. This survival activity is dependent on STAT1 in T cells, indicating that it must involve direct IFNαβ signaling in T cells. Marack,
P. , J .; Kappler, and T.K. Mitchell. 1999. Type I
interferons keep activated T cells alive
. J Exp Med 189: 521. Recently, IFNα has been shown to act directly on naive CD8 + T cells in response to antigen and B7-mediated costimulation to promote proliferation, effector function and memory progression. Curtsinger, J. et al. M.M.
, J .; O. Valenzuela, P.A. Agarwal, D.A. Lins, and M.M. F
. Mescher. 2005.
[00119] Type I IFN provides a third signal to CD8 T cells to stimulate clonal expansion and differentiation. J Immunol 174: 4465. Conversely, there is the theory that the effect of IFNαβ on the proliferation of CD8 + memory T cells is indirect. This proliferation is
It arises from the production of IL-15 from other cell types and selectively induces the proliferation of memory CD8 but not CD4 T cells. Zhang, X. et al. S. Sun, I. et al. Hwang, D.H. F. T
ough, and J.M. Srent. 1998. Potent and select
eve stimulation of memory-phenotype CD8 +
T cells in vivo by IL-15. Immunity 8: 591;
Srent, J .; , X. Zhang, S.M. Sun, and D.D. Tough. 1999
. T-cell turnover in vivo and the role of
cytokines. Immunol Lett 65:21. Thus, both indirect and direct effects of IFNαβ on T cells have been observed in T cell activity and initiation of proliferation.

[00120] Conversely, there is little data on the effects of type I IFNs on the control of T cell growth or function. There are reports showing that human regulatory cells can be generated in vitro using conjugates of IFNα and IL-10. Levings, M.M. K. , R. San
gregorio, F.M. Galbiati, S.M. Squadrone, R.A. de Waa
1 Malefyt, and M.M. G. Roncarolo. 2001. IFNα an
d IL-10 induction the differentiation of hu
man type 1 T regulatory cells. J Immunol
166: 5530.
[00121] As noted above and in support of the data in the examples following the discovery of the invention, conjugates of type I interferon and CD40 agonists elicited a synergistic effect on immune cells and CD70 on dendritic cells. By providing an exponential increase in CD8 + T cells, the amount of and nature of cellular immunity elicited by the subject's novel adjuvant conjugate in therapy is considered necessary for the treatment. Enabling the development of more powerful vaccines.

[00122] The following examples are provided for illustrative purposes. However, it will be appreciated that the scope of the invention is defined by the claims.
Materials and methods used in some of the examples below.

[00123] C57BL / 6, IFNαβRKO, or CD4 deleted IFNαβR
KO mice are immunized with model antigen. That is, 0.1-0.5 mg total protein (
Ovalbumin or HSV glycoprotein B [HSVgB]) or 50 μg peptide (SIINFEKL of ovalbumin, SSIFFARRL of HSVgB, TSYKSEFV of vaccinia virus B8R) and TLR agonist (50 μg Pam3Cys, 25
μg MALP-2, 100 μg PolyIC, 150 μg 27609, 50 μg
CpG 1826, or 25 μg flagellin), anti-CD40 antibody FGK45 (50
μg) or in combination with both. Ovalbumin is a Sigma Cor
purchased from Portion (St. Louis, MO) and TritonX as described above
-114 Remove contaminating LPS using LPS detoxification method. Adam, O .; A. V
ercellone, F.M. Paul, P.M. F. Monsan, and G.M. Puzo. 1
995. A nondegradative route for the remove
al of endotoxin from exopolysaccharides.
Anal Biochem 225: 321. Total HSVgB protein was formed by expression in baculovirus and purification on a nickel column as described above, and was obtained from Dr. University of Pennsylvania. Courtesy of Roselyn Eisenberg. Ben
der, F.D. C, J. et al. C. Whitbeck, M .; Ponce de Leon, H.C. L
ou, R.A. J. et al. Eisenberg, and G.C. H. Cohen. 2003. Spec
if association of glycoprotein B with
lipid rafts DURING HERPS SIMPLEX VIRUS
entry. J Virol 77: 9542. The TLR agonist used is purchased (Pam3Cys-InVivogen, MALP-2-Alexis Bioche
micals, PolyIC-Amersham / GE Healthcare, CpG
1826-Invitrogen), provided through material transfer agreement (27609-
3M Pharmaceuticals) or synthesized in-house (flagellin).
For each TLR agonist, LPS contamination was tested by the Limulus assay and found to have less than 5 IU of LPS activity (approximately 50-300 ng) injected in vivo. Injection of this amount of LPS has no observable effect on splenic dendritic cells in vivo (data not shown). For flagellin isolated in-house, the same protocol described above for ovalbumin detoxification was used to remove contaminating LPS.

[00124] These TLR agonists were selected for use in this experiment for two main reasons. First, the major DC subsets in secondary lymphoid tissues are CD8 + and CD11b + DC, both expressing common and unique TLRs. Selected TLR
Agonists are CD8 + DC (poly IC-TLR3), CD11b + DC (27609-
TLR7 and flagellin-TLR5), or both DC subsets (Pam3Cys /
MALP-2, TLR2 stimulation) is stimulated directly. Second, the selected molecule is IFNαβ dependent (Poly IC, 27609) for inducing a CD8 + T cell response in combination with anti-CD40.
, CpG 1826) or independent (Malp-2, Pam3Cys, flagellin)
Represents a TLR agonist.

[00125] The immunization described in CD0012 (FR70), OX40L / CD134 (
RM134L) or 41BBL / CD137L (TKS-1) is administered in combination with or without an antibody. Intraperitoneal administration of 250 μg of antibody every 2 days sufficiently blocks the interaction of each of these ligand / receptor interactions (see FIG. 5). Blocking experiments were performed using this dosing regimen, and subsequent similar experiments were used to identify the minimum amount of blocking antibody required to produce an effect on the CD8 + T cell response, if any.

[00126] To monitor antigen-specific CD8 + T cell responses, 5-7 days after immunization, peripheral blood and / or spleen cells were isolated as described above and H-2 Kb / SIINFEKL
Or stained with H-2Kb / SSIFARRL MHC tetramer. Kedl, R.A. M.M. ,
M.M. Jordan, T .; Potter, J.M. Kappler, P.M. Marrack, an
d S.M. Dow. 2001. CD40 stimulation accelerate
s deletion of tutor specific CD8 (+) T cel
ls in the absence of tumor-antigen vacci
nation. Proc Natl Acad Sci USA 98: 10811; Ke
dl, R.D. M.M. , W .; A. Rees, D.D. A. Hildeman, B.M. Schaefer
, T. Mitchell, J.M. Kappler, and P.K. Marack. 2000
. T cells Compete for Access to Antigen-b
earing Antigen-presenting Cells. J. et al. Exp. Me
d. 192: 1105; Kedl, R .; M.M. , B. C. Schaefer, J. et al. W. Ka
ppler, and p. Marack. 2002. T cells down-mo
duplicate peptide-MHC complexes on APCs in
vivo. 3:27. CD8 + T cells are analyzed by intracellular interferon γ (IC IFNγ) staining as an indicator of the ability of the cells to produce effector cytokines. IC IF
Nγ staining is widely used in the literature and is performed as described above. Furthermore, CD107a expression after antigen stimulation is analyzed as an index of antigen-specific cell lysis function. CD107a (
LAMP-1) is a membrane protein component of cytolytic granules, and its discrimination on the T cell plasma membrane after antigen stimulation is an indication of exocytosis of cytolytic granules. Bound tetramer and CD107a staining is performed as described above. That is, the cells are cultured for 30 minutes at 37 ° C. using the MHC tetramer. Then antigen peptide (1 μg / ml) and anti-CD107a
-Add FITC antibody for an additional hour, then add 1 μg / ml monensin to cells to suppress disruption of integrated FITC fluorescence as antibody-bound CD107a is absorbed by lysosomes. Cells are further cultured for 3-4 hours at 37 ° C., stained with antibodies against CD8, washed, fixed and analyzed by FACS. As described above, bound TLR2- or -5 / CD4
CD70, 41BBL, OX-40L, and CD30L during immunization with 0 agonist
A blocking antibody against is injected into IFNαβRKO mice as well. The magnitude and function of the CD8 + T cell response is identified by FACS analysis of tetramers, IC IFNγ staining, and PBL and / or spleen cells, as described above.

[00127] To determine the effect of TNF ligand blocking during primary immunization on increase in memory CD8 + T cells, the immunized mice were rested for at least 60 days,
Sensitization again with the same immunization and secondary reactions are analyzed as described above. The experiment was conducted with IFNα
βRKO mice, CD4 deleted IFNαβRKO mice, and normal and CD as controls
Perform in 4 deletion B6 mice. In healthy IFNαβRKO mice, IFN
TLR / CD40 conjugates that generate αβ-independent CD8 + T cell responses are analyzed. CD4
In deletion IFNαβRKO mice, IFNαβ-dependent and independent TLR / CD
Both conjugates are tested. Representative CD4 deficient and immunized mice are allowed to rest for at least 60 days after primary immunization and then sensitized again by combined TLR / CD40 agonist immunization. Using these experiments, primary and memory CD8 + T cell responses following immunization of IFNαβ-deficient, CD4 deficient or non-deleted host cells are
Determine if it depends on 0 and / or other TNF ligands.

Example 1:
The increase in CD8 + T cells following conjugated TLR / CD40 agonist immunization indicates a variable dependence on IFNαβ.

[00128] All TLR agonists interact with anti-CD40 and CD8 + T cells
Although the present inventor has derived from certain TLR agonist / anti-CD40 conjugates
It was noticed that the CD8 + T cell response made was completely dependent on IFNαβ. Based on it
In the experiment contained in FIGS. 1 and 2 as described above, the inventor
Interferon αβ receptor knockout (IFN) related to R / CD40 agonist
αβRKO) mice were immunized with peptide antigens.

[00129] In the experiment included in FIG. 1, the ovalbumin peptide, anti-CD40,
And the increase in CD8 + T cells following administration of the bound TLR / CD40 agonist was measured in an agonized mouse (bottom row) immunized with the indicated TLR agonist. Seven days later, ovalbumin-specific T cell responses in the spleen were measured by tetramer staining and FACS analysis. The numbers in the upper right quadrant indicate the percentage of tetramer stained cells in the total CD8 + cells.

[00130] The experiments included in FIG. 2 reveal that CD4 deletion of IFNαβRKO host cells restores CD8 + T cell responses following immunization with an IFNαβ-dependent TLR agonist in combination with agonist anti-CD40. It was. WT and IFNαβR
KO mice, CD4 deficient or non-deficient mice as shown in FIG. 2, were immunized with HSV-1 peptide, agonist anti-CD40 antibody, and poly IC. 7 days later, HSV
-1 specific responses were identified by tetramer (A) and IC IFNγ (B) stained PBL cells.

[00131] As can be seen from the results contained in FIG. 2, in these mice, TL
The CD8 + T cell response to immunization combined with R3, 7, or 9 agonist and anti-CD40 was completely abolished (FIG. 2). Conversely, is the CD8 + T cell response to the remaining TLR / CD40 agonist conjugate dependent only in part on IFNαβ (TLR4 / CD40)?
Or it was relatively independent (TLR2 / 6 / CD40 agonist) (FIG. 1). In other experiments, the TLR1 / 2 agonist Pam3Cys and the TLR5 agonist flagellin also generated a CD8 + T cell response corresponding to wt mice when combined with anti-CD40 in IFNαβRKO (data not shown). These results show that anti-CD40
When combined with a TLR2 or 5 agonist, an IFNαβ-independent CD8 + T cell response is elicited, but when combined with a TLR3, 7, or 9 agonist, an IFNαβ-dependent C
Deriving a D8 + T cell response. For this reason, TLR2 or 5 agonists and CD
Interaction with the 40 pathway is thought to be independent of IFNαβ. Conversely, TLR3, 7
Alternatively, the interaction between the 9 agonist and the CD40 pathway is believed to depend on IFNαβ. This data suggested to the inventor the role of IFNαβ in generating CD8 + T cell responses by signaling directly through T cells, antigen-containing APC, or both.

[00132] Example 2
The increase in CD8 + T cells following combined TLR / CD40 agonist immunization is restored in CD4 deficient IFNαβRKO host cells.

[00133] CD8 + T cell dysfunction in IFNαβRKO mice appeared to suggest to the inventor a mandatory role for IFNαβ in the responses elicited by the specific TLR / CD40 agonist conjugates described above. As shown in the experiment of FIG. 2, Wt and IFNαβRKO mice lacked CD4 + T cells by injecting anti-CD4 antibody GK1.5 one day prior to peptide immunization with a combined TLR / CD40 agonist ( Figure 2). Seven days after conjugated TLR / CD40 agonist immunization, mice were sacrificed,
PBL and spleen cells were isolated and analyzed by tetramer and intracellular IFNγ staining. Immunization with peptide and poly IC / anti-CD40 failed to generate a CD8 + T cell response in IFNαβRKO mice. However, CD4 deletion restored CD8 + T cell responses, both in terms of the number of antigen-specific T cells in IFNαβRKO mice (percent of total CD8 + T cells, FIG. 2A) and function (FIG. 2B). This is also true for all TLR / CD40 agonist conjugates tested (TLR2, 5, and 7) and IF
CD for Nαβ-independent TLR / CD40 agonist conjugates (ie, TLR2)
Even the 8+ T cell response was enhanced compared to the non-CD4 deletion control (data not shown).
Therefore, the CD8 + T cell response in IFNαβRKO mice following combined TLR / CD40 agonist immunization is generally enhanced after CD4 deletion.

[00134] One of the inventor's concerns regarding these discoveries was whether they were physiologically related or simply unique to IFNαβRKO host cells. Therefore, experiments were performed in wt host cells using polyclonal rabbit anti-IFN antibodies, blocking IFNαβ and not CD4 deletion and deletion.

[00135] As shown in FIG. 3, anti-IFN blocks the poly IC / CD40-mediated CD8 response recovered by CD4 deletion. In this experiment, anti-IFN and / or
Alternatively, CD4 deletion and non-deletion mice were immunized against anti-IFN and / or CD-deleted ovalbumin (conjugated poly IC / anti-CD40). On day 7, antigen-specific T
PBL were analyzed by tetramer staining as described above in the Materials and Methods section for percent cells.

[00136] As shown in FIG. 3, in wt mice immunized with conjugated poly IC / αCD40, anti-IFNαβ antibody significantly reduced the extent of CD8 + T cell responses (FIG. 3).
Consistent with the results seen in IFNαβRKO mice, CD4 deletion in anti-IFN treated mice completely restored CD8 + T cell responses. Therefore, in both IFNαβRKO host cells and wt host cells injected with IFNαβ-deficient antibody, deletion of CD4 is associated with bound T
It appeared to reduce the dependence of the CD8 + T cell response on IFNαβ following LR / CD40 agonist immunization. These results show that: 1) CD4 + T cell subpopulations of C of IFNαβRKO mice following immunization with specific TLR / CD40 agonist conjugates.
Controls the D8 + T cell response 2) IFNαβ may serve to inhibit the regulatory ability of this CD4 + T cell population after immunization with these TLR / CD40 agonist conjugates, and 3) Other TLRs / CD40 agonist conjugates (eg, TLR2
Or 5) suggested to the inventors that inhibition by regulatory CD4 + T cells could be avoided in an IFNαβ-independent manner.

[00137] These results show that combined TLR / CD40 agonist immunization can elicit strong primary and secondary CD8 + T cell responses that show variable dependence of interest on IFNαβ depending on the TLR agonist used. These findings suggested to the inventor a role for IFNαβ in the direct CD8 + T cell response than previously elucidated. It has also been shown that combined TLR / CD40 agonist immunization inherently induces CD70 upregulation on DCs, and the reliable CD8 + T cell response in WT mice appears to be highly dependent on this. This preliminary data relates to enhanced CD70 expression on activated APCs and subsequent stimulation of antigen-specific T cells via CD27 is related to the formation and survival of CD8 + T cell responses in response to conjugated TLR / CD40 agonist immunization. Suggested to be a major checkpoint. However, more surprisingly, IFNαβRKO (FIG. 2
IFNαβ-dependent CD8 + T cell responses in both) and WT mice (FIG. 3)
It is the inventor's observation that rescue can be achieved by deleting host cells of D4 + T cells. These results suggest that IFNαβ may affect CD8 + T cell responses for the following reasons. 1) control CD8 + T cell responses to TNFL-expressing APCs,
2) Control APC activity and TNF ligand expression 3) Inhibit CD4 + T cell regulatory function that suppresses ANF expression of TNF ligand or CD8 + T cell increase against TNFL-bearing APC 4) Any combination described above. The following examples are all combined TLRs
I) IFNα in mediating the CD8 + T cell response following / CD40 agonist immunization
The accuracy of these hypotheses is ultimately determined by systematically examining the role of β, ii) the role of IFNαβ in DC activation, and iii) the role of IFNαβ in CD4 + regulatory cell function.

[00138] Example 3
Role of TNF ligand on CD8 + response in IFNαβRKO [00139] As shown in the experiment of FIG. 4, the CD8 + T cell response in WT mice generated by combined TLR / CD40 agonist immunization is dependent on CD70 (see FIG. 4) ). In this experiment, in a CD4 deficient IFNαβRKO host cell, the bound T
CD8 + T cell responses following LR / CD40 immunization were evaluated. From the results, it was found that it was highly dependent on CD70. IFNαβRKO mice lacked CD4 cells and were immunized with HSV-1 peptide, poly IC, and anti-CD40 antibody as described above. Next, mice were injected with anti-TNF ligand antibody as shown in FIG. On day 7, PBL was analyzed again by tetramer staining.

[00140] As evident from the above experiments, CD in IFNαβRKO mice
The 8+ T cell response is unique in that it can only be elicited by TLR / CD40 agonist conjugates that do not stimulate IFNαβ or IFNαβRKO host cells lacking CD4 prior to TLR / CD40 agonist immunization. The result of FIG. 4 shows that CD70 is IFNαβR.
It is further shown that it plays a necessary role in the CD8 + T cell response of KO mice. In this experiment, anti-CD70 blocked the reaction approximately 10-fold, whereas other TNFL antibodies
Note that the D8 + T cell response was inhibited up to 2-fold. This is a wt mouse (Figure 1)
On the contrary, it is suggested that multiple TNF ligands may have at least some effect on the extent of CD8 + T cell response in IFNαβRKO mice. FIG.
The data shown in was obtained by injection of a minimal blocking antibody.

[00141] Example 4
Materials and methods.
[00142] Injection of soluble CD70 / lg fusion protein (sCD70lg)
Southampton General Hospital Dr. Aymen Al-
As originally described by Shamkhani (Rowley, TF, and
A. Al-Shamkhani. 2004. Stimulation by solu
ble CD70 promotes strong primary and sec
ondary CD8 + cytotoxic T cells responses
in vivo. J Immunol 172: 6039), via CD27 in vivo
Provides good agonist stimulation to T cells. Dr. This reagent, kindly provided by Al-Shamkhani, is injected into IFNαβRKO host cells in combination with TLR and CD40 stimulation. First, we attempt to rescue the CD8 + T cell response to the IFNαβ-dependent TLR / CD40 agonist conjugate by boosting with sCD70lg reagent. Seven days after initial antigen sensitization, CD8 + T cell responses are analyzed again. Dr. Al-Shamkha
Data obtained from ni laboratories show that daily injections of 250 μg sCD70 lg on days 2-4 after antigen sensitization provide an optimal CD70-mediated signal for CD8 + T cell expansion ( Personal communication). The inventor has shown that the time course of this CD70lg injection is W
It was confirmed that CD8 + T cell responses to TLR agonist alone were increased in Tuus (data not shown). Mice are sensitized intraperitoneally on day zero with antigen and TLR agonist, anti-CD40, or both. On days 2, 3, and 4 after antigen injection, 25
After intraperitoneal injection of 0 μg sCD70 lg, CD8 + T cell responses in blood and / or spleen were analyzed 7 days after the original antigen sensitization.

[00143] From the data shown in FIG. 4, it is clear that IFNαβRKO host cells lacking CD4 become responsive to any combination of TLR / CD40 stimulation. As shown in FIG. 4, the blockade of CD70 is a T8 to induce CD8 + T cell responses.
Eliminate the interaction between LR agonist and CD40 agonist. In this experiment, anti-C
Mice were sensitized with the indicated conjugate of D40 + / TLR-agonist. A representative subset of mice was injected with anti-CD70 blocking antibody FR70 (lower dot plot). FIG.
A shows representative tetramer staining, FIG. 4B shows the mean and standard deviation of 3 mice per group, and FIG. 4C shows mice immunized but not given anti-CD70 as in 5A ,
The case of one injection or two injections is shown. DCs were separated after 24 hours and the number of DCs in each subset (upper panel) and CD70 staining (lower panel) were analyzed.

[00144] In WT mice, the CD8 + T cell response following immunization with combined TLR / CD40 agonist is found to be dependent on CD70 (FIG. 4). The data described above and shown in FIG. 4 suggests that this also applies to IFNαβRKO host cells that are at least CD4 deleted. The results in FIG. 4 also suggest that multiple TNF ligands may be involved to some extent in the CD8 + T cell response of IFNαβRKO host cells.

[00145] Example 5
[00146] Immune cell response following recombinant IFNα +/− anti-CD40 in wt mice.

[00147] Experiments were performed using the following materials and methods to determine IFNαβ-dependent TLRs
After immunization with the / CD40 agonist conjugate, it is determined whether the action of IFNαβ alone sufficiently leads to an increase in CD8 + T cells.
Materials and Methods [00148] Briefly, novel IFNα sequences were cloned from Poly IC stimulated B cell cDNA. Of the induced subtypes, this IFNα subtype was chosen because it lacks the glycosylation sequence and can therefore be expressed in insect cells and there is no concern for abnormal glycosylation. A TCR Cα epitope tag was added to the C-terminus for affinity purification purposes and the sequence was cloned into the p10 promoter site of the pBac vector (Invitrogen).
Recombinant baculovirus was generated and after infection of Hi5 cells, recombinant IFNα was purified from the supernatant by affinity and size chromatography. IFNα activity was confirmed in vitro and in vivo based on upregulation of class I MHC on APC (data not shown).

[00149] The use of recombinant IFNα in a vaccine environment has been published (Le Bon, A., and DF Tough. 2002. Links betw.
een innate and adaptive immunity via type
e I interferon. Curr Opin Immunol 14: 432)
Also, similar protocols are first used in the studies proposed here. As described above, antigen and anti-CD40 are combined with 104-106 units of IFNα to prepare wild type mice. The resulting CD8 + T cell response is then expressed as a bound TLR (3, 7, or 9
) / IFNα and anti-CD40 are TL compared to mice immunized with CD40 agonist
It is determined whether it interacts to the same extent as R stimulation and leads to an increase in CD8 + T cells. Other control mice are injected with IFNα or anti-CD40 only. CD8 + T cell responses are analyzed as described above.

[00150] As can be seen from the experiment in FIG. 5, the data obtained showed a synergistic effect on immunization with recombinant IFNα and anti-CD40. 1x105 unit IFN 3 times, 1x106 unit IFN once, anti-CD40 alone, or anti-CD40 IF
Mice were immunized with the antigen in the context of injection in combination with either N regimen. IFN or CD40 alone stimulated a detectable CD8 + T cell response, but bound IF
N / CD40 interacted and produced a CD8 + T cell response similar to that seen in response to poly IC / CD40 immunization (FIG. 5).

[00151] In particular, this experiment shows that combined administration of type I interferon and agonist CD40 antibody induces an exponential increase in antigen-specific CD8 + and T cells compared to either administration alone. To clarify. Ovalbumin and anti-CD40
Mice were injected intraperitoneally with the indicated conjugate of Poly IC, or recombinant IFN. In the case of IFN injection, mice were injected with 1 × 10 5 units of IFN for 3 consecutive days starting from the day of antigen injection, or with 1 × 10 6 units of IFN at the same time as the antigen injection. After 7 days, mice were sacrificed and cells from either peripheral blood or spleen were stained with tetramer to identify the extent of increase in ovalbumin-specific CD8 + T cells. Analyze the cells by FACS,
The indicated data was gated on the CD8 + B220 event. In the figure, 5- (A) is a tetramer-stained dot plate, 5 (B) is the mean and standard deviation of CD8 + cells in blood occupying 2% tetramer and total CD8 + T cells (2 individual mice) It is.

[00152] The data shown in FIG. 5 shows that recombinant type I interferon is TLR /
These results indicate that it interacts with CD40 to the same extent as CD40 stimulation, and these results further indicate that recombinant IFN produced in baculovirus acts in vivo as well. Furthermore, these results indicate that bound IFNα / CD40 stimulation can interact to the same extent as TLR / CD40 stimulation in promoting CD8 + T cell proliferation.

[00153] Example 6
Combined administration of type I interferon and CD40 antibody induces CD70 + expression on DC [00154] Data included in previous experiments suggests that DC and / or CD4 + Tregs response to IFNαβ identifies IFNαβ dependence To do. The present inventor hypothesized that CD70 is involved in the mechanism by which IFNαβ elicits such strong CD8 + T cell immunity in the case of combined IFNα / CD40 agonist immunization. From the results of the previous examples, it can be seen that CD40 agonist and type I interferon in particular elicit a synergistic effect on CD8 + immunity (see FIG. 5). This data shows the net effect of bound IFNα / CD40 stimulation on responding CD8 + T cells but not APC. The following experiment was performed to show that bound IFNα / CD40 stimulation was induced by CD7 on the antigen-containing DC subset.
Whether to induce expression of 0 and / or other TNF ligands.

[00155] iWTB6 mice were prepared using antigen and anti-CD40 as described above using the recombinant IFNα described above in combination with 104-106 units of IFNα. As a control, mice are immunized with anti-CD40 alone, IFNα alone, or conjugated poly IC / anti-CD40 positive controls to increase DC CD70 expression. Representative mice were sacrificed 6-48 hours after adjustment, spleen collagenase digested, DCs stained and analyzed by FACS. TNF ligands CD70, 41 BBL, OX-40L, CD30L, and G
DCs are evaluated for expression of ITRL. The resulting DC expression type is expressed as bound TLR3
, 7, or 9 / CD40 agonist compared to mice immunized with IFNα and anti-CD
Determine if 40 can interact to the same extent as TLR stimulation and derive an increase in CD8 + T cells. Other control mice are injected with IFNα or anti-CD40 only. To determine the effect of IFNα on antigen processing and presentation of various subsets, mice are sensitized with fluorescent antigen in combination with recombinant IFNα V − / − anti-CD40 as described above.
Antigen uptake, antigen presentation, and DC activity and TNFL expression are determined as described above. These experiments show how independent of IFNα and in combination with anti-CD40, antigen presentation, DC
Determine whether it affects TNFL expression and CD8 + T cell proliferation.

[00156] As can be seen from the experiment of FIG. 6, co-administration of type I interferon and agonist CD40 antibody induces CD70 expression on CD8 + T cells in vivo, but not when either is administered alone. In this experiment, mice were injected with anti-CD40 antibody alone, poly IC (positive control), recombinant alpha interferon or anti-CD40 antibody and type I interferon. After 18 hours, splenic DCs were isolated and analyzed for their CD70 expression. The numbers in the upper right quadrant of FIG. 7 indicate the standard fluorescence intensity of CD70 staining. This data shows that, similar to the administration of poly IC / CD40 agonist, CD / IFN is also CD on CD8 + DC.
It is also shown to increase the expression of 70.

[00157] Therefore, the data (FIG. 6) show that the IF8
DCs from mice that showed successful Nα / CD40 immunization and injected with IFNα / anti-CD40 were able to bind TLR / CD40 with respect to antigen uptake, antigen presentation, and / or TNFL upregulation.
Shown is similar to DCs obtained from controls immunized with agonists. In particular, CD70 increases on one or more DC subsets following combined IFNα / αCD40 immunization, but not on sensitization with either stimulus alone.

[00158] Example 7
[00159] Co-administration of increasing amounts of αIFN with or without CD40 agonist antibody [00160] In the experiment of FIG. 7, the above example except that a large amount of type I interferon is used regardless of the presence or absence of agonist CD40 antibody Mice were injected in the same manner. The data in the figure is expressed as the average CD70MFI between two individual mice and error bars represent standard deviation. Similarly from these results, combined administration of type I interferon and CD40 agonist increased CD70 expression on DC in vivo, but did not increase when type I interferon and CD40 agonist were each administered alone. I understood.

[00161] Example 8
[00162] Percentage of antigen-specific T cells in mice immunized with decreasing doses of IFNα and CD40 agonist or anti-CD70 [00163] In the experiment of FIG. 8, anti-CD40 antibody, IFNα and anti-CD40 antibody, poly IC and CD40 antibody Mice were immunized with different decreasing doses as described therein and with alpha interferon and anti-CD40 antibody. From the data shown in the figure, there is an exponential decrease in the number of antigen (ovalbumin) specific T cells at low doses of IFNα and the number of plateau specific cells with IFN / Poly IC and IFNα / CD40 agonists. Can be seen to be almost the same (see FIG. 8). Therefore, FIGS. 6, 7 and 8
These data indicate that IFNα added from outside the body interacts with anti-CD40 and upregulates CD70 expression on DC, resulting in an increase in antigen-specific T cells.

[00164] Example 9
[00165] CD70 expression on DCs obtained from IFNαβRKO mice with a TLR / CD40 agonist conjugate [00166] To demonstrate the results seen in Figures 6 and 7, IFNα related to endogenous IFN was added from outside the body, Experiments were performed in IFNαβ mice as shown in FIG. Experiments are performed as shown. Here, mice were reconstituted normally with metastatic bone marrow (in this case, BM-expressing GFP +/− Bcl-2) (FIG. 9) and immunized 8 weeks after reconstitution and then generated an immune response. (Not shown).

[00167] As shown in the experiment of FIG. 9, sensitization with bound TLR / CD40 agonist administration induces CD70 expression only on DCs expressing the target TLR in IFNαβRKO mice. In this experiment, anti-CD40 antibody alone, or poly IC or Pam3
IFNαβR mice were injected in combination with Cys. Pam3Cys is a TLR2 agonist and Poly IC is a TLR3 agonist. After 24 hours, splenic DCs were isolated and stained for CD70 expression as described above. CD8 + DC expresses TLR2 and TLR3,
CD11b + DC expresses TLR2, not TLR3. These data are IFNα
DCs stimulated directly through both TLR and CD40 in the absence of β signaling
Suggests that only can increase CD70 expression.

[00168] Combined with previous data, this data indicates that increased CD70 expression indicates CD8 +
It further suggests that it is involved in the simultaneous increase of T cells.
[00169] Example 10
[00170] Effect of type I IFN / CD40 conjugate and number of IL-2 / CD40 agonist conjugates on the number of antigen-specific T cells [00171] The experiment of FIG. It was designed to compare the effects of two different cytokines and type I interferons. As mentioned above, the interaction obtained with the IFNα / CD40 agonist conjugate is quite unexpected and may not be seen with other cytokine / CD40 agonist conjugates.

[00172] In this experiment, the effects of type I IFN / CD40 antibody, IL-2 / CD40 antibody, IL-2 alone, IFNα alone, and CD40 agonist alone were compared. From this result shown in FIG. 10, the IL-2 and IFN / CD40 conjugates are antigen specific T
It can be seen that it does not have a similar effect on the percentage of cellular immune cells. Here, ovalbumin (300 mg) is treated with anti-CD40 (50 mg), recombinant IFNα (1 ×
106 U), IL-2 (1 × 106 U), IL-2 and CD40, or the same dose of IFN and CD40 agonist alone were injected into mice. Seven days later, peripheral blood was collected and stained with Kb / ova tetramer to identify the percentage of antigen-specific T cells. The numbers in the dot plot are the percentage of CD8 + T cells in the displayed elliptical gate (tetramer +). The bar graph is the average and standard deviation of 2 mice per injection. The results show that the number of antigen-specific T cells is equivalent to the IL-2 / CD40 conjugate in the same amount of CD.
It is shown that it was much higher in the animals that received the IFN / CD40 conjugate when administered in combination with the 40 agonist and when both cytokines were administered at the same activity level. This further demonstrates that the synergistic effect obtained with the IFN / CD40 agonist conjugate is not expected.

[00173] Example 11
[00174] Effect of IFNα and CD40 agonist on survival time in metastatic melanoma [00175] In this experiment, C57BI / 6 mice were treated with 100,000
0 B16. F10 melanoma cells were administered intravenously. Four days later, mice were administered 100 μg tumor peptide (Delta V), 100 μg anti-CD40 and 1 × 10 6 units alpha interferon. As shown in the figure, mice that received anti-CD40 / IFN conjugates had significantly longer survival times. This data further supports possible applications of the subject adjuvant conjugates in tumor vaccines and cancer therapy.

[00176] Example 12
[00177] Effect of CD40 agonist / IFN alpha conjugate on metastatic lung cancer t [00178] The experiment of Figure 12 shows that the subject CD40 agonist / IFN alpha conjugate protects mice from metastatic lung cancer. In this experiment, on day zero, C57BI
/ 6 mice with 100,000 B16. F10 melanoma cells were administered intravenously. 4
One day later, mice were administered 100 μg tumor peptide, Delta V, 100 μg anti-CD40 antibody, 100 μg S-27609 (TLR7 agonist) and 1 × 10 6 units alpha interferon. Mice were sacrificed 21 days after tumor sensitization, the lungs were removed, and metastatic nodules were counted with a dissecting microscope. Panel A in the figure shows a digital photograph of a representative lung on the day the lung was removed. Panel B shows lung metastasis counts. Here, N = 7-8 mice / group. These results show the protective effect of the CD40 agonist / IFN alpha conjugate compared to other treatments.

[00179] Example 13
[00180] Lung Invasion Analysis from Tumored Lung [00181] The experiment shown in FIG. 13 performed a TIL (Tumor Invasion Lymphocyte) analysis from the tumored lung. On day zero, 100,000 B16. F10 melanoma cells in C5
7BI / 6 mice were administered intravenously. Five days later, as shown in the figure, 100 μg of tumor peptide (Delta V), 100 μg of anti-CD40, and 1 × 10 6 units of interferon alpha were administered to the mice. Mice were sacrificed 20 days after tumor sensitization. The lungs were removed and TIL was separated by Percoll gradient centrifugation. The cells were then analyzed by flow cytometry to determine the relative and absolute numbers of invading CD4 (13A and 13D), CD8 (13B and 13E), and FoxP3 + cells (13C and 13F). In this experiment, N = 4 mice / group.

[00182] Example 14
[00183] Effect of Combined Immunotherapy on CD8 + T Cells Invading the Lung in Tumored Mice [00184] In this experiment included in FIG. 14, on the generation of antigen-specific effector CD8 + T cells that invade the lung of tumored mice The impact of subject combination immunotherapy was analyzed. In this experiment, on day zero, 100,000 B16. F10 melanoma cells
57BI / 6 mice were administered intravenously. Five days later, as shown in the figure, three mice were administered 100 μg tumor peptide (Delta V), 100 μg anti-CD40, and 1 × 10 6 units alpha interferon. On day 20 after tumor sensitization, the mice were sacrificed, the lungs were removed, and TIL was separated again by Percoll gradient centrifugation. Next, 1 μg / ml r
Cells were stimulated with hlL-2 and brefeldin A for 12-18 hours before intracellular cytokine staining. Prior to staining with IFNg, the cells were first treated with antibodies with CD8 and C8.
Labeled D44, then fixed and allowed to penetrate. Positive cells are calculated by subtracting the background observed with an irrelevant (SIINFEKL) peptide control and then CD8
Expressed as positive percentage of CD44 + IFNg + T cells (14A) or absolute number (14B). In this experiment, N = 4 mice / group.

[00185] From the results shown in the figure, it is clear that administration of the subject IFN / CD40 agonist conjugate increases the number of antigen-specific CD8 + T cells. These results further demonstrate the effectiveness of the subject adjuvant conjugates in cancer vaccines and other treatments where such immune enhancement is desired.

[00186] Finally, for further explanation of the present invention, the present application describes FIG. 15 which shows the sequence of a typical agonist antibody used in the examples, and in accordance with the present invention, for example, a DNA construct using a baculovirus expression system. And FIGS. 16 and 17 schematically illustrate suitable methods and materials for producing polypeptide complexes.

[00187] Of course, the present invention is not limited to the embodiments listed above, and its rights are reserved for all modifications included in the exemplary embodiments and the scope of the following claims. .

[00188] Various references to academic journals, patents, and other publications cited herein, including the latest, are incorporated by reference as a full description.

[0031] FIG. 1 shows that the increase in CD8 + T cells following conjugated TLR / CD40 agonist immunization is variably dependent on IFNα / β. WT (top row) and IFNαβRKO (bottom row) were immunized with ovalbumin peptide, anti-CD40 and the indicated TLR agonist. Seven days later, ovalbumin-specific T cell responses in the spleen were measured by tetramer staining and FACS analysis. The number in the upper right quadrant indicates the percentage of tetramer stained cells in the total CD8 + T cells. [0032] FIG. 2 shows that CD4 deleted IFNαβRKO host cells recover CD8 + T cell responses after immunization with an IFNαβ-dependent TLR agonist in combination with anti-CD40. WT and IFNαβRKO mice lacking or not CD40 as shown were immunized with HSV-1 peptide, anti-CD40, and poly IC as described above. Seven days later, HSV-1 specific responses were identified by tetramer (A) and poly IC IFNγ (B) stained PBL. [0033] FIG. 3 is a graph showing the CD8 response mediated by anti-IFN blocked poly IC / CD40 recovered by CD4 deletion. Mice were immunized against ovalbumin (conjugated poly IC / alpha CD40) with or without anti-IFN and / or CD4 deletion. Day 7 PBL of antigen-specific T cells was analyzed by tetramer staining as described above. [0034] FIG. 4 shows that the CD8 + T cell response in CD4 deleted IFNαβRKO host cells following combined TLR / CD40 immunization is mainly dependent on CD70. IFNαβRKO mice lacked CD4 cells and were immunized with HSV-1 peptide, poly IC, and anti-CD40 as described above. As shown in FIG. 6, mice were injected with anti-TNF ligand antibody. Day 7 PBL was analyzed by tetramer staining. [0035] FIG. 5 shows the interaction of IFN and CD40 leading to an exponential increase in CD8 + T cells. Mice were sensitized as described above. Seven days after the first antigen sensitization, PBL was analyzed by tetramer staining. [0036] FIG. 6 includes experimental results for combined administration of type I interferon and agonist antibody, which conjugate induces CD70 expression on CD8 + dendritic cells in vivo but not by any single administration It shows that. Mice were injected with anti-CD40 antibody alone, poly IC, recombinant type I interferon (1X107U) or anti-CD40 + IFN as positive controls. After 18 hours, pancreatic DCs were isolated and analyzed for CD70 expression. The numbers in the upper right quadrant indicate the average fluorescence intensity of CD70 staining. The data shows that CD40 / IFN increases CD70 expression on CD8 + DC as well as CD40 / Poly IC injection. [0037] FIG. 7 includes experiments showing the effect of administration of bound type I interferon and agonist CD40 antibody on CD70 expression on CD8 + DC in vivo. The results are graphs showing that only the immunostimulatory conjugate induces CD70 expression on DC, but not CD40 agonist or IFN alone. [0038] FIG. 8 analyzes the percentage of antigen specificity (Ovalbumin T cells) in mice administered anti-CD40, IFNα, poly IC / CD40, IFNα and anti-CD70 or IFNα / CD40 at various reduced IFN doses. It is a graph including the experiment performed. [0039] FIG. 9 is a graph showing that sensitized conjugated TLR / CD40 agonist induces CD70 only on DCs expressing target TLRs in IFNαβRKO mice, similar to the experiment in FIG. IFNαβRKO mice were injected with anti-CD40 alone (aCD40) or Poly IC (+ Poly IC) or Pam3Cys (+ Pam3Cys). Pam3Cys is a TLR2 agonist and PolyIC is a TLR3 agonist. After 24 hours, pancreatic DC were isolated and stained for CD70 expression as described above. CD8 + DC expressed TLR2 and 3, whereas CD11b + DC expressed TLR2 but not TLR3. This data suggests that in the absence of IFNαβ signaling, only DCs directly stimulated through both TLR and CD40 can increase CD70 expression. [0040] FIG. 10 includes experiments comparing the effect of IL-2 / CD40 agonist conjugates and IFNα / CD40 agonist conjugates on the percentage of antigen-specific (ovalbumin) T cells obtained from PBL. The results contained therein show that IL-2 / CD40 agonist conjugates do not elicit similar synergistic effects on CD8 + T cell immunity as IFNα / CD40 agonist conjugates. [0040] FIG. 10 includes experiments comparing the effect of IL-2 / CD40 agonist conjugates and IFNα / CD40 agonist conjugates on the percentage of antigen-specific (ovalbumin) T cells obtained from PBL. The results contained therein show that IL-2 / CD40 agonist conjugates do not elicit similar synergistic effects on CD8 + T cell immunity as IFNα / CD40 agonist conjugates. [0041] FIG. 11 is a graph containing experiments in which C57BI / 6 mice were injected with melanoma cells, and the IFNα / CD40 agonist conjugate increased survival time in this metastatic melanoma animal model. [0042] The experiment of FIG. 12 shows that subject binding adjuvant treatment of metastatic lung cancer with CD40 agonist and IFNα in the C57BI / 6 animal model reduced the number of metastatic nodules in animals treated with adjuvant conjugates. As can be seen from the figure, mice are protected from metastatic lung cancer. [0042] The experiment of FIG. 12 shows that subject binding adjuvant treatment of metastatic lung cancer with CD40 agonist and IFNα in the C57BI / 6 animal model reduced the number of metastatic nodules in animals treated with adjuvant conjugates. As can be seen from the figure, mice are protected from metastatic lung cancer. [0043] In the experiment of FIG. 13, B16. Treated with the subject adjuvant conjugate and appropriate controls. The figure which performed the TIL analysis in the C57BI / 6 mouse which inoculated F10 melanoma cell. Mice that received the subject adjuvant conjugates were found to have an increased number of TILs, as shown in the data in the figure. [0044] FIG. 14 shows that subject CD40 agonist / IFN conjugate treatment generates antigen-specific effector T cells that invade the lungs of tumor-bearing mice (C57BI / 6 mice inoculated with B16.F10 melanoma cells). It is a figure including the experiment which shows. [0045] FIGS. 15A and 15B are diagrams comprising the light and heavy chain sequences of a typical CD40 agonist antibody (FGK.45) used in the examples. [0045] FIGS. 15A and 15B are diagrams comprising the light and heavy chain sequences of a typical CD40 agonist antibody (FGK.45) used in the examples. [0046] FIG. 16 is a view showing a schematic structure of a DNA structure for expression of a CD40 agonist antibody-antigen-type I IFN complex in a baculovirus expression system according to the present invention. This construct results in the expression of an anti-CD40 antibody linked to a selected antigen (eg, HIV gag) and type I interferon (alpha interferon). [0047] FIG. 17 is a diagram showing a structure for generating a CD40ab antibody type I IFN complex according to the present invention and a structure for generating a vector used for DNA immunization in a baculovirus expression system. is there.

Claims (79)

(I) at least one nucleic acid sequence encoding an agonist of CD40;
(Ii) a nucleic acid sequence encoding an optionally desired antigen;
(Iii) a nucleic acid sequence encoding type I interferon;
A nucleic acid structure comprising
Said sequences (i), (ii) (if present) and (iii) are operatively linked to the same or different transcription regulatory sequences, and said sequences (i), (ii) and (iii) are
A nucleic acid structure which is optionally separated by a linker sequence and / or an IRES. Polypeptide type I interferon is interferon alpha, beta, tao, epsilon,
The nucleic acid structure according to claim 1, wherein the nucleic acid structure is selected from zeta and omega. The nucleic acid structure according to claim 1, wherein the type I interferon is alpha interferon. The nucleic acid structure according to claim 3, wherein the type I interferon is beta interferon. The nucleic acid structure according to claim 1, wherein the CD40 agonist is an anti-CD40 antibody or an agonist CD40 antibody fragment. The nucleic acid structure according to claim 1, wherein the CD40 agonist is CD40L. The nucleic acid structure according to claim 6, wherein the CD40L is human, mouse, rat, or primate CD40L. The nucleic acid structure according to claim 7, wherein the CD40L is a human CD40L or a soluble human CD40L fragment, a soluble CD40L oligomer, or a mutant or complex of CD40L that binds human CD40.   The nucleic acid structure according to claim 5, wherein the antibody is a chimeric antibody.   The nucleic acid structure according to claim 5, wherein the antibody is a humanized antibody.   The nucleic acid structure according to claim 5, wherein the antibody is a human antibody. 6. The nucleic acid structure according to claim 5, wherein the antibody is a single chain immunoglobulin. 6. The nucleic acid structure according to claim 5, wherein the antibody comprises human heavy chain and light chain constant regions. 6. The nucleic acid structure according to claim 5, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. 6. The antibody of claim 5, wherein the antibody is encoded by an immunoglobulin light chain encoding a nucleic acid sequence encoding a nucleic acid sequence and a nucleic acid sequence operably linked to the same promoter. The described nucleic acid structure. 16. The nucleic acid structure according to claim 15, wherein the immunoglobulin light chain and immunoglobulin heavy chain sequences are mediated by IRES. The nucleic acid structure according to claim 1, wherein the antigen sequence (ii) encodes a viral antigen, a bacterial antigen, a fungal antigen, or a parasitic antigen. The nucleic acid structure according to claim 1, wherein the antigen sequence (ii) encodes a human antigen. 19. The nucleic acid structure according to claim 18, wherein the human antigen is a cancer antigen, a self-antigen, or other human antigens whose expression is related or involved in chronic human diseases. The viral antigens include HIV, herpes, papillomavirus, Ebola, picorna, enterovirus, measles virus, mumps virus, avian influenza virus,
Rabies virus, VSV, dengue virus, hepatitis virus, rhinovirus, yellow fever virus, bunga virus, polyoma virus, coronavirus, rubella virus, echovirus, poxvirus, varicella-zoster virus, African swine fever virus, influenza virus, and para 18. Nucleic acid structure according to claim 17, which is specific for a virus selected from the group consisting of influenza viruses. The nucleic acid structure according to claim 3, wherein the alpha interferon is human alpha interferon which may optionally be PEGylated. The bacterial antigen is Salmonella, Escherichia coli, Pseudomonas, Bacillus, Vibrio, Campylobacter, Helicobacter, Erbinia, Borrelia, Perovacter, Clostridium, Celacia, Xanthomonas, Yersinia, Burkholderia, Listeria, Shigella,
The nucleic acid structure according to claim 17, wherein the nucleic acid structure is derived from a bacterium selected from the group consisting of Pasteurella, Enterobacteriaceae, Corynebacterium, and Streptococcus. The nucleic acid structure according to claim 17, wherein the parasitic antigen is derived from a parasite selected from Babesia, Entoameba, Leishmania, Malaria parasite, Trypanosoma, Toxoplasma, Giardia, Plasmodium and Roundworm. . The nucleic acid structure according to claim 17, wherein the fungal antigen is derived from a fungus selected from the group consisting of Aspergillus, Coccidioides, Cryptococca, Candidanocardia, Pneumocystis, and Chlamydia.   The nucleic acid structure according to claim 1, wherein the antigen is a tumor antigen.   26. The nucleic acid structure according to claim 25, wherein the tumor antigen is a lung tumor antigen. The antigen is prostate cancer, pancreatic cancer, brain tumor, lung cancer (small cell or large cell), osteosarcoma, stomach cancer, liver cancer, breast cancer, ovarian cancer, testicular cancer, skin cancer, lymphoma, leukemia, colon cancer, thyroid cancer, The nucleic acid structure according to claim 1, which is a cancer antigen expressed by a human cancer selected from the group consisting of cervical cancer, head and neck cancer, sarcoma, glial cancer, and gallbladder cancer. The nucleic acid structure according to claim 1, wherein the antigen is an autoantigen whose expression is associated with an autoimmune disease.   An expression vector comprising the nucleic acid construct according to claim 1. 30. The expression vector of claim 29, wherein the expression vector is selected from a plasmid, a recombinant virus, and an episomal vector. A recombinant host cell or non-human animal, characterized in that it expresses the nucleic acid structure according to claim 1. 32. Recombinant host cell cell according to claim 31, characterized in that it is selected from bacterial cells, yeast cells, mammalian cells, insect cells, bird cells and amphibian cells.   32. A recombinant host cell cell according to claim 31 which is a human cell. A protein complex produced during expression of the nucleic acid structure or vector according to any one of claims 1 to 30. 35. The protein complex of claim 34, comprising an anti-CD40 antibody, human alpha interferon, and an antigen whose expression is associated with a disease state. 36. The protein complex of claim 35, wherein the disease is selected from cancer, allergies, autoimmune diseases, infectious diseases, and inflammatory conditions.   37. A protein complex according to claim 36 comprising an HIV antigen. 38. The protein complex according to claim 37, wherein the HIV antigen is Gag. An enhanced cellular immune response is derived by administering the nucleic acid structure according to any one of claims 1 to 28, or a vector or host cell cell containing the nucleic acid structure. Method. Said administration enhances primary and memory CD8 + T cell responses to the following (i) administration of DNA encoding only a CD40 agonist or type I interferon:
(Ii) induction of an exponential increase in antigen-specific CD8 + T cells;
(Iii) generation of a protective immune response in CD4 deficient host cells corresponding to normal (not CD40 deficient) host cells, and (iv) induction of CD70 expression on dendritic cells,
40. The method of claim 39, wherein at least one of the following occurs. The enhanced cellular immune response includes viral antigens, bacterial antigens, fungal antigens, self antigens,
4. Specificity for an allergen and an antigen selected from cancer antigens
9. The method according to 9.   42. The method of claim 41, wherein the antigen is an HIV antigen. 43. The method of claim 42, wherein the HIV antigen is gag or env. 42. The method of claim 41, wherein the antigen is an antigen expressed by a human tumor. A method of deriving an enhanced CD8 + T cell immune response in a subject in need thereof, wherein the synergistically effective amount of (i) at least one CD40 agonist, (ii) at least one type I interferon, and Optionally (iii) administering a polypeptide complex comprising at least one antigen whose expression is associated with a particular disease, or these portions included in the same or separate pharmaceutically acceptable compositions. A method characterized by. 46. The method of claim 45, wherein the CD40 agonist, the type I interferon, and the antigen, if present, are contained in the same composition. 46. The method of claim 45, wherein the CD40 agonist and the type I interferon are included in separate compositions. 46. The method of claim 45, wherein the CD40 agonist is an anti-CD40 antibody. 5. The CD40 agonist is a CD40L polypeptide.
5. The method according to 5. 50. The method of claim 49, wherein the CD40L polypeptide comprises a soluble CD40L polypeptide, a fragment or oligomer CD40L polypeptide thereof, or a complex comprising any of the foregoing. 51. The method of claim 50, wherein the CD40L comprises human CD40L or a soluble fragment, oligomer, or a complex that binds human CD40. 46. The type I interferon is selected from alpha, beta, alpha / beta, epsilon, tau, omega, or zeta interferon, or variants, fragments, or PEGylated forms thereof. Method. 46. The method of claim 45, wherein the disease is selected from cancer, allergies, inflammatory diseases, infectious diseases, and autoimmune diseases. 54. The method of claim 53, wherein the infection is caused by a virus, bacterium, fungus, or parasite.   55. The method of claim 54, wherein the virus is HIV. The administration results in the following (i) substantial induction of enhanced primary and memory CD8 + T cell responses to the administration of the CD40 agonist or the type I interferon alone:
(Ii) induction of an exponential increase of antigen-specific CD8 + T cells, and (iii) generation of protective immune responses in CD4 deficient host cells corresponding to normal (non-CD4 deficient) host cells, and (iv) trees Induction of CD70 expression on dendritic cells,
46. The method of claim 45, wherein at least one of the following occurs.   57. The method of claim 56, wherein the method is used for the treatment of viral infection or cancer. The nucleic acid structure is administered mucosally, topically, orally, intravenously, intramuscularly, intranasally, vaginally, nasally, intratumorally, intrathecally, or intraocularly. 40. The method of claim 39. The polypeptide complex is administered mucosally, topically, orally, intravenously, intramuscularly, intranasally, transvaginally, transanally, intratumorally, intrathecally, or intraocularly. Claim 4
2. The method according to 2. A human that elicits an enhanced CD8 + T cell immune response comprising a synergistically effective amount of (i) at least one CD40 agonist, (ii) at least one type I interferon, and (iii) optionally at least one antigen. A composition characterized in that it is suitable for use in the treatment of. 61. The composition of claim 60, wherein the CD40 agonist is an agonist anti-CD40 antibody or agonist anti-CD40 antibody fragment. 62. The composition of claim 61, wherein the agonist anti-CD40 antibody is a human, chimeric, humanized, or single chain antibody. 62. The composition of claim 61, wherein the agonist anti-CD40 antibody is IgG1, IgG2, IgG3, or IgG4. 61. The CD40 agonist is a CD40L polypeptide.
A composition according to 1. 65. The composition of claim 64, wherein the CD40L polypeptide is a soluble human CD40L fragment, an oligomer thereof, or a complex comprising the same.   61. The composition of claim 60, comprising a human tumor antigen or autoantigen. 61. The composition of claim 60, comprising a bacterial antigen, a viral antigen or a fungal antigen. 61. The composition of claim 60, wherein the type I interferon is human alpha or beta interferon. 69. The composition of claim 68, wherein the interferon is a consensus alpha interferon or a PEGylated alpha or beta interferon.   61. The composition of claim 60, comprising an allergen. A method comprising enhancing the effectiveness of a vaccine composition comprising adding or administering said vaccine in combination with at least one CD40 agonist and at least one type I interferon. 72. The method of claim 71, wherein the CD40 agonist is a CD40 agonist antibody or fragment thereof. 72. The method of claim 71, wherein the type I interferon is human alpha interferon or human beta interferon. The addition or combination administration is enhanced C specific for the antigen contained in the vaccine.
72. The method of claim 71, wherein the method induces D8 + T cell immunity.   75. The method of claim 74, wherein CD70 expression is induced on dendritic cells. Where the CD40 agonist is administered in the absence of the type I interferon or TLR agonist, the agonist is administered in combination with a sufficient amount of type I interferon or TLR agonist to reduce or eliminate hepatotoxicity CD
40. A method comprising reducing the toxicity of an agonist. The CD40 agonist is a CD40 agonist antibody or fragment, or C
77. The method of claim 76, wherein the method is a D40L polypeptide. 77. The method of claim 76, wherein the reduction in liver toxicity is determined based on liver transaminase levels. Providing a maximum tolerated dose (MTD) of said CD40 agonist about 1.5 to 10 times greater than MTD in the absence of type I interferon or TLR agonists, resulting in a similar increase in liver transaminase levels 77. The method of claim 76.

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