WO2006091720A2 - Compositions and methods for targeted delivery of immune response modifiers - Google Patents

Compositions and methods for targeted delivery of immune response modifiers Download PDF

Info

Publication number
WO2006091720A2
WO2006091720A2 PCT/US2006/006387 US2006006387W WO2006091720A2 WO 2006091720 A2 WO2006091720 A2 WO 2006091720A2 US 2006006387 W US2006006387 W US 2006006387W WO 2006091720 A2 WO2006091720 A2 WO 2006091720A2
Authority
WO
WIPO (PCT)
Prior art keywords
irm
alkyl
immunomodulatory composition
moiety
targeting moiety
Prior art date
Application number
PCT/US2006/006387
Other languages
French (fr)
Other versions
WO2006091720A3 (en
Inventor
Sefik Alkan
William C. Kieper
John P. Vasilakos
Jason D. Bonk
George W. Griesgraber
Kenneth E. Lipson
Jie J. Liu
James D. Mendoza
Doris Stoermer
Paul D. Wightman
Naiyong Jing
William J. Schultz
Original Assignee
3M Innovative Properties Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 3M Innovative Properties Company filed Critical 3M Innovative Properties Company
Priority to CA002598144A priority Critical patent/CA2598144A1/en
Priority to AU2006216669A priority patent/AU2006216669A1/en
Priority to EP06735874A priority patent/EP1850850A4/en
Priority to JP2007557149A priority patent/JP2008531580A/en
Publication of WO2006091720A2 publication Critical patent/WO2006091720A2/en
Publication of WO2006091720A3 publication Critical patent/WO2006091720A3/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • IRMs immune response modifiers
  • TLRs Toll-like receptors
  • certain IRMs may be useful for treating viral diseases (e.g., human papilloma virus, hepatitis, herpes), neoplasias (e.g., basal cell carcinoma, squamous cell carcinoma, actinic keratosis, melanoma), and T H 2-mediated diseases (e.g., asthma, allergic rhinitis, atopic dermatitis), and are also useful as vaccine adjuvants.
  • Immune response modifiers include compounds that possess potent immunomodulating activity including but not limited to antiviral and antitumor activity. Certain IRMs modulate the production and secretion of cytokines.
  • certain IRM compounds induce the production and secretion of cytokines such as, e.g., Type I interferons, TNF- ⁇ , IL-I, IL-6, IL-8, IL-10, IL-12, MIP-I, and/or MCP-I.
  • cytokines such as, e.g., Type I interferons, TNF- ⁇ , IL-I, IL-6, IL-8, IL-10, IL-12, MIP-I, and/or MCP-I.
  • certain IRM compounds can inhibit production and secretion of certain TH2 cytokines, such as IL-4 and IL- 5.
  • some IRM compounds are said to suppress IL-I and TNF (U.S. Patent No. 6,518,265).
  • IRMs are small organic molecules (e.g., molecular weight under about 1000 Daltons, preferably under about 500 Daltons, as opposed to large biological molecules such as proteins, peptides, nucleic acids, and the like) such as those disclosed in, for example, U.S. Patent Nos.
  • IRMs include certain purine derivatives (such as those described in U.S. Patent Nos. 6,376,501, and 6,028,076), certain imidazoquinoline amide derivatives (such as those described in U.S. Patent No. 6,069,149), certain imidazopyridine derivatives (such as those described in U.S. Patent No. 6,518,265), certain benzimidazole derivatives (such as those described in U.S. Patent 6,387,938), certain derivatives of a 4-aminopyrimidine fused to a five membered nitrogen containing heterocyclic ring (such as adenine derivatives described in U. S. Patent Nos.
  • IRM oligonucleotide sequences contain cytosine-guanine dinucleotides (CpG) and are described, for example, in U.S. Patent Nos. 6,194,388; 6,207,646; 6,239,116; 6,339,068; and 6,406,705.
  • CpG-containing oligonucleotides can include synthetic immunomodulatory structural motifs such as those described, for example, in U.S. Patent Nos. 6,426,334 and 6,476,000.
  • Other IRM nucleotide sequences lack CpG sequences and are described, for example, in International Patent Publication No. WO 00/75304.
  • IRMs include biological molecules such as aminoalkyl glucosaminide phosphates (AGPs) and are described, for example, in U.S. Patent Nos. 6,113,918; 6,303,347; 6,525,028; and 6,649,172.
  • AGPs aminoalkyl glucosaminide phosphates
  • the immunostimulatory effects of IRMs may be increased by co-delivery of an IRM compound and an antigen to cells of the immune system.
  • Co-delivery may be accomplished by, for example, covalent or non-covalent chemical coupling of the IRM and antigen, or physically confining the IRM and antigen to a defined space.
  • Methods for co-delivery of IRM and an antigen are described, for example, in U.S. Patent Publication NO. US2004/0091491.
  • an immune response modifier material can be coupled to a target-specific material and each portion can retain its respective function.
  • the targeting moiety of the resulting immunomodulatory composition can provide targeted delivery of the immune response modifier moiety.
  • the present invention provides an immunomodulatory composition that includes an immune response modifier moiety coupled to a targeting moiety.
  • the present invention also provides method of targeted delivery of an immune response modifier compound.
  • the method includes administering to a subject an immunomodulatory composition that includes an immune response modifier moiety coupled to a targeting moiety that recognizes a delivery target.
  • the present invention also provides a method of inducing a localized immune response.
  • the method includes administering to a subject an immunomodulatory composition that includes an immune response modifier moiety coupled to a targeting moiety that recognizes a delivery target in an amount effective to induce an immune response.
  • the present invention provides a method of treating a condition in a subject that is treatable by inducing an immune response.
  • the method includes administering to the subject an immunomodulatory composition that includes an immune response modifier moiety coupled to a targeting moiety that recognizes a delivery target in an amount effective to treat at least one symptom or sign of the condition.
  • Fig. 1 is a line graph that demonstrates anti-CD20 activity of an IRM/anti-CD20 antibody immunomodulatory composition.
  • Fig. 2 is a line graph that demonstrates anti-CD20 activity of a control compound/anti-CD20 antibody composition.
  • Fig. 3 is a line graph that demonstrates anti-CD20 activity of a control compound/anti-CD20 antibody composition.
  • Fig. 4 is a line graph that demonstrates anti-CD20 activity of an IRM/anti-CD20 antibody immunomodulatory composition.
  • Fig. 5 is a line graph that demonstrates anti-CD20 activity of an IRM/anti-CD20 antibody immunomodulatory composition.
  • Fig. 6 is a line graph that demonstrates anti-CD20 activity of an IRM/anti-CD20 antibody immunomodulatory composition.
  • Fig. 7 is a bar graph showing cytokine induction by IRM/anti-CD20 antibody immunomodulatory compositions.
  • Fig. 8 is a line graph demonstrating anti-CD40 activity of an IRM/anti-CD40 antibody immunomodulatory composition.
  • Fig. 9 is a line graph demonstrating anti-CD40 activity of an IRM/anti-CD40 antibody immunomodulatory composition.
  • Fig. 10 is a line graph demonstrating anti-CD40 activity of an IRM/anti-CD40 antibody immunomodulatory composition.
  • Fig. 11 is a bar graph demonstrating cytokine induction by IRM/anti-CD40 antibody immunomodulatory compositions.
  • Fig. 12 is a line graph demonstrating anti-CD8 activity of an IRM/anti-CD8 antibody immunomodulatory composition.
  • Fig. 13 is a line graph demonstrating anti-CD8 activity of an IRM/anti-CD8 antibody immunomodulatory composition.
  • Fig. 14 is a line graph demonstrating anti-CD8 activity of an IRM/anti-CD8 antibody immunomodulatory composition.
  • Fig. 15 is a bar graph demonstrating cytokine induction by IRM/anti-CD8 antibody immunomodulatory compositions.
  • Fig. 16 is a line graph demonstrating anti-HER2 activity of an IRM/anti-HER2 antibody immunomodulatory composition.
  • Fig. 17 is a line graph demonstrating anti-HER2 activity of an IRM/anti-HER2 antibody immunomodulatory composition.
  • Fig. 18 is a bar graph demonstrating cytokine induction by IRM/anti-HER2 antibody immunomodulatory compositions.
  • Fig. 19 is a line graph demonstrating anti-HER2 activity of an IRM/anti-HER2 antibody immunomodulatory composition.
  • Fig. 20 is a line graph showing IFN- ⁇ induction by an IRM/anti-HER2 antibody immunomodulatory composition.
  • Fig. 21 is a line graph showing TNF- ⁇ induction by an IRM/anti-HER2 antibody immunomodulatory composition.
  • Fig. 22 is a line graph showing IFN- ⁇ induction by an IRM/anti-HER2 antibody immunomodulatory composition.
  • Fig. 23 is a line graph showing TNF- ⁇ induction by an IRM/anti-HER2 antibody immunomodulatory composition.
  • Fig. 24 a line graph that demonstrates the immunospecificity of an IRM/anti-CD8 antibody immunomodulatory composition.
  • Fig. 25 is a line graph that shows induction of IFN- ⁇ in peripheral blood mononuclear cells (PBMCs) by an IRM/anti-CD8 antibody immunomodulatory composition.
  • Fig. 26 is a line graph that shows induction of TNF- ⁇ in PBMCs by an IRM/anti- CD8 antibody immunomodulatory composition.
  • the invention provides immunomodulatory compositions in which an immune response modifier (IRM) moiety is coupled to a targeting moiety for targeted delivery of the IRM moiety.
  • IRM immune response modifier
  • the targeting moiety can direct, sequester, retain, or otherwise actively target delivery of the IRM moiety, thereby concentrating the IRM moiety at the target site. Concentrating the IRM moiety at a target site may result in various benefits such as, for example, reducing the amount of the IRM moiety that is available systemically, thereby reducing — perhaps even eliminating — systemic side effects associated with administration of the IRM moiety.
  • the IRM moiety is concentrated at the target site, a smaller dose of the IRM moiety - at least as compared to an uncoupled form of the IRM moiety (i.e., the uncoupled IRM compound) - may be needed to provide effective treatment, which may provide cost and resource benefits as well as further limit the extent, severity, and/or duration of undesirable side effects.
  • Antagonist refers to a compound that can combine with a receptor (e.g., a TLR) to induce a cellular activity.
  • a receptor e.g., a TLR
  • An agonist may be a ligand that directly binds to the receptor.
  • an agonist may combine with a receptor indirectly by, for example, (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise results in the modification of another compound so that the other compound directly binds to the receptor.
  • An agonist may be referred to as an agonist of a particular TLR (e.g., a TLR6 agonist) or a particular combination of TLRs (e.g., a TLR 7/8 agonist - an agonist of both TLR7 and TLR8).
  • a particular TLR e.g., a TLR6 agonist
  • a particular combination of TLRs e.g., a TLR 7/8 agonist - an agonist of both TLR7 and TLR8.
  • “Ameliorate” refers to any reduction in the extent, severity, frequency, and/or likelihood of a symptom or clinical sign characteristic of a particular condition.
  • Antigen refers to any substance that may be bound by an antibody in a manner that is immunospecific to some degree.
  • Immunocell refers to cell of the immune system, i.e., a cell directly or indirectly involved in the generation or maintenance of an immune response, whether the immune response is innate, acquired, humoral, or cell-mediated.
  • Immunomodulatory and variations thereof refer to any increase or decrease (i.e., induction or inhibition) of immune activity.
  • Induce and variations thereof refer to any measurable increase in cellular activity.
  • induction of an immune response may include, for example, an increase in the production of a cytokine, activation, proliferation, or maturation of a population of immune cells, and/or other indicator of increased immune function.
  • Inhibit and variations thereof refer to any measurable reduction of cellular activity.
  • inhibition of a particular cytokine refers to a decrease in production of the cytokine. The extent of inhibition may be characterized as a percentage of a normal level of activity.
  • IRM compound refers generally to an immune response modifier compound that alters the level of one or more immune regulatory molecules, e.g., cytokines or co- stimulatory markers, when administered to an IRM-responsive cell.
  • IRM compounds include, for example, the small organic molecules, purine derivatives, small heterocyclic compounds, amide derivatives, and oligonucleotide sequences described above.
  • IRM moiety refers to that portion of an immunomodulatory composition that possesses immunomodulatory activity. The IRM moiety may be, or be derived from, an IRM compound, but may, alternatively, be or be derived from some other immunomodulatory material.
  • the term “IRM moiety” may refer to an uncoupled compound prior to coupling to, or after uncoupling from, a targeting moiety.
  • Marker and variations thereof refer to any substance on a cell surface that may be bound by a ligand in a manner that is specific to some degree. As used herein, a marker-ligand interaction explicitly excludes immunological affinity - i.e., antibody- antigen affinity binding. Thus, some substances on the cell surface may be considered a marker (i.e., it may be capable of non-immunological receptor-ligand binding) in one context and an antigen in another context (i.e., it may be the target of an antibody).
  • prophylactic and variations thereof refer to a treatment that limits, to any extent, the development and/or appearance of a symptom or clinical sign of a condition.
  • Selective and variations thereof refer to having a differential or a non-general impact on biological activity.
  • An agonist that selectively modulates biological activity through a particular TLR may be a TLR-selective agonist.
  • TLR-selectivity may be described with respect to a particular TLR (e.g., TLR8-selective or TLR7-selective) or with respect to a particular combination of TLRs (e.g., TLR 7/9-selective).
  • “Sign” or “clinical sign” refers to an objective physical finding relating to a particular condition capable of being found by one other than the patient.
  • Specific and variations thereof refer to having a differential or a non-general affinity, to any degree, for a particular target.
  • Symptom refers to any subjective evidence of disease or of a patient's condition.
  • Targeting moiety refers to that portion of an immunomodulatory composition that possesses target-specific affinity.
  • the targeting moiety may be, or be derived from, an antibody, but may, alternatively, be or be derived from a non-antibody protein or peptide, or non-protein material including, for example, small molecules and/or nanoparticles.
  • targeting moiety may refer to an uncoupled compound prior to coupling to, or after uncoupling from, an IRM moiety.
  • “Therapeutic” and variations thereof refer to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition.
  • Treatment or variations thereof refer to reducing, limiting progression, ameliorating, preventing, or resolving, to any extent, the symptoms or signs related to a condition.
  • an immunomodulatory composition comprising “an” IRM compound can be interpreted to mean that the composition includes at least one IRM compound.
  • the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
  • IRM compounds have been shown to stimulate certain aspects of the immune system. Often, this may occur through activation of dendritic cells (DCs), which are potent antigen presenting cells.
  • DCs dendritic cells
  • Topical application of certain IRM compounds have been shown to be effective in several animal disease models and in human clinical trials (e.g., genital warts, actinic keratosis, superficial basal cell carcinoma, etc.).
  • some IRM compounds also can induce undesirable side effects in some patients when administered systemically. This may be so, in part, because systemically administered IRM compounds may activate immune cells that are irrelevant to ameliorating a particular condition. For example, the activation of B cells may be irrelevant to treating certain forms of cancer.
  • the present invention provides immunomodulatory compositions that include an IRM moiety coupled to a targeting moiety.
  • the IRM moiety may be, or be derived from, an IRM compound.
  • the targeting moiety may be an antibody or be derived from an antibody (i.e., at least enough of the immunospecific portion of an antibody — e.g., enough of a light chain — to provide some degree of immunospecificity.
  • the targeting moiety may be, or be derived from, an agent that recognizes at least a portion of a tumor-specific marker such as, for example, a ligand that binds to a receptor that is, to some extent, specifically expressed by the target cell population.
  • the receptor may be considered a tumor- specific marker.
  • the IRM moiety and the targeting moiety may be coupled directly or indirectly, For example, direct coupling of the IRM moiety and the targeting moiety may be accomplished through a covalent bond between the IRM moiety and the targeting moiety. Direct coupling also can be accomplished noncovalently by, for example, avidin-biotin affinity.
  • One moiety may be biotinylated and the other moiety may be modified to contain an avidin moiety use known methods. The moieties so modified may be directly coupled by exploiting avidin-biotin affinity
  • the IRM moiety and the targeting moiety may be coupled indirectly by coupling each moiety to an intervening component such as, for example, a solid support or a spacer arm.
  • an intervening component such as, for example, a solid support or a spacer arm.
  • An intervening component e.g., a solid support
  • the IRM moieties may be derived from the same IRM compound or from different IRM compounds.
  • the targeting moieties may be the same or different, offering the opportunity to design a composition having a high number of a particular targeting moiety (e.g., to increase the likelihood of finding a target) and/or the ability to bind to multiple targets (e.g., by having a number of different targeting moieties).
  • an IRM moiety may be coupled to an anti-tumor targeting moiety such as, for example, a ligand of a tumor-specific marker or an anti-tumor antibody.
  • an anti-tumor antibody refers to an antibody (Ab) that recognizes cells of a tumor with some degree of specificity over normal tissue cells.
  • the coupled IRM-Ab composition exploits the tumor specificity provided by the antibody to target delivery of the coupled IRM moiety to the vicinity of tumor antigens.
  • dendritic cells in the vicinity of the tumor - as opposed to dendritic cells throughout the patient - are preferentially activated, thereby generating a localized tumor-specific, DC- mediated immune response while limiting systemic activation of dendritic cells that can induce general DC-mediated side effects.
  • Therapy employing a coupled IRM/tumor- specific composition may be particularly desirable for treatment of cancers (e.g., metastatic cancers) that are difficult or impossible to treat by other therapies such as, for example, surgery, radiotherapy, etc.
  • tumor-specific targeting moieties include certain non-protein materials such as, for example, nanoparticles and certain small molecules.
  • Nanoparticles that are about 1 nm to 200 nm in diameter may be used to provide tumor-specific delivery of IRM moieties to tumors.
  • an IRM compound may be attached to a nanoparticle by any suitable means such as, for example, covalent and noncovalent chemical interactions.
  • Noncovalent chemical interactions can include affinity (e.g., avidin/biotin, antigen/antibody, receptor/ligand), ionic interaction, and/or hydrophobic interaction.
  • Nanoparticles can possess tumor-specific targeting activity in at least two ways.
  • the nanoparticle may be coated with a targeting moiety that directs the nanoparticle to a tumor.
  • Methods for attaching targeting moieties e.g., antibodies, receptor ligands, etc.
  • nanoparticles may provide tumor-specific targeted delivery of an IRM moiety even without a having one or more targeting moieties attached.
  • Nanoparticles having a diameter of from about 50 nm to about 200 nm may be delivered systemically and reside in bloodstream until they reach tumor vasculature. Localized changes in the porosity or permeability of the circulatory system permit the nanoparticles to escape the bloodstream, leave the circulatory system, and be deposited in the vicinity of the tumor.
  • One example of a small molecule moiety that can provide tumor-specific targeted delivery of an IRM moiety is bis-phosphonate.
  • Bis-phosphonate functionality imparts high affinity, long-term association to the hydroxyapetite components of bone.
  • Bis- phosphonates are known to be useful for targeted delivery and sequestering of diagnostic and/or therapeutic agents in bone.
  • bis-phosphonate drugs are used diagnostically for the delivery of bone imaging agents and therapeutically in osteoporosis, tumor osteolysis, and bone metastasis.
  • An IRM/bis-phosphonate immunomodulatory composition could provide a depot of IRM within a common site of metastasis.
  • LHRH receptors are significantly elevated on breast cancer, prostate cancer, endometrial cancer, ovarian cancer, and melanoma cells.
  • ligands of LHRH receptors may be used as targeting moieties in immunomodulatory compositions to provide tumor-specific targeted delivery of the IRM moiety to a tumor site.
  • LHRH- directed therapeutics selectively home to the affected tissues. Coupling an IRM to a ligand of the LHRH receptor (e.g., LHRH or a synthetic analog) can provide targeted delivery of the IRM to tumor cells of these cancers, thereby concentrating the IRM at the site of the tumor and increasing the therapeutic index over that observed with the IRM compound alone.
  • LHRH receptor ligands could include LHRH decapeptide, an analog with agonist or antagonist activity, or a small molecule receptor ligand.
  • LHRH receptor is known to be overexpressed on many tumor cells (e.g., breast, prostate, melanoma) compared to normal organ tissues.
  • a single IRM-LHRH receptor ligand coupled composition could be used for treating more than one cancer.
  • LHRH receptor ligands may be coupled directly to an IRM moiety or may be attached to nanoparticles to which one or more IRM moieties are also attached. Nanoparticles bearing LHRH receptor ligands have been shown to target breast cancer cells, whether within the breast or within metastases to the lung. By comparison, nanoparticles with LHRH preferentially traffic to the liver of normal animals.
  • Folic acid receptor ligands also may be useful as targeting moieties that may be coupled to an IRM moiety and provide tumor-specific targeted delivery of the IRM.
  • the expression of folic acid receptors is increased on the surface of many tumor cells.
  • coupling a folic acid receptor ligand to an IRM moiety can result in selective accumulation of the IRM at a tumor site, reducing systemic availability of the IRM moiety, and increasing the therapeutic index of the IRM moiety.
  • Suitable folic acid receptor ligands include folic acid, an analog with agonist or antagonist activity, or a small molecule receptor ligand.
  • an IRM moiety may be coupled to a dendritic cell targeting moiety.
  • the targeting moiety may be an antibody (e.g., an anti-DC antibody) or a non-antibody ligand that recognizes a DC-specific marker.
  • Suitable DC-specific markers may include, for example, a co-stimulatory marker such as, for example, any member of the TNFR Superfamily (e.g., CD40), CD70, CD80, CD86, B7-CD, B7.1, B7.2, etc.
  • An immunomodulatory composition that includes a targeting moiety that recognizes a co-stimulatory marker may be used to deliver two DC- activating stimuli (i.e., IRM moiety and co-stimulation) in a single chemical entity.
  • an anti-DC antibody refers to an antibody that recognizes a dendritic cell antigen.
  • a suitable dendritic cell targeting moiety may bind to any antigen that is differentially expressed, either qualitatively or quantitatively, by dendritic cells.
  • Suitable dendritic cell targeting moieties may bind to such antigens as, for example,
  • DEC205 DEC205, BDCA-I, BDCA-2, BDCA-3, BDCA-4, DC-SIGN, L-SIGN, HLR-DR, CDl Ic, CD13, CD14, CD21, CD33, CD35, CD123, C-type lectins, integrins (e.g., ⁇ 4, ⁇ 6, ⁇ l ⁇ l), and/or any one of the Toll-like receptors (TLRs), etc.
  • TLRs Toll-like receptors
  • the targeting moiety recognized a DC-specific marker or antigen
  • coupling the IRM moiety to the targeting moiety can limit systemic availability of the IRM moiety, even when administered via a systemic delivery route.
  • the IRM moiety may be concentrated in the vicinity of dendritic cells, thereby maturing and activating dendritic cells more effectively.
  • Dendritic cells activated at the site of a tumor - or even inside a tumor mass - may be able to utilize a tumor antigen present on the surface of the tumor cells to initiate an immune response against the tumor. This method could provide a generalized anti-tumor therapy without the need for tumor-specific antibodies.
  • an IRM moiety may be coupled to an anti- macrophage targeting moiety. Macrophages are often localized in the vicinity of tumor cells. Thus, again, systemic availability of the IRM moiety can be limited, and the IRM moiety may be concentrated in the vicinity of the target cells (i.e., macrophages), thereby activating macrophages more efficiently. Activated macrophages are known to possess anti-tumor activity. Thus, this method could provide a generalized tumor therapy without the need for tumor-specific antibodies.
  • an IRM moiety may be coupled to a target specific moiety that recognizes a surface antigen on a cell type that can directly kill tumor cells such as, for example, CD8 + cytotoxic T cells, NK cells, or NKT cells.
  • tumor cells such as, for example, CD8 + cytotoxic T cells, NK cells, or NKT cells.
  • the IRM moiety may be concentrated in the vicinity of the tumor-killing cells, thereby (a) activating tumor- killing cells more effectively, and/or (b) limiting the systemic availability of the IRM moiety.
  • Tumor-killing cells activated at the site of a tumor - or even inside a tumor mass - may be able to utilize a tumor antigen present on the surface of the tumor cells to initiate an immune response against the tumor. This method could provide a generalized tumor therapy without the need for tumor-specific antibodies.
  • the IRM moiety may be coupled to a targeting moiety that recognizes, for example, an endothelial target.
  • a targeting moiety that recognizes, for example, an endothelial target.
  • Suitable anti-angiogenesis reagents include, for example, anti-CD 105 antibodies (CD 105 is overexpressed in tumor endothelium), anti-ED-B antibodies (ED-B is a fibronectin isoform found in tumor masses), peptides recognized by endothelial integrins associated with tumors, and growth factors whose receptors are upregulated on tumor endothelium (e.g., vascular endothelial growth factor).
  • anti-angiogenic reagents in this way may offer the promise of combined anti-angiogenesis and immunotherapy. Additionally, targeted delivery of an IRM to the tumor endothelium, as opposed to the tumor itself, may provide more effective long-term treatment since, generally, the endothelium is a less mutagenic tissue than a tumor mass. Therefore, therapy directed toward the endothelium may be far less likely to cause drug resistance. Also, a therapy directed toward the endothelium may be effective against virtually any vascularized tumor (e.g., breast cancer, prostate cancer, lung cancer) without the need for tumor-specific reagents.
  • vascularized tumor e.g., breast cancer, prostate cancer, lung cancer
  • the targeting moiety may include two or more targeting moieties, each of which could bind to a different target.
  • a targeting moiety may include one targeting moiety that recognizes, for example, an immune cell antigen or co-stimulatory marker (e.g., a dendritic cell target) and a second targeting moiety (e.g., an anti-tumor antigen) that recognizes, for example, target tumor cells.
  • an immune cell antigen or co-stimulatory marker e.g., a dendritic cell target
  • a second targeting moiety e.g., an anti-tumor antigen
  • Such a composition may not only target delivery of the IRM moiety to either or both target cell populations, but also may provide targeted delivery of the target immune cell (e.g., dendritic cell or tumor-killing cell) and IRM moiety to the vicinity of the target tumor cells (e.g., a tumor).
  • the targeting moiety of the composition may be any material that can provide targeted delivery of the composition.
  • the targeting portion may provide immunospecific targeting, i.e., may be a sufficient portion of an immunoglobulin (i.e., an antibody) to promote immunospecific binding of the composition to a target antigen.
  • the invention may be practiced using non-immunoglobulin targeting materials as well such as, for example, receptor ligands such as, for example, hormones (natural or synthetic), lipids, etc. Because immunoglobulins are proteins, it is understood that modifications can be made to a particular immunoglobulin without rendering the modified immunoglobulin unsuitable for use as a targeting moiety.
  • one or more portions of the immunoglobulin amino acid sequence may be deleted or substituted, or additional amino acids may be added to an immunoglobulin, and the immunoglobulin can still retain sufficient immunospecific character to be suitable for practicing the invention. Therefore, in the description that follows, reference to a particular antibody includes modified immunoglobulins that have such modifications (e.g., amino acid additions, deletions, and/or substitutions) as are possible while retaining a sufficient amount of immunospecific character.
  • Suitable antibodies may be specific for microbial antigens (e.g., bacterial, viral, parasitic or fungal antigens), cancer or tumor-associated antigens, and/or self antigens.
  • a suitable antibody is one that recognizes and binds to an antigen present on or in a cell.
  • An antibody that binds to a particular material i.e., Antigen
  • Antigen may be referred to, interchangeably, as "anti- Antigen” or an "Antigen antibody”.
  • an antibody may be referred to by a generic name or commercial tradename.
  • suitable antibodies include, but are not limited to, RITUXAN (rituximab, anti-CD20 antibody), HERCEPTIN (trastuzumab), QUADRAMET,
  • antibodies similarly useful for the invention include alemtuzumab (B cell chronic lymphocytic leukemia), gemtuzumab ozogamicin (CD33+acute myeloid leukemia), hP67.6 (CD33+ acute myeloid leukemia), infliximab (inflammatory bowel disease and rheumatoid arthritis), ET ANERCEPT (rheumatoid arthritis), tositumomab, MDX-210, oregovomab, anti-EGF receptor mAb, MDX-447, anti-tissue factor protein (TF), (Sunol); ior-c5, c5, edrecolomab, ibritumomab tiuxetan, anti-idiotypic mAb mimic of ganglioside GD3 epitope, anti-HLA-DrlO mAb, anti-CD33 humanized mAb, anti- CD52 humAb, anti-CDl mAb (ior t
  • Fas/Fas Ligand antibodies including, but not limited to, anti-human Fas/Fas Ligand antibodies, anti-murine Fas/Fas Ligand antibodies, Granzyme antibodies, Granzyme B antibodies;
  • BcI Antibodies including, but not limited to, anti-cytochrome C antibodies, anti-human BcI antibodies (monoclonal), anti-human BcI antibodies (polyclonal), anti-murine BcI Antibodies
  • Miscellaneous apoptosis antibodies such as, for example, anti-TRADD, anti- TRAIL, and anti-DR3 antibodies;
  • Bim antibodies including, but not limited to, anti-human, murine bim antibodies
  • Caspase antibodies such as, for example, anti-human caspase antibodies (monoclonal), and anti-murine caspase antibodies;
  • Anti-CD antibodies such as, for example, anti-CD25, anti-CD29, anti-CD29, anti-CD41 a, anti-CD42b, anti-CD42b, anti-CD42b, anti-CD43 , anti-CD46, anti-
  • Human chemokine antibodies such as, for example, human CNTF antibodies, human eotaxin antibodies, human epithelial neutrophil activating peptide-78 (ENA-78) antibodies, human exodus antibodies, human GRO antibodies, human HCC-I antibodies, human 1-309 antibodies, human IP-IO antibodies, human I-
  • TAC antibodies human LIF antibodies, human liver-expressed chemokine (LEC) antibodies, human lymphotaxin antibodies, human MCP antibodies, human MIP antibodies, human monokine induced by IFN- ⁇ (MIG/CXCL9) antibodies, human NAP-2 antibodies, human NP-I antibodies, human platelet factor-4 antibodies, human RANTES antibodies, human SDF antibodies, and human TECK antibodies;
  • Murine chemokine antibodies such as, for example, human B-cell attracting murine chemokine antibodies, chemokine- 1 antibodies, murine eotaxin antibodies, murine exodus antibodies, murine GCP-2 antibodies, murine KC antibodies, murine MCP antibodies, murine MIP antibodies, and murine RANTES antibodies;
  • Rat Chemokine Antibodies such as, for example, rat CNTF antibodies, rat
  • GRO antibodies rat MCP antibodies, rat MIP antibodies, and rat RANTES antibodies
  • Cytokine/cytokine receptor antibodies such as, for example, human biotinylated cytokine/cytokine receptor antibodies, human interferon (IFN) antibodies, human interleukin (IL) antibodies, human leptin antibodies, human oncostatin antibodies, human tumor necrosis factor (TNF) antibodies, human TNF receptor family antibodies, murine biotinylated cytokine/cytokine receptor antibodies, murine IFN antibodies, murine IL antibodies, murine TNF antibodies, murine TNF receptor antibodies, rat biotinylated cytokine/cytokine receptor antibodies, rat IFN antibodies, rat IL antibodies, and rat TNF antibodies;
  • human biotinylated cytokine/cytokine receptor antibodies such as, for example, human biotinylated cytokine/cytokine receptor antibodies, human interferon (IFN) antibodies, human interleukin (IL) antibodies, human leptin antibodies, human oncostatin antibodies, human tumor necrosis factor (TN
  • Extracellular matrix antibodies such as, for example, collagen/procollagen antibodies, laminin antibodies, human collagen antibodies, human laminin antibodies, human procollagen antibodies, vitronectin/vitronectin receptor antibodies, hukman vitronectin antibodies, human vitronectin receptor antibodies, fibronectin/fibronectin receptor antibodies, human fibronectin antibodies, and human fibronectin receptor antibodies;
  • Growth factor antibodies such as, for example, human growth factor antibodies, murine growth factor antibodies, and porcine growth factor antibodies;
  • Miscellaneous antibodies such as, for example, baculovirus antibodies, cadherin antibodies, complement antibodies, CIq antibodies, VonWillebrand factor antibodies, Cre Antibodies, HIV Antibodies, influenza antibodies, human leptin antibodies, murine leptin antibodies, murine CTLA-4 antibodies, P450 antibodies, and RNA polymerase antibodies;
  • Neurobiological antibodies such as, for example, amyloid antibodies, GFAP antibodies, human NGF antibodies, human NT-3 antibodies, and human NT-4 antibodies.
  • Additional antibodies suitable for use in the invention include, for example, antibodies listed in references such as the MSRS Catalog of Primary Antibodies and Linscott's Directory.
  • the immune response modifier moiety of the composition may be, or may be derived from, any suitable IRM compound.
  • reference to a compound can include the compound in any pharmaceutically acceptable form, including any isomer (e.g., diastereomer or enantiomer), salt, solvate, polymorph, and the like.
  • reference to the compound can include each of the compound's enantiomers as well as racemic mixtures of the enantiomers.
  • the IRM compound may be a small molecule immune response modifier (e.g., molecular weight of less than about 1000 Daltons).
  • the IRM compound may include a 2-aminopyridine fused to a five membered nitrogen-containing heterocyclic ring, or a 4-aminopyrimidine fused to a five membered nitrogen-containing heterocyclic ring.
  • IRM compounds suitable for use in the invention include compounds having a 2- aminopyridine fused to a five membered nitrogen-containing heterocyclic ring.
  • Such compounds include, for example, imidazoquinoline amines including but not limited to substituted imidazoquinoline amines such as, for example, amide substituted imidazoquinoline amines, sulfonamide substituted imidazoquinoline amines, urea substituted imidazoquinoline amines, aryl ether substituted imidazoquinoline amines, heterocyclic ether substituted imidazoquinoline amines, amido ether substituted imidazoquinoline amines, sulfonamido ether substituted imidazoquinoline amines, urea substituted imidazoquinoline ethers, thioether substituted imidazoquinoline amines, 6-, 7-, 8-, or 9-aryl, heteroaryl, aryloxy or arylalkyleneoxy substituted imidazoquinoline amines, and imidazoquinoline diamines; tetrahydroimidazoquinoline amines including but not limited to amide substituted te
  • the IRM compound may be an imidazonaphthyridine amine, a tetrahydroimidazonaphthyridine amine, an oxazoloquinoline amine, a thiazoloquinoline amine, an oxazolopyridine amine, a thiazolopyridine amine, an oxazolonaphthyridine amine, or a thiazolonaphthyridine amine.
  • the IRM compound may be a substituted imidazoquinoline amine, a tetrahydroimidazoquinoline amine, an imidazopyridine amine, a 1 ,2-bridged imidazoquinoline amine, a 6,7-fused cycloalkylimidazopyridine amine, an imidazonaphthyridine amine, a tetrahydroimidazonaphthyridine amine, an oxazoloquinoline amine, a thiazoloquinoline amine, an oxazolopyridine amine, a thiazolopyridine amine, an oxazolonaphthyridine amine, or a thiazolonaphthyridine amine.
  • a substituted imidazoquinoline amine refers to an amide substituted imidazoquinoline amine, a sulfonamide substituted imidazoquinoline amine, a urea substituted imidazoquinoline amine, an aryl ether substituted imidazoquinoline amine, a heterocyclic ether substituted imidazoquinoline amine, an amido ether substituted imidazoquinoline amine, a sulfonamido ether substituted imidazoquinoline amine, a urea substituted imidazoquinoline ether, a thioether substituted imidazoquinoline amine, a 6-, 7-, 8-, or 9-aryl, heteroaryl, aryloxy or arylalkyleneoxy substituted imidazoquinoline amine, or an imidazoquinoline diamine.
  • substituted imidazoquinoline amines specifically and expressly exclude l-(2-methylpropyl)-l/i-imidazo[4,5-c]quinolin- 4-amine and 4-amino- ⁇ , ⁇ -dimethyl-2-ethoxymethyl- 1 H-imidazo [4,5 -cjquinolin- 1 -ethanol.
  • Suitable IRM compounds also may include the purine derivatives, imidazoquinoline amide derivatives, benzimidazole derivatives, adenine derivatives, aminoalkyl glucosaminide phosphates, and oligonucleotide sequences described above.
  • the immune response modifier moiety of the compound is derived from N-[6-( ⁇ 2-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin- 1 -yl]- 1 , 1 -dimethylethyl ⁇ amino)-6-oxohexyl]-4-azido-2-hydroxybenzamide.
  • the IRM compound may be an agonist of at least one TLR such as, for example, an agonist of TLR6, TLR7, or TLR8.
  • the IRM may in some cases be an agonist of TLR9.
  • the TLR agonism for a particular compound may be assessed in any suitable manner.
  • assays and recombinant cell lines suitable for detecting TLR agonism of test compounds are described, for example, in U.S. Patent Publication Nos. US2004/0014779, US2004/0132079, US2004/0162309, US2004/0171086, US2004/0191833, and US2004/0197865.
  • a compound can be identified as an agonist of a particular TLR if performing the assay with a compound results in at least a threshold increase of some biological activity mediated by the particular TLR.
  • a compound may be identified as not acting as an agonist of a specified TLR if, when used to perform an assay designed to detect biological activity mediated by the specified TLR, the compound fails to elicit a threshold increase in the biological activity.
  • an increase in biological activity refers to an increase in the same biological activity over that observed in an appropriate control. An assay may or may not be performed in conjunction with the appropriate control.
  • the precise threshold increase of TLR-mediated biological activity for determining whether a particular compound is or is not an agonist of a particular TLR in a given assay may vary according to factors known in the art including but not limited to the biological activity observed as the endpoint of the assay, the method used to measure or detect the endpoint of the assay, the signal-to-noise ratio of the assay, the precision of the assay, and whether the same assay is being used to determine the agonism of a compound for both TLRs. Accordingly it is not practical to set forth generally the threshold increase of TLR- mediated biological activity required to identify a compound as being an agonist or a non- agonist of a particular TLR for all possible assays. Those of ordinary skill in the art, however, can readily determine the appropriate threshold with due consideration of such factors.
  • Assays employing HEK293 cells transfected with an expressible TLR structural gene may use a threshold of, for example, at least a three-fold increase in a TLR-mediated biological activity (e.g., NFKB activation) when the compound is provided at a concentration of, for example, from about 1 ⁇ M to about 10 ⁇ M for identifying a compound as an agonist of the TLR transfected into the cell.
  • a thresholds and/or different concentration ranges may be suitable in certain circumstances.
  • different thresholds may be appropriate for different assays.
  • the immunomodulatory composition may be formulated in any manner suitable for administration to a subject. Suitable types of formulations are described, for example, in U.S. Pat. Nos. 5,736,553; 5,238,944; 5,939,090; 6,365,166; 6,245,776; and 6,486,186; European Patent No. EP 0 394 026; and U.S. Patent Publication No. US2003/0199538.
  • the compound may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, or any form of mixture.
  • the compound may be delivered in formulation with any pharmaceutically acceptable excipient, carrier, or vehicle.
  • the formulation may be delivered in a conventional topical dosage form such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like.
  • the formulation may further include one or more additives including but not limited to adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, moisturizers, thickeners, and the like.
  • a formulation containing an immunomodulatory composition may be administered in any suitable manner such as, for example, non-parenterally or parenterally.
  • non-parenterally refers to administration through the digestive tract, including by oral ingestion.
  • Parenterally refers to administration other than through the digestive tract such as, for example, intravenously, intramuscularly, transdermally, subcutaneously, transmucosally (e.g., by inhalation), or topically.
  • composition of a formulation suitable for practicing the invention will vary according to factors known in the art including but not limited to the physical and chemical nature of the immunomodulatory composition, the nature of the carrier, the intended dosing regimen, the state of the subject's immune system (e.g., suppressed, compromised, stimulated), the method of administering the immunomodulatory composition, and the species to which the formulation is being administered.
  • the present invention includes a method of targeted delivery of an immune response modifier.
  • the method includes administering to a subject an immunomodulatory composition that includes an immune response modifier coupled to a targeting moiety that recognizes a delivery target.
  • the present invention provides a method of inducing a localized immune response.
  • the method includes administering to a subject an immunomodulatory composition that includes an immune response modifier coupled to a targeting moiety that recognizes a delivery target in an amount effective to induce an immune response.
  • the present invention provides a method of treating a condition in a subject treatable by inducing an immune response.
  • the method includes administering to the subject an immunomodulatory composition that includes an immune response modifier coupled to a targeting moiety that recognizes a delivery target in an amount effective to treat at least one symptom or sign of the condition.
  • suitable immunomodulatory compositions include the immunomodulatory composition described above.
  • the delivery target includes a tumor cell.
  • the delivery target includes an immune cell.
  • the targeting moiety may recognize more than one delivery target. In one such case, one delivery target can include a tumor cell and a second delivery target can include an immune cell.
  • the methods of the invention include administering the immunomodulatory composition to a subject in a formulation of, for example, from about 0.001% to about 10% (unless otherwise indicated, all percentages provided herein are weight/weight with respect to the total formulation) to the subject, although in some embodiments the immunomodulatory composition may be administered using a formulation that provides the immunomodulatory composition in a concentration outside of this range.
  • the method includes administering to a subject a formulation that includes from about 0.01% to about 1% immunomodulatory composition, for example, a formulation that includes from about 0.1 % to about 0.5% immunomodulatory composition.
  • An amount of an immunomodulatory composition effective for practicing the invention is an amount sufficient to generate a target-specific immune response.
  • the precise amount of an immunomodulatory composition needed to practice the invention will vary according to factors known in the art including but not limited to the physical and chemical nature of the immunomodulatory composition, the nature of the carrier, the intended dosing regimen, the state of the subject's immune system (e.g., suppressed, compromised, stimulated), the method of administering the immunomodulatory composition, and the species to which the immunomodulatory composition is being administered. Accordingly, it is not practical to set forth generally the amount that constitutes an amount of immunomodulatory composition effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.
  • the methods of the present invention include administering sufficient immunomodulatory composition to provide a dose of the IRM moiety of, for example, from about 100 ng/kg to about 50 mg/kg to the subject, although in some embodiments the methods may be performed by administering immunomodulatory composition to provide a dose of the IRM moiety outside this range.
  • the method includes administering sufficient immunomodulatory composition to provide a dose of the IRM moiety of from about 10 ⁇ g/kg to about 5 mg/kg to the subject, for example, a dose of from about 100 ⁇ g/kg to about 1 mg/kg.
  • the dose may be calculated using actual body weight obtained just prior to the beginning of a treatment course.
  • the methods of the present invention may include administering sufficient IRM conjugate to provide a dose of, for example, from about 0.01 mg/m 2 to about 10 mg/m 2 .
  • the methods of the present invention include administering sufficient immunomodulatory composition to provide a dose of the targeting moiety of, for example, from about 50 ng/kg to about 100 mg/kg to the subject, although in some embodiments the methods may be performed by administering immunomodulatory composition to provide a dose of the targeting moiety outside this range.
  • the method includes administering sufficient immunomodulatory composition to provide a dose of the targeting moiety of from about 10 ⁇ g/kg to about 50 mg/kg to the subject, for example, a dose of from about 1 mg/kg to about 20 mg/kg.
  • the dosing regimen may depend at least in part on many factors known in the art including but not limited to the physical and chemical nature of the immunomodulatory composition, the nature of the carrier, the amount of immunomodulatory composition being administered, the state of the subject's immune system (e.g., suppressed, compromised, stimulated), the method of administering the immunomodulatory composition, and the species to which the immunomodulatory composition is being administered. Accordingly it is not practical to set forth generally the dosing regimen effective for all possible applications. Those of ordinary skill in the art, however, can readily determine an appropriate dosing regimen with due consideration of such factors.
  • the immunomodulatory composition may be administered, for example, from a single dose to multiple doses.
  • the immunomodulatory composition may be administered from about once per day to about once every three months, although in some embodiments the methods of the present invention may be performed by administering the immunomodulatory composition at a frequency outside this range.
  • the immunomodulatory composition is administered from about once per week to about once per month.
  • the immunomodulatory composition is administered once daily, two days per week.
  • the immunomodulatory composition is administered once daily three times per week.
  • Conditions that may be treated by administering an immunomodulatory composition include, but are not limited to: (a) viral diseases such as, for example, diseases resulting from infection by an adenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), a poxvirus (e.g., an orthopoxvirus such as variola or vaccinia, or molluscum contagiosum), a picornavirus (e.g., rhinovirus or enterovirus), an orthomyxovirus (e.g., influenzavirus), a paramyxovirus (e.g., parainfluenzavirus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), a coronavirus (e.g., SARS), a papovavirus (e.g., papillomaviruses, such as those that cause genital warts, common warts, or plantar warts),
  • bacterial diseases such as, for example, diseases resulting from infection by bacteria of, for example, the genus Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, or Bordetella; (c) other infectious diseases, such chlamydia, fungal diseases including but not limited to candidiasis, aspergillosis, histoplasmosis, cryptococcal meningitis, or parasitic diseases including but not limited to malaria, Pneumocystis car
  • neoplastic diseases such as solid tumor cancers (including, but not limited to breast cancer, colon cancer, pancreatic cancer, prostate cancer, lung cancer, prostate cancer, liver cancer, etc.), intraepithelial neoplasias, cervical dysplasia, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, Kaposi's sarcoma, melanoma, leukemias including but not limited to myelogeous leukemia, chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, B -cell lymphoma, and hairy cell leukemia, and other cancers;
  • solid tumor cancers including, but not limited to breast cancer, colon cancer, pancreatic cancer, prostate cancer, lung cancer, prostate cancer, liver cancer, etc.
  • intraepithelial neoplasias including, but not limited to breast cancer, colon cancer, pan
  • atopic diseases such as atopic dermatitis or eczema, eosinophilia, asthma, allergy, allergic rhinitis, and Ommen's syndrome
  • certain autoimmune diseases such as systemic lupus erythematosus, essential thrombocythaemia, multiple sclerosis, discoid lupus, alopecia areata; and
  • diseases associated with wound repair such as, for example, inhibition of keloid formation and other types of scarring (e.g., enhancing wound healing, including chronic wounds).
  • Certain immunomodulatory compositions may be particularly helpful in individuals having compromised immune function.
  • certain immunomodulatory compositions may be used for treating the opportunistic infections and tumors that occur after suppression of cell mediated immunity in, for example, transplant patients, cancer patients and HIV patients.
  • the IRM moiety and the targeting moiety may be coupled by any suitable means including, for example, covalent and certain types of non-covalent coupling.
  • Methods of covalently and non-covalently coupling an IRM compound and an antigen are described, for example, in U.S. Patent Publication No. US2004/0091491.
  • Such methods also may be used to covalently or non-covalently couple an IRM moiety and a targeting moiety so that each moiety of the resulting composition retains its functional character.
  • an IRM and antigen could be coupled (e.g., a pH of greater than 9.0 and UV irradiation) would destroy the target-specific character of certain targeting moieties (e.g., destroy the antigen recognition sites of antibody light chains).
  • steric considerations and the possibility that one or more IRM moieties would bind to and, therefore, block the target-binding portion of the targeting moiety were considered obstacles that would preclude using the methods of coupling an IRM and antigen for coupling an IRM and a targeting moiety.
  • a targeting moiety may be coupled to an IRM moiety using chemistry that does not depend upon UV irradiation to couple the IRM moiety and the targeting moiety. Such methods use chemistry that may make it easier to control the conjugation reaction, control the ratio of IRM moiety to targeting moiety, characterize the final composition, and obtain a more uniform product. Additional methods for coupling an IRM moiety and a targeting moiety are described, for example, in U.S. Provisional Patent Application entitled IMMUNE RESPONSE MODIFIER CONJUGATES, filed February 22, 2006. As noted above, an IRM moiety may be coupled to a targeting moiety using affinity interactions rather than covalent bonds. One example noted above exploits affinity between avidin and biotin. Alternative affinity interactions that may be useful for coupling an IRM moiety and a targeting moiety include, for example, glycoprotein/lectin interaction.
  • an immunomodulatory composition may be prepared by covalently coupling an IRM moiety and a targeting moiety.
  • An immunomodulatory composition generally may be prepared by reacting an immune response modifier with a crosslinker and then reacting the resulting intermediate with a targeting moiety such as, for example, a sufficient portion of an antibody to provide the desired amount of target-specific delivery function.
  • a targeting moiety such as, for example, a sufficient portion of an antibody to provide the desired amount of target-specific delivery function.
  • Many crosslinkers suitable for preparing bioconjugates are known and many are commercially available. See for example, Hermanson, G. (1996) Bioconjugate Techniques, Academic Press.
  • an immunomodulatory composition may be prepared, for example, according to the method shown in Reaction Scheme I in which a targeting moiety is linked to an IRM moiety through Ri of the IRM moiety.
  • a targeting moiety is linked to an IRM moiety through Ri of the IRM moiety.
  • a heterobifunctional cross-linker of Formula IV to provide a compound of II.
  • R A and RB each contain a functional group that is selected to react with the other.
  • R A contains a primary amine
  • a heterobifunctional cross-linker may be selected in which R B contains an amine-reactive functional group such as an N-hydroxysulfosuccinimidyl ester.
  • R A and R B may be selected so that they react to provide the desired linker group in the conjugate.
  • Patent Publication Nos. US2004/0147543 and US2004/0176367 Many heterobifunctional cross-linkers are known and many are commercially available. See for example, Hermanson, G. (1996), Bioconjugate Techniques, Academic Press, Chapter 5 "Heterobifunctional Cross-Linkers", 229-285.
  • the reaction generally can be carried out by combining a solution of the compound of Formula III in a suitable solvent such as N,N-dimethylformamide with a solution of the heterobifunctional cross- linker of Formula IV in a suitable solvent such as N,N-dimethylformamide.
  • the reaction may be run at ambient temperature.
  • the product of Formula II may then be isolated using conventional techniques.
  • step (2) of Reaction Scheme I a compound of Formula II that contains reactive group Z A is reacted with the targeting moiety to provide the immunomodulatory conjugate of Formula I.
  • the reaction generally can be carried out by combining a solution of the compound of Formula II in a suitable solvent such as dimethyl sulfoxide with a solution of the targeting moiety in a suitable buffer such as PBS. The reaction may be run at ambient temperature or at a reduced temperature (-4 0 C). If Z A is a photoreactive group such as a phenyl azide then the reaction mixture will be exposed to long wave UV light for a length of time .adequate to effect cross-linking (e.g., 10 — 20 minutes). The average number of IRM moieties per targeting moiety may be controlled by adjusting the amount of compound of Formula II used in the reaction.
  • the immunomodulatory conjugate of Formula I may be isolated and purified using conventional techniques.
  • a compound of Formula II may be synthesized without using a heterobifunctional cross-linker. So long as the compound of Formula II contains the reactive group ZA, it may be reacted with a targeting moiety using the method of step (2) above to provide an immunomodulatory conjugate.
  • the terms "alkyl”, “alkenyl” and the prefix “alk-” include straight chain, branched chain, and cyclic groups, i.e. cycloalkyl and cycloalkenyl. Unless otherwise specified, these groups contain from 1 to 20 carbon atoms, with alkenyl groups containing from 2 to 20 carbon atoms. Preferred groups have a total of up to 10 carbon atoms.
  • Cyclic groups can be monocyclic or poly cyclic and preferably have from 3 to 10 ring carbon atoms.
  • Exemplary cyclic groups include cyclopropyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, and adamantyl.
  • haloalkyl is inclusive of groups that are substituted by one or more halogen atoms, including perfluorinated groups. This is also true of groups that include the prefix "halo-”. Examples of suitable haloalkyl groups are chloromethyl, trifluoromethyl, and the like.
  • aryl as used herein includes carbocyclic aromatic rings or ring systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl and indenyl.
  • heteroaryl includes aromatic rings or ring systems that contain at least one ring hetero atom (e.g., O, S, N).
  • Suitable heteroaryl groups include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl, pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl, naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl, and so on.
  • Heterocyclyl includes non-aromatic rings or ring systems that contain at least one ring hetero atom (e.g., O, S, N) and includes all of the fully saturated and partially unsaturated derivatives of the above mentioned heteroaryl groups.
  • exemplary heterocyclic groups include pyrrolidinyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, thiazolidinyl, isothiazolidinyl, and imidazolidinyl.
  • the aryl, heteroaryl, and heterocyclyl groups can be unsubstituted or substituted by one or more substituents independently selected from the group consisting of alkyl, alkoxy, methylenedioxy, ethylenedioxy, alkylthio, haloalkyl, haloalkoxy, haloalkylthio, halogen, nitro, hydroxy, mercapto, cyano, carboxy, formyl, aryl, aryloxy, arylthio, arylalkoxy, arylalkylthio, heteroaryl, heteroaryloxy, heteroarylthio, heteroarylalkoxy, heteroarylalkylthio, amino, alkylamino, dialkylamino, heterocyclyl, heterocycloalkyl, alkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, haloalkylcarbonyl, haloalkoxycarbonyl, alkylthiocarbonyl
  • R 2 groups include hydrogen, alkyl groups having 1 to 4 carbon atoms (i.e., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, and cyclopropylmethyl), and alkoxyalkyl groups (e.g., methoxy ethyl and ethoxymethyl).
  • R 3 and R 4 are independently hydrogen or methyl or R 3 and R 4 join together to form a benzene ring, a pyridine ring, a 6- membered saturated ring or a 6-membered saturated ring containing a nitrogen atom.
  • R 3 and R 4 are independently hydrogen or methyl or R 3 and R 4 join together to form a benzene ring, a pyridine ring, a 6- membered saturated ring or a 6-membered saturated ring containing a nitrogen atom.
  • an immunomodulatory composition may include an intervening component such as, for example, a spacer arm or a solid support.
  • an intervening component such as, for example, a spacer arm or a solid support.
  • Certain spacer arms such as, for example, those having a length of from about 20 A to about 100 A, may improve solubility of the composition, thereby increasing the level of IRM activity obtainable using the composition. Suitable spacers are commercially available
  • an immunomodulatory composition may include a macromolecular support to which both the targeting moiety and the IRM moiety are attached.
  • the macromolecular support may be a solid support.
  • the IRM moiety, targeting moiety, or both may be covalently attached to the macromolecular support using a linking group such as those described above.
  • the macromolecular support may include, for example, supports such as those described in United States Patent Publication Nos. US2004/0202720 and US2004/0258698 such as, for example, agarose beads, gold particles, etc.
  • the methods of the present invention may be performed on any suitable subject.
  • Suitable subjects include but are not limited to animals such as but not limited to humans, non-human primates, rodents, dogs, cats, horses, pigs, sheep, goats, or cows.
  • HPFC high performance flash chromatography
  • IRM Compound 1 N- ⁇ 2-[4-Amino-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin- 1 -yl]- 1 , 1 -dimethylethyl ⁇ -6-[(3-mercaptopropanoyl)amino]hexanamide
  • the crude product was purified by HPFC on silica gel three times (gradient elution with CMA in chloroform) to provide 4.90 g of benzyl 6-( ⁇ 2-[4-amino-2-(ethoxymethyl)-l/i-imidazo[4,5-c]quinolin-l-yl]-l,l- dimethylethyl ⁇ amino)-6-oxohexylcarbamate as a white foam.
  • the material from Part B (8.75 mmol) was dissolved in DMF (20 mL), cooled to 0°C, and the cold solution of the activated diacid was added in one portion, with two DMF rinses (10 mL each). The reaction was allowed to warm slowly to room temperature overnight. Several more portions of EDC were added to the reaction at O 0 C over the next two days. The reaction was allowed to stir at room temperature for several days more, then was diluted with water and saturated aqueous sodium bicarbonate and was extracted with ethyl acetate several times. The combined organic extracts were washed with water and brine, and were concentrated under reduced pressure.
  • the crude product was purified by HPFC to give 3.8 g of the disulfide dimer of N- ⁇ 2-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin- 1 -yl]- 1 , 1 -dimethylethyl ⁇ -6-[(3-mercaptopropanoyl)arnino]hexananiide.
  • Part D The material from Part C (3.8 g, 3.7 mmol) was dissolved in methanol (30 mL) at room temperature. Tris(2-carboxyethyl)phosphine hydrochloride (1.38 g, 4.81 mmol) was added, followed by water (3 mL), and 12.5 M aqueous sodium hydroxide (1.12 mL, 14.1 mmol). The solution was stirred at room temperature for 2 hours and then was cooled to 0 0 C. The solution was adjusted to pH 6 with 1 M aqueous hydrochloric acid (approximately 14 mL). The methanol was removed under reduced pressure and aqueous sodium bicarbonate was added. The mixture was extracted with dichloromethane (3 x).
  • IRM2 N- ⁇ 2-[4-Amino-2-(ethoxymethyl)-lH-imidazo[4,5- cjquinolin- 1 -yl]- 1 , 1 -dimethylethyl ⁇ -6- ⁇ [3 -(pyridin-2-yldithio)propanoyl] amino ⁇ hexanamide
  • N- ⁇ 2- [4-amino-2-(ethoxymethyl)- 1 H-imidazo [4,5 -c] quinolin- 1 -yl]- 1 , 1 -dimethylethyl ⁇ -6- ⁇ [3 -(pyridin-2-yldithio)propanoyl]amino ⁇ hexanamide was synthesized from 6-amino-N- ⁇ 2-[4-amino-2-(ethoxymethyl)-l/i-imidazo[4,5-c]quinolin-l- yl]-l,l-dimethylethyl ⁇ hexanamide hydrochloride in one step.
  • the aqueous phase was extracted with ethyl acetate.
  • the organic layers were combined, washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure.
  • the crude product was purified by chromatography on silica gel to provide 36 mg of iV- ⁇ 2-[4-amino-2- (ethoxyniethyO-l/f-imidazo ⁇ j S-cjquinolin-l-ylj-ljl-dimethylethylJ- ⁇ -IP- ⁇ yridin ⁇ - yldithio)propanoyl] amino ⁇ hexanamide as a colorless oil.
  • IRM3 N-[4-(4-Amino-2-butyl-lH-imidazo[4,5-c]quinolin-l- yl)butyl] -3 -mercaptopropanamide
  • IRM4 N-[4-(4-Amino-2-butyl-lH-imidazo[4,5-c]quinolin-l- yl)butyl] -3 -(pyridin-2-y ldithio)propanamide
  • N- [4-(4-amino-2-butyl- 1 H-imidazo [4,5 -c]quinolin- 1 -yl)butyl] -3 -mercaptopropanamide (0.20 g, 0.50 mmol) was converted into 47 mg of iV-[4-(4-amino-2-butyl-l//-imidazo[4,5- c]quinolin-l-yl)butyl]-3-(pyridin-2-yldithio)propanamide.
  • IRM5 N- ⁇ 2-[4-Amino-2-(ethoxymethyl)-l#-imidazo[4,5- c]quinolin- 1 -yl]- 1 , 1 -dimethylethyl ⁇ -4-hydrazino-4-oxobutanamide
  • Succinic anhydride (3.20 g, 32.0 mmol) was added to a 100 0 C solution of l-(2- amino-2-methylpropyl)-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-4-amine (prepared as described in U.S. Patent Publication No. US2004/0091491, 2.00 g, 6.39 mmol) in DMF (20 mL). After 2 days, the reaction mixture was concentrated under reduced pressure to give an off-white solid. The solid was stirred with 100 mL of dichloromethane and was isolated by filtration. The filtrate was concentrated, stirred with dichloromethane (25 mL), and filtered to yield additional solid.
  • the reaction mixture was stirred for 4 days and then was treated with 100 mL of water. The layers were separated and the aqueous portion was extracted with chloroform (50 mL). The combined organic layers were washed successively with water (50 mL) and brine (50 mL). The organic portion was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a white foam. The white foam was dissolved in dichloromethane (50 mL) and treated with ethylene diamine (1 mL). After stirring for 4 hours, the reaction mixture was treated with water (50 mL) and chloroform (50 mL) and the layers were separated. The aqueous portion was extracted with chloroform (2 x 25 mL).
  • IRM Compound 6 N- ⁇ 2-[4-Amino-2-(ethoxymethyl)-l/f-imidazo[4,5- c]quinolin-l-yl]-l,l-dimethylethyl ⁇ -6- ⁇ [3-(2,5-dioxo-2,5-dihydro-lH- ⁇ yrrol-l- yl)propanoyl] amino ⁇ hexanamide
  • the foam was purified by reverse phase HPLC using 0.05% formic acid/acetonitrile in 0.05% formic acid/water as the eluent to yield 11 mg of N- ⁇ 2-[4-amino-2-(ethoxymethyl)- 1 H-imidazo[4,5 -cjquinolin- 1 -yl]- 1 , 1 - dimethylethyl ⁇ -6- ⁇ [3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)propanoyl] amino ⁇ hexanamide as the monoformate salt.
  • IRM Compound 7 N- ⁇ 6-[(4-Amino-2-(ethoxymethyl)-l- ⁇ 2-methyl-2-
  • N- ⁇ 2-[4-Amino-7-[(6-aminohexyl)oxy]-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin-l-yl]-l,l-dimethylethyl ⁇ methanesulfonamide (prepared as described in Parts A-J of Example 45 in WO 2005/032484, 457 mg, 0.903 mmol) was dissolved in anhydrous DMF (9 mL) and treated with N-hydroxysuccinamidyl-4-azidosalacylic acid (248 mg, 0.903 mmol). The mixture was stirred under a nitrogen atmosphere overnight. The reaction mixture was then concentrated under reduced pressure.
  • IRM Compound 8 N- ⁇ 6-[(4-Amino-l- ⁇ 4-[(methylsulfonyl)amino]butyl ⁇ -2- propyl- 1 H-imidazo [4,5 -c]quinolin-7-yl)oxy]hexyl ⁇ -4-azido-2-hydroxybenzamide
  • N-(4- ⁇ 4- Amino-7- [(6-aminohexy l)oxy] -2-propy 1- 1 H-imidazo [4, 5 -c] quinolin- 1 - yl ⁇ butyl)methanesulfonamide (prepared as described in Parts A-J of Example 47 in WO 2005/032484, 490 mg, 1.00 mmol) was dissolved in anhydrous DMF (10 mL) and treated with iV-hydroxysuccinamidyl-4-azidosalacylic acid (274 mg, 1.00 mmol) and the mixture was stirred under nitrogen overnight. The reaction mixture was then concentrated under reduced pressure.
  • IRM Compound 9 (IRM9): iV- ⁇ 6-[(4-Amino-2-propyl[l,3]thiazolo[4 5 5-c]quinolin-7- yl)oxy]hexyl ⁇ -4-azido-2-hydroxybenzamide
  • Control Compound 1 (CCl): iV- ⁇ 2-[2-(Ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]- 1,1 -dimethylethyl ⁇ -6- [(3 -mercaptopropanoyl)amino]hexanamide
  • Trifluoroacetic acid (30 mL) was added slowly to a solution of tert-butyl 6-( ⁇ 2-[2- (ethoxymethyl)-l/f-imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethyl ⁇ amino)-6- oxohexylcarbamate (3.99 g, 7.82 mmol) in dichloromethane (80 mL) at room temperature. After 1.5 hours, the solution was concentrated under pressure to afford an oil. The oil was dissolved in a small amount of water and concentrated ammonium hydroxide. The resulting basic mixture was extracted with dichloromethane multiple times.
  • the solution was concentrated under reduced pressure and partitioned between saturated aqueous sodium bicarbonate and ethyl acetate/methanol. The organic layers were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure.
  • the crude product was purified by HPFC on silica gel (gradient elution with CMA/chloroform) to provide the disulfide dimer of N- ⁇ 2- [2-(ethoxymethyl> 1 H-imidazo [4,5 -c]quinolin- 1 -yl] -1,1 -dimethylethyl ⁇ -6- [(3 - mercaptopropanoyl)amino]hexanamide.
  • the solution was adjusted to pH 6 with 1 M HCl and the resulting mixture was concentrated under reduced pressure to remove the methanol.
  • the mixture was partitioned between saturated aqueous sodium bicarbonate and dichloromethane. The aqueous layer was extracted multiple times with dichloromethane.
  • Control Compound 2 iV- ⁇ 2-[2-(Ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]- 1 , 1 -dimethylethyl ⁇ -6- ⁇ [3 -(pyridin-2-yldithio)propanoyl] amino ⁇ hexanamide
  • iV- ⁇ 2-[2-(Ethoxymethyl)-li/-imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethyl ⁇ -6- ⁇ [3-(pyridin-2-yldithio)propanoyl]amino ⁇ hexanamide (0.55 g) was prepared from iV- ⁇ 2- [2-(ethoxymethyl)- 1 /f-imidazo[4,5 -cjquinolin- 1 -yl] -1,1 -dimethylethyl ⁇ -6- [(3 - mercaptopropanoyl)amino]hexanamide (0.83 g) using the procedure described for the preparation of IRM Compound 2.
  • the final product was purified by HPFC on silica gel (gradient elution with 1-10% methanol in dichloromethane) and was isolated as a colorless glassy solid after heating under vacuum.
  • Control Compound 3 N- ⁇ 2-[2-(Ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]- 1 , 1 -dimethylethyl ⁇ -4-hydrazino-4-oxobutanamide
  • the solution from Part A was cooled to 0°C and was tert-bntyl carbazate (0.489 g, 3.70 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.709 g, 3.70 mmol) were added. The mixture was allowed to warm to room temperature and was stirred overnight. More tert-butyl carbazate and l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride were added. After 1 hour, the solution was diluted with water (60 mL) and extracted with ethyl acetate (3 x 75 mL).
  • Trifluoroacetic acid (3 mL) was added slowly to a solution of tert-butyl 2-[4-( ⁇ 2- [2-(ethoxymethyl)-l//-imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethyl ⁇ amino)-4- oxobutanoyljhydrazinecarboxylate (0.688 g, 1.34 mmol) in dichloromethane (7 mL). The solution was stirred for 2.5 hours, then was concentrated under reduced pressure.
  • the trifluoroacetic acid salt of N- ⁇ 2-[2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]-l 5 l- dimethylethyl ⁇ -4-hydrazino-4-oxobutanamide was applied to anion exchange resins, which were eluted with pyridine in methanol to provide iV- ⁇ 2-[2-(ethoxymethyl)-lH " - imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethyl ⁇ -4-hydrazino-4-oxobutanamide as a free base, which was purified by HPFC on silica gel (gradient elution, 2-50% CMA in chloroform).
  • Control Compound 4 iV- ⁇ 2-[2-(Ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]- 1,1 -dimethylethyl ⁇ -6- ⁇ [3-(2,5-dioxo-2,5-dihydro- lH-pyrrol- 1 -yl)propanoyl] amino ⁇ hexanamide
  • the antibody is adjusted to a concentration of 5 to 10 mg/mL in PBS, pH 7.4 containing 5 mM EDTA.
  • a 5 mg/mL solution of 2-IT is prepared by dissolving it in PBS, pH 7.4 containing 5 mM EDTA.
  • the 2-IT solution is slowly added to the antibodysolution at the desired molar excess while mixing, and incubated for one hour at room temperature.
  • the thiolated antibody is purified by applying the mixture to a desalting column equilibrated with PBS, pH 7.2 containing 5 raM EDTA. 1 mL fractions are collected and the fractions containing thiolated antibody, determined by measuring the absorbance at a wavelength of 280 nm, are pooled together.
  • the level of activated antibody is determined using a thiol detection reagent, (for example Ellman's reagent).
  • the antibody is adjusted to a concentration of 5 to 10 mg/mL in PBS, pH 7.4.
  • the crosslinker for example, sSMCC, SMPT, or NHS-PEO 8 -Maleimide
  • sSMCC for example, sSMCC, SMPT, or NHS-PEO 8 -Maleimide
  • the crosslinker solution is slowly added to the antibody solution at the desired molar excess while mixing, and incubated for one hour at room temperature.
  • the modified antibody is purified by applying the mixture to a desalting column equilibrated with PBS, pH 7.2 containing 5 mM EDTA. One-milliliter fractions are collected and the fractions containing modified IgG, determined by measuring the absorbance at a wavelength of 280 nm, are pooled together.
  • the level of activated antibody is determined using an analytical method appropriate for the crosslinker.
  • the antibody was dissolved in PBS, pH 7.4 at a concentration of 5 to 10 mg/mL. In some instances, the antibody solution was adjusted to pH 10 with IN NaOH.
  • the pIRM e.g., IRMlO
  • the pIRM solution was slowly added to the antibody solution at the desired molar excess while mixing. The mixture is added to a 12- well plate (500 mL/well) and a long-wave UV light (366 nm) is placed directly over the plate for 15 minutes while on ice. The reaction was quenched by adding 1 M Tris, pH 8.0 to at 1/20 the reaction volume (v/v).
  • the IRM-antibody conjugate was purified by size exclusion chromatography using PBS, pH 7.4 as the column running buffer at a flow rate of 1 mL/min. One-milliliter fractions were collected and the absorbance of each fraction was measured at 280 nm. Fractions containing the IRM-antibody conjugate were pooled and filtered under sterile conditions through a 0.2-micron filter. The concentration of the IRM-antibody conjugate was determined by BCA assay using bovine gamma globulin as a standard. The filtered conjugate was stored at 4 0 C for future testing in biological assays.
  • the antibody is adjusted to a concentration of 3 to 10 mg/niLin PBS, pH 7.4.
  • the amine reactive dye for example, Alexa 488, is dissolved in DMSO to a concentration of 5 mg/niL.
  • the amine reactive dye solution is slowly added to the antibody solution at the desired molar excess (for example, 8-fold molar excess of dye to antibody) while mixing, and incubated for one hour at room temperature.
  • the labeled antibody is purified by applying the mixture to a desalting column equilibrated with PBS, pH 7.4. 1 mL fractions are collected and the labeled antibody is pooled.
  • the absorbance of the antibody-dye conjugate is measured at 280 nm and at the absorbance maximum for the dye (A max ) to determine the dye:antibody ratio.
  • size exclusion chromatography may be performed to purify the antibody-dye conjugate. The fractions containing dye labeled antibody are pooled together.
  • pdIRM Pyridyl Disulfide Modified IRM
  • the pdIRM is added to the thiolated antibody, as prepared above, at one half the molar excess of 2-IT used to thiolate the antibody.
  • the mixture is incubated overnight at room temperature.
  • a 500 mM L-cysteine solution (dissolved in 1 M Tris, pH 8.0) is added at 0.01 times (v/v) the reaction mixture.
  • the IRM- antibody conjugate is purified by size exclusion chromatography using PBS, pH 7.4 as the column running bufferat a flow rate of 1 mL/min. 1 mL fractions are collected and the absorbance is measured at a wavelength of 280 nm.
  • IRM-antibody conjugate Fractions containing the IRM-antibody conjugate are pooled and filtered under sterile conditions through a 0.2-micron filter. The concentration of the IRM- antibody conjugate is determined by BCA assay using bovine gamma globulin as a standard. The filtered conjugate is stored at 4 0 C for future testing in biological assays.
  • the sIRM (e.g., IRMl) is dissolved in DMSO at a concentration of 10 mg/mL.
  • the sIRM is added to the modified antibody, as prepared above, at a four-fold molar excess of sIRM to the amount of crosslinker used to modify the antibody.
  • the mixture is incubated overnight at room temperature.
  • a 500 mM L-cysteine solution (dissolved in 1 M Tris, pH 8.0) is added at 0.01 times (v/v) the reaction mixture.
  • the IRM- antibody conjugate is purified by size exclusion chromatography using PBS, pH 7.4 as the column running buffer, at a flow rate of 1 niL/min.
  • fractions of mixture are collected and measured at 280 run.
  • Fractions containing the IRM-antibody conjugate are pooled and filtered under sterile conditions through a 0.2-micron filter.
  • concentration of the IRM-antibody conjugate is determined by BCA assay using bovine gamma globulin as a standard.
  • the filtered conjugate is stored at 4 0 C for future testing in biological assays.
  • IRMl, IRM2, IRMlO, CCl and CC2 were conjugated to a human anti-CD20 antibody (RITUXAN, Genentech, San Francisco, CA).
  • Conjugates prepared with IRMl and CCl used the general methods described above for conjugation of antibodies modified with heterobifunctional crosslinkers to sulfhydryl modified IRMs (sIRM). Specifically, the SMPT crosslinker was mixed with the antibody at a 12-fold molar excess of SMPT to antibody.
  • Conjugates prepared with IRM2 and CC2 used the general methods described above for conjugation of thiolated antibodies to pyridyl disulfide modified IRM (pdIRM)..
  • the 2-IT was mixed with the antibody at a 60-fold molar excess of 2-IT to antibody.
  • the antibody conjugate prepared with IRMl 0 used the photo-reactive methods described in the above general method.
  • IRMlO was mixed with the antibody at a 20-fold (20X) and 40-fold (40X) molar excess of IRMlO to antibody.
  • PBMC Peripheral blood mononuclear cells
  • HISTOPAQUE-1077 or Ficoll-Paque Plus The PBMC layer is collected and washed twice with DPBS and resuspended in flow cytometry staining buffer (FACS buffer, Biosource).
  • FACS buffer flow cytometry staining buffer, Biosource.
  • the PBMCs were added to a 96-well flat bottom sterile tissue culture plate (Costar, Cambridge, MA or Becton Dickinson Labware, Lincoln Park, NJ) to a final PBMC concentration of 1 x 10 6 cells/well.
  • Anti- CD20 or the above prepared IRM/anti-CD20 antibody conjugates were added to each well at three fold dilutions from 9 ⁇ g/mL to 0.004 ug/mL, final concentration in combination with FcR blocking reagent (BD Pharmigen, San Diego, CA).
  • FcR blocking reagent BD Pharmigen, San Diego, CA.
  • the plate was incubated on ice for 15 minutes and then treated with 0.3- ⁇ g/mL anti-CD20-Alexa 488 per well.
  • An Isotype (IgGl) negative control (15 ⁇ g/mL, Control, BD Pharmigen) and 0.3 ⁇ g/mL anti- CD20-Alexa 488 were placed in individual wells. The plate was incubated for 30 minutes on ice in the dark.
  • the plate was centrifuged for 10 minutes at 1350 rpm, and cells were resuspended and washed with FACS buffer twice, resuspended in 200 ⁇ L FACS buffer and filtered through a multi-well filter plate (Pall Corporation). Cells were resuspended in 100 ⁇ L CYTOFIX buffer (BD Pharmingen) for 15 minutes at room temperature in the dark. Samples were stored overnight at 4 0 C and run on a FACSCalibur (Becton Dickenson) the following day. Antibody activities of the conjugates are shown in Figures 1 through 6. Activity was measured by the conjugates ability to inhibit the anti-CD20-
  • PBMC Peripheral blood mononuclear cells
  • HISTOPAQUE-1077 or Ficoll-Paque Plus The PBMC layer is collected and washed twice with DPBS or HBSS and resuspended at 4 x 10 6 cells/mL in RPMI complete media.
  • the PBMCs were added to a 96-well flat bottom sterile tissue culture plate (Costar, Cambridge, MA or Becton Dickinson Labware, Lincoln Park, NJ) to a final PBMC concentration of 2 x 10 6 cells/mL.
  • PBMCs were stimulated overnight at 37°C in a 5% carbon dioxide atmosphere with 0.33 ⁇ M , 0.420 ⁇ M , 0.830 ⁇ M, or 1.67 ⁇ M anti-CD20 or the anti-CD20 conjugates prepared in Example 1, based on final antibody concentration.
  • Culture supernatants were analyzed for IFN- ⁇ and TNF production using a human IFN- ⁇ ELISA (PBL Biomedical Laboratories, Piscataway, NJ) and human-specific TNF BVTM immunoassay (BioVeris Corp., Gaithersburg, MD), respectively, with results expressed in pg/mL. Cytokine induction by the conjugates is shown in Figure 7.
  • Example 3 Example 3
  • IRM2 and IRMlO were conjugated to a mouse anti-CD40 antibody (FGK4.5).
  • the method used to conjugate anti-CD40 to IRM2 was the same as described in Example 1; however, a 70-fold molar excess of 2-IT to anti-CD40 was used in the preparation of the antibody conjugate.
  • the method used to conjugate anti-CD40 to IRMlO was the same as described in Example 1; however, an 8-fold (8X) or 25-fold (25X) molar excess of IRMlO to anti-CD40 was used in the preparation of these antibody conjugates.
  • Splenocytes were removed from sacrificed C57BL6 mice and splenocytes were isolated from the mice by homogenizing the spleens. Splenocytes were homogenized in Hanks Balance Salt Solution media (Biosource International, Camarillo, CA) containing 1% FCS, washed and resuspended in FACS buffer (Biosource International). Splenocytes were plated in a 96-well round bottom sterile tissue culture plate (Costar, Cambridge, MA or Becton Dickinson Labware, Lincoln Park, NJ) to a final cell concentration of 2 x 10 6 cells/well.
  • Hanks Balance Salt Solution media Biosource International, Camarillo, CA
  • FACS buffer Biosource International
  • Anti-CD40 or the above-prepared conjugate were added to each well at three fold dilutions from 300 ⁇ g/mL to 0.14 ⁇ g/mL, final concentration in combination with mouse FcR blocking reagent (2.4G2).
  • the plate was incubated on ice for 15 minutes and then treated with 25-ug/mL anti-CD40-Alexa 488 per well.
  • An Isotype (IgG2a) negative control (15 ⁇ g/mL, Control, BD Pharmigen) and 25 ⁇ g/mL anti-CD40-Alexa 488 were placed in individual wells. The plate was incubated for 30 minutes on ice in the dark.
  • the plate was centrifuged for 10 minutes at 1500 rpm, and cells were resuspended and washed with FACS buffer twice, resuspended in 100 ⁇ L CYTOFIX buffer (BD Pharmigen) for 15 minutes at room temperature in the dark. Cells were washed and resuspended in 200 ⁇ L FACS buffer and filtered through a Multi-well filter plate. Samples were stored overnight at 4 0 C and run on a FACSCalibur (Becton Dickenson) the following day. Antibody activities of the conjugates are shown in Figures 8 through 10. Activity was measured by the conjugates ability to inhibit the anti-CD40-Alexa 488 to bind to the cells.
  • Example 4 The anti-CD40 antibody and conjugate, as prepared in Example 3, were tested for cytokine induction as described in Example 2. Cytokine induction by the conjugate is shown in Figure 11.
  • Example 5 The anti-CD40 antibody and conjugate, as prepared in Example 3, were tested for cytokine induction as described in Example 2. Cytokine induction by the conjugate is shown in Figure 11.
  • IRM2 and IRMlO were conjugated to a mouse anti-CD8 antibody (53.6.72; ATCC, Manassas, VA).
  • the methods used to conjugate anti-CD8 to IRM2 or IRMlO were the same as those described in Example 3.
  • the conjugate was tested for antibody activity, as described in Example 3, using three fold dilutions of the conjugate from 12 ⁇ g/mL to 0.006 ⁇ g/mL and 0.40 ⁇ g/mL for the anti-CD8-Alexa 488.
  • the anti-CD8 antibody and conjugates were also tested for cytokine induction as described in Example 2.
  • Antibody activities of the conjugates are shown in Figures 12 through 14. Activity was measured by the conjugates ability to inhibit the anti-CD8-Alexa 488 to bind to the cells. Cytokine induction by the conjugate is shown in Figure 15.
  • IRMlO was conjugated to the HERCEPTIN antibody (HER2; Genentech) using the same method that was described in Example 1 except that a 28.4-fold (pH 7.4), 28.4- fold (pH 10), or 42.6-fold (pH 10) molar excess of IRMlO to HER2 was used in the preparation of the antibody conjugate.
  • the HER2 antibody and conjugate were tested for antibody activity as described in Example 2; however, her2 positive human breast cancer cells (HCC2218; ATCC, Manassas, VA) were used in the assay (2.5 X lO 5 cells/well) instead of human PBMCs.
  • the conjugate was tested for antibody activity using three fold dilutions of the conjugate from 45 ⁇ g/mL to 0.007 ⁇ g/mL and 0.30 ⁇ g/mL for the HER2-Alexa 488.
  • Antibody activities of the conjugates are shown in Figures 16 and 17.
  • the HER2 antibody and conjugate were tested for cytokine induction as described in Example 2. Cytokine induction by the conjugate is shown in Figure 18.
  • IRMl was conjugated to HER2 as described in Example 1; however, a NHS-PEO 8 - Maleimide crosslinker was mixed with the antibody at a 15-fold molar excess of NHS- PEO 8 -Maleimide to antibody.
  • the HER2 antibody and conjugate were tested for antibody activity as described in Example 6. Activity was measured by the conjugates ability to inhibit the HER2-Alexa 488 to bind to the cells. Antibody activities of the conjugates are shown in Figure 19. The HER2 antibody and conjugate were tested for cytokine induction as described in Example 2. Cytokine induction by the conjugate is shown in Figures 20 and 21.
  • IRMl was conjugated to the anti-CD 8 antibody as described in Example 1; however, a NHS-PEOs-Maleimide crosslinker was mixed with the antibody at a 15-fold molar excess of NHS-PEO 8 -Maleimide to antibody.
  • the anti-CD8 antibody and conjugate were tested for cytokine induction as described in Example 2. Cytokine induction by the conjugate is shown in Figures 22 and 23.
  • IRMlO was suspended in dimethyl sulfoxide (DMSO) to 10 mg/mL.
  • Rat anti- mouse CD8 antibody (53.6.72, BioExpress, Inc., West Lebanon, NH) was suspended in phosphate buffered saline (PBS) to 7.8 mg/mL and the pH adjusted to > 9.0 by the addition of NaOH.
  • a 1 :10 ratio of IRM: antibody (volume:volume) was mixed together by adding 60 ⁇ L of the IRMlO solution (0.6 mg IRMlIRMlO) with 540 ⁇ L of the anti-CD8 antibody solution (4.21 mg anti-CD8).
  • the antibody control was 60 ⁇ L of PBS mixed with 540 ⁇ L of the anti-CD8 antibody solution.
  • IRM:antibody, and antibody control were each placed in single wells of a 24-well tissue culture plate.
  • the plate was placed on ice and a long wavelength UV light source was placed directly over the plate as close to the well containing the IRMlO/antibody mixture as possible. The mixtures were irradiated for 15 minutes.
  • the resulting conjugate and antibody control were removed from the wells and resuspended in PBS to a final concentration of 0.5 mg/mL IRMlO, 3.51 mg/mL anti-CD8; 0.05 mg/mL IRMlO, 3.86 mg/mL anti-CD8; and 3.51 mg/mL anti-CD8 for the 1 :10, 1:100, and antibody control, respectively, and dialyzed using a 10,000 molecular weight cutoff S lide-a-Lyzer (Pierce, Rockford, IL) against PBS to remove any unconjugated IRM.
  • a 10,000 molecular weight cutoff S lide-a-Lyzer Pierce, Rockford, IL
  • Splenocytes were removed from sacrificed C57BL6 mice and splenocytes were isolated from the mice by homogenizing the spleens. Splenocytes were homogenized in EHAA media (Biosource International, Camarillo, CA) containing 1% FCS, washed and resuspended in FACS buffer (Biosource International). Splenocytes were plated in a 96 well round bottom sterile tissue culture plate (Costar, Cambridge, MA or Becton Dickinson Labware, Lincoln Park, NJ) to a final cell concentration of 1 x 10 6 cells/well.
  • EHAA media Biosource International, Camarillo, CA
  • FACS buffer Biosource International
  • Splenocytes were treated for 30 minutes at 4 0 C with the IRM:antibody (1 : 10), antibody control, both as prepared in Example 9, or non-UV treated antibody at five fold dilution concentrations ranging from 766 ⁇ g/mL to 0.002 ⁇ g/mL.
  • FITC-labeled mouse anti-CD8 53.6.7, BD Pharmingen, San Diego, CA
  • PE-labeled mouse anti-CD3 BD Pharmingen
  • Cells were then washed two times with FACS buffer and fixed with CYTOFIX buffer (BD Pharmingen, San Diego, CA).
  • Flow cytometry analysis was performed by gating on the CD3+ lymphocytes and measuring the mean fluorescence intensity (MFI) of the FITC-labeled antibody. Results are shown in Figure 24.
  • PBMC Peripheral blood mononuclear cells
  • DPBS Dulbecco's Phosphate Buffered Saline
  • HBSS Hank's Balanced Salts Solution
  • the PBMC layer was collected and washed twice with DPBS or HBSS and resuspended at 4 x 10 6 cells/mL in RPMI complete media.
  • the PBMCs were added to a 96 well flat bottom sterile tissue culture plate (Costar, Cambridge, MA or Becton
  • PBMCs were stimulated overnight at 37 0 C in a 5% carbon dioxide atmosphere with the rat anti-mouse CD8 antibody alone or the 1 : 10 IRM/antibody conjugate as described in Example 9 in 3 -fold antibody dilutions (292-0.13 ⁇ g/mL).
  • Culture supernatants were analyzed for IFN- ⁇ and TNF production using a human IFN- ⁇ ELISA (PBL Biomedical Laboratories, Piscataway, NJ) and human-specific TNF BVTM immunoassay (BioVeris Corp., Gaithersburg, MD), respectively, with results expressed in pg/mL. Results are shown in Figure 25 and Figure 26.

Abstract

The present invention provides immunomodulatory compositions include an immune response modifier moiety coupled to a targeting moiety. In another aspect, the invention provides methods of providing targeted delivery of an IRM, generating a localized immune response, and treating a condition in a subject. Generally, the methods include administering to the subject an immunomodulatory composition that includes an immune response modifier moiety coupled to a targeting moiety that recognizes a delivery target.

Description

COMPOSITIONS AND METHODS FOR TARGETED DELIVERY OF IMMUNE
RESPONSE MODIFIERS
Cross-reference to Related Applications This application is a CIP of U.S. Patent Application Serial No. 11/220,235, filed
August 6, 2005, which is a continuation of U.S. Patent Application Ser. No, 10/013,193, filed December 6, 2001, now abandoned, which claims priority to U.S. Provisional Patent Application Serial No. 60/254,229, filed December 28, 2000. In addition, this application claims priority to U.S. Provisional Application Serial No. 60/655,713 filed February 23, 2005 and U.S. Provisional Patent Application entitled, "Immune Response Modifier Conjugates," filed February 22, 2006.
Background
There has been a major effort in recent years, with significant success, to discover new drug compounds that act by stimulating certain key aspects of the immune system, as well as by suppressing certain other aspects (see, e.g., U.S. Pat. Nos. 6,039,969 and 6,200,592). These compounds, referred to herein as immune response modifiers (IRMs), appear to act through basic immune system mechanisms known as Toll-like receptors (TLRs) to induce selected cytokine biosynthesis. They may be useful for treating a wide variety of diseases and conditions. For example, certain IRMs may be useful for treating viral diseases (e.g., human papilloma virus, hepatitis, herpes), neoplasias (e.g., basal cell carcinoma, squamous cell carcinoma, actinic keratosis, melanoma), and TH2-mediated diseases (e.g., asthma, allergic rhinitis, atopic dermatitis), and are also useful as vaccine adjuvants. Immune response modifiers include compounds that possess potent immunomodulating activity including but not limited to antiviral and antitumor activity. Certain IRMs modulate the production and secretion of cytokines. For example, certain IRM compounds induce the production and secretion of cytokines such as, e.g., Type I interferons, TNF-α, IL-I, IL-6, IL-8, IL-10, IL-12, MIP-I, and/or MCP-I. As another example, certain IRM compounds can inhibit production and secretion of certain TH2 cytokines, such as IL-4 and IL- 5. Additionally, some IRM compounds are said to suppress IL-I and TNF (U.S. Patent No. 6,518,265). Certain IRMs are small organic molecules (e.g., molecular weight under about 1000 Daltons, preferably under about 500 Daltons, as opposed to large biological molecules such as proteins, peptides, nucleic acids, and the like) such as those disclosed in, for example, U.S. Patent Nos. 4,689,338; 4,929,624; 5,266,575; 5,268,376; 5,346,905; 5,352,784; 5,389,640; 5,446,153; 5,482,936; 5,756,747; 6,110,929; 6,194,425; 6,331,539; 6,376,669; 6,451,810; 6,525,064; 6,541,485; 6,545,016; 6,545,017; 6,573,273; 6,656,938; 6,660,735; 6,660,747; 6,664,260; 6,664,264; 6,664,265; 6,667,312; 6,670,372; 6,677,347; 6,677,348; 6,677,349; 6,683,088; 6,756,382; 6,797,718; and 6,818,650; U.S. Patent Publication Nos. US2004/0091491; US2004/0147543; and US2004/0176367; and International Publication Nos. WO2005/18551, WO2005/18556, WO2005/20999, WO2005/032484, WO2005/048933, WO2005/048945, WO2005/051317, WO2005/051324, WO2005/066169, WO2005/066170, WO2005/066172, WO2005/076783, and WO2005/079195.
Additional examples of small molecule IRMs include certain purine derivatives (such as those described in U.S. Patent Nos. 6,376,501, and 6,028,076), certain imidazoquinoline amide derivatives (such as those described in U.S. Patent No. 6,069,149), certain imidazopyridine derivatives (such as those described in U.S. Patent No. 6,518,265), certain benzimidazole derivatives (such as those described in U.S. Patent 6,387,938), certain derivatives of a 4-aminopyrimidine fused to a five membered nitrogen containing heterocyclic ring (such as adenine derivatives described in U. S. Patent Nos. 6,376,501; 6,028,076 and 6,329,381; and in WO 02/08905), certain 3-β-D- ribofuranosylthiazolo[4,5-d]pyrimidine derivatives (such as those described in U.S. Publication No. US2003/0199461), and certain small molecule immuno-potentiator compounds such as those described, for example, in US2005/0136065. Other IRMs include large biological molecules such as oligonucleotide sequences.
Some IRM oligonucleotide sequences contain cytosine-guanine dinucleotides (CpG) and are described, for example, in U.S. Patent Nos. 6,194,388; 6,207,646; 6,239,116; 6,339,068; and 6,406,705. Some CpG-containing oligonucleotides can include synthetic immunomodulatory structural motifs such as those described, for example, in U.S. Patent Nos. 6,426,334 and 6,476,000. Other IRM nucleotide sequences lack CpG sequences and are described, for example, in International Patent Publication No. WO 00/75304. Other IRMs include biological molecules such as aminoalkyl glucosaminide phosphates (AGPs) and are described, for example, in U.S. Patent Nos. 6,113,918; 6,303,347; 6,525,028; and 6,649,172.
The immunostimulatory effects of IRMs may be increased by co-delivery of an IRM compound and an antigen to cells of the immune system. Co-delivery may be accomplished by, for example, covalent or non-covalent chemical coupling of the IRM and antigen, or physically confining the IRM and antigen to a defined space. Methods for co-delivery of IRM and an antigen are described, for example, in U.S. Patent Publication NO. US2004/0091491. In view of the great therapeutic potential for IRMs, and despite the important work that has already been done, there is a substantial ongoing need to expand their uses and therapeutic benefits.
Summary It has been found that an immune response modifier material can be coupled to a target-specific material and each portion can retain its respective function. When administered to a subject, the targeting moiety of the resulting immunomodulatory composition can provide targeted delivery of the immune response modifier moiety.
Accordingly, in one aspect, the present invention provides an immunomodulatory composition that includes an immune response modifier moiety coupled to a targeting moiety.
In another aspect, the present invention also provides method of targeted delivery of an immune response modifier compound. Generally, the method includes administering to a subject an immunomodulatory composition that includes an immune response modifier moiety coupled to a targeting moiety that recognizes a delivery target.
In another aspect, the present invention also provides a method of inducing a localized immune response. Generally, the method includes administering to a subject an immunomodulatory composition that includes an immune response modifier moiety coupled to a targeting moiety that recognizes a delivery target in an amount effective to induce an immune response.
In yet another aspect, the present invention provides a method of treating a condition in a subject that is treatable by inducing an immune response. Generally, the method includes administering to the subject an immunomodulatory composition that includes an immune response modifier moiety coupled to a targeting moiety that recognizes a delivery target in an amount effective to treat at least one symptom or sign of the condition. Various other features and advantages of the present invention should become readily apparent with reference to the following detailed description, examples, claims and appended drawings. In several places throughout the specification, guidance is provided through lists of examples. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
Brief Description of the Drawings
Fig. 1 is a line graph that demonstrates anti-CD20 activity of an IRM/anti-CD20 antibody immunomodulatory composition.
Fig. 2 is a line graph that demonstrates anti-CD20 activity of a control compound/anti-CD20 antibody composition.
Fig. 3 is a line graph that demonstrates anti-CD20 activity of a control compound/anti-CD20 antibody composition.
Fig. 4 is a line graph that demonstrates anti-CD20 activity of an IRM/anti-CD20 antibody immunomodulatory composition. Fig. 5 is a line graph that demonstrates anti-CD20 activity of an IRM/anti-CD20 antibody immunomodulatory composition.
Fig. 6 is a line graph that demonstrates anti-CD20 activity of an IRM/anti-CD20 antibody immunomodulatory composition.
Fig. 7 is a bar graph showing cytokine induction by IRM/anti-CD20 antibody immunomodulatory compositions.
Fig. 8 is a line graph demonstrating anti-CD40 activity of an IRM/anti-CD40 antibody immunomodulatory composition.
Fig. 9 is a line graph demonstrating anti-CD40 activity of an IRM/anti-CD40 antibody immunomodulatory composition. Fig. 10 is a line graph demonstrating anti-CD40 activity of an IRM/anti-CD40 antibody immunomodulatory composition. Fig. 11 is a bar graph demonstrating cytokine induction by IRM/anti-CD40 antibody immunomodulatory compositions.
Fig. 12 is a line graph demonstrating anti-CD8 activity of an IRM/anti-CD8 antibody immunomodulatory composition. Fig. 13 is a line graph demonstrating anti-CD8 activity of an IRM/anti-CD8 antibody immunomodulatory composition.
Fig. 14 is a line graph demonstrating anti-CD8 activity of an IRM/anti-CD8 antibody immunomodulatory composition.
Fig. 15 is a bar graph demonstrating cytokine induction by IRM/anti-CD8 antibody immunomodulatory compositions.
Fig. 16 is a line graph demonstrating anti-HER2 activity of an IRM/anti-HER2 antibody immunomodulatory composition.
Fig. 17 is a line graph demonstrating anti-HER2 activity of an IRM/anti-HER2 antibody immunomodulatory composition. Fig. 18 is a bar graph demonstrating cytokine induction by IRM/anti-HER2 antibody immunomodulatory compositions.
Fig. 19 is a line graph demonstrating anti-HER2 activity of an IRM/anti-HER2 antibody immunomodulatory composition.
Fig. 20 is a line graph showing IFN-α induction by an IRM/anti-HER2 antibody immunomodulatory composition.
Fig. 21 is a line graph showing TNF-α induction by an IRM/anti-HER2 antibody immunomodulatory composition.
Fig. 22 is a line graph showing IFN-α induction by an IRM/anti-HER2 antibody immunomodulatory composition. Fig. 23 is a line graph showing TNF-α induction by an IRM/anti-HER2 antibody immunomodulatory composition.
Fig. 24 a line graph that demonstrates the immunospecificity of an IRM/anti-CD8 antibody immunomodulatory composition.
Fig. 25 is a line graph that shows induction of IFN-α in peripheral blood mononuclear cells (PBMCs) by an IRM/anti-CD8 antibody immunomodulatory composition. Fig. 26 is a line graph that shows induction of TNF-α in PBMCs by an IRM/anti- CD8 antibody immunomodulatory composition.
Detailed Description of Illustrative Embodiments of the Invention The invention provides immunomodulatory compositions in which an immune response modifier (IRM) moiety is coupled to a targeting moiety for targeted delivery of the IRM moiety. Thus, even though the composition may be administered systemically, the targeting moiety can direct, sequester, retain, or otherwise actively target delivery of the IRM moiety, thereby concentrating the IRM moiety at the target site. Concentrating the IRM moiety at a target site may result in various benefits such as, for example, reducing the amount of the IRM moiety that is available systemically, thereby reducing — perhaps even eliminating — systemic side effects associated with administration of the IRM moiety. Also, because the IRM moiety is concentrated at the target site, a smaller dose of the IRM moiety - at least as compared to an uncoupled form of the IRM moiety (i.e., the uncoupled IRM compound) - may be needed to provide effective treatment, which may provide cost and resource benefits as well as further limit the extent, severity, and/or duration of undesirable side effects.
For the purposes of the present invention, the following terms shall have the indicated meanings: "Agonist" refers to a compound that can combine with a receptor (e.g., a TLR) to induce a cellular activity. An agonist may be a ligand that directly binds to the receptor. Alternatively, an agonist may combine with a receptor indirectly by, for example, (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise results in the modification of another compound so that the other compound directly binds to the receptor. An agonist may be referred to as an agonist of a particular TLR (e.g., a TLR6 agonist) or a particular combination of TLRs (e.g., a TLR 7/8 agonist - an agonist of both TLR7 and TLR8).
"Ameliorate" refers to any reduction in the extent, severity, frequency, and/or likelihood of a symptom or clinical sign characteristic of a particular condition. "Antigen" refers to any substance that may be bound by an antibody in a manner that is immunospecific to some degree. "Immune cell" refers to cell of the immune system, i.e., a cell directly or indirectly involved in the generation or maintenance of an immune response, whether the immune response is innate, acquired, humoral, or cell-mediated.
"Immunomodulatory" and variations thereof refer to any increase or decrease (i.e., induction or inhibition) of immune activity.
"Induce" and variations thereof refer to any measurable increase in cellular activity. For example, induction of an immune response may include, for example, an increase in the production of a cytokine, activation, proliferation, or maturation of a population of immune cells, and/or other indicator of increased immune function. "Inhibit" and variations thereof refer to any measurable reduction of cellular activity. For example, inhibition of a particular cytokine refers to a decrease in production of the cytokine. The extent of inhibition may be characterized as a percentage of a normal level of activity.
"IRM compound" refers generally to an immune response modifier compound that alters the level of one or more immune regulatory molecules, e.g., cytokines or co- stimulatory markers, when administered to an IRM-responsive cell. Representative IRM compounds include, for example, the small organic molecules, purine derivatives, small heterocyclic compounds, amide derivatives, and oligonucleotide sequences described above. "IRM moiety" refers to that portion of an immunomodulatory composition that possesses immunomodulatory activity. The IRM moiety may be, or be derived from, an IRM compound, but may, alternatively, be or be derived from some other immunomodulatory material. In some cases, the term "IRM moiety" may refer to an uncoupled compound prior to coupling to, or after uncoupling from, a targeting moiety. "Marker" and variations thereof refer to any substance on a cell surface that may be bound by a ligand in a manner that is specific to some degree. As used herein, a marker-ligand interaction explicitly excludes immunological affinity - i.e., antibody- antigen affinity binding. Thus, some substances on the cell surface may be considered a marker (i.e., it may be capable of non-immunological receptor-ligand binding) in one context and an antigen in another context (i.e., it may be the target of an antibody).
"Prophylactic" and variations thereof refer to a treatment that limits, to any extent, the development and/or appearance of a symptom or clinical sign of a condition. "Selective" and variations thereof refer to having a differential or a non-general impact on biological activity. An agonist that selectively modulates biological activity through a particular TLR may be a TLR-selective agonist. TLR-selectivity may be described with respect to a particular TLR (e.g., TLR8-selective or TLR7-selective) or with respect to a particular combination of TLRs (e.g., TLR 7/9-selective).
"Sign" or "clinical sign" refers to an objective physical finding relating to a particular condition capable of being found by one other than the patient.
"Specific" and variations thereof refer to having a differential or a non-general affinity, to any degree, for a particular target. "Symptom" refers to any subjective evidence of disease or of a patient's condition.
"Targeting moiety" refers to that portion of an immunomodulatory composition that possesses target-specific affinity. The targeting moiety may be, or be derived from, an antibody, but may, alternatively, be or be derived from a non-antibody protein or peptide, or non-protein material including, for example, small molecules and/or nanoparticles. In some cases, the term "targeting moiety" may refer to an uncoupled compound prior to coupling to, or after uncoupling from, an IRM moiety.
"Therapeutic" and variations thereof refer to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition.
"Treat" or variations thereof refer to reducing, limiting progression, ameliorating, preventing, or resolving, to any extent, the symptoms or signs related to a condition.
As used herein, "a," "an," "the," "at least one," and "one or more" are used interchangeably. Thus, for example, an immunomodulatory composition comprising "an" IRM compound can be interpreted to mean that the composition includes at least one IRM compound. Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
Many IRM compounds have been shown to stimulate certain aspects of the immune system. Often, this may occur through activation of dendritic cells (DCs), which are potent antigen presenting cells. Topical application of certain IRM compounds have been shown to be effective in several animal disease models and in human clinical trials (e.g., genital warts, actinic keratosis, superficial basal cell carcinoma, etc.). However, some IRM compounds also can induce undesirable side effects in some patients when administered systemically. This may be so, in part, because systemically administered IRM compounds may activate immune cells that are irrelevant to ameliorating a particular condition. For example, the activation of B cells may be irrelevant to treating certain forms of cancer. Treatment of such cancers with an IRM that is administered systemically may cause activation of B cells, which could lead to such side effects as, for example, nonspecific immunoglobulin production, certain chronic condition (such as, for example, auto-immunity), and/or B cell depletion (which could lead to greater susceptibility of disease upon subsequent exposure to a pathogen). Thus, in one aspect, the present invention provides immunomodulatory compositions that include an IRM moiety coupled to a targeting moiety. In some cases, the IRM moiety may be, or be derived from, an IRM compound. In some cases, the targeting moiety may be an antibody or be derived from an antibody (i.e., at least enough of the immunospecific portion of an antibody — e.g., enough of a light chain — to provide some degree of immunospecificity. However, in other cases, the targeting moiety may be, or be derived from, an agent that recognizes at least a portion of a tumor-specific marker such as, for example, a ligand that binds to a receptor that is, to some extent, specifically expressed by the target cell population. In this example, the receptor may be considered a tumor- specific marker.
The IRM moiety and the targeting moiety may be coupled directly or indirectly, For example, direct coupling of the IRM moiety and the targeting moiety may be accomplished through a covalent bond between the IRM moiety and the targeting moiety. Direct coupling also can be accomplished noncovalently by, for example, avidin-biotin affinity. One moiety may be biotinylated and the other moiety may be modified to contain an avidin moiety use known methods. The moieties so modified may be directly coupled by exploiting avidin-biotin affinity
Alternatively, the IRM moiety and the targeting moiety may be coupled indirectly by coupling each moiety to an intervening component such as, for example, a solid support or a spacer arm. Examples of, and methods for attaching IRM compounds to, suitable solid supports are described, for example, in U.S. Patent Publication No. US2004/0258698.
An intervening component (e.g., a solid support) may include a plurality of functional groups, thereby permitting the indirect coupling of a plurality of IRM moieties and/or a plurality of targeting moieties. When a plurality of IRM moieties is attached to an intervening component, the IRM moieties may be derived from the same IRM compound or from different IRM compounds. Likewise, when a plurality of targeting moieties is attached to an intervening component, the targeting moieties may be the same or different, offering the opportunity to design a composition having a high number of a particular targeting moiety (e.g., to increase the likelihood of finding a target) and/or the ability to bind to multiple targets (e.g., by having a number of different targeting moieties).
In some embodiments, an IRM moiety may be coupled to an anti-tumor targeting moiety such as, for example, a ligand of a tumor-specific marker or an anti-tumor antibody. As used herein, an anti-tumor antibody refers to an antibody (Ab) that recognizes cells of a tumor with some degree of specificity over normal tissue cells. The coupled IRM-Ab composition exploits the tumor specificity provided by the antibody to target delivery of the coupled IRM moiety to the vicinity of tumor antigens. Thus, dendritic cells in the vicinity of the tumor - as opposed to dendritic cells throughout the patient - are preferentially activated, thereby generating a localized tumor-specific, DC- mediated immune response while limiting systemic activation of dendritic cells that can induce general DC-mediated side effects. Therapy employing a coupled IRM/tumor- specific composition may be particularly desirable for treatment of cancers (e.g., metastatic cancers) that are difficult or impossible to treat by other therapies such as, for example, surgery, radiotherapy, etc.
Other examples of tumor-specific targeting moieties include certain non-protein materials such as, for example, nanoparticles and certain small molecules.
Nanoparticles that are about 1 nm to 200 nm in diameter may be used to provide tumor-specific delivery of IRM moieties to tumors. As noted above, an IRM compound may be attached to a nanoparticle by any suitable means such as, for example, covalent and noncovalent chemical interactions. Noncovalent chemical interactions can include affinity (e.g., avidin/biotin, antigen/antibody, receptor/ligand), ionic interaction, and/or hydrophobic interaction. Methods for attaching IRM compounds to solid supports such as nanoparticles are described, for example, in U.S. Patent Publication No. US2004/0258698.
Nanoparticles can possess tumor-specific targeting activity in at least two ways. First, as described above, the nanoparticle may be coated with a targeting moiety that directs the nanoparticle to a tumor. Methods for attaching targeting moieties (e.g., antibodies, receptor ligands, etc.) are well known. Second, nanoparticles may provide tumor-specific targeted delivery of an IRM moiety even without a having one or more targeting moieties attached. Nanoparticles having a diameter of from about 50 nm to about 200 nm may be delivered systemically and reside in bloodstream until they reach tumor vasculature. Localized changes in the porosity or permeability of the circulatory system permit the nanoparticles to escape the bloodstream, leave the circulatory system, and be deposited in the vicinity of the tumor.
One example of a small molecule moiety that can provide tumor-specific targeted delivery of an IRM moiety is bis-phosphonate. Bis-phosphonate functionality imparts high affinity, long-term association to the hydroxyapetite components of bone. Bis- phosphonates are known to be useful for targeted delivery and sequestering of diagnostic and/or therapeutic agents in bone. For example, bis-phosphonate drugs are used diagnostically for the delivery of bone imaging agents and therapeutically in osteoporosis, tumor osteolysis, and bone metastasis. An IRM/bis-phosphonate immunomodulatory composition could provide a depot of IRM within a common site of metastasis.
Leuteinizing hormone releasing hormone (LHRH) receptors are significantly elevated on breast cancer, prostate cancer, endometrial cancer, ovarian cancer, and melanoma cells. Thus, ligands of LHRH receptors may be used as targeting moieties in immunomodulatory compositions to provide tumor-specific targeted delivery of the IRM moiety to a tumor site. In animal models for the human cancers noted above, LHRH- directed therapeutics selectively home to the affected tissues. Coupling an IRM to a ligand of the LHRH receptor (e.g., LHRH or a synthetic analog) can provide targeted delivery of the IRM to tumor cells of these cancers, thereby concentrating the IRM at the site of the tumor and increasing the therapeutic index over that observed with the IRM compound alone. In studies comparing free Dox to LHRH-coupled Dox, approximately 200 times more free Dox was required to demonstrate an antitumor activity equal to the LHRH conjugate. Suitable LHRH receptor ligands could include LHRH decapeptide, an analog with agonist or antagonist activity, or a small molecule receptor ligand. LHRH receptor is known to be overexpressed on many tumor cells (e.g., breast, prostate, melanoma) compared to normal organ tissues. Thus, a single IRM-LHRH receptor ligand coupled composition could be used for treating more than one cancer. LHRH receptor ligands may be coupled directly to an IRM moiety or may be attached to nanoparticles to which one or more IRM moieties are also attached. Nanoparticles bearing LHRH receptor ligands have been shown to target breast cancer cells, whether within the breast or within metastases to the lung. By comparison, nanoparticles with LHRH preferentially traffic to the liver of normal animals.
Folic acid receptor ligands also may be useful as targeting moieties that may be coupled to an IRM moiety and provide tumor-specific targeted delivery of the IRM. The expression of folic acid receptors is increased on the surface of many tumor cells. Once again, coupling a folic acid receptor ligand to an IRM moiety can result in selective accumulation of the IRM at a tumor site, reducing systemic availability of the IRM moiety, and increasing the therapeutic index of the IRM moiety. Suitable folic acid receptor ligands include folic acid, an analog with agonist or antagonist activity, or a small molecule receptor ligand.
In some alternative embodiments, an IRM moiety may be coupled to a dendritic cell targeting moiety. The targeting moiety may be an antibody (e.g., an anti-DC antibody) or a non-antibody ligand that recognizes a DC-specific marker.
Suitable DC-specific markers may include, for example, a co-stimulatory marker such as, for example, any member of the TNFR Superfamily (e.g., CD40), CD70, CD80, CD86, B7-CD, B7.1, B7.2, etc. An immunomodulatory composition that includes a targeting moiety that recognizes a co-stimulatory marker may be used to deliver two DC- activating stimuli (i.e., IRM moiety and co-stimulation) in a single chemical entity.
As used herein, an anti-DC antibody refers to an antibody that recognizes a dendritic cell antigen. A suitable dendritic cell targeting moiety may bind to any antigen that is differentially expressed, either qualitatively or quantitatively, by dendritic cells. Suitable dendritic cell targeting moieties may bind to such antigens as, for example,
DEC205, BDCA-I, BDCA-2, BDCA-3, BDCA-4, DC-SIGN, L-SIGN, HLR-DR, CDl Ic, CD13, CD14, CD21, CD33, CD35, CD123, C-type lectins, integrins (e.g., α4, α6, αlβl), and/or any one of the Toll-like receptors (TLRs), etc.
Regardless of whether the targeting moiety recognized a DC-specific marker or antigen, coupling the IRM moiety to the targeting moiety can limit systemic availability of the IRM moiety, even when administered via a systemic delivery route. Moreover, the IRM moiety may be concentrated in the vicinity of dendritic cells, thereby maturing and activating dendritic cells more effectively. Dendritic cells activated at the site of a tumor - or even inside a tumor mass - may be able to utilize a tumor antigen present on the surface of the tumor cells to initiate an immune response against the tumor. This method could provide a generalized anti-tumor therapy without the need for tumor-specific antibodies. ' In other alternative embodiments, an IRM moiety may be coupled to an anti- macrophage targeting moiety. Macrophages are often localized in the vicinity of tumor cells. Thus, again, systemic availability of the IRM moiety can be limited, and the IRM moiety may be concentrated in the vicinity of the target cells (i.e., macrophages), thereby activating macrophages more efficiently. Activated macrophages are known to possess anti-tumor activity. Thus, this method could provide a generalized tumor therapy without the need for tumor-specific antibodies.
In other alternative embodiments, an IRM moiety may be coupled to a target specific moiety that recognizes a surface antigen on a cell type that can directly kill tumor cells such as, for example, CD8+ cytotoxic T cells, NK cells, or NKT cells. Once again, even if the immunomodulatory composition is administered systemically, the IRM moiety may be concentrated in the vicinity of the tumor-killing cells, thereby (a) activating tumor- killing cells more effectively, and/or (b) limiting the systemic availability of the IRM moiety. Tumor-killing cells activated at the site of a tumor - or even inside a tumor mass - may be able to utilize a tumor antigen present on the surface of the tumor cells to initiate an immune response against the tumor. This method could provide a generalized tumor therapy without the need for tumor-specific antibodies.
In other alternative embodiments, the IRM moiety may be coupled to a targeting moiety that recognizes, for example, an endothelial target. Significant differences exist in the endothelium environments of tumor masses compared to normal capillary beds. Differences exist, for example, in the identity and extent to which certain endothelial surface proteins, adhesion molecules (e.g., integrins), extracellular matrix proteins, growth factor receptors, etc. are expressed. These differences can be exploited to target delivery of an IRM moiety to tumor-related endothelium. Some reagents that specifically target such differences have been demonstrated to be useful as anti-angiogenic therapies. Coupling such an agent, as a targeting moiety, to an IRM moiety can combine two effective anti-tumor therapies: immunotherapy and anti-angiogenesis therapy. Suitable anti-angiogenesis reagents include, for example, anti-CD 105 antibodies (CD 105 is overexpressed in tumor endothelium), anti-ED-B antibodies (ED-B is a fibronectin isoform found in tumor masses), peptides recognized by endothelial integrins associated with tumors, and growth factors whose receptors are upregulated on tumor endothelium (e.g., vascular endothelial growth factor).
The use of anti-angiogenic reagents in this way may offer the promise of combined anti-angiogenesis and immunotherapy. Additionally, targeted delivery of an IRM to the tumor endothelium, as opposed to the tumor itself, may provide more effective long-term treatment since, generally, the endothelium is a less mutagenic tissue than a tumor mass. Therefore, therapy directed toward the endothelium may be far less likely to cause drug resistance. Also, a therapy directed toward the endothelium may be effective against virtually any vascularized tumor (e.g., breast cancer, prostate cancer, lung cancer) without the need for tumor-specific reagents.
In still other alternative embodiments, the targeting moiety may include two or more targeting moieties, each of which could bind to a different target. Thus, for example, a targeting moiety may include one targeting moiety that recognizes, for example, an immune cell antigen or co-stimulatory marker (e.g., a dendritic cell target) and a second targeting moiety (e.g., an anti-tumor antigen) that recognizes, for example, target tumor cells. Such a composition may not only target delivery of the IRM moiety to either or both target cell populations, but also may provide targeted delivery of the target immune cell (e.g., dendritic cell or tumor-killing cell) and IRM moiety to the vicinity of the target tumor cells (e.g., a tumor).
The targeting moiety of the composition may be any material that can provide targeted delivery of the composition. In many embodiments, the targeting portion may provide immunospecific targeting, i.e., may be a sufficient portion of an immunoglobulin (i.e., an antibody) to promote immunospecific binding of the composition to a target antigen. However, the invention may be practiced using non-immunoglobulin targeting materials as well such as, for example, receptor ligands such as, for example, hormones (natural or synthetic), lipids, etc. Because immunoglobulins are proteins, it is understood that modifications can be made to a particular immunoglobulin without rendering the modified immunoglobulin unsuitable for use as a targeting moiety. For example, one or more portions of the immunoglobulin amino acid sequence may be deleted or substituted, or additional amino acids may be added to an immunoglobulin, and the immunoglobulin can still retain sufficient immunospecific character to be suitable for practicing the invention. Therefore, in the description that follows, reference to a particular antibody includes modified immunoglobulins that have such modifications (e.g., amino acid additions, deletions, and/or substitutions) as are possible while retaining a sufficient amount of immunospecific character.
Suitable antibodies may be specific for microbial antigens (e.g., bacterial, viral, parasitic or fungal antigens), cancer or tumor-associated antigens, and/or self antigens. In many embodiments, a suitable antibody is one that recognizes and binds to an antigen present on or in a cell. An antibody that binds to a particular material (i.e., Antigen) may be referred to, interchangeably, as "anti- Antigen" or an "Antigen antibody". In some instances, an antibody may be referred to by a generic name or commercial tradename. Examples of suitable antibodies include, but are not limited to, RITUXAN (rituximab, anti-CD20 antibody), HERCEPTIN (trastuzumab), QUADRAMET,
PANOREX5 IDEC- Y2B8, BEC2, C225, ONCOLYM, SMART M195, ATRAGEN, OVAREX, BEXXAR, LDP-03, ior t6, MDX-210, MDX-Il, MDX-22, OV103, 3622W94, anti- VEGF, ZENAPAX, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-I, CEACIDE [google], PRETARGET, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD- 72000, LYMPHOCIDE, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti- FLK-2, MDX-260, ANA Ab, SMART IDlOAb, SMART ABL 364 Ab, CC49 (mAb B72.3), ImmuRAIT-CEA, anti-IL-4 antibody, an anti-IL-5 antibody, an anti- IL-9 antibody, an anti-Ig antibody, an anti-IgE antibody, serum-derived hepatitis B antibodies, recombinant hepatitis B antibodies, anti-CD40 antibody, anti-OX40 antibody, anti- Cytokine Receptor antibodies, and the like.
Other antibodies similarly useful for the invention include alemtuzumab (B cell chronic lymphocytic leukemia), gemtuzumab ozogamicin (CD33+acute myeloid leukemia), hP67.6 (CD33+ acute myeloid leukemia), infliximab (inflammatory bowel disease and rheumatoid arthritis), ET ANERCEPT (rheumatoid arthritis), tositumomab, MDX-210, oregovomab, anti-EGF receptor mAb, MDX-447, anti-tissue factor protein (TF), (Sunol); ior-c5, c5, edrecolomab, ibritumomab tiuxetan, anti-idiotypic mAb mimic of ganglioside GD3 epitope, anti-HLA-DrlO mAb, anti-CD33 humanized mAb, anti- CD52 humAb, anti-CDl mAb (ior t6), MDX-22, celogovab, anti-17-lA mAb, bevacizumab, daclizumab, anti-TAG-72 (MDX-220), anti-idiotypic mAb mimic of high molecular weight proteoglycan (I-Mel-1), anti-idiotypic mAb mimic of high molecular weight proteoglycan (I-Mel-2), anti-CEA Ab, hmAbHl 1, anti-DNA or DNA-associated proteins (histones) mAb, Gliomab-H mAb, GNI- 250 mAb, anti-CD22, CMA 676), anti- idiotypic human mAb to GD2 ganglioside, ior egf/r3, anti-ior c2 glycoprotein mAb, ior c5, anti-FLK- 2/FLT-3 mAb, anti-GD-2 bispecific mAb, antinuclear autoantibodies, anti- HLA-DR Ab, anti-CEA mAb, palivizumab, bevacizumab, alemtuzumab, BLyS-mAb, anti-VEGF2, anti-Trail receptor; B3 mAb, mAb BR96, breast cancer; and Abx- CbI mAb. Suitable antibodies also include the following:
Apoptosis antibodies such as, for example, Fas/Fas Ligand antibodies including, but not limited to, anti-human Fas/Fas Ligand antibodies, anti-murine Fas/Fas Ligand antibodies, Granzyme antibodies, Granzyme B antibodies; BcI Antibodies including, but not limited to, anti-cytochrome C antibodies, anti-human BcI antibodies (monoclonal), anti-human BcI antibodies (polyclonal), anti-murine BcI Antibodies
(monoclonal), and anti-murine BcI antibodies (polyclonal);
Miscellaneous apoptosis antibodies such as, for example, anti-TRADD, anti- TRAIL, and anti-DR3 antibodies;
Miscellaneous apoptosis related antibodies such as, for example, Bim antibodies including, but not limited to, anti-human, murine bim antibodies
(polyclonal), anti-human, murine bim antibodies (monoclonal);
Caspase antibodies such as, for example, anti-human caspase antibodies (monoclonal), and anti-murine caspase antibodies;
Anti-CD antibodies such as, for example, anti-CD25, anti-CD29, anti-CD29, anti-CD41 a, anti-CD42b, anti-CD42b, anti-CD42b, anti-CD43 , anti-CD46, anti-
CD61, anti-CD61, anti-CD62/P-slctn, anti-CD62/P-slctn, and anti-CD 154;
Human chemokine antibodies such as, for example, human CNTF antibodies, human eotaxin antibodies, human epithelial neutrophil activating peptide-78 (ENA-78) antibodies, human exodus antibodies, human GRO antibodies, human HCC-I antibodies, human 1-309 antibodies, human IP-IO antibodies, human I-
TAC antibodies, human LIF antibodies, human liver-expressed chemokine (LEC) antibodies, human lymphotaxin antibodies, human MCP antibodies, human MIP antibodies, human monokine induced by IFN-γ (MIG/CXCL9) antibodies, human NAP-2 antibodies, human NP-I antibodies, human platelet factor-4 antibodies, human RANTES antibodies, human SDF antibodies, and human TECK antibodies; Murine chemokine antibodies such as, for example, human B-cell attracting murine chemokine antibodies, chemokine- 1 antibodies, murine eotaxin antibodies, murine exodus antibodies, murine GCP-2 antibodies, murine KC antibodies, murine MCP antibodies, murine MIP antibodies, and murine RANTES antibodies; Rat Chemokine Antibodies such as, for example, rat CNTF antibodies, rat
GRO antibodies, rat MCP antibodies, rat MIP antibodies, and rat RANTES antibodies;
Cytokine/cytokine receptor antibodies such as, for example, human biotinylated cytokine/cytokine receptor antibodies, human interferon (IFN) antibodies, human interleukin (IL) antibodies, human leptin antibodies, human oncostatin antibodies, human tumor necrosis factor (TNF) antibodies, human TNF receptor family antibodies, murine biotinylated cytokine/cytokine receptor antibodies, murine IFN antibodies, murine IL antibodies, murine TNF antibodies, murine TNF receptor antibodies, rat biotinylated cytokine/cytokine receptor antibodies, rat IFN antibodies, rat IL antibodies, and rat TNF antibodies;
Extracellular matrix antibodies such as, for example, collagen/procollagen antibodies, laminin antibodies, human collagen antibodies, human laminin antibodies, human procollagen antibodies, vitronectin/vitronectin receptor antibodies, hukman vitronectin antibodies, human vitronectin receptor antibodies, fibronectin/fibronectin receptor antibodies, human fibronectin antibodies, and human fibronectin receptor antibodies;
Growth factor antibodies such as, for example, human growth factor antibodies, murine growth factor antibodies, and porcine growth factor antibodies;
Miscellaneous antibodies such as, for example, baculovirus antibodies, cadherin antibodies, complement antibodies, CIq antibodies, VonWillebrand factor antibodies, Cre Antibodies, HIV Antibodies, influenza antibodies, human leptin antibodies, murine leptin antibodies, murine CTLA-4 antibodies, P450 antibodies, and RNA polymerase antibodies;
Neurobiological antibodies such as, for example, amyloid antibodies, GFAP antibodies, human NGF antibodies, human NT-3 antibodies, and human NT-4 antibodies.
Additional antibodies suitable for use in the invention include, for example, antibodies listed in references such as the MSRS Catalog of Primary Antibodies and Linscott's Directory.
The immune response modifier moiety of the composition may be, or may be derived from, any suitable IRM compound. Unless otherwise indicated, reference to a compound can include the compound in any pharmaceutically acceptable form, including any isomer (e.g., diastereomer or enantiomer), salt, solvate, polymorph, and the like. In particular, if a compound is optically active, reference to the compound can include each of the compound's enantiomers as well as racemic mixtures of the enantiomers. In some embodiments, the IRM compound may be a small molecule immune response modifier (e.g., molecular weight of less than about 1000 Daltons). In some embodiments, the IRM compound may include a 2-aminopyridine fused to a five membered nitrogen-containing heterocyclic ring, or a 4-aminopyrimidine fused to a five membered nitrogen-containing heterocyclic ring. IRM compounds suitable for use in the invention include compounds having a 2- aminopyridine fused to a five membered nitrogen-containing heterocyclic ring. Such compounds include, for example, imidazoquinoline amines including but not limited to substituted imidazoquinoline amines such as, for example, amide substituted imidazoquinoline amines, sulfonamide substituted imidazoquinoline amines, urea substituted imidazoquinoline amines, aryl ether substituted imidazoquinoline amines, heterocyclic ether substituted imidazoquinoline amines, amido ether substituted imidazoquinoline amines, sulfonamido ether substituted imidazoquinoline amines, urea substituted imidazoquinoline ethers, thioether substituted imidazoquinoline amines, 6-, 7-, 8-, or 9-aryl, heteroaryl, aryloxy or arylalkyleneoxy substituted imidazoquinoline amines, and imidazoquinoline diamines; tetrahydroimidazoquinoline amines including but not limited to amide substituted tetrahydroimidazoquinoline amines, sulfonamide substituted tetrahydroimidazoquinoline amines, urea substituted tetrahydroimidazoquinoline amines, aryl ether substituted tetrahydroimidazoquinoline amines, heterocyclic ether substituted tetrahydroimidazoquinoline amines, amido ether substituted tetrahydroimidazoquinoline amines, sulfonamido ether substituted tetrahydroimidazoquinoline amines, urea substituted tetrahydroimidazoquinoline ethers, thioether substituted tetrahydroimidazoquinoline amines, and tetrahydroimidazoquinoline diamines; imidazopyridine amines including but not limited to amide substituted imidazopyridine amines, sulfonamide substituted imidazopyridine amines, urea substituted imidazopyridine amines, aryl ether substituted imidazopyridine amines, heterocyclic ether substituted imidazopyridine amines, amido ether substituted imidazopyridine amines, sulfonamido ether substituted imidazopyridine amines, urea substituted imidazopyridine ethers, and thioether substituted imidazopyridine amines; 1,2-bridged imidazoquinoline amines; 6,7-fused cycloalkylimidazopyridine amines; imidazonaphthyridine amines; tetrahydroimidazonaphthyridine amines; oxazoloquinoline amines; thiazoloquinoline amines; oxazolopyridine amines; thiazolopyridine amines; oxazolonaphthyridine amines; thiazolonaphthyridine amines; and lH-imidazo dimers fused to pyridine amines, quinoline amines, tetrahydroquinoline amines, naphthyridine amines, or tetrahydronaphthyridine amines.
In certain embodiments, the IRM compound may be an imidazonaphthyridine amine, a tetrahydroimidazonaphthyridine amine, an oxazoloquinoline amine, a thiazoloquinoline amine, an oxazolopyridine amine, a thiazolopyridine amine, an oxazolonaphthyridine amine, or a thiazolonaphthyridine amine.
In certain embodiments, the IRM compound may be a substituted imidazoquinoline amine, a tetrahydroimidazoquinoline amine, an imidazopyridine amine, a 1 ,2-bridged imidazoquinoline amine, a 6,7-fused cycloalkylimidazopyridine amine, an imidazonaphthyridine amine, a tetrahydroimidazonaphthyridine amine, an oxazoloquinoline amine, a thiazoloquinoline amine, an oxazolopyridine amine, a thiazolopyridine amine, an oxazolonaphthyridine amine, or a thiazolonaphthyridine amine.
As used herein, a substituted imidazoquinoline amine refers to an amide substituted imidazoquinoline amine, a sulfonamide substituted imidazoquinoline amine, a urea substituted imidazoquinoline amine, an aryl ether substituted imidazoquinoline amine, a heterocyclic ether substituted imidazoquinoline amine, an amido ether substituted imidazoquinoline amine, a sulfonamido ether substituted imidazoquinoline amine, a urea substituted imidazoquinoline ether, a thioether substituted imidazoquinoline amine, a 6-, 7-, 8-, or 9-aryl, heteroaryl, aryloxy or arylalkyleneoxy substituted imidazoquinoline amine, or an imidazoquinoline diamine. As used herein, substituted imidazoquinoline amines specifically and expressly exclude l-(2-methylpropyl)-l/i-imidazo[4,5-c]quinolin- 4-amine and 4-amino-α,α-dimethyl-2-ethoxymethyl- 1 H-imidazo [4,5 -cjquinolin- 1 -ethanol. Suitable IRM compounds also may include the purine derivatives, imidazoquinoline amide derivatives, benzimidazole derivatives, adenine derivatives, aminoalkyl glucosaminide phosphates, and oligonucleotide sequences described above.
In one particular embodiment, the immune response modifier moiety of the compound is derived from N-[6-({2-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin- 1 -yl]- 1 , 1 -dimethylethyl } amino)-6-oxohexyl]-4-azido-2-hydroxybenzamide. In some embodiments of the present invention, the IRM compound may be an agonist of at least one TLR such as, for example, an agonist of TLR6, TLR7, or TLR8. The IRM may in some cases be an agonist of TLR9.
The TLR agonism for a particular compound may be assessed in any suitable manner. For example, assays and recombinant cell lines suitable for detecting TLR agonism of test compounds are described, for example, in U.S. Patent Publication Nos. US2004/0014779, US2004/0132079, US2004/0162309, US2004/0171086, US2004/0191833, and US2004/0197865.
Regardless of the particular assay employed, a compound can be identified as an agonist of a particular TLR if performing the assay with a compound results in at least a threshold increase of some biological activity mediated by the particular TLR. Conversely, a compound may be identified as not acting as an agonist of a specified TLR if, when used to perform an assay designed to detect biological activity mediated by the specified TLR, the compound fails to elicit a threshold increase in the biological activity. Unless otherwise indicated, an increase in biological activity refers to an increase in the same biological activity over that observed in an appropriate control. An assay may or may not be performed in conjunction with the appropriate control. With experience, one skilled in the art may develop sufficient familiarity with a particular assay (e.g., the range of values observed in an appropriate control under specific assay conditions) that performing a control may not always be necessary to determine the TLR agonism of a compound in a particular assay. The precise threshold increase of TLR-mediated biological activity for determining whether a particular compound is or is not an agonist of a particular TLR in a given assay may vary according to factors known in the art including but not limited to the biological activity observed as the endpoint of the assay, the method used to measure or detect the endpoint of the assay, the signal-to-noise ratio of the assay, the precision of the assay, and whether the same assay is being used to determine the agonism of a compound for both TLRs. Accordingly it is not practical to set forth generally the threshold increase of TLR- mediated biological activity required to identify a compound as being an agonist or a non- agonist of a particular TLR for all possible assays. Those of ordinary skill in the art, however, can readily determine the appropriate threshold with due consideration of such factors.
Assays employing HEK293 cells transfected with an expressible TLR structural gene may use a threshold of, for example, at least a three-fold increase in a TLR-mediated biological activity (e.g., NFKB activation) when the compound is provided at a concentration of, for example, from about 1 μM to about 10 μM for identifying a compound as an agonist of the TLR transfected into the cell. However, different thresholds and/or different concentration ranges may be suitable in certain circumstances. Also, different thresholds may be appropriate for different assays.
The immunomodulatory composition may be formulated in any manner suitable for administration to a subject. Suitable types of formulations are described, for example, in U.S. Pat. Nos. 5,736,553; 5,238,944; 5,939,090; 6,365,166; 6,245,776; and 6,486,186; European Patent No. EP 0 394 026; and U.S. Patent Publication No. US2003/0199538. The compound may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, or any form of mixture. The compound may be delivered in formulation with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be delivered in a conventional topical dosage form such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like. The formulation may further include one or more additives including but not limited to adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, moisturizers, thickeners, and the like.
A formulation containing an immunomodulatory composition may be administered in any suitable manner such as, for example, non-parenterally or parenterally. As used herein, non-parenterally refers to administration through the digestive tract, including by oral ingestion. Parenterally refers to administration other than through the digestive tract such as, for example, intravenously, intramuscularly, transdermally, subcutaneously, transmucosally (e.g., by inhalation), or topically. The composition of a formulation suitable for practicing the invention will vary according to factors known in the art including but not limited to the physical and chemical nature of the immunomodulatory composition, the nature of the carrier, the intended dosing regimen, the state of the subject's immune system (e.g., suppressed, compromised, stimulated), the method of administering the immunomodulatory composition, and the species to which the formulation is being administered.
Accordingly, it is not practical to set forth generally the composition of a formulation effective for all possible applications. Those of ordinary skill in the art, however, can readily determine an appropriate formulation with due consideration of such factors.
In another aspect, the present invention includes a method of targeted delivery of an immune response modifier. Generally, the method includes administering to a subject an immunomodulatory composition that includes an immune response modifier coupled to a targeting moiety that recognizes a delivery target.
In another aspect, the present invention provides a method of inducing a localized immune response. Generally, the method includes administering to a subject an immunomodulatory composition that includes an immune response modifier coupled to a targeting moiety that recognizes a delivery target in an amount effective to induce an immune response.
In yet another aspect, the present invention provides a method of treating a condition in a subject treatable by inducing an immune response. Generally, the method includes administering to the subject an immunomodulatory composition that includes an immune response modifier coupled to a targeting moiety that recognizes a delivery target in an amount effective to treat at least one symptom or sign of the condition.
For each of the methods, suitable immunomodulatory compositions include the immunomodulatory composition described above. In some embodiments, the delivery target includes a tumor cell. In other embodiments, the delivery target includes an immune cell. In certain embodiments, the targeting moiety may recognize more than one delivery target. In one such case, one delivery target can include a tumor cell and a second delivery target can include an immune cell.
In some embodiments, the methods of the invention include administering the immunomodulatory composition to a subject in a formulation of, for example, from about 0.001% to about 10% (unless otherwise indicated, all percentages provided herein are weight/weight with respect to the total formulation) to the subject, although in some embodiments the immunomodulatory composition may be administered using a formulation that provides the immunomodulatory composition in a concentration outside of this range. In certain embodiments, the method includes administering to a subject a formulation that includes from about 0.01% to about 1% immunomodulatory composition, for example, a formulation that includes from about 0.1 % to about 0.5% immunomodulatory composition.
An amount of an immunomodulatory composition effective for practicing the invention is an amount sufficient to generate a target-specific immune response. The precise amount of an immunomodulatory composition needed to practice the invention will vary according to factors known in the art including but not limited to the physical and chemical nature of the immunomodulatory composition, the nature of the carrier, the intended dosing regimen, the state of the subject's immune system (e.g., suppressed, compromised, stimulated), the method of administering the immunomodulatory composition, and the species to which the immunomodulatory composition is being administered. Accordingly, it is not practical to set forth generally the amount that constitutes an amount of immunomodulatory composition effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors. In some embodiments, the methods of the present invention include administering sufficient immunomodulatory composition to provide a dose of the IRM moiety of, for example, from about 100 ng/kg to about 50 mg/kg to the subject, although in some embodiments the methods may be performed by administering immunomodulatory composition to provide a dose of the IRM moiety outside this range. In some of these embodiments, the method includes administering sufficient immunomodulatory composition to provide a dose of the IRM moiety of from about 10 μg/kg to about 5 mg/kg to the subject, for example, a dose of from about 100 μg/kg to about 1 mg/kg. Alternatively, the dose may be calculated using actual body weight obtained just prior to the beginning of a treatment course. For the dosages calculated in this way, body surface area (m2) is calculated prior to the beginning of the treatment course using the Dubois method: m2 = (wt kg0425 x height cm0 725) x 0.007184. In some embodiments, the methods of the present invention may include administering sufficient IRM conjugate to provide a dose of, for example, from about 0.01 mg/m2 to about 10 mg/m2.
In some embodiments, the methods of the present invention include administering sufficient immunomodulatory composition to provide a dose of the targeting moiety of, for example, from about 50 ng/kg to about 100 mg/kg to the subject, although in some embodiments the methods may be performed by administering immunomodulatory composition to provide a dose of the targeting moiety outside this range. In some of these embodiments, the method includes administering sufficient immunomodulatory composition to provide a dose of the targeting moiety of from about 10 μg/kg to about 50 mg/kg to the subject, for example, a dose of from about 1 mg/kg to about 20 mg/kg.
The dosing regimen may depend at least in part on many factors known in the art including but not limited to the physical and chemical nature of the immunomodulatory composition, the nature of the carrier, the amount of immunomodulatory composition being administered, the state of the subject's immune system (e.g., suppressed, compromised, stimulated), the method of administering the immunomodulatory composition, and the species to which the immunomodulatory composition is being administered. Accordingly it is not practical to set forth generally the dosing regimen effective for all possible applications. Those of ordinary skill in the art, however, can readily determine an appropriate dosing regimen with due consideration of such factors. In some embodiments of the invention, the immunomodulatory composition may be administered, for example, from a single dose to multiple doses. In certain embodiments, the immunomodulatory composition may be administered from about once per day to about once every three months, although in some embodiments the methods of the present invention may be performed by administering the immunomodulatory composition at a frequency outside this range. In one particular embodiment, the immunomodulatory composition is administered from about once per week to about once per month. In another embodiment, the immunomodulatory composition is administered once daily, two days per week. In yet another embodiment, the immunomodulatory composition is administered once daily three times per week.
Conditions that may be treated by administering an immunomodulatory composition include, but are not limited to: (a) viral diseases such as, for example, diseases resulting from infection by an adenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), a poxvirus (e.g., an orthopoxvirus such as variola or vaccinia, or molluscum contagiosum), a picornavirus (e.g., rhinovirus or enterovirus), an orthomyxovirus (e.g., influenzavirus), a paramyxovirus (e.g., parainfluenzavirus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), a coronavirus (e.g., SARS), a papovavirus (e.g., papillomaviruses, such as those that cause genital warts, common warts, or plantar warts), a hepadnavirus (e.g., hepatitis B virus), a flavivirus (e.g., hepatitis C virus or Dengue virus), or a retrovirus (e.g., a lentivirus such as HIV);
(b) bacterial diseases such as, for example, diseases resulting from infection by bacteria of, for example, the genus Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, or Bordetella; (c) other infectious diseases, such chlamydia, fungal diseases including but not limited to candidiasis, aspergillosis, histoplasmosis, cryptococcal meningitis, or parasitic diseases including but not limited to malaria, Pneumocystis carnii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosome infection;
(d) neoplastic diseases, such as solid tumor cancers (including, but not limited to breast cancer, colon cancer, pancreatic cancer, prostate cancer, lung cancer, prostate cancer, liver cancer, etc.), intraepithelial neoplasias, cervical dysplasia, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, Kaposi's sarcoma, melanoma, leukemias including but not limited to myelogeous leukemia, chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, B -cell lymphoma, and hairy cell leukemia, and other cancers;
(e) TH2 -mediated, atopic diseases, such as atopic dermatitis or eczema, eosinophilia, asthma, allergy, allergic rhinitis, and Ommen's syndrome; (f) certain autoimmune diseases such as systemic lupus erythematosus, essential thrombocythaemia, multiple sclerosis, discoid lupus, alopecia areata; and
(g) diseases associated with wound repair such as, for example, inhibition of keloid formation and other types of scarring (e.g., enhancing wound healing, including chronic wounds).
Certain immunomodulatory compositions may be particularly helpful in individuals having compromised immune function. For example, certain immunomodulatory compositions may be used for treating the opportunistic infections and tumors that occur after suppression of cell mediated immunity in, for example, transplant patients, cancer patients and HIV patients.
The IRM moiety and the targeting moiety may be coupled by any suitable means including, for example, covalent and certain types of non-covalent coupling. Methods of covalently and non-covalently coupling an IRM compound and an antigen are described, for example, in U.S. Patent Publication No. US2004/0091491. Such methods, surprisingly, also may be used to covalently or non-covalently couple an IRM moiety and a targeting moiety so that each moiety of the resulting composition retains its functional character. It was believed that some of the conditions under which an IRM and antigen could be coupled (e.g., a pH of greater than 9.0 and UV irradiation) would destroy the target-specific character of certain targeting moieties (e.g., destroy the antigen recognition sites of antibody light chains). Moreover, steric considerations and the possibility that one or more IRM moieties would bind to and, therefore, block the target-binding portion of the targeting moiety were considered obstacles that would preclude using the methods of coupling an IRM and antigen for coupling an IRM and a targeting moiety.
Alternatively, a targeting moiety may be coupled to an IRM moiety using chemistry that does not depend upon UV irradiation to couple the IRM moiety and the targeting moiety. Such methods use chemistry that may make it easier to control the conjugation reaction, control the ratio of IRM moiety to targeting moiety, characterize the final composition, and obtain a more uniform product. Additional methods for coupling an IRM moiety and a targeting moiety are described, for example, in U.S. Provisional Patent Application entitled IMMUNE RESPONSE MODIFIER CONJUGATES, filed February 22, 2006. As noted above, an IRM moiety may be coupled to a targeting moiety using affinity interactions rather than covalent bonds. One example noted above exploits affinity between avidin and biotin. Alternative affinity interactions that may be useful for coupling an IRM moiety and a targeting moiety include, for example, glycoprotein/lectin interaction.
Alternatively, an immunomodulatory composition may be prepared by covalently coupling an IRM moiety and a targeting moiety. An immunomodulatory composition generally may be prepared by reacting an immune response modifier with a crosslinker and then reacting the resulting intermediate with a targeting moiety such as, for example, a sufficient portion of an antibody to provide the desired amount of target-specific delivery function. Many crosslinkers suitable for preparing bioconjugates are known and many are commercially available. See for example, Hermanson, G. (1996) Bioconjugate Techniques, Academic Press.
Alternatively, an immunomodulatory composition may be prepared, for example, according to the method shown in Reaction Scheme I in which a targeting moiety is linked to an IRM moiety through Ri of the IRM moiety. In step (1) of Reaction Scheme I an IRM compound of Formula III is reacted with a heterobifunctional cross-linker of Formula IV to provide a compound of II. RA and RB each contain a functional group that is selected to react with the other. For example, if RA contains a primary amine, then a heterobifunctional cross-linker may be selected in which RB contains an amine-reactive functional group such as an N-hydroxysulfosuccinimidyl ester. RA and RB may be selected so that they react to provide the desired linker group in the conjugate.
Methods for preparing compounds of Formula III where RA contains a functional group are known. See for example, U.S. Patent Nos. 4,689,338; 4,929,624; U.S. Patent Nos. 5,268,376; 5,389,640; 5,352,784; 5,494,916; 4,988,815; 5,367,076; 5,175,296; 5,395,937; 5,741,908; 5,693,811; 6,069,149; 6,194,425; 6,331,539; 6,451,810; 6,525,064;6,541,485; 6,545,016; 6,545,017; 6,656,938; 6,660,747; 6,664,260; 6,664,264; 6,670,372; 6,677,349; 6,683, 088; and 6,797,718; and U.S. Patent Publication Nos. US2004/0147543 and US2004/0176367. Many heterobifunctional cross-linkers are known and many are commercially available. See for example, Hermanson, G. (1996), Bioconjugate Techniques, Academic Press, Chapter 5 "Heterobifunctional Cross-Linkers", 229-285. The reaction generally can be carried out by combining a solution of the compound of Formula III in a suitable solvent such as N,N-dimethylformamide with a solution of the heterobifunctional cross- linker of Formula IV in a suitable solvent such as N,N-dimethylformamide. The reaction may be run at ambient temperature. The product of Formula II may then be isolated using conventional techniques.
In step (2) of Reaction Scheme I a compound of Formula II that contains reactive group ZA is reacted with the targeting moiety to provide the immunomodulatory conjugate of Formula I. The reaction generally can be carried out by combining a solution of the compound of Formula II in a suitable solvent such as dimethyl sulfoxide with a solution of the targeting moiety in a suitable buffer such as PBS. The reaction may be run at ambient temperature or at a reduced temperature (-40C). If ZA is a photoreactive group such as a phenyl azide then the reaction mixture will be exposed to long wave UV light for a length of time .adequate to effect cross-linking (e.g., 10 — 20 minutes). The average number of IRM moieties per targeting moiety may be controlled by adjusting the amount of compound of Formula II used in the reaction. The immunomodulatory conjugate of Formula I may be isolated and purified using conventional techniques.
Reaction Scheme I
Figure imgf000031_0001
target-specific moiety
Figure imgf000031_0002
Alternatively, a compound of Formula II may be synthesized without using a heterobifunctional cross-linker. So long as the compound of Formula II contains the reactive group ZA, it may be reacted with a targeting moiety using the method of step (2) above to provide an immunomodulatory conjugate. As used herein, the terms "alkyl", "alkenyl" and the prefix "alk-" include straight chain, branched chain, and cyclic groups, i.e. cycloalkyl and cycloalkenyl. Unless otherwise specified, these groups contain from 1 to 20 carbon atoms, with alkenyl groups containing from 2 to 20 carbon atoms. Preferred groups have a total of up to 10 carbon atoms. Cyclic groups can be monocyclic or poly cyclic and preferably have from 3 to 10 ring carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, and adamantyl.
The term "haloalkyl" is inclusive of groups that are substituted by one or more halogen atoms, including perfluorinated groups. This is also true of groups that include the prefix "halo-". Examples of suitable haloalkyl groups are chloromethyl, trifluoromethyl, and the like.
The term "aryl" as used herein includes carbocyclic aromatic rings or ring systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl and indenyl. The term "heteroaryl" includes aromatic rings or ring systems that contain at least one ring hetero atom (e.g., O, S, N). Suitable heteroaryl groups include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl, pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl, naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl, and so on.
"Heterocyclyl" includes non-aromatic rings or ring systems that contain at least one ring hetero atom (e.g., O, S, N) and includes all of the fully saturated and partially unsaturated derivatives of the above mentioned heteroaryl groups. Exemplary heterocyclic groups include pyrrolidinyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, thiazolidinyl, isothiazolidinyl, and imidazolidinyl.
The aryl, heteroaryl, and heterocyclyl groups can be unsubstituted or substituted by one or more substituents independently selected from the group consisting of alkyl, alkoxy, methylenedioxy, ethylenedioxy, alkylthio, haloalkyl, haloalkoxy, haloalkylthio, halogen, nitro, hydroxy, mercapto, cyano, carboxy, formyl, aryl, aryloxy, arylthio, arylalkoxy, arylalkylthio, heteroaryl, heteroaryloxy, heteroarylthio, heteroarylalkoxy, heteroarylalkylthio, amino, alkylamino, dialkylamino, heterocyclyl, heterocycloalkyl, alkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, haloalkylcarbonyl, haloalkoxycarbonyl, alkylthiocarbonyl, arylcarbonyl, heteroarylcarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, arylthiocarbonyl, heteroarylthiocarbonyl, alkanoyloxy, alkanoylthio, alkanoylamino, arylcarbonyloxy, arylcarbonythio, alkylaminosulfonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aryldiazinyl, alkylsulfonylamino, arylsulfonylamino, arylalkylsulfonylamino, alkylcarbonylamino, alkenylcarbonylamino, arylcarbonylamino, arylalkylcarbonylamino, heteroarylcarbonylamino, heteroarylalkycarbonylamino, alkylsulfonylamino, alkenylsulfonylamino, arylsulfonylamino, arylalkylsulfonylamino, heteroarylsulfonylamino, heteroarylalkylsulfonylamino, alkylaminocarbonylamino, alkenylaminocarbonylamino, arylaminocarbonylamino, arylalkylaminocarbonylamino, heteroarylaminocarbonylamino, heteroarylalkylaminocarbonylamino and, in the case of heterocyclyl, oxo. If other groups are described as being "substituted" or "optionally substituted", then those groups can also be substituted by one or more of the above-enumerated substituents.
Certain substituents are generally preferred. For example, preferred R2 groups include hydrogen, alkyl groups having 1 to 4 carbon atoms (i.e., methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, and cyclopropylmethyl), and alkoxyalkyl groups (e.g., methoxy ethyl and ethoxymethyl). Preferably R3 and R4 are independently hydrogen or methyl or R3 and R4 join together to form a benzene ring, a pyridine ring, a 6- membered saturated ring or a 6-membered saturated ring containing a nitrogen atom. One or more of these preferred substituents, if present, can be present in any combination.
Regardless of whether the IRM moiety and the targeting moiety are coupled covalently or noncovalently, an immunomodulatory composition may include an intervening component such as, for example, a spacer arm or a solid support. Certain spacer arms such as, for example, those having a length of from about 20 A to about 100 A, may improve solubility of the composition, thereby increasing the level of IRM activity obtainable using the composition. Suitable spacers are commercially available
In some embodiments, an immunomodulatory composition may include a macromolecular support to which both the targeting moiety and the IRM moiety are attached. In certain embodiments, the macromolecular support may be a solid support. The IRM moiety, targeting moiety, or both may be covalently attached to the macromolecular support using a linking group such as those described above. The macromolecular support may include, for example, supports such as those described in United States Patent Publication Nos. US2004/0202720 and US2004/0258698 such as, for example, agarose beads, gold particles, etc. The methods of the present invention may be performed on any suitable subject.
Suitable subjects include but are not limited to animals such as but not limited to humans, non-human primates, rodents, dogs, cats, horses, pigs, sheep, goats, or cows.
Examples The following examples have been selected merely to further illustrate features, advantages, and other details of the invention. It is to be expressly understood, however, that while the examples serve this purpose, the particular materials and amounts used as well as other conditions and details are not to be construed in a matter that would unduly limit the scope of this invention.
In the examples below normal high performance flash chromatography (HPFC) was carried out using a HORIZON HPFC system (an automated high-performance flash purification product available from Biotage, Inc, Charlottesville, Virginia, USA) or an INTELLIFLASH Flash Chromatography System (an automated flash purification system available from AnaLogix, Inc, Burlington, Wisconsin, USA). The eluent used for each purification is given in the example. In some chromatographic separations, the solvent mixture 80/18/2 v/v/v chloroform/methanol/concentrated ammonium hydroxide (CMA) was used as the polar component of the eluent. In these separations, CMA was mixed with chloroform in the indicated ratio.
Preparation of the IRM Compounds
IRM Compound 1 (IRMl): N-{2-[4-Amino-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin- 1 -yl]- 1 , 1 -dimethylethyl} -6-[(3-mercaptopropanoyl)amino]hexanamide
Figure imgf000034_0001
Part A
To a solution of ό-(carbobenzyloxyamino) caproic acid (8.49 grams (g), 32.0 millimole (mmol)) in DMF (50 mL) at O0C was added N-hydroxysuccinimide and l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). After a short period, the solution was added to a O0C solution of l-(2~amino~2-methylpropyl)-2- (ethoxymethyl)-lH-imidazo[4,5-c]quinolin-4-amine (prepared as described in U.S. Patent Publication No. US2004/0091491, 10 g, 32 mmol) in DMF (100 niL). The mixture was allowed to warm to room temperature and was stirred for 3 days. The solution was diluted with water (400 mL) and extracted with ethyl acetate (3x). The organic layers were combined and washed with water (2x) and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by HPFC on silica gel three times (gradient elution with CMA in chloroform) to provide 4.90 g of benzyl 6-({2-[4-amino-2-(ethoxymethyl)-l/i-imidazo[4,5-c]quinolin-l-yl]-l,l- dimethylethyl}amino)-6-oxohexylcarbamate as a white foam.
Part B
A mixture of benzyl 6-({2-[4-amino-2-(ethoxymethyl)-lJi-imidazo[4,5-c]quinolin- l-yl]-l,l-dimethylethyl}amino)-6-oxohexylcarbamate (4.90 g, 8.75 mmol) and 10% palladium on carbon (0.5 g) in ethanol (100 mL) was hydrogenated on a Parr apparatus at 20-40 psi (1.4 x 105 - 2.8 x 105 Pa) for 1 day, during which time fresh hydrogen was introduced several times. The mixture was filtered through CELITE filter agent. The filtrate was concentrated under reduced pressure to yield a white foam that was used directly in the next step.
Part C To a mixture of 3,3'-dithiodipropionic acid (920 mg, 4.38 mmol) and 1- hydroxybenzotriazole (HOBT) (1.42 g, 10.5 mmol) in dimethylformamide (DMF) (50 mL) at O0C was added l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.85 g, 9.63 mmol). The mixture was stirred at O0C for 4 hours. The material from Part B (8.75 mmol) was dissolved in DMF (20 mL), cooled to 0°C, and the cold solution of the activated diacid was added in one portion, with two DMF rinses (10 mL each). The reaction was allowed to warm slowly to room temperature overnight. Several more portions of EDC were added to the reaction at O0C over the next two days. The reaction was allowed to stir at room temperature for several days more, then was diluted with water and saturated aqueous sodium bicarbonate and was extracted with ethyl acetate several times. The combined organic extracts were washed with water and brine, and were concentrated under reduced pressure. The crude product was purified by HPFC to give 3.8 g of the disulfide dimer of N-{2-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin- 1 -yl]- 1 , 1 -dimethylethyl} -6-[(3-mercaptopropanoyl)arnino]hexananiide.
Part D The material from Part C (3.8 g, 3.7 mmol) was dissolved in methanol (30 mL) at room temperature. Tris(2-carboxyethyl)phosphine hydrochloride (1.38 g, 4.81 mmol) was added, followed by water (3 mL), and 12.5 M aqueous sodium hydroxide (1.12 mL, 14.1 mmol). The solution was stirred at room temperature for 2 hours and then was cooled to 00C. The solution was adjusted to pH 6 with 1 M aqueous hydrochloric acid (approximately 14 mL). The methanol was removed under reduced pressure and aqueous sodium bicarbonate was added. The mixture was extracted with dichloromethane (3 x). The organic extracts were combined, washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by HPFC on silica gel (gradient elution with 0 - 50% CMA in chloroform). The appropriate fractions were concentrated to provide 2.36 g of iV-{2-[4-amino-2- (ethoxy methyl)- 1 /i-imidazo [4, 5 -c] quinolin- 1 -yl] - 1 , 1 -dimethylethyl } -6- [(3 - mercaptopropanoyl)amino]hexanamide.
White foam, MS (ESI) m/z 515 (M + H)+. 1U NMR (300 MHz, CDCl3) δ 8.22 (dd, IH), 7.80 (dd, IH), 7.51 (ddd, IH), 7.32 (ddd, IH), 5.96 (m, IH), 5.59 (s, IH), 5.51 (br s, 2H), 5.06 (s, 2H), 4.84 (br s, 2H), 3.62 (q, J= 6.9 Hz, 2H), 3.25 (q, J= 6.9 Hz, 2H), 2.81 (m, 2H), 2.50 (t, J= 6.9 Hz5 2H), 1.98 (m, 2H), 1.61-1.18 (m, 13H), 1.24 (t, J= 6.9 Hz, 3H). Anal, calcd for C26H38N6O3SO.5H2O: C, 59.63; H, 7.51; N, 16.05; S, 6.12. Found: C, 59.89; H, 7.66; N, 16.22; S, 6.25.
IRM Compound 2 (IRM2): N-{2-[4-Amino-2-(ethoxymethyl)-lH-imidazo[4,5- cjquinolin- 1 -yl]- 1 , 1 -dimethylethyl } -6- { [3 -(pyridin-2-yldithio)propanoyl] amino } hexanamide
Figure imgf000037_0001
A solution of N-{2-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]- l,l-dimethylethyl}-6-[(3-mercaptopropanoyl)amino]hexanamide (1.33 g, 2.58 mmol) in dichloromethane (16 mL) was added dropwise over 1.5 hours to a solution of 2,2'- dipyridyl disulfide (2.27 g, 10.3 mmol) in dichloromethane (10 mL). The solution was stirred at room temperature for 18 hours, then was concentrated under reduced pressure. The residue was purified by HPFC on silica gel twice (gradient elution with 1-10% methanol in dichloromethane) to yield 800 mg of N-{2-[4-amino-2-(ethoxymethyl)-l/f- imidazo [4,5-c]quinolin- 1 -yl] -1,1 -dimethylethyl } -6- { [3 -(pyridin-2- yldithio)propanoyl] amino }hexanamide as a white foam.
Alternatively, N- { 2- [4-amino-2-(ethoxymethyl)- 1 H-imidazo [4,5 -c] quinolin- 1 -yl]- 1 , 1 -dimethylethyl } -6- { [3 -(pyridin-2-yldithio)propanoyl]amino} hexanamide was synthesized from 6-amino-N-{2-[4-amino-2-(ethoxymethyl)-l/i-imidazo[4,5-c]quinolin-l- yl]-l,l-dimethylethyl}hexanamide hydrochloride in one step. A mixture of 6-amino-iV- {2- [4-amino-2-(ethoxymethyl)- 1 H-imidazo [4,5 -c]quinolin- 1 -yl] -1,1- dimethylethyl} hexanamide hydrochloride (68 mg, 0.16 mmol), triethylamine (0.046 mL, 0.32 mmol), and iV-succinimidyl 3-(2-pyridyldithio)propionate in tetrahydrofuran (1.6 mL) and DMF (0.5 mL) was stirred at room temperature for 5 hours. The reaction mixture was partitioned between water and ethyl acetate. The aqueous phase was extracted with ethyl acetate. The organic layers were combined, washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel to provide 36 mg of iV-{2-[4-amino-2- (ethoxyniethyO-l/f-imidazo^jS-cjquinolin-l-ylj-ljl-dimethylethylJ-ό-IP-^yridin^- yldithio)propanoyl] amino }hexanamide as a colorless oil.
MS (ESI) m/z 624 (M + H)+. 1H NMR (300 MHz5 CDCl3) δ 8.43 (m, IH)5 8.23 (m, IH)5 7.80 (m5 IH)5 7.63 (m, 2H)5 7.50 (m, IH), 7.32 (m, IH), 7.11 sextet, J= 4.4 Hz, 1 H)5 6.52 (m, IH)5 5.55 (s, IH)5 5.47 (br s, 2H), 5.07 (s, 2H)5 4.83 (br s, 2H), 3.62 (q, J= 6.9 Hz5
2H)5 3.27 (q, 2H)5 3.08 (t5 J- 6.9 Hz5 2H)5 2.61 (t5 2H), 2.04-1.99 (m, 2H), 1.67-1.22 (m, 12H)5 1.24 (t, J= 7.0 Hz5 3H). Anal, calcd for C3iH41N7O3S2«1.0 H2O: C, 58.01; H5 6.75; N, 15.28; S5 9.99. Found: C5 58.37; H5 6.69; N5 15.24; S, 9.99.
IRM Compound 3 (IRM3): N-[4-(4-Amino-2-butyl-lH-imidazo[4,5-c]quinolin-l- yl)butyl] -3 -mercaptopropanamide
Figure imgf000038_0001
Part A
Following a procedure similar to that described above in Part C of IRM Compound
1, l-(4-aminobutyl)-2-butyl-lH-imidazo[4,5-c]quinolin-4-amine (U.S. Patent No.
6,451,810 and references cited therein, 1.00 g, 3.21 mmol) was converted into 1.05 g of the disulfide dimer of iV-[4-(4-amino-2-butyl-l/i-imidazo[4,5-c]quinolin-l-yl)butyl]-3- mercaptopropanamide.
Part B
Following a procedure similar to that described above in Part D of IRM Compound 1, the disulfide dimer of N-[4-(4-amino-2-butyl-lH-imidazo[4,5-c]quinolin-l-yl)butyl]-3- mercaptopropanamide (0.78 g, 0.98 mmol) was converted into 600 mg of iV-[4-(4-amino- 2-butyl-lH-imidazo[4,5-c]quinolin-l-yl)butyl]-3-mercaptopropanamide after purification by HPFC on silica gel (gradient elution with 2-30% CMA in chloroform) White solid, mp 133.0-135.0°C. MS (ESI) m/z 400 (M + H)+. 1H NMR (SOO MHZ, CDCl3) δ 7.90 (dd, J= 8.1, 1.2 Hz, IH), 7.82 (dd, J= 8.1, 1.2 Hz, IH), 7.50 (m, IH)3 7.32 (m, IH), 5.50 (m, IH), 5.34 (br s, 2H), 4.49 (t, J= 7.5 Hz, 2H), 3.34 (q, J= 6.8 Hz, 2H), 2.90 (m, 2H), 2.77 (q, J= 6.9 Hz, 2H), 2.41 (t, J= 6.8 Hz, 2H), 2.03-1.82 (m, 4H), 1.73- 1.45 (m, 5H), 1.01 (t, J- 7.5 Hz, 3H). Anal, calcd for C2iH29N5OS: C, 63.13; H, 7.32; N, 17.53. Found: C, 63.14; H, 7.36; N, 17.84.
IRM Compound 4 (IRM4): N-[4-(4-Amino-2-butyl-lH-imidazo[4,5-c]quinolin-l- yl)butyl] -3 -(pyridin-2-y ldithio)propanamide
Figure imgf000039_0001
Part A
Following a procedure similar to that described above in Part A of IRM Compound 2, N- [4-(4-amino-2-butyl- 1 H-imidazo [4,5 -c]quinolin- 1 -yl)butyl] -3 -mercaptopropanamide (0.20 g, 0.50 mmol) was converted into 47 mg of iV-[4-(4-amino-2-butyl-l//-imidazo[4,5- c]quinolin-l-yl)butyl]-3-(pyridin-2-yldithio)propanamide.
Yellow glassy solid, mp 63.0-73.0°C. MS (ESI) m/z 509 (M + H)+. 1H NMR (300 MHz, CDCl3) δ 8.33 (m, IH), 7.89 (m, IH), 7.81 (m, IH), 7.61-7.46 (m, 3H), 7.30 (m, IH), 7.05 (m, IH), 6.77 (m, IH), 5.49 (br s, 2H), 4.47 (t, J= 7.5 Hz, 2H), 3.37 (q, J= 6.5 Hz, 2H), 3.04 (m, 2H), 2.88 (m, 2H), 2.55 (t, J= 6.5 Hz, 2H), 2.05-1.66 (m, 6H), 1.49 (sextet, J = 7.5 Hz, 2H), 0.99 (t, J= 7.5 Hz, 3H). Anal, calcd for C26H32N6OS2O1O H2O: C, 60.11; H, 6.44; N, 16.18. Found: C, 59.80; H, 6.23; N, 16.25.
IRM Compound 5 (IRM5): N-{2-[4-Amino-2-(ethoxymethyl)-l#-imidazo[4,5- c]quinolin- 1 -yl]- 1 , 1 -dimethylethyl} -4-hydrazino-4-oxobutanamide
Figure imgf000040_0001
Part A
Succinic anhydride (3.20 g, 32.0 mmol) was added to a 1000C solution of l-(2- amino-2-methylpropyl)-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-4-amine (prepared as described in U.S. Patent Publication No. US2004/0091491, 2.00 g, 6.39 mmol) in DMF (20 mL). After 2 days, the reaction mixture was concentrated under reduced pressure to give an off-white solid. The solid was stirred with 100 mL of dichloromethane and was isolated by filtration. The filtrate was concentrated, stirred with dichloromethane (25 mL), and filtered to yield additional solid. The combined solids were dried under vacuum to give 3.16 g of 4-({2-[4-(2,5-dioxopyrrolidin-l-yl)-2-(ethoxymethyl)-l//-imidazo[4,5- c]quinolin-l-yl]-l,l-dimethylethyl}amino)-4-oxobutanoic acid as a white solid that was used without further purification.
Part B
A solution of 4-({2-[4-(2,5-dioxopyrrolidin-l-yl)-2-(ethoxymethyl)-li:/- imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethyl}amino)-4-oxobutanoic acid (3.16 g, 6.39 mmol) in dichloromethane (100 mL) was treated with triethylamine (2.67 mL, 19.2 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.67 g, 19.2 mmol), ført-butylcarbazate (2.46 g, 19.2 mmol) and iV,N-dimethylpyridin-4-amine (78 mg, 0.64 mmol). The reaction mixture was stirred for 4 days and then was treated with 100 mL of water. The layers were separated and the aqueous portion was extracted with chloroform (50 mL). The combined organic layers were washed successively with water (50 mL) and brine (50 mL). The organic portion was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a white foam. The white foam was dissolved in dichloromethane (50 mL) and treated with ethylene diamine (1 mL). After stirring for 4 hours, the reaction mixture was treated with water (50 mL) and chloroform (50 mL) and the layers were separated. The aqueous portion was extracted with chloroform (2 x 25 mL). The combined organic layers were washed water (50 mL) and brine (50 mL). The organic layer was concentrated and purification of the crude product by chromatography on silica gel (gradient elution, 25%- 100% CMA in chloroform) followed by crystallization from dichloromethane gave 1.67 g of tert-butyl 2-[4-({2-[4- amino-2-(ethoxymethyl)-l/7-imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethyl}amino)-4- oxobutanoyljhydrazinecarboxylate as a white powder.
Part C
A solution of tert-butyl 2-[4-({2-[4-amino-2-(ethoxymethyl)-lH-imidazo[4,5- cjquinolin- 1 -yl]- 1 , 1 -dimethylethyl} amino)-4-oxobutanoyl]hydrazinecarboxylate (792 mg, 1.50 mmol) in dichloromethane (30 mL) was treated with trifluoroacetic acid (3 mL). After stirring for 2 hours, an additional trifluoroacetic acid (3 mL) was added to the reaction mixture and stirring was continued for 1 hour. The reaction mixture was concentrated under reduced pressure and the resulting syrup was dissolved in water. The solution was made basic by the addition concentrated ammonium hydroxide and then was extracted repeatedly with 10% methanol/chloroform. The combined organic portions were and dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude white solid was purified by chromatography on silica gel (gradient elution, 25%- 75% CMA in chloroform) gave 340 mg of N-{2-[4-amino-2-(ethoxymethyl)-l/f- imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethyl}-4-hydrazino-4-oxobutanamide. White solid, mp 203.0-204.6 °C. 1H NMR (300 MHz5 OMSO-d6) δ 8.98 (s, IH), 8.32 (d, J= 7.4 Hz, IH)3 7.74 (s, IH), 7.60 (dd, J= 8.3, 1.2, Hz, IH), 7.41 (m, IH), 7.23 (ddd, J= 8.2, 7.0, 1.2 Hz, IH), 6.59 (s, 2H), 4.99 (br s, 2H), 4.72 (br s, 2H), 4.16 (br s, 2H), 3.51 (q, J= 7.0 Hz, 2H), 2.34-2.24 (m, 4H), 1.20 (br s, 6H), 1.13 (t, J= 7.0 Hz, 3H); 13C NMR (125 MHz, OMSO-dβ) δ 172.3, 171.2, 152.4, 150.7, 145.8, 134.5, 127.0, 126.8, 126.7, 121.5, 121.0, 115.6, 65.8, 64.6, 55.1, 51.4, 31.8, 29.1, 25.9, 15.3; MS (ESI) m/z 428 (M + H)+; Anal. Calcd for C21H29N7O3.0.5 H2O: C, 57.78; H, 6.93; N, 22.46. Found: C, 58.00; H, 6.69; N, 22.36.
IRM Compound 6 (IRM6): N-{2-[4-Amino-2-(ethoxymethyl)-l/f-imidazo[4,5- c]quinolin-l-yl]-l,l-dimethylethyl}-6-{[3-(2,5-dioxo-2,5-dihydro-lH-ρyrrol-l- yl)propanoyl] amino } hexanamide
Figure imgf000042_0001
Part A
To a solution of 6-amino-N-{2-[4-amino-2-(ethoxymethyl)-l/f-imidazo[4,5- c]quinolm-l-yl]-l,l-dimethylethyl}hexanarnide (28 mg, 0.66 mmol, prepared as generally described in Part A-B of IRM Compound 1) in dichloromethane (0.5 rnL) at room temperature was added N-succinimidyl-3-maleimidopropionate (18 mg, 0.68 mmol). The mixture was shaken until the reagent dissolved, allowed to stand for 30 minutes, then concentrated under reduced pressure. The foam was purified by reverse phase HPLC using 0.05% formic acid/acetonitrile in 0.05% formic acid/water as the eluent to yield 11 mg of N- {2-[4-amino-2-(ethoxymethyl)- 1 H-imidazo[4,5 -cjquinolin- 1 -yl]- 1 , 1 - dimethylethyl}-6-{[3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)propanoyl] amino} hexanamide as the monoformate salt.
Off white glassy solid, MS (ESI) m/z 578 (M + H)+. 1H NMR (300 MHz, DMSO-d6) δ 8.34 (d, J= 7.5 Hz, IH), 8.22 (s, 2H), 7.90 (t, J= 5.3 Hz, IH), 7.65 (s, IH), 7.60 (dd, J= 8.4, 1.2 Hz, IH), 7.42 (m, IH)5 7.22 (m, IH), 7.00 (s, 2H), 6.67 (br s, 2H), 5.00 (br s, 2H), 4.74 (br, 2H), 3.59 (t, J= 7.3 Hz, 2H), 3.51 (q, J= 7.0 Hz, 2H), 2.98 (q, J= 6.3 Hz, 2H), 2.31 (t, J= 7.2 Hz, 2H), 2.04 (t, J= 7.4 Hz, 2H), 1.47 (m, 2H), 1.36 (m, 2H), 1.20 (m, 8H), 1.13 (t, J= 7.0 Hz, 3H).
IRM Compound 7 (IRM7): N-{6-[(4-Amino-2-(ethoxymethyl)-l-{2-methyl-2-
[(methylsulfonyl)amino]propyl}-lH-imidazo[4,5-c]quinolin-7-yl)oxy]hexyl}-4-azido-2- hydroxybenzamide
Figure imgf000043_0001
N-{2-[4-Amino-7-[(6-aminohexyl)oxy]-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin-l-yl]-l,l-dimethylethyl}methanesulfonamide (prepared as described in Parts A-J of Example 45 in WO 2005/032484, 457 mg, 0.903 mmol) was dissolved in anhydrous DMF (9 mL) and treated with N-hydroxysuccinamidyl-4-azidosalacylic acid (248 mg, 0.903 mmol). The mixture was stirred under a nitrogen atmosphere overnight. The reaction mixture was then concentrated under reduced pressure. The resulting syrup was dissolved in dichloromethane (25 mL) and then washed with water (4 x 25 mL) and brine. The organic portion was dried over sodium sulfate, filtered, and concentrated under reduced pressure. Chromatography on silica gel (5% methanol/chloroform) gave a sticky, white solid that was concentrated from a mixture of dichloromethane and hexanes to give a white solid. The material was dried under vacuum at 50 °C for several days to give 380 mg ofiV-{6-[(4-amino-2-(ethoxymethyl)-l-{2-methyl-2-[(methylsulfonyl)amino]propyl}- lH-imidazo[4,5-c]quinolin-7-yl)oxy]hexyl}-4-azido-2 -hydroxybenzamide as a white powder. 1H NMR (300 MHz, OMSO-d6) δ 8.54 (br s, IH), 8.16 (d, J= 9.1 Hz, IH), 7.86 (d, J= 8.5 Hz, IH), 7.06 (d, J= 2.6 Hz, IH), 7.00 (s, IH), 6.85 (dd, J= 9.1, 2.6 Hz, IH), 6.60 (dd, J = 8.5, 2.3 Hz, IH), 6.55 (d, J= 2.1 Hz, IH), 6.21 (s, 2H), 4.86 (s, 2H), 4.82 (s, 2H), 4.06 (t, J= 6.4 Hz, 2H), 3.57 (q, J= 7.0 Hz, 2H), 3.31 (q, J= 6.5 Hz, 2H), 2.96 (s, 3H), 1.78 (m, 2H), 1.60 (m, 2H), 1.52-1.39 (m, 4H), 1.29 (s, 6H), 1.15 (t, J= 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-^) δ 168.8, 162.1, 157.9, 152.5, 150.1, 147.3, 144.7, 135.0, 129.7, 125.3, 122.7, 112.6, 111.5, 110.0, 109.5, 108.2, 107.5, 67.6, 65.7, 65.1, 57.7, 54.6, 44.7, 39.3, 29.1, 29.0, 26.6, 25.8, 25.7, 15.3; MS m/z 668 (M + H)+; Anal, calcd for C31H4]N9O6S: C, 55.76; H, 6.19; N, 18.88; S, 4.80. Found: C, 55.87; H, 6.28; N, 18.18; S, 4.66.
IRM Compound 8 (IRM8): N-{6-[(4-Amino-l-{4-[(methylsulfonyl)amino]butyl}-2- propyl- 1 H-imidazo [4,5 -c]quinolin-7-yl)oxy]hexyl } -4-azido-2-hydroxybenzamide
Figure imgf000044_0001
N-(4- {4- Amino-7- [(6-aminohexy l)oxy] -2-propy 1- 1 H-imidazo [4, 5 -c] quinolin- 1 - yl}butyl)methanesulfonamide (prepared as described in Parts A-J of Example 47 in WO 2005/032484, 490 mg, 1.00 mmol) was dissolved in anhydrous DMF (10 mL) and treated with iV-hydroxysuccinamidyl-4-azidosalacylic acid (274 mg, 1.00 mmol) and the mixture was stirred under nitrogen overnight. The reaction mixture was then concentrated under reduced pressure. The resulting syrup was dissolved in dichloromethane (25 mL) and then washed with water (3 x 25 mL) and brine. The organic portion was dried over sodium sulfate, filtered, and concentrated under reduced pressure. Chromatography on silica gel (5% methanol/chloroform) gave a sticky, white solid. The material was dried under vacuum at 50 °C for several days to give 400 mg of N-{6-[(4-amino-l-{4- [(methylsulfonyl)amino]butyl}-2-propyl-l/i-imidazo[4,5-c]quinolin-7-yl)oxy]hexyl}-4- azido-2-hydroxybenzamide.
White powder. 1H NMR (300 MHz, DMS(W,?) δ 8.83 (t, J= 5.3 Hz, IH), 7.91 (d, J= 9.0 Hz, IH), 7.89 (d, J= 8.6 Hz, IH), 7.05 (d, J= 2.6 Hz, IH), 6.98 (t, J= 5.8 Hz5 IH), 6.89 (dd, J= 8.9, 2.6 Hz5 IH)5 6.64 (dd, J= 8.55 2.3 Hz, IH)5 6.57 (d, J= 2.3 Hz5 IH)5 6.38 (s, 2H)5 4.46 (t, J= 7.2 Hz, 2H)5 4.04 (t, J= 6.4 Hz, 2H), 3.30 (q, J= 6.5 Hz, 2H)5 2.98 (q, J= 6.4 Hz, 2H)5 2.87 (t, J= 7.4 Hz, 2H), 2.86 (s, 3H), 1.91-1.75 (m, 6H), 1.66-1.54 (m, 4H), 1.53-1.39 (m, 4H)5 1.03 (t, J= 7.4 Hz, 3H); 13C NMR (125 MHz5 DMS(W*) δ 168.7, 162.1, 157.7, 152.5, 152.2, 146.7, 144.7, 133.1, 129.8, 125.3, 121.4, 112.7, 112.2, 109.9, 109.2, 108.3, 107.5, 44.7, 44.2, 39.5, 39.3, 29.1, 29.0, 28.7, 27.4, 26.7, 26.6, 25.7, 21.3, 14.2; MS m/z 652 (M + H)+; Anal, calcd for C31H41N9O5S: C, 57.13; H, 6.34; N, 19.34; S, 4.92. Found: C5 56.79; H5 6.05; N5 19.09; S5 4.79.
IRM Compound 9 (IRM9): iV-{6-[(4-Amino-2-propyl[l,3]thiazolo[455-c]quinolin-7- yl)oxy]hexyl}-4-azido-2-hydroxybenzamide
Figure imgf000045_0001
7-[(6-Aminohexyl)oxy]-2-propyl[l ,3]thiazolo[4,5-c]quinolin-4-amine (prepared as described in Part A of Example 9 of U.S. Provisional Patent Application No. 60/733036, 350 mg, 0.978 mmol) was dissolved in anhydrous DMF (10 mL) and treated with N- hydroxysuccinamidyl-4-azidosalacylic acid (268 mg, 0.978 mmol) and the mixture was stirred under a nitrogen atmosphere for 3 hours. The reaction mixture was then concentrated under reduced pressure. The resulting syrup was dissolved in dichloromethane (50 mL) and then washed with water (4 x 25 niL) and brine. The organic portion was dried over sodium sulfate, filtered, and concentrated under reduced pressure. Chromatography on silica gel (3% methanol/chloroform saturated with ammonium hydroxide) gave a sticky, light-yellow solid that still contained traces of DMF. The material was dissolved in dichloromethane (25 mL) and then washed with water (3 x 25 mL). The organic portion was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give 330 mg of Λ/-{6-[(4-amino-2-propyl[l,3]thiazolo[4,5-c]quinolin- 7-yl)oxy]hexyl}-4-azido-2-hydroxybenzamide. Light-yellow foam, mp 127-128 °C. 1H NMR (300 MHz, DMSO-^) δ 13.12 (s, IH), 8.80 (t, J= 5.3 Hz, IH), 7.88 (d, J= 8.6 Hz, IH), 7.64 (d, J= 8.7 Hz, IH), 7.02 (d, J= 2.4 Hz, IH), 6.88 (dd, J= 8.8, 2.5 Hz, IH), 6.78 (s, 2H), 6.64 (dd, J= 8.5, 2.3 Hz, IH), 6.57 (d, J = 2.3 Hz, IH), 4.06 (t, J= 6.4 Hz, 2H), 3.29 (t, J= 6.9 Hz, 2H), 3.11 (t, J= 7.5 Hz, 2H) 1.86 (m, 2H), 1.77 (m, 2H), 1.58 (m, 2H), 1.51-1.35 (m, 4H), 1.02 (t, J= 7.3 Hz, 3H); 13C NMR (125 MHz, DMSO-^) δ 169.4, 168.8, 162.0, 159.8, 152.7, 146.9, 144.7, 139.8, 136.6, 129.8, 125.9, 113.8, 113.7, 113.1, 109.9, 108.4, 107.5, 68.2, 39.4, 35.6, 29.1, 29.0, 26.6, 25.6, 22.8, 13.6; MS m/z 520 (M + H)+; Anal, calcd for C26H29N7O3S: C, 60.10; H, 5.63; N, 18.87; S, 6.17. Found: C, 59.97; H, 5.34; N, 18.63; S, 6.21.
Control Compound 1 (CCl): iV-{2-[2-(Ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]- 1,1 -dimethylethyl } -6- [(3 -mercaptopropanoyl)amino]hexanamide
Figure imgf000046_0001
Part A 6-[(ført-Butoxycarbonyl)amino]hexanoic acid (6.81 g, 29.5 mmol), 1- hydroxybenzotriazole (3.98 g, 29.5 mmol), and l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDC) (5.66 g, 29.5 mmol) were added to a 0°C solution of 2-[2-(ethoxymethyl)-l/f-imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethylamine (which was prepared by acid-mediated deprotection of fert-butyl 2-[2-(ethoxymethyl)-lH- imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethylcarbamate, which is described in U.S. Patent Publication No. US2004/0091491, 8.00 g, 26.8 mmol) in DMF (80 mL). The solution was stirred at room temperature overnight. More 6-[(tert- butoxycarbonyl)amino]hexanoic acid and EDC were added and the solution was stirred for an additional hour. The solution was partitioned between aqueous sodium bicarbonate and ethyl acetate. The aqueous layer was extracted with ethyl acetate (2x). The organic layers were combined, washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel to provide 3.99 g of tert-butyl 6-({2-[2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin-l-yl]-l,l-dimethylethyl}amino)-6-oxohexylcarbamate as a foam.
Part B
Trifluoroacetic acid (30 mL) was added slowly to a solution of tert-butyl 6-({2-[2- (ethoxymethyl)-l/f-imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethyl}amino)-6- oxohexylcarbamate (3.99 g, 7.82 mmol) in dichloromethane (80 mL) at room temperature. After 1.5 hours, the solution was concentrated under pressure to afford an oil. The oil was dissolved in a small amount of water and concentrated ammonium hydroxide. The resulting basic mixture was extracted with dichloromethane multiple times. The combined organic extracts were dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford 3.4 g of 6-amino-N-{2-[2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l- yl]-l,l-dimethylethyl}hexanamide, which was used in the next step without further purification.
Part C
A solution of 6-amino-N-{2-[2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]- l,l-dimethylethyl}hexanamide (1.35 g, 3.28 mmol), 3,3'-dithiodipropionic acid (0.345 g, 1.64 mmol), 1 -hydroxybenzotriazole (0.443 g, 3.28 mmol), and 1 -(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloroide (0.692, 3.61 mmol) in DMF (10 mL) was stirred at room temperature overnight. The solution was concentrated under reduced pressure and partitioned between saturated aqueous sodium bicarbonate and ethyl acetate/methanol. The organic layers were combined, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by HPFC on silica gel (gradient elution with CMA/chloroform) to provide the disulfide dimer of N-{2- [2-(ethoxymethyl> 1 H-imidazo [4,5 -c]quinolin- 1 -yl] -1,1 -dimethylethyl} -6- [(3 - mercaptopropanoyl)amino]hexanamide.
Part D
N- {2-[2-(Ethoxymethyl)~ 1 /f-imidazo [4,5 -c]quinolin- 1 -yl] -1,1 -dimethylethyl } -6- [(3-mercaptopropanoyl)amino]hexanamide (2.14 g, 2.15 mmol) was dissolved in methanol (20 mL). Tris(2-carboxyethyl)phosphine (0.800 g, 2.79 mmol), water (2 mL), and 12.5 M NaOH (0.65 mL, 8.17 mmol) were added. The solution was allowed to stir for 1.5 hours at room temperature, then was cooled in an ice bath. The solution was adjusted to pH 6 with 1 M HCl and the resulting mixture was concentrated under reduced pressure to remove the methanol. The mixture was partitioned between saturated aqueous sodium bicarbonate and dichloromethane. The aqueous layer was extracted multiple times with dichloromethane. The organic phases were combined, washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide 1.69 g of N- {2- [2-(ethoxymethyl)- 1 /f-imidazo [4,5 -c]quinolin- 1 -yl]- 1 , 1 -dimethylethyl } -6- [(3 - rnercaptopropanoyl)amino]hexanamide as a white foam that was heated under vacuum to produce a glassy solid.
Colorless glassy solid. MS (ESI) m/z 500 (M + H)+. 1H NMR (300 MHz, CDCl3) δ 9.30 (s, IH), 8.52 (m, IH), 8.26 (m, IH), 7.72-7.62 (m, 2H), 5.84 (m, IH), 5.59 (br s, IH), 5.18 (br s, 2H), 4.91 (br s, 2H) 3.63 (m, 2H), 3.27 (m, 2H), 2.81 (m, 2H), 2.49 (t, J= 6.9 Hz, 2H), 2.05 (t, J= 7.5 Hz, 2H), 1.63-1.22 (m, 16H). Anal, calcd for C26H37N5O3S: C, 62.50; H, 7.46; N, 14.02; S, 6.42. Found: C, 62.23; H, 7.54; N, 13.90; S, 6.65.
Control Compound 2 (CC2): iV-{2-[2-(Ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]- 1 , 1 -dimethylethyl } -6- { [3 -(pyridin-2-yldithio)propanoyl] amino } hexanamide
Figure imgf000049_0001
iV-{2-[2-(Ethoxymethyl)-li/-imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethyl}-6- {[3-(pyridin-2-yldithio)propanoyl]amino}hexanamide (0.55 g) was prepared from iV-{2- [2-(ethoxymethyl)- 1 /f-imidazo[4,5 -cjquinolin- 1 -yl] -1,1 -dimethylethyl} -6- [(3 - mercaptopropanoyl)amino]hexanamide (0.83 g) using the procedure described for the preparation of IRM Compound 2. The final product was purified by HPFC on silica gel (gradient elution with 1-10% methanol in dichloromethane) and was isolated as a colorless glassy solid after heating under vacuum.
Colorless glassy solid. MS (ESI) m/z 609 (M + H)+. 1H NMR (300 MHz, CDCl3) δ 9.30 (s, IH), 8.53 (m, IH), 8.43 (m, IH), 8.26 (m, IH), 7.71-7.60 (m, 4H), 7.11 (m, IH), 6.50 (m, IH), 5.58 (br s, IH), 5.19 (br s, 2H), 4.90 (br s, 2H) 3.63 (q, J= 6.9 Hz, 2H), 3.28 (m, 2H), 3.07 (m, 2H), 2.60 (m, 2H), 2.07 (t, J= 7.5 Hz, 2H), 1.65-1.22 (m, 12H), 1.24 (t, J= 6.9 Hz, 3H). Anal, calcd for C31H4oN603S3«0.15CH3OH: C, 60.97; H, 6.67; N, 13.70; S, 10.43. Found: C, 60.57; H, 6.75; N, 13.61; S, 10.62.
Control Compound 3 (CC3): N-{2-[2-(Ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]- 1 , 1 -dimethylethyl } -4-hydrazino-4-oxobutanamide
Figure imgf000049_0002
Part A
DMF (10 niL) was added to a mixture of 2-[2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin-l-yl]-l,l-dimethylethylamine (which was prepared by acid-mediated deprotection of tert-butyl 2-[2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]-l,l- dimethylethylcarbamate, which is described in U.S. Patent Publication No. US2004/0091491, 1.00 g, 3.36 mmol) and succinic anhydride (0.336 g, 3.36 mmol) at room temperature. The mixture was sonicated briefly until a solution formed. The solution was allowed to stand at room temperature for 3 days and then was used in the next step.
Part B
The solution from Part A was cooled to 0°C and was tert-bntyl carbazate (0.489 g, 3.70 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.709 g, 3.70 mmol) were added. The mixture was allowed to warm to room temperature and was stirred overnight. More tert-butyl carbazate and l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride were added. After 1 hour, the solution was diluted with water (60 mL) and extracted with ethyl acetate (3 x 75 mL). The combined organic layers were washed with water, saturated aqueous sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to yield a yellow foam. Chloroform was added to the foam causing a fine white solid to form. The solid was isolated by filtration to provide 0.758 g of tert-butyl 2-[4-({2-[2- (ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethyl}amino)-4- oxobutanoyl]hydrazinecarboxylate.
Part C
Trifluoroacetic acid (3 mL) was added slowly to a solution of tert-butyl 2-[4-({2- [2-(ethoxymethyl)-l//-imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethyl}amino)-4- oxobutanoyljhydrazinecarboxylate (0.688 g, 1.34 mmol) in dichloromethane (7 mL). The solution was stirred for 2.5 hours, then was concentrated under reduced pressure. The trifluoroacetic acid salt of N-{2-[2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]-l5l- dimethylethyl}-4-hydrazino-4-oxobutanamide was applied to anion exchange resins, which were eluted with pyridine in methanol to provide iV-{2-[2-(ethoxymethyl)-lH"- imidazo[4,5-c]quinolin-l-yl]-l,l-dimethylethyl}-4-hydrazino-4-oxobutanamide as a free base, which was purified by HPFC on silica gel (gradient elution, 2-50% CMA in chloroform). The appropriate fractions were concentrated under reduced pressure to yield a foam that was heated under vacuum to afford 0.31 g of N-{2-[2-(ethoxymethyl)-l/i- imidazo [4,5 -c] quinolin- 1 -yl] -1,1 -dimethylethyl } -4-hydrazino-4-oxobutanamide as a glassy solid.
Glassy solid. MS (ESI) m/z 413 (M + H)+. 1H NMR (300 MHz, CDCl3) δ 9.30 (s, IH), 8.50 (m, IH), 8.26 (m, IH), 7.71-7.61 (m, 2H), 7.14 (br s, IH), 6.13 (br s, IH), 5.16 (brs, 2H), 4.90 (br s, 2H), 3.88 (br s, 2H), 3.63 (q, J= 6.9 Hz, 2H), 2.41 (s, 4H), 1.37 (br s, 6H), 1.24 (t, J= 6.9 Hz, 3H). Anal. Calcd for C21H28N6O3O^ H2O: C, 60.10; H, 6.92; N, 20.02. Found: C, 60.37; H, 7.01; N, 20.01.
Control Compound 4 (CC4): iV-{2-[2-(Ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]- 1,1 -dimethylethyl} -6- { [3-(2,5-dioxo-2,5-dihydro- lH-pyrrol- 1 -yl)propanoyl] amino} hexanamide
Figure imgf000051_0001
To a solution of 6-amino-iV-{2-[2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l- yl]- 1,1 -dimethylethyl} hexanamide (which was prepared as described above in Parts A and B of Control Compound 1, 163 mg, 0.40 mmol) in dichloromethane (4 niL) at room temperature was added iV-succinimidyl-3-maleimidopropionate (111 mg, 0.42 mmol). The mixture was shaken until the reagent dissolved and allowed to stand overnight. The mixture was diluted with dichloromethane (25 mL), washed with 2M aqueous ammonia (10 niL), dried over magnesium sulfate and concentrated under reduced pressure to provide 158 mg of N-{2-[2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-l-yl]-l,l- dimethylethyl} -6- { [3 -(2,5-dioxo-2,5-dihydro-lH-pyrrol- 1 - yl)propanoyl] amino } hexanamide.
Light yellow foam, MS (ESI) m/z 563 (M + H)+. 1H NMR (300 MHz, CDCl3) δ 9.30 (s, IH), 8.55 (d, J= 7.0 Hz, IH), 8.31 (m, IH), 7.69 (m, 2H), 6.68 (s, 2H), 5.79 (m, IH), 5.57 (br s, IH), 5.20 (m, 2H), 4.92 (m, 2H), 3.83 (t, J- 7.1 Hz, 2H), 3.63 (q, J= 6.9 Hz, 2H), 3.22 (q, J= 6.7 Hz, 2H), 2.52 (t, J= 7.2 Hz, 2H), 2.05 (t, J= 7.3 Hz, 2H), 1.55 (m, 12H), 1.25 (t, J= 7.0 Hz, 3H).
AdditionalThe IRM compounds used in the examples are shown in Table 1.
Table 1
Figure imgf000052_0001
PREPARATION OF MODIFIED ANTIBODIES
Materials used to prepare the antibody-IRM conjugates can be found in Table 2 below.
Table 2
Figure imgf000052_0002
Figure imgf000053_0001
Thiolation of Antibodies:
The antibody is adjusted to a concentration of 5 to 10 mg/mL in PBS, pH 7.4 containing 5 mM EDTA. A 5 mg/mL solution of 2-IT is prepared by dissolving it in PBS, pH 7.4 containing 5 mM EDTA. The 2-IT solution is slowly added to the antibodysolution at the desired molar excess while mixing, and incubated for one hour at room temperature. The thiolated antibody is purified by applying the mixture to a desalting column equilibrated with PBS, pH 7.2 containing 5 raM EDTA. 1 mL fractions are collected and the fractions containing thiolated antibody, determined by measuring the absorbance at a wavelength of 280 nm, are pooled together. Optionally, the level of activated antibody is determined using a thiol detection reagent, (for example Ellman's reagent).
Modification of Antibodies using heterobifunctional crosslinkers:
The antibody is adjusted to a concentration of 5 to 10 mg/mL in PBS, pH 7.4. The crosslinker (for example, sSMCC, SMPT, or NHS-PEO8-Maleimide) is dissolved in
DMSO to a concentration of 5 mg/mL. The crosslinker solution is slowly added to the antibody solution at the desired molar excess while mixing, and incubated for one hour at room temperature. The modified antibody is purified by applying the mixture to a desalting column equilibrated with PBS, pH 7.2 containing 5 mM EDTA. One-milliliter fractions are collected and the fractions containing modified IgG, determined by measuring the absorbance at a wavelength of 280 nm, are pooled together. Optionally, the level of activated antibody is determined using an analytical method appropriate for the crosslinker.
Conjugation of Antibodies to Photo-reactive IRJVl (pIRM)
The antibody was dissolved in PBS, pH 7.4 at a concentration of 5 to 10 mg/mL. In some instances, the antibody solution was adjusted to pH 10 with IN NaOH. The pIRM (e.g., IRMlO) was dissolved in DMSO. The pIRM solution was slowly added to the antibody solution at the desired molar excess while mixing. The mixture is added to a 12- well plate (500 mL/well) and a long-wave UV light (366 nm) is placed directly over the plate for 15 minutes while on ice. The reaction was quenched by adding 1 M Tris, pH 8.0 to at 1/20 the reaction volume (v/v). The IRM-antibody conjugate was purified by size exclusion chromatography using PBS, pH 7.4 as the column running buffer at a flow rate of 1 mL/min. One-milliliter fractions were collected and the absorbance of each fraction was measured at 280 nm. Fractions containing the IRM-antibody conjugate were pooled and filtered under sterile conditions through a 0.2-micron filter. The concentration of the IRM-antibody conjugate was determined by BCA assay using bovine gamma globulin as a standard. The filtered conjugate was stored at 4 0C for future testing in biological assays.
Dye Labeled Antibodies The antibody is adjusted to a concentration of 3 to 10 mg/niLin PBS, pH 7.4. The amine reactive dye (for example, Alexa 488) is dissolved in DMSO to a concentration of 5 mg/niL. The amine reactive dye solution is slowly added to the antibody solution at the desired molar excess (for example, 8-fold molar excess of dye to antibody) while mixing, and incubated for one hour at room temperature. The labeled antibody is purified by applying the mixture to a desalting column equilibrated with PBS, pH 7.4. 1 mL fractions are collected and the labeled antibody is pooled. The absorbance of the antibody-dye conjugate is measured at 280 nm and at the absorbance maximum for the dye (Amax) to determine the dye:antibody ratio. Alternately, size exclusion chromatography may be performed to purify the antibody-dye conjugate. The fractions containing dye labeled antibody are pooled together.
PREPARATION OF IRM- ANTIBODY CONJUGATES
Conjugation of thiolated Antibodies to Pyridyl Disulfide Modified IRM (pdIRM) The pdIRM (e.g., IRM2) is dissolved in DMSO at a concentration of 10 mg/mL.
The pdIRM is added to the thiolated antibody, as prepared above, at one half the molar excess of 2-IT used to thiolate the antibody. The mixture is incubated overnight at room temperature. To quench the reaction, a 500 mM L-cysteine solution (dissolved in 1 M Tris, pH 8.0) is added at 0.01 times (v/v) the reaction mixture. The IRM- antibody conjugate is purified by size exclusion chromatography using PBS, pH 7.4 as the column running bufferat a flow rate of 1 mL/min. 1 mL fractions are collected and the absorbance is measured at a wavelength of 280 nm. Fractions containing the IRM-antibody conjugate are pooled and filtered under sterile conditions through a 0.2-micron filter. The concentration of the IRM- antibody conjugate is determined by BCA assay using bovine gamma globulin as a standard. The filtered conjugate is stored at 4 0C for future testing in biological assays. Conjugation of Antibodies Modified with Heterobifunctional Crosslinkers to sulfhydryl modified IRMs (sIRM)
The sIRM (e.g., IRMl) is dissolved in DMSO at a concentration of 10 mg/mL. The sIRM is added to the modified antibody, as prepared above, at a four-fold molar excess of sIRM to the amount of crosslinker used to modify the antibody. The mixture is incubated overnight at room temperature. To quench the reaction, a 500 mM L-cysteine solution (dissolved in 1 M Tris, pH 8.0) is added at 0.01 times (v/v) the reaction mixture. The IRM- antibody conjugate is purified by size exclusion chromatography using PBS, pH 7.4 as the column running buffer, at a flow rate of 1 niL/min. 1 mL fractions of mixture are collected and measured at 280 run. Fractions containing the IRM-antibody conjugate are pooled and filtered under sterile conditions through a 0.2-micron filter. The concentration of the IRM-antibody conjugate is determined by BCA assay using bovine gamma globulin as a standard. The filtered conjugate is stored at 4 0C for future testing in biological assays.
Example 1
IRMl, IRM2, IRMlO, CCl and CC2 were conjugated to a human anti-CD20 antibody (RITUXAN, Genentech, San Francisco, CA). Conjugates prepared with IRMl and CCl used the general methods described above for conjugation of antibodies modified with heterobifunctional crosslinkers to sulfhydryl modified IRMs (sIRM). Specifically, the SMPT crosslinker was mixed with the antibody at a 12-fold molar excess of SMPT to antibody. Conjugates prepared with IRM2 and CC2 used the general methods described above for conjugation of thiolated antibodies to pyridyl disulfide modified IRM (pdIRM).. Specifically, the 2-IT was mixed with the antibody at a 60-fold molar excess of 2-IT to antibody. The antibody conjugate prepared with IRMl 0 used the photo-reactive methods described in the above general method. Specifically, IRMlO was mixed with the antibody at a 20-fold (20X) and 40-fold (40X) molar excess of IRMlO to antibody.
Whole blood from healthy human donors is collected by venipuncture into EDTA containing tubes. Peripheral blood mononuclear cells (PBMC) are separated from whole blood by density gradient centrifugation using HISTOPAQUE-1077 or Ficoll-Paque Plus. The PBMC layer is collected and washed twice with DPBS and resuspended in flow cytometry staining buffer (FACS buffer, Biosource). The PBMCs were added to a 96-well flat bottom sterile tissue culture plate (Costar, Cambridge, MA or Becton Dickinson Labware, Lincoln Park, NJ) to a final PBMC concentration of 1 x 106 cells/well. Anti- CD20 or the above prepared IRM/anti-CD20 antibody conjugates were added to each well at three fold dilutions from 9 μg/mL to 0.004 ug/mL, final concentration in combination with FcR blocking reagent (BD Pharmigen, San Diego, CA). The plate was incubated on ice for 15 minutes and then treated with 0.3-μg/mL anti-CD20-Alexa 488 per well. An Isotype (IgGl) negative control (15 μg/mL, Control, BD Pharmigen) and 0.3 μg/mL anti- CD20-Alexa 488 were placed in individual wells. The plate was incubated for 30 minutes on ice in the dark. The plate was centrifuged for 10 minutes at 1350 rpm, and cells were resuspended and washed with FACS buffer twice, resuspended in 200 μL FACS buffer and filtered through a multi-well filter plate (Pall Corporation). Cells were resuspended in 100 μL CYTOFIX buffer (BD Pharmingen) for 15 minutes at room temperature in the dark. Samples were stored overnight at 40C and run on a FACSCalibur (Becton Dickenson) the following day. Antibody activities of the conjugates are shown in Figures 1 through 6. Activity was measured by the conjugates ability to inhibit the anti-CD20-
Alexa 488 to bind to the cells.
Example 2
Whole blood from healthy human donors is collected by venipuncture into EDTA containing tubes. Peripheral blood mononuclear cells (PBMC) are separated from whole blood by density gradient centrifugation using HISTOPAQUE-1077 or Ficoll-Paque Plus. The PBMC layer is collected and washed twice with DPBS or HBSS and resuspended at 4 x 106 cells/mL in RPMI complete media. The PBMCs were added to a 96-well flat bottom sterile tissue culture plate (Costar, Cambridge, MA or Becton Dickinson Labware, Lincoln Park, NJ) to a final PBMC concentration of 2 x 106 cells/mL. PBMCs were stimulated overnight at 37°C in a 5% carbon dioxide atmosphere with 0.33 μM , 0.420 μM , 0.830 μM, or 1.67 μM anti-CD20 or the anti-CD20 conjugates prepared in Example 1, based on final antibody concentration. Culture supernatants were analyzed for IFN-α and TNF production using a human IFN-α ELISA (PBL Biomedical Laboratories, Piscataway, NJ) and human-specific TNF BV™ immunoassay (BioVeris Corp., Gaithersburg, MD), respectively, with results expressed in pg/mL. Cytokine induction by the conjugates is shown in Figure 7. Example 3
IRM2 and IRMlO were conjugated to a mouse anti-CD40 antibody (FGK4.5). The method used to conjugate anti-CD40 to IRM2 was the same as described in Example 1; however, a 70-fold molar excess of 2-IT to anti-CD40 was used in the preparation of the antibody conjugate. The method used to conjugate anti-CD40 to IRMlO was the same as described in Example 1; however, an 8-fold (8X) or 25-fold (25X) molar excess of IRMlO to anti-CD40 was used in the preparation of these antibody conjugates.
Mouse spleens were removed from sacrificed C57BL6 mice and splenocytes were isolated from the mice by homogenizing the spleens. Splenocytes were homogenized in Hanks Balance Salt Solution media (Biosource International, Camarillo, CA) containing 1% FCS, washed and resuspended in FACS buffer (Biosource International). Splenocytes were plated in a 96-well round bottom sterile tissue culture plate (Costar, Cambridge, MA or Becton Dickinson Labware, Lincoln Park, NJ) to a final cell concentration of 2 x 106 cells/well. Anti-CD40 or the above-prepared conjugate were added to each well at three fold dilutions from 300 μg/mL to 0.14 μg/mL, final concentration in combination with mouse FcR blocking reagent (2.4G2). The plate was incubated on ice for 15 minutes and then treated with 25-ug/mL anti-CD40-Alexa 488 per well. An Isotype (IgG2a) negative control (15 μg/mL, Control, BD Pharmigen) and 25 μg/mL anti-CD40-Alexa 488 were placed in individual wells. The plate was incubated for 30 minutes on ice in the dark. The plate was centrifuged for 10 minutes at 1500 rpm, and cells were resuspended and washed with FACS buffer twice, resuspended in 100 μL CYTOFIX buffer (BD Pharmigen) for 15 minutes at room temperature in the dark. Cells were washed and resuspended in 200 μL FACS buffer and filtered through a Multi-well filter plate. Samples were stored overnight at 40C and run on a FACSCalibur (Becton Dickenson) the following day. Antibody activities of the conjugates are shown in Figures 8 through 10. Activity was measured by the conjugates ability to inhibit the anti-CD40-Alexa 488 to bind to the cells.
Example 4 The anti-CD40 antibody and conjugate, as prepared in Example 3, were tested for cytokine induction as described in Example 2. Cytokine induction by the conjugate is shown in Figure 11. Example 5
IRM2 and IRMlO were conjugated to a mouse anti-CD8 antibody (53.6.72; ATCC, Manassas, VA). The methods used to conjugate anti-CD8 to IRM2 or IRMlO were the same as those described in Example 3. The conjugate was tested for antibody activity, as described in Example 3, using three fold dilutions of the conjugate from 12 μg/mL to 0.006 μg/mL and 0.40 μg/mL for the anti-CD8-Alexa 488. The anti-CD8 antibody and conjugates were also tested for cytokine induction as described in Example 2. Antibody activities of the conjugates are shown in Figures 12 through 14. Activity was measured by the conjugates ability to inhibit the anti-CD8-Alexa 488 to bind to the cells. Cytokine induction by the conjugate is shown in Figure 15.
Example 6
IRMlO was conjugated to the HERCEPTIN antibody (HER2; Genentech) using the same method that was described in Example 1 except that a 28.4-fold (pH 7.4), 28.4- fold (pH 10), or 42.6-fold (pH 10) molar excess of IRMlO to HER2 was used in the preparation of the antibody conjugate.
The HER2 antibody and conjugate were tested for antibody activity as described in Example 2; however, her2 positive human breast cancer cells (HCC2218; ATCC, Manassas, VA) were used in the assay (2.5 X lO5 cells/well) instead of human PBMCs. The conjugate was tested for antibody activity using three fold dilutions of the conjugate from 45 μg/mL to 0.007 μg/mL and 0.30 μg/mL for the HER2-Alexa 488. Antibody activities of the conjugates are shown in Figures 16 and 17. The HER2 antibody and conjugate were tested for cytokine induction as described in Example 2. Cytokine induction by the conjugate is shown in Figure 18.
Example 7
IRMl was conjugated to HER2 as described in Example 1; however, a NHS-PEO8- Maleimide crosslinker was mixed with the antibody at a 15-fold molar excess of NHS- PEO8-Maleimide to antibody.
The HER2 antibody and conjugate were tested for antibody activity as described in Example 6. Activity was measured by the conjugates ability to inhibit the HER2-Alexa 488 to bind to the cells. Antibody activities of the conjugates are shown in Figure 19. The HER2 antibody and conjugate were tested for cytokine induction as described in Example 2. Cytokine induction by the conjugate is shown in Figures 20 and 21.
Example 8
IRMl was conjugated to the anti-CD 8 antibody as described in Example 1; however, a NHS-PEOs-Maleimide crosslinker was mixed with the antibody at a 15-fold molar excess of NHS-PEO8-Maleimide to antibody.
The anti-CD8 antibody and conjugate were tested for cytokine induction as described in Example 2. Cytokine induction by the conjugate is shown in Figures 22 and 23.
Example 9
IRMlO was suspended in dimethyl sulfoxide (DMSO) to 10 mg/mL. Rat anti- mouse CD8 antibody (53.6.72, BioExpress, Inc., West Lebanon, NH) was suspended in phosphate buffered saline (PBS) to 7.8 mg/mL and the pH adjusted to > 9.0 by the addition of NaOH. A 1 :10 ratio of IRM: antibody (volume:volume) was mixed together by adding 60 μL of the IRMlO solution (0.6 mg IRMlIRMlO) with 540 μL of the anti-CD8 antibody solution (4.21 mg anti-CD8). The antibody control was 60 μL of PBS mixed with 540 μL of the anti-CD8 antibody solution. The 1 :10 IRM:antibody, 1 :100
IRM:antibody, and antibody control were each placed in single wells of a 24-well tissue culture plate. The plate was placed on ice and a long wavelength UV light source was placed directly over the plate as close to the well containing the IRMlO/antibody mixture as possible. The mixtures were irradiated for 15 minutes. The resulting conjugate and antibody control were removed from the wells and resuspended in PBS to a final concentration of 0.5 mg/mL IRMlO, 3.51 mg/mL anti-CD8; 0.05 mg/mL IRMlO, 3.86 mg/mL anti-CD8; and 3.51 mg/mL anti-CD8 for the 1 :10, 1:100, and antibody control, respectively, and dialyzed using a 10,000 molecular weight cutoff S lide-a-Lyzer (Pierce, Rockford, IL) against PBS to remove any unconjugated IRM. Example 10
Mouse spleens were removed from sacrificed C57BL6 mice and splenocytes were isolated from the mice by homogenizing the spleens. Splenocytes were homogenized in EHAA media (Biosource International, Camarillo, CA) containing 1% FCS, washed and resuspended in FACS buffer (Biosource International). Splenocytes were plated in a 96 well round bottom sterile tissue culture plate (Costar, Cambridge, MA or Becton Dickinson Labware, Lincoln Park, NJ) to a final cell concentration of 1 x 106 cells/well.
Splenocytes were treated for 30 minutes at 40C with the IRM:antibody (1 : 10), antibody control, both as prepared in Example 9, or non-UV treated antibody at five fold dilution concentrations ranging from 766 μg/mL to 0.002 μg/mL. After the 30-minute treatment time, FITC-labeled mouse anti-CD8 (53.6.7, BD Pharmingen, San Diego, CA) and PE-labeled mouse anti-CD3 (BD Pharmingen) were added to all the wells and incubated for 30 minutes at 4°C. Cells were then washed two times with FACS buffer and fixed with CYTOFIX buffer (BD Pharmingen, San Diego, CA). Flow cytometry analysis was performed by gating on the CD3+ lymphocytes and measuring the mean fluorescence intensity (MFI) of the FITC-labeled antibody. Results are shown in Figure 24.
Example 11
Whole blood from healthy human donors was collected by venipuncture into EDTA vacutainer tubes. Peripheral blood mononuclear cells (PBMC) were separated from whole blood by density gradient centrifugation using Histopaque®-1077. Blood was diluted 1 :1 with Dulbecco's Phosphate Buffered Saline (DPBS) or Hank's Balanced Salts Solution (HBSS). The PBMC layer was collected and washed twice with DPBS or HBSS and resuspended at 4 x 106 cells/mL in RPMI complete media. The PBMCs were added to a 96 well flat bottom sterile tissue culture plate (Costar, Cambridge, MA or Becton
Dickinson Labware, Lincoln Park, NJ) to a final PBMC concentration of 2 x 106 cells/mL. PBMCs were stimulated overnight at 370C in a 5% carbon dioxide atmosphere with the rat anti-mouse CD8 antibody alone or the 1 : 10 IRM/antibody conjugate as described in Example 9 in 3 -fold antibody dilutions (292-0.13 μg/mL). Culture supernatants were analyzed for IFN-α and TNF production using a human IFN-α ELISA (PBL Biomedical Laboratories, Piscataway, NJ) and human-specific TNF BV™ immunoassay (BioVeris Corp., Gaithersburg, MD), respectively, with results expressed in pg/mL. Results are shown in Figure 25 and Figure 26.
The complete disclosures of the patents, patent documents, and publications cited herein are incorporated by reference in their entirety as if each were individually incorporated. In case of conflict, the present specification, including definitions, shall control.
Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. Illustrative embodiments and examples are provided as examples only and are not intended to limit the scope of the present invention. The scope of the invention is limited only by the claims set forth as follows.

Claims

What is Claimed is:
1. An immunomodulatory composition comprising: an IRM moiety coupled to a targeting moiety.
2. The immunomodulatory composition of claim 1 wherein the IRM moiety is an agonist of at least one TLR.
3. The immunomodulatory composition of claim 1 further comprising a spacer arm or solid support to which the IRM moiety and the targeting moiety are attached.
4. The immunomodulatory composition of claim 1 wherein the spacer arm length is from about 20 A to about 100 A.
5. The immunomodulatory composition of claim 1 wherein the solid support comprises a particle having a diameter of from 1 nm to about 200 nm.
6. The immunomodulatory composition of claim 1 wherein IRM moiety and the targeting moiety are affinity coupled.
7. The immunomodulatory composition of claim 1 wherein IRM moiety and the targeting moiety are covalently coupled.
8. The immunomodulatory composition of claim 1 wherein the targeting moiety recognizes at least a portion of a tumor-specific antigen or marker.
9. The immunomodulatory composition of claim 8 wherein the targeting moiety recognizes at least a portion of at least one antigen or marker specific for breast cancer, colon cancer, pancreatic cancer, prostate cancer, lung cancer, prostate cancer, liver cancer, or melanoma.
10. The immunomodulatory composition of claim 1 wherein the targeting moiety comprises a ligand of a tumor-specific marker.
11. The immunomodulatory composition of claim 10 wherein the targeting moiety comprises a Leuteinizing hormone releasing hormone (LHRH) receptor ligand.
12. The immunomodulatory composition of claim 10 wherein the targeting moiety comprises a folic acid receptor ligand.
13. The immunomodulatory composition of claim 1 wherein the targeting moiety comprises bis-phosphonate.
14. The immunomodulatory composition of claim 1 wherein the targeting moiety recognizes at least a portion of at least one endothelial antigen or marker.
15. The immunomodulatory composition of claim 1 wherein the targeting moiety recognizes at least a portion of a dendritic cell surface antigen or marker.
16. The immunomodulatory composition of claim 1 wherein the targeting moiety recognizes at least a portion of a surface antigen or marker of a cell that, when activated, is capable of killing a tumor cell.
17. The immunomodulatory composition of claim 16 wherein the targeting moiety recognizes at least a portion of a surface antigen or marker of a cytotoxic T lymphocyte, an NKT cell, or an NK cell.
18. The immunomodulatory composition of claim 1 further comprising a second targeting moiety.
19. The immunomodulatory composition of claim 18 wherein one target specific moiety recognizes at least a portion of an antigen or marker specific for an immune cell and the second targeting moiety recognizes an antigen or marker specific for a tumor cell.
20. The immunomodulatory composition of claim 18 wherein one target specific moiety recognizes at least a portion of an antigen or marker specific for an immune cell and the second targeting moiety recognizes at least a portion of an endothelial antigen or marker.
21. A method of targeted delivery of an IRM compound, the method comprising: administering to a subject an immunomodulatory composition that includes an
IRM moiety coupled to a targeting moiety that recognizes a delivery target.
22. The method of claim 21 wherein the delivery target comprises a tumor cell.
23. The method of claim 21 wherein the delivery target comprises an immune cell.
24. A method of inducing a localized immune response, the method comprising: administering to a subject an immunomodulatory composition that includes an
IRM moiety coupled to a targeting moiety that recognizes a delivery target in an amount effective to induce an immune response.
25. The method of claim 24 wherein the delivery target comprises a tumor cell and the immune response is directed against the delivery target.
26. The method of claim 24 wherein the delivery target is an immune cell and the immune response is at least partially generated by the delivery target.
27. A method of treating a condition in a subject that is treatable by inducing an immune response, the method comprising: administering to the subject an immunomodulatory composition that includes an
IRM moiety coupled to a targeting moiety that recognizes a delivery target in an amount effective to treat at least one symptom or sign of the condition.
28. The method of claim 27 wherein the amount effective to treat at least one symptom or sign of the condition is an amount effective to ameliorate at least one symptom or sign of the condition.
29. The method of claim 27 wherein the amount effective to treat at least one symptom or sign of the condition is an amount effective to reduce an increase of at least one symptom or sign of the condition.
30. An immunomodulatory composition comprising: an IRM moiety coupled to a targeting moiety, wherein the IRM moiety is a compound of the formula:
Figure imgf000066_0001
wherein:
Ri is a linker group;
R2 is selected from the group consisting of:
-hydrogen;
-alkyl; -alkenyl;
-aryl;
-substituted aryl;
-heteroaryl;
-substituted heteroaryl; -alkyl-O-alkyl;
-alkyl-S-alkyl;
-alkyl-O-aryl;
-alkyl-S-aryl: -alkyl-O- alkenyl; -alkyl-S- alkenyl; and
-alkyl or alkenyl substituted by one or more substituents selected from the group consisting of: -OH;
-halogen; -N(R5)2; -CO-N(Rs)2; -CS-N(Rs)2; -SO2-N(Rs)2;
-NR5-CO-C1-I0 alkyl; -NR5-CS-C1-10 alkyl; -NR5- SO2-C1-10 alkyl; -CO-C1-I0 alkyl; -CO-O-Ci-10 alkyl;
-N3; -aryl;
-substituted aryl; -heteroaryl; -substituted heteroaryl;
-heterocyclyl; -substituted heterocyclyl; -CO-aryl;
-CO-(substituted aryl); -CO-heteroaryl; and
-CO-(substituted heteroaryl); R3 and R4 are each independently: -hydrogen; -halogen; -alkyl;
-alkenyl; -O-alkyl; -S-alkyl; and -N(R5)2; or when taken together, R3 and R4 form a fused aryl or heteroaryl group that is optionally substituted by one or more substituents selected from the group consisting of;
-halogen; -alkyl; -alkenyl; -O-alkyl; -S-alkyl; and
-N(R5)2; or when taken together, R3 and R4 form a fused 5 to 7 membered saturated ring, optionally containing one or more heteroatoms and optionally substituted by one or more substituents selected from the group consisting of;
-halogen; -alkyl; -alkenyl; -O-alkyl; -S-alkyl; and
-N(R5)2; and each R5 is independently hydrogen or C1-10 alkyl.
PCT/US2006/006387 2000-12-08 2006-02-23 Compositions and methods for targeted delivery of immune response modifiers WO2006091720A2 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002598144A CA2598144A1 (en) 2000-12-08 2006-02-23 Compositions and methods for targeted delivery of immune response modifiers
AU2006216669A AU2006216669A1 (en) 2000-12-08 2006-02-23 Compositions and methods for targeted delivery of immune response modifiers
EP06735874A EP1850850A4 (en) 2000-12-08 2006-02-23 Compositions and methods for targeted delivery of immune response modifiers
JP2007557149A JP2008531580A (en) 2000-12-08 2006-02-23 Compositions and methods for targeted delivery of immune response modifiers

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US25422900P 2000-12-08 2000-12-08
US65571305P 2005-02-23 2005-02-23
US60/655,713 2005-02-23
US11/360,071 US20060142202A1 (en) 2000-12-08 2006-02-23 Compositions and methods for targeted delivery of immune response modifiers

Publications (2)

Publication Number Publication Date
WO2006091720A2 true WO2006091720A2 (en) 2006-08-31
WO2006091720A3 WO2006091720A3 (en) 2007-08-23

Family

ID=55086882

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2006/006387 WO2006091720A2 (en) 2000-12-08 2006-02-23 Compositions and methods for targeted delivery of immune response modifiers

Country Status (6)

Country Link
US (1) US20060142202A1 (en)
EP (1) EP1850850A4 (en)
JP (1) JP2008531580A (en)
AU (1) AU2006216669A1 (en)
CA (1) CA2598144A1 (en)
WO (1) WO2006091720A2 (en)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014012479A1 (en) 2012-07-18 2014-01-23 Shanghai Birdie Biotech, Inc. Compounds for targeted immunotherapy
EP3085373A1 (en) 2006-02-22 2016-10-26 3M Innovative Properties Company Immune response modifier conjugates
US9827329B2 (en) 2014-01-10 2017-11-28 Birdie Biopharmaceuticals, Inc. Compounds and compositions for immunotherapy
US11046781B2 (en) 2016-01-07 2021-06-29 Birdie Biopharmaceuticals, Inc. Anti-HER2 combinations for treating tumors
US11053240B2 (en) 2017-04-27 2021-07-06 Birdie Biopharmaceuticals, Inc. 2-amino-quinoline derivatives
US11110178B2 (en) 2016-07-07 2021-09-07 The Board Of Trustees Of The Leland Standford Junior University Antibody adjuvant conjugates
US11130812B2 (en) 2014-09-01 2021-09-28 Birdie Biopharmaceuticals, Inc. Anti PD-L1 conjugates for treating tumors
US11136397B2 (en) 2016-01-07 2021-10-05 Birdie Pharmaceuticals, Inc. Anti-EGFR combinations for treating tumors
US11220552B2 (en) 2016-01-07 2022-01-11 Birdie Biopharmaceuticals, Inc. Anti-CD20 combinations for treating tumors
US11279761B2 (en) 2014-07-09 2022-03-22 Birdie Biopharmaceuticals, Inc. Anti-PD-L1 combinations for treating tumors
US11400164B2 (en) 2019-03-15 2022-08-02 Bolt Biotherapeutics, Inc. Immunoconjugates targeting HER2
US11517567B2 (en) 2017-06-23 2022-12-06 Birdie Biopharmaceuticals, Inc. Pharmaceutical compositions

Families Citing this family (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040265351A1 (en) 2003-04-10 2004-12-30 Miller Richard L. Methods and compositions for enhancing immune response
RU2006105101A (en) 2003-08-27 2007-10-10 3М Инновейтив Пропертиз Компани (US) Aryloxy and arylalkylene-substituted substituted imidazoquinolines
AU2004315876B2 (en) 2003-10-03 2011-05-26 3M Innovative Properties Company Pyrazolopyridines and analogs thereof
KR101154101B1 (en) 2003-10-03 2012-06-11 쓰리엠 이노베이티브 프로퍼티즈 컴파니 Alkoxy substituted imidazoquinolines
NZ547467A (en) 2003-11-25 2010-06-25 3M Innovative Properties Co Substituted imidazo ring system and methods
EP1755665A4 (en) * 2004-04-28 2010-03-03 3M Innovative Properties Co Compositions and methods for mucosal vaccination
US8541438B2 (en) 2004-06-18 2013-09-24 3M Innovative Properties Company Substituted imidazoquinolines, imidazopyridines, and imidazonaphthyridines
AU2005326708C1 (en) 2004-12-30 2012-08-30 3M Innovative Properties Company Substituted chiral fused [1,2]imidazo[4,5-c] ring compounds
JP5543068B2 (en) 2004-12-30 2014-07-09 スリーエム イノベイティブ プロパティズ カンパニー Chiral fused [1,2] imidazo [4,5-c] cyclic compound
KR101415864B1 (en) * 2005-01-28 2014-07-09 갈렌바이오 인코포레이티드 Immunologically active compositions
WO2006086449A2 (en) 2005-02-09 2006-08-17 Coley Pharmaceutical Group, Inc. Alkyloxy substituted thiazoloquinolines and thiazolonaphthyridines
AU2006338521A1 (en) 2005-02-09 2007-10-11 Coley Pharmaceutical Group, Inc. Oxime and hydroxylamine substituted thiazolo(4,5-c) ring compounds and methods
WO2006091394A2 (en) 2005-02-11 2006-08-31 Coley Pharmaceutical Group, Inc. Substituted imidazoquinolines and imidazonaphthyridines
AU2006223634A1 (en) 2005-02-23 2006-09-21 Coley Pharmaceutical Group, Inc. Hydroxyalkyl substituted imidazoquinolines
US8846710B2 (en) 2005-02-23 2014-09-30 3M Innovative Properties Company Method of preferentially inducing the biosynthesis of interferon
CA2598639A1 (en) 2005-02-23 2006-08-31 Coley Pharmaceutical Group, Inc. Hydroxyalkyl substituted imidazonaphthyridines
AU2006216798A1 (en) 2005-02-23 2006-08-31 Coley Pharmaceutical Group, Inc. Hydroxyalkyl substituted imidazoquinoline compounds and methods
AU2006287270A1 (en) 2005-09-09 2007-03-15 Coley Pharmaceutical Group, Inc. Amide and carbamate derivatives of N-{2-[4-amino-2- (ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}methanesulfonamide and methods
ZA200803029B (en) 2005-09-09 2009-02-25 Coley Pharm Group Inc Amide and carbamate derivatives of alkyl substituted /V-[4-(4-amino-1H-imidazo[4,5-c] quinolin-1-yl)butyl] methane-sulfonamides and methods
KR20080083270A (en) 2005-11-04 2008-09-17 콜레이 파마시티컬 그룹, 인코포레이티드 Hydroxy and alkoxy substituted 1h-imidazoquinolines and methods
WO2007106854A2 (en) 2006-03-15 2007-09-20 Coley Pharmaceutical Group, Inc. Hydroxy and alkoxy substituted 1h-imidazonaphthyridines and methods
WO2008005509A2 (en) * 2006-07-06 2008-01-10 Massachusetts Institute Of Technology Methods and compositions for altering biological surfaces
US8178539B2 (en) 2006-09-06 2012-05-15 3M Innovative Properties Company Substituted 3,4,6,7-tetrahydro-5H-1,2a,4a,8-tetraazacyclopenta[cd]phenalenes and methods
US20080149123A1 (en) 2006-12-22 2008-06-26 Mckay William D Particulate material dispensing hairbrush with combination bristles
KR20180099900A (en) 2010-05-26 2018-09-05 셀렉타 바이오사이언시즈, 인크. Dose selection of adjuvanted synthetic nanocarriers
EP3222621B1 (en) 2010-08-17 2023-03-08 3M Innovative Properties Company Lipidated immune response modifier compound and its medical use
US9994443B2 (en) 2010-11-05 2018-06-12 Selecta Biosciences, Inc. Modified nicotinic compounds and related methods
WO2012167088A1 (en) * 2011-06-03 2012-12-06 3M Innovative Properties Company Heterobifunctional linkers with polyethylene glycol segments and immune response modifier conjugates made therefrom
AU2012261959B2 (en) * 2011-06-03 2015-12-03 Solventum Intellectual Properties Company Hydrazino 1H-imidazoquinolin-4-amines and conjugates made therefrom
US20130039954A1 (en) 2011-07-29 2013-02-14 Selecta Biosciences, Inc. Control of antibody responses to synthetic nanocarriers
MA44334A (en) 2015-10-29 2018-09-05 Novartis Ag ANTIBODY CONJUGATES INCLUDING A TOLL-TYPE RECEPTOR AGONIST
WO2019118884A1 (en) * 2017-12-15 2019-06-20 Silverback Therapeutics, Inc. Antibody construct-drug conjugate for the treatment of hepatitis
EP3728255B1 (en) 2017-12-20 2022-01-26 3M Innovative Properties Company Amide substituted imidazo[4,5-c]quinoline compounds with a branched chain linking group for use as an immune response modifier
US11655269B2 (en) * 2018-03-03 2023-05-23 The University Of Tokyo Prodrug-type anticancer agent using cancer-specific enzymatic activity
JP2022539804A (en) * 2019-07-08 2022-09-13 パーデュー・リサーチ・ファウンデーション Compounds and methods for the treatment and prevention of fibrotic disease states and cancer
US11179473B2 (en) 2020-02-21 2021-11-23 Silverback Therapeutics, Inc. Nectin-4 antibody conjugates and uses thereof
JP2023532304A (en) 2020-07-01 2023-07-27 エーアールエス ファーマシューティカルズ オペレーションズ，インク． Anti-ASGR1 antibody conjugates and uses thereof
CN116368135A (en) 2020-07-08 2023-06-30 3M创新有限公司 N-1 branched imidazoquinolines, conjugates thereof and methods
AU2021366287A1 (en) 2020-10-20 2023-04-13 Kantonsspital St. Gallen Antibodies or antigen-binding fragments specifically binding to Gremlin-1 and uses thereof
WO2022130046A1 (en) 2020-12-16 2022-06-23 3M Innovative Properties Company N-1 branched imidazoquinolines, conjugates thereof, and methods
WO2023111726A1 (en) 2021-12-14 2023-06-22 3M Innovative Properties Company N-1 triazole substituted imidazoquinolines, conjugates thereof, and methods
WO2023203177A1 (en) 2022-04-20 2023-10-26 Kantonsspital St. Gallen Antibodies or antigen-binding fragments pan-specifically binding to gremlin-1 and gremlin-2 and uses thereof

Family Cites Families (94)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL73534A (en) * 1983-11-18 1990-12-23 Riker Laboratories Inc 1h-imidazo(4,5-c)quinoline-4-amines,their preparation and pharmaceutical compositions containing certain such compounds
ZA848968B (en) * 1983-11-18 1986-06-25 Riker Laboratories Inc 1h-imidazo(4,5-c)quinolines and 1h-imidazo(4,5-c)quinolin-4-amines
US5238944A (en) * 1988-12-15 1993-08-24 Riker Laboratories, Inc. Topical formulations and transdermal delivery systems containing 1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine
US5756747A (en) * 1989-02-27 1998-05-26 Riker Laboratories, Inc. 1H-imidazo 4,5-c!quinolin-4-amines
US4929624A (en) * 1989-03-23 1990-05-29 Minnesota Mining And Manufacturing Company Olefinic 1H-imidazo(4,5-c)quinolin-4-amines
US5037986A (en) * 1989-03-23 1991-08-06 Minnesota Mining And Manufacturing Company Olefinic 1H-imidazo[4,5-c]quinolin-4-amines
US4988815A (en) * 1989-10-26 1991-01-29 Riker Laboratories, Inc. 3-Amino or 3-nitro quinoline compounds which are intermediates in preparing 1H-imidazo[4,5-c]quinolines
WO1992006093A1 (en) * 1990-10-05 1992-04-16 Minnesota Mining And Manufacturing Company Process for the preparation of imidazo[4,5-c]quinolin-4-amines
US5389640A (en) * 1991-03-01 1995-02-14 Minnesota Mining And Manufacturing Company 1-substituted, 2-substituted 1H-imidazo[4,5-c]quinolin-4-amines
US5175296A (en) * 1991-03-01 1992-12-29 Minnesota Mining And Manufacturing Company Imidazo[4,5-c]quinolin-4-amines and processes for their preparation
US5268376A (en) * 1991-09-04 1993-12-07 Minnesota Mining And Manufacturing Company 1-substituted 1H-imidazo[4,5-c]quinolin-4-amines
US5266575A (en) * 1991-11-06 1993-11-30 Minnesota Mining And Manufacturing Company 2-ethyl 1H-imidazo[4,5-ciquinolin-4-amines
IL105325A (en) * 1992-04-16 1996-11-14 Minnesota Mining & Mfg Immunogen/vaccine adjuvant composition
US5395937A (en) * 1993-01-29 1995-03-07 Minnesota Mining And Manufacturing Company Process for preparing quinoline amines
DE69314318T2 (en) * 1993-04-27 1998-04-09 Agfa Gevaert Nv Method for inserting a water-soluble compound into a hydrophilic layer
US5352784A (en) * 1993-07-15 1994-10-04 Minnesota Mining And Manufacturing Company Fused cycloalkylimidazopyridines
KR100341341B1 (en) * 1993-07-15 2002-11-25 미네소타 마이닝 앤드 매뉴팩춰링 캄파니 IMIDAZO[4,5-c]PYRIDIN-4-AMINES
US6239116B1 (en) * 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
PT772619E (en) * 1994-07-15 2006-10-31 Univ Iowa Res Found OLIGONUCLEOTIDOS IMUNOMODULADORES
US6207646B1 (en) * 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
EP0772776B1 (en) * 1994-07-27 2000-03-22 Herbert Dr. Pilgrimm Superparamagnetic particles, process for their production and their use
US5482936A (en) * 1995-01-12 1996-01-09 Minnesota Mining And Manufacturing Company Imidazo[4,5-C]quinoline amines
US5547668A (en) * 1995-05-05 1996-08-20 The Board Of Trustees Of The University Of Illinois Conjugates of folate anti-effector cell antibodies
US5693811A (en) * 1996-06-21 1997-12-02 Minnesota Mining And Manufacturing Company Process for preparing tetrahdroimidazoquinolinamines
US5741908A (en) * 1996-06-21 1998-04-21 Minnesota Mining And Manufacturing Company Process for reparing imidazoquinolinamines
KR19990044383A (en) * 1996-07-03 1999-06-25 나가시마 카쭈시게, 노미야마 아키히코 Novel purine derivative
US6387938B1 (en) * 1996-07-05 2002-05-14 Mochida Pharmaceutical Co., Ltd. Benzimidazole derivatives
JP4391592B2 (en) * 1996-10-25 2009-12-24 スリーエム カンパニー Immune response modifying compounds for the treatment of TH2-mediated and related diseases
US5939090A (en) * 1996-12-03 1999-08-17 3M Innovative Properties Company Gel formulations for topical drug delivery
US6069149A (en) * 1997-01-09 2000-05-30 Terumo Kabushiki Kaisha Amide derivatives and intermediates for the synthesis thereof
US6406705B1 (en) * 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
US6426334B1 (en) * 1997-04-30 2002-07-30 Hybridon, Inc. Oligonucleotide mediated specific cytokine induction and reduction of tumor growth in a mammal
US6113918A (en) * 1997-05-08 2000-09-05 Ribi Immunochem Research, Inc. Aminoalkyl glucosamine phosphate compounds and their use as adjuvants and immunoeffectors
US6303347B1 (en) * 1997-05-08 2001-10-16 Corixa Corporation Aminoalkyl glucosaminide phosphate compounds and their use as adjuvants and immunoeffectors
ATE370740T1 (en) * 1997-05-20 2007-09-15 Ottawa Health Research Inst METHOD FOR PRODUCING NUCLEIC ACID CONSTRUCTS
DE69817393T2 (en) * 1997-11-28 2004-06-17 Sumitomo Pharmaceuticals Co., Ltd. NEW HETEROCYCLIC CONNECTIONS
UA67760C2 (en) * 1997-12-11 2004-07-15 Міннесота Майнінг Енд Мануфакчурінг Компані Imidazonaphthyridines and use thereof to induce the biosynthesis of cytokines
TW572758B (en) * 1997-12-22 2004-01-21 Sumitomo Pharma Type 2 helper T cell-selective immune response inhibitors comprising purine derivatives
US6110929A (en) * 1998-07-28 2000-08-29 3M Innovative Properties Company Oxazolo, thiazolo and selenazolo [4,5-c]-quinolin-4-amines and analogs thereof
JP2000119271A (en) * 1998-08-12 2000-04-25 Hokuriku Seiyaku Co Ltd 1h-imidazopyridine derivative
US20020058674A1 (en) * 1999-01-08 2002-05-16 Hedenstrom John C. Systems and methods for treating a mucosal surface
EP1140091B1 (en) * 1999-01-08 2005-09-21 3M Innovative Properties Company Formulations comprising imiquimod or other immune response modifiers for treating cervical dysplasia
US6558951B1 (en) * 1999-02-11 2003-05-06 3M Innovative Properties Company Maturation of dendritic cells with immune response modifying compounds
US6756382B2 (en) * 1999-06-10 2004-06-29 3M Innovative Properties Company Amide substituted imidazoquinolines
US6541485B1 (en) * 1999-06-10 2003-04-01 3M Innovative Properties Company Urea substituted imidazoquinolines
US6331539B1 (en) * 1999-06-10 2001-12-18 3M Innovative Properties Company Sulfonamide and sulfamide substituted imidazoquinolines
US6451810B1 (en) * 1999-06-10 2002-09-17 3M Innovative Properties Company Amide substituted imidazoquinolines
US6573273B1 (en) * 1999-06-10 2003-06-03 3M Innovative Properties Company Urea substituted imidazoquinolines
MXPA02001610A (en) * 1999-08-13 2003-07-21 Hybridon Inc MODULATION OF OLIGONUCLEOTIDE CpG-MEDIATED IMMUNE STIMULATION BY POSITIONAL MODIFICATION OF NUCLEOSIDES.
US6376669B1 (en) * 1999-11-05 2002-04-23 3M Innovative Properties Company Dye labeled imidazoquinoline compounds
US20040023870A1 (en) * 2000-01-21 2004-02-05 Douglas Dedera Methods of therapy and diagnosis using targeting of cells that express toll-like receptor proteins
GB0001704D0 (en) * 2000-01-25 2000-03-15 Glaxo Group Ltd Protein
WO2001070663A2 (en) * 2000-03-17 2001-09-27 Corixa Corporation Novel amphipathic aldehydes and their use as adjuvants and immunoeffectors
US6894060B2 (en) * 2000-03-30 2005-05-17 3M Innovative Properties Company Method for the treatment of dermal lesions caused by envenomation
US20020055517A1 (en) * 2000-09-15 2002-05-09 3M Innovative Properties Company Methods for delaying recurrence of herpes virus symptoms
AU3249802A (en) * 2000-12-08 2002-06-18 3M Innovative Properties Co Screening method for identifying compounds that selectively induce interferon alpha
US6677347B2 (en) * 2000-12-08 2004-01-13 3M Innovative Properties Company Sulfonamido ether substituted imidazoquinolines
US6525064B1 (en) * 2000-12-08 2003-02-25 3M Innovative Properties Company Sulfonamido substituted imidazopyridines
US6677348B2 (en) * 2000-12-08 2004-01-13 3M Innovative Properties Company Aryl ether substituted imidazoquinolines
US6660735B2 (en) * 2000-12-08 2003-12-09 3M Innovative Properties Company Urea substituted imidazoquinoline ethers
US6545017B1 (en) * 2000-12-08 2003-04-08 3M Innovative Properties Company Urea substituted imidazopyridines
US6545016B1 (en) * 2000-12-08 2003-04-08 3M Innovative Properties Company Amide substituted imidazopyridines
JP4331944B2 (en) * 2001-04-17 2009-09-16 大日本住友製薬株式会社 New adenine derivatives
CA2458876A1 (en) * 2001-08-30 2003-03-13 3M Innovative Properties Company Methods of maturing plasmacytoid dendritic cells using immune response modifier molecules
US20030139364A1 (en) * 2001-10-12 2003-07-24 University Of Iowa Research Foundation Methods and products for enhancing immune responses using imidazoquinoline compounds
ATE416771T1 (en) * 2001-11-16 2008-12-15 3M Innovative Properties Co N-Ä4-(4-AMINO-2-ETHYL-1H-IMIDAZOÄ4,5-CUCHINOLINE 1-YL)BUTYLUMETHANESULFONAMIDE, PHARMACEUTICAL COMPOSITION CONTAINING SAME AND USE THEREOF
AP2004003069A0 (en) * 2001-11-27 2004-06-30 Anadys Pharmaceuticals Inc 3-beta-d-ribofuranosynthiazolo [4-5-d] pyridimine nucleosides and uses thereof.
ES2312659T3 (en) * 2001-11-29 2009-03-01 3M Innovative Properties Company PHARMACEUTICAL FORMULATIONS THAT INCLUDE A MODIFIER OF THE IMMUNE RESPONSE.
US6677349B1 (en) * 2001-12-21 2004-01-13 3M Innovative Properties Company Sulfonamide and sulfamide substituted imidazoquinolines
US6525028B1 (en) * 2002-02-04 2003-02-25 Corixa Corporation Immunoeffector compounds
JP2005518433A (en) * 2002-02-22 2005-06-23 スリーエム イノベイティブ プロパティズ カンパニー Methods for reducing and treating UVB-induced immunosuppression
US7344700B2 (en) * 2002-02-28 2008-03-18 University Of Tennessee Research Corporation Radiolabeled selective androgen receptor modulators and their use in prostate cancer imaging and therapy
AU2003233519A1 (en) * 2002-05-29 2003-12-19 3M Innovative Properties Company Process for imidazo(4,5-c)pyridin-4-amines
JP2005538057A (en) * 2002-06-07 2005-12-15 スリーエム イノベイティブ プロパティズ カンパニー Ether-substituted imidazopyridine
AU2003299863B2 (en) * 2002-08-15 2009-09-24 3M Innovative Properties Company Immunostimulatory compositions and methods of stimulating an immune response
US6818650B2 (en) * 2002-09-26 2004-11-16 3M Innovative Properties Company 1H-imidazo dimers
AU2003287316A1 (en) * 2002-12-11 2004-06-30 3M Innovative Properties Company Assays relating to toll-like receptor activity
CA2510375A1 (en) * 2002-12-20 2004-07-15 3M Innovative Properties Company Aryl / hetaryl substituted imidazoquinolines
WO2004060319A2 (en) * 2002-12-30 2004-07-22 3M Innovative Properties Company Immunostimulatory combinations
WO2004071459A2 (en) * 2003-02-13 2004-08-26 3M Innovative Properties Company Methods and compositions related to irm compounds and toll-like receptor 8
JP2006519020A (en) * 2003-02-27 2006-08-24 スリーエム イノベイティブ プロパティズ カンパニー Selective regulation of TLR-mediated biological activity
WO2004078138A2 (en) * 2003-03-04 2004-09-16 3M Innovative Properties Company Prophylactic treatment of uv-induced epidermal neoplasia
US20040176367A1 (en) * 2003-03-07 2004-09-09 3M Innovative Properties Company 1-Amino 1H-imidazoquinolines
US7699057B2 (en) * 2003-03-13 2010-04-20 3M Innovative Properties Company Methods for treating skin lesions
EP1603476A4 (en) * 2003-03-13 2010-01-13 3M Innovative Properties Co Method of tattoo removal
JP4891066B2 (en) * 2003-03-13 2012-03-07 スリーエム イノベイティブ プロパティズ カンパニー How to improve skin quality
US20040192585A1 (en) * 2003-03-25 2004-09-30 3M Innovative Properties Company Treatment for basal cell carcinoma
EP1613956A2 (en) * 2003-03-25 2006-01-11 3M Innovative Properties Company Selective activation of cellular activities mediated through a common toll-like receptor
WO2004087153A2 (en) * 2003-03-28 2004-10-14 Chiron Corporation Use of organic compounds for immunopotentiation
WO2004108072A2 (en) * 2003-04-10 2004-12-16 3M Innovative Properties Company Delivery of immune response modifier compounds using metal-containing particulate support materials
US20040214851A1 (en) * 2003-04-28 2004-10-28 3M Innovative Properties Company Compositions and methods for induction of opioid receptors
NZ545536A (en) * 2003-09-05 2010-04-30 Anadys Pharmaceuticals Inc TLR7 ligands for the treatment of hepatitis C
WO2005065678A1 (en) * 2003-12-30 2005-07-21 3M Innovative Properties Company Immunomodulatory combinations
JP2007532572A (en) * 2004-04-09 2007-11-15 スリーエム イノベイティブ プロパティズ カンパニー Methods, compositions and preparations for delivering immune response modifiers

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of EP1850850A4 *

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3085373A1 (en) 2006-02-22 2016-10-26 3M Innovative Properties Company Immune response modifier conjugates
US10548988B2 (en) 2012-07-18 2020-02-04 Birdie Biopharmaceuticals, Inc. Compounds for targeted immunotherapy
CN103566377A (en) * 2012-07-18 2014-02-12 上海博笛生物科技有限公司 Targeted immunotherapy for cancer
EP2874651A4 (en) * 2012-07-18 2016-04-27 Shanghai Birdie Biotech Inc Compounds for targeted immunotherapy
WO2014012479A1 (en) 2012-07-18 2014-01-23 Shanghai Birdie Biotech, Inc. Compounds for targeted immunotherapy
US9878052B2 (en) 2012-07-18 2018-01-30 Birdie Biopharmaceuticals Inc. Compounds for targeted immunotherapy
US10660971B2 (en) 2012-07-18 2020-05-26 Birdie Biopharmaceuticals, Inc. Compounds for targeted immunotherapy
US10548985B2 (en) 2014-01-10 2020-02-04 Birdie Biopharmaceuticals, Inc. Compounds and compositions for treating EGFR expressing tumors
TWI691338B (en) * 2014-01-10 2020-04-21 英屬開曼群島商博笛生物科技有限公司 Compounds and compositions for treating her2 positive tumors
TWI691337B (en) * 2014-01-10 2020-04-21 英屬開曼群島商博笛生物科技有限公司 Compounds and compositions for treating egfr expressing tumors
US10328158B2 (en) 2014-01-10 2019-06-25 Birdie Biopharmaceuticals, Inc. Compounds and compositions for immunotherapy
US10744206B2 (en) 2014-01-10 2020-08-18 Birdie Biopharmaceuticals, Inc. Compounds and compositions for immunotherapy
US10780180B2 (en) 2014-01-10 2020-09-22 Birdie Biopharmaceuticals, Inc. Compounds and compositions for immunotherapy
US9827329B2 (en) 2014-01-10 2017-11-28 Birdie Biopharmaceuticals, Inc. Compounds and compositions for immunotherapy
US11786604B2 (en) 2014-01-10 2023-10-17 Birdie Biopharmaceuticals, Inc. Compounds and compositions for treating HER2 positive tumors
US11633495B2 (en) 2014-01-10 2023-04-25 Birdie Biopharmaceuticals, Inc. Compounds and compositions for immunotherapy
US11633494B2 (en) 2014-01-10 2023-04-25 Birdie Biopharmaceuticals, Inc. Compounds and compositions for immunotherapy
US11279761B2 (en) 2014-07-09 2022-03-22 Birdie Biopharmaceuticals, Inc. Anti-PD-L1 combinations for treating tumors
US11130812B2 (en) 2014-09-01 2021-09-28 Birdie Biopharmaceuticals, Inc. Anti PD-L1 conjugates for treating tumors
US11046781B2 (en) 2016-01-07 2021-06-29 Birdie Biopharmaceuticals, Inc. Anti-HER2 combinations for treating tumors
US11220552B2 (en) 2016-01-07 2022-01-11 Birdie Biopharmaceuticals, Inc. Anti-CD20 combinations for treating tumors
US11136397B2 (en) 2016-01-07 2021-10-05 Birdie Pharmaceuticals, Inc. Anti-EGFR combinations for treating tumors
US11702476B2 (en) 2016-01-07 2023-07-18 Birdie Biopharmaceuticals, Inc. Anti-EGFR combinations for treating tumors
US11547761B1 (en) 2016-07-07 2023-01-10 The Board Of Trustees Of The Leland Stanford Junior University Antibody adjuvant conjugates
US11110178B2 (en) 2016-07-07 2021-09-07 The Board Of Trustees Of The Leland Standford Junior University Antibody adjuvant conjugates
US11053240B2 (en) 2017-04-27 2021-07-06 Birdie Biopharmaceuticals, Inc. 2-amino-quinoline derivatives
US11834448B2 (en) 2017-04-27 2023-12-05 Birdie Biopharmaceuticals, Inc. 2-amino-quinoline derivatives
US11517567B2 (en) 2017-06-23 2022-12-06 Birdie Biopharmaceuticals, Inc. Pharmaceutical compositions
US11400164B2 (en) 2019-03-15 2022-08-02 Bolt Biotherapeutics, Inc. Immunoconjugates targeting HER2

Also Published As

Publication number Publication date
AU2006216669A1 (en) 2006-08-31
EP1850850A4 (en) 2011-06-15
JP2008531580A (en) 2008-08-14
WO2006091720A3 (en) 2007-08-23
EP1850850A2 (en) 2007-11-07
US20060142202A1 (en) 2006-06-29
CA2598144A1 (en) 2006-08-31

Similar Documents

Publication Publication Date Title
US20060142202A1 (en) Compositions and methods for targeted delivery of immune response modifiers
US10144735B2 (en) Immune response modifier compositions and methods
US10472420B2 (en) Immune response modifier conjugates
JP6921745B2 (en) Anti-PD-L1 conjugate for treating tumors
JP6957154B2 (en) Compounds and compositions for immunotherapy
JP5491364B2 (en) Immunostimulatory composition and method of stimulating immune response
JP2020007346A (en) Compounds for targeted immunotherapy
AU2005244260B2 (en) Methods, compositions, and preparations for delivery of immune response modifiers

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2598144

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2006735874

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2007557149

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2006216669

Country of ref document: AU

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2006216669

Country of ref document: AU

Date of ref document: 20060223

Kind code of ref document: A