WO2018213747A1 - Molécules bispécifiques agonistes de 4-1bb et de cd40 - Google Patents

Molécules bispécifiques agonistes de 4-1bb et de cd40 Download PDF

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Publication number
WO2018213747A1
WO2018213747A1 PCT/US2018/033456 US2018033456W WO2018213747A1 WO 2018213747 A1 WO2018213747 A1 WO 2018213747A1 US 2018033456 W US2018033456 W US 2018033456W WO 2018213747 A1 WO2018213747 A1 WO 2018213747A1
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amino acid
agonist
seq
bispecific molecule
moiety
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PCT/US2018/033456
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English (en)
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Klaus RAUE
Eric M. Tam
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Merrimack Pharmaceuticals, Inc.
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Publication of WO2018213747A1 publication Critical patent/WO2018213747A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen

Definitions

  • Targeted treatment of diseases, such as cancer that benefit from enhanced immune responses and T-cell response and compositions that comprise a 4-1 BB agonist moiety connected to a dendritic cell binding moiety (e.g. , a 4-1 BB agonist bispecific molecule) or that comprise a CD-40 agonist moiety connected to a dendritic cell binding moiety (e.g. , a CD40 agonist bispecific molecule).
  • a 4-1 BB agonist moiety connected to a dendritic cell binding moiety e.g. , a 4-1 BB agonist bispecific molecule
  • CD-40 agonist moiety connected to a dendritic cell binding moiety
  • T-cells recognize peptide antigens in the context of the major histocompatibility complex (MHC) molecule complexes depending on the specificity of their T-cell antigen receptor (TCR).
  • MHC major histocompatibility complex
  • T-cells require an additional co-stimulatory signal to ensure full activation, clonal expansion and concomitant effector differentiation. This co-stimulatory signal can also be found at the interface of the antigen presenting cells (APC) and the T-cells and involve several members of TNF
  • 4-1 BB also known as CD137 or "tumor necrosis factor superfamily, member 9" (TNFRSF9)
  • TNFRSF9 tumor necrosis factor superfamily, member 9
  • 4-1 BB signaling is critical for formation of immunological memory and T-cell proliferation.
  • 4-1 BB signaling induces maturation of dendritic cells and production of
  • CD40 (TNFRSF5) is a stimulatory receptor that has been found to be essential in mediating a broad variety of immune and inflammatory responses, including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation. CD40 also regulates activity of dendritic cells (DCs), macrophages and B cells (see, e.g. , Beatty GL, et al. , Expert Rev. Anticancer Ther. 2017 Feb; 17(2): 175- 186). CD40 signals can promote survival in these cell types and induce production of inflammatory cytokines in macrophages and DCs. CD40 can also upregulate molecules involved in antigen presentation and T cell stimulation. CD40 is constitutively expressed on B cells, dendritic cells and macrophages.
  • the present invention provides such improved strategies.
  • the present invention relates to compositions that include a 4-1 BB agonist moiety connected to a dendritic cell binding moiety (e.g. , a 4-1 BB agonist bispecific molecule) and method for the therapeutic use of the 4-1 BB agonist bispecific molecules.
  • the present invention also relates to compositions that include a CD40 agonist moiety connected to a dendritic cell binding moiety (e.g. , a CD40 agonist bispecific molecule) and method for the therapeutic use of the CD40 agonist bispecific molecules.
  • the present disclosure provides 4-1 BB agonist bispecific molecules comprising a 4-1 BB agonist moiety; and a dendritic cell binding moiety.
  • 4-1 BB agonist bispecific molecule is used interchangeably with the term "bispecific molecule”.
  • the 4-1 BB agonist moiety is connected to the dendritic cell binding moiety by a linker.
  • the linker is a polypeptide linker that is 15-20 amino acid residues in length.
  • the 4-1 BB agonist moiety is an anti-4-1 BB agonist antibody or a 4-1 BB binding fragment thereof, a 4-1 BBL moiety, or a 4-1 BB agonist aptamer.
  • the 4-1 BBL moiety comprises two or more 4-1 BBL domains where the 4-1 BBL moiety comprises intra-domain linkers connecting the two or more 4-1 BBL domains.
  • the 4-1 BBL moiety comprises three 4-1 BBL domains where the 4-1 BBL moiety comprises intra-domain linkers connecting the two or more 4-1 BBL domains.
  • intra-domain linkers are polypeptide linkers that are each 15- 20 amino acid residues in length.
  • the bispecific molecule comprising one or more 4-1 BB domains, each 4-1 BBL domain, independently, comprises an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: (a) residues 50-254 of SEQ I D NO: 4; (b) residues 71-254 of SEQ I D NO: 4; and (c) residues 85-254 of SEQ
  • the bispecific molecule comprising one or more 4- 1 BB domains, each 4-1 BBL domain, independently, comprises an amino acid sequence identical to a sequence selected from the group consisting of: (a) residues 50-254 of SEQ I D NO: 4; (b) residues 71-254 of SEQ I D NO: 4; and (c) residues 85-254 of SEQ I D NO: 4.
  • the present disclosure also provides for bispecific molecules where the dendritic cell binding moiety binds to a dendritic cell surface antigen.
  • the bispecific molecule comprises a 4-1 BB agonist moiety and the dendritic cell surface antigen is LI LRA4, LAMP5, CLEC4C, I L3RA, CLEC9A, XCR1 , FLT3, or SIGLEC6.
  • the bispecific molecule comprises a 4-1 BB agonist moiety and the dendritic cell surface antigen is human LI LRA4 (SEQ I D NO: 15), human LAMP5 (SEQ I D NO: 17), human CLEC4C (SEQ I D NO: 18), human I L3RA (SEQ I D NO: 19), human CLEC9A (SEQ I D NO: 21), human XCR1 (SEQ ID NO: 22), human FLT3 (SEQ I D NO: 25), or human SIGLEC6 (SEQ I D NO: 27).
  • human LI LRA4 SEQ I D NO: 15
  • human LAMP5 SEQ I D NO: 17
  • human CLEC4C SEQ I D NO: 18
  • human I L3RA SEQ I D NO: 19
  • human CLEC9A SEQ I D NO: 21
  • human XCR1 SEQ ID NO: 22
  • human FLT3 SEQ I D NO: 25
  • human SIGLEC6 SEQ I D NO: 27
  • the bispecific molecule of the present disclosure comprises a 4-1 BB agonist moiety and the dendritic cell binding moiety is an antibody that binds to the dendritic cell surface antigen or an antigen binding fragment thereof, or an aptamer that binds to the antigen.
  • the bispecific molecule of the present disclosure comprises a 4-1 BB agonist moiety and the dendritic cell surface antigen is a receptor and the dendritic cell binding moiety is a ligand of the receptor.
  • the disclosure provides a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of a 4-1 BB agonist bispecific molecule according to the disclosure.
  • the contacting is in vitro.
  • the contacting is in vivo.
  • the T-cell is a tumor- associated T-cell.
  • the method is a method for treating cancer in a subject in need thereof, the method comprising administering to the patient an effective amount of a bispecific molecule according to the present disclosure.
  • the cancer is a solid tumor.
  • the 4-1 BB bispecific molecule is administered in
  • the present disclosure provides CD40 agonist bispecific molecules comprising a CD40 agonist moiety; and a dendritic cell binding moiety.
  • CD40 agonist bispecific molecule is used interchangeably with the term "bispecific molecule”.
  • the CD40 agonist moiety is connected to the dendritic cell binding moiety by a linker.
  • the linker is a polypeptide linker that is 15-20 amino acid residues in length. Also provided herein are methods of treating disease (e.g. , cancer or autoimmune disease) in a human patient by administering to the patient an effective amount of a CD40 agonist bispecific molecules described herein.
  • disease e.g. , cancer or autoimmune disease
  • the CD40 agonist moiety is an anti- CD40 agonist antibody or a CD40 binding fragment thereof, a CD40L moiety, or an agonist aptamer.
  • the CD40L moiety comprises two or more CD40L domains where the CD40L moiety comprises intra-domain linkers connecting the two or more CD40L domains.
  • the CD40L moiety comprises three CD40L domains where the CD40L moiety comprises intra-domain linkers connecting the two or more CD40L domains.
  • intra-domain linkers are polypeptide linkers that are each 15- 20 amino acid residues in length.
  • the bispecific molecule comprising one or more CD40L domains, each CD40L domain, independently, comprises an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: (a) amino acid residues 47-261 of SEQ I D NO: 32; (b) amino acid residues 108-261 of SEQ I D NO: 32; and (c) amino acid residues 1 16-261 of SEQ I D NO: 32.
  • the bispecific molecule comprising one or more CD40L domains, each CD40L domain, independently, comprises an amino acid sequence selected from the group consisting of: (a) amino acid residues 47-261 of SEQ I D NO: 32; (b) amino acid residues 108-261 of SEQ I D NO: 32; and (c) amino acid residues 1 16-261 of SEQ I D NO: 32.
  • the present disclosure also provides for bispecific molecules where the dendritic cell binding moiety binds to a dendritic cell surface antigen.
  • the bispecific molecule comprises a CD40 agonist moiety and the dendritic cell surface antigen is LI LRA4, LAMP5, CLEC4C, I L3RA, CLEC9A, XCR1 , FLT3, or SIGLEC6.
  • the bispecific molecule comprises a CD40 agonist moiety and the dendritic cell surface antigen is human LI LRA4 (SEQ I D NO: 15), human LAMP5 (SEQ I D NO: 17), human CLEC4C (SEQ I D NO: 18), human I L3RA (SEQ I D NO: 19), human CLEC9A (SEQ I D NO: 21), human XCR1 (SEQ ID NO: 22), human FLT3 (SEQ I D NO:25), or human SIGLEC6 (SEQ I D NO: 27).
  • human LI LRA4 SEQ I D NO: 15
  • human LAMP5 SEQ I D NO: 17
  • human CLEC4C SEQ I D NO: 18
  • human I L3RA SEQ I D NO: 19
  • human CLEC9A SEQ I D NO: 21
  • human XCR1 SEQ ID NO: 22
  • human FLT3 SEQ I D NO:25
  • human SIGLEC6 SEQ I D NO: 27
  • the bispecific molecule of the present disclosure comprises a CD40 agonist moiety and the dendritic cell binding moiety is an antibody that binds to the dendritic cell surface antigen or an antigen binding fragment thereof, or an aptamer that binds to the antigen.
  • the bispecific molecule of the present disclosure comprises a CD40 agonist moiety and the dendritic cell surface antigen is a receptor and the dendritic cell binding moiety is a ligand of the receptor.
  • the disclosure provides a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of a CD40 agonist bispecific molecule according to the disclosure.
  • the contacting is in vitro.
  • the contacting is in vivo.
  • the T-cell is a tumor- associated T-cell.
  • the method is a method for treating cancer in a subject in need thereof, the method comprising administering to the patient an effective amount of a bispecific molecule according to the present disclosure.
  • the cancer is a solid tumor.
  • the CD40 bispecific molecule is administered in
  • compositions disclosed herein provide 4-1 BB agonists that can synapse dendritic cells and T cells, and CD40 agonists that can bind to antigen presenting cells with increased specificity. These properties can result in increased efficacy, decreased toxicity, and an increased therapeutic window.
  • compositions comprising A or B
  • the method must comprise at least one of A and B but may also comprise both A and B.
  • a composition comprising "A, B, C or D" must comprise at least one of the group of A, B, C and D, but may also comprise all or any combination of A, B, C and D.
  • non-human animals includes all vertebrates, e.g. , mammals, such as non-human primates, (particularly higher primates and monkeys), sheep, dogs, rodents (e.g. mouse or rat), guinea pigs, goats, pigs, cats, rabbits, cows, and non- mammals such as chickens, amphibians, reptiles etc.
  • the subject is a human.
  • treatment includes therapeutic treatment and prophylactic treatment.
  • Therapeutic treatment is treatment of a subject that has signs or symptoms of the disease, condition or disorder to be treated.
  • Prophylactic treatments refers to treatment of a subject that is predisposed to the disease, condition or disorder that does not show overt signs of the disease, condition or disorder.
  • treatment may result in stasis of, partial or total alleviation, or reduction of signs or symptoms of illness, and specifically includes, without limitation, prolongation of survival and cure.
  • Peptide refers to any peptide comprising two or more amino acids joined by peptide bonds or modified peptide bonds (e.g. , peptide isosteres).
  • Peptides can contain amino acids other than the 20 naturally occurring nucleic acid encoded amino acids, and include amino acid sequences modified either by natural processes, such as post- translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in a peptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification can be present in the same or varying degrees at several sites in a given peptide.
  • polypeptides can contain many types of modifications.
  • Polypeptides can be branched as a result of ubiquitination, and they can be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides can result from natural posttranslational processes or can be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • isolated protein or "isolated polypeptide” (e.g. , an isolated antibody or isolated antigen binding fragment) is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature.
  • a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components.
  • a protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • variant as used herein is defined as a modified or altered form of a wildtype sequence, e.g. where one or more amino acids may be replaced by other amino acid(s) or non-amino acid(s) which do not substantially affect function.
  • the variant may contain an altered side chain for at least one amino acid residue.
  • antigen as used herein is defined as an entity that can stimulate the production of antibodies and specifically combine with them and/or an entity which elicits an immune system response.
  • a cell surface protein or a specific linear or non-linear portion thereof For example, a cell surface protein or a specific linear or non-linear portion thereof.
  • the term herein may be abbreviated to "Ag.”
  • an "effective amount" of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • Binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y may generally be represented by the equilibrium dissociation constant (KD), a ratio of koff/kon, between the antibody and its antigen. KD and affinity are inversely related.
  • the KD value relates to the concentration of antibody (the amount of antibody needed for a particular experiment) and so the lower the KD value (lower concentration) and thus the higher the affinity of the antibody. Affinity may be measured by common methods known in the art, including those described herein. Specific, illustrative, and exemplary embodiments for measuring binding affinity may be measured by
  • RIA radioimmunoassays
  • SPR Surface Plasmon Resonance
  • the Kinetic Exclusion Assay is a general purpose immunoassay platform that is capable of measuring equilibrium dissociation constants, and association and dissociation rate constants for antigen/anti-body interactions.
  • human antibody refers to an antibody which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any technique for making human antibodies. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.
  • an "antigen binding antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fd fragments, dAb fragments, Fab'-SH, F(ab')2; diabodies; triabodies; linear antibodies; single- chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments, minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide).
  • CDR complementarity determining region
  • An antigen-binding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
  • full length antibody “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region.
  • immune cell refers to cells that play a role in the immune response, including lymphocytes, such as B cells and T-cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • An "immune response” refers to a biological response within a vertebrate against foreign agents, which response protects the organism against these agents and diseases caused by them.
  • An immune response is mediated by the action of a cell of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • a cell of the immune system for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutr
  • An immune reaction includes, e.g., activation or inhibition of a T-cell, e.g. , an effector T-cell or a Th cell, such as a CD4+ or CD8+ T-cell, or the inhibition of a Treg cell.
  • a T-cell e.g., an effector T-cell or a Th cell, such as a CD4+ or CD8+ T-cell, or the inhibition of a Treg cell.
  • an “immunomodulator” or “immunoregulator” refers to an agent, e.g. , a component of a signaling pathway that may be involved in modulating, regulating, or modifying an immune response.
  • “Modulating,” “regulating,” or “modifying” an immune response refers to any alteration in a cell of the immune system or in the activity of such cell (e.g. , an effector T-cell).
  • modulation includes stimulation or suppression of the immune system which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other changes which can occur within the immune system.
  • immunomodulator is located on the surface of a T-cell.
  • An "immunomodulatory target” or “immunoregulatory target” is an immunomodulator that is targeted for binding by, and whose activity is altered by the binding of, a substance, agent, moiety, compound or molecule.
  • Immunomodulatory targets include, for example, receptors on the surface of a cell
  • immunomodulatory receptors and receptor ligands
  • immunomodulatory ligands receptor ligands
  • T-cell-mediated response refers to a response mediated by T-cells, including effector T-cells (e.g. , CD8+ cells) and helper T-cells (e.g. , CD4+ cells).
  • T- cell mediated responses include, for example, T-cell cytotoxicity and proliferation.
  • cytotoxic T lymphocyte (CTL) response refers to an immune response induced by cytotoxic T-cells. CTL responses are mediated primarily by CD8+ T-cells.
  • inhibitor means to reduce by a measurable amount.
  • Inhibitors and “antagonists,” or “activators” and “agonists,” refer to inhibitory or activating molecules, respectively, e.g., for the activation of, e.g. , a ligand, receptor, cofactor, a gene, cell, tissue, or organ.
  • a modulator of, e.g., a gene, a receptor, a ligand, or a cell is a molecule that alters an activity of the gene, receptor, ligand, or cell, where activity can be activated, inhibited, or altered in its regulatory properties.
  • the modulator may act alone, or it may use a cofactor, e.g., a protein, metal ion, or small molecule.
  • Inhibitors are compounds that decrease, block, prevent, delay activation, inactivate, desensitize, or down regulate, e.g., a gene, protein, ligand, receptor, or cell.
  • Activators are compounds that increase, activate, facilitate, enhance activation, sensitize, or up regulate, e.g., a gene, protein, ligand, receptor, or cell.
  • An inhibitor may also be defined as a compound that reduces, blocks, or inactivates a constitutive activity.
  • An "agonist” is a compound that interacts with a target to cause or promote an increase in the activation of the target (e.g., a polypeptide which agonizes (promotes) 4-1 BB signaling to B and T-cells).
  • An "aptamer” is a single chain RNA or DNA oligonucleotide molecules, generally 15 to 60 bases in length with high affinity binding to a specific target molecule such as a protein, nucleic acid or small molecule compound. Aptamers can be designed and selected in vitro, by an enrichment process to develop a unique structure. Aptamers are often identified by a technique called Systematic evolution of ligands by exponential selection (SELEX). Several aptamer selection kits are available commercially.
  • An "antagonist” is a compound that opposes the actions of an agonist.
  • An antagonist prevents, reduces, inhibits, or neutralizes the activity of an agonist.
  • An antagonist can also prevent, inhibit, or reduce constitutive activity of a target, e.g., a target receptor, even where there is no identified agonist.
  • 4-1 BB also known as CD137 or "tumor necrosis factor superfamily, member 9" (TNFRSF9)
  • TNFRSF9 tumor necrosis factor superfamily, member 9
  • This receptor contributes to the clonal expansion, survival, and development of T-cells. It can also induce proliferation in peripheral monocytes, enhance T-cell apoptosis induced by TCR/CD3 triggered activation, and regulate CD28 co-stimulation to promote Th1 cell responses. The expression of this receptor is induced by lymphocyte activation.
  • 4-1 BB was first identified in mice by a modified differential screening procedure (see Kwon BS, Weissman SM, Proc. Natl. Acad. Sci. USA, 1989;
  • An exemplary human 4- 1 BB sequence is provided as SEQ ID NO: 3. Residues 24-186 of SEQ ID NO: 3 comprise an extracellular domain. Agonists of 4-1 BB are known, and include agonist antibodies against 4- 1 BB, soluble 4-1 BB ligand molecules, and aptamers (see Bartkowiak and Curran, 2015, Front Oncol., 5: 1 17).
  • CD40 refers to a receptor that is a member of the TNF-receptor superfamily, which binds to ligand CD40L (also referred to as CD154).
  • CD40 is mediates a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, and memory B cell development.
  • the term "CD40” includes any variants or isoforms of CD40 which are naturally expressed by cells (e.g., human CD40 isoform 1 deposited with GENBANK® having accession no. NP_001241). CD40 or any variants and isoforms thereof, may either be isolated from cells or tissues that naturally express them or be recombinantly produced using well-known techniques in the art and/or those described herein.
  • CD40 is found on antigen presenting cells, such as dendritic cells, macrophages and B cells, CD40 is a key activator of the immune response and is expressed on many cancer cells.
  • CD40L refers to the ligand for CD40 (see, for example, Schonbeck and Libby (2001) Cell Mol Life Sci, 58(1):4-43). CD40L is primarily expressed on activated T cells and is a member of the TNF superfamily of molecules. It binds to CD40 on antigen-presenting cells (APC), which leads to many effects depending on the target cell type (Parham, Peter (2004). The Immune System (2nd ed.). Garland Science. Pp. 169-173).
  • APC antigen-presenting cells
  • PBMC is an abbreviation for peripheral blood mononuclear cells.
  • a G 4 S linker is the peptide GGGGS (SEQ ID NO: 12).
  • the present invention relates to bispecific molecules that comprise a 4-1 BB agonist moiety connected to a dendritic cell binding moiety (a 4-1 BB agonist bispecific molecule).
  • a 4-1 BB agonist bispecific molecule As used herein connected means the same as fused when used in reference to the fusion proteins of the invention.
  • the dendritic cell binding moiety binds to a dendritic cell surface protein or other dendritic cell surface molecule either of which may be referred to as a dendritic cell surface antigen herein.
  • 4-1 BB agonistic bispecific molecules which are molecules that comprise a 4-1 BB agonist moiety and a dendritic cell binding moiety.
  • the 4-1 BB agonist moiety is an anti-4-1 BB agonist antibody, or a 4-1 BB binding fragment thereof, a 4-1 BBL moiety, or a 4-1 BB agonist aptamer.
  • the 4-1 BB agonist moiety is a 4-1 BBL moiety that comprises one 4-1 BBL domain (monomer). In another embodiment, the 4-1 BB agonist moiety is a 4-1 BBL moiety that comprises two 4-1 BBL domains (dimer). In another embodiment, the 4-1 BB agonist moiety is a 4-1 BBL moiety that comprises three 4-1 BBL domains (trimer). In one embodiment, the bispecific molecule comprises a 4-1 BB agonist moiety comprising a set of three human 4- 1 BBL domains to form a single-chain 4-1 BBL trimer.
  • the single-chain 4- 1 BBL trimer comprises, in amino- to carboxyl-terminal order, a first 4-1 BBL domain, an inter- domain linker, a second 4-1 BBL domain, a second inter-domain linker, and a third 4-1 BBL domain.
  • each inter-domain linker consists of 15-20 amino acids.
  • each of the two inter-domain 4-1 BBL monomer linkers comprises three G 4 S domains.
  • the 4-1 BBL domain comprises full-length human 4-1 BBL (i.e. , amino acid residues 1-254 of SEQ I D NO: 4).
  • the 4-1 BBL domain comprises a portion of the amino acid sequence set forth in SEQ I D NO: 4.
  • the 4-1 BBL domain comprises amino acid residues 30-254 of SEQ I D NO:4.
  • the 4-1 BBL domain consists of amino acid residues 30-254 of SEQ I D NO: 4.
  • the 4-1 BBL domain comprises amino acid residues 50-254 of SEQ I D NO: 4.
  • the 4-1 BBL domain consists of amino acid residues 50- 254 of SEQ I D NO: 4.
  • the 4-1 BBL domain comprises amino acid residues 71-254 of SEQ I D NO: 4. In another embodiment, the 4-1 BBL domain consists of amino acid residues 71-254 of SEQ I D NO: 4. In another embodiment, the 4-1 BBL domain comprises amino acid residues 85-254 of SEQ I D NO: 4. In another embodiment, the 4-1 BBL domain consists of amino acid residues 85-254 of SEQ I D NO: 4.
  • the 4-1 BBL domain comprises or consists of a sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence having an N-terminus at any one of amino acid residues 31-84 of SEQ I D NO: 4 and a C terminus at any one of amino acid residues 234-254 of SEQ ID NO: 4.
  • the 4-1 BBL domain comprises no more than about 200 amino acid residues, preferably no more than about 150 amino acid residues, and more preferably no more than about 100 amino acid residues. In another embodiment, the 4-1 BBL domain consists of no more than about 200 amino acid residues, preferably no more than about 150 amino acid residues, and more preferably no more than about 100 amino acid residues.
  • the 4-1 BBL domain comprises the amino acid sequence of SEQ I D NO: 5 or a portion thereof. In another embodiment, the 4-1 BBL domain consists of the amino acid sequence of SEQ I D NO: 5. In another embodiment, the 4-1 BBL domain comprises the amino acid sequence of 6 or a portion thereof. In another embodiment, the 4-1 BBL domain consists of the amino acid sequence of SEQ I D NO: 6. In another embodiment, the 4-1 BBL domain comprises the amino acid sequence of SEQ I D NO:7 or a portion thereof. In another embodiment, the 4-1 BBL domain consists of the amino acid sequence of SEQ I D NO: 7.
  • the 4-1 BBL domain comprises an amino acid sequence that is highly identical to any one of the sequences set forth herein. For example, in one
  • the 4-1 BBL domain comprises an amino acid sequences at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-254 of SEQ I D NO: 4.
  • the 4-1 BBL domain comprises an amino acid sequences at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 30-254, 50-254, 71-254, or 85-254 of SEQ I D NO: 4.
  • the 4-1 BBL domain comprises an amino acid sequences at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ I D NO: 5, 6, or 7.
  • the 4-1 BBL domain comprises an amino acid sequence at least 95% identical to residues 30-254, 50-254, 71 -254, or 85-254 of SEQ I D NO: 4.
  • the 4-1 BBL domain comprises an amino acid sequence at least 95% identical to SEQ I D NO: 5, 6, or 7.
  • the present invention also relates to bispecific molecules that comprise a CD40 agonist moiety connected to a dendritic cell binding moiety (a CD40 agonist bispecific molecule).
  • a CD40 agonist bispecific molecule As used herein connected means the same as fused when used in reference to the fusion proteins of the invention.
  • the dendritic cell binding moiety binds to a dendritic cell surface protein or other dendritic cell surface molecule either of which may be referred to as a dendritic cell surface antigen herein.
  • CD40 agonistic bispecific molecules which are molecules that comprise a CD40 agonist moiety and a dendritic cell binding moiety.
  • the CD40 agonist moiety is an anti-CD40 agonist antibody, or a CD40 binding fragment thereof, a CD40 moiety, or a CD40 agonist aptamer.
  • the CD40 agonist moiety is a CD40 moiety that comprises one CD40L domain (monomer). In another embodiment, the CD40 agonist moiety is a CD40L moiety that comprises two CD40L domains (dimer). In another embodiment, the CD40 agonist moiety is a CD40L moiety that comprises three CD40L domains (trimer). In one embodiment, the bispecific molecule comprises a CD40 agonist moiety comprising a set of three human CD40L domains to form a single-chain CD40L trimer.
  • the single-chain CD40L trimer comprises, in amino- to carboxyl-terminal order, a first CD40L domain, an inter- domain linker, a second CD40L domain, a second inter-domain linker, and a third CD40L domain.
  • each inter-domain linker consists of 15-20 amino acids.
  • each of the two inter-domain CD40L monomer linkers comprises three G4S domains.
  • the CD40L moiety is a fusion polypeptide wherein the CD40L moiety is linked to an antibody Fc region or fragment thereof (Fc-CD40L moiety fusion), Fab-Fc (Fab-Fc-CD40L moiety fusion), Fab fragment (CD40L moiety-antibody Fab fragment fusion), antibody (CD40L moiety-antibody fusion) or albumin (CD40L moiety albumin fusion).
  • the polypeptide comprises one or more CD40L domains linked (e.g., fused) to an antibody Fc region or a fragment thereof and/or a Fab or fragment thereof and/or an antibody and/or an albumin (e.g., HSA). Examples of such fusion polypeptides are disclosed in US 62/453,354.
  • the CD40L domain comprises full-length human CD40L (i.e., amino acid residues 1-261 of SEQ ID NO:32). In another embodiment, the CD40L domain comprises a portion of the amino acid sequence set forth in SEQ ID NO:32. In another embodiment, the CD40L domain comprises amino acid residues 47-261 of SEQ ID NO:32 (SEQ ID NO:33). In another embodiment, the CD40L domain consists of amino acid residues 47-261 of SEQ ID NO:32 (SEQ ID NO:33). In another embodiment, the CD40L domain comprises amino acid residues 108-261 of SEQ ID NO: 32 (SEQ ID NO:34).
  • the CD40L domain consists of amino acid residues 108-261 of SEQ ID NO: 32 (SEQ ID NO:34). In another embodiment, the CD40L domain comprises amino acid residues 1 13-261 of SEQ ID NO: 32 (SEQ ID NO:35). In another embodiment, the CD40L domain consists of amino acid residues 113-261 of SEQ ID NO: 4 (SEQ ID NO:35). In another embodiment, the CD40L domain comprises amino acid residues 116-261 of SEQ ID NO: 32 (SEQ ID NO:36). In another embodiment, the CD40L domain consists of amino acid residues 1 16-261 of SEQ ID NO: 32 (SEQ ID NO:36).
  • the CD40L domain comprises or consists of a sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence having an N-terminus at any one of amino acid residues 31-120 of SEQ ID NO: 32 and a C-terminus at any one of amino acid residues 240-261 of SEQ ID NO: 32.
  • the CD40L domain comprises no more than about 200 amino acid residues, preferably no more than about 150 amino acid residues, and more preferably no more than about 100 amino acid residues. In another embodiment, the CD40L domain consists of no more than about 200 amino acid residues, preferably no more than about 150 amino acid residues, and more preferably no more than about 100 amino acid residues.
  • the CD40L domain comprises the amino acid sequence of SEQ ID NO:33 or a portion thereof. In another embodiment, the CD40L domain consists of the amino acid sequence of SEQ ID NO:33. In another embodiment, the CD40L domain comprises the amino acid sequence of SEQ ID NO:34 or a portion thereof. In another embodiment, the CD40L domain consists of the amino acid sequence of SEQ ID NO:34. In another embodiment, the CD40L domain comprises the amino acid sequence of SEQ ID NO:35 or a portion thereof. In another embodiment, the CD40L domain consists of the amino acid sequence of SEQ ID NO:35. In another embodiment, the CD40L domain comprises the amino acid sequence of SEQ ID NO:36or a portion thereof. In another embodiment, the CD40L domain consists of the amino acid sequence of SEQ ID NO:36.
  • the CD40L domain comprises an amino acid sequence that is highly identical to any one of the sequences set forth herein. For example, in one
  • the CD40L domain comprises an amino acid sequences at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-261 of SEQ I D NO:32.
  • the CD40L domain comprises an amino acid sequences at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 47-261 , 108-261 , 1 13-261 , or 1 16-261 of SEQ I D NO:32.
  • the CD40L domain comprises an amino acid sequences at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ I D NO: 33, 34, 35, or 36.
  • the CD40L domain comprises an amino acid sequence at least 95% identical to residues 47-261 , 108-261 , 1 13-261 , or 1 16-261 of SEQ I D NO:32.
  • the CD40L domain comprises an amino acid sequence at least 95% identical to SEQ I D NO: 33, 34, 35, or 36.
  • the percentages for global identity are calculated using Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • GAP program in the GCG software package.
  • the 4-1 BB agonist moiety is an anti-4-1 BB agonist antibody or 4- 1 BB binding fragment thereof.
  • the anti-4-1 BB agonist antibody is an lgG1 , antibody.
  • the anti-4-1 BB agonist antibody is an lgG2 antibody.
  • the anti-4-1 BB agonist antibody is an lgG3 antibody.
  • the anti-4-1 BB agonist antibody is an lgG4 antibody.
  • the 4- 1 BB binding fragment thereof is an scFv.
  • the anti-4-1 BB agonist antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody and the 4-1 BB binding fragment thereof is an Fab.
  • the anti-4-1 BB agonist antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody and the 4-1 BB binding fragment thereof is an F(ab')2.
  • the anti-4-1 BB agonist antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody and the 4-1 BB binding fragment thereof is a minibody.
  • the anti-4-1 BB agonist antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody and the 4-1 BB binding fragment thereof is a triabody.
  • the anti-4-1 BB agonist antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody and the 4-1 BB binding fragment thereof is a scFvFc.
  • the anti-4-1 BB agonist antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody and the 4-1 BB binding fragment thereof is an hclgG.
  • Antibodies e.g., agonist antibodies
  • 4-1 BB are known for example, urelumab (see e.g., US8137667), utomilumab (see e.g. , US20120237498), ATOR-1017 (see, e.g.,
  • WO2017049452 WO2017205745, WO20171 1281 1 , WO2017077085, WO2017182672, WO2018017761 , US20130149301 , US20160244528, US20160083474, US20120076722, US201701 14141 , US20170247467, and US7829088.
  • WO2007/035518 describes aptamers, for example M 12-22 (5"-GCACAGCAACACCACGACCCCCCCUAGGC UUCCGCCCGCG-3') (SEQ I D NO: 13)), that can stimulate 4-1 BB.
  • WO201 1/109642 describes 4-1 BB aptamers, for example, 5'-CCTGCACCCAGTGTCCCCGACGGGGCCCTCTAGCCGTA CTCTGTAATGGC GGATGCTGACGGAGAGGAGGACGG-3' (SEQ I D NO: 14).
  • WO2010/144295 describes aptamers specific to 4-1 BB and bi-specific aptamers. Also see Blind, M. , Molecular Therapy -Nucleic Acids (2015) 4.e223.
  • the CD40 agonist moiety is an anti-CD40 agonist antibody or CD40 binding fragment thereof.
  • the anti- CD40 agonist antibody is an lgG1 , antibody.
  • the anti-CD40 agonist antibody is an lgG2 antibody.
  • the anti-CD40 agonist antibody is an lgG3 antibody.
  • the anti- CD40 agonist antibody is an lgG4 antibody.
  • the CD40 binding fragment thereof is an scFv.
  • the anti- CD40 agonist antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody and the CD40 binding fragment thereof is an Fab.
  • the anti- CD40 agonist antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody and the CD40 binding fragment thereof is an F(ab')2.
  • the anti- CD40 agonist antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody and the CD40 binding fragment thereof is a minibody.
  • the anti-CD40 agonist antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody and the CD40 binding fragment thereof is a triabody.
  • the anti-CD40 agonist antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody and the CD40 binding fragment thereof is a scFvFc.
  • the anti- CD40 agonist antibody is an lgG1 , lgG2, lgG3, or lgG4 antibody and the CD40 binding fragment thereof is an hclgG.
  • Aptamers useful in the present disclosure are agonists aptamers specific to CD40 for example those disclosed in US20130209514 and in Soldevilla et al., 2015 Biomaterials 67:274-285 Epub Jul 14, 2015.
  • Antibodies useful in the present disclosure are agonist antibodies to CD40 for example dacetuzumab (see e.g. , WO2000075348, WO2006128103, WO2016069919), selicrelumab (see e.g., US8,388,971 ), ABBV-428 (see e.g., US20160347850, ADC-1013 (see e.g., US9, 676,862 and Mangsbo et al.
  • the 4- 1 BB agonist moiety is connected to a dendritic cell binding moiety.
  • the 4-1 BB agonist moiety is connected to the C-terminus of the dendritic cell binding moiety.
  • the 4-1 BB agonist moiety is connected to the N-terminus of the dendritic cell binding moiety.
  • a 4-1 BB agonist moiety can be connected to the dendritic cell binding moiety by a linker. Additionally, each 4-1 BBL domain of the 4-1 BBL agonist moiety can be separated by an inter-domain linker.
  • the CD40 agonist moiety is connected to a dendritic cell binding moiety. In one embodiment of the CD40 agonist bispecific molecule the CD40 agonist moiety is connected to the C-terminus of the dendritic cell binding moiety. In one embodiment of the CD40 agonist bispecific molecule the CD40 agonist moiety is connected to the N-terminus of the dendritic cell binding moiety.
  • a CD40 agonist moiety can be connected to the dendritic cell binding moiety by a linker. Additionally, each CD40L domain of the CD40L agonist moiety can be separated by an inter-domain linker.
  • each linker or inter-domain linker comprises 5-25 amino acids.
  • the linker or inter-domain linker comprises 5-10, 5-15, 5-20, 5-25, 10-15, 10-20, 10-25, 15-20, 15-25, or 20-25 amino acids.
  • the linker or inter-domain linker comprises 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 amino acids.
  • the linker or inter-domain linker comprises 15-20 amino acids.
  • the linker or inter-domain linker comprises at least one, two, or three G 4 S motifs.
  • a G 4 S motif comprises four glycine residues followed by one serine residue (i.e., amino acid sequence GGGGS).
  • the linker or inter-domain linker comprises three G 4 S motifs.
  • the dendritic cell binding moiety binds to a dendritic cell surface antigen (e.g. , an extracellular region of a dendritic cell surface antigen).
  • the dendritic cell binding moiety is an antibody that binds to the dendritic cell surface antigen or an antigen binding fragment thereof.
  • the dendritic cell binding moiety binds to a dendritic cell surface antigen (e.g., an extracellular region of a dendritic cell surface antigen).
  • the dendritic cell binding moiety is an antibody that binds to the dendritic cell surface antigen or an antigen binding fragment thereof.
  • the dendritic cell binding moiety is an antibody that binds to the antigen or an antigen binding fragment thereof and the antibody is an lgG1 antibody.
  • the antibody is an lgG2 antibody.
  • the antibody is an lgG3 antibody.
  • the antibody is an lgG4 antibody.
  • antigen binding fragment include Fv, Fab, Fab', Fd fragments, dAb fragments, Fab'-SH, F(ab')2; diabodies; triabodies; linear antibodies; single-chain antibody molecules.
  • the antigen binding fragment that binds to the dendritic cell surface antigen is an scFv.
  • the antibody to the dendritic cell surface antigen is an lgG1 , lgG2, lgG3, or lgG4 antibody and the antigen binding fragment thereof is an Fab.
  • the antibody to the dendritic cell surface antigen is an lgG1 , lgG2, lgG3, or lgG4 antibody and the antigen binding fragment thereof is an F(ab')2.
  • the antibody to the dendritic cell surface antigen is an lgG1 , lgG2, lgG3, or lgG4 antibody and the antigen binding fragment thereof is a minibody.
  • the antibody to the dendritic cell surface antigen an lgG1 , lgG2, lgG3, or lgG4 antibody and the antigen binding fragment thereof is a triabody.
  • the antibody to the dendritic cell surface antigen is an lgG1 , lgG2, lgG3, or lgG4 antibody and the antigen binding fragment thereof is a minibody.
  • the antibody to the dendritic cell surface antigen an lgG1 , lgG2, lgG3, or lgG4 antibody and the antigen binding fragment thereof is a scFvFc.
  • the antibody to the dendritic cell surface antigen is an lgG1 , lgG2, lgG3, or lgG4 antibody and the antigen binding fragment thereof is a minibody.
  • the antibody to the dendritic cell surface antigen an lgG1 , lgG2, lgG3, or lgG4 antibody and the antigen binding fragment thereof is an hclgG.
  • the dendritic cell binding moiety is an antibody that binds to the antigen or an antigen binding fragment thereof as described herein, where the 4-1 BB agonist moiety comprises three 4-1 BBL domains and the 4-1 BB agonist moiety is connected to the C-terminus of the Fc region of the antibody or antigen binding fragment thereof.
  • the dendritic cell binding moiety is an antibody to the dendritic cell surface antigen or antigen binding fragment thereof that is agonistic. In another embodiment the antibody or the antigen binding fragment is antagonistic. In still another embodiment the antibody or the antigen binding fragment is a neutral activity antibody or antigen binding fragment thereof. In some embodiments the dendritic cell binding moiety is an antibody to the dendritic cell surface antigen or antigen binding fragment thereof that is a ligand blocking antibody or antigen binding fragment thereof.
  • the dendritic cell binding moiety is an antibody that binds to the antigen or an antigen binding fragment thereof as described herein, where the CD40 agonist moiety comprises three CD40L domains and the CD40 agonist moiety is connected to the C terminus of the Fc region of the antibody or antigen binding fragment thereof.
  • the dendritic cell binding moiety is an antibody to the dendritic cell surface antigen or antigen binding fragment thereof that is agonistic. In another embodiment the antibody or the antigen binding fragment is antagonistic. In still another embodiment the antibody or the antigen binding fragment is a neutral activity antibody or antigen binding fragment thereof. In some embodiments the dendritic cell binding moiety is an antibody to the dendritic cell surface antigen or antigen binding fragment thereof that is a ligand blocking antibody or antigen binding fragment thereof.
  • the dendritic cell binding moiety is an aptamer that binds to the dendritic cell surface antigen.
  • the dendritic cell surface antigen is a cell surface receptor. In some embodiments the dendritic cell surface antigen is a cell surface receptor ligand. In one embodiment of the 4-1 BB agonist bispecific molecule the dendritic cell surface antigen is LILRA4. In one embodiment of the CD40 agonist bispecific molecule the dendritic cell surface antigen is LI LRA4. In other embodiment the dendritic cell surface antigen is LAMP5. In still another embodiment the dendritic cell surface antigen is CLEC4C. In yet another embodiment the dendritic cell surface antigen is I L3RA. In another embodiment, the dendritic cell surface antigen is CLEC9A.
  • the dendritic cell surface antigen is XCR1. In yet another embodiment the dendritic cell surface antigen is FLT3. In yet another embodiment the dendritic cell surface antigen is SIGLEC6. In one embodiment the dendritic cell surface antigen is human LI LRA4 (SEQ I D NO: 15). In another embodiment the dendritic cell surface antigen is human LAMP5 (SEQ I D NO: 17). In still another embodiment the dendritic cell surface antigen is human CLEC4C (SEQ I D NO: 18). In yet another embodiment the dendritic cell surface antigen is human I L3RA (SEQ I D NO: 19). In another embodiment, the dendritic cell surface antigen is human CLEC9A (SEQ I D NO: 21).
  • the dendritic cell surface antigen is human XCR1 (SEQ I D NO: 22). In yet another embodiment the dendritic cell surface antigen is human FLT3 (SEQ I D NO: 25). In yet another embodiment the dendritic cell surface antigen is human SIGLEC6 (SEQ I D NO: 27).
  • the LI LRA4 gene encodes an immunoglobulin-like cell surface protein (LILRA4 or I LT7) that is expressed predominantly on plasmacytoid dendritic cells (PDCs) and modulates the function of these cells in the immune response. Expression of the gene is downregulated by interleukin 3 (I L3). LI LRA4 is one of a cluster of highly related genes located at chromosomal region 19q13.4. An exemplary human LI LRA4 sequence is provide as SEQ I D NO: 15.
  • Residues 24-446 of SEQ I D NO: 15 comprise an extracellular domain, and residues 468-499 comprise a cytoplasmic domain.
  • An exemplary ligand of LI LRA4 is bone marrow stromal cell antigen 2 (BST2 or CD317; SEQ I D NO: 16).
  • Residues 49-161 of SEQ I D NO: 16 comprise an extracellular domain of BST2.
  • LAMP5 has an extracellular domain with 4 highly conserved cysteines, followed by a transmembrane domain and a 24-amino acid cytoplasmic tail with a C-terminal endosomal targeting motif.
  • An exemplary human LAMP5 sequence is provided as SEQ I D NO: 17
  • Residues 30-235 of SEQ I D NO: 17 comprise an extracellular domain, and residues 257-280 comprise a cytoplasmic domain.
  • C-type lectin domain family 4 member C (CLEC4C, BDCA-2, or CD303), is a member of the C-type lectin/C-type lectin-like domain (CTUCTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune response. Two transcript variants encoding distinct isoforms have been identified for this gene.
  • CLEC4C binds to complex carbohydrates containing a terminal galactose in a beta 1 -4 or beta 1-3 linkage.
  • An exemplary human CLEC4C sequence is provided as SEQ I D NO: 18. Residues 45-213 of SEQ I D NO: 18 comprise an extracellular domain, and residues 1-21 of SEQ I D NO: 18 comprise an intracellular domain.
  • I L-3 receptor subunit alpha is an interleukin 3 specific subunit of a heterodimeric cytokine receptor.
  • the receptor is comprised of a ligand specific alpha subunit and a signal transducing beta subunit shared by the receptors for interleukin 3 (I L-3), colony stimulating factor 2 (CSF2/GM-CSF), and interleukin 5 (I L-5).
  • I L-3 interleukin 3
  • CSF2/GM-CSF colony stimulating factor 2
  • I L-5 interleukin 5
  • This gene and the gene encoding the colony stimulating factor 2 receptor alpha chain (CSF2RA) form a cytokine receptor gene cluster in an X-Y pseudoautosomal region on chromosomes X or Y.
  • CSF2RA colony stimulating factor 2 receptor alpha chain
  • An exemplary human I L3RA sequence is provided as SEQ I D NO: 19. Residues 19-305 of SEQ I D NO: 19 comprise an extracellular domain, and residues 326-278 comprise a cytoplasmic domain.
  • An exemplary ligand of I L3RA is interleukin 3 (I L-3; SEQ I D NO: 20). Residues 20-152 of SEQ I D NO: 20 comprise the secreted interleukin 3.
  • C-type lectin domain containing 9A (CLEC9A, CD370, or DNGR1) is a group V C- type lectin-like receptor (CTLR) that functions as an activation receptor and is expressed on myeloid lineage cells.
  • CLR C- type lectin-like receptor
  • CLEC9A binds to filamentous actin in association with particular actin- binding domains of cytoskeletal proteins.
  • An exemplary human CLEC9A sequence is provided as SEQ I D NO: 21. Residues 57-241 of SEQ I D NO: 21 comprise an extracellular domain, and residues 1-35 of SEQ I D NO: 21 comprise an intracellular domain.
  • X-C Motif Chemokine Receptor 1 is a chemokine receptor belonging to the G protein-coupled receptor superfamily.
  • An exemplary human XCR1 sequence is provided as SEQ I D NO: 22. Extracellular regions of XCR1 include residues 1-31 , 90-103, 168-190, and 251-267 of SEQ I D NO: 22.
  • Exemplary ligands of XCR1 include XCL1
  • Residues 22-1 14 of SEQ I D NO: 23 comprise the secreted XCL1
  • residues 22-1 14 of SEQ I D NO: 24 comprise the secreted XCL2.
  • Fms like tyrosine kinase 3 is a class I I I receptor tyrosine kinase that regulates hematopoiesis. This receptor is activated by binding of the fms-related tyrosine kinase 3 ligand to the extracellular domain, which induces homodimer formation in the plasma membrane leading to autophosphorylation of the receptor. The activated receptor kinase subsequently phosphorylates and activates multiple cytoplasmic effector molecules in pathways involved in apoptosis, proliferation, and differentiation of hematopoietic cells in bone marrow.
  • An exemplary human FLT3 sequence is provided as SEQ I D NO: 25. Residues 27-543 of SEQ I D NO: 25 comprise an extracellular domain, and residues 564-993 of SEQ I D NO: 25 comprise an intracellular domain.
  • An exemplary ligand of FTL3 is FLT3LG (SEQ I D NO: 26). Residues 27-184 of SEQ I D NO: 26 comprise an extracellular domain of FLT3LG.
  • SIGLEC6 sialic acid binding Ig like lectin 6
  • CD327, CD33L, or OB-BP1 is a member of the SIGLEC (sialic acid binding immunoglobulin-like lectin) family of proteins.
  • the transmembrane receptor binds sialyl-TN glycans and leptin.
  • An exemplary human SIGLEC6 sequence is provided as SEQ I D NO: 27. Residues 27-347 of SEQ I D NO: 27 comprise an extracellular domain, and residues 369-453 of SEQ I D NO: 27 comprise an intracellular domain.
  • An exemplary ligand of SIGLEC6 is leptin (SEQ I D NO: 28). Residues 22-167 of SEQ I D NO: 28 comprise secreted leptin.
  • the dendritic cell binding moiety is a receptor ligand, e.g., an extracellular or secreted portion of a receptor ligand disclosed herein.
  • the dendritic cell binding moiety is a receptor ligand, e.g. , an extracellular or secreted portion of a receptor ligand disclosed herein.
  • the receptor ligand is BST2 (SEQ I D NO: 16).
  • the receptor ligand is I L-3 (SEQ I D NO: 20).
  • the receptor ligand is XCL1 (SEQ I D NO: 23).
  • the receptor ligand is XCL2 (SEQ I D NO: 24). In another embodiment the receptor ligand is FLT3LG (SEQ I D NO: 26). In another embodiment the receptor ligand is leptin (SEQ I D NO: 28).
  • the dendritic cell binding moiety is a receptor ligand, e.g. , an extracellular or secreted portion of a receptor ligand disclosed herein, that contains one or more mutations that abrogate agonist activity on its cognate receptor but do not significantly affect receptor binding.
  • One embodiment of the invention is a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of any embodiment of a 4-1 BB agonist bispecific molecule disclosed herein. Another embodiment is a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of any embodiment of a 4-1 BB agonist bispecific molecule disclosed herein, where the contacting is in vitro. Another embodiment is a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of any embodiment of a 4-1 BB agonist bispecific molecule disclosed herein, where the contacting is in vivo.
  • in yet another embodiment of the invention is a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of any embodiment of a 4-1 BB agonist bispecific molecule disclosed herein, where the T-cell is a tumor-associated T-cell.
  • Another embodiment is a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of any embodiment of a 4-1 BB agonist bispecific molecule disclosed herein, where the T-cell is a tumor-associated T-cell and where the contacting is in vitro.
  • Another embodiment is a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of any embodiment of a 4-1 BB agonist bispecific molecule disclose
  • One embodiment of the invention is a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of any embodiment of a CD40 agonist bispecific molecule disclosed herein. Another embodiment is a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of any embodiment of a CD40 agonist bispecific molecule disclosed herein, where the contacting is in vitro. Another embodiment is a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of any embodiment of a CD40 agonist bispecific molecule disclosed herein, where the contacting is in vivo.
  • in yet another embodiment of the invention is a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of any embodiment of a CD40 agonist bispecific molecule disclosed herein, where the T-cell is a tumor-associated T-cell.
  • Another embodiment is a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of any embodiment of a CD40 agonist bispecific molecule disclosed herein, where the T-cell is a tumor-associated T-cell and where the contacting is in vitro.
  • Another embodiment is a method of activating a T-cell comprising, contacting a dendritic cell with an effective amount of any embodiment of a CD40 agonist bispecific molecule disclosed herein, where the T-cell is a tumor-associated T-cell and where the contacting is in vivo.
  • the present disclosure provides bispecific molecules where one portion of the molecule is a 4-1 BB agonist and the other portion binds to the extracellular portion of a dendritic cell surface antigen.
  • the present disclosure provides bispecific molecules where one portion of the molecule is a CD40 agonist and the other portion binds to the extracellular portion of a dendritic cell surface antigen.
  • the bispecific molecule can comprise a number of options.
  • Bispecific molecules include, but are not limited to, bispecific antibodies, bispecific antibody fragment, antibodies coupled to an aptamer, full length antibodies couple to an antibody fragment.
  • a bispecific IgG (BslgG) is a commonly used bispecific antibody (BsAb) format that is monovalent for one of two antigens.
  • Bispecific and multi-specific antibodies can also be made by appending either the amino or carboxy termini of either a light or heavy chain with additional antigen-binding molecules including, but not limited to, single domain antibodies (unpaired VL or VH) paired antibody variable domains such as F v or scFv.
  • additional antigen-binding molecules including, but not limited to, single domain antibodies (unpaired VL or VH) paired antibody variable domains such as F v or scFv.
  • BslgGs A potential advantage of BslgGs is that they can enable simultaneous binding of antigen to more than one variable domain and provide higher specific binding capacity.
  • Bispecific antibodies can also be bispecific antibody fragments. Many BsAb fragments are connected with short peptide linker sequences that can allow efficient expression of the BsAb in a single host cell. scFv fragments are commonly used fragments in generating BsAb molecules.
  • An scFv is constructed by using short amino acid linkers to provide inter-chain pairing of VH and VL domains. Co-expression of two such scFv fragments can be used to form a bispecific fragment known as a diabody.
  • the bispecific T cell Engager (BiTE) is a type of tandem scFv in which the component scFv fragments are designed to bind CD3 on T cells and a surface antigen on tumor cells to redirect T cells to kill tumor cells.
  • Single domain antibody fragments (dAbs) can reduce molecular size.
  • Another type of bispecific molecule is a fusion protein in which antibody fragments are linked to other proteins such as a receptor ligand. Such bispecific molecules can improve the targeting of the bispecific molecule.
  • Clinical stage BsAbs include Tiromab, BiTes, TandAbs, ImmTac, DAF, HAS-body, IgG-scFv, CrossMab, dock-and-lock, DVD-lg and nanobodies.
  • bispecific molecules include, but are not limited to, Catumaxomab (Removab) which targets CD3 and EpCAM, Ertumaxomab which binds to CD3 and HER2, Blinatumomab which binds to CD3 and CD19.
  • Solitomab which binds to CD3 and EpCAM, Medi 565 which binds to CD3 and CEA, Duligotuzumabe which binds to EGFR, HER3, GSK2434735 (GSK) which binds to IL-13, and IL-4.
  • ALX-0061 AbbVie, Ablynx
  • SAR156697 Sanofi
  • ALX-0761 MerckSerono, Ablynx
  • ALX-0761 MerckSerono, Ablynx
  • HAS and RG6013/ACE910 Chougai, Roche
  • a disease e.g., a cancer, infectious disease, inflammatory or autoimmune disorder
  • One embodiment of the present invention is a method for treating cancer in a subject in need thereof, the method comprising administering to the patient an effective amount of any 4-1 BB agonist bispecific molecule as disclosed herein.
  • the cancer is a solid tumor.
  • a disease e.g., a cancer, infectious disease, inflammatory or autoimmune disorder
  • On embodiment of the present invention is a method for treating cancer in a subject in need thereof, the method comprising administering to the patient an effective amount of any CD40 agonist bispecific molecule as disclosed herein.
  • the cancer is a solid tumor.
  • the method for treating cancer in a subject in need thereof comprising administering to the patient an effective amount of any 4-1 BB agonist bispecific molecule as disclosed herein, and further comprises administering an additional antineoplastic agent.
  • the method for treating cancer in a subject in need thereof comprising administering to the patient an effective amount of any CD40 agonist bispecific molecule as disclosed herein, and further comprises administering an additional antineoplastic agent.
  • the term "inhibits growth" of a tumor includes any measurable decrease in the growth of a tumor, e.g., the inhibition of growth of a tumor by at least about 10%, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%.
  • Cancers can be cancers with solid tumors or blood malignancies (liquid tumors).
  • cancers for treatment include lung cancer, renal cancer, breast cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, lung carcinoma, cervical cancer, prostate cancer, melanoma, head and neck cancer, lymphoma, glioma, and colorectal cancer.
  • the methods described herein may also be used for treatment of metastatic cancers, unresectable and/or refractory cancers (e.g. , cancers refractory to previous immunotherapy), and recurrent cancers.
  • the disease is an autoimmune disease.
  • An "autoimmune disease” herein is a disease or disorder arising from and directed against an individual's own tissues or a co-segregate or manifestation thereof or resulting condition therefrom.
  • autoimmune diseases or disorders include, but are not limited to arthritis (e.g. , rheumatoid arthritis and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis, dermatitis urticaria , multiple sclerosis (MS), inflammatory bowel disease (I BD), and juvenile onset (Type I) diabetes mellitus, .
  • the disease is an infectious disease.
  • the infectious disease relates to an agent selected from the group consisting of: a virus, a bacterium, a fungus, and a protozoan parasite.
  • infectious diseases include, but are not limited to human immunodeficiency viruses (GIV), hepatitis viruses class A, B and C, human cytomegalovirus, human papilloma viruses, leishmaniasis, toxoplasmosis,
  • herpes virus e.g., VZV, HSV-1 , HAV-6, HSV-I I, and CMV, Epstein Barr virus
  • adenovirus e.g., VZV, HSV-1 , HAV-6, HSV-I I, and CMV, Epstein Barr virus
  • influenza virus flaviviruses
  • echovirus e.g., adenovirus
  • rhinovirus e.g., adenovirus
  • coronavirus e.g., coronavirus
  • respiratory syncytial virus e.g., mumps virus, rotavirus, measles virus, rubella virus, parvovirus
  • vaccinia virus HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.
  • pathogenic bacteria causing infections treatable by methods described herein include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria.
  • pathogenic fungi causing infections treatable by methods described herein include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
  • pathogenic parasites causing infections treatable by methods described herein include Entamoeba histolytica, Balantidium coli, Naegleriafowleri,
  • the 4-1 BB agonist moiety and/or the dendritic cell binding moiety may be a humanized antibody or antigen binding fragment thereof.
  • embodiments of the CD40 agonist moiety and/or the dendritic cell binding moiety may be a humanized antibody or antigen binding fragment thereof.
  • Humanized antibodies are antibodies produced from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. The process of
  • humanization is usually applied to monoclonal antibodies developed for administration to humans (for example, antibodies developed as anti-cancer drugs). Humanization can be necessary when the process of developing a specific antibody involves generation in a non- human immune system (such as that in mice, rabbits, dogs or non-human primates).
  • the protein sequences of antibodies produced in this way are partially distinct from homologous antibodies occurring naturally in humans, and are therefore potentially immunogenic when administered to human. There are other types of antibodies developed.
  • the International Nonproprietary Names of humanized antibodies end in -zumab, as in omalizumab.
  • the humanization processes takes advantage of the fact that production of monoclonal antibodies can be accomplished using recombinant DNA to create constructs capable of expression in mammalian cell culture. That is, gene segments capable of producing antibodies are isolated and cloned into cells that can be grown in a bioreactor such that antibody proteins produced from the DNA of the cloned genes can be harvested in amounts necessary for therapeutic use.
  • the step involving recombinant DNA provides an intervention point that can be readily exploited to alter the protein sequence of the expressed antibody.
  • the alterations to antibody structure that are achieved in the humanization process are therefore all effectuated through techniques at the DNA level. Not all methods for deriving antibodies intended for human therapy require a humanization step (e.g. phage display) but essentially all are dependent on techniques that similarly allow the "insertion" or "swapping-out" of portions of the antibody molecule.
  • Direct creation of a humanized antibody can be accomplished by inserting the appropriate CDR coding segments (responsible for the desired binding properties) into a human antibody "scaffold". This is achieved through recombinant DNA methods using an appropriate vector and expression in mammalian cells. That is, after an antibody is developed to have the desired properties in a mouse (or other non-human), the DNA coding for that antibody can be isolated, cloned into a vector and sequenced. The DNA sequence corresponding to the antibody CDRs can then be determined. Once the precise sequence of the desired CDRs are known, a strategy can be devised for inserting these sequences appropriately into a construct containing the DNA for a human antibody variant. The strategy may also employ synthesis of linear DNA fragments based on the reading of CDR sequences.
  • the 4-1 BB agonist moiety and/or the dendritic cell binding moiety may be a fully human antibody or antigen binding fragment thereof.
  • embodiments of the CD40 agonist moiety and/or the dendritic cell binding moiety may be a humanized antibody or antigen binding fragment thereof.
  • Fully human antibodies can be produced in a number of ways including production in transgenic animals, mice that have been engineered to express fully human antibodies in mice.
  • humanized transgenic mice are immunized with specific target immunogen(s). After immunization and enough time to allow the mice to make antibodies, mRNA and cDNA derived from PBMCs, splenocytes or bone marrow cells are collected. Next the cDNA of the human antibody can be PCR amplified and cloned into vectors for expression.
  • Other systems for generating human antibodies is a combination of hybridoma technology and phage display.
  • the humanized transgenic mice are used to create antibodies as described above but the cDNA is cloned into phage display vectors to generate a phage display library which can be screened for human antibodies with high specificity and affinity.
  • Examples of such technology are XenomouseTM from Abgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse®, Fully Human Antibody TechnologyTM (Creative Biolabs, Shirley NY) and TC MouseTM from Medarex, Inc. (Princeton, N.J.).
  • Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and usually purification to a pharmaceutically acceptable purity.
  • nucleic acids encoding the respective polypeptides are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells (such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells), and the binding protein is recovered from the cells (supernatant or cells after lysis).
  • polypeptides may be suitably separated from the culture medium by
  • Purification can be performed in order to eliminate cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCI banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F., et al., ed.
  • affinity chromatography with microbial proteins e.g. protein A or protein G affinity chromatography
  • ion exchange chromatography e.g. cation exchange (carboxylmethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange
  • thiophilic adsorption e.g. with beta-mercaptoethanol and other SH ligands
  • hydrophobic interaction or aromatic adsorption chromatography e.g.
  • linkers can be used in the bispecific molecules described herein.
  • Linker refers to one or more amino acids connecting two domains or regions together.
  • linker polypeptides are well known in the art (see e.g. , Holliger, P. , et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J. , et al. (1994) Structure 2: 1 121-1 123). Additional linkers suitable for use can be found in the Registry of Standard Biological Parts at partsregistry.org/Protein_domains/Linker (see also, e.g.
  • a linker may be 1-10, 10-20, 20- 30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or at least 90-100 amino acids long.
  • Linkers also included non-polypeptide linkers.
  • Linkers can be comprised of nucleotides, or non-nucleotides.
  • Non-nucleotide linkers include, but are not limited to, abasic nucleotides polyethers, polyamines, polyamides, peptides, carbohydrates, lipids,
  • polyhydrocarbons or other polymeric compounds include, for example, polyethylene glycols such as those having between 2 and 100 ethylene glycol units.
  • linkers are particularly useful for linking aptamers that are linked to one or more other aptamers with similar or varying specificities.
  • Such linked aptamers e.g. dimers or trimers, may increase the affinities of the aptamers for their targets, (see Hasegawa, H. , et al. , Sensors (Basel) 2008 Feb 8(2): 1090-1098.)
  • An aptamer 4-1 BB agonist moiety or dendritic cell binding moiety may be conjugated to a protein 4-1 BB agonist moiety or dendritic cell binding moiety to create a bispecific molecule disclosed herein.
  • Exemplary methods of conjugating aptamers and other nucleic acids to proteins are described in U.S. Pat. Nos. 7,910,297, 8,318,920, and 8,389,710.
  • compositions provided herein contain one or more of the 4-1 BB agonist bispecific molecules and/or CD40 agonist bispecific molecule disclosed herein, formulated together with a carrier (e.g., a "pharmaceutically acceptable carrier").
  • a carrier e.g., a "pharmaceutically acceptable carrier”.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • Saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid carriers, particularly for injectable solutions.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for
  • compositions are known in the art. Except insofar as any excipient, diluent or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions provided herein is contemplated. Supplementary active compounds (e.g., additional anti-cancer agents) can also be incorporated into the compositions.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. , by injection or infusion).
  • the polypeptide may be coated in a material to protect them from the action of acids and other natural conditions that may inactivate proteins.
  • the polypeptide may be administered to a patient in an appropriate carrier, for example, in liposomes, or a diluent.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Liposomes include water-in-oil-in-water CGF emulsions, as well as conventional liposomes.
  • the composition can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated.
  • an exemplary dose comprising the 4- 1 BB agonist bispecific molecule, or the CD40 agonist bispecific molecule, to be administered to a patient in need thereof may include a single dose of about 0.01 to about 100 mg/kg body weight, about 0.03 to about 50 mg/kg body weight, or about 0.05 to about 25 mg/kg body weight dosed, once or more times per day, and/or one or more times per week, for example, for one to four weeks, or one to eight weeks, or one to twelve weeks, one to fourteen weeks, or one to 26 weeks or more.
  • the 4-1 BB agonist bispecific molecule, or the CD40 agonist bispecific molecule may be administered for periods ranging from 4 weeks, 8 weeks, 12 weeks, 24 weeks, 26 weeks, 28 weeks, 3 months, 6 months, 12 months or indefinitely.
  • an exemplary dosing regimen may include administration of a maximal dose or dosing frequency that avoids significant undesirable side effects.
  • a total daily dose may be at least 0.05 ⁇ g/kg body weight, at least 0.2 ⁇ g/kg, at least 0.5 ⁇ g/kg, at least 1 ⁇ g/kg, at least 10 ⁇ g/kg, at least 100 ⁇ g/kg, at least 0.2 mg/kg, at least 0.5 mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg, or at least 10 mg/kg, or at least 25 mg/kg, or at least 50 mg/kg, or at least 75 mg/kg, or at least 100 mg/kg, or at least 200 mg/kg.
  • a therapeutically effective dose may also equate to a daily dose as provided herein.
  • the dose, frequency and the duration of the treatment may be adjusted accordingly, in view of proper medical standards known to those of skill in the art.
  • the 4-1 BB agonist bispecific molecule of the disclosure, or the CD40 agonist bispecific molecules of the disclosure may be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, to about 100 mg, or to about 50 mg.
  • the first dose may be an initial loading dose, to be followed subsequently by a plurality of maintenance doses
  • an exemplary loading dose may include 500 mg/kg, 450 mg/kg, 400 mg/kg, 350 mg/kg, 300 mg/kg, 250 mg/kg, 200 mg/kg, 150 mg/kg, 100 mg/kg, 50 mg/kg, 40 mg/kg, 30 mg/kg, 20 mg/kg, 18 mg/kg, 16 mg/kg, 14 mg/kg, 12 mg/kg, 10 mg/kg, 8 mg/kg, 4 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, or 0.05 mg/kg to be dosed on day 1 , or in the first week of a 1 , 2, 3, or 4 week cycle, and wherein the maintenance dose is 200 mg/kg, 150 mg/kg, 100 mg/kg, 50 mg/kg, 40 mg/kg, 30 mg/kg, 20 mg/kg, 18 mg/kg, 16 mg/kg, 14 mg/kg, 12 mg/kg, 10 mg/
  • the initial dose may be followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that may be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks, or doses of the 4-1 BB agonist bispecific molecules of the disclosure, or the CD40 agonist bispecific molecules of the disclosure, may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.
  • compositions of the present disclosure may be by, e.g., topical or cutaneous application, injection or infusion by intravenous, intraperitoneal, subcutaneous, intracerebral, intramuscular, intraocular, intraarterial, intradermal,
  • the injectable preparations may include dosage forms for intravenous,
  • the polypeptides and compositions described herein can be administered alone or in combination, i.e., combined with other agents.
  • the combination therapy can include a polypeptide described herein with at least one additional therapeutic agent (e.g., an antineoplastic (anti-cancer) agent).
  • additional therapeutic agent e.g., an antineoplastic (anti-cancer) agent.
  • the polypeptides and compositions described herein can also be administered in conjunction with an anti-cancer treatment modality, such as radiation therapy and/or surgery.
  • Adjunctive or combined administration includes simultaneous administration of any of the polypeptides described herein and one or more agents in the same or different dosage form, or separate administration of the polypeptide and one or more agents (e.g., sequential administration). Such concurrent or sequential administration preferably results in both the polypeptide and the one or more agents being simultaneously present in treated patients.
  • anti-plastic agent refers to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia.
  • Inhibition of metastasis is frequently a property of antineoplastic agents.
  • the 4-1 BB agonist bispecific molecules described herein are administered in combination with an additional antineoplastic agent. In another embodiment, no more than three antineoplastic agents are administered in combination with the 4-1 BB agonist bispecific molecules described herein. In another embodiment, no more than two other antineoplastic agents are administered in combination with the 4-1 BB agonist bispecific molecules described herein. In another embodiment, no more than one other antineoplastic agent is administered in combination with the 4-1 BB agonist bispecific molecules described herein. In another embodiment, no other antineoplastic agent is administered in combination with the 4-1 BB agonist bispecific molecules described herein.
  • the CD40 agonist bispecific molecules described herein are administered in combination with an additional antineoplastic agent.
  • no more than three antineoplastic agents are administered in combination with the CD40 agonist bispecific molecules described herein.
  • no more than two other antineoplastic agents are administered in combination with the CD40 agonist bispecific molecules described herein.
  • no more than one other antineoplastic agent is administered in combination with the CD40 agonist bispecific molecules described herein.
  • no other antineoplastic agent is administered in combination with the 40 agonist bispecific molecules described herein.
  • the 4-1 BB agonist bispecific molecules described herein can be combined with a vaccination protocol.
  • the CD40 agonist bispecific molecules described herein can be combined with a vaccination protocol.
  • Many experimental strategies for vaccination against tumors have been devised (see Rosenberg, S. , 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M. , Cancer Vaccines, Ch. 61 , pp.
  • a vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A. 90: 3539-43).
  • the bispecific molecules described herein can be used in combination (e.g. , simultaneously or separately) with an additional treatment, such as irradiation, chemotherapy (e.g. , using camptothecin (CPT-1 1), 5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, 5-fu, or camptothecin + apo2l/TRAI L (a 6X combo)), one or more proteasome inhibitors (e.g.
  • Bcl-2 inhibitors e.g. , BH3I-2' (bcl-xl inhibitor), indoleamine dioxygenase-1 inhibitor (e.g. , I NCB24360, indoximod, NLG-919, or F001287), AT-101 (R-(-)-gossypol derivative), ABT-263 (small molecule), GX-15-070 (obatoclax), or MCL-1 (myeloid leukemia cell differentiation protein-1 ) antagonists), iAP
  • Bcl-2 inhibitors e.g. , BH3I-2' (bcl-xl inhibitor), indoleamine dioxygenase-1 inhibitor (e.g. , I NCB24360, indoximod, NLG-919, or F001287), AT-101 (R-(-)-gossypol derivative), ABT-263 (small molecule), GX-15-070 (obatoclax), or MCL-1 (myeloid leukemia cell differentiation
  • inhibitor of apoptosis protein antagonists
  • smac7, smac4, small molecule smac mimetic, synthetic smac peptides see Fulda et al. , Nat Med 2002;8:808-15), ISIS23722 (LY2181308), or AEG-35156 (GEM-640)
  • HDAC histone deacetylase
  • anti-CD20 antibodies e.g. , rituximab
  • angiogenesis inhibitors e.g., bevacizumab
  • anti-angiogenic agents targeting VEGF and VEGFR e.g. , Avastin
  • synthetic triterpenoids see Hyer et al., Cancer Research
  • c-FLI P cellular FLICE-inhibitory protein modulators
  • PPARv peroxisome proliferator-activated receptor ⁇
  • kinase inhibitors e.g. , Sorafenib
  • Trastuzumab Cetuximab
  • Temsirolimus mTOR inhibitors such as rapamycin and temsirolimus
  • mTOR inhibitors such as rapamycin and temsirolimus
  • Bortezomib JAK2 inhibitors
  • HSP90 inhibitors PI3K-AKT inhibitors
  • Lenalildomide ⁇ 5 ⁇ 3 ⁇ inhibitors
  • IAP inhibitors and/or genotoxic drugs.
  • bispecific molecules described herein can further be used in combination with one or more anti-proliferative cytotoxic agents.
  • Classes of compounds that may be used as anti-proliferative cytotoxic agents include, but are not limited to, the following:
  • Alkylating agents including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes: Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXANTM) fosfamide, Melphalan, Chlorambucil, Pipobroman,
  • Triethylenemelamine Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, dacarbazine, and Temozolomide.
  • Antimetabolites including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors: Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate,
  • Suitable anti-proliferative agents for combining with the bispecific molecules described herein include without limitation, taxanes, paclitaxel (paclitaxel is commercially available as TAXOLTM), docetaxel, discodermolide (DDM), dictyostatin (DCT), Peloruside A, epothilones, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanoepothilone D, desoxyepothilone Bl, [17]-dehydrodesoxyepothilone B,
  • hormones and steroids include synthetic analogs, such as 17a-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate, Methylprednisolone, Methyl-testosterone, Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine,
  • Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, ZOLADEXTM can also be administered to the patient.
  • other agents used in the modulation of tumor growth or metastasis in a clinical setting such as antimimetics, can also be administered as desired.
  • chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is described in the Physicians' Desk Reference (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, N.J. 07645-1742, USA); the disclosure of which is incorporated herein by reference thereto.
  • PDR Physicians' Desk Reference
  • the chemotherapeutic agent(s) and/or radiation therapy can be administered according to therapeutic protocols well known in the art. It will be apparent to those skilled in the art that the administration of the chemotherapeutic agent(s) and/or radiation therapy can be varied depending on the disease being treated and the known effects of the chemotherapeutic agent(s) and/or radiation therapy on that disease. Also, in accordance with the knowledge of the skilled clinician, the therapeutic protocols (e.g., dosage amounts and times of
  • administration can be varied in view of the observed effects of the administered therapeutic agents on the patient, and in view of the observed responses of the disease to the administered therapeutic agents.
  • kits containing the bispecific molecule compositions described herein and instructions for use typically include a packaged combination of reagents in predetermined amounts with instructions and a label indicating the intended use of the contents of the kit.
  • the term label or instruction includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit at any time during its manufacture, transport, sale or use. It can be in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of the manufacture, use or sale for administration to a human or for veterinary use.
  • the label or instruction can also encompass advertising leaflets and brochures, packaging materials, and audio or video instructions.
  • the kit contains the bispecific molecule in suitable containers and instructions for administration in accordance with the treatment regimens described herein.
  • the kit further comprises an additional antineoplastic agent.
  • the bispecific molecules are provided in suitable containers as a dosage unit for administration. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic.
  • the bispecific molecules are provided in lyophilized form, and the kit may optionally contain a sterile and physiologically acceptable reconstitution medium such as water, saline, buffered saline, and the like. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use, for example, comprising administration schedules, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein.
  • a sterile and physiologically acceptable reconstitution medium such as water, saline, buffered saline, and the like. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use, for example, comprising administration schedules, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition
  • Anti-LI LRA4/I LT7 antibodies are being developed, including but not limited to, those disclosed in US2009/0280128 to Medimmune and US8084585 to SBI Biotech.
  • Anti-LAMP5 antibodies are also being developed, including but not limited to, those disclosed in WO2013147153 and US201501 18184 to Chugai Pharmaceutical Co. and
  • anti-CLEC4C/BDCA-2 antibodies are being developed, these antibodies include, but are not limited to, BI I B059 (see, e.g., US20150299325), and antibodies disclosed in US20130315820 and WO2016156450 to LFB Biotechnologies, US20160280790 to the
  • Anti-I L3RA/CD123 antibodies are also being developed. These antibodies include, but are not limited to, talacotuzumab (JNJ-56022473) (see, e.g. , WO2012021934 and
  • IMGN632 see, e.g. , WO2017004026, WO2017091745 to Immunogen, Inc.
  • KHK2823 see, e.g., US84921 19 to Kyowa Hakko Kirin Co.
  • MGD006 see, e.g., WO2015026892 to MacroGenics, Inc.
  • SGN-CD123A see WO2016201065 to Seattle Genetics
  • SL-101 see, e.g., US8163279 and WO2008127735 to Stemline Therapeutics
  • XmAb14045 see, e.g.
  • IMGN632 see, e.g. , US20170029514 to ImmunoGen
  • UB-221 see, e.g.
  • Anti-CLEC9A antibodies are also being developed, including but not limited to those disclosed in WO2017134301 to Ononis Biosciences NV.
  • Anti-XCR1 antibodies are also being developed, including but not limited to, those disclosed in WO2013032032 and US9371389 to Eisai, Inc.
  • Anti-FLT3 antibodies are also being developed. These antibodies include, but are not limited to, IMC-EB10 (see, e.g. , US8071099 and WO2009155015 to Imclone, LLC), AGS62P1 (see, e.g. , WO2016145099 and US20160272716 to Agensys), and 4G8SDI EM (see Hoffman et al., 2012, Leukemia, 26: 1228-37), as well as antibodies and variable domains disclosed in WO2017205747 to United States Dept HHS, WO201 1076922 and US9023996 to Synimmune GmbH, WO2017176760 to Hemogenyx, WO2017023162 to Amgen, and
  • Table 3 below provides the SEQ I D NOs for various polypeptides relating to the present invention.
  • Amino Acids 50-254 ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMF 5 of Human 4-1 BBL AQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVV
  • NP_036408.4 YGGQYRCYGAHNVSSEWSAPSDPLDILIAGQISDRPSLSVQPGPTVTS
  • Murine CD40 - MVSLPRLCALWGCLLTAVHLGQCVTCSDKQYLHDGQCCDLCQP 29 isoform 1 GSRLTSHCTALEKTQCHPCDSGEFSAQWNREIRCHQHRHCEPN
  • CD40L (ligand) GRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNV
  • GRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNV DPSQVSHGTGF SFGLLKLGGGGSGGGGSGGGGSMQKGDQNP QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTV KRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILL RAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNV DPSQVSH GTGFTSFGLLKLGGGGSGGGGSGGSMQKGDQNPQIAAHVIS EASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYI YAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSS AKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFG LLKL
  • Single-cell melanoma data were obtained from Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/gds) under accession number GSE72056 in a pre-processed format.
  • GEO Gene Expression Omnibus
  • To characterize the baseline of immune cells we accessed data from 4000 cells derived from peripheral blood of four healthy subjects (Zheng et al. 2016, Nature Communication, 10: 14049). Single-cell RNA-seq data of PBMCs from patient blood samples were downloaded from the 10x Genomics website
  • dendritic cells and all other cells.
  • 4-1 BB was observed to be selectively upregulated in the tumor context and expressed mostly on T-cells.
  • CD40 was observed to be expressed more broadly on antigen presenting cell, including cross-presenting dendritic cells (see also Roberts et al. , 2016, Cancer Cell 30:324-336).
  • Table 4 Ranking of secondary targets for 4-1 BB agonistic bispecific molecules and CD40 agonistic bispecific molecules.

Abstract

L'invention concerne des molécules bispécifiques qui comprennent une fraction agoniste de 4-1BB reliée à une fraction de liaison de cellule dendritique et des procédés d'utilisation de telles molécules bispécifiques pour traiter des troubles liés à 4-1BB tels que le cancer. L'invention concerne également des molécules bispécifiques qui comprennent une fraction agoniste de CD40 reliée à une fraction de liaison de cellule dendritique et des procédés d'utilisation de telles molécules bispécifiques pour traiter des troubles liés à CD40 tels que le cancer.
PCT/US2018/033456 2017-05-19 2018-05-18 Molécules bispécifiques agonistes de 4-1bb et de cd40 WO2018213747A1 (fr)

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WO2020185628A1 (fr) * 2019-03-08 2020-09-17 Obsidian Therapeutics, Inc. Compositions de cd40l et procédés de régulation accordable
US10968280B2 (en) 2017-08-04 2021-04-06 Genmab A/S Binding agents binding to PD-L1 and CD137 and use thereof
US11058725B2 (en) 2019-09-10 2021-07-13 Obsidian Therapeutics, Inc. CA2 compositions and methods for tunable regulation
WO2021149053A1 (fr) 2020-01-22 2021-07-29 Yeda Research And Development Co. Ltd. Anticorps multispécifiques destinés à être utilisés dans le traitement de maladies
EP3706786A4 (fr) * 2017-11-09 2021-09-01 Medimmune, LLC Polypeptides de fusion bispécifiques et leurs procédés d'utilisation
WO2021239968A1 (fr) * 2020-05-28 2021-12-02 Strike Pharma Ab Protéine de liaison au cd40
WO2023036041A1 (fr) * 2021-09-09 2023-03-16 广东东阳光药业有限公司 Anticorps agoniste anti-4-1bb et son utilisation
IL286430A (en) * 2021-09-14 2023-04-01 Yeda Res & Dev Multispecific antibodies for use in the treatment of diseases
US11667723B2 (en) 2020-08-17 2023-06-06 Utc Therapeutics (Shanghai) Co., Ltd. Lymphocytes-antigen presenting cells co-stimulators and uses thereof
US11939388B2 (en) 2016-07-14 2024-03-26 Genmab A/S Multispecific antibodies against CD40 and CD137

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Cited By (11)

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Publication number Priority date Publication date Assignee Title
US11939388B2 (en) 2016-07-14 2024-03-26 Genmab A/S Multispecific antibodies against CD40 and CD137
US10968280B2 (en) 2017-08-04 2021-04-06 Genmab A/S Binding agents binding to PD-L1 and CD137 and use thereof
US11459395B2 (en) 2017-08-04 2022-10-04 Genmab A/S Binding agents binding to PD-L1 and CD137 and use thereof
EP3706786A4 (fr) * 2017-11-09 2021-09-01 Medimmune, LLC Polypeptides de fusion bispécifiques et leurs procédés d'utilisation
WO2020185628A1 (fr) * 2019-03-08 2020-09-17 Obsidian Therapeutics, Inc. Compositions de cd40l et procédés de régulation accordable
US11058725B2 (en) 2019-09-10 2021-07-13 Obsidian Therapeutics, Inc. CA2 compositions and methods for tunable regulation
WO2021149053A1 (fr) 2020-01-22 2021-07-29 Yeda Research And Development Co. Ltd. Anticorps multispécifiques destinés à être utilisés dans le traitement de maladies
WO2021239968A1 (fr) * 2020-05-28 2021-12-02 Strike Pharma Ab Protéine de liaison au cd40
US11667723B2 (en) 2020-08-17 2023-06-06 Utc Therapeutics (Shanghai) Co., Ltd. Lymphocytes-antigen presenting cells co-stimulators and uses thereof
WO2023036041A1 (fr) * 2021-09-09 2023-03-16 广东东阳光药业有限公司 Anticorps agoniste anti-4-1bb et son utilisation
IL286430A (en) * 2021-09-14 2023-04-01 Yeda Res & Dev Multispecific antibodies for use in the treatment of diseases

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