FLT3L-FC fusion proteins and methods of use

Abstract

Provided are FLT3L-Fc fusion proteins, polynucleotides encoding such fusion proteins, expression cassettes, vectors, cells and kits comprising such fusion proteins, and methods of use.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 62 / 866,584, filed on June 25, 2019, which is hereby incorporated by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on May 27, 2020, is named 1290_PF_SL.txt, and is 195,679 bytes in size. Background Art

[0005] Dendritic cells (DCs) are the most potent antigen-presenting cells in the body. Their function is to process antigenic material and present it to T cells on their cell surface. DCs act as messengers between the innate and adaptive immune systems. Fms-related tyrosine kinase 3 ligand (FLT3LG, FLT3L, NCBI Gene ID: 2323) selectively expands DCs from bone marrow precursors and promotes the proliferation of terminally differentiated DCs in lymphoid and tumor tissues.

[0006] The soluble recombinant human protein form of FLT3L has a serum half-life of about 12-28 hours in humans after five consecutive subcutaneous (SC) administrations and needs to be administered to patients every day within a 28-day treatment cycle. Daily administration is undesirable for both patients and clinicians, and the dosage schedule is inconsistent with other approved immuno-oncology therapeutics (usually once every 2 to 3 weeks). Long-acting antiviral therapeutics are also considered desirable. Summary of the Invention

[0007] Provided are FLT3L-Fc fusion proteins having prolonged serum half-life in human subjects relative to soluble FLT3L.

[0008] Therefore, in one aspect, a fusion protein is provided, which comprises: a human fms-related tyrosine kinase 3 ligand (FLT3L) extracellular domain, which is operably linked to an immunoglobulin fragment crystallizable region (Fc region), wherein: at least 5 amino acids are truncated from the C-terminus of the FLT3L extracellular domain; and / or the Fc region does not comprise a hinge region. In some embodiments, the FLT3L extracellular domain is derived from a human FLT3L extracellular domain. In some embodiments, the FLT3L extracellular domain is a human FLT3L extracellular domain. In some embodiments, the fusion protein is capable of binding to human FLT3. In some embodiments, the FLT3L extracellular domain is from FLT3L isoform 1 or from FLT3L isoform 2. In some embodiments, at least 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids are truncated from the C-terminus of the FLT3L extracellular domain. In some embodiments, the FLT3L extracellular domain does not comprise (e.g., is deleted, removed, or excluded) the amino acid sequence PTAPQ (SEQ ID NO: 85), APTAPQ (SEQ ID NO: 86), TAPTAPQ (SEQ ID NO: 87), ATAPTAPQ (SEQ ID NO: 88), EATAPTAPQ (SEQ ID NO: 89), or LEATAPTAPQ (SEQ ID NO: 90), PTAPQPP (SEQ ID NO: 91), APTAPQPP (SEQ ID NO: 92), TAPTAPQPP (SEQ ID NO: 93), ATAPTAPQPP (SEQ ID NO: 94), EATAPTAPQPP (SEQ ID NO: 95), or LEATAPTAPQPP (SEQ ID NO: 96). In some embodiments, the FLT3L extracellular domain comprises an N-terminal signal peptide. In some embodiments, the FLT3L extracellular domain comprises one or more of the following amino acid substitutions: H8Y; K84E; S102A; and / or S125A; wherein the amino acid residue positions are referenced to SEQ ID NOs: 1-18, 21-27, or 71-81. In some embodiments, one or both of the serine residues at positions 102 and 125 are substituted with alanine, wherein the amino acid residue positions are referenced to SEQ ID NOs: 1-18, 21-27, or 71-81. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is derived from human IgG1 or IgG4.In some embodiments, the Fc region comprises a human IgG1 isotype and comprises one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of: N297A, N297G, N297Q, N297G, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, P329G, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, M428L, N434S, T366W, T366S, L368A, Y407V, and any combination thereof, wherein the numbering of the residues is according to EU numbering. In some embodiments, the Fc region comprises a human IgG1 isotype and comprises one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of: L234A, L234V, L234F, L235A, L235E, P331S, and any combination thereof, wherein the numbering of the residues is according to EU numbering. In some embodiments, the Fc region comprises a human IgG4 isotype and comprises one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of: E233P, F234V, F234A, L235A, G237A, E318A, S228P, L235E, T394D, M252Y, S254T, T256E, N297A, N297G, N297Q, T366W, T366S, L368A, Y407V, M428L, N434S, and any combination thereof, wherein the numbering of the residues is according to EU numbering. In some embodiments, the Fc region comprises a human IgG4 isotype and comprises one or more amino acid substitutions at a residue position selected from the group consisting of: F234V, F234A, L235A, L235E, S228P, and any combination thereof, wherein the numbering of the residues is according to EU numbering. In some embodiments, the Fc region comprises the following amino acids at specified positions (EU index numbering): (i) tyrosine at position 252, threonine at position 254, and glutamic acid (YTE) at position 256; or (ii) leucine at position 428 and serine (LS) at position 434. In some embodiments, the FLT3L extracellular domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:71-81.In some embodiments, the Fc region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 103-107. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-18 and 21-27. In some embodiments, the Fc region is from human IgG1 and does not comprise a hinge region.

[0009] In some embodiments, the C-terminus of the FLT3L extracellular domain is not truncated.In some embodiments, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 1.

[0010] In some embodiments, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the fusion protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 5, 7, 9, 10, 13, 15, 22, 23, and 24, or comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 5, 7, 9, 10, 13, 15, 22, 23, and 24, wherein the Fc region is derived from a human IgG1 isotype and does not comprise a hinge region, e.g., does not comprise the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 101) or EPKSCDKTHTCPPCPAPELL (SEQ ID NO: 110). In some embodiments, the Fc region is derived from human IgG4 and is truncated by at least 5 amino acids from the C-terminus of the FLT3L extracellular domain. In some embodiments, the Fc region comprises a hinge region. In some embodiments, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the fusion protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 6, 8, 11, 12, 14, 16, 17, 18, 25, and 26, or comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 6, 8, 11, 12, 14, 16, 17, 18, 25, and 26, wherein the Fc region is derived from a human IgG4 isotype, and wherein at least 5 amino acids are truncated from the C-terminus of the FLT3L extracellular domain, e.g., wherein the FLT3L extracellular domain does not comprise the amino acid sequence PTAPQ (SEQ ID NO: 85).

[0011] In another aspect, a fusion protein is provided comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-20.

[0012] In another aspect, a fusion protein is provided, comprising: (i) a FLT3L-Fc fusion protein comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-27; and (ii) a second polypeptide. In some embodiments, the second polypeptide comprises a targeting moiety or domain, a growth factor, a cytokine, a chemokine, or a TNF superfamily (TNFSF) member. In some embodiments, the second polypeptide is the N-terminus of the FLT3L extracellular domain. In some embodiments, the second polypeptide is the C-terminus of the Fc region. In some embodiments, the second polypeptide is between the FLT3L extracellular domain and the Fc region. In some embodiments, the targeting moiety domain binds to a target protein or antigen identified in Table B below, such as, but not limited to, CD19, MS4A1 (CD20), CD22, IL2RA (CD25), CD27, TNFRSF8 (CD30), CD33, CD37, CD38, CD40, CD44, CD48, CD52, CD70, NT5E (CD73), ENTPD1 (CD39), CD74, CD79b, CD80, CD 86, IL3RA (CD123), PROM1 (CD133), CD137, SDC1 (CD138), α-fetoprotein (AFP), c-Met; c-Kit; C-type lectin domain family 12 member A (CLEC12A, CLL1, CD371); C-type lectin domain-containing 9A (CLEC9A, CD370); cadherin 3 (CDH3, p-cadherin, PCAD); carbonic anhydrase 6 (CA6); carbonic anhydrase 9 (C A9, CAIX); carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3); carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5); carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6, CD66c); chorionic villus growth hormone 1 (CSH1, CS1); coagulation factor III tissue factor (F3, TF); collagen lectin subfamily member 10 (COLEC10); delta-like canonical Notch ligand 3 (DLL3); ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor (EGFR); EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2); epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine kinase 2 (ERBB2; HER2); fibroblast activation protein alpha (FAP); fibroblast growth factor receptor 2 (FGFR2); fibroblast growth factor receptor 3 (FGFR3);Folate hydrolase 1 (FOLH1, PSMA); folate receptor 1 (FOLR1, FRα); GD2 ganglioside; glycoprotein NMB (GPNMB, osteoactivin); guanylate cyclase 2C (GUCY2C, GCC); human papillomavirus (HPV) E6; HPV E7; major histocompatibility complex (MHC) class I presenting neoantigens; major histocompatibility complex (MHC) class II presenting neoantigens; major histocompatibility complex class I E (HLA-E); major histocompatibility complex class I F (HLA-F); major histocompatibility complex class I G (HLA-G, MHC-G); integrin subunit β7 (ITGB7); leukocyte immunoglobulin-like receptor B1 (LILRB1, ILT2); leukocyte immunoglobulin-like receptor B2 (LILRB2, ILT4); LY6 / PLAUR domain-containing 3 (LYPD3, C 4.4A); glypican 3 (GPC3); KRAS proto-oncogene GTPase (KRAS); MAGE family member A1 (MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member D1 (MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin 1 (MUC 1) and its splice variants (e.g., MUC1 / C, D, and Z); mucin 16 (MUC16); necdin (NDN); nectin cell adhesion molecule 4 (NECTIN4); SLIT and NTRK-like family member 6 (SLITRK6); promyelocytic leukemia (PML, TRIM19); protein tyrosine kinase 7 (inactive) (PTK7); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, 19A, CD319, CRACC, CS1); sialic acid-binding Ig-like lectin 7 (SIGLEC7); sialic acid-binding Ig-like lectin 9 (SIGLEC9); solute carrier family 34 (sodium phosphate) member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6; LIV1); STEAP family member 1 (STEAP1); TNF receptor superfamily member 4 (TNFRSF4, OX40, or CD134); TNF superfamily member 9 (TNFSF9; 4-1BB-L, CD137L); TNF receptor superfamily member 10a (TNFRSF10A, DR4, CD261, TRAILR1);TNF receptor superfamily member 10b (TNFRSF10B, DR5, CD262, TRAILR2); TNF receptor superfamily member 13B (TNFRSF13B; CD267, TACI, IGAD2); TNF receptor superfamily member 17 (TNFRSF17, BCMA, CD269); TNF receptor superfamily member 18 (TNFRSF18, GITR or CD357); transferrin (TF); transforming growth factor beta 1 (TGFB1); trophoblast glycoprotein (TPBG, 5T4); trophinin (TRO, MAGED3); tumor-associated calcium signal transducer 2 (TACSTD2, TROP2, EGP1); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); and Lewis Y antigen.

[0013] In a related aspect, a homodimer is provided, comprising two (i.e., a first and a second) identical FLT3L-Fc fusion proteins as described above and herein. In a related aspect, a heterodimer is provided, comprising two (i.e., a first and a second) non-identical FLT3L-Fc fusion proteins as described above and herein. In various embodiments of such heterodimers, the FLT3L domains between the first and second FLT3L-Fc fusion proteins are identical, but the Fc regions are different.

[0014] In another aspect, heterodimers are provided that comprise a FLT3L-Fc fusion protein as described above and herein and a second fusion protein comprising a targeting moiety domain fused to a second Fc region. In some embodiments, the targeting moiety domain binds to a target protein or antigen identified in Table B below, such as, but not limited to, CD19, MS4A1 (CD20), CD22, IL2RA (CD25), CD27, TNFRSF8 (CD30), CD33, CD37, CD38, CD40, CD44, CD48, CD52, CD70, NT5E (CD73), ENTPD1 (CD39), CD74, CD79b, CD80, CD86, IL3RA (CD123), PROM1 (CD13 3), CD137, SDC1 (CD138), alpha-fetoprotein (AFP), c-Met; c-Kit; C-type lectin domain family 12 member A (CLEC12A, CLL1, CD371); C-type lectin domain-containing 9A (CLEC9A, CD370); cadherin 3 (CDH3, p-cadherin, PCAD); carbonic anhydrase 6 (CA6); carbonic anhydrase 9 (CA9, CAIX); carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3); carcinoembryonic antigen-related cell adhesion molecule 5 (CEACA M5); carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6, CD66c); chorionic villus growth hormone 1 (CSH1, CS1); coagulation factor III tissue factor (F3, TF); collagen lectin subfamily member 10 (COLEC10); delta-like canonical Notch ligand 3 (DLL3); ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor (EGFR); EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2); Epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine kinase 2 (ERBB2; HER2); fibroblast activation protein alpha (FAP); fibroblast growth factor receptor 2 (FGFR2); fibroblast growth factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1, PSMA); folate receptor 1 (FOLR1, FRα); GD2 ganglioside; glycoprotein NMB (GPNMB, osteoactivin); guanylate cyclase 2C (GUCY2C, GCC); human papillomavirus (HPV) E6; HPV E7; major histocompatibility complex (MHC) class I presenting neoantigens, major histocompatibility complex (MHC) class II presenting neoantigens, major histocompatibility complex class I E (HLA-E); major histocompatibility complex class I F (HLA-F); major histocompatibility complex class I G (HLA-G, MHC-G);Integrin subunit beta 7 (ITGB7); leukocyte immunoglobulin-like receptor B1 (LILRB1, ILT2); leukocyte immunoglobulin-like receptor B2 (LILRB2, ILT4); LY6 / PLAUR domain-containing 3 (LYPD3, C4.4A); glypican 3 (GPC3); KRAS proto-oncogene GTPase (KRAS); MAGE family member A1 (MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC 2); MAGE family member D1 (MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin 1 (MUC1) and its splice variants (e.g., MUC1 / C, D, and Z); mucin 16 (MUC16); necdin (NDN); nectin cell adhesion molecule 4 (NECTIN4); SLIT and NTRK-like family member 6 (SLITRK6); promyelocytic leukemia (PML, TRIM19); protein tyrosine kinase 7 (inactive) (PTK7); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, 19A, CD31 9, CRACC, CS1); sialic acid-binding Ig-like lectin 7 (SIGLEC7); sialic acid-binding Ig-like lectin 9 (SIGLEC9); solute carrier family 34 (sodium phosphate) member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6; LIV1); STEAP family member 1 (STEAP1); TNF receptor superfamily member 4 (TNFRSF4, OX40 or CD134); TNF superfamily member 9 (TNFSF9; 4-1BB-L, CD137L); TNF receptor superfamily member 10a (TNFRSF10A, DR4, CD261, TRAILR1); TNF receptor superfamily members 10b (TNFRSF10B, DR5, CD262, TRAILR2); TNF receptor superfamily member 13B (TNFRSF13B; CD267, TACI, IGAD2); TNF receptor superfamily member 17 (TNFRSF17, BCMA, CD269); TNF receptor superfamily member 18 (TNFRSF18, GITR or CD357); transferrin (TF); transforming growth factor beta 1 (TGFB1); trophoblast glycoprotein (TPBG, 5T4); trophinin (TRO, MAGED3); tumor-associated calcium signal transducer 2 (TACSTD2, TROP2, EGP1); fucosyl GM1;Sialyl Lewis adhesion molecule (sLe); and Lewis Y antigen. In some embodiments, the targeting moiety domain comprises an antigen-binding antibody fragment. In some embodiments, the antibody fragment comprises a Fab or a single-chain variable fragment (scFv). In some embodiments, the targeting moiety domain comprises a non-immunoglobulin binding portion or an antibody mimetic protein. In some embodiments, the non-immunoglobulin antigen binding domain or antibody mimetic protein is selected from the group consisting of: adnectin, affibody molecule, affilin, affimer, affitin, alphabody, anticalin, peptide aptamer, armadillo repeat protein (ARM), atrimer, avimer, designed ankyrin repeat protein; fynomers, knottins, Kunitz domain peptides, monobodies, and nanoCLAMPs. In some embodiments, neither the first Fc region nor the second Fc region comprises a hinge region. In some embodiments, the heterodimer is stabilized by the interaction between the first Fc region and the second Fc region. In some embodiments, the heterodimer comprises a heterodimeric human IgG1 or human IgG4. In some embodiments, the heterodimeric human IgG1 or human IgG4 comprises a first Fc region and a second Fc region, wherein the Fc regions comprise the following amino acids (EU numbering) at the specified positions: (i) the first Fc region comprises a tryptophan (T366W) at position 366; and the second Fc region comprises a serine (T366S) at position 366, an alanine (L368A) at position 368, and a valine (Y407V) at position 407; (ii) the first Fc region comprises a serine (T366S) at position 366, an alanine (L368A) at position 368, and a valine (Y407V) at position 407; and the second Fc region comprises a tryptophan (T366W) at position 366; (iii) the first Fc region comprises a serine (T366S) at position 366, an alanine (L368A) at position 368, and a valine (Y407V) at position 407; wherein the first Fc region comprises a cysteine ​​at position 349 (Y349C), a serine at position 366 (T366S), an alanine at position 368 and a valine at position 407 (Y407V); or (iv) the first Fc region comprises a cysteine ​​at position 349 (Y349C), a serine at position 366 (T366S), an alanine at position 368 and a valine at position 407 (Y407V); and the second Fc region comprises a cysteine ​​at position 354 (S354C), a tryptophan at position 366 (T366W).

[0015] On the other hand, a conjugate is provided, comprising: (i) a FLT3L-Fc fusion protein as described above and herein, or a homodimer or heterodimer of such a FLT3L-Fc protein; the conjugate is attached to a therapeutic agent or a detectable label. In some embodiments, the therapeutic agent is covalently linked. In some embodiments, the therapeutic agent is a small organic compound. In some embodiments, the therapeutic agent is an agonist or activator of a toll-like receptor (TLR) or a stimulator of interferon genes (STING) receptor. In some embodiments, the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. In some embodiments, the TLR7 agonist is selected from the group consisting of GS 9620, DS-0509, LHC-165 and TMX-101 (imiquimod), and / or the TLR8 agonist is selected from the group consisting of GS-9688 and NKTR-262 (dual TLR7 / TLR8 agonist). In some embodiments, the STING receptor agonist or activator is selected from the group consisting of ADU-S100 (MIW-815), SB-11285, MK-1454, SR-8291, AdVCA0848, GSK-532, SYN-STING, MSA-1, SR-8291, 5,6-dimethylxanthone-4-acetic acid (DMXAA), cyclic GAMP (cGAMP) and cyclic diAMP. In some embodiments, the therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a small molecule inhibitor of CD274 (PDL1, PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1), or CTLA4. In some embodiments, the small molecule inhibitor of CD274 or PDCD1 is selected from the group consisting of GS-4224, GS-4416, INCB086550, and MAX10181. In some embodiments, the small molecule inhibitor of CTLA4 is BPI-002.

[0016] In another aspect, polynucleotides are provided that encode a FLT3L-Fc fusion protein as described above and herein. In some embodiments, the polynucleotide is selected from the group consisting of DNA, cDNA, RNA, or mRNA. In some embodiments, the polynucleotide comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-70. An expression cassette is also provided that comprises one or more regulatory sequences operably linked to a FLT3L-Fc encoding polynucleotide as described herein.

[0017] In another aspect, a vector is provided comprising a polynucleotide encoding a FLT3L-Fc fusion protein as described herein, or an expression cassette comprising such a FLT3L-Fc encoding polynucleotide. In some embodiments, the vector is a plasmid vector or a viral vector. In some embodiments, the viral vector comprises an oncolytic viral vector. In some embodiments, the viral vector comprises a DNA virus or an RNA virus. In some embodiments, the viral vector is from a family of viruses selected from the group consisting of: Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., lymphocytic choriomeningitis mammalian arenavirus, Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus)), Poxviridae (e.g., vaccinia virus), Herpesviridae (e.g., herpesvirus, such as HSV-1), Parvoviridae (e.g., Parvovirus H1), Reoviridae (e.g., reovirus), Picornaviridae (e.g., coxsackievirus, Seneca Valley virus, poliovirus), Paramyxoviridae (e.g., measles virus, Newcastle disease virus (NDV)), Rhabdoviridae (e.g., vesicular stomatitis virus (VSV)), Togaviridae (e.g., alphavirus, Sindbis virus), Enteroviridae (e.g., echovirus).

[0018] In another aspect, a lipoplex, such as a lipid nanoparticle (LNP), comprising a polynucleotide encoding a FLT3L-Fc fusion protein as described herein, or an expression cassette or vector comprising such a FLT3L-Fc encoding polynucleotide is provided.

[0019] In another aspect, a cell or cell population is provided, comprising a polynucleotide encoding a FLT3L-Fc fusion protein as described herein, or an expression cassette or vector comprising such a FLT3L-Fc encoding polynucleotide, wherein the cell expresses the FLT3L-Fc fusion protein, or a homodimer or heterodimer comprising such a FLT3L-Fc fusion protein. In some embodiments, the cell or cell population is a eukaryotic cell. In some embodiments, the cell or cell population comprises a mammalian cell, an insect cell, a plant cell, or a yeast cell. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell or a human cell. In some embodiments, the human cell is a human embryonic kidney cell. In some embodiments, the cell primarily sialylates N-linked and / or O-linked glycosylation sites in the fusion protein. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or more of the N-linked and / or O-linked glycosylation sites in the fusion protein are sialylated. In some embodiments, the sialylated N-linked and / or O-linked glycosylation site in the fusion protein comprises 2 to 7 sialic acid residues, such as 3 to 6 sialic acid residues, such as 4 to 5 sialic acid residues.

[0020] In another aspect, a pharmaceutical composition is provided, comprising a FLT3L-Fc fusion protein as described herein, or a fusion protein, homodimer, heterodimer, or conjugate comprising such a FLT3L-Fc fusion protein; a polynucleotide encoding a FLT3L-Fc fusion protein as described herein, or an expression cassette, vector, or lipoplex (such as LNP) comprising such a FLT3L-Fc encoding polynucleotide; and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises an aqueous formulation. In some embodiments, the composition comprises a FLT3L-Fc fusion protein or a fusion protein, homodimer, heterodimer, or conjugate comprising such a FLT3L-Fc fusion protein at a concentration ranging from about 1 mg / ml to about 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, or 20 mg / ml. In some embodiments, the composition is lyophilized. In some embodiments, the composition further comprises one or more additional therapeutic agents, such as a second therapeutic agent, a third therapeutic agent, or a fourth therapeutic agent.

[0021] Also provided is a method for promoting, inducing and / or increasing the amplification and / or proliferation of cells or cell groups expressing fms-related tyrosine kinase 3 (FLT3, CD135). In some embodiments, the method includes contacting the cell or cell group with an effective amount of the following items: a FLT3L-Fc fusion protein as described herein or a fusion protein, homodimer, heterodimer, conjugate or pharmaceutical composition comprising such a FLT3L-Fc fusion protein; a polynucleotide encoding a FLT3L-Fc fusion protein as described herein or an expression cassette, vector, lipoplex (such as LNP) or pharmaceutical composition comprising such a FLT3L-Fc encoding polynucleotide. In some embodiments, cells or cell groups expressing FLT3 include dendritic cells (e.g., cDC1 cells and / or cDC2 cells), monocyte-derived dendritic cells (moDC) and / or their progenitor cells. In some embodiments, cells or cell groups expressing FLT3 include hematopoietic progenitor cells. In some embodiments, hematopoietic progenitor cells are selected from the group consisting of common lymphoid progenitor cells (CLP), early progenitor cells with lymphocyte and myeloid potential (EPLM), granulocyte-monocyte (GM) progenitor cells (GMP), monocyte-derived dendritic cell (moDC) progenitor cells, and early multipotent progenitor cells (MPP) within the Lineage-kit+Sca1 (LSK) compartment. In various embodiments, cells or cell groups are contacted in vitro or in vivo. In some embodiments, cells or cell groups are proliferated or expanded within solid tumors. In some embodiments, conventional dendritic cells (e.g., cDC1 and / or cDC2) are expanded or induced to proliferate. In some embodiments, cDC1 dendritic cells (e.g., positive for surface expression of XC motif chemokine receptor 1 (XCR1), thrombomodulin (THBD, CD141), and 9A (CLEC9A) containing a C-type lectin domain) are expanded or induced to proliferate. In some embodiments, dendritic cells expressing CC motif chemokine receptor 5 (CCR5, CD195) and / or XC motif chemokine receptor 1 (XCR1) on their cell surface are expanded or their proliferation is induced. In some embodiments, dendritic cells expressing one or more cell surface proteins selected from the group consisting of the following items on their cell surface are expanded or their proliferation is induced: XCR1, cell adhesion molecule 1 (CADM1), C-type lectin domain-containing 9A (CLEC9A, CD370) and thrombomodulin (THBD) are expanded or their proliferation is induced. In some embodiments, dendritic cells expressing one or more cell surface proteins selected from the group consisting of the following items on their cell surface are expanded or their proliferation is induced.In some embodiments, dendritic cells expressing one or more proteins selected from the group consisting of the following items on their cell surface are expanded or their proliferation is induced: basic leucine zipper ATF-like transcription factor 3 (BATF3) and interferon regulatory factor 8 (IRF8). In some embodiments, dendritic cells expressing one or more proteins selected from the group consisting of the following items on their cell surface are expanded or their proliferation is induced: BATF3, IRF8, THBD, CLEC9A and XCR1. In some embodiments, cDC2 dendritic cells (e.g., positive for surface expression of CD1c molecule (BDCA)) are expanded or their proliferation is induced. In some embodiments, FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate, polynucleotide or pharmaceutical composition is administered to a subject or patient, e.g., a mammal, e.g., a human.

[0022] Also provided is a method for expanding hematopoietic stem cells (HSC) in vitro, comprising culturing HSC in the presence of mesenchymal lineage precursors or stem cells (MLPSC) and an effective amount of a FLT3L-Fc fusion protein as described herein, or a fusion protein, homodimer, heterodimer, conjugate, or pharmaceutical composition comprising such a FLT3L-Fc fusion protein; a polynucleotide encoding a FLT3L-Fc fusion protein as described herein, or an expression cassette, vector, or lipid complex (such as LNP) comprising such a FLT3L-Fc encoding polynucleotide, such that HSC with a phenotype of CD34+ is expanded. In some embodiments, HSC is further cultured in the presence of at least one histone deacetylase inhibitor (HDACi). In some embodiments, HDACi is selected from the group consisting of valproic acid (VPA), trichostatin A (TSA), DLS3, MS275, and SAHA. In some embodiments, the HSCs have the phenotype CD34+, CD90+ or ​​CD34+, CD45RA-, CD49f+ and are expanded at least 5-fold, at least 10-fold, at least 20-fold, or at least 40-fold. In some embodiments, the method further comprises isolating cells having the phenotype CD34+, CD90+ or ​​CD34+, CD45RA-, CD49f+ to provide an enriched population of cells having the phenotype CD34+, CD90+ or ​​CD34+, CD45RA-, CD49f+.

[0023] In related aspects, methods are provided for preventing, reducing and / or inhibiting the recurrence, growth, proliferation, migration and / or metastasis of cancer cells or cancer cell groups in subjects in need thereof. On the other hand, methods are provided for enhancing, promoting and / or increasing tumor infiltration of T cells and / or NK cells in subjects in need thereof. Methods for enhancing, promoting and / or accelerating the recovery of lymphopenia in subjects in need thereof or reversing its effects are also provided. Methods for treating or preventing viral infections are also provided. Methods for inhibiting viral replication, treating viral infections or delaying the onset of symptoms of viral infections in subjects in need thereof are also provided. Methods for enhancing, improving and / or increasing the response of subjects in need thereof to anti-cancer therapy or antiviral therapy are also provided. Methods for promoting, increasing, supplementing and / or strengthening the immune response induced by a vaccine are also provided. Methods for enhancing, improving and / or increasing the response of subjects in need thereof to immune checkpoint proteins are also provided. In some embodiments, the method includes administering to the subject an effective amount of a FLT3L-Fc fusion protein as described herein, or a fusion protein, homodimer, heterodimer, conjugate, or pharmaceutical composition comprising such a FLT3L-Fc fusion protein; a polynucleotide encoding a FLT3L-Fc fusion protein as described herein, or an expression cassette, vector, lipoplex (such as LNP), or pharmaceutical composition comprising such a FLT3L-Fc encoding polynucleotide. In some embodiments, one or more additional therapeutic agents (e.g., a second therapeutic agent, a third therapeutic agent, and / or a fourth therapeutic agent) are co-administered. In some embodiments, one or more additional therapeutic agents include one or more of the following: AGEN1884 (zalifrelimab), AGEN1181, AGEN2034 (balstilimab), AGEN1307, AGEN2373, AGEN1223, and GS-1423 (AGEN1423; see WO2019 / 173692). In some embodiments, the one or more additional therapeutic agents include a vaccine. In some embodiments, the vaccine is selected from the group consisting of: an antiviral vaccine, an antibacterial vaccine, and an anticancer vaccine (e.g., a neoantigen vaccine). In some embodiments, the vaccine includes an antiviral vaccine against a virus selected from the group consisting of: hepatitis A virus (HAV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), human orthopneumovirus or human respiratory syncytial virus (RSV), human papillomavirus (HPV), varicella zoster virus, measles virus, mumps virus, poliovirus vaccine, influenza virus, paramyxovirus, rotavirus, Zika virus, dengue virus,Ebola virus and coronavirus. In some embodiments, the vaccine comprises an antibacterial vaccine against a bacterium selected from the group consisting of Mycobacterium tuberculosis, pertussis, tetanus, diphtheria, meningococcus, pneumococcus, Haemophilus influenzae, cholera, typhoid and anthrax. In some embodiments, the one or more additional therapeutic agents comprise an oncolytic viral vector. In some embodiments, the oncolytic viral vector comprises a DNA virus or an RNA virus. In some embodiments, the viral vector is from a family of viruses selected from the group consisting of Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., lymphocytic choriomeningitis mammalian arenavirus, Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus)), Poxviridae (e.g., vaccinia virus), Herpesviridae (e.g., herpesvirus, such as HSV-1), Parvoviridae (e.g.,

[00135] The invention also includes but is not limited to viruses such as Parvovirus H1, Reoviridae (e.g., Reovirus), Picornaviridae (e.g., Coxsackievirus, Seneca Valley virus, poliovirus), Paramyxoviridae (e.g., Measles virus, Newcastle disease virus (NDV)), Rhabdoviridae (e.g., Vesicular Stomatitis Virus (VSV)), Togaviridae (e.g., Alphavirus, Sindbis virus), Enteroviridae (e.g., Echovirus). In some embodiments, the one or more additional therapeutic agents include immunotherapy, immunostimulatory therapy, cytokine therapy, chemokine therapy, cell therapy, gene therapy, and combinations thereof. In some embodiments, the immunotherapy comprises co-administering one or more antibodies or antigen-binding antibody fragments thereof or antibody-drug conjugates thereof, multispecific molecules targeting CD3, multispecific molecules targeting CD16, or non-immunoglobulin antigen binding domains or antibody mimetic proteins to one or more targets or tumor-associated antigens (TAAs) selected from the group consisting of CD19, MS4A1 (CD20), CD22, IL2RA (CD25), CD27, TNFRSF8 (CD30), CD33, CD37, CD38, CD40, CD44, CD48, CD52, CD70, NT5E (CD73), ENTPD1 (CD39), CD74, CD79b, CD80, CD86, IL3RA (CD123), PROM1 (CD133), CD137, SDC1 (CD138), alpha fetoprotein (AFP),c-Met; c-Kit; C-type lectin domain-containing family 12, member A (CLEC12A, CLL1, CD371); C-type lectin domain-containing family 9A (CLEC9A, CD370); cadherin 3 (CDH3, p-cadherin, PCAD); carbonic anhydrase 6 (CA6); carbonic anhydrase 9 (CA9, CAIX); carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3); carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5); carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6, CD66c); chorionic villus growth hormone 1 (CSH1, CS1); coagulation factor III tissue factor (F3, TF); collectin subfamily member 10 (COLEC10); delta-like canonical Notch ligand 3 (DLL3); exonucleoside Pyrophosphatase / phosphodiesterase 3 (ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor (EGFR); EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2); epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine kinase 2 (ERBB2, HER2); fibroblast activation protein alpha (FAP); fibroblast growth factor receptor 2 (FGFR2); fibroblast growth factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1, PSMA); folate receptor 1 (FOLR1, FRα); GD2 ganglioside; glycoprotein NMB (GPNMB, osteoactivin); guanylate cyclase 2C (GUCY2C, GCC); human papillomavirus (HPV) E6; HPV E7; major histocompatibility complex (MHC) class I presenting neoantigens, major histocompatibility complex (MHC) class II presenting neoantigens, major histocompatibility complex class I E (HLA-E); major histocompatibility complex class I F (HLA-F); major histocompatibility complex class I G (HLA-G, MHC-G); integrin subunit β7 (ITGB7); leukocyte immunoglobulin-like receptor B1 (LILRB1, ILT2); leukocyte immunoglobulin-like receptor B2 (LILRB2, ILT4); LY6 / PLAUR domain-containing 3 (LYPD3,C4.4A); glypican 3 (GPC3); KRAS proto-oncogene GTPase (KRAS); MAGE family member A1 (MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member D1 (MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin 1 (MUC1) and its splice variants (e.g., MUC1 / C, D, and Z); mucin 16 (MUC16); necdin (NDN); nectin cell adhesion molecule 4 (NECTIN4); SLIT and NTRK-like family member 6 (SLITRK6); promyelocytic leukemia (PML, TRIM19); protein tyrosine kinase 7 (inactive) (PTK7); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, 19A, CD319, CRACC, CS1); sialic acid-binding Ig-like lectin 7 (SIGLEC7); sialic acid-binding Ig-like lectin 9 (SIGLEC9); solute Carrier family 34 (sodium phosphate) member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6, LIV1); STEAP family member 1 (STEAP1); TNF receptor superfamily member 4 (TNFRSF4, OX40 or CD134); TNF superfamily member 9 (TNFSF9, 4-1BB-L, CD137L); TNF receptor superfamily member 10a (TNFRSF10A, DR4, CD261, TRAILR1); TNF receptor superfamily member 10b (TNFRSF10B, DR5, CD262, TRAILR2); TNF receptor superfamily members 13B (TNFRSF13B; CD267, TACI, IGAD2); TNF receptor superfamily member 17 (TNFRSF17, BCMA, CD269); TNF receptor superfamily member 18 (TNFRSF18, GITR, CD357); transferrin (TF); transforming growth factor beta 1 (TGFB1); trophoblast glycoprotein (TPBG, 5T4); trophinin (TRO, MAGED3); tumor-associated calcium signal transducer 2 (TACSTD2, TROP2, EGP1); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); and Lewis Y antigen. In some embodiments, one or more antibodies or antigen-binding antibody fragments thereof or antibody-drug conjugates thereof, multispecific molecules targeting CD3,The CD16 multispecific molecule or non-immunoglobulin antigen binding domain or antibody mimetic protein binds to an epitope of a target or tumor-associated antigen (TAA) (e.g., a neoantigen) presented in a major histocompatibility complex (MHC) molecule. In some embodiments, the TAA is a cancer testis antigen. In some embodiments, the cancer testis antigen is selected from the group consisting of: sperm nodule protein binding protein (ACRBP, CT23, OY-TES-1, SP32), alpha fetoprotein (AFP, AFPD, FETA, HPAFP); A kinase anchoring protein 4 (AKAP4, AKAP82, AKAP-4, AKAP82, CT99, FSC1, HI, PRKA4, hAKAP82, p82), ATPase family AAA domain-containing 2 (ATAD2, ANCCA, CT137, PRO2000), centromere scaffold 1 (KNL1, AF15Q14, CA SC5, CT29, D40, MCPH4, PPP1R55, Spc7, hKNL-1, hSpc105), centrosomal protein 55 (CEP55, C10orf3, CT111, MARCH, URCC6), cancer / testis antigen 1A (CTAG1A, ESO1, CT6.1, LAGE-2, LAGE2A, NY-ESO-1), cancer / testis antigen 1B (CTAG1B, CT6.1, CTAG, CTAG1, ESO1, LAGE-2, LAGE2B, NY-ESO-1), cancer / testis antigen 2 (CTAG2, CAME L, CT2, CT6.2, CT6.2a, CT6.2b, ESO2, LAGE-1, LAGE2B), CCCTC-binding factor-like (CTCFL, BORIS, CT27, CTCF-T, HMGB1L1, dJ579F20.2), catenin α2 (CTNNA2, CAP-R, CAPR, CDCBM9, CT114, CTNR), cancer / testis antigen 83 (CT83, CXorf61, KK-LC-1, KKLC1), cyclin A1 (CCNA1, CT146), DEAD box helicase 43 (DDX43, C T13, HAGE), developmental pluripotency-related 2 (DPPA2, CT100, ECAT15-2, PESCRG1), fetal and adult testis expressed 1 (FATE1, CT43, FATE), FMR1 neighbor (FMR1NB, CT37, NY-SAR-35, NYSAR35), HORMA domain-containing 1 (HORMAD1, CT46, NOHMA), insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3, CT98, IMP-3, IMP3, KOC, KOC1, VICKZ3), leucine zipper protein 4 (LUZP4,CT-28, CT-8, CT28, HOM-TES-85), lymphocyte antigen 6 family member K (LY6K, CT97, HSJ001348, URLC10, ly-6K), whirlpool spermatogenesis transposon silencer (MAEL, CT128, SPATA35), MAGE family member A1 (MAGEA1, CT1.1, MAGE1); MAGE family member A3 (MAGEA3, CT1.3, HIP8, HYPD, MAGE3, MAGEA6); MAGE family member A4 (MAGEA4, CT1.4, MAGE-41, MAGE-X2, MAGE4, ​​MAGE4A, MAGE4B); M AGE family member A11 (MAGEA11, CT1.11, MAGE-11, MAGE11, MAGEA-11); MAGE family member C1 (MAGEC1, CT7, CT7.1); MAGE family member C2 (MAGEC2, CT10, HCA587, MAGEE1); MAGE family member D1 (MAGED1, DLXIN-1, NRAGE); MAGE family member D2 (MAGED2, 11B6, BARTS5, BCG-1, BCG1, HCA10, MAGE-D2); kinesin family member 20B (KIF20B, CT90, KRMP1, MPHOSPH1, MPP- 1, MPP1); NUF2 components of the NDC80 centromere complex (NUF2, CDCA1, CT106, NUF2R), nuclear RNA export factor 2 (NXF2, CT39, TAPL-2, TCP11X2); PAS domain-containing repressor protein 1 (PASD1, CT63, CT64, OXTES1), PDZ-binding kinases (PBK, CT84, HEL164, Nori-3, SPK, TOPK); piwi-like RNA-mediated gene silencing 2 (PIWIL2, CT80, HILI, PIWIL1L, mili); antigens preferentially expressed in melanoma (PRAME, CT130, MAPE, OIP-4, O Sperm-associated antigen 9 (SPAG9, CT89, HLC-6, HLC4, HLC6, JIP-4, JIP4, JLP, PHET, PIG6), sperm protein X-linked family member A1 associated with the nucleus (SPANXA1, CT11.1, CT11.3, NAP-X, SPAN-X, SPAN-Xa, SPAN-Xb, SPANX, SPANX-A), SPANX family member A2 (SPANXA2, CT11.1, CT11.3, SPANX, SPANX-A, SPANX-C, SPANXA, SPANXC), SPANX family member C (SPANXC, CT11.3,CTp11, SPANX-C, SPANX-E, SPANXE), SPANX family member D (SPANXD, CT11.3, CT11.4, SPANX-C, SPANX-D, SPANX-E, SPANXC, SPANXE, dJ171K16.1), SSX family member 1 (SSX1, CT5.1, SSRC), SSX family member 2 (SSX2, CT5.2, CT5.2A, HD21, HOM-MEL-40, SSX), synaptonemal complex protein 3 (SYCP 3, COR1, RPRGL4, SCP3, SPGF4), 14 intercellular bridge-forming factors expressed in testis (TEX14, CT113, SPGF23), transcription factor Dp family member 3 (TFDP3, CT30, DP4, HCA661), serine protease 50 (PRSS50, CT20, TSP50), TTK protein kinase (TTK, CT96, ESK, MPH1, MPS1, MPS1L1, PYT) and zinc finger protein 165 (ZNF165, CT53, LD65, ZSCAN7). In some embodiments, the non-immunoglobulin antigen binding domain or antibody mimetic protein is selected from the group consisting of: adnectin, affinity molecule, affilin, affimer, affitin, alphabody, anticalin, peptide aptamer, armadillo repeat protein (ARM), atrimer, avimer, designed ankyrin repeat protein, fynomers, knottins, Kunitz domain peptides, monobodies, and nanoCLAMPs. In some embodiments, the immunotherapy comprises co-administering one or more antagonists or inhibitors of inhibitory immune checkpoint proteins or receptors and / or one or more activators or agonists of stimulatory immune checkpoint proteins or receptors. In some embodiments, the one or more immune checkpoint proteins or receptors are selected from the group consisting of: CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain-containing 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244 (SLAMF4); CD276 (B7H3); V-set domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4); V-set immunomodulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR-associated 2 (HHLA2, B7H7); inducible T cell costimulator (ICOS, CD278); inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137 ), TNFSF9 (CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF); TNFRSF18 (GITR), TNFSF18 (GITRL); MHC Class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR,CD155); PVR-associated immunoglobulin domain-containing (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); T-cell immunoglobulin and mucin domain-containing 4 (TIMD4; TIM4); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); lymphocyte activation 3 (LAG3, CD223); signal transducer lymphocyte activation molecule family member 1 (SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9 , CD229, SLAMF3); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, CD319); UL16 binding protein 1 (ULBP1); UL16 binding protein 2 (ULBP2); UL16 binding protein 3 (ULBP3); retinoic acid early transcript 1E (RAET1E; ULBP4); retinoic acid early transcript 1G (RAET1G; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314); killer cell lectin-like receptor C2 (KLRC2, CD159c, NKG2C); killer cell lectin-like receptor C3 (KLRC3, NKG2E); killer cell lectin-like receptor C4 (KLRC4, NKG2F); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell Killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); Killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); Killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); Killer cell lectin-like receptor D1 (KLRD1); Killer cell lectin-like receptor G1 (KLRG1; CLEC15A, MAFA, 2F1); Sialic acid-binding Ig-like lectin 7 (SIGLEC7); and Sialic acid-binding Ig-like lectin 9 (SIGLEC9). In some embodiments, immunotherapy includes co-administration of one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the T cell inhibitory immune checkpoint protein or receptor is selected from the group consisting of: CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PD1,PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4); V-set immunoregulatory receptors (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); PVR-related immunoglobulin domain-containing (PVRIG, CD112R); T cell immunoreceptor with Ig and ITIM domains (TIGIT); Lymphocyte activation 3 (LAG3, CD223); Hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); Galectin 9 (LGALS9); Killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); Killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); Killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); Killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and Killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1). In some embodiments, immunotherapy includes co-administration of one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors. In some embodiments, the T cell stimulatory immune checkpoint protein or receptor is selected from the group consisting of: CD27, CD70; CD40, CD40LG; inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). In some embodiments, immunotherapy includes co-administration of one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the NK cell inhibitory immune checkpoint protein or receptor is selected from the group consisting of: killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR,CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); killer cell lectin-like receptor D1 (KLRD1, CD94); killer cell lectin-like receptor G1 (KLRG1; CLEC15A, MAFA, 2F1); sialic acid-binding Ig-like lectin 7 (SIGLEC7); and sialic acid-binding Ig-like lectin 9 (SIGLEC9). In some embodiments, immunotherapy includes co-administration of one or more agonists or activators of one or more NK cell stimulating immune checkpoint proteins or receptors. In some embodiments, NK cell stimulating immune checkpoint proteins or receptors include but are not limited to CD16, CD226 (DNAM-1); Killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314); and SLAM family member 7 (SLAMF7). In some embodiments, one or more immune checkpoint inhibitors include protein inhibitors of PD-L1 (CD274), PD-1 (PDCD1) or CTLA4 (e.g., antibodies or antigen-binding fragments thereof, or non-immunoglobulin antibody mimetic proteins). In some embodiments, the protein inhibitor of CTLA4 is selected from the group consisting of ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884 (zeflimab), BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA- 3071, ONC-392, AGEN-2041, JHL-1155, KN-044, CG-0161, ATOR-1144, PBI-5D3H5, FPT-155 (CTLA4 / PD-L1 / CD 28), PF-06936308(PD-1 / CTLA4), MGD-019(PD-1 / CTLA4), KN-046(PD-1 / CTLA4), MEDI-5752(CTLA4 / PD-1),XmAb-20717 (PD-1 / CTLA4) and AK-104 (CTLA4 / PD-1). In some embodiments, the protein inhibitor of PD-L1 (CD274) or PD-1 (PDCD1) is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, pidilizumab, AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab, durvalumab, BMS-93 6559, CK-301, PF-06801591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055), AK-103 (HX-008), AK-105, CS-1003, HLX-10, MGA-012, BI-754091, AGEN-2034, JS-001 (toripalimab), JNJ-63723283, genolimzumab (CBT-501), LZM-009, BCD-100, LY-3300054, S HR-1201, SHR-1210 (camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306 (MSB0010718C), CX-072, CBT-502, TSR-042 (dostarlimab), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308 (sintilimab), HLX-20, KL-A167, STI -A1014, STI-A1015(IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, FPT-155(CTLA4 / PD-L1 / CD28), PF-06936308(PD-1 / CTLA4), MGD-0 13(PD-1 / LAG-3), FS-118(LAG-3 / PD-L1)MGD-019(PD-1 / CTLA4), KN-046(PD-1 / CTLA4), MEDI-5752(CTLA4 / PD-1), RO-7121661(PD-1 / TIM-3),XmAb-20717 (PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), M7824 (PD-L1 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM3 / PDL1) and INBRX-105 (4-1BB / PDL1). In some embodiments, one or more immune checkpoint inhibitors include small molecule inhibitors of CD274 (PDL1, PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1) or CTLA4. In some embodiments, the small molecule inhibitor of CD274 or PDCD1 is selected from the group consisting of GS-4224, GS-4416, INCB086550 and MAX10181. In some embodiments, the small molecule inhibitor of CTLA4 includes BPI-002. In some embodiments, the immunotherapy comprises co-administering one or more cell therapies selected from the group consisting of natural killer (NK) cells, NK-T cells, T cells, cytokine-induced killer (CIK) cells, macrophages (MAC) cells, tumor infiltrating lymphocytes (TIL), and dendritic cells (DC). In some embodiments, the one or more cell therapies comprise T cell therapies selected from the group consisting of α / β TCR T cells, γ / δ TCR T cells, regulatory T (Treg) cells, and TRuC, TMT cells. In some embodiments, one or more cell therapies include NK cell therapies containing NK-92 cells. In some embodiments, one or more cell therapies include cells that are autologous, isogenic, or allogeneic to the subject. In some embodiments, one or more cell therapies include cells containing a chimeric antigen receptor (CAR). In some embodiments, the cells in the cell therapy bind to a target or tumor-associated antigen (TAA) selected from the group consisting of the following items (e.g., via a chimeric antigen receptor (CAR)): CD19, MS4A1 (CD20), CD22, IL2RA (CD25), CD27, TNFRSF8 (CD30), CD33, CD37, CD38, CD40, CD44, CD48, CD52, CD70, NT5E (CD73), ENTPD1 (CD39), CD74, CD79b, CD80, CD86, IL3R A (CD123), PROM1 (CD133), CD137, SDC1 (CD138), alpha-fetoprotein (AFP), c-Met; c-Kit; C-type lectin domain family 12 member A (CLEC12A, CLL1, CD371); C-type lectin domain-containing 9A (CLEC9A, CD370); cadherin 3 (CDH3, p-cadherin, PCAD); carbonic anhydrase 6 (CA6); carbonic anhydrase 9 (CA9, CAIX); carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3); carcinoembryonic antigen-related Cell adhesion molecule 5 (CEACAM5); carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6, CD66c); chorionic villus growth hormone 1 (CSH1, CS1); coagulation factor III tissue factor (F3, TF); collagen lectin subfamily member 10 (COLEC10); delta-like canonical Notch ligand 3 (DLL3); ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor (EGFR); EGFR variant III (EGFRvIII); EPH receptor A2 (E PHA2); epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine kinase 2 (ERBB2; HER2); fibroblast activation protein alpha (FAP); fibroblast growth factor receptor 2 (FGFR2); fibroblast growth factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1, PSMA); folate receptor 1 (FOLR1, FRα); GD2 ganglioside; glycoprotein NMB (GPNMB, osteoactivin); guanylate cyclase 2C (GUCY2C, GCC); human papillomavirus (HPV) E6; HPV E7; major histocompatibility complex (MHC) class I presenting neoantigens, major histocompatibility complex (MHC) class II presenting neoantigens,Major histocompatibility complex class I E (HLA-E); major histocompatibility complex class I F (HLA-F); major histocompatibility complex class I G (HLA-G, MHC-G); integrin subunit β7 (ITGB7); leukocyte immunoglobulin-like receptor B1 (LILRB1, ILT2); leukocyte immunoglobulin-like receptor B2 (LILRB2, ILT4); LY6 / PLAUR domain-containing 3 (LYPD3, C4.4A); glypican 3 (GPC3); KRAS proto-oncogene GTPase (KRAS); MAGE family member A1 (MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member D1 (MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin 1 (MUC1) and its splice variants (e.g., MUC1 / C, D, and Z); mucin 16 (MUC16); necdin (NDN); nectin cell adhesion molecule 4 (NECTIN4); SLIT and NTRK-like family member 6 (SLITRK6); promyelocytic leukemia (PML, TRIM19); protein White tyrosine kinase 7 (inactive) (PTK7); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, 19A, CD319, CRACC, CS1); sialic acid-binding Ig-like lectin 7 (SIGLEC7); sialic acid-binding Ig-like lectin 9 (SIGLEC9); solute carrier family 34 (sodium phosphate) member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6; LIV1); STEAP family member 1 (STEAP1); TNF receptor superfamily member 4 (TNFRSF4, OX40 or CD134); TNF superfamily member 9 (TNFSF9; 4- 1BB-L, CD137L); TNF receptor superfamily member 10a (TNFRSF10A, DR4, CD261, TRAILR1); TNF receptor superfamily member 10b (TNFRSF10B, DR5, CD262, TRAILR2); TNF receptor superfamily member 13B (TNFRSF13B; CD267, TACI, IGAD2); TNF receptor superfamily member 17 (TNFRSF17, BCMA, CD269); TNF receptor superfamily member 18 (TNFRSF18, GITR or CD357); transferrin (TF); transforming growth factor β1 (TGFB1); trophoblast glycoprotein (TPBG,5T4); trophinin (TRO, MAGED3); tumor-associated calcium signal transducer 2 (TACSTD2, TROP2, EGP1); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); and Lewis Y antigen. In some embodiments, the cells in the cell therapy bind to an epitope of a target or tumor-associated antigen (TAA) (e.g., a neoantigen) presented in a major histocompatibility complex (MHC) molecule. In some embodiments, the TAA is a cancer testis antigen. In some embodiments, the cancer testis antigen is selected from the group consisting of: sperm nodule protein binding protein (ACRBP, CT23, OY-TES-1, SP32), alpha fetoprotein (AFP, AFPD, FETA, HPAFP); A kinase anchoring protein 4 (AKAP4, AKAP 82, AKAP-4, AKAP82, CT99, FSC1, HI, PRKA4, hAKAP82, p82), ATPase family AAA domain-containing 2 (ATAD2, ANCCA, CT137, PRO2000), centromere scaffold 1 (KNL1, AF15Q14, CASC5, CT29, D40, MCPH4, PPP1R55, Spc7, hKNL-1, hSpc105), centrosomal protein 55 (CEP55 , C10orf3, CT111, MARCH, URCC6), cancer / testis antigen 1A (CTAG1A, ESO1, CT6.1, LAGE-2, LAGE2A, NY-ESO-1), cancer / testis antigen 1B (CTAG1B, CT6.1, CTAG, CTAG1, ESO1, LAGE-2, LAGE2B, NY-ESO-1), cancer / testis antigen 2 (CTAG2, CAMEL, CT2, CT6.2, C T6.2a, CT6.2b, ESO2, LAGE-1, LAGE2B), CCCTC-binding factor-like (CTCFL, BORIS, CT27, CTCF-T, HMGB1L1, dJ579F20.2), catenin α2 (CTNNA2, CAP-R, CAPR, CDCBM9, CT114, CTNR), cancer / testis antigen 83 (CT83, CXorf61, KK-LC-1, KKLC1), cell cycle A1 (CCNA1, CT146), DEAD box helicase 43 (DDX43, CT13, HAGE), developmental pluripotency-associated 2 (DPPA2, CT100, ECAT15-2, PESCRG1), fetal and adult testis expressed 1 (FATE1, CT43, FATE), FMR1 neighbor (FMR1NB, CT37, NY-SAR-35, NYSAR35), HORMA domain-containing 1 (HORMAD1, CT46,NOHMA), insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3, CT98, IMP-3, IMP3, KOC, KOC1, VICKZ3), leucine zipper protein 4 (LUZP4, CT-28, CT-8, CT28, HOM-TES-85), lymphocyte antigen 6 family member K (LY6K, CT97, HSJ001348, URLC10, ly-6K), whirlpool spermatogenesis transposon silencer (MAEL, CT128, SPATA35), MAGE family member A1 (MAGEA1, CT1.1, MAGE1); MAGE family member A3 (MAGEA3, CT1.3, H MAGE family member A4 (MAGEA4, CT1.4, MAGE-41, MAGE-X2, MAGE4, ​​MAGE4A, MAGE4B); MAGE family member A11 (MAGEA11, CT1.11, MAGE-11, MAGE11, MAGEA-11); MAGE family member C1 (MAGEC1, CT7, CT7.1); MAGE family member C2 (MAGEC2, CT10, HCA587, MAGEE1); MAGE family member D1 (MAGED1, DLXIN-1, NRAGE); MAGE family member D2 (MAGED2, 11B6, BARTS5, BCG-1, BCG1, HCA10, MAGE-D2); kinesin family member 20B (KIF20B, CT90, KRMP1, MPHOSPH1, MPP-1, MPP1); NUF2 components of the NDC80 centromere complex (NUF2, CDCA1, CT106, NUF2R), nuclear RNA export factor 2 (NXF2, CT39, TAPL-2, TCP11X2); PAS domain-containing repressor 1 (PASD1, CT63, CT64, OXTES1), PDZ-binding kinases (PBK, CT84, HEL164, Nori-3, S PK, TOPK); piwi-like RNA-mediated gene silencing 2 (PIWIL2, CT80, HILI, PIWIL1L, mili); antigens preferentially expressed in melanoma (PRAME, CT130, MAPE, OIP-4, OIP4); sperm-associated antigen 9 (SPAG9, CT89, HLC-6, HLC4, HLC6, JIP-4, JIP4, JLP, PHET, PIG6); sperm protein associated with the nucleus, X-linked family member A1 (SPANXA1, CT11.1, CT11.3, NAP-X, SPAN-X, SPAN-Xa, SPAN-Xb, SPANX, SPANX-A),SPANX family member A2 (SPANXA2, CT11.1, CT11.3, SPANX, SPANX-A, SPANX-C, SPANXA, SPANXC), SPANX family member C (SPANXC, CT11.3, CTp11, SPANX-C, SPANX-E, SPANXE), SPANX family member D (SPANXD, CT11.3, CT11.4, SPANX-C, SPANX-D, SPANX-E, SPANXC, SPANXE, dJ171K16.1), SSX family member 1 (SSX1, CT5.1, SSRC), SSX family member 2 (SSX2, CT5.2, CT5.2A, HD21, HOM-MEL-40, SSX), synaptonemal complex protein 3 (SYCP3, COR1, RPRGL4, SCP3, SPGF4), testis-expressed 14-cell intercellular bridge-forming factor (TEX14, CT113, SPGF23), transcription factor Dp family member 3 (TFDP3, CT30, DP4, HCA661), serine protease 50 (PRSS50, CT20, TSP50), TTK protein kinase (TTK, CT96, ESK, MPH1, MPS1, MPS1L1, PYT), and zinc finger protein 165 (ZNF165, CT53, LD65, ZSCAN7). In some embodiments, cytokine or chemokine therapy comprises co-administering one or more immunostimulatory cytokines or chemokines that promote or increase the proliferation or activation of T cells (including α / β TCR T cells and γ / δ TCR T cells), NK-T cells, NK cells and / or dendritic cells. In some embodiments, the one or more immunostimulatory cytokines or chemokines are selected from the group consisting of IL 2, IL-12, IL-15, IL-18, IL-21, interferon (IFN)-α, IFN-β, IFN-γ, CXCL9 / Mig (monokine induced by interferon-γ), CXCL10 / IP10 (interferon-γ inducible 10 kDa protein) and CXCL11 / I-TAC (interferon-inducible T cell α-chemoattractant), CXCL4 / PF4 (platelet factor 4), monocyte chemoattractant protein 2 (MCP-2), macrophage inflammatory protein 1 α (MIP-1α), macrophage inflammatory protein 1 β (MIP-1β), and regulated activated normal T expressed and secreted protein (RANTES). In some embodiments, the one or more additional therapeutic agents include an activator or agonist of: a toll-like receptor (TLR); a stimulator of interferon genes (STING) receptor; an inducible T-cell costimulator (ICOS,CD278); and / or a TNF receptor superfamily (TNFRSF) member. In some embodiments, the TNF receptor superfamily (TNFRSF) member is selected from the group consisting of TNFRSF1A, TNFRSF1B, TNFRSF4 (OX40), TNFRSF5 (CD40), TNFRSF6 (FAS), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB, CD137), TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10C (CD263, T RAILR3), TNFRSF10D (CD264, TRAILR4), TNFRSF11A (CD265, RANK), TNFRSF11B, TNFRSF12A (CD266), TNFRSF13B (CD267), TNFRSF13C (CD 268), TNFRSF16 (NGFR, CD271), TNFRSF17 (BCMA, CD269), TNFRSF18 (GITR, CD357), TNFRSF19, TNFRSF21 (CD358, DR6) and TNFRSF25 (DR3). In some embodiments, TNFRSF4 (OX40 or CD134) activators or agonists include INCAGN1949, tavolimab (MEDI0562), pogalizumab (MOXR0916 / RG7888), MEDI6469, BMS 986178, PF-04518600, GSK3174998, IBI101, ATOR-1015, ABBV-368, or SL-279252. In some embodiments, TNFRSF9 (4-1BB or CD137) activators or agonists include urelumab, BMS-663513, utomilumab (PF-05082566), CTX-471, MP-0310, ADG-106, ATOR-1017 or AGEN2373. In some embodiments, TNFRSF18 (GITR or CD357) activators or agonists include GWN323, MEDI1873, MK-1248, MK-4166, TRX518, INCAGN1876, BMS-986156, BMS-986256, AMG-228, ASP1951 (PTZ 522), FPA-154 or OMP-336B11. In some embodiments, the one or more additional therapeutic agents include a TNF receptor superfamily member 4 (TNFRSF4,In some embodiments, the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. In some embodiments, the TLR7 agonist is selected from the group consisting of GS 9620, DS-0509, LHC-165, and TMX-101 (imiquimod), and / or the TLR8 agonist is selected from the group consisting of GS-9688 and NKTR-262 (dual TLR7 / TLR8 agonists). In some embodiments, the STING receptor agonist or activator is selected from the group consisting of ADU-S100 (MIW-815), SB-11285, MK-1454, SR-8291, AdVCA0848, GSK-532, SYN-STING, MSA-1, SR-8291, 5,6-dimethylxanthone-4-acetic acid (DMXAA), cyclic GAMP (cGAMP) and cyclic diAMP. In some embodiments, one or more additional therapeutic agents include an anti-CD47 antibody. In some embodiments, the anti-CD47 antibody is magrolimab. In some embodiments, one or more additional therapeutic agents include an inhibitor of SIRPα. In some embodiments, the SIRPα inhibitor is selected from the group consisting of AL-008, RRx-001, CTX-5861, FSI-189 (GS-0189), ES-004, BI765063, ADU1805, and CC-95251. In some embodiments, the one or more additional therapeutic agents include inhibitors or antagonists of the following: non-receptor protein tyrosine phosphatase 11 (PTPN11 or SHP2), myeloid cell leukemia sequence 1 (MCL1) apoptosis regulator, mitogen-activated protein kinase kinase kinase kinase kinase 1 (MAP4K1) (also known as hematopoietic progenitor cell kinase 1 (HPK1)), phosphatidylinositol-4,5-bisphosphate 3-kinase (including catalytic subunit α (PIK3CA), catalytic subunit β (PIK3CB), catalytic subunit γ (PIK3CG) ) and catalytic subunit delta (PIK3CD)), diacylglycerol kinase alpha (DGKA, DAGK, DAGK1 or DGK-α), 5'-ectonucleotidase (NT5E or CD73), ectonucleotide triphosphate diphosphohydrolase 1 (ENTPD1 or CD39), transforming growth factor beta 1 (TGFB1 or TGFβ), heme oxygenase 1 (HMOX1, HO-1 or HO1), heme oxygenase 2 (HMOX2, HO-2 or HO2), vascular endothelial growth factor A (VEGFA or VEGF),erb-b2 receptor tyrosine kinase 2 (ERBB2, HER2, HER2 / neu or CD340), epidermal growth factor receptor (EGFR, ERBB, ERBB1 or HER1), ALK receptor tyrosine kinase (ALK, CD246), poly (ADP-ribose) polymerase 1 (PARP1), poly (ADP-ribose) polymerase 2 (PARP2), TCDD-inducible poly (ADP-ribose) polymerase (TIPARP, PARP7), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), TNF receptor superfamily member 14 (TNFRSF14, HVEM, CD270), T cell immunoreceptor with Ig and ITIM domains (TIGIT), X-linked inhibitor of apoptosis (XIAP, BIRC4, IAP-3), baculoviral IAP repeat-containing 2 (BIRC2, cIAP1), baculoviral IAP repeat-containing 3 (BIRC3, cIAP2), baculoviral IAP repeat-containing 5 (BIRC5, survivin), C-C motif chemokine receptor 2 (CCR2, CD192), C-C motif chemokine receptor 5 (CCR5, CD195), C-C motif chemokine receptor 8 (CCR8, CDw198), CXC motif chemokine receptor 2 (CX CR2, CD182), CXC motif chemokine receptor 3 (CXCR3, CD182, CD183), CXC motif chemokine receptor 4 (CXCR4, CD184), cytokine-inducible SH2-containing protein (CISH), arginase (ARG1, ARG2), carbonic anhydrase (CA1, CA2, CA3, CA4, CA5A, CA5B, CA6, CA7, CA8, CA9, CA10, CA11, CA12, CA13, CA14), prostaglandin-endoperoxide synthase 1 (PTGS1, COX-1), prostaglandin-endoperoxide synthase 2 (PTGS2, COX-2), secretion Type phospholipase A2, prostaglandin E synthase (PTGES, PGES), arachidonic acid 5-lipoxygenase (ALOX5, 5-LOX), soluble epoxide hydrolase 2 (EPHX2), indoleamine 2,3-dioxygenase 1 (IDO1), indoleamine 2,3-dioxygenase 2 (IDO2), hypoxia-inducible factor 1 subunit alpha (HIF1A), angiopoietin 1 (ANGPT1), endothelial TEK tyrosine kinase (TIE-2, TEK), Janus kinase 1 (JAK1), catenin beta 1 (CTNNB1), histone deacetylase 9 (HDAC9), 5'-3' exoribonuclease 1 (XRN1) and / or WRN RecQ-like helicase (WRN). In some embodiments, the inhibitor comprises an antibody or antigen-binding fragment thereof or an antibody-drug conjugate thereof,CD3 targeting multispecific molecules, CD16 targeting multispecific molecules, non-immunoglobulin antigen binding molecules or antibody mimicking proteins. In some embodiments, the inhibitor includes inhibitory nucleic acids (e.g., siRNA). In some embodiments, the inhibitor includes organic small molecules. In some embodiments, the inhibitor of 5'-ectonucleotidase (NT5E or CD73) is selected from the group consisting of the following items: MEDI9447 (oleclumab), CPI-006, BMS-986179, IPH5301, TJ4309 (TJD5), NZV-930, AB-680, PSB-12379, PSB-12441, PSB-12425, CB-708, GS-1423 (AGEN1423) and PBF-1662. In some embodiments, the inhibitor of CCR2 and / or CCR5 is selected from the group consisting of: BMS-813160, PF-04136309, and CCX-872. In some embodiments, the inhibitor of MCL1 is selected from the group consisting of: AMG-176, AMG-397, S-64315, AZD-5991, 483-LM, A 1210477, UMI-77, and JKY-5-037. In some embodiments, the inhibitor of PTPN11 or SHP2 is selected from the group consisting of: TNO155 (SHP-099), RMC-4550, JAB-3068, and RMC-4630. In some embodiments, the inhibitor of Janus kinase 1 (JAK1) is selected from the group consisting of filgotinib, tofacitinib, baricitinib, and ABT-494. In some embodiments, the one or more additional therapeutic agents include a regulatory T cell (Treg) inhibitor. In some embodiments, the subject further receives radiation therapy. In some embodiments, radiation therapy includes stereotactic body radiation therapy (SBRT). In some embodiments, the one or more additional therapeutic agents include one or more anti-tumor agents or chemotherapeutic agents. In some embodiments, the one or more anti-tumor agents or chemotherapeutic agents are selected from the group consisting of nucleoside analogs (e.g., 5-fluorouracil, gemcitabine, cytarabine, cladribine, pentostatin, fludarabine), taxanes (e.g., paclitaxel, nab-paclitaxel, docetaxel, cabazitaxel), platinum coordination complexes (cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenoplatin, picoplatin, satraplatin, dicycloplatin, etoposide, lobaplatin, miplatin), dihydrofolate reductase (DHFR) inhibitors (e.g., methotrexate, trimesapatite, pemetrexed), topoisomerase inhibitors (e.g., doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin,

[0010] Irinotecan, mitoxantrone, pixanol, sobuzoxane, topotecan, irinotecan, MM-398 (liposomal irinotecan), vosaroxin and GPX-150, aclarubicin, AR-67, mavelertinib, AST-2818, avitinib (ACEA-0010), ilofofen (MGI-114)), alkylating agents (e.g., nitrogen mustards (e.g., cyclophosphamide, mechlorethamine, uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, temozolomide, carmustine), nitrosoureas (e.g., carmustine, lomustine, streptozotocin), alkyl sulfonates (e.g., busulfan)), and mixtures thereof. In some embodiments, the one or more additional therapeutic agents include the FOLFOX regimen, the FOLFIRI regimen, the FOLFOXIRI regimen, or the FOLFIRINOX regimen. In some embodiments, the one or more additional therapeutic agents include antiviral therapy. In some embodiments, antiviral therapy includes co-administration of a hepatitis B virus (HBV) therapeutic agent. In some embodiments, the HBV therapeutic agent is selected from HBV vaccines, HBV polymerase inhibitors, immunomodulators, interferon alpha receptor ligands, hyaluronidase inhibitors, hepatitis B surface antigen (HBsAg) inhibitors, cyclophilin inhibitors, antisense oligonucleotides, short interfering RNA (siRNA) or DNA-guided RNA interference (ddRNAi) targeting HBV viral mRNA, endonuclease modulators (e.g., PGN-514), ribonucleotide reductase inhibitors (e.g., Trimidox), HBV replication inhibitors, non-classical RNA polymerase PAPD5 and PAPD7 inhibitors (e.g., siRNA), covalently closed circular DNA inhibitors (cccDNA), caspase 9 stimulators (e.g., ENOB-HB-01), CD3 modulators (e.g., IMC-I109V), Ff Ar2 and Ffar3 agonists (e.g., SFA-001), additional HBV antibodies, CCR2 chemokines (e.g., propagermanium), FXR agonists, thymosin antagonists, nuclear protein modulators, retinoic acid-inducible gene stimulator 1, arginase inhibitors (e.g., astodrimer, CB-1158, C-201, resminostat), endonuclease inhibitors (e.g., PGN-154), ribonuclease reductase inhibitors (e.g., Trimidox), non-nucleoside reverse transcriptase inhibitors (NNRTIs), HBV replication inhibitors, capsid inhibitors, transcript inhibitors, CAR-T cell therapy, TCR-T cell therapy, and inhibitors of HCV nonstructural proteins (e.g., NS5A inhibitors (e.g., ledipasvir,velpatasvir), NS5B inhibitors (e.g., sofosbuvir, mecitabine), NS3 inhibitors (e.g., voxilaprevir). In some embodiments, the HBV vaccine is selected from the group consisting of: HBsAG-HBIG complex, ARB-1598, Bio-Hep-B, NASVAC, abi-HB (intravenous), ABX-203, Tetrabhay, GX-110E, GS-4774, peptide vaccine (εPA-44), Hepatrol-07, NASVAC (NASTERAP), IMP-321, BEVAC, RevacB mcf, Revac B+, MGN-1333, KW-2, CVI-HBV-002, AltraHepB, VGX-6200, FP-02, FP-02.2 (HepTcell), NU-500, HBVax, im / TriGrid / antigen vaccine, Mega-CD40L-adjuvant vaccine, HepB-v, RG7944 (INO-1800), recombinant VLP-based therapeutic vaccine (HBV infection, VLP Biotech), hepatitis B therapeutic DNA vaccine, AdTG-17909, AdTG-17910AdTG-18202, ChronVac-B, TG-1050, VVX-001, GSK-3528869A (ChAd155-hli-HBV+MVA-HBV+Hbc-HBs / AS01B-4), VBI-2601, VTP-300 (ChAdOx1-SIi-HBV-CPmut-TPA-Ssh prime and MVA-SIi-HBV-CPmut-TPA-Ssh boost), MVA-BN, AVA-2100, HBV-ADV311, YS-HBV-002 and Lm HBV. In some embodiments, the HBV polymerase inhibitor is selected from the group consisting of: adefovir, Emtricitabine Tenofovir disoproxil fumarate Tenofovir alafenamide, tenofovir, tenofovir disoproxil, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir disoproxil fumarate, tenofovir disoproxil fumarate, tenofovir octadecyloxyethyl ester, CMX-157, tenofovir exalidex, besifovir, entecavir Entecavir maleate, telbivudine Feloxivir, pradefovir, clavudine, ribavirin, lamivudine Phosphorus azide, famciclovir, fusolin, metacavir, ATI-2173, SNC-019754, FMCA, AGX-1009, AR-II-04-26, HIP-1302, tenofovir disoproxil aspartate, tenofovir disoproxil orotate, and HS-10234. In some embodiments, the immunomodulator is selected from the group consisting of retasimid, imidol hydrochloride, ingaron, dermaVir, hydroxychloroquine, proleukin, hydroxyurea, mycophenolate mofetil (MPA) and its ester derivative mycophenolate mofetil (MMF), JNJ-440, WF-10, AB-452, ribavirin, IL-12, INO-9112, polymer polyethyleneimine (PEI), Gepon, VGV-1, MOR-22, CRV-431, JNJ-0535, TG-1050, ABI-H2158, BMS-936559, GS-9688, RO-7011785, RG-7854, RO-6871765, AIC-649 and IR-103. In some embodiments, the interferon alpha receptor ligand is selected from the group consisting of interferon alpha-2b (INTRON ), pegylated interferon α-2a Pegylated interferon α-1b, interferon α-1b Veldona, Infradure, Roferon-A, YPEG-interferon alpha-2a (YPEG-rhIFNα-2a), P-1101, Algeron, Alfarona, Ingaron (interferon gamma), rSIFN-co (recombinant super compound interferon), Ypeg interferon alpha-2b (YPEG-rhIFNα-2b), MOR-22, pegylated interferon alpha-2b Bioferon, Novaferon, Inmutag (Inferon), Interferon α-n1 Interferon beta-1a Shaferon, interferon alpha-2b (Axxo), Alfaferone, interferon alpha-2b (BioGenericPharma), interferon-alpha 2 (CJ), Laferonum, VIPEG, BLAUFERON-A, BLAUFERON-B, Intermax alpha, Realdiron, Lanstion, Pegaferon, PDferon-B PDferon-B, interferon alpha-2b (IFN, LaboratoriosBioprofarma), alpha interferon a 2b, Kalferon, Pegnano, Feronsure, PegiHep, interferon alpha 2b (Zydus-Cadila), interferon alpha 2a, Optipeg A, Realfa In some embodiments, the hyaluronidase inhibitor is sterol-2a, sterol-2b, sterol-2b, sterol-3, sterol-4, sterol-5, sterol-6, sterol-7, sterol-8, sterol-9, sterol-10, sterol-11, sterol-12, sterol-13, sterol-14, sterol-15, sterol-16, sterol-17, sterol-18, sterol-19, sterol-20, sterol-21, sterol-22, sterol-23, sterol-24, sterol-25, sterol-26, sterol-27, sterol-28, sterol-29, sterol-30, sterol-31, sterol-32, sterol-33, sterol-34, sterol-35, sterol-36, sterol-37, sterol-38, sterol-39, sterol-40, sterol-41, sterol-42, sterol-43, sterol-44, sterol-45, sterol-47, sterol-48, sterol-49, sterol-51, sterol-52, sterol-53, sterol-54, sterol-55, sterol-56, sterol-57, sterol-58, sterol-59, sterol-61, sterol-62, sterol-63, sterol-64, sterol-65 In some embodiments, the hepatitis B surface antigen (HBsAg) inhibitor is selected from the group consisting of AK-074, HBF-0259, PBHBV-001, PBHBV-2-15, PBHBV-2-1, REP-9AC, REP-9C, REP-9, REP-2139, REP-2139-Ca, REP-2055, REP-2163, REP-2165, REP-2053, REP-2031, REP-006, and REP-9AC'. In some embodiments, the HBsAg inhibitor is an HBsAg secretion inhibitor selected from the group consisting of BM601, GST-HG-131, and AB-452.In some embodiments, the cyclophilin inhibitor is selected from the group consisting of: CPI-431-32, EDP-494, OCB-030, SCY-635, NVP-015, NVP-018, NVP-019, and STG- 175. In some embodiments, the antisense oligonucleotide targeting viral mRNA is selected from the group consisting of: ISIS-HBVRx, IONIS-HBVRx, IONIS-HBV-LRx, IONIS-GSK6-LRx, GSK-3389404, and RG-6004. In some embodiments, short interfering RNA (siRNA) or DNA-guided RNA interference (ddRNAi) is selected from the group consisting of TKM-HBV (TKM-HepB), ALN-HBV (e.g., ALN-HBV02), SR-008, HepB-nRNA, ARC-520, ARC-521, ARB-1740, ARB-1467, AB-729, DCR-HBVS, RG-6084 (PD-L1), RG-6217, ALN-HBV-02, JNJ-3989 (ARO-HBV), STSG-0002, ALG-010133, ALG-ASO, LUNAR-HBV, and DCR-HBVS (DCR-S219). In some embodiments, the ddRNAi is BB-HB-331. In some embodiments, the HBV replication inhibitor is selected from the group consisting of GP-31502, isothiafludine, IQP-HBV, RM-5038, and Xingantie. In some embodiments, cccDNA is selected from the group consisting of BSBI-25, ccc-R08, and CHR-101. In some embodiments, the additional HBV antibody targets the surface antigen of the hepatitis B virus. In some embodiments, the additional HBV antibody is selected from lenvervimab (GC-1102), XTL-17, XTL-19, KN-003, IV Hepabulin SN, VIR-3434, Omri-Hep-B, Nabi-HB, Hepatect CP, HepaGam B, igantibe, Niuliva, CT-P24, Fovepta (BT-088), and HBC-34. In some embodiments, the FXR agonist is selected from the group consisting of EYP-001, GS-9674, EDP-305, MET-409, Tropifexor, AKN-083, RDX-023, BWD-100, LMB-763, INV-3, NTX-023-1, EP-024297, and GS-8670.In some embodiments, the thymosin antagonist is selected from thymosin alpha 1, recombinant thymosin alpha 1 (GeneScience), NL-004, and pegylated thymosin alpha-1. In some embodiments, the nuclear protein modulator is selected from GS-4882, AB-423, AB-836, AT-130, ALG-001075, ALG-001024, ALG-000184, EDP-514, GLS4, NVR-1221, NVR-3778, AL-3778, BAY 41-4109, mofecillin mesylate, ARB-168786, ARB-880, ARB-1820, GST-HG-141, JNJ-379, JNJ-632, RG-7907, GST-HG-141, HEC-72702, KL-060332, AB-506, ABI-H0731, ABI-H3733, JNJ-440, ABI-H2158, CB-HBV-001, AK-0605, SOC-10, SOC-11, and DVR-23. In some embodiments, retinoic acid-inducible gene stimulator 1 is selected from the group consisting of: inarigivir soproxil (SB-9200), SB-40, SB-44, ORI-7246, ORI-9350, ORI-7537, ORI-9020, ORI-9198, ORI-7170 and RGT-100. In some embodiments, arginase inhibitors are selected from the group consisting of: CB-1158, C-201 and Ramuci. In some embodiments, CAR-T cell therapy for HBV therapy includes an immune effector cell population engineered to express a chimeric antigen receptor (CAR), wherein the CAR includes an HBV antigen binding domain (e.g., HbsAg-CART). In some embodiments, TCR-T cell therapy includes T cells expressing HBV-specific T cell receptors (e.g., (HBsAg)-specific TCR).In some embodiments, the HBV therapeutic agent is selected from α-hydroxytropolone, amidosvir, antroquinol, β-hydroxycytidine, ARB-199, CCC-0975, ccc-R08, efcitabine, ezetimibe, cyclosporine A, gentioside (gentiopicroside), HH-003, hepalatide, JNJ-56136379, nitazoxanide, birenapam, NJK14047, NOV-205 (molixan, BAM-205), oligonucleotides, mivotifen, feron, GST-HG-131, levamisole, Ka Shu Ning, alloferon, WS-007, Y-101 (Ti Fen Tai), rSIFN-co, PEG-IIFNm, KW-3, BP-Inter-014, caryophyllin, HepB-nRNA, cTP-5(rTP-5), HSK-II-2, HEISCO-106-1, HEISCO-106, Hepbarna, IBPB-006IA, Hepuyinfen, DasKloster 0014-01, ISA-204, Jiangantai (Ganxikang), MIV-210, OB-AI-004, PF-06, picroside, DasKloster-0039, hepulantai, IMB-2613, NCO-48 fumarate, TCM-800B, reduced glutathione, RO-6864018, RG-7834, QL-007 sofosbuvir, ledipasvir, UB-551, PA-1010, HPN-BV1, STSG-0002 and ZH-2N. In some embodiments, the antiviral therapy comprises co-administering a hepatitis C virus (HCV) therapeutic agent. In some embodiments, the HCV therapeutic agent is selected from daclatasvir, ledipasvir, ombitasvir, elbasvir, sofosbuvir, dasabuvir, ribavirin, anaprevir, simeprevir, paritaprevir, ritonavir, elbasvir, and gezoprevir. In some embodiments, the antiviral therapy comprises co-administering a human immunodeficiency virus (HIV) therapeutic agent. In some embodiments, the HIV therapeutic agent comprises an HIV protease inhibitor, an HIV RNase H inhibitor, an HIV Nef inhibitor, an HIV reverse transcriptase inhibitor, an HIV integrase inhibitor, an HIV entry inhibitor, an HIV maturation inhibitor, a latency reversal agent, an HIV capsid inhibitor, an HIV-targeted antibody, an HIV vaccine, or a birth control or contraceptive regimen.In some embodiments, the HIV protease inhibitor is selected from the group consisting of amprenavir, atazanavir, brecanavir, darunavir, fosamprenavir, fosamprenavir calcium, indinavir, indinavir sulfate, lopinavir, nelfinavir, nelfinavir mesylate, ritonavir, saquinavir, saquinavir mesylate, tipranavir, AEBL-2, DG-17, GS-1156, TMB-657 (PPL-100), T-169, BL-008, MK-8122, TMB-607, GRL-02031, and TMC-310911. In some embodiments, the HIV RNase H inhibitor is NSC-727447. In some embodiments, the HIV Nef inhibitor is FP-1. In some embodiments, the HIV reverse transcriptase inhibitor is a non-nucleoside / non-nucleotide reverse transcriptase inhibitor. In some embodiments, the non-nucleoside / non-nucleotide inhibitor is selected from the group consisting of dapivirine, delavirdine, delavirdine mesylate, doravirine, efavirenz, etravirine, lentinan, nevirapine, rilpivirine, ACC-007, ACC-008, AIC-292, F-18, KM-023, PC-1005, VM-1500A-LAI, PF-3450074, esxavirine (sustained release oral, HIV infection), esxavirine (long-acting injectable nanosuspension, HIV infection) and esxavirine (VM-1500). In some embodiments, the HIV reverse transcriptase inhibitor is a nucleoside or nucleotide inhibitor. In some embodiments, the nucleoside or nucleotide inhibitor is selected from the group consisting of adefovir, adefovir dipivoxil, azvudine, emtricitabine, tenofovir, tenofovir alafenamide, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir disoproxil fumarate, tenofovir disoproxil fumarate, tenofovir octadecyloxyethyl ester (AGX-1009), tenofovir disoproxil fumarate, . and VIDEX (didanosine, ddl), abacavir, abacavir sulfate, alovudine, aritibine, sinavudine, didanosine, efavirenz, festinavir, fosalvudine tidoxil, CMX-157, dapivirine, doravirine, etravirine, OCR-5753, tenofovir disoproxil orotate, fosalvudine tidoxil, lamivudine, phosphazid, stavudine, zalcitabine, zidovudine, rovafovir-etafoetida (GS-9131), GS-9148, MK-8504, MK-8591, MK-8583, VM-2500, and KP-1461. In some embodiments, the HIV integrase inhibitor is selected from the group consisting of elvitegravir, elvitegravir (extended release microcapsules), curcumin, a derivative of curcumin, chicoric acid, a derivative of chicoric acid, 3,5-dicaffeoylquinic acid, a derivative of 3,5-dicaffeoylquinic acid, aurintricarboxylic acid, a derivative of aurintricarboxylic acid, caffeic acid phenethyl ester, a derivative of caffeic acid phenethyl ester, a tyrosine kinase inhibitor, a derivative of a tyrosine kinase inhibitor, quercetin, a derivative of quercetin, raltegravir, pegylated raltegravir, dolutegravir, JTK-351, bicagvir, AVX-15567, cabotegravir (long-acting injection), diketoquinoline-4-1 derivatives, integrase-LEDGF inhibitors, ledgins, M-522, M-532, MK-0536, NSC-310217, NSC-371056, NSC-48240, NSC-642710, NSC-699171, NSC-699172, NSC-699173, NSC-699174, stilbene disulfonic acid, T-169, STP-0404, VM-3500 and cabotegravir. In some embodiments, the HIV integrase inhibitor is an HIV non-catalytic site or allosteric integrase inhibitor (NCINI). In some embodiments, NCINI is selected from the group consisting of CX-05045, CX-05168 and CX-14442. In some embodiments, the HIV entry inhibitor is AAR-501, LBT-5001, seneviroc, a CCR5 inhibitor, a gp41 inhibitor, a CD4 attachment inhibitor, a gp120 inhibitor, a gp160 inhibitor, and a CXCR4 inhibitor. In some embodiments, the CCR5 inhibitor is selected from the group consisting of apraviroc, vicriviroc, maraviroc, maraviroc (long-acting injectable nanoemulsion), seneviroc, leronlimab (PRO-140), adaptavir (RAP-101),Nifeviroc (TD-0232), anti-GP120 / CD4 or CCR5 bispecific antibodies, B-07, MB-66, polypeptide C25P, TD-0680, thioraviroc and vMIP (Haimipu). In some embodiments, the gp41 inhibitor is selected from the group consisting of albuvirtide, enfuvirtide, griffithsin (gp41 / gp120 / gp160 inhibitor), BMS-986197, enfuvirtide biosimilars, enfuvirtide biosimilars, HIV-1 fusion inhibitor (P26-Bapc), ITV-1, ITV-2, ITV-3, ITV-4, CPT-31, Cl3hmAb, PIE-12 trimer and sifuvirtide. In some embodiments, the CD4 attachment inhibitor is ibalizumab or a CADA analog. In some embodiments, the gp120 inhibitor is selected from the group consisting of anti-HIV microbicides, Radha-108 (receptol) 3B3-PE38, BanLec, bentonite-based nanomedicines, fostemsavir tromethamine, IQP-0831, VVX-004, and BMS-663068. In some embodiments, the gp160 inhibitor is fangchinoline. In some embodiments, the CXCR4 inhibitor is selected from the group consisting of plerixafor, ALT-1188, N15 peptide, and vMIP (Haimipu). In some embodiments, the HIV entry inhibitor is selected from docosanol, enfuvirtide, maraviroc, palivizumab, respiratory syncytial virus immune globulin, intravenous [RSV-IGIV], varicella zoster immune globulin [VariZIG] and varicella zoster immune globulin [VZIG]. In some embodiments, the HIV maturation inhibitor is selected from the group consisting of: BMS-955176, GSK-3640254 and GSK-2838232. In some embodiments, the latency reversal agent is selected from the group consisting of a toll-like receptor (TLR) agonist (including a TLR7 agonist, such as GS-9620 (vesatolimod), vesatolimod analogs), a histone deacetylase (HDAC) inhibitor, a proteasome inhibitor (such as velcade), a protein kinase C (PKC) activator (such as indole lactams, prostaglandins, ingenol B, DAG lactone), a Smyd2 inhibitor, a BET-bromodomain 4 (BRD4) inhibitor,Ionomycin, IAP antagonists (inhibitors of apoptosis proteins; e.g., APG-1387, LBW-242), SMAC mimetics (including TL32711, LCL161, GDC-0917, HGS1029, AT-406, Debio-1143), PMA, SAHA (suberylamine hydroxamic acid or suberoyl, aniline and hydroxamic acid), NIZ-985, IL-15 modulating antibodies, IL-15, IL-15 fusion proteins, IL-15 receptor agonists, JQ1, disulfiram, amphotericin B, and ubiquitin inhibitors (e.g., largazole analogs, APH-0812, GSK-343). In some embodiments, the HIV capsid inhibitor is selected from the group consisting of: capsid polymerization inhibitors, capsid disrupting compounds, HIV nucleocapsid p7 (NCp7) inhibitors (e.g., azodicarbonamide), and HIV p24 capsid protein inhibitors (e.g., GS-6207, GS-CA1, AVI-621, AVI-101, AVI-201, AVI-301, AVI-CAN1-15 series, and PF-3450074). In some embodiments, the HIV targeting antibody is selected from bNAbs (broadly neutralizing HIV-1 antibodies), TMB-360, antibodies targeting HIV gp120 or gp41, antibody recruitment molecules targeting HIV, anti-CD63 monoclonal antibodies, anti-GB virus C antibodies, anti-GP120 / CD4, gp120 bispecific monoclonal antibodies, CCR5 bispecific antibodies, anti-Nef single domain antibodies, anti-Rev antibodies, camelid-derived anti-CD18 antibodies, camelid-derived anti-ICAM-1 antibodies, DCVax-001, gp140 targeting antibodies, gp41-based HIV therapeutic antibodies, human recombinant mAb (PGT-121), PGT121.414.LS, ibalizumab, Immuglo, MB-66, and VRC-HIVMAB091-00-AB. In some embodiments, the HIV targeting antibody is selected from the group consisting of: UB-421, BF520.1, CHO1, CH59, C2F5, C4E10, C2F5+C2G12+C4E10, 3BNC117, 3BNC117-LS, 3BNC60, DH270.1, DH270.6, D1D2, 10-1074-LS, Cl3hmAb, GS-9722( elipovimab), DH411-2, BG18, GS-9721, PGT145, PGT121, PGT-121.60, PGT-121.66, PGT122, PGT-123, PGT-124, PGT-125, PGT-126, PGT-151, PGT-130, PGT-133, PGT-134, PGT-135,PGT-128, PGT-136, PGT-137, PGT-138, PGT-139, MDX010 (ipilimumab), DH511, DH511-2, N6, N6LS, N49P6, N49P7, N49P7.1, N49P9, N49P11, N60P1.1, N60P25.1, N60P2.1, N60P31.1, N60P22, NIH 45-46, PGC14, PGG14, PGT-142, PGT-143, PGT-144, PGDM1400, PGDM12, PGDM21, PCD N-33A, 2Dm2m, 4Dm2m, 6Dm2m, PGDM1400, VRC01, VRC-01-LS, A32, 7B2, 10E8, VRC-07 -523, VRC07-523LS, VRC24, VRC41.01, 10E8VLS, 3810109, 10E8v4, IMC-HIV, iMabm36, eCD4-Ig, IOMA, CAP256-VRC26.25, DRVIA7, VRC-HIVMAB080-00-AB, VRC-HIVMA B060-00-AB, P2G12, VRC07, 354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411, 354BG426, VRC29.03, CAP256, CAP256-VRC26.08, CAP256-VRC26.0 9, CAP256-VRC26.25, PCT64-24E and VRC38.01, PGT-151, CAP248-2B, 35O22, ACS202, VRC34 and VRC34.01, 10E8, 10E8v4, 10E8-5R-100cF, 4E10, DH511.11P, 2F5, 7b2 and LN01. In some embodiments, the HIV targeting antibody is a bispecific or trispecific antibody selected from the group consisting of MGD014, B12BiTe, BiIA-SG, TMB-bispecific, SAR-441236, VRC-01 / PGDM-1400 / 10E8v4, 10E8.4 / iMab and 10E8v4 / PGT121-VRC01. In some embodiments, the HIV-targeting antibody is a bNAb delivered in vivo (e.g., AAV8-VRC07; mRNA encoding the anti-HIV antibody VRC01; or engineered B cells encoding 3BNC117). In some embodiments, the HIV vaccine is selected from the group consisting of: a peptide vaccine, a recombinant subunit protein vaccine, a live vector vaccine, a DNA vaccine, an HIV MAG DNA vaccine, a CD4-derived peptide vaccine, a vaccine combination,Adenovirus vector vaccines (adenovirus vectors such as Ad5, Ad26 or Ad35), monkey adenovirus (chimpanzee, gorilla, rhesus monkey, i.e., rhAd), adeno-associated virus vector vaccines, chimpanzee adenovirus vaccines (e.g., ChAdOX1, ChAd68, ChAd3, ChAd63, ChAd83, ChAd155, ChAd157, Pan5, Pan6, Pan7, Pan9), coxsackievirus-based vaccines, enterovirus-based vaccines, gorilla adenovirus vaccines, lentivirus-based vaccines, arenavirus vaccines (e.g., LCMV, Pichind), vaccines based on two or three segmented arenaviruses, trimer-based HIV-1 vaccines, measles virus-based vaccines, flavivirus-based vaccines, Tobacco mosaic virus vectored vaccines, varicella zoster virus-based vaccines, human parainfluenza virus 3 (PIV3)-based vaccines, poxvirus-based vaccines (modified vaccinia virus Ankara (MVA), orthopoxvirus-derived NYVAC, and fowlpox virus-derived ALVAC (canarypox virus) strains); fowlpox virus-based vaccines, rhabdovirus-based vaccines such as VSV and Maraba virus; recombinant human CMV (rhCMV)-based vaccines, alphavirus-based vaccines such as Semliki Forest virus, Venezuelan equine encephalitis virus, and Sindbis virus; mRNA-based therapeutic vaccines formulated in lipoplexes (e.g., LNPs); and self-replicating RNA / self-amplifying RNA vaccines formulated in lipoplexes (e.g., LNPs). In some embodiments, the HIV vaccine is selected from the group consisting of: anti-CD40.Env-gp140 vaccine, Ad4-EnvC150, BG505 SOSIP.664 gp140 adjuvant vaccine, BG505 SOSIP.GT1.1 gp140 adjuvant vaccine, Chimigen HIV vaccine, ConM SOSIP.v7 gp140, rgp120 (AIDSVAX), ALVAC HIV (vCP1521) / AIDSVAX B / E (gp120) (RV144), monomeric gp120 HIV-1 subtype C vaccine, MPER-656 liposomal subunit vaccine, Remune, ITV-1, Contre Vir, Ad5-ENVA-48, DCVax-001 (CDX-2401), Vacc-4x, Vacc-C5, VAC-3S, multistage DNA recombinant adenovirus-5 (rAd5), rAd5 gag-pol env A / B / C vaccines, Pennvax-G, Pennvax-GP, Pennvax-G / MVA-CMDR, HIV-TriMix-mRNA vaccine, HIV-LAMP-vax, Ad35, Ad35-GRIN, NAcGM3 / VSSP ISA-51,poly-ICLC adjuvanted vaccines, TatImmune, GTU-multiHIV (FIT-06), ChAdV63.HIVconsv, gp140[delta]V2.TV1+MF-59, rVSVIN HIV-1 gag vaccine, SeV-EnvF, SeV-Gag vaccine, AT-20, DNK-4, ad35-Grin / ENV, TBC-M4, HIVAX, HIVAX-2, HIV vaccine based on N123-VRC-34.01 inducible antigen, NYVAC-HIV-PT1, NYVAC-HIV-PT4, DNA-HIV-PT123, rAAV1-PG9DP, GOVX-B11, GOVX-B21, GOVX-C55, TVI-HIV-1, Ad-4 (Ad4-env Clade C+Ad4-mGag), Paxvax, EN41-UGR7C, EN41-FPA2, ENOB-HV-11, PreVaxTat, AE-H, MYM-V101, CombiHIVvac, ADVAX, MYM-V201, MVA-CMDR, MagaVax, DNA-Ad5 gag / pol / nef / nev (HVTN505), MVATG-17401, ETV-01, CDX-1401, DNA and Sev vector vaccines expressing SCaVII, rcAD26.MOS1.HIV-Env, Ad26.Mod.HIV vaccine, Ad26.Mod.HIV+MVA mosaic vaccine+gp140, AGS-004, AVX-101, AVX-201, PEP-6409, SAV-001, ThV-01, TL-01, TUTI-16, VGX-3300, VIR-1111, IHV-001 and viral oxygen particle vaccines (such as pseudovirus vaccines), CombiVICHvac, LFn-p24B / C fusion vaccine, GTU-based DNA vaccine, HIV gag / pol / nef / env DNA vaccine, anti-TAT HIV vaccine, conjugated peptide vaccine, dendritic cell vaccine (such as DermaVir), gag-based DNA vaccine, GI-2010, gp41 HIV-1 vaccine, HIV vaccine (PIKA adjuvant), i-key / MHC class II antigen hybrid peptide vaccine, ITV-2, ITV-3, ITV-4, LIPO-5, multi-stage Env vaccine, MVA vaccine, Pennvax-GP, pp71-deficient HCMV vector HIV gag vaccine, rgp160 HIV vaccine, RNActive HIV vaccine, SCB-703, Tat Oyi vaccine, TBC-M4, UBI HIV gp120, Vacc-4x+romidepsin,Variant gp120 peptide vaccine, rAd5 gag-pol env A / B / C vaccine, DNA.HTI and MVA.HTI, VRC-HIVDNA016-00-VP+VRC-HIVADV014-00-VP, INO-6145, JNJ-9220, gp145 C.6980; eOD-GT860mer-based vaccine, PD-201401, env(A, B, C, A / E) / gag(C) DNA vaccine, gp120(A, B, C, A / E) protein vaccine, PDPHV-201401, Ad4-EnvCN54, EnvSeq-1 Envs HIV-1 vaccine (GLA-SE adjuvant), HIV p24gag Prime-boost plasmid DNA vaccine, HIV-1iglb12 neutralizing VRC-01 antibody-stimulating anti-CD4 vaccine, vaccines based on arenavirus vectors (Vaxwave, TheraT), MVA-BN HIV-1 vaccine regimen, UBI HIV gp120, mRNA-based prophylactic vaccines, VPI-211 and TBL-1203HI. In some embodiments, the birth control or contraceptive regimen is selected from the group consisting of cyproterone acetate, desogestrel, dienogest, drospirenone, estradiol valerate, ethinyl estradiol, norethindrone, etonogestrel, levonorgestrel, levonorgestrel, linegestrel, medroxyprogesterone acetate, ethinylestradiol methyl ether, mifepristone, misoprostol, nomegestrol acetate, norgestrel, norethindrone, norgestimate, ormeloxifene, sigsone acetate, ulipristal acetate, and any combination thereof. In some embodiments, antiviral therapy includes the co-administration of influenza therapeutic agents. In some embodiments, influenza therapeutic agents are selected from matrix 2 inhibitors (such as amantadine, rimantadine), neuraminidase inhibitors (such as zanamivir, oseltamivir, peramivir, laninamivir octanoate) and polymerase inhibitors (such as ribavirin, favipiravir). In some embodiments, influenza virus inhibitors are selected from amantadine, rimantadine, arbidol (uminovir), baloxavir mapositate, oseltamivir, peramivir, ingavirin (ingavirin), laninamivir octanoate, zanamivir, favipiravir, ribavirin and combinations thereof. In some embodiments, influenza virus inhibitors are selected from amantadine, rimantadine, zanamivir, oseltamivir, peramivir, laninamivir octanoate, ribavirin and favipiravir. In some embodiments, antiviral therapy includes the co-administration of respiratory syncytial virus (RSV) therapeutic agents. In some embodiments, the RSV therapeutic agent is selected from ribavirin, ALS-8112, and pusatovir. In some embodiments, the antiviral therapy comprises co-administering a picornavirus therapeutic agent. In some embodiments, the picornavirus therapeutic agent is selected from hydantoin, guanidine hydrochloride, L-buthionine sulfoximine,Py-11 and lupinquvir. In some embodiments, the antiviral therapy comprises co-administering an Ebola virus therapeutic agent. In some embodiments, the Ebola virus therapeutic agent is selected from ribavirin, palivizumab, motavizumab, RSV-IGIV, MEDI-557, A-60444, MDT-637, BMS-433771, amiodarone, dronedarone, verapamil, Ebola convalescent plasma (ECP), TKM-100201, BCX4430 ((2S,3S,4R,5R)-2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol), favipiravir (also known as T-705 or Avigan), T-705 monophosphate, T-705 diphosphate, T-705 triphosphate, FGI-106 (1-N,7-N-bis[3-(dimethylamino)propyl]-3,9-dimethylquinolino[8,7-h]quinolone-1,7-diamine), JK-05, TKM-Ebola, ZMapp, rNAPc2, VRC-EBOADC076-00-VP, OS-2966, MVA-BN filo, bromcidofovir, Vaxart adenovirus vector 5-based Ebola vaccine, Ad26-ZEBOV, FiloVax vaccine, GOVX-E301, GOVX-E302, Ebola virus entry inhibitors (NPC1 inhibitors), rVSV-EBOV. In some embodiments, the Ebola virus therapeutic agent is selected from ZMapp, mAB114, and REGEN-EB3. In some embodiments, antiviral therapy includes the co-administration of a coronavirus therapeutic agent. In some embodiments, the coronavirus is a severe acute respiratory syndrome (SARS)-related coronavirus. In some embodiments, the coronavirus is a Middle East respiratory syndrome (MERS)-related coronavirus. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the SARS-CoV-2 (COVID-19) therapeutic agent is an RNA polymerase inhibitor (e.g., redcivir, galidesivir). In some embodiments, the SARS-CoV-2 (COVID-19) therapeutic agent is redcivir (GS-5734). In some embodiments, the SARS-CoV-2 (COVID-19) therapeutic agent is an anti-SARS-CoV-2 hyperimmune globulin therapy (plasma from recovered COVID-19 patients, for example, processed into hyperimmune globulin) (e.g., TAK-888).In some embodiments, the SARS-CoV-2 (COVID-19) therapeutic agent is selected from COVID-19 vaccines (e.g., BN162, Ad5-nCoV, INO-4800, mRN1273), anti-IL6 receptor antibodies (e.g., tocilizumab, sarilumab, TZLS-501), anti-IL6 antibodies (e.g., siltuximab), RNA-dependent RNA polymerase (RdRp) inhibitors (e.g., favipiravir, remdesivir), anti-CCR5 antibodies (e.g., lerolizumab (PRO 140)), broadly neutralizing antibodies (e.g., anti-ACE2 receptor antibodies, SAB-185, COVID-HIG, COVID-EIG), ACE2 (angiotensin converting enzyme 2)-Fc fusion protein (COVIDTRAP) or recombinant human ACE2 protein (APN1), ACE-MAB designed to bind to the spike protein of coronaviruses (including SARS-CoV-2 and SARS-CoV). TM Bispecific fusion proteins (e.g., STI-4920, CMAB020), Janus kinase (JAK1 / JAK2) inhibitors (e.g., ruxolitinib, baricitinib), siRNA (e.g., targeting angiotensin-converting enzyme 2 (ACE2) or transmembrane protease, serine 2 (TMPRSS2)), HIV-1 protease inhibitors (e.g., lopinavir / ritonavir; darunavir alone or in combination with cobicistat), complement inhibitors (e.g., eculizumab), HCV protease inhibitors (e.g., danoprevir), stem cell therapy (e.g., Remestemcel-L, CYNK-001), NK cell therapy (NKG2D-ACE2CAR-NK cells), neutralizing antibodies to human granulocyte-macrophage colony-stimulating factor (GM-CSF) (e.g., IZN-101, gimsilumab), vasoconstrictors (e.g., angiotensin II), selective inhibitors of nuclear export (SINEs) (such as XPO1 inhibitors (e.g., selinexor)), NSAIDs (including COX inhibitors (e.g., ibuprofen, aspirin, diclofenac, naxopren) and selective COX2 inhibitors (e.g., celecoxib, rofecoxib, etoricoxib, lumiracoxib, valdecoxib)) and other antiviral agents (e.g., ENU200, lopinavir / ritonavir combination). In some embodiments, the COVID-19 vaccine is an mRNA vaccine (e.g., BN162), including vaccines encapsulated in lipoplexes (e.g., lipid nanoparticles (LNPs)) (e.g., mRNA1273). In some embodiments, the COVID-19 vaccine is a DNA vaccine (e.g., INO-4800). In some embodiments, the COVID-19 vaccine encodes a pre-fusion stable form of the spike (S) protein (e.g., mRNA1273). In some embodiments, the COVID-19 vaccine is a recombinant protein-based vaccine composed of the receptor binding domain (RBD) of the spike protein of the coronavirus. In some embodiments, the COVID-19 vaccine uses a ligand antigen epitope presentation system (LEAPS) peptide comprising a conserved region of a coronavirus protein to stimulate protective cell-mediated T cell responses and reduce viral load. In some embodiments, the COVID-19 vaccine is a microneedle array (MNA)-delivered vaccine. In some embodiments, the vaccine is based on an influenza vector expressing a surface antigen of SARS-CoV-2. In some embodiments, the COVID-19 vaccine is an intranasal vaccine (e.g., AdCOVID). In some embodiments, the COVID-19 vaccine is NVX-CoV2373, INO4800, or BNT-162.In some embodiments, the SARS-CoV-2 (COVID-19) therapeutic agent is selected from PIKfyve kinase inhibitors (e.g., apimod), immunomodulators (e.g., retasimid), T cell immunotherapy, recombinant sialidase (e.g., DAS181), CRAC channel inhibitors (e.g., CM-4620-IE), cardiac cell therapy using allogeneic cardiosphere-derived cells (e.g., CAP-1002), cardioprotective drugs (e.g., aspirin, plavix, lipitor, opremazole), S1P receptor antagonists (e.g., fingolimod), cyclooxygenase 2 (COX-2) inhibitors (e.g., celecoxib), phosphodiesterase inhibitors (e.g., dapoxetine), dapoxetine, ... Phosphodiesterase 5 (PDE5) inhibitors (e.g., sildenafil citrate), serine protease TMPRSS2 inhibitors (camostat mesylate), anti-human complement 5a antibodies (e.g., IFX-1), macrophage migration inhibitory factor (MIF) inhibitors, phosphodiesterase (PDE)-4 and -10 inhibitors (e.g., ibudilast), eEF1A2 inhibitors (e.g., plitidepsin), sphingosine kinase 2 (SK2) inhibitors (e.g., ABC294640, RHB-107), galectin inhibitors (e.g., BXT-10), membrane fusion inhibitors (e.g., uminovir), anti-PD1 antibodies, thymosin, antimalarials (e.g., chloroquine, hydroxychloroquine), and Other antiviral therapeutic agents (e.g., HTCC (N-(2-hydroxypropyl)-3-trimethylammonium 47 chitosan chloride, OYA1). In some embodiments, the subject has cancer. In some embodiments, the subject is in remission from cancer. In some embodiments, the subject has a hematological cancer, such as a leukemia (e.g., acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), B-cell ALL, myelodysplastic syndrome (MDS), myeloproliferative disorders (MPD), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), undifferentiated leukemia), lymphoma (e.g., small lymphocytic lymphoma (SLL), mantle cell Lymphoma (MCL), follicular lymphoma (FL), T cell lymphoma, B cell lymphoma, diffuse large B cell lymphoma (DLBCL), marginal zone lymphoma (MZL), Waldenstrom's macroglobulinemia (WM)) and / or myeloma (e.g., multiple myeloma (MM)). In some embodiments, the subject has a solid tumor. In some embodiments, the tumor or cancer is malignant or metastatic. In some embodiments, the subject has a tumor infiltrated by conventional dendritic cells (cDC1). In some embodiments, the tumor-infiltrating dendritic cells express CC motif chemokine receptor 5 (CCR5, CD195) and / or XC motif chemokine receptor 1 (XCR1) on their cell surface.In some embodiments, the tumor infiltrating dendritic cells express one or more cell surface proteins selected from the group consisting of: XCR1, cell adhesion molecule 1 (CADM1), C-type lectin domain-containing 9A (CLEC9A, CD370), and thrombomodulin (THBD).

[0024] In some embodiments, tumor infiltrating dendritic cells express one or more cell surface proteins selected from the group consisting of: CD1A, CD1C, CD1E, signal regulatory protein alpha (SIRPA; CD172A), CD207 and Fc fragment of IgE receptor Ia (FCER1A). In some embodiments, tumor infiltrating dendritic cells express one or more proteins selected from the group consisting of: basic leucine zipper ATF-like transcription factor 3 (BATF3) and interferon regulatory factor 8 (IRF8). In some embodiments, tumor infiltrating dendritic cells express one or more proteins selected from the group consisting of: BATF3, IRF8, THBD, CLEC9A and XCR1. In some embodiments, the subject has a cancer that detectably expresses or overexpresses one or more cell surface immune checkpoint receptors. In some embodiments, the one or more cell surface immune checkpoint receptors are selected from the group consisting of: CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain-containing 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244 (SLAMF4); CD276 (B7H3); V-set domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4); V-set immunomodulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR-associated 2 (HHLA2, B7H7); inducible T cell costimulator (ICOS, CD278); inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFR SF9 (CD137), TNFSF9 (CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF); TNFRSF18 (GITR),TNFSF18 (GITRL); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155); PVR-related immunoglobulin domain-containing (PVRIG, CD112R); T cell immunoreceptor with Ig and ITIM domains (TIGIT) ; T cell immunoglobulin and mucin domain-containing 4 (TIMD4; TIM4); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); lymphocyte activation 3 (LAG3, CD223); signal transducer lymphocyte activation molecule family member 1 (SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, CD319); UL16 binding protein 1 (ULBP1); UL16 binding protein 2 (ULBP2 ); UL16 binding protein 3 (ULBP3); retinoic acid early transcript 1E (RAET1E; ULBP4); retinoic acid early transcript 1G (RAET1G; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314); killer cell lectin-like receptor C2 (KLRC2, CD159c, NKG2C); killer cell lectin-like Receptor C3 (KLRC3, NKG2E); killer cell lectin-like receptor C4 (KLRC4, NKG2F); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor D1 (KLRD1); killer cell lectin-like receptor G1 (KLRG1; CLEC15A, MAFA,2F1); sialic acid-binding Ig-like lectin 7 (SIGLEC7); and sialic acid-binding Ig-like lectin 9 (SIGLEC9). In some embodiments, greater than about 50% of cancer or tumor cells detectably express one or more cell surface immune checkpoint receptor proteins (e.g., PD1 or PD-L1; so-called "hot" cancers or tumors). In some embodiments, greater than about 1% and less than about 50% of cancer or tumor cells detectably express one or more cell surface immune checkpoint receptor proteins (e.g., PD1 or PD-L1; so-called "warm" cancers or tumors). In some embodiments, less than about 1% of cancer cells detectably express one or more cell surface immune checkpoint receptor proteins (e.g., PD1 or PD-L1; so-called "cold" cancers or tumors). In some embodiments, the subject has a cancer or tumor selected from the group consisting of: an epithelial tumor (e.g., carcinoma, squamous cell carcinoma, basal cell carcinoma, squamous intraepithelial neoplasia), a glandular tumor (e.g., adenocarcinoma, adenoma, adenomyoma), a mesenchymal or soft tissue tumor (e.g., sarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, dermatofibrosarcoma, neurofibrosarcoma, fibrohistiocytoma, angiosarcoma, angiomyxoma, leiomyomas, chondromas, chondrosarcomas, alveolar soft part sarcomas, epithelioid hemangioendothelioma, Spitz tumor, synovial sarcoma, and lymphoma. In some embodiments, the subject has a solid tumor in or derived from a tissue or organ selected from the group consisting of: bone (e.g., adamantoma, aneurysmal bone cyst, angiosarcoma, chondroblastoma, enchondroma, chondromyxoid fibroma, chondrosarcoma, chordoma, dedifferentiated chondrosarcoma, enchondroma, epithelioid hemangioendothelioma, Spitz tumor, synovial sarcoma, and lymphoma). Dermoid hemangioendothelioma, fibrous dysplasia of bone, giant cell tumor of bone, hemangioma and related lesions, osteoblastoma, osteochondroma, osteosarcoma, osteoid osteoma, osteoma, periosteal chondroma, desmoid tumor, Ewing sarcoma); lips and oral cavity (e.g., odontogenic ameloblastoma, oral leukoplakia, oral squamous cell carcinoma, primary oral mucosal melanoma); salivary glands (e.g., pleomorphic salivary adenoma, salivary adenoid cystic carcinoma, salivary mucoepidermoid carcinoma, salivary Warthin's tumor); esophagus (e.g., Barrett's esophagus, dysplasia, and adenocarcinoma); gastrointestinal tract, including stomach (e.g., gastric adenocarcinoma, primary gastric lymphoma, gastrointestinal stromal tumor (GIST), metastatic deposits, gastric cancer, gastric sarcoma, neuroendocrine carcinoma, primary squamous cell carcinoma of the stomach, gastric adenoacanthocarcinoma), intestine and smooth muscle (e.g., intravenous leiomyomas), colon (e.g., colorectal adenocarcinoma), rectum, anus; pancreas (e.g., serous tumors, including microcystic or macrocystic serous cystadenomas, solid serous cystadenomas, Von Hippel-Landau (VHL)-associated serous cystic tumors, serous cystadenocarcinomas; mucinous cystic neoplasms (MCNs), intraductal papillary mucinous neoplasms (IPMNs),Intraductal oncocytic papillary neoplasm (IOPN), intraductal tubular neoplasm, cystic acinar neoplasm (including acinar cell cystadenoma and acinar cell cystadenocarcinoma), pancreatic cancer, invasive pancreatic ductal adenocarcinoma (including tubular adenocarcinoma and adenosquamous carcinoma), colloid carcinoma, medullary carcinoma, hepatoid carcinoma, signet ring cell carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with osteoclast-like giant cells, acinar cell carcinoma, neuroendocrine tumor, neuroendocrine microadenoma, neuroendocrine tumor (NET), neuroendocrine carcinoma (NEC) (including small cell or large cell NEC), insulinoma, gastrinoma, glucagonoma, serotonin-producing NET, somatostatinoma, VIPoma, solid pseudopapillary neoplasm (SPN), pancreatoblastoma); gallbladder (e.g., gallbladder carcinoma, extrahepatic bile duct carcinoma, intrahepatic bile duct carcinoma); neuroendocrine glands (e.g., adrenal cortical carcinoma, carcinoid, pheochromocytoma, pituitary adenoma); thyroid (e.g., anaplastic (undifferentiated) carcinoma, medullary carcinoma, oncocytic tumor, papillary carcinoma, adenocarcinoma); liver (e.g., adenoma, combined hepatocellular and cholangiocarcinoma, fibrolamellar carcinoma, hepatoblastoma, hepatocellular carcinoma, mesenchymal nested stromal epithelial tumor, undifferentiated carcinoma; hepatocellular carcinoma, intrahepatic bile duct carcinoma, biliary cystadenocarcinoma, epithelioid hemangioendothelioma, angiosarcoma, embryonal sarcoma, rhabdomyosarcoma, solitary fibrous tumor, teratoma, yolk sac tumor, carcinosarcoma, rhabdoid tumor); kidney (e.g., ALK-rearranged renal cell carcinoma, chromophobe renal cell carcinoma, clear cell renal cell carcinoma, clear cell sarcoma, metanephric adenoma, metanephric fibroma, mucinous tubular and spindle cell carcinoma, nephroma, nephroblastoma (Wilms tumor), papillary adenoma, papillary renal cell carcinoma, renal oncocytoma, renal cell carcinoma, succinate dehydrogenase-deficient renal cell carcinoma, collecting duct carcinoma); breast (e.g., invasive ductal carcinoma, including but not limited to acinar cell carcinoma, adenoid cystic carcinoma, apocrine carcinoma, cribriform carcinoma, glycogen-rich / clear cell inflammatory carcinoma, lipid-rich carcinoma, medullary carcinoma, metaplastic carcinoma, micropapillary carcinoma, mucinous carcinoma, neuroendocrine carcinoma, oncocytic carcinoma, papillary carcinoma, sebaceous gland carcinoma, secretory breast carcinoma, tubular carcinoma; lobular carcinoma, including but not limited to pleomorphic carcinoma, signet ring cell carcinoma); peritoneum (e.g., mesothelioma; primary peritoneal carcinoma); tissues of the female sex organs, including ovary (e.g., chorionic villi carcinoma, epithelial cell tumor, germ cell tumor, sex cord-stromal tumor), fallopian tube (e.g., serous adenocarcinoma, mucinous adenocarcinoma, endometrioid adenocarcinoma, clear cell adenocarcinoma, transitional cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, Müllerian tumor, adenosarcoma, leiomyosarcoma, teratoma, germ cell tumor, choriocarcinoma, trophoblastoma), uterus (e.g., cervical cancer, endometrial polyps, endometrial hyperplasia, intraepithelial carcinoma (EIC), endometrial cancer (e.g., endometrioid carcinoma, serous carcinoma, clear cell carcinoma, mucinous carcinoma, squamous cell carcinoma, transitional carcinoma, small cell carcinoma, undifferentiated carcinoma, mesenchymal tumor), leiomyoma (e.g., endometrial stromal nodules, leiomyosarcoma, endometrial stromal sarcoma (ESS), mesenchymal tumor),Mixed epithelial and mesenchymal tumors (e.g., adenofibroma, carcinofibroma, adenosarcoma, carcinosarcoma (malignant mixed mesodermal sarcoma - MMMT), endometrial stromal tumor, malignant mixed Müllerian tumor of the endometrium, gestational trophoblastic tumor (partial hygroma, complete hygroma, invasive hygroma, placental site tumor)), vulva, vagina; male sex organ tissues, including prostate, testis (e.g., germ cell tumor, spermatocytic seminoma), penis; bladder (e.g., squamous cell carcinoma, urothelial carcinoma, urothelial bladder carcinoma); brain (e.g., gliomas (e.g., astrocytomas (including non-invasive, low-grade, anaplastic), glioblastoma; oligodendroglioma, ependymoma), meningioma, ganglion neuroblastoma Glioma, Schwannoma (neurilemmoma), Craniopharyngioma, Chordoma, Non-Hodgkin Lymphoma (NHL), Indolent Non-Hodgkin Lymphoma (iNHL), Refractory iNHL, Pituitary Tumor; Eye (e.g., retinoblastoma, retinoblastoma, ocular melanoma, choroidal melanoma, iris hamartoma); Head and Neck (e.g., nasopharyngeal carcinoma, endolymphatic sac tumor (ELST), epidermoid carcinoma, Laryngeal cancer (including squamous cell carcinoma (SCC) (e.g., glottic carcinoma, supraglottic carcinoma, subglottic carcinoma, transglottic carcinoma)), Carcinoma in situ, Verrucous Hemangioma, Spindle Cell and Basal Cell SCC, Undifferentiated Carcinoma, Laryngeal Adenocarcinoma, Adenoid Cystic Carcinoma, Neuroendocrine Carcinoma, Laryngeal Tumor); Paraganglioma of the Head and Neck (e.g., carotid body tumor, cervicotympanic tumor, vagus nerve tumor); neuroma); thymus (e.g., thymoma); heart (e.g., cardiac myxoma); lung (e.g., small cell carcinoma (SCLC), non-small cell lung cancer (NSCLC) (including squamous cell carcinoma (SCC), adenocarcinoma, and large cell carcinoma), carcinoid (typical or atypical), carcinosarcoma, pulmonary blastoma, giant cell carcinoma, spindle cell carcinoma, pleuropulmonary blastoma); lymphoid (e.g., lymphoma (including Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), indolent non-Hodgkin lymphoma (iNHL), refractory iNHL), Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (including B-cell lymphoma and T-cell lymphoma) (e.g., Burkitt lymphoma; large B-cell lymphoma, diffuse large B-cell lymphoma); lymphoma (DLBCL), mantle cell lymphoma, indolent B-cell lymphoma, low-grade B-cell lymphoma, fibrin-associated diffuse large cell lymphoma; primary effusion lymphoma; plasmablastic lymphoma; extranodal NK / T-cell lymphoma, nasal type; peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma; follicular T-cell lymphoma; systemic T-cell lymphoma), lymphangioleiomyomatosis); central nervous system (CNS) (e.g., gliomas (including astrocytic tumors (e.g., pilocytic astrocytoma, fluid-like astrocytoma, subependymal giant cell astrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma, fibrillary astrocytoma, fat cell astrocytoma,astrocytic tumors (e.g., protoplasmic astrocytoma, anaplastic astrocytoma), glioblastomas (e.g., giant cell glioblastoma, gliosarcoma, glioblastoma multiforme), and gliomatosis cerebri), oligodendroglial tumors (e.g., oligodendroglioma, anaplastic oligodendroglioma), oligoastrocytic tumors (e.g., oligoastrocytoma, anaplastic oligoastrocytoma), ependymal tumors (e.g., perependymoma, myxopapillary ependymoma, ependymomas (e.g., cellular, papillary, clear cell, elongated cell), anaplastic ependymoma), optic nerve gliomas, and nonglial tumors (e.g., choroid plexus tumors, neuronal and mixed neuronal-glial tumors). fibroblastoma, pineal region tumor, embryonal tumor, medulloblastoma, meningeal tumor, primary CNS lymphoma, germ cell tumor, pituitary adenoma, skull and paraspinal nerve tumor, stellate region tumor); neurofibroma, meningioma, peripheral nerve sheath tumor, peripheral neuroblastoma (including but not limited to neuroblastoma, ganglioneuroma, ganglioneuroma), trisomy 19 ependymoma); neuroendocrine tissue (e.g., paraganglioma, including adrenal medullary (pheochromocytoma) and extraadrenal paraganglioma); skin (e.g., clear cell hidradenoma, benign fibromatosis of the skin); histiocytoma, cylindroma, sweat adenoma, melanoma (including cutaneous melanoma, mucosal melanoma), pilomatricoma, Spitz tumor); and soft tissue (e.g., aggressive angiomyxoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, angiofibroma, angiomatoid fibrous histiocytoma, synovial sarcoma, biphasic synovial sarcoma, clear cell sarcoma, dermatofibrosarcoma protuberans, desmoid fibroma, small round cell tumor, desmoplastic small round cell tumor, fibroelastoma, embryonal rhabdomyosarcoma, Ewing tumor / primitive neuroectodermal tumor (PNET), extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, paraspinal sarcoma, inflammatory In some embodiments, the subject has a cancer selected from the group consisting of lung cancer, colorectal cancer, breast cancer, prostate cancer, fibrosarcoma, myxoid liposarcoma, fibromyxoid sarcoma, lymphoangioleiomyomas, malignant myoepithelioma, malignant melanoma of the soft part, myoepithelial carcinoma, myoepithelioma, myxoinflammatory fibroblastic sarcoma, undifferentiated sarcoma, pericytoma, rhabdomyosarcoma, non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), soft tissue leiomyosarcoma, undifferentiated sarcoma, well-differentiated liposarcoma. In some embodiments, the subject has a cancer selected from the group consisting of lung cancer, colorectal cancer, breast cancer, prostate cancer,Cervical cancer and head and neck cancer. In some embodiments, the subject suffers from neutropenia or lymphopenia. In some embodiments, the subject has received a lymphocyte depletion chemotherapy regimen. In some embodiments, the subject is chemotherapy-naive or has not received chemotherapy. In some embodiments, the subject has bone marrow cells, or has not depleted bone marrow cells. In some embodiments, the subject does not have a mutation that causes or leads to cancer or is associated with cancer in the gene encoding the FLT3 receptor. In some embodiments, the subject suffers from a viral infection. In some embodiments, the subject is at risk of viral infection. In certain embodiments, the subject may not have received antiviral treatment before (treatment-naive type). In certain embodiments, the subject may have received antiviral treatment before (treatment-experienced type). In some embodiments, the viral infection is HBV infection. In some embodiments, the viral infection is HIV infection. In some embodiments, the viral infection is coronavirus infection. In some embodiments, coronavirus is a MERS-related virus. In some embodiments, coronavirus is a SARS-related virus. In some embodiments, coronavirus is a COVID-19-related virus (SARS-CoV-2). In certain embodiments, the subject may have previously received antiviral therapy and developed resistance to the antiviral therapy previously received. In some embodiments, FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNP) and / or pharmaceutical composition are administered systemically or locally. In some embodiments, FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNP) and / or pharmaceutical composition are administered intravenously, intratumorally, subcutaneously, intradermally, intramuscularly, intraperitoneally, intravesically, intracranially, intrathecally, intracavitarily or intraventricularly. In some embodiments, FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNP) and / or pharmaceutical composition are administered intravenously, intratumorally, subcutaneously, intradermally, intramuscularly, intraperitoneally, intravesically, intracranially, intrathecally, intracavitarily or intraventricularly. In some embodiments, FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNP) and / or pharmaceutical composition and one or more additional therapeutic agents are administered by the same route or by different routes of administration. In some embodiments, the FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (such as LNP) and / or pharmaceutical composition and one or more additional therapeutic agents are administered simultaneously or sequentially. In some embodiments, the FLT3L-Fc fusion protein has a serum half-life of at least about 7 days (e.g., at least about 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 days or longer). In some embodiments, the method requires multiple administrations of the FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, optionally with one or more additional therapeutic agents at predetermined time intervals.Lipoplexes (such as LNPs) and / or pharmaceutical compositions. In some embodiments, the fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNPs) and / or pharmaceutical compositions are administered once a week (i.e., QW), once every two weeks (i.e., once every other week or once every two weeks or Q2W), once every three weeks (i.e., once every three weeks or Q3W), once a month (i.e., QM), or once every two months (i.e., once every other month or once every two months or Q2M), or administered less frequently. In some embodiments, the FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNPs) and / or pharmaceutical compositions and one or more additional therapeutic agents are co-administered according to the same schedule (e.g., co-administered at the same time intervals). In some embodiments, the FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (such as LNP) and / or pharmaceutical composition and one or more additional therapeutic agents are co-administered according to different schedules (e.g., co-administered at different time intervals). In some embodiments, the FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (such as LNP) and / or pharmaceutical composition and one or more additional therapeutic agents are co-administered according to different schedules (e.g., co-administered at different time intervals). In some embodiments, the FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (such as LNP) and / or pharmaceutical composition and one or more additional therapeutic agents are co-administered at a dose ranging from about 0.5 μg / kg to about 5000 μg / kg (e.g., at least about 0.5 μg / kg per dose and up to about 1 μg / kg, 2 μg / kg, 3 μg / kg, 4 μg / kg, 5 μg / kg, 6 μg / kg, 7 μg / kg, 8 μg / kg, 9 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 30 μg / kg, 50 μg / kg, 100 μg / kg, 150 μg / kg, 300 μg / kg per dose). , 400 μg / kg, 500 μg / kg, 600 μg / kg, 700 μg / kg, 800 μg / kg, 900 μg / kg, 1000 μg / kg, 1500 μg / kg, 2000 μg / kg, 2500 μg / kg, 3000 μg / kg, 3500 μg / kg, 4000 μg / kg or 5000 μg / kg) of FLT3L fusion proteins, homodimers, heterodimers, conjugates, polynucleotides, vectors, lipoplexes (such as LNPs) and / or pharmaceutical compositions are administered at a dose of 100 μg / kg, 2000 μg / kg, 2500 μg / kg, 3000 μg / kg, 3500 μg / kg, 4000 μg / kg or 5000 μg / kg. In some embodiments, the FLT3L fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (such as LNP), and / or pharmaceutical composition is administered at a dose ranging from about 1 μg / kg to about 100 μg / kg (e.g., at least about 1 μg / kg per dose and up to about 100 μg / kg per dose). In some embodiments, the FLT3L fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (such as LNP), and / or pharmaceutical composition is administered at a dose of 1 μg / kg per dose, 3 μg / kg per dose, 10 μg / kg per dose, 30 μg / kg per dose, 60 μg / kg per dose, or 100 μg / kg per dose.In some embodiments, the FLT3L fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNP) and / or pharmaceutical composition is administered at a dose of 1 μg / kg per dose. In some embodiments, the FLT3L fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNP) and / or pharmaceutical composition is administered at a dose of 3 μg / kg per dose. In some embodiments, the FLT3L fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNP) and / or pharmaceutical composition is administered at a dose of 10 μg / kg per dose. In some embodiments, the FLT3L fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNP) and / or pharmaceutical composition is administered at a dose of 30 μg / kg per dose. In some embodiments, the FLT3L fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNP), and / or pharmaceutical composition is administered at a dose of 60 μg / kg per dose. In some embodiments, the FLT3L fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNP), and / or pharmaceutical composition is administered at a dose of 100 μg / kg per dose. In some embodiments, the FLT3L fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNP), and / or pharmaceutical composition is administered at a dose ranging from about 0.5 mg to about 50 mg (e.g., at least about 0.5 mg per dose and at most about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg per dose). In some embodiments, the FLT3L fusion protein, homodimer, heterodimer, conjugate, polynucleotide, carrier, lipoplex (such as LNP), and / or pharmaceutical composition is administered at a dose of 10 mg per dose. In some embodiments, within 3 weeks of a single administration of the fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (such as LNP) and / or pharmaceutical composition, cells expressing FLT3 (e.g., dendritic cells) expand at least about 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold or more. In some embodiments, cells expressing FLT3 (e.g., dendritic cells) expand in the bone marrow and / or solid tumor of a subject.

[0025] On the other hand, a test kit is provided. In various embodiments, the test kit includes one or more overall doses of a FLT3L-Fc fusion protein, homodimer, heterodimer, conjugate, polynucleotide, expression cassette, vector, lipoplex (such as LNP) and / or pharmaceutical composition as described above and herein. In some embodiments, one or more overall doses are in a single container. In some embodiments, one or more overall doses are in two or more separate containers. In some embodiments, one or more containers may include vials, ampoules, prefilled syringes, and combinations thereof. In some embodiments, the test kit includes one or more containers comprising an aqueous solution of a FLT3L-Fc fusion protein, homodimer, heterodimer, or conjugate. In some embodiments, the aqueous solution comprises a FLT3L-Fc fusion protein, homodimer, heterodimer, or conjugate at a concentration in the range of about 1-20 mg / ml (e.g., about 1 mg / ml to about 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, or 20 mg / ml). In some embodiments, the aqueous solution comprises a FLT3L-Fc fusion protein, homodimer, heterodimer, or conjugate at a concentration of about 2 mg / ml. In various embodiments, one or more overall doses may be the same or different. In some embodiments, each overall dose is in the range of about 0.5 mg to about 50 mg, for example, at least about 0.5 mg per dose and at most about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg per dose. In some embodiments, each overall dose is about 10 mg per dose. In some embodiments, the kit further comprises one or more overall doses of one or more additional therapeutic agents. In some embodiments, the kit further comprises one or more overall doses of one or more therapeutic agents selected from the group consisting of: AGEN1884 (zeflimab), AGEN1181, AGEN2034 (batilizumab), AGEN1307, AGEN2373,AGEN1223 and GS-1423 (AGEN1423; see WO2019 / 173692). In some embodiments, the kit further comprises one or more oncolytic viral vectors. In some embodiments, the viral vector is from a family of viruses selected from the group consisting of: Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., lymphocytic choriomeningitis mammalian arenavirus, Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus)), Poxviridae (e.g., vaccinia virus), Herpesviridae (e.g., herpesvirus, such as HSV-1), Parvoviridae (e.g., Parvovirus H1), Reoviridae (e.g., reovirus), Picornaviridae (e.g., coxsackievirus, Seneca Valley virus, poliovirus), Paramyxoviridae (e.g., measles virus, Newcastle disease virus (NDV)), Rhabdoviridae (e.g., vesicular stomatitis virus (VSV)), Togaviridae (e.g., alphavirus, Sindbis virus), Enteroviridae (e.g., echovirus). In some embodiments, the kit comprises one or more antibodies or antigen-binding antibody fragments thereof or antibody-drug conjugates thereof, multispecific molecules targeting CD3, multispecific molecules targeting CD16, or non-immunoglobulin antigen binding domains or antibody mimetic proteins to one or more targets selected from the group consisting of: CD19, MS4A1 (CD20), CD22, IL2RA (CD25), CD27, TNFRSF8 (CD30), CD33, CD37, CD38, CD40, CD44, CD48, CD52, CD70, NT5E (CD73), ENTPD1 (CD39), CD74, CD79b, CD80, CD86, IL3RA (CD123), PROM1 (CD133), CD137, SDC1 (CD138), α-fetoprotein (AFP), c-Met; c-Kit; C-type lectin domain family 12 member A (CLEC12A, CLL1, CD371); C-type lectin domain-containing 9A (CLEC9A, CD370); cadherin 3 (CDH3, p-cadherin, PCAD); carbonic anhydrase 6 (CA6); carbonic anhydrase 9 (CA9,Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3); Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5); Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6, CD66c); Chorionic somatostatin 1 (CSH1, CS1); Coagulation factor III tissue factor (F3, TF); Collectin subfamily member 10 (COLEC10); Delta-like canonical Notch ligand 3 (DLL3); Ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3); Ephrin A1 (EFNA1); Epidermal growth factor receptor (EGFR); EGFR variant III (EGFR) FRvIII); EPH receptor A2 (EPHA2); epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine kinase 2 (ERBB2; HER2); fibroblast activation protein alpha (FAP); fibroblast growth factor receptor 2 (FGFR2); fibroblast growth factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1, PSMA); folate receptor 1 (FOLR1, FRα); GD2 ganglioside; glycoprotein NMB (GPNMB, osteoactivin); guanylate cyclase 2C (GUCY2C, GCC); human papillomavirus (HPV) E6; HPV E7; major histocompatibility complex (MHC) class I presenting neoantigens, major histocompatibility complex (MHC) class II presenting neoantigens, major histocompatibility complex class I E (HLA-E); major histocompatibility complex class I F (HLA-F); major histocompatibility complex class I G (HLA-G, MHC-G); integrin subunit β7 (ITGB7); leukocyte immunoglobulin-like receptor B1 (LILRB1, ILT2); leukocyte immunoglobulin-like receptor B2 (LILRB2, ILT4); LY6 / PLAUR domain-containing 3 (LYPD3, C4.4A); glypican 3 (GPC3); KRAS proto-oncogene GTPase (KRAS); MAGE family member A1 (MAGE MAGE family member A1); MAGE family member A3 (MAGEA3); MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member D1 (MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin 1 (MUC1) and its splice variants (e.g., MUC1 / C, D, and Z); mucin 16 (MUC16); necdin (NDN); nectin cell adhesion molecule 4 (NECTIN4); SLIT and NTRK-like family member 6 (SLITRK6); promyelocytic leukemia (PML,TRIM19); protein tyrosine kinase 7 (inactive) (PTK7); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, 19A, CD319, CRACC, CS1); sialic acid-binding Ig-like lectin 7 (SIGLEC7); sialic acid-binding Ig-like lectin 9 (SIGLEC9); solute carrier family 34 (sodium phosphate) member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6; LIV1); STEAP family member 1 (STEAP1); TNF receptor superfamily member 4 (TNFRSF4, OX40 or CD134); TNF superfamily member 9 (TNFSF9; 4-1BB-L, CD137L); TNF receptor superfamily member 10a (TNFRSF10A, DR4, TNF receptor superfamily member 13B (TNFRSF13B; CD267, TACI, IGAD2); TNF receptor superfamily member 17 (TNFRSF17, BCMA, CD269); TNF receptor superfamily member 18 (TNFRSF18, GITR or CD357); transferrin (TF); transforming growth factor beta 1 (TGFB1); trophoblast glycoprotein (TPBG, 5T4); trophinin (TRO, MAGED3); tumor-associated calcium signal transducer 2 (TACSTD2, TROP2, EGP1); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); and Lewis Y antigen. In some embodiments, the kit further comprises one or more antagonists or inhibitors of inhibitory immune checkpoint proteins or receptors and / or one or more activators or agonists of stimulatory immune checkpoint proteins or receptors. In some embodiments, the one or more immune checkpoint proteins or receptors are selected from the group consisting of: CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain-containing 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244 (SLAMF4); CD276 (B7H3); V-set domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4); V-set immunomodulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR associated 2 (HHLA2,B7H7); inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFS F9 (CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF); TNFRSF18 (GITR), TNFSF18 (GITRL); MHC class I polypeptide-related sequence A (MICA); MHC Class I polypeptide-related sequence B (MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155); PVR-associated immunoglobulin domain-containing (PVRIG, CD112R); T cell immunoreceptor with Ig and ITIM domains (TIGIT); T cell immunoglobulin and mucin domain-containing 4 (TIMD4; TIM4); hepatitis A virus cell-mediated immune receptor 2 (HAVCR2, TIMD3, TIM3) ; galectin 9 (LGALS9); lymphocyte activation 3 (LAG3, CD223); signal transducer lymphocyte activation molecule family member 1 (SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, CD319); UL16 binding protein 1 (ULBP1); UL16 binding protein 2 (ULBP2); UL16 binding protein 3 (ULBP3); retinoic acid early transcript 1E (RAET1E; ULBP4); retinoic acid early transcript 1G (RAET1G; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6); killer cell immunoglobulin-like receptor with three Ig domains and long cytoplasmic tail 1 (KIR,CD158E1); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314); killer cell lectin-like receptor C2 (KLRC2, CD159c, NKG2C); killer cell lectin-like receptor C3 (KLRC3, NKG2E); killer cell lectin-like receptor C4 (KLRC4, NKG2F); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin Killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); Killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); Killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); Killer cell lectin-like receptor D1 (KLRD1); Killer cell lectin-like receptor G1 (KLRG1; CLEC15A, MAFA, 2F1); Sialic acid-binding Ig-like lectin 7 (SIGLEC7); and Sialic acid-binding Ig-like lectin 9 (SIGLEC9). In some embodiments, the kit further includes one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the T cell inhibitory immune checkpoint protein or receptor is selected from the group consisting of: CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4); V-set immunomodulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HV EM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); PVR-related immunoglobulin domain-containing (PVRIG, CD112R); T cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor,Two Ig domains and a long cytoplasmic tail 2 (KIR2DL2); Killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3); and Killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1). In some embodiments, the kit further includes one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors. In some embodiments, the T cell stimulatory immune checkpoint protein or receptor is selected from the group consisting of: CD27, CD70; CD40, CD40LG; inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). In some embodiments, the kit further includes one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the NK cell inhibitory immune checkpoint protein or receptor is selected from the group consisting of: killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3); L3); Killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); Killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); Killer cell lectin-like receptor D1 (KLRD1, CD94); Killer cell lectin-like receptor G1 (KLRG1; CLEC15A, MAFA, 2F1); Sialic acid-binding Ig-like lectin 7 (SIGLEC7); and Sialic acid-binding Ig-like lectin 9 (SIGLEC9). In some embodiments, the kit further comprises one or more agonists or activators of one or more NK cell stimulatory immune checkpoint proteins or receptors. In some embodiments, NK cell stimulatory immune checkpoint proteins or receptors include but are not limited to CD16, CD226 (DNAM-1); Killer cell lectin-like receptor K1 (KLRK1, NKG2D,CD314); and SLAM family member 7 (SLAMF7). In some embodiments, the kit further comprises one or more inhibitors of CD274, PDCD1 or CTLA4. In some embodiments, the kit further comprises one or more inhibitors of CD274, PDCD1 or CTLA4 selected from the group consisting of: ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884 (zeflimab), BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044, CG-0161, ATOR-1144, PB I-5D3H5, FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), MEDI-5752 (CTLA4 / PD-1), XmAb-20717 (PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), pembrolizumab, nivolumab, cimetuzumab, pidilizumab, AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab, durvalumab Rivolumab, BMS-936559, CK-301, PF-06801591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055), AK-103 (HX-008), AK-105, CS-1003, HLX-10, MGA-012, BI-754091, AGEN-2034, JS-001 (toripalimab), JNJ-63723283, genoluzumab (CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR-1210 (camrelizumab), S ym-021, ABBV-181, PD1-PIK, BAT-1306 (MSB0010718C), CX-072, CBT-502, TSR-042 (dotalizumab), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308 (sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01,MGD-013 (PD-1 / LAG-3), FS-118 (LAG-3 / PD-L1), RO-7121661 (PD-1 / TIM-3), M7824 (PD-L1 / TGFβ-EC domain) and CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM3 / PDL1), INBRX-105 (4-1BB / PDL1), GS-4224, GS-4416, INCB086550, MAX10181 and BPI-002. In some embodiments, the kit further comprises one or more containers comprising one or more immune cell populations selected from the group consisting of natural killer (NK) cells, NK-T cells, T cells, cytokine-induced killer (CIK) cells, macrophages (MAC) cells, tumor infiltrating lymphocytes (TIL) and dendritic cells (DC). In some embodiments, the T cell population is selected from the group consisting of α / β TCR T cells, γ / δ TCR T cells, regulatory T (Treg) cells and TRuC, TMT cells. In some embodiments, the kit further comprises an NK-92 cell population. In some embodiments, one or more immune cell populations comprise one or more chimeric antigen receptors (CARs). In some embodiments, the cells are allogeneic to the intended recipient. In some embodiments, the kit further comprises one or more cytokines or chemokines selected from the group consisting of: IL 2, IL-12, IL-15, IL-18, IL-21, interferon (IFN)-α, IFN-β, IFN-γ, CXCL9 / Mig (monokine induced by interferon-γ), CXCL10 / IP10 (interferon-γ inducible 10kDa protein) and CXCL11 / I-TAC (interferon-inducible T cell α-chemoattractant), CXCL4 / PF4 (platelet factor 4), monocyte chemoattractant protein 2 (MCP-2), macrophage inflammatory protein 1α (MIP-1α), macrophage inflammatory protein In some embodiments, the kit further comprises one or more activators or agonists of the following: toll-like receptors (TLRs); stimulator of interferon genes (STING) receptors; inducible T cell co-stimulators (ICOS, CD278); and / or TNF receptor superfamily (TNFRSF) members. In some embodiments, the TNF receptor superfamily (TNFRSF) member is selected from the group consisting of TNFRSF1A, TNFRSF1B, TNFRSF4 (OX40), TNFRSF5 (CD40), TNFRSF6 (FAS), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB, CD137), TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10C (CD263, TRAILR3), TNFRSF10D (CD264, TRAILR4), TNFRSF11A (CD265, RANK), TNFRSF11B, TNFRSF12A (CD266), TNFRSF13B (CD267), TNFRSF13C (CD268), TNFRSF16 (NGFR,In some embodiments, the TNFRSF4 activator or agonist comprises INCAGN1949, tadalafil (MEDI0562), poloxamer (MOXR0916 / RG7888), MEDI6469, BMS 986178, PF-04518600, GSK3174998, IBI101, ATOR-1015, ABBV-368 or SL-279252. In some embodiments, TNFRSF9 (4-1BB or CD137) activators or agonists include urelumab, BMS-663513, utomilumab (PF-05082566), CTX-471, MP-0310, ADG-106, ATOR-1017 or AGEN2373. In some embodiments, TNFRSF18 (GITR or CD357) agonists include GWN323, MEDI1873, MK-1248, MK-4166, TRX518, INCAGN1876, BMS-986156, BMS-986256, AMG-228, ASP1951 (PTZ 522), FPA-154 or OMP-336B11. In some embodiments, the kit includes molecules that simultaneously bind to TNF receptor superfamily member 4 (TNFRSF4, OX40, or CD134) and TNF receptor superfamily member 18 (TNFRSF18, GITR, or CD357). In some embodiments, the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. In some embodiments, the TLR7 activator or agonist is selected from the group consisting of GS9620, DS-0509, LHC-165, and TMX-101 (imiquimod), and / or wherein the TLR8 agonist is selected from the group consisting of GS-9688 and NKTR-262 (dual TLR7 / TLR8 agonist). In some embodiments, the STING receptor activator or agonist or activator is selected from ADU-S100 (MIW-815), SB-11285, MK-1454, SR-8291, AdVCA0848, GSK-532, SYN-STING, MSA-1, SR-8291, 5,6-dimethylxanthone-4-acetic acid (DMXAA),In some embodiments, the kit comprises an anti-CD47 antibody. In some embodiments, the anti-CD47 antibody is magrolimab. In some embodiments, the kit comprises a SIRPα inhibitor. In some embodiments, the SIRPα inhibitor is selected from the group consisting of: AL-008, RRx-001, CTX-5861, FSI-189 (GS-0189), ES-004, BI765063, ADU1805, and CC-95251. In some embodiments, the kit includes one or more inhibitors or antagonists of: non-receptor protein tyrosine phosphatase 11 (PTPN11 or SHP2), myeloid cell leukemia sequence 1 (MCL1) apoptosis regulator, mitogen-activated protein kinase kinase kinase kinase kinase 1 (MAP4K1) (also known as hematopoietic progenitor cell kinase 1 (HPK1)), phosphatidylinositol-4,5-bisphosphate 3-kinase (including catalytic subunit alpha (PIK3CA), catalytic subunit beta (PIK3CB), catalytic subunit gamma (PIK3CG), and catalytic subunit delta (PIK3C). kinases (PIK3CD), diacylglycerol kinase α (DGKA, DAGK, DAGK1, or DGK-α), 5'-ectonucleotidase (NT5E or CD73), ectonucleotide triphosphate diphosphohydrolase 1 (ENTPD1 or CD39), transforming growth factor β1 (TGFB1 or TGFβ), heme oxygenase 1 (HMOX1, HO-1, or HO1), heme oxygenase 2 (HMOX2, HO-2, or HO2), vascular endothelial growth factor A (VEGFA or VEGF), erb-b2 receptor tyrosine kinase 2 (ERBB2, HER2, HER2 / neu or CD340), epidermal growth factor receptor (EGFR, ERBB, ERBB1 or HER1), ALK receptor tyrosine kinase (ALK, CD246), poly (ADP-ribose) polymerase 1 (PARP1), poly (ADP-ribose) polymerase 2 (PARP2), TCDD-inducible poly (ADP-ribose) polymerase (TIPARP, PARP7), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), T NF receptor superfamily member 14 (TNFRSF14, HVEM, CD270), T cell immunoreceptor with Ig and ITIM domains (TIGIT), X-linked inhibitor of apoptosis (XIAP, BIRC4, IAP-3), baculovirus IAP repeat-containing 2 (BIRC2, cIAP1), baculovirus IAP repeat-containing 3 (BIRC3, cIAP2), baculovirus IAP repeat-containing 5 (BIRC5, survivin), CC motif chemokine receptor 2 (CCR2, CD192),C-C motif chemokine receptor 5 (CCR5, CD195), C-C motif chemokine receptor 8 (CCR8, CDw198), C-X-C motif chemokine receptor 2 (CXCR2, CD182), C-X-C motif chemokine receptor 3 (CXCR3, CD182, CD183), C-X-C motif chemokine receptor 4 (CXCR4, CD184), cytokine-inducible SH2-containing protein (CISH), arginase (ARG1, ARG2), carbonic anhydrase (CA1, CA2, CA3, CA4, CA5A, CA5B, CA6, CA7, CA8, CA9, CA10, CA11, CA12, CA13, CA14), prostaglandin-endoperoxide synthase 1 (PTGS1, COX- 1), prostaglandin-endoperoxide synthase 2 (PTGS2, COX-2), secreted phospholipase A2, prostaglandin E synthase (PTGES, PGES), arachidonic acid 5-lipoxygenase (ALOX5, 5-LOX), soluble epoxide hydrolase 2 (EPHX2), indoleamine 2,3-dioxygenase 1 (IDO1), indoleamine 2,3-dioxygenase 2 (IDO2), hypoxia-inducible factor 1 subunit alpha (HIF1A), angiopoietin 1 (ANGPT1), endothelial TEK tyrosine kinase (TIE-2, TEK), Janus kinase 1 (JAK1), catenin beta 1 (CTNNB1), histone deacetylase 9 (HDAC9), 5'-3' exoribonuclease 1 (XRN1), and / or WRN RecQ-like helicase (WRN). In some embodiments, activators / agonists or blockers / inhibitors include antibodies or their antigen-binding fragments or their antibody-drug conjugates, CD3-targeted multispecific molecules, CD16-targeted multispecific molecules, non-immunoglobulin antigen-binding molecules or antibody-mimetic proteins. In some embodiments, activators / agonists or blockers / inhibitors include small organic molecules. In some embodiments, inhibitors of 5'-ectonucleotidase (NT5E or CD73) are selected from the group consisting of MEDI9447 (oleclumab), CPI-006, BMS-986179, IPH5301, TJ4309 (TJD5), NZV-930, AB-680, PSB-12379, PSB-12441, PSB-12425, CB-708, GS-1423 (AGEN1423) and PBF-1662. In some embodiments, the inhibitor of CCR2 and / or CCR5 is selected from the group consisting of: BMS-813160, PF-04136309, and CCX-872. In some embodiments, the inhibitor of MCL1 is selected from the group consisting of: AMG-176, AMG-397, S-64315, AZD-5991,483-LM, A1210477, UMI-77, and JKY-5-037. In some embodiments, the inhibitor of PTPN11 or SHP2 is selected from the group consisting of TNO155 (SHP-099), RMC-4550, JAB-3068, and RMC-4630. In some embodiments, the kit further comprises an inhibitor or antagonist of regulatory T cells (Tregs). In some embodiments, the kit further comprises one or more anti-tumor agents or chemotherapeutic agents. In some embodiments, the one or more anti-tumor agents or chemotherapeutic agents are selected from the group consisting of nucleoside analogs (e.g., 5-fluorouracil, gemcitabine, cytarabine, cladribine, pentostatin, fludarabine), taxanes (e.g., paclitaxel, nab-paclitaxel, docetaxel, cabazitaxel), platinum coordination complexes (cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenoplatin, picoplatin, satraplatin, dicycloplatin, etoposide, lobaplatin, mitoplatin), dihydrofolate reductase (DHFR) inhibitors (e.g., methotrexate, trimesapatite, pemetrexed), topoisomerase inhibitors (e.g., doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone, , pixenolone, sobuzosine, topotecan, irinotecan, MM-398 (liposomal irinotecan), vosaroxin and GPX-150, aclarubicin, AR-67, mavelertinib, AST-2818, avitinib (ACEA-0010), ilofofen (MGI-114)), alkylating agents (e.g., nitrogen mustards (e.g., cyclophosphamide, mechlorethamine, uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine), nitrosoureas (e.g., carmustine, lomustine, streptozotocin), alkyl sulfonates (e.g., busulfan)), and mixtures thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Shown is the induction of mouse IL-6 in the M1 cell line expressing mouse FLT3 by titration of recombinant human FLT3-ligand (recombinant huFLT3L, solid circles), recombinant human FLT3-ligand human IgG1 fusion protein (recombinant huFLT3L-Fc, hollow triangles), anti-mouse FLT3 agonist antibody (comparator 1, solid triangles) or human IgG1 isotype antibody (isotype negative control, hollow squares). The x-axis shows protein concentration (nM) and the y-axis shows mouse IL-6 concentration (pg / mL). The cross symbol represents the IL-6 baseline level in untreated cells. The figure is a combination of two independent experiments. The experiment was performed in duplicate. The error bars represent the standard deviation of the mean. The EC50 values ​​are shown in Table 1.

[0027] Figure 2 The proliferation of the AML5 cell line expressing human FLT3 in response to titration (100-0.0025 nM) of recombinant human FLT3-ligand (recombinant huFLT3L, open squares), human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand human IgG1 fusion protein (SEQ ID NO: 21, open circles), or human IgG1 isotype antibody (hIgG1 isotype, crosses) is shown. The x-axis shows protein concentration (nM) and the y-axis shows relative luminescence units (RLU). The graph is the result of one experiment. The experiment was performed in triplicate. The error bars represent the standard deviation of the mean. EC50 values ​​are shown in Table 2.

[0028] Figure 3 The proliferation of AML5 cell lines expressing human FLT3 induced by titration of human wild-type FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (H8Y) human hingeless IgG1 fusion protein (SEQ ID NO: 22, open circles), human FLT3-ligand (K84E) human hingeless IgG1 fusion protein (SEQ ID NO: 23, open squares), human FLT3-ligand (H8Y+K84E) human hingeless IgG1 fusion protein (SEQ ID NO: 24, closed circles) or human IgG1 isotype antibody (hIgG1 isotype, cross) is shown. The x-axis shows protein concentration (nM) and the y-axis shows relative luminescence units (RLU). The figure is a combination of two independent experiments. The experiment was performed in triplicate. The error bars represent the standard deviation of the mean. EC50 values ​​are shown in Table 3.

[0029] Figure 4The proliferation of the AML5 cell line expressing human FLT3 induced by titration (10-0.0004 nM) of human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO: 1, open triangles), mouse FLT3-ligand mouse IgG2a (LALA-PG) fusion protein (SEQ ID NO: 19, open circles), mouse FLT3-ligand mouse IgG2a (C136S LALA-PG) fusion protein (SEQ ID NO: 20, open squares) or human IgG1 isotype antibody (hIgG1 isotype, cross) is shown. The x-axis shows protein concentration (nM) and the y-axis shows relative luminescence units (RLU). The figure is the result of one experiment. The experiment was performed in triplicate. The error bars represent the standard deviation of the mean. The EC50 values ​​are shown in Table 4.

[0030] Figure 5 The results of titration (10-0.0004 nM) of human FLT3-ligand human no-hinge IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human no-hinge IgG1 fusion protein (SEQ ID NO: 2, closed triangles), human FLT3-ligand human IgG4 (S228P / L235E) fusion protein (SEQ ID NO: 3, open circles), human FLT3-ligand human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 4, closed circles), human FLT3-ligand (S128A / S151A) human no-hinge IgG1 fusion protein (SEQ ID NO: 5, open squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open squares) are shown. Proliferation of the AML5 cell line expressing human FLT3 induced by either human FLT3-ligand (Δ10 amino acids) human hingeless IgG1 fusion protein (SEQ ID NO: 6, solid squares), human FLT3-ligand (Δ10 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 8, solid diamonds). The x-axis shows protein concentration (nM) and the y-axis shows relative luminescence units (RLU). The graph is a combination of two independent experiments. Experiments were performed in triplicate. Error bars represent the standard deviation of the mean. EC50 values ​​are shown in Table 5.

[0031] Figure 6Titration (35-0.0001 nM) of human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human hingeless IgG1 fusion protein (SEQ ID NO: 2, closed triangles), human FLT3-ligand human IgG4 (S228P / L235E) fusion protein (SEQ ID NO: 3, open circles), human FLT3-ligand human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 4, closed circles), human FLT3-ligand (S128A / S151A) human hingeless IgG1 fusion protein (SEQ ID NO: 5, open squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open squares) are shown. Binding of recombinant human FLT3 to human FLT3 ligand (Δ10 amino acids) human hingeless IgG1 fusion protein (SEQ ID NO: 6, solid squares), human FLT3 ligand (Δ10 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 8, solid diamonds) was performed. The x-axis shows protein concentration (nM), and the y-axis shows optical density (OD) at 450 nm. The graph is the result of one experiment. EC50 values ​​are shown in Table 6.

[0032] Figure 7Shown are the interactions of recombinant human FcRn with dose titration (235-0.035 nM) of human FLT3-ligand human no-hinge IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human no-hinge IgG1 fusion protein (SEQ ID NO: 2, closed triangles), human FLT3-ligand human IgG4 (S228P / L235E) fusion protein (SEQ ID NO: 3, open circles), human FLT3-ligand human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 4, closed circles), human FLT3-ligand (S128A / S151A) human no-hinge IgG1 fusion protein (SEQ ID NO: 5, open squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open squares). Binding of human FLT3-ligand (Δ10 amino acids) human hingeless IgG1 fusion protein (SEQ ID NO: 6, solid squares), human FLT3-ligand (Δ10 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 8, solid diamonds), human IgG1 isotype antibody (open diamonds), or human IgG4 isotype antibody (open stars). The x-axis shows protein concentration (nM) and the y-axis shows optical density (OD) at 450-650 nm. The graph is the result of one experiment. Experiments were performed in duplicate. Error bars represent the standard deviation of the mean. Estimated EC50 values ​​are shown in Table 7.

[0033] Figure 8Shown are titrated (294-0.48 nM) human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human hingeless IgG1 fusion protein (SEQ ID NO: 2, closed triangles), human FLT3-ligand human IgG4 (S228P / L235E) fusion protein (SEQ ID NO: 3, open circles), human FLT3-ligand human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 4, closed circles), human FLT3-ligand (S128A / S151A) human hingeless IgG1 fusion protein (SEQ ID NO: 5, open squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open squares). Binding of human IgG to recombinant human FcγRI was compared with competition of human FLT3-ligand (Δ10 amino acids) human hingeless IgG1 fusion protein (SEQ ID NO: 6, solid squares), human FLT3-ligand (Δ10 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 8, solid diamonds), human IgG1 isotype antibodies (open diamonds), or human IgG4 isotype antibodies (open stars). The x-axis shows protein concentration (nM), and the y-axis shows relative fluorescence units (RFU). The graph represents the results of one experiment. Experiments were performed in duplicate. Error bars represent the standard deviation of the mean. EC50 values ​​are shown in Table 8.

[0034] Figure 9Shown are dose titrations (1176-1.92 nM) of human FLT3-ligand human no-hinge IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human no-hinge IgG1 fusion protein (SEQ ID NO: 2, closed triangles), human FLT3-ligand human IgG4 (S228P / L235E) fusion protein (SEQ ID NO: 3, open circles), human FLT3-ligand human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 4, closed circles), human FLT3-ligand (S128A / S151A) human no-hinge IgG1 fusion protein (SEQ ID NO: 5, open squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: Binding of human IgG to recombinant human FcγRIIIa (V-variant) was compared with competition of human FLT3-ligand (Δ10 amino acids) human hingeless IgG1 fusion protein (SEQ ID NO: 6, solid squares), human FLT3-ligand (Δ10 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 8, solid diamonds), human IgG1 isotype antibodies (open diamonds), or human IgG4 isotype antibodies (open stars). The x-axis shows protein concentration (nM), and the y-axis shows relative fluorescence units (RFU). The graph represents the results of one experiment. Experiments were performed in duplicate. Error bars represent the standard deviation of the mean. EC50 values ​​are shown in Table 9.

[0035] Figure 10Shown are the interactions of recombinant human C1q with dose titration (94-0.74 nM) of human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human hingeless IgG1 fusion protein (SEQ ID NO: 2, closed triangles), human FLT3-ligand human IgG4 (S228P / L235E) fusion protein (SEQ ID NO: 3, open circles), human FLT3-ligand human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 4, closed circles), human FLT3-ligand (S128A / S151A) human hingeless IgG1 fusion protein (SEQ ID NO: 5, open squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: Binding of human FLT3-ligand (Δ10 amino acids) human hingeless IgG1 fusion protein (SEQ ID NO: 6, solid squares), human FLT3-ligand (Δ10 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 8, solid diamonds), human IgG1 isotype antibody (open diamonds), or human IgG4 isotype antibody (open stars). The x-axis shows protein concentration (nM), and the y-axis shows optical density (OD) at 450-650 nm. The graph represents the results of one experiment. Experiments were performed in duplicate. Error bars represent the standard deviation of the mean. EC50 values ​​are shown in Table 10.

[0036] Figures 11A-11BShown are mouse serum concentration-time curves relative to recombinant FLT3-ligand after intraperitoneal administration of 8 FLT3-ligand fusion proteins at 5 mg / kg. Panel A: linear scale; Panel B: single dose intravenous administration (5 mg / kg) of human FLT3-ligand human hingeless IgG1 fusion protein produced in the Expi293 expression system (SEQ ID NO: 1Expi293, open triangles), human FLT3-ligand human hingeless IgG1 fusion protein produced in the ExpiCHO expression system (SEQ ID NO: 1ExpiCHO, closed triangles), human FLT3-ligand (Δ5 amino acids) human hingeless IgG1 fusion protein (SEQ ID NO: 2, open circles), human FLT3-ligand human IgG4 (S228P / L235E) fusion protein (SEQ ID NO: 3, closed circles), human FLT3-ligand human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 4, open squares), human FLT3-ligand (S128A / S151A) human hingeless IgG1 fusion protein (SEQ ID NO: 5, open circles), human FLT3-ligand human IgG4 (S228P / L235E) fusion protein (SEQ ID NO: 6, closed circles), human FLT3-ligand human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 7, open squares), human FLT3-ligand human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 8, open squares), human FLT3-ligand human IgG4 (S128A / S151A) human hingeless IgG1 fusion protein (SEQ ID NO: 9, open squares), human FLT3-ligand human IgG4 (S228P / L234A / L235A) fusion protein (SEQ ID NO: 10, open squares), human FLT3-ligand human IgG4 (S128A / S151A) human hingeless IgG1 5, filled squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, crosses), human FLT3-ligand (Δ10 amino acids) human hingeless IgG1 fusion protein (SEQ ID NO: 7, open diamonds), human FLT3-ligand (Δ10 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 8, filled diamonds), or recombinant human FLT3-ligand (recombinant huFLT3L, filled stars). The graph is the result of one experiment. The x-axis shows the number of days after injection, and the y-axis shows the protein concentration in serum (μg / mL). Each data point represents the mean of 4 animals. The error bars represent the standard deviation (SD) of the mean. The mean pharmacokinetic values ​​± SD are shown in Table 11.

[0037] Figure 12Shown are 5 mg / kg of human FLT3-ligand human non-hinge IgG1 fusion protein produced in the Expi293 expression system (SEQ ID NO: 1Expi293, open triangles), human FLT3-ligand human non-hinge IgG1 fusion protein produced in the ExpiCHO expression system (SEQ ID NO: 1ExpiCHO, closed triangles), human FLT3-ligand (Δ5 amino acids) human non-hinge IgG1 fusion protein (SEQ ID NO: 2, open circles), human FLT3-ligand human IgG4 (S228P / L235E) fusion protein (SEQ ID NO: 3, closed circles), human FLT3-ligand human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 4, open squares), human FLT3-ligand (S128A / S151A) human non-hinge IgG1 fusion protein (SEQ ID NO: 5, open circles), and human FLT3-ligand human IgG4 (S228P / L235E) fusion protein (SEQ ID NO: 6, open circles). Figure 11 shows the frequency of conventional dendritic cell subset 1 (cDC1) in the spleens of C57BL / 6 mice injected with human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 5, solid squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, crosses), human FLT3-ligand (Δ10 amino acids) human hingeless IgG1 fusion protein (SEQ ID NO: 7, open diamonds), human FLT3-ligand (Δ10 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 8, solid diamonds), or recombinant human FLT3-ligand (recombinant huFLT3L, solid stars). Baseline cDC1 frequency (solid stars) is shown. The x-axis shows the percentage of splenic cDC1 in total mononuclear cells (MNCs). The graph shows the results of one experiment. Each individual symbol represents a data point for a single mouse. The horizontal bars represent the mean and the error bars represent the standard deviation of the mean. The mean frequency for each group is shown in Table 12.

[0038] Figure 13Figure 1 shows the proliferation of AML5 cell lines expressing human FLT3 stimulated by dose titration (10-0.0004 nM) of human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open circles), human FLT3-ligand human hingeless IgG1 (M252Y / S254T / T256E) fusion protein (SEQ ID NO: 9, open squares), or human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A / M252Y / S254T / T256E) fusion protein (SEQ ID NO: 14, crosses). The x-axis shows protein concentration (nM) and the y-axis shows relative luminescence units (RLU). The graph is the result of one experiment. The experiment was performed in duplicate. Error bars represent the standard deviation of the mean. EC50 values ​​are shown in Table 13.

[0039] Figure 14 Shown is the differentiation of conventional dendritic cell subtype 1 (cDC1) cells from human bone marrow CD34+ stem cells by dose titration (10-0.002 nM) of human FLT3-ligand human non-hinge IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open circles), human FLT3-ligand human non-hinge IgG1 (M252Y / S254T / T256E) fusion protein (SEQ ID NO: 9, open squares), or human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A / M252Y / S254T / T256E) fusion protein (SEQ ID NO: 14, cross). The x-axis shows protein concentration (nM) and the y-axis shows the percentage of cDC1 in total mononuclear cells (MNC). The graph is a summary of 13 bone marrow donors. Error bars represent the standard error of the mean. EC50 values ​​are shown in Table 14.

[0040] Figure 15Shown are dose-titration (10-0.002 nM) efficacy of enhancing the survival of PBMC-derived conventional dendritic cell subtype 1 (cDC1) cells by human FLT3-ligand human no-hinge IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open circles), human FLT3-ligand human no-hinge IgG1 (M252Y / S254T / T256E) fusion protein (SEQ ID NO: 9, open squares), or human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A / M252Y / S254T / T256E) fusion protein (SEQ ID NO: 14, cross). The x-axis shows protein concentration (nM) and the y-axis shows the percentage of cDC1 in total mononuclear cells (MNC). The graph is a summary of 16 PBMC donors. Error bars represent the standard error of the mean. EC50 values ​​are shown in Table 15.

[0041] Figure 16 Figure 2 shows the binding of recombinant human FLT3 to titrated (15-0.007 nM) human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open circles), human FLT3-ligand human hingeless IgG1 (M252Y / S254T / T256E) fusion protein (SEQ ID NO: 9, open squares), or human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A / M252Y / S254T / T256E) fusion protein (SEQ ID NO: 14, crosses). The x-axis shows protein concentration (nM) and the y-axis shows optical density (OD) at 450 nm. The graph is the result of one experiment. The experiment was performed in duplicate. Error bars represent standard deviation of the mean. EC50 values ​​are shown in Table 16.

[0042] Figure 17Figure 2 shows the binding of recombinant human FcRn to dose titration (3529-0.55 nM) of human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open circles), human FLT3-ligand human hingeless IgG1 (M252Y / S254T / T256E) fusion protein (SEQ ID NO: 9, open squares), or human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A / M252Y / S254T / T256E) fusion protein (SEQ ID NO: 14, crosses). The x-axis shows protein concentration (nM) and the y-axis shows optical density (OD) at 450-650 nm. The graph is the result of one experiment. The experiment was performed in duplicate. Error bars represent the standard deviation of the mean. EC50 values ​​are shown in Table 17.

[0043] Figure 18 Shown is the binding of human IgG to recombinant human FcγRI in competition with titrated (294-0.48 nM) human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open circles), human FLT3-ligand human hingeless IgG1 (M252Y / S254T / T256E) fusion protein (SEQ ID NO: 9, open squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A / M252Y / S254T / T256E) fusion protein (SEQ ID NO: 14, crosses), human IgG1 isotype antibodies (open diamonds), or human IgG4 isotype antibodies (open stars). The x-axis shows protein concentration (nM) and the y-axis shows relative fluorescence units (RFU). The graph is the result of one experiment. The experiment was performed in duplicate. The error bars represent the standard deviation of the mean. EC50 values ​​are shown in Table 18.

[0044] Figure 19The results are shown by titration (1176-1.92 nM) of human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open circles), human FLT3-ligand human hingeless IgG1 (M252Y / S254T / T256E) fusion protein (SEQ ID NO: 9, open squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A / M252Y / S254T / T256E) fusion protein (SEQ ID NO: 10, open squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A / M252Y / S254T / T256E) fusion protein (SEQ ID NO: 11, open squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A / M252Y / S254T / T256E) fusion protein (SEQ ID NO: 12, open squares). Binding of human IgG to recombinant human FcγRIIIa (V-variant) competed with a human IgG1 isotype antibody (open diamonds), or a human IgG4 isotype antibody (open stars). The x-axis shows protein concentration (nM), and the y-axis shows relative fluorescence units (RFU). The graph represents the results of one experiment. Experiments were performed in duplicate. Error bars represent the standard deviation of the mean. EC50 values ​​are shown in Table 19.

[0045] Figure 20 Shown is binding of recombinant human CIq to titrated (94-0.74 nM) human FLT3-ligand human non-hinge IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open circles), human FLT3-ligand human non-hinge IgG1 (M252Y / S254T / T256E) fusion protein (SEQ ID NO: 9, open squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A / M252Y / S254T / T256E) fusion protein (SEQ ID NO: 14, crosses), human IgG1 isotype antibodies (open diamonds), or human IgG4 isotype antibodies (open stars). The x-axis shows protein concentration (nM) and the y-axis shows optical density (OD) at 450-650 nm. The graph is the result of one experiment. The experiment was performed in duplicate. The error bars represent the standard deviation of the mean. EC50 values ​​are shown in Table 20.

[0046] Figures 21A-21BShown are the serum concentration-time curves of cynomolgus monkeys relative to recombinant FLT3 ligand following intravenous and subcutaneous administration of four FLT3-ligand fusion proteins at 500 μg / kg. Mean serum concentration-time curves after intravenous (Panel A) or subcutaneous (Panel B) administration of human FLT3-ligand human hingeless IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open circles), human FLT3-ligand human hingeless IgG1 (M252Y / S254T / T256E) fusion protein (SEQ ID NO: 9, open squares), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A / M252Y / S254T / T256E) fusion protein (SEQ ID NO: 14, crosses). The x-axis shows the number of days after injection and the y-axis shows the protein concentration in serum (μg / mL). Each data point represents the average of 3 animals. The error bars represent the standard deviation of the mean. The mean pharmacokinetic values ​​are shown in Table 21.

[0047] Figures 22A-22B Shown are the kinetics of fold change in conventional dendritic cell subset 1 (cDC1) in the peripheral blood of cynomolgus macaques administered 500 μg / kg of human FLT3-ligand human non-hinge IgG1 fusion protein (SEQ ID NO: 1, open triangles), human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A) fusion protein (SEQ ID NO: 6, open circles), human FLT3-ligand human non-hinge IgG1 (M252Y / S254T / T256E) fusion protein (SEQ ID NO: 9, open squares), or human FLT3-ligand (Δ5 amino acids) human IgG4 (S228P / F234A / L235A / M252Y / S254T / T256E) fusion protein (SEQ ID NO: 14, crosses) intravenously (Panel A) or subcutaneously (Panel B) on day 0. The graph is the result of one experiment. Each data point represents the mean of 3 animals. Error bars represent the standard deviation of the mean.

[0048] Figure 23 The results show that the tumor volume reached 50 mm on day 0. 3Figure 2 Tumor growth kinetics of MC38 tumor-bearing C57BL / 6 mice administered intravenously at 3750 μg / kg (open triangles), 750 μg / kg (open circles), 150 μg / kg (open squares), 30 μg / kg (crosses) of mouse FLT3-ligand mouse IgG2a (C136S, LALA-PG), or 3753 μg / kg of mouse IgG2a isotype (open stars) at 4 hr (time of dosing). The x-axis is the number of days after dosing. The y-axis is the tumor volume in mm. 3 The data are presented in units of . The graph represents two independent experiments. Each data point represents the mean of seven animals. Error bars represent the standard deviation of the mean. Statistical differences in tumor growth rate for each dose group compared to the isotype group were determined by repeated mixed ANOVA. Data were fitted with a linear mixed-effects model.

[0049] Figures 24A-24B The results show that the tumor volume reached 50 mm on day 0. 3 Quantification of conventional dendritic cell subset 1 (cDC1) numbers in tumors (Panel A) or spleens (Panel B) of MC38-bearing C57BL / 6 mice intravenously administered 3750 μg / kg (open triangles), 750 μg / kg (open circles), 150 μg / kg (open squares), 30 μg / kg (crosses), or 3753 μg / kg of mouse FLT3-ligand mouse IgG2a (C136S, LALA-PG) isotype (open stars) at day 7. The x-axis represents the dose group. The y-axis shows the number of cDC1s per gram of tumor (Panel A) or per spleen (Panel B). The graphs are the results of one experiment. Each individual symbol represents a data point from a single mouse. Horizontal bars represent the mean, and error bars represent the standard deviation of the mean. Statistical differences were determined using one-way ANOVA with a Dunnett post hoc test. ****p-value < 0.0001; ***p-value < 0.001; **p-value < 0.01.

[0050] Figure 25 The results show that the tumor volume reached 50 mm on day 0. 3 Tumor growth kinetics of MC38 tumor-bearing C57BL / 6 mice administered intravenously QWx2 doses of 3.75 μg / kg (open circles) mouse FLT3-ligand mouse IgG2a (C136S, LALA-PG), Q3W doses of 3 mg / kg (open triangles) anti-mouse PD-1 (clone RMP1-14), a combination of both agents (closed circles), or QWx2 doses of 10 mg / kg mouse IgG2a isotype control (open stars). Each data point represents the mean of 10 animals. Error bars represent the standard deviation of the mean.

[0051] Figure 26The results show that the tumor volume reached 65 mm on day 0. 3 Figure 2. Tumor growth kinetics of CT26 tumor-bearing BALB / c mice administered intravenously (qw x 2) at 3.75 μg / kg (open circles) mouse FLT3-ligand mouse IgG2a (C136S, LALA-PG), 3 mg / kg (open triangles) anti-mouse CTLA4 (clone 9D9) at q3w, a combination of both agents (closed circles), or 10 mg / kg mouse IgG2a isotype control (open stars) at q3w. Each data point represents the mean of 10 animals. Error bars represent the standard deviation of the mean.

[0052] Figure 27 Figure 3 shows an immunogenicity study in C57BL / 6 mice transduced with an adeno-associated virus (AAV) vector encoding a 1.2-fold longer hepatitis B virus (HBV) genome (AAV-HBV mice). At the indicated times (asterisks), AAV-HBV mice were administered 3 doses of HBV vaccine and treated with saline, mouse FLT3L, anti-mouse inhibitory PD-1, anti-mouse inhibitory CTLA-4, or anti-mouse stimulatory CD137 antibodies. A control group of mice received the HBV vaccine alone but not AAV-HBV. HBV-specific IFN-γ ELISPOT was performed using spleens from all animals on day 105 after the first vaccination.

[0053] Figure 28A -C shows the Figure 27 The immunogenicity observed at the end of the study for the indicated treatment and control groups is shown in the table below. Figure 28A )、HBV nucleus( Figure 28B ) and HBV polymerase ( Figure 28C )-specific IFN-γ ELISPOT responses in AAV-HBV mice. DETAILED DESCRIPTION

[0054] 1. Introduction

[0055] Provided is a fms-related tyrosine kinase 3 ligand (FLT3L) extracellular domain-immunoglobulin fragment crystallizable region (Fc region) fusion protein having a serum half-life of an administration interval (e.g., approximately once every 1, 2, 3, or 4 weeks or longer intervals) that allows coordination with other approved immuno-oncology therapeutic agents. FLT3L-Fc fusion protein shows good expression yield in in vitro culture, has structural properties that allow efficient large-scale purification and long-term storage. Compared with soluble FLT3L, the FLT3L-Fc fusion protein described herein has an increased serum half-life. In view of the fact that soluble FLT3L must be administered every day, the FLT3L-Fc fusion protein described herein can be administered approximately once every 1 to 8 weeks or at longer intervals.

[0056] The FLT3L-Fc fusion proteins described herein differ from commercially available FLT3L-Fc fusion proteins (e.g., available from BioXCell or described in Kreiter et al., Cancer Research (2011) 71(19): 6132-42) in that they have several structural modifications that improve functionality and can be used for administration to mammalian subjects and have functional efficacy in mammalian subjects. For example, the FLT3L-Fc fusion proteins described herein have been engineered and formulated to have improved glycosylation characteristics, thereby allowing for predictable and consistent serum half-life or pharmacokinetics (PK). The linker region between the ligand and the Fc fusion partner has reduced or eliminated glycosylation sites. In some FLT3L-Fc fusion variants described herein, the IgG hinge region is truncated or eliminated. For example, in a FLT3L-Fc fusion variant comprising a human IgG4 Fc, the N-terminal five amino acid residues of the IgG4 hinge (i.e., ESKYG (SEQ ID NO: 97)) are truncated or removed. Furthermore, mutations have been incorporated to stabilize the amino acids retained in the entire or truncated hinge region (e.g., S228P in the human IgG4 hinge), thereby eliminating Fab arm exchange or IgG half-chain exchange and allowing for favorable chemical manufacturing controls. Additionally, mutations have been introduced to reduce or eliminate Fcγ receptor (FcRγ) binding (e.g., 234A / 235A substitutions in human IgG4), effectively reducing or eliminating antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Additionally, alterations have been made to enhance FcRn binding (e.g., 252Y / 254T / 256E substitutions in human IgG4), resulting in an increased serum half-life of the FLT3L-Fc fusion protein (e.g., compared to wild-type Fc).

[0057] In certain embodiments, the FLT3L-Fc fusion protein described herein is not in the form of an antibody and therefore only binds to Flt3 and FcRn receptors. In such embodiments, the FLT3L-Fc fusion protein described herein is structurally different from WO 2005 / 001048 and WO 2006 / 060021 (describing FLT3L fused to HER2, CD20, TRAIL, CD3 or SM5-1). In such embodiments, the lack of antigen-guided binding allows systemic exposure of FLT3L-Fc, which can lead to a systemic increase in cDC1 cells, thereby allowing the pharmacodynamics of FLT3L-Fc to be monitored by liquid biopsy.

[0058] 2. FLT3L-Fc fusion protein composition

[0059] A fusion protein is provided, comprising: an fms-related tyrosine kinase 3 ligand (FLT3L) extracellular domain operably linked to an immunoglobulin fragment crystallizable region (Fc region), wherein: at least 5 amino acids are truncated from the C-terminus of the FLT3L extracellular domain; and / or wherein the Fc region does not comprise a hinge region.

[0060] In some embodiments, the FLT3L fusion proteins provided herein are capable of binding to human fms-related tyrosine kinase 3 ligand (FLT3). Human fms-related tyrosine kinase 3 is identified as NCBI gene ID 2322 and is also known as human CD135, FLK-2, FLK2, or STK1. The binding of FLT3L fusion proteins to FLT3L can be analyzed, for example, by FACS, SPR, ELISA, immunoprecipitation-western blotting, and other assays known in the art.

[0061] Fusion protein and its homodimer

[0062] FLT3L extracellular domain

[0063] In certain embodiments, the FLT3L extracellular domain comprises or is derived from the human FLT3L sequence. Human fms-related tyrosine kinase 3 ligand is identified as NCBI gene ID 2323, and the alternative symbols are FLT3LG, FLT3L, FL, and FLG3L. NCBI identifies two isoforms and five transcript variants. Exemplary polynucleotide and polypeptide sequences of FLT3L include Ref Seq No. NM_001204502.1→NP_001191431.1 (isoform 1, transcript variant 1); NM_001204503.1→NP_001191432.1 (isoform 1, transcript variant 2); NM_001459.4→NP_001450.2 (isoform 1, transcript variant 3); NM_001278637.1→NP_001265566.1 (isoform 2, transcript variant 4); and NM_001278638.1→NP_001265567.1 (isoform 2, transcript variant 5). In some embodiments, the FLT3L extracellular domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of NP_001191431.1, NP_001191432.1, NP_001450.2, NP_001265566.1 or NP_001265567.1, wherein the FLT3L extracellular domain binds to fms-related tyrosine kinase 3 (FLT3, CD135, FLK2, STK1) and activates signaling through the kinase. In some embodiments, the FLT3L extracellular domain comprises or is derived from human FLT3L isoform 1. In some embodiments, the FLT3L extracellular domain comprises or is derived from human FLT3L isoform 2.

[0064] In some embodiments, the FLT3L portion of the fusion protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the following amino acid sequence:

[0065] TQDCSFQ H SPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVT K CAFQPPPSCLRFVQTNI SRLLQETSEQLVALKPWITRQNF S RCLELQCQPDSSTLPPPWSPRP(SEQ ID NO:71);

[0066] TQDCSFQ H SPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVT K CAFQPPPSCLRFVQTNI S RLLQETSEQLVALKPWITRQNF S RCLELQCQPDSSTLPPPWSPRPL(SEQ ID NO:72);

[0067] TQDCSFQ H SPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVT K CAFQPPPSCLRFVQTNI S RLLQETSEQLVALKPWITRQNF S RCLELQCQPDSSTLPPPWSPRPLE(SEQ ID NO:73);

[0068] TQDCSFQ H SPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVT K CAFQPPPSCLRFVQTNI S RLLQETSEQLVALKPWITRQNF S RCLELQCQPDSSTLPPPWSPRPLEA(SEQ ID NO:74);

[0069] TQDCSFQ H SPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVT K CAFQPPPSCLRFVQTNI S RLLQETSEQLVALKPWITRQNF SRCLELQCQPDSSTLPPPWSPRPLEAT(SEQ ID NO:75);

[0070] TQDCSFQ H SPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVT K CAFQPPPSCLRFVQTNI S RLLQETSEQLVALKPWITRQNF S RCLELQCQPDSSTLPPPWSPRPLEATA(SEQ ID NO:76);

[0071] TQDCSFQ H SPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVT K CAFQPPPSCLRFVQTNI S RLLQETSEQLVALKPWITRQNF S RCLELQCQPDSSTLPPPWSPRPLEATAP(SEQ ID NO:77);

[0072] TQDCSFQ H SPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVT K CAFQPPPSCLRFVQTNI S RLLQETSEQLVALKPWITRQNF S RCLELQCQPDSSTLPPPWSPRPLEATAPT(SEQ ID NO:78);

[0073] TQDCSFQ H SPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVT K CAFQPPPSCLRFVQTNI S RLLQETSEQLVALKPWITRQNF SRCLELQCQPDSSTLPPPWSPRPLEATAPTA(SEQ ID NO:79);

[0074] TQDCSFQ H SPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVT K CAFQPPPSCLRFVQTNI S RLLQETSEQLVALKPWITRQNF S RCLELQCQPDSSTLPPPWSPRPLEATAPTAP(SEQ ID NO:80); or

[0075] TQDCSFQ H SPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVT K CAFQPPPSCLRFVQTNI S RLLQETSEQLVALKPWITRQNF SRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQ (SEQ ID NO: 81); wherein the FLT3L extracellular domain binds to fms-related tyrosine kinase 3 (gene ID: 2322; FLT3, CD135, FLK2, STK1) and activates signal transduction through the kinase, and promotes or increases the proliferation of cells expressing FLT3 on their cell surface. In some embodiments, one or more FLT3L domain amino acid residues N100, S102, N123, and S125 are substituted (e.g., to remove the NXS / T motif and potential N-linked and / or O-linked glycosylation sites), for example, with an amino acid residue selected from the group consisting of glycine (G), alanine (A), or valine (V), wherein the amino acid residue positions are referenced to SEQ ID NOs: 1-18, 21-27, or 71-81. In some embodiments, one or both of the serine residues at positions 102 and 125 are substituted with alanine (A), wherein the amino acid residue positions are referenced to SEQ ID NOs: 1-18, 21-27, or 71-81. In some embodiments, the FLT3L extracellular domain comprises one or more amino acid substitutions at the following positions: H8, K84, S102, and / or S125, wherein the amino acid residue positions are referenced to SEQ ID NOs: 1-18, 21-27, or 71-81. In some embodiments, the FLT3L extracellular domain comprises one or more of the following amino acid substitutions: H8Y; K84E; S102A; and / or S125A; wherein the amino acid residue positions are referenced to SEQ ID NOs: 1-18, 21-27, or 71-81.

[0076] Modifications can be made in the structure of the FLT3L-Fc polynucleotides and polypeptides described herein, and these modifications still result in functional molecules encoding variant or derivative polypeptides having the desired characteristics. When it is desired to alter the amino acid sequence of a polypeptide to produce an equivalent or even improved variant or portion of a polypeptide described herein, one skilled in the art will typically alter one or more of the codons encoding the DNA sequence.

[0077] For example, certain amino acids can be substituted for other amino acids in a protein structure without significantly losing its ability to bind to other polypeptides (e.g., antigens) or cells. Since it is the binding ability and properties of a protein that define the biological functional activity of that protein, certain amino acid sequence substitutions can be made in a protein sequence, and certainly in its underlying DNA coding sequence, while still obtaining a protein with similar properties. It is therefore conceivable that various changes can be made in the polypeptide sequences of the disclosed antibodies and antigen-binding fragments thereof, or in the corresponding DNA sequences encoding the polypeptides, without significantly losing their biological utility or activity.

[0078] In many cases, polypeptide variants will contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid with similar properties, such that one skilled in the art of peptide chemistry would expect that the secondary structure and hydrophilicity of the polypeptide would be substantially unchanged.

[0079] When comparing polynucleotide and polypeptide sequences, the two sequences are considered "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions (typically 30 to about 75, 40 to about 50) or over the full length of a sequence, wherein the sequence can be compared to a reference sequence after optimal alignment with a reference sequence having the same number of contiguous positions.

[0080] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene bioinformatics software package (DNASTAR, Inc., Madison, WI) using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM (1989) CABIOS 5: 151-153; Myers, EW and Muller W. (1988) CABIOS 4: 11-17; Robinson, ED (1971) Comb. Theor 77:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4: 406-425; Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJand Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0081] Alternatively, optimal alignment of sequences for comparison can be performed by the local identity algorithm (Smith and Waterman (1981) Add. APL. Math 2:482), the identity alignment algorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48:443), the search similarity method (Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444), computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or inspection.

[0082] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 methods, which are described in Altschul et al. (1977) Nucl. Acids Res. 25: 3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein to determine percent sequence identity for the polynucleotides and polypeptides described herein. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0083] In an illustrative example, for nucleotide sequences, the cumulative score can be calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). Extension of the word hits in each direction is stopped when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11 and an expectation (E) of 10 as defaults, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) for alignments, (B) of 50, an expectation (E) of 10, M = 5, N = -4, and a comparison of both chains.

[0084] For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score goes to zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0085] In one method, "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less (usually 5% to 15%, or 10% to 12%) additions or deletions (i.e., gaps) compared to a reference sequence for optimal alignment of the two sequences (not comprising additions or deletions). The percentage is calculated by determining the number of positions where the identical nucleic acid base or amino acid residue occurs in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to produce the percentage of sequence identity.

[0086] In some embodiments, the FLT3L extracellular domain does not include a signal peptide. In some embodiments, the FLT3L extracellular domain includes an N-terminal signal peptide. The signal peptide can be an endogenous signal peptide (e.g., from a natural or wild-type FLT3L protein) or from a heterologous polypeptide. In some embodiments, the heterologous signal peptide is from a secreted protein, such as a serum protein, an immunoglobulin, or a cytokine. In some embodiments, the signal peptide is from a serum albumin signal peptide (e.g., with the amino acid sequence KWVTFISLLFLFSSAYS (SEQ ID NO: 82)). In some embodiments, the signal peptide is from a FLT3L protein (e.g., with the amino acid sequence MTVLAPAWSPTTYLLLLLLLSSGLSG (SEQ ID NO: 83) or MTVLAPAWSPNSSLLLLLLLLSPCLRG (SEQ ID NO: 84)). The signal peptide can be designed to be, for example, cut off after secretion from the cell to form a mature fusion protein. Modified human serum albumin signal peptides for secreting proteins in cells that can be used to express the fusion proteins of the present invention are described, for example, in Attallah et al., Protein Expr Purif. (2017) 132: 27-33. Additional signal peptide sequences for expressing the fusion proteins described herein are described, for example, in Kober et al., Biotechnol Bioeng. (2013) 110 (4): 1164-73.

[0087] In some embodiments, at least five amino acids are truncated from the C-terminus of the FLT3L extracellular domain. For example, in various embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues are truncated or removed from the C-terminus of the FLT3L extracellular domain. In some embodiments, the length of the FLT3L extracellular domain in the fusion protein is no longer than 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, or 157 amino acid residues. In some embodiments, the FLT3L extracellular domain does not comprise the amino acid sequence PTAPQ (SEQ ID NO: 85), APTAPQ (SEQ ID NO: 86), TAPTAPQ (SEQ ID NO: 87), ATAPTAPQ (SEQ ID NO: 88), EATAPTAPQ (SEQ ID NO: 89), or LEATAPTAPQ (SEQ ID NO: 90). In some embodiments, the FLT3L extracellular domain does not comprise the amino acid sequence PTAPQPP (SEQ ID NO:91), APTAPQPP (SEQ ID NO:92), TAPTAPQPP (SEQ ID NO:93), ATAPTAPQPP (SEQ ID NO:94), EATAPTAPQPP (SEQ ID NO:95), or LEATAPTAPQPP (SEQ ID NO:96).

[0088] In certain embodiments, the FLT3L extracellular domain comprises or is derived from a mouse or murine FLT3L sequence. Mus musculus fms-related tyrosine kinase 3 ligand is identified as NCBI gene ID 14256 and has alternative symbols Flt31, Ly72L, and Flt31g. NCBI has identified one validated isoform and three unvalidated isoforms (X1, X2, and X3). Exemplary polynucleotide and polypeptide sequences of FLT3L include RefSeq No.NM_013520.3→NP_038548.3 (confirmed isoform 1); XM_006540607.3→XP_006540670.1 (isoform X1); XM_006540608.3→XP_006540671.1 (isoform X1); XM_006540606.2→XP_006540669.1 (isoform X1); XM_011250793.1→XP_0 11249095.1 (isoform X1); XM_006540609.3→XP_006540672.1 (isoform X2); XM_006540610.3→XP_006540673.1 (isoform X2); XM_006540612.3→XP_006540675.1 (isoform X3); and XM_011250794.2→XP_011249096.1 (isoform X3). In some embodiments, the FLT3L extracellular domain comprises an amino acid sequence that is at least 80% identical to that of NP_038548.3, XP_006540670.1, XP_006540671.1, XP_006540669.1, XP_011249095.1, XP_006540672.1, XP_006540673.1, XP_006540675.1, XP_011249096.1. In some embodiments, the FLT3L extracellular domain comprises or is derived from mouse FLT3L isoform 1, X1, X2 or X3. In some embodiments, at least five amino acids are truncated from the C-terminus of the mouse FLT3L extracellular domain.For example, in various embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues are truncated or removed from the C-terminus of the mouse FLT3L extracellular domain. In some embodiments, the length of the FLT3L extracellular domain in the fusion protein is no longer than 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, or 159 amino acid residues.

[0089] In some embodiments, the mouse FLT3L portion of the fusion protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the following amino acid sequence: TPDCYFSHSPISSNFKVKFRELTDHLLKDYPVTVAVNLQDEKHCKALWSLFLAQRWIEQLKTVAGSKMQTLLEDVNTEIHFVTSCTFQPLPECLRFVQTNISHLLKDT C TQLLALKPCIGKACQNFSRCLEVQCQPDSSTLLPPRSPIALEATELPEPR (SEQ ID NO: 98), wherein the mouse FLT3L extracellular domain binds to mouse fms-related tyrosine kinase 3 (NCBI human gene ID: 14255; Flt3, Flk2; Ly72; wmfl; CD135; Flk-2; Flt-3; B230315G04) and activates signal transduction through the kinase. In some embodiments, the cysteine ​​at position 109 is substituted with an amino acid residue selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T) or valine (V), wherein the amino acid residue positions are referenced to SEQ ID NOs: 19, 20 and 42.

[0090] In certain embodiments, the FLT3L extracellular domain comprises or is derived from a macaque or cynomolgus FLT3L sequence. Common macaque (Macaca mulatta) (rhesus monkey) fms-related tyrosine kinase 3 ligand is identified as NCBI gene ID 719239, and the alternative symbols are FLT3L and FLT3LG. NCBI has identified five unverified isoforms (X1, X2, X3, X4, X5). Exemplary polynucleotide and polypeptide sequences of FLT3L include RefSeq No. XM_015124576.1→XP_014980062.1 (isoform X1), XM_015124578.1→XP_014980064.1 (isoform X2), XM_015124579.1→XP_014980065.1 (isoform X3), XM_015124580.1→XP_014980066.1 (isoform X4), and XM_015124581.1→XP_014980067.1 (isoform X5). In some embodiments, the FLT3L extracellular domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence of XP_014980062.1, XP_014980064.1, XP_014980065.1, XP_014980066.1, or XP_014980067.1, wherein the FLT3L extracellular domain binds to and activates signaling through fms-related tyrosine kinase 3 (FLT3, CD135, FLK2, STK1) and promotes or increases proliferation of cells expressing FLT3 on their cell surface. In some embodiments, the FLT3L extracellular domain comprises or is derived from macaque FLT3L isoform X1, X2, X3, X4 or X5. In some embodiments, at least five amino acids are truncated from the C-terminus of the macaque FLT3L extracellular domain. For example, in various embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues are truncated or removed from the C-terminus of the macaque FLT3L extracellular domain. In some embodiments, the length of the FLT3L extracellular domain in the fusion protein is no longer than 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 amino acid residues.

[0091] In some embodiments, the FLT3L extracellular domain comprises or is derived from a canine or feline FLT3L extracellular domain. In some embodiments, the dog or wolf (Canis lupus) FLT3L portion of the fusion protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the following amino acid sequence: NP_001003350.1, XP_005615795.1, or XP_022273164.1. In some embodiments, the feline or domestic cat (Felis catus) FLT3L portion of the fusion protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the following amino acid sequence: NP_001009842.1 or XP_011287950.1.

[0092] As used herein, the term "polypeptide variant" is a polypeptide that typically differs from the polypeptides specifically disclosed herein by one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be generated synthetically, for example, by modifying one or more of the above-described polypeptide sequences described herein and evaluating one or more biological activities of the polypeptides as described herein and / or using any of a variety of techniques well known in the art.

[0093] The term "variant" may also refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations. In one embodiment, the multispecific antigen-binding molecule is a bispecific antigen-binding molecule. In one embodiment, the multispecific antigen-binding molecule is a bispecific antibody. For example, somatic variants may encompass all related naturally occurring antibodies that are part of or derived from the same B-cell lineage. Engineered variants may encompass all single or combined mutations made to an antibody.

[0094] Fc region

[0095] FLT3L extracellular domain or its truncated fragment are operably connected to Fc domain.Generally, Fc domain includes or is derived from the species identical with FLT3L extracellular domain (for example, people, dog, cat, mouse or monkey).In some embodiments, FLT3L extracellular domain or its truncated fragment directly connects or is continuously connected or is adjacent to Fc domain.In some embodiments, FLT3L extracellular domain or its truncated fragment is operably connected to Fc domain via joint.As the case may be, joint can be flexible joint, for example, comprising GGGGS motif or " GS joint " (SEQ ID NO:99) 3 or 4 repeated sequences (Desplancq et al. 1994, Protein Engineering 7:1027-1033).

[0096] In some embodiments, the Fc region is from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is from human IgG1 or IgG4.

[0097] In certain embodiments, the FLT3L extracellular domain or a truncated fragment thereof is directly connected to or connected via an intermediate amino acid sequence (e.g., a GS linker) to a human IgG1 (e.g., a mutant IgG1m3 sequence), IgG2, IgG3, or IgG4 having 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions. In some embodiments, the Fc modification can promote one or more of an increase in the serum half-life of the molecule or a decrease in the antibody effector function. In other embodiments, some of these modifications reduce the antibody effector function and increase the half-life of the antibody. In some embodiments, the FLT3L-Fc fusion protein described herein comprises two or more, three or more, four or more, five or more, six or more, six or less, five or less, four or less, three or less, two or less, or one modified Fc amino acid residue. Exemplary amino acid substitutions are described below.

[0098] In some embodiments, the Fc domain of the fusion protein does not include a hinge region; The hinge region is truncated or missing in whole or in part. The structural hinge region of human IgG1, IgG2, and IgG4 antibodies is a peptide linker of about 19 to 23 amino acids containing two to four cysteine ​​residues, which is genetically encoded together with the 5' end of the CH2 exon on the hinge exon and allows the formation of a disulfide bridge between the first Fc domain and the second Fc domain (Roux et al., J. Immunol. (1998) 161: 4083). The structural hinge region is composed of amino acid residue positions 216-238 (EU numbering) or 226-251 (Kabat numbering) (identified on imgt.org). In some embodiments, the Fc region comprises or is derived from a human IgG4 isotype and does not comprise the amino acid sequence ESKYGPPCPPCP (SEQ ID NO: 100). In some embodiments, the Fc region comprises or is derived from a human IgG1 isotype and does not comprise the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 101) or EPKSCDKTHTCPPCPAPELL (SEQ ID NO: 110).

[0099] Fc mutations that increase serum half-life

[0100] In some embodiments, the Fc region includes amino acid modifications that promote the serum half-life of the fusion protein to increase. Mutations that increase the half-life of antibodies have been described. In one embodiment, the constant region of the FLT3L-Fc fusion protein described herein includes methionine to tyrosine substitutions at position 252 (EU numbering), serine to threonine substitutions at position 254 (EU numbering), and threonine to glutamic acid substitutions at position 256 (EU numbering). See, for example, U.S. Patent No. 7,658,921. This type of mutant (named "YTE mutant") shows a four-fold increased half-life relative to the wild-type version of the same antibody (Dall'Acqua et al., J Biol Chem, 281: 23514-24 (2006); Robbie et al., Antimicrob Agents Chemotherap., 57 (12): 6147-6153 (2013)). In certain embodiments, FLT3L-Fc fusion protein as described herein includes IgG constant domain, and this IgG constant domain includes one, two, three or more amino acid replacements of the amino acid residues at positions 251-257, 285-290, 308-314, 385-389 and 428-436 (EU numbering). Alternatively, M428L and N434S (" LS ") replacements can increase the pharmacokinetic half-life of the fusion protein. In other embodiments, FLT3L-Fc fusion protein as described herein includes M428L and N434S replacements (EU numbering). In other embodiments, FLT3L-Fc fusion protein as described herein includes T250Q and M428L (EU numbering) mutations. In other embodiments, FLT3L-Fc fusion protein as described herein includes H433K and N434F (EU numbering) mutations.

[0101] Fc mutations that reduce or eliminate effector activity

[0102] In some embodiments, the FLT3L-Fc fusion proteins described herein can have an Fc domain with amino acid substitutions that reduce or eliminate Fc effector functions, including, for example, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0103] In some embodiments, the Fc region is changed by replacing at least one amino acid residue with different amino acid residues to reduce or eliminate the effector functions of the antibody.For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 (EU numbering) can be replaced with different amino acid residues so that the affinity of the fusion protein to the effector ligand is reduced. The effector ligand for which affinity is changed can be, for example, Fc receptors (for example, residue positions 234, 235, 236, 237, 297 (EU numbering)) or the C1 components of complement (for example, residue positions 297, 318, 320, 322 (EU numbering)) (U.S. Patent number 5,624,821 and 5,648,260 of Winter et al.).

[0104] Fc modifications that reduce or eliminate effector function include substitutions, insertions, and deletions at one or more positions (e.g., including 234, 235, 236, 237, 267, 269, 325, and 328, such as 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R (EU numbering). In addition, the Fc variant may include 236R / 328R. Other modifications for reducing FcγR and complement interactions include substitutions at positions 297A, 234A, 235A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V (EU numbering). These modifications and others are reviewed in Strohl (2009) Current Opinion in Biotechnology 20: 685-691. Effector function (both ADCC and complement activation) can be reduced while maintaining nascent FcR binding (maintaining half-life) by mutating IgG residues at one or more positions in positions 233-236 and 327-331 (such as E233P, L234V, L235A, optionally G236A, A327G, A330S, and P331S in IgG1; E233P, F234V, L235A, optionally G236A in IgG4; and A330S and P331S in IgG2 (EU numbering). See Armour et al. (1999) Eur. J. Immunol. 29:2613; WO 99 / 58572. Other mutations that reduce effector function include L234A and L235A in IgG1 (Alegre et al. (1994) Transplantation 57:1537); V234A and G237A in IgG2 (Cole et al. (1997) J. Immunol. 159:3613; see also U.S. Patent No. 5,834,597); and S228P and L235E of IgG4 (Reddy et al. (2000) J. Immunol. 164:1925). Another combination of mutations used to reduce effector function in human IgG1 includes L234F, L235E, and P331S (Oganesyan et al. (2008) Acta Crystallogr. D. Biol. Crystallogr. 64: 700). See generally Labrijn et al. (2008) Curr. Op. Immunol. 20: 479. Other mutations found to reduce effector function in the context of Fc (IgG1) fusion proteins (abatacept) include C226S, C229S, and P238S (EU numbering).Davis et al. (2007) J. Immunol. 34:2204.

[0105] ADCC activity can be reduced by modifying the Fc region. In certain embodiments, sites that affect binding to Fc receptors, such as sites other than the salvage receptor binding site, can be removed. In other embodiments, the Fc region can be modified to remove ADCC sites. Exemplary ADCC sites have been described for ADCC sites in IgG1 (Sarmay et al., (1992) Molec. Immunol. 29(5): 633-9). In one embodiment, the G236R and L328R variants of human IgG1 effectively eliminate FcγR binding (Horton et al. (2011) J. Immunol. 186: 4223 and Chu et al. (2008) Mol. Immunol. 45: 3926). In other embodiments, the Fc region that reduces binding to FcγRs comprises amino acid substitutions L234A, L235E, and G237A. Gross et al. (2001) Immunity 15: 289. Modifications in the IgG Fc region identified in WO 88 / 007089 that reduce binding to FcγRI to reduce ADCC (e.g., 234A; 235E; 236A; G237A) can be used in the fusion proteins of the present invention. See also Duncan & Winter (1988) Nature 332:563; Chappel et al. (1991) Proc. Nat'l Acad. Sci. (USA) 88:9036; and Sondermann et al. (2000) Nature 406:267 (discussing the effects of these mutations on FcγRIII binding).

[0106] CDC activity can also be reduced by modifying the Fc region. Mutations at positions D270, K322, P329, and P331 of IgG1 (particularly the alanine mutations D270A, K322A, P329A, and P331A) significantly reduced the ability of the corresponding antibodies to bind to C1q and activate complement (Idusogie et al. (2000) J. Immunol. 164:4178; WO 99 / 51642). Modifications at position 331 of IgG1 (e.g., P331S) have been shown to reduce complement binding (Tao et al. (1993) J. Exp. Med. 178:661; Xu Y et al. J. Biol. Chem. 1994. 269:3469-74; and Canfield & Morrison (1991) J. Exp. Med. 173:1483). In another example, one or more amino acid residues within amino acid positions 231 to 239 are altered to reduce the ability of the antibody to fix complement (WO 94 / 29351). Modifications in the IgG Fc region identified in WO 88 / 007089 that reduce or eliminate binding to complement component C1q and thus reduce or eliminate CDC (e.g., E318A or V / K320A and K322A / Q) can be used in the fusion proteins of the present invention.

[0107] In some embodiments, the Fc with reduced complement fixation has the amino acid substitutions A330S and P331 S. Gross et al. (2001) Immunity 15:289.

[0108] Other Fc variants with reduced ADCC and / or CDC are disclosed in Glaesner et al. (2010) Diabetes Metab. Res. Rev. 26:287 (F234A and L235A in IgG4 that reduce ADCC and ADCP); Hutchins et al. (1995) Proc. Nat'l Acad. Sci. (USA) 92:11980 (F234A, G237A, and E318A in IgG4); An et al. (2009) MAbs 1:572 and U.S. Patent Application Publication 2007 / 0148167 (H268Q, V309L, A330S, and P331S in IgG2); McEarchern et al. (2007) Blood 109: 1185 (C226S, C229S, E233P, L234V, L235A in IgG1); Vafa et al. (2014) Methods 65: 114 (V234A, G237A, P238S, H268A, V309L, A330S, P331S in IgG2) (EU numbering).

[0109] In certain embodiments, the fusion protein has an Fc that is substantially devoid of effector function, e.g., reduced or abolished binding of the Fc to FcγRs and reduced or abolished complement fixation, e.g., effector-free. An exemplary effector-free IgG1 Fc comprises the following five mutations: L234A, L235E, G237A, A330S, and P331S (EU numbering) (Gross et al. (2001) Immunity 15:289). These five substitutions can be combined with N297A to eliminate glycosylation.

[0110] IgG1 isotype Fc

[0111] In one embodiment, the Fc region comprises or is derived from human IgGl.In some embodiments, the antibody has a chimeric heavy chain constant region (eg, having the CHl, hinge, CH2 regions of IgG4 and the CH3 region of IgGl).

[0112] IgG1 antibodies exist in various allotypes and heteroallelotypes. In certain embodiments, the FLT3L-Fc fusion proteins described herein include IgG1 heavy chains having the following allotypes: G1m1; nG1m2; G1m3; G1m17,1; G1m17,1,2; G1m3,1; or G1m17. Each of these allotypes or heteroallelotypes is characterized by having the following amino acid residues (EU numbering) at the specified positions within the IgG1 heavy chain constant region (Fc):

[0113] G1m1: D356, L358;

[0114] nG1m1:E356,M358;

[0115] G1m3: R214, E356, M358, A431;

[0116] G1m17,1: K214, D356, L358, A431;

[0117] G1m17,1,2: K214, D356, L358, G431;

[0118] G1m3,1: R214, D356, L358, A431; and

[0119] G1m17: K214, E356, M358, A431.

[0120] In a specific embodiment, the FLT3L extracellular domain or a truncated fragment thereof is linked directly or via an intervening amino acid sequence (eg, a GS linker) to the wild-type IgG1m3 sequence or a fragment thereof provided below.

[0121] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:102)。

[0122] In certain embodiments, the FLT3L-Fc fusion protein has an IgG1 isotype. In some embodiments, the FLT3L-Fc fusion protein contains a human IgG1 constant region. In some embodiments, the human IgG1 Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from N297A, N297Q (Bolt S et al. (1993) Eur J Immunol 23:403-411), D265A, L234A, L235A (McEarchem et al. (2007) Blood, 109: 1185-1192), C226S, C229S (McEarchem et al. (2007) Blood. 109: 1185-1192), P238S (Davis et al. (2007) J Rheumatol, 34: 2204-2210), E233P, L234V (McEarchern et al. (2007) Blood, 109: 1185-1192), P238A, A327Q, A327G, P329A (Shields R et al. (2007) Blood, 109: 1185-1192), L. et al., (2001) J Biol Chem. 276(9):6591-604), K322A, L234F, L235E (Hezareh et al., (2001) J Virol 75, 12161-12168; Oganesyan et al., (2008) Acta Crystallographica 64, 700-704), P331S (Oganesyan et al., (2008) Acta Crystallographica 64, 700-704), T394D (Wilkinson et al. (2013) MAbs 5(3):406-417), A330L, M252Y, S254T and / or T256E, wherein amino acid positions are according to the EU numbering convention. In certain embodiments, the Fc region further comprises an amino acid deletion at the position corresponding to glycine 236 according to the EU numbering convention. As used herein, when the position of any given polymer component (e.g., amino acid, nucleotide, also collectively referred to as "residue") is designated by reference to the same or equivalent position in a selected amino acid or nucleic acid polymer (e.g., based on an optimal alignment or consensus sequence), rather than by the actual numerical position of the component in the given polymer, the numbering of a given amino acid polymer or nucleic acid polymer "corresponds to," "corresponds to," or "is relative to" the numbering of a selected or reference amino acid polymer or nucleic acid polymer.

[0123] In some embodiments, the FLT3L-Fc fusion protein is of the IgG1 isotype with a heavy chain constant region containing a C220S amino acid substitution according to the EU numbering convention.

[0124] In some embodiments, the Fc region comprises a human IgG1 isotype and comprises one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of: N297A, N297G, N297Q, N297G, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, P329G, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, M428L, N434S, T366W, T366S, L368A, Y407V, and any combination thereof, wherein the numbering of the residues is according to EU numbering. In some embodiments, the Fc region comprises a human IgG1 isotype and comprises one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of: L234A, L234V, L234F, L235A, L235E, A330L, P331S, and any combination thereof, wherein the numbering of the residues is according to EU numbering.

[0125] IgG4 isotype Fc

[0126] For uses where effector function is to be avoided entirely, such as when antigen binding alone is sufficient to produce the desired therapeutic benefit and effector function only results in (or increases the risk of) undesirable side effects, IgG4 antibodies can be used, or antibodies or fragments lacking the Fc region or a substantial portion thereof can be designed, or the Fc can be mutated to completely eliminate glycosylation (e.g., N297A). Alternatively, hybrid constructs of human IgG2 (CH1 domain and hinge region) and human IgG4 (CH2 and CH3 domains) have been generated that lack effector function, the ability to bind FcγRs (e.g., IgG2), and the inability to activate complement (e.g., IgG4). (See Rother et al. (2007) Nat. Biotechnol. 25: 1256; Mueller et al. (1997) Mol. Immunol. 34: 441; and Labrijn et al. (2008) Curr. Op. Immunol. 20: 479, which discuss Fc modifications that generally reduce effector function).

[0127] In one embodiment, the Fc region comprises or is derived from human IgG4. In certain embodiments, the FLT3L-Fc fusion protein has an IgG4 isotype. In some embodiments, the FLT3L-Fc fusion protein contains a human IgG4 constant region. In some embodiments, the human IgG4 constant region includes an Fc region. In some embodiments, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from E233P, F234V, F234A, L235A, G237A, E318A, S228P, L235E, T394D, M252Y, S254T, T256E, N297A, N297G, N297Q, T366W, T366S, L368A, Y407V, M428L, N434S, and any combination thereof, where the amino acid positions are according to the EU numbering convention. See, e.g., Hutchins et al. (1995) Proc Natl Acad Sci USA, 92: 11980-11984; Reddy et al. (2000) J Immunol, 164: 1925-1933; Angal et al. (1993) Mol Immunol. 30(1): 105-8; U.S. Patent No. 8,614,299 B2; Vafa O. et al. (2014) Methods 65: 114-126; and Jacobsen et al., J. Biol. Chem. (2017) 292(5): 1865-1875. In some embodiments, the Fc region comprises a human IgG4 isotype and comprises one or more amino acid substitutions in the Fc region at a residue position selected from the group consisting of: F234V, F234A, L235A, L235E, S228P, and any combination thereof, wherein the numbering of the residues is according to EU numbering.

[0128] In some embodiments, the IgG4 variants of the present disclosure may be combined with an S228P mutation according to the EU numbering convention (Angal et al., (1993) Mol Immunol, 30: 105-108) and / or with one or more mutations (Peters et al., (2012) J Biol Chem. 13; 287(29): 24525-33) to enhance antibody stability.

[0129] IgG2 isotype Fc

[0130] In certain embodiments, the FLT3L-Fc fusion protein has an IgG2 isotype. In some embodiments, the FLT3L-Fc fusion protein contains a human IgG2 constant region. In some embodiments, the human IgG2 constant region includes an Fc region. In some embodiments, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (for example, relative to a wild-type Fc region of the same isotype). In some embodiments, one or more amino acid substitutions are selected from P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297G, N297Q, V309L, A330S, P331S, C232S, C233S, M252Y, S254T and / or T256E, wherein the amino acid position is according to EU numbering convention (Vafa et al., (2014) Methods 65: 114-126).

[0131] In certain embodiments, the FLT3L-Fc fusion protein described herein comprises L234F, L235E, D265A mutations, collectively referred to as "FEA". FEA mutations can reduce or eliminate effector function. In certain embodiments, the FLT3L-Fc fusion protein described herein comprises L234F, L235E, D265A, and F405L mutations, collectively referred to as "FEAL". In certain embodiments, the FLT3L-Fc fusion protein described herein comprises L234F, L235E, D265A, and a mutation selected from the group consisting of F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y. In certain embodiments, the FLT3L-Fc fusion proteins described herein comprise L234F, L235E, D265A, and K409R mutations, collectively referred to as "FEAR". In certain embodiments, FEAL and FEAR are included in the fusion proteins described herein. In certain embodiments, the FLT3L-Fc fusion proteins described herein further comprise M428L and N434S mutations, collectively referred to as LS. In certain embodiments, the FLT3L-Fc fusion proteins described herein comprise L234F, L235E, D265A, F405L, M428L, and N434S mutations, collectively referred to as "FEALLS". In certain embodiments, the FLT3L-Fc fusion proteins described herein comprise L234F, L235E, D265A, M428L, and N434S mutations and one additional mutation selected from the group consisting of F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y. In certain embodiments, the FLT3L-Fc fusion proteins described herein comprise L234F, L235E, D265A, K409R, M428L, and N434S mutations, collectively referred to as "FEARLS." In certain embodiments, FEALLS and FEARLS are included in the fusion proteins described herein. By reducing or eliminating effector functions on the Fc domain of the FLT3L-Fc fusion protein, cells bound by this molecule are not killed by innate effector cells (eg, NK cells, macrophages).

[0132] In certain embodiments, the one or more modifications are selected from the following Fc amino acid substitutions (EU numbering), or a combination thereof: L234F; L235E; G236A; S239D; F243L; D265E; D265A; S267E; H268F; R292P; N297Q; N297G, N297A; S298A; S324T; I332E; S239D; A330L; L234F; L235E; P33 1S; F243L; Y300L; V305I; P396L; S298A; E333A; K334A; E345R; L235V; F243L; R292P; Y300L; P396L; M428L; E430G; N434S; G236A, S267E, H268F, S324T, and I332E; G236A, S239D, and I332E; S239D, A330L, and I332E ; L234F, L235E and P331S; F243L, R292P, Y300L, V305I and P396L; G236A, H268F, S324T and I332E; S239D, H268F, S324T and I332E; S298A, E333A and K334A; L235V, F243L, R292P, Y300L and P396L; S239D, I332E; S239D, S In certain embodiments, one or more modifications are selected from the group consisting of D265A, L234F, L235E, N297A, N297G, N297Q, and P331S. In certain embodiments, one or more modifications are selected from N297A and D265A. In certain embodiments, one or more modifications are selected from L234F and L235E. In certain embodiments, one or more modifications are selected from L234F, L234E, and D265A. In certain embodiments, one or more modifications are selected from L234F, L234E, and N297Q. In certain embodiments, one or more modifications are selected from L234F, L235E, and P331S. In certain embodiments, one or more modifications are selected from D265A and N297Q. In certain embodiments, one or more modifications are selected from L234F, L235E, D265A, N297A, N297G, N297Q, and P331S.

[0133] Mutations that reduce Fc-receptor binding and are useful in fusion proteins described herein include, for example, N297A; N297G; N297Q; D265A; L234F / L235E; L234F / L235E / N297Q; L234F / L235E / P331S; D265A / N297Q; and L234F / L235E / D265A / N297Q / P331S (all EU numbering). In certain embodiments, the FLT3L-Fc fusion protein described herein comprises L234F and L235E mutations. In certain embodiments, the FLT3L-Fc fusion protein described herein comprises L234F, L235E, and D265A mutations. In certain embodiments, the FLT3L-Fc fusion protein described herein comprises L234F, L235E, and N297Q mutations. In certain embodiments, FLT3L-Fc fusion protein as described herein includes N297A or N297Q mutation. In certain embodiments, FLT3L-Fc fusion protein as described herein includes N297A, N297G or N297Q mutation and L234F, L235E and D265A mutation. In certain embodiments, one, two, three, four or more amino acid substitutions are introduced into the Fc region to change the effector function of the antigen binding molecules. For example, these substitutions are located at the position of the group selected from the following items: amino acid residues 234, 235, 236, 237, 265, 297, 318, 320 and 322 (according to EU numbering). These positions can be replaced by different amino acid residues so that the affinity of the antigen binding molecules to effector ligands (for example, Fc receptors or complement C1 components) changes (for example, reduces), but retains the antigen binding ability of the parent antibody. In certain embodiments, FLT3L-Fc fusion protein as described herein comprises E233P, L234V, L235A and / or G236A mutations (EU numbering). In some embodiments, FLT3L-Fc fusion protein as described herein comprises A327G, A330S and / or P331S mutations (EU numbering). In some embodiments, FLT3L-Fc fusion protein as described herein comprises K322A mutation (EU numbering). In some embodiments, FLT3L-Fc fusion protein as described herein comprises E318A, K320A and K322A mutations (EU numbering). In certain embodiments, FLT3L-Fc fusion protein as described herein comprises L235E mutation (EU numbering).

[0134] In some embodiments, the Fc portion of the fusion protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence of:

[0135] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:103);

[0136] GGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:104);

[0137] ESKYGPPCP P CPAPEF E GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:105);

[0138] ESKYGPPCP PCPAPE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:106); or ESKYGPPCP P CPAPEF E GGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:107).

[0139] Exemplary polypeptide sequences of FLT3L-Fc fusion proteins described herein are provided in Table A. In some embodiments, the FLT3-Fc fusion protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-18 and 21-27. In some embodiments, the FLT3-Fc fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-18 and 21-27. In some embodiments, the FLT3-Fc fusion protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-20. In some embodiments, the FLT3-Fc fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-20.

[0140] In various embodiments, the FLT3L-Fc fusion protein can be glycosylated or non-glycosylated. In certain embodiments where the FLT3L-Fc fusion protein is glycosylated, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or more of the N-linked and / or O-linked glycosylation sites in the fusion protein are sialylated. In certain embodiments where the FLT3L-Fc fusion protein is sialylated, the sialylated N-linked and / or O-linked glycosylation sites in the fusion protein comprise 2 to 7 sialic acid residues, e.g., 3 to 6 sialic acid residues, e.g., 4 to 5 sialic acid residues.

[0141] In some embodiments, the FLT3L-Fc fusion protein has a serum half-life of at least about 7 days in a mammal (e.g., in a human, monkey, mouse, cat, or dog). In some embodiments, the FLT3L-Fc fusion protein has a serum half-life of at least about 7 days in a mammal (e.g., in a human, monkey, mouse, cat, or dog). In general, a shorter serum half-life is observed at a relatively low dose. A longer serum half-life is observed at a relatively high dose.

[0142] Functionally, FLT3L-Fc fusion proteins as described herein induce, promote and / or increase the growth, proliferation and / or amplification of cells or cell groups expressing or overexpressing FLT3 on their cell surfaces.Expression or overexpression of exemplary cells or cell groups of FLT3 include dendritic cells (e.g., cDC1 cells and / or cDC2 cells), monocyte-derived dendritic cells (moDC) and / or their progenitor cells. In some embodiments, cells or cell groups expressing FLT3 include hematopoietic progenitor cells, such as common lymphocyte progenitor cells (CLP), early progenitor cells (EPLM) with lymphocyte and bone marrow potential, granulocyte-monocyte (GM) progenitor cells (GMP), monocyte-derived dendritic cells (moDC) progenitor cells and early multipotent progenitor cells (MPP) in the Lineage-kit+Sca1 (LSK) compartment.

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150] Heterodimers and fusion proteins comprising a FLT3L-Fc fusion protein and a second polypeptide

[0151] Also provided are fusion proteins comprising (i) a FLT3L-Fc fusion protein as described herein, e.g., having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-20; and (ii) a second polypeptide. In some embodiments, the second polypeptide comprises a targeting moiety or domain, a growth factor, a cytokine, a chemokine, or a member of the TNF superfamily (TNFSF). In some embodiments, the second polypeptide is the N-terminus of the FLT3L extracellular domain. In some embodiments, the second polypeptide is the C-terminus of the Fc region. In some embodiments, the second polypeptide is between the FLT3L extracellular domain and the Fc region. In various embodiments, the targeting moiety binds to a protein target in Table B.

[0152] Also provided are heterodimeric molecules comprising (i) a FLT3L-Fc fusion protein as described herein, e.g., having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-20; and (ii) a second polypeptide fused to a second Fc region. In certain embodiments, the first and second Fc regions of the heterodimeric molecule are different, e.g., having complementary "knob (W) and hole (S)" amino acid substitutions at position 366 (EU numbering). In some embodiments, the second polypeptide comprises a targeting moiety or domain, a growth factor, a cytokine, a chemokine, or a TNF superfamily (TNFSF) member. In various embodiments, the targeting moiety binds to a protein target in Table B.

[0153] In some embodiments, the targeting moiety or domain comprises an antibody fragment (e.g., scFv, sc(Fv)2, Fab, F(ab)2, Fab', F(ab')2, Facb, and Fv). In some embodiments, the antibody fragment comprises a Fab or a single-chain variable fragment (scFv). In some embodiments, neither the first Fc region nor the second Fc region comprises a hinge region. In some embodiments, the heterodimer is stabilized by an interaction between the first Fc region and the second Fc region. Exemplary interactions that can stabilize heterodimers by the Fc region include, but are not limited to, disulfide bonds and complementary amino acid substitutions (e.g., knob-into-hole mutations) in the first Fc region and the second Fc region.

[0154] In some embodiments, the targeting moiety or domain comprises a non-immunoglobulin or antibody mimetic protein. Examples of non-immunoglobulin or antibody mimetic protein targeting moieties or domains include, but are not limited to, adnectin, affibody molecule, affilin, affimer, affitin, alphabody, anticalin, peptide aptamer, armadillo repeat protein (ARM), atrimer, avimer, designed ankyrin repeat protein, Fynomers, knottins, Kunitz structures, monobodies, and nanoCLAMPs. Non-immunoglobulin or antibody-mimicking protein targeting moieties or domains used in the FLT3L-Fc fusion protein heterodimers described herein are described in, for example, Zhang et al., Methods Mol Biol. 2017; 1575: 3-13; Ta et al., Future Med Chem. 2017 Aug; 9(12): 1301-1304; Yu et al., Annu Rev Anal Chem (Palo Alto Calif). 2017 Jun 12; 10(1): 293-320; Baloch et al., Crit Rev Biotechnol. 2016; 36(2): 268-75; and Bruce et al., Chembiochem. 2016 Oct 17; 17(20): 1892-1899.

[0155] In some embodiments, the targeting moiety or domain has T cell receptor (TCR)-like binding properties and binds to an epitope of the target or tumor associated antigen (TAA) presented in a major histocompatibility complex (MHC) molecule.

[0156] In some embodiments, the targeting moiety or domain comprises a binding partner domain, such as a soluble or extracellular domain of a binding partner or ligand of a protein target or antigen. For example, in some embodiments, the targeting moiety or domain comprises a binding partner or ligand of any of the protein or antigen targets listed in Table B. In one embodiment, the targeting moiety or domain comprises the extracellular domain of the TGFB1 receptor (e.g., a "TGFβ trap").

[0157] In the homodimer or heterodimer form of the FLT3L-Fc fusion protein, the dimer molecule comprises a first Fc domain and a second Fc domain. In certain embodiments, amino acid substitutions may be in one or both of the first Fc domain and the second Fc domain. In certain embodiments, one or both of the first Fc domain and the second Fc domain have one or more (1, 2, 3, 4 or 5) of the following mutations (EU numbering). In some embodiments, the heterodimerization of the Fc region of two different immunoadhesins (Fc fusion proteins) can be promoted by so-called "knob into hole" mutations (Atwell et al., 1997. JMB 270: 26-35). The "hole" mutation (T366S, L368A and Y407V) is incorporated into one Fc-containing chain, and the T366W "knob" mutation is incorporated into another chain. The knob and hole amino acid substitutions can be incorporated into human IgG1 or human IgG4 Fc domains. In addition, the C220S mutation can be incorporated into the IgG1 hinge region of the scFv-containing arm to eliminate free cysteine, which originally forms a disulfide bond with the corresponding cysteine ​​in the light chain in wild-type IgG1. Co-transfection of such constructs leads to preferential formation of homodimeric Fc, with low levels of homodimeric contaminants. In addition, incorporating the S354C mutation into the Fc containing the "knob" mutation and the Y349C mutation into the Fc containing the "hole" mutation can optionally be used to create a covalent bond between the two halves of the heterodimeric Fc when additional thermodynamic stability is required (Merchant et al. 1998. Nat. Biotechnol. 16: 677-81). In certain embodiments, R409D and K370E mutations are introduced into the "knob chain" and D399K and E357K mutations are introduced into the "hole chain." In other embodiments, Y349C and T366W mutations are introduced into one of the chains, and E356C, T366S, L368A, and Y407V mutations are introduced into the corresponding chain. In some embodiments, Y349C and T366W mutations are introduced into one chain, and S354C, T366S, L368A, and Y407V mutations are introduced into the corresponding chain. In some embodiments, Y349C and T366W mutations are introduced into one chain, and S354C, T366S, L368A, and Y407V mutations are introduced into the corresponding chain. In other embodiments, Y349C and T366W mutations are introduced into one chain, and S354C, T366S, L368A, and Y407V mutations are introduced into the corresponding chain (all EU numbering).

[0158] To facilitate purification of heterodimeric molecules from contaminated homodimeric products, H435R or H435R+Y436F mutations that reduce or eliminate protein A binding can be introduced into one of the Fc-containing chains instead of both (Jendeberg, L. et al. 1997 J. Immunol. Methods 201: 25-34). This reduces or eliminates protein A binding of homodimeric contaminants containing these mutations and greatly simplifies the process of purifying the desired heterodimer from the remaining homodimeric contaminants via an additional chromatography step (e.g., ion exchange). In embodiments where the H435R (or H435R+Y436F) mutation is incorporated into the first or second Fc region of the heavy chain, if the VH region in the same heavy chain is from a VH3 family variable region, the VH region may also include amino acid substitutions as described herein to reduce or eliminate protein A binding of the entire heavy chain.

[0159] By using a trifunctional hybrid antibody platform Another exemplary method for preparing bispecific antibodies is to use a chimeric construct composed of two halves of a full-length antibody of different isotypes, namely mouse IgG2a and rat IgG2b. This technology relies on species-preferred heavy chain / light chain pairing associations. See Lindhofer et al., J. Immunol., 155:219-225 (1995).

[0160] Another method for preparing bispecific antibodies is CrossMab technology. CrossMab is a chimeric antibody consisting of half of two full-length antibodies. For correct chain pairing, it combines two technologies: (i) knob-into-hole, which facilitates the correct pairing between the two heavy chains; and (ii) exchange between the heavy chain and the light chain of one of the two Fabs to introduce asymmetry that avoids light chain mispairing. See Ridgway et al., Protein Eng., 9: 617-621 (1996); Schaefer et al., PNAS, 108: 11187-11192 (2011). CrossMab can combine two or more antigen binding domains to target two or more targets, or be used to introduce bivalence to one target, such as a 2: 1 format.

[0161] In some embodiments, the targeting moiety or domain targets or binds to effector cells, such as engaging or activating T cells or NK cells. In certain embodiments, the targeting moiety or domain binds to CD3. In some embodiments, the targeting moiety binds to CD16. Exemplary proteins and antigens (including tumor-associated antigens, immune checkpoint proteins, and dendritic cell surface proteins) that can be targeted or combined by the targeting moiety or domain include but are not limited to those listed in Table B. The target names, symbols (official and alternative) and gene IDs determined in Table B are from ncbi.nlm.nih.gov / gene.

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] In some embodiments, the target antigen comprises a tumor-associated carbohydrate antigen (TACA). Exemplary carbohydrate antigen targets include, for example, mucin TACA, including truncated glycans Thomsen-nouveau (Tn) (GalNAcα1-Ser / Thr) and STn (Neu5Acα2,6GalNAcα1-Ser / Thr), RM2 antigen hexasaccharide, carbocyclic sugar, C-glycoside, ganglioside GM2, GD2 and GD3; globo-H, sialyl Lewis (a), sialyl Lewis (x) and sialyl Lewis (y) antigens. TACAs are described, for example, in Sadraei et al., Adv Carbohydr Chem Biochem. (2017) 74: 137-237; Sletmoen et al., Glycobiology. (2018) 28(7): 437-442; Chuang et al., J Am Chem Soc. (2013) 135(30): 11140-50; Ragupathi, Cancer Immunol Immunother. (1996) 43(3): 152-7; Ugorski et al., Acta Biochim Pol. 2002; 49(2): 303-11; Takada et al., Cancer Res. 1993 Jan 15; 53(2): 354-61.

[0175] In some embodiments, the target antigen comprises a neoantigen presented within a major histocompatibility complex (MHC) class I or class II molecule. See, e.g., Ott et al., Nature. (2017) 547(7662):217-221; Capietto et al., Curr Opin Immunol. (2017) 46:58-65; Sun et al., Cancer Lett. (2017) 392:17-25; Khodadoust et al., Nature. (2017) 543(7647):723-727; Kreiter et al., Nature. (2015) 520(7549):692-6; Marty et al., Cell. (2017) 171(6):1272-1283; and Kochin et al., Oncoimmunology. (2017) 6(4):e1293214 (describing SUV39H2 peptide presented in HLA-A24).

[0176] Conjugated FLT3L-Fc fusion protein

[0177] Any of the FLT3L-Fc fusion proteins disclosed herein or their homodimers or heterodimers may be conjugated. FLT3L-Fc fusion proteins conjugated to various molecules (e.g., labels) include, but are not limited to, macromolecular substances such as polymers (e.g., polyethylene glycol (PEG), polyethyleneimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer)), hyaluronic acid, radioactive materials (e.g., 90 Y. 131 I. 125 I. 35 S. 3 H. 121 In, 99 Tc), fluorescent substances (e.g., fluorescein and rhodamine), fluorescent proteins, luminescent substances (e.g., luminol), quantum dots, haptens, enzymes (e.g., glucose oxidase), metal chelates, biotin, avidin, and drugs.

[0178] The conjugated FLT3L-Fc fusion protein can be prepared according to known methods, for example, by chemically modifying the FLT3L-Fc fusion protein described herein. In certain embodiments, a labeling moiety or therapeutic moiety is conjugated to the Fc portion of the fusion protein. Methods for modifying the Fc region of an antibody are well known in the art (e.g., US 5,057,313 and US 5,156,840).

[0179] In some embodiments, the FLT3L-Fc fusion protein or its homodimer or heterodimer is conjugated to a drug or therapeutic agent. In various embodiments, the drug is an organic compound or an inhibitory nucleic acid, such as a short inhibitory RNA (siRNA), a microRNA (miRNA). In some embodiments, the drug or therapeutic agent is an anti-tumor agent or chemotherapeutic agent as known in the art and described herein. In a specific embodiment, the drug or therapeutic agent is selected from the group consisting of monomethyl rioxetine E (MMAE), monomethyl rioxetine F (MMAF), calicheamicin, ansamitocin, maytansine or its analogs (e.g., mertansine / emtansine (DM1), ravtansine / soravtansine (DM4)), anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin), pyrrolobenzodiazepine (PBD) DNA cross-linker SC-DR002 (D6.5), duocarmycins, microtubule inhibitors (MTIs) (e.g., taxanes, vinca alkaloids, epothilones), pyrrolobenzodiazepines (PBD) or dimers thereof, and duocarmycins (A, B1, B2, C1, C2, D, SA, CC-1065).

[0180] 3. Polynucleotide encoding FLT3L-Fc fusion protein

[0181] Provided herein are polynucleotides encoding the FLT3L-Fc fusion proteins described herein, vectors comprising such polynucleotides, and host cells (e.g., human cells, mammalian cells, yeast cells, plant cells, insect cells, bacterial cells, such as E. coli) comprising such polynucleotides or expression vectors. Provided herein are polynucleotides comprising a nucleotide sequence encoding any of the FLT3L-Fc fusion proteins provided herein, as well as expression cassettes and vectors comprising such polynucleotide sequences, such as expression vectors for efficient expression in host cells (e.g., mammalian cells). In various embodiments, the polynucleotide is DNA, cDNA, or mRNA.

[0182] The terms "polynucleotide" and "nucleic acid molecule" refer interchangeably to polymeric forms of nucleotides, and include sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers thereof. As used herein, the term "nucleic acid molecule" is interchangeable with the term "polynucleotide." In some embodiments, nucleotides refer to ribonucleotides, deoxyribonucleotides, or modified forms of any type of nucleotide, and combinations thereof. The term also includes, but is not limited to, single-stranded and double-stranded forms of DNA. In addition, a polynucleotide, such as a cDNA or mRNA, may include one or both of naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds. As will be readily understood by those skilled in the art, nucleic acid molecules may be chemically or biochemically modified, or may contain non-natural or derived nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more of the naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged bonds (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), side chain moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralens, etc.), chelating agents, alkylates, and modified bonds (e.g., α-anomeric nucleic acids, etc.). The above terms are also intended to include any topological conformation, including single-stranded conformation, double-stranded conformation, partially double-stranded conformation, triple-stranded conformation, hairpin conformation, circular conformation, and padlock conformation. Unless otherwise indicated, reference to a nucleic acid sequence encompasses its complementary sequence. Therefore, reference to a nucleic acid molecule with a specific sequence is understood to encompass its complementary chain and its complementary sequence. The term also includes codon-biased polynucleotides for improving expression in desired host cells.

[0183] As used herein, "substitution" means that one or more amino acids or nucleotides are replaced by different amino acids or nucleotides, respectively.

[0184] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. An "isolated nucleic acid encoding a FLT3L-Fc fusion protein" refers to one or more nucleic acid molecules encoding a first antigen-binding domain and optionally a second antigen-binding domain, an antibody heavy chain, and a light chain (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

[0185] An "isolated" polypeptide (such as the isolated FLT3L-Fc fusion protein provided herein) is a polypeptide that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the isolated polypeptide will be purified (1) to greater than 95% by weight (e.g., greater than 99% by weight) of the polypeptide as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. An isolated polypeptide includes the polypeptide in situ within recombinant cells, as at least one component of the antibody's natural environment will not be present. Generally, however, the isolated polypeptide will be prepared by at least one purification step.

[0186] As used herein, the term polynucleotide "variant" is a polynucleotide that typically differs from the polynucleotides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be generated synthetically, for example, by modifying one or more of the polynucleotide sequences described herein and evaluating one or more biological activities of the encoded polypeptides as described herein and / or using any of a variety of techniques well known in the art.

[0187] In some embodiments, the nucleic acid molecule has a codon preference to enhance expression in a desired host cell (e.g., a human cell, a mammalian cell, a yeast cell, a plant cell, an insect cell, or a bacterial cell, such as an E. coli cell). Thus, a polynucleotide encoding a FLT3L-Fc fusion protein is provided, wherein the polynucleotide has a codon preference, comprises a replacement heterologous signal sequence, and / or eliminates an mRNA instability element. Methods for generating codon-biased nucleic acids can be performed by adapting the methods described in the following documents: for example, U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498. Preferred codon usage for expressing FLT3L-Fc fusion proteins in desired host cells is provided, for example, at kazusa.or.jp / codon / and genscript.com / tools / codon-frequency-table.

[0188] In some embodiments, the polynucleotide encoding a FLT3L-Fc fusion protein as described herein has a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-70 as provided in Table C.

[0189] Optionally, in certain embodiments, the 3' end of the polynucleotide encoding the FLT3L-Fc fusion protein comprises multiple tandem stop codons, such as two or more tandem TAG ("amber"), TAA ("ochre"), or TGA ("opal" or "umber") stop codons. The multiple tandem stop codons can be the same or different.

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[0219] 4. Vectors and Host Cells

[0220] Also provided are vectors comprising one or more polynucleotides encoding one or more of the FLT3L-Fc fusion proteins described herein. The vector can be of any type, such as a recombinant vector, such as an expression vector. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs), as well as vectors derived from bacteriophages or plants or animals (including humans) viruses. The vector may include a replication origin recognized by the proposed host cell, and, with respect to expression vectors, includes a promoter and other regulatory regions recognized by the host cell. In another embodiment, the vector comprises a polynucleotide encoding an antibody of the present disclosure, which is operably linked to a promoter and optionally other regulatory elements. Certain vectors are capable of autonomous replication in the host into which they are introduced (for example, vectors with a bacterial replication origin can replicate in bacteria). Other vectors can be integrated into the host's genome when introduced into the host, thereby replicating together with the host genome. Vectors include, but are not limited to, those suitable for recombinant production of the antibodies disclosed herein.

[0221] The choice of vector depends on the subsequent recombination procedure and the host used. The introduction of the vector into the host cell is particularly affected by calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofection or electroporation. The vector can replicate autonomously or can replicate together with the chromosome into which it is integrated. In certain embodiments, the vector contains one or more selection markers. The choice of marker can depend on the host cell selected. These include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, zeocin, thymidine kinase gene from herpes simplex virus (HSV-TK) and dihydrofolate reductase gene (dhfr) from mouse. The present disclosure also encompasses vectors comprising one or more nucleic acid molecules encoding the FLT3L-Fc fusion protein described herein, which are operably linked to one or more nucleic acid molecules encoding proteins or peptides that can be used to isolate the FLT3L-Fc fusion protein. These proteins or peptides include, but are not limited to, glutathione-S-transferase, maltose binding protein, metal binding polyhistidine, green fluorescent protein, luciferase and β-galactosidase.

[0222] In other embodiments, the vector used is pcDNA TM 3.1+ (ThermoFisher, MA).

[0223] In some embodiments, the viral vector comprises an oncolytic viral vector. Depending on the circumstances, the oncolytic viral vector can be a DNA virus or an RNA virus. In some embodiments, the viral vector is from a family of viruses selected from the group consisting of: Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., lymphocytic choriomeningitis mammalian arenavirus, Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus)), Poxviridae (e.g., vaccinia virus), Herpesviridae (e.g., herpesvirus, such as HSV-1), Parvoviridae (e.g., Parvovirus H1), Reoviridae (e.g., reovirus), Picornaviridae (e.g., coxsackievirus, Seneca Valley virus, poliovirus), Paramyxoviridae (e.g., measles virus, Newcastle disease virus (NDV)), Rhabdoviridae (e.g., vesicular stomatitis virus (VSV)), Togaviridae (e.g., alphavirus, Sindbis virus), Enteroviridae (e.g., echovirus). The use of oncolytic viruses in cancer therapy is described, for example, in Fukuhara et al., Cancer Sci. (2016) 107(10): 1373-1379; Kaufman et al., Nat Rev Drug Discov. (2015) 14(9): 642-62; Hamid et al., Cancer Immunol Immunother. (2017) 66(10): 1249-1264; Taguchi et al., Int JUrol. (2017) 24(5): 342-351; and Buijs et al., Hum Vaccin Immunother. (2015) 11(7): 1573-84.

[0224] The present disclosure also provides host cells comprising nucleic acids or vectors as described herein. Any of a variety of host cells can be used. In one embodiment, the host cell is a prokaryotic cell, such as Escherichia coli. In another embodiment, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, a mammalian cell, such as a cell line based on Chinese hamster ovary (CHO) or CHO-derived cells (e.g., CHO-S, CHO DG44, ExpiCHO, TM 、 ZFN-modified GS- / - CHO cell lines, CHO-K1, CHO-K1a), COS cells, BHK cells, NSO cells or Bowes melanoma cells. Examples of human host cells include HeLa, 911, AT1080, A549 and HEK293 (e.g., HEK293E, HEK293T, Expi293 TM In addition, the FLT3L-Fc fusion protein can be expressed in yeast cells such as Pichia pastoris (see, eg, Powers et al., J Immunol Methods. 251: 123-35 (2001)), Hansenula or Saccharomyces cerevisiae.

[0225] In some embodiments, the host cell predominantly sialylates the N-linked glycosylation sites of the fusion protein. In some embodiments, a polynucleotide encoding a FLT3L-Fc fusion protein as described herein is expressed in a host cell, and the host cell sialylates at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or more of the N-linked glycosylation sites in the expressed FLT3L-Fc fusion protein.

[0226] As used herein, the term "vector" refers to a nucleic acid molecule that is capable of propagating another nucleic acid to which it is attached. The term includes vectors that are self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which the vector has been introduced. Some vectors are suitable for delivering the nucleic acid molecules or polynucleotides of the present application. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively attached. Such vectors are referred to herein as expression vectors.

[0227] The term "operably linked" refers to two or more nucleic acid sequences or polypeptide elements that are typically physically linked and in a functional relationship with each other. For example, a promoter is operably linked to a coding sequence if it is capable of initiating or regulating the transcription or expression of the coding sequence, in which case the coding sequence is understood to be "under the control of the promoter."

[0228] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0229] Host cells can be stably or transiently transfected with a polynucleotide encoding a FLT3L-Fc fusion protein as described herein, as appropriate.

[0230] 5. Methods for Producing FLT3L-Fc Fusion Protein

[0231] The FLT3L-Fc fusion proteins described herein can be produced by any method known in the art for synthesizing fusion proteins, such as by chemical synthesis or by recombinant expression techniques.

[0232] Methods for recombinantly expressing fusion proteins are known and can be applied to the recombinant production and separation / purification of FLT3L-Fc fusion proteins. Methods for recombinantly expressing proteins (including fusion proteins) are described, for example, in Green and Sambrook, "Molecular Cloning: A Laboratory Manual," 4th edition, 2012, Cold Spring Harbor Laboratory Press; Current Protocols in Protein Science, Wiley, 1995-2109 (currentprotocols.onlinelibrary.wiley.com / journal / 19343663 / ); and Current Protocols in Molecular Biology, Wiley, 1987-2019 (currentprotocols.onlinelibrary.wiley.com / journal / 19343647 / ). Additionally, other publications related to the production of recombinantly expressed fusion proteins include, for example, Argelia Lorence (ed.), “Recombinant Gene Expression” (Methods in Molecular Biology) 2012, Humana Press; James L Hartley (ed.), “Protein Expression in Mammalian Cells: Methods and Protocols” (Methods in Molecular Biology) 2012, Humana Press; Roslyn M. Bill (ed.), “Recombinant Protein Production in Yeast: Methods and Protocols” (Methods in Molecular Biology) 2012, Humana Press; and MacDonald, Kolotilin and Menassa (eds.), “Recombinant Proteins from Plants: Methods and Protocols” (Methods in Molecular Biology), 2nd ed., 2016, Humana Press.

[0233] In various embodiments, the FLT3L-Fc fusion proteins described herein can be produced in bacteria or eukaryotic cells. The FLT3L-Fc fusion proteins can also be produced in eukaryotic cells such as transformed cell lines (e.g., CHO, CHO-S, CHO DG44, ExpiCHO TM 、 In one embodiment, the FLT3L-Fc fusion proteins described herein are produced in CHO cell lines (e.g., CHO-S, CHO DG44, ExpiCHO, CHO-S, CHO-DG4 ... TM 、 CHO-K1, CHO-K1a cell lines or HEK293 (e.g., HEK293E, HEK293T, Expi293 TM ) cell line). In order to produce the FLT3L-Fc fusion protein of interest (including heterodimers containing FLT3-Fc fusion proteins), one or more polynucleotides encoding the FLT3L-Fc fusion proteins are constructed, introduced into expression vectors, and then expressed in one or more suitable host cells. In some embodiments, three polynucleotides encoding the FLT3L-Fc fusion, the Fab heavy chain comprising the second antigen binding domain, and the Fab light chain are co-expressed in a single host cell. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells, and recover the FLT3L-Fc fusion proteins.

[0234] In some embodiments, the host cell predominantly sialylates the N-linked glycosylation sites of the fusion protein. In some embodiments, a polynucleotide encoding a FLT3L-Fc fusion protein as described herein is expressed in a host cell, and the host cell sialylates at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or more of the N-linked glycosylation sites in the expressed FLT3L-Fc fusion protein.

[0235] If the FLT3L-Fc fusion protein is to be expressed in bacterial cells (e.g., E. coli), the expression vector should have features that allow the vector to be amplified in bacterial cells. In addition, when E. coli (such as JM109, DH5α, HB101, or XL1-Blue) is used as a host, the vector must have a promoter that allows efficient expression in E. coli, such as the lacZ promoter (Ward et al., 341:544-546 (1989)), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or the T7 promoter. Examples of such vectors include, for example, the M13 series vectors, the pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), the "QIAexpress system" (QIAGEN), pEGFP, and pET (when using this expression vector, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may contain a signal sequence for secretion of the FLT3L-Fc fusion protein. For production into the periplasm of E. coli, the pelB signal sequence (Lei et al., J. Bacteriol., 169:4379 (1987)) can be used as a signal sequence for secretion of the FLT3L-Fc fusion protein. For bacterial expression, the expression vector can be introduced into bacterial cells using the calcium chloride method or electroporation method.

[0236] If the FLT3L-Fc fusion protein will be expressed in mammalian cells (e.g., CHO-S, CHO DG44, ExpiCHO TM 、 CHO-K1, CHO-K1a, 293E, 293T, Expi293 TM, COS, NIH3T3 cells), the expression vector includes a promoter that promotes expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277: 108 (1979)), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res., 18: 5322 (1990)) or CMV promoter. In addition to the nucleic acid sequence encoding the immunoglobulin or its domain, the recombinant expression vector can also carry additional sequences, such as sequences (e.g., replication origins) and selective marker genes that regulate the replication of the vector in the host cell. Selective marker genes help select host cells into which the vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017). For example, selective marker genes typically confer resistance to drugs (such as G418, hygromycin or methotrexate) in host cells into which the vector has been introduced. Examples of vectors having a selectable marker include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0237] In one embodiment, the FLT3L-Fc fusion protein is produced in mammalian cells. Exemplary mammalian host cells for expressing the FLT3L-Fc fusion protein include Chinese Hamster Ovary (e.g., CHO, CHO-S, CHO DG44, ExpiCHO TM 、 CHO-K1, CHO-K1a) (including dhfr-CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selection marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T, Expi293 TM ), COS cells, NIH3T3 cells, lymphocyte lines (e.g., NS0 myeloma cells and SP2 cells) and cells from transgenic animals (e.g., transgenic mammals). For example, in some embodiments, the cell is a mammary epithelial cell.

[0238] In an exemplary system for expressing a FLT3L-Fc fusion protein, a recombinant expression vector encoding the FLT3L-Fc fusion protein is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. In a specific embodiment, the dhfr-CHO cells are cells of the DG44 cell line, such as DG44i (see, for example, Derouaz et al., Biochem Biophys Res Commun., 340(4):1069-77(2006)). Within the recombinant expression vector, a polynucleotide encoding the FLT3L-Fc fusion protein and, optionally, a second polynucleotide encoding a second Fc fusion protein for forming a heterodimer are operably linked to an enhancer / promoter regulatory element (e.g., derived from SV40, CMV, adenovirus, etc., such as a CMV enhancer / AdMLP promoter regulatory element or an SV40 enhancer / AdMLP promoter regulatory element) to drive high-level transcription of the gene. The recombinant expression vector also carries the DHFR gene, which allows selection of CHO cells that have been transfected with the methotrexate selection / amplification vector. The selected transformant host cells are cultured to allow expression and secretion of the FLT3L-Fc fusion protein, and the fusion protein is recovered from the culture medium.

[0239] FLT3L-Fc fusion proteins can also be produced by transgenic animals. For example, U.S. Patent No. 5,849,992 describes a method for expressing antibodies in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and one or more polynucleotides encoding the FLT3L-Fc fusion protein of interest, as well as a signal sequence for secretion. The milk produced by females of such transgenic mammals includes the secreted FLT3L-Fc fusion protein of interest. The FLT3L-Fc fusion protein can be purified from the milk or, for some applications, used directly. Animals containing one or more of the nucleic acids encoding FLT3L-Fc described herein are also provided.

[0240] FLT3L-Fc fusion proteins can be isolated from the inside or outside of host cells (such as the culture medium) and purified as substantially pure and homogeneous non-aggregated FLT3L-Fc fusion proteins (e.g., including monomers, homodimers, and / or heterodimers of bispecific FLT3L-Fc fusion proteins). Commonly used separation and purification methods for protein purification (including antibody purification) can be used to separate and purify the FLT3L-Fc fusion proteins described herein, and are not limited to any particular method. Suitable protein purification techniques are described, for example, in Labrou, Chronopoulou and Ataya (Editors), "Handbook on Protein Purification: Industry Challenges and Technological Developments, 2018, Nova Science Pub Inc; Gottschalk (Editor), "Process Scale Purification of Antibodies," 2nd Edition, 2017, Wiley; Staby, Rathore and Ahuja (Editors), "Preparative Chromatography for Separation of Proteins, 2017, Wiley; and Labrou (Editor), "Protein Downstream Processing: Design, Development and Application of High and Low-Resolution Methods, 2014, Human Press. The FLT3L-Fc fusion protein can be separated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Liquid chromatography such as HPLC and FPLC can be used for chromatography. Columns used for affinity chromatography include protein A columns and protein G columns.Examples of columns using a Protein A column include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). The present disclosure also includes FLT3L-Fc fusion proteins that are highly purified using these purification methods.

[0241] 6. Pharmaceutical Compositions

[0242] Provided are pharmaceutical compositions comprising a FLT3L-Fc fusion protein as described herein or a polynucleotide encoding a FLT3L-Fc fusion protein as described herein, and a pharmaceutically acceptable diluent, carrier, or excipient. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a FLT3L-Fc fusion protein or a polynucleotide encoding such a FLT3L-Fc fusion protein.

[0243] In light of the present disclosure, various pharmaceutically acceptable diluents, carriers, and excipients, as well as techniques for preparing and using pharmaceutical compositions, will be known to those skilled in the art. Exemplary pharmaceutical compositions and pharmaceutically acceptable diluents, carriers, and excipients are also described, for example, in the following references: Loyd V.Allen Jr (Editor), "Remington: The Science and Practice of Pharmacy," 22nd Edition, 2012, Pharmaceutical Press; Brunton, Knollman and Hilal-Dandan, "Goodman and Gilman's The Pharmacological Basis of Therapeutics," 13th Edition, 2017, McGraw-Hill Education / Medical; McNally and Hastedt (Editors), "Protein Formulation and Delivery," 2nd Edition, 2007, CRC Press; Banga, "Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems," 3rd Edition, 2015, CRC Press; Lars Hovgaard, Frokjaer and van de Weert (Editors), "Pharmaceutical Formulation Development of Peptides and Proteins," 2nd Edition, 2012, CRC Press; Carpenter and Manning (Editors), "Rational Design of Stable Protein Formulations: Theory and Practice," 2002, Springer (Pharmaceutical Biotechnology (Book 13)); Meyer (Editor), "Therapeutic Protein Drug Products: Practical Approaches to Formulation in the Laboratory, Manufacturing, and the Clinic," 2012, Woodhead Publishing; and Shire, "Monoclonal Antibodies: Meeting the Challenges in Manufacturing, Formulation, Delivery and Stability of Final Drug Product," 2015, Woodhead Publishing.

[0244] In some embodiments, each carrier, diluent or excipient is "acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical composition and not injurious to the subject. Typically, a pharmaceutically acceptable carrier is an aqueous pH buffer. Some examples of materials that can be used as pharmaceutically acceptable carriers, diluents or excipients include: water; buffers such as phosphate-buffered saline; sugars such as lactose, trehalose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, and malt starch. Rice oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; amino acids (e.g., charged amino acids, including but not limited to aspartic acid, asparagine, glutamic acid, glutamine, histidine, lysine); and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants (such as sodium lauryl sulfate and magnesium stearate) as well as colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives and antioxidants may also be present in the composition.

[0245] The preparation and delivery method of the pharmaceutical composition will generally be adjusted according to the site to be treated and the disease. Exemplary preparations include, but are not limited to, those suitable for parenteral administration (e.g., intratumoral, intravenous, intraarterial, intramuscular or subcutaneous administration), including preparations encapsulated in micelles, liposomes or drug release capsules (incorporated into active agents in biocompatible coatings designed for slow release); ingestible preparations; preparations for topical use, such as creams, ointments and gels; and other preparations, such as inhalants, aerosols and sprays. In some embodiments, the pharmaceutical composition is formulated for parenteral (e.g., intravenous, subcutaneous or oral) administration. In some embodiments, the pharmaceutical composition is formulated for intratumoral administration.

[0246] In certain embodiments, the pharmaceutical composition is sterile. In certain embodiments, the pharmaceutical composition has a pH in the range of 4.5 to 8.5, 4.5 to 6.5, 6.5 to 8.5, or a pH of about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.5. In some embodiments, the pharmaceutical composition has a pH of about 5.9. In one embodiment, the pharmaceutical composition has an osmotic concentration in the range of 240-260 or 250-330 mOsmol / L. In certain embodiments, the pharmaceutical composition is isotonic or nearly isotonic.

[0247] In some embodiments, the pharmaceutical composition is a liquid or solid. In some embodiments, the pharmaceutical composition comprises, for example, an aqueous solution having a concentration of about 1 mg / ml to about 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml or 20 mg / ml. In some embodiments, the pharmaceutical composition comprises an aqueous solution of the FLT3L-Fc fusion protein having a concentration of about 2 mg / ml. In some embodiments, the pharmaceutical composition comprises an aqueous solution of the FLT3L-Fc fusion protein having a concentration of 2 mg / ml. In some embodiments, the pharmaceutical composition is lyophilized. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration and has a FLT3L-Fc fusion protein concentration of about 1-100 mg / ml, 1-10 mg / ml, 2-20 mg / ml, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / ml. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration and has a FLT3L-Fc fusion protein concentration of about 2 mg / ml. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration and has a FLT3L-Fc fusion protein concentration of 2 mg / ml. In some embodiments, the pharmaceutical composition is formulated for subcutaneous injection and has a FLT3L-Fc fusion protein concentration of 1-100 mg / ml, 1-10 mg / ml, 2-20 mg / ml, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / ml and a viscosity of less than 50 cP, less than 30 cP, less than 20 cP, or about 10 cP.

[0248] In some embodiments, the pharmaceutical composition is an aqueous solution containing 2 mg / mL FLT3L-Fc fusion protein in 20 mM histidine, 90 g / L sucrose, 0.2 g / L polysorbate 80 (pH 5.9).

[0249] In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents, eg, a second therapeutic agent or a second and a third therapeutic agent.

[0250] 7. Treatment Methods

[0251] The FLT3L-Fc fusion proteins described herein can be used to treat cancer or another proliferative disorder. In some embodiments, these methods entail administering a FLT3L-Fc fusion protein, a homodimer or heterodimer comprising such a fusion protein, a polynucleotide encoding such a fusion protein, or a pharmaceutical composition comprising such a fusion protein or polynucleotide to a patient suffering from cancer or another proliferative disorder. In certain embodiments, the method of treating cancer or another proliferative disorder comprises administering a provided compound or composition thereof to a mammal.

[0252] In another aspect, provided herein are methods for inducing the immune system in a subject in need thereof, comprising administering to the subject a FLT3L-Fc fusion protein or lipoplex (such as LNP) provided herein, or a polynucleotide or vector encoding a FLT3L-Fc fusion protein provided herein.

[0253] In another aspect, provided herein are compounds for treating cancer, comprising a FLT3L-Fc fusion protein provided herein, or a polynucleotide or vector encoding a FLT3L-Fc fusion protein provided herein.

[0254] On the other hand, the FLT3L-Fc fusion protein described herein can be used to treat or prevent viral infection. In some embodiments, the viral infection is an infection caused by HIV. In some embodiments, the viral infection is an infection caused by HBV. In some embodiments, the viral infection is an infection caused by a coronavirus. In some embodiments, the coronavirus infection is caused by SARS virus, MERS virus, or 2019-nCoV (COVID-19) virus. In some embodiments, these methods require administering a FLT3L-Fc fusion protein, a homodimer or heterodimer comprising such a fusion protein, a polynucleotide encoding such a fusion protein, or a pharmaceutical composition comprising such a fusion protein or polynucleotide to a patient suffering from an infection caused by a virus or at risk of such an infection. In some embodiments, the patient suffers from an infection caused by HIV, HBV, or a coronavirus (e.g., SARS virus, MERS virus, or COVID-19 virus) or is at risk of such an infection. In certain embodiments, a method for treating or preventing a viral infection (such as an infection caused by a virus such as HIV, HBV, or a coronavirus (e.g., SARS virus, MERS virus, or COVID-19 virus)) comprises administering a provided compound or composition thereof to a mammal.

[0255] As used herein, the terms "inhibit cancer" and "inhibit cancer cell proliferation" refer to inhibiting the growth, division, maturation or vitality of cancer cells, and / or causing the death of cancer cells alone or in aggregate with other cancer cells through cytotoxicity, nutrient depletion or apoptosis induction.

[0256] As used herein, the term "treatment" refers to reversing, alleviating a disease or disorder as described herein or one or more symptoms thereof, delaying its onset or inhibiting its progression. In some embodiments, treatment may be administered after one or more symptoms occur. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to susceptible individuals before the onset of symptoms (e.g., based on a history of symptoms and / or based on genetic factors or other predisposing factors). Treatment may also be continued after symptoms subside, for example to prevent or delay their recurrence.

[0257] As used herein, "delaying" the development of a disease or disorder or one or more symptoms thereof means postponing, hindering, slowing down, retarding, stabilizing and / or delaying the development of a disease, disorder or its symptoms. The delay can have different lengths of time, depending on the history of the disease and / or the subject to be treated. It will be apparent to those skilled in the art that sufficient or significant delays can actually encompass prevention, as the subject will not develop the disease, disorder or its symptoms. For example, a method of "delaying" the development of AIDS is a method of reducing the likelihood of disease development and / or reducing the extent of the disease within a given timeframe (compared to not using the method). Such comparisons can be based on clinical studies using statistically significant numbers of subjects. For example, known methods can be used to detect the development of AIDS, such as confirming that the subject's HIV + The subject's status and T cell count or other indicators of AIDS progression, such as extreme fatigue, weight loss, persistent diarrhea, high fever, swollen lymph nodes in the neck, armpits, and groin, or the presence of opportunistic conditions known to be associated with AIDS (e.g., conditions that are not normally present in subjects with a functioning immune system but do occur in AIDS patients). Progression can also refer to disease progression that may not be detected initially, including onset, relapse, and flare-ups.

[0258] As used herein, " prevention " refers to preventing the onset of disease or disorder so that the clinical symptoms of disease do not develop. Therefore, " prevention " relates to administering therapy (for example, administering therapeutic substance) to the subject before signs of disease can be detected in the subject (for example, in the absence of a detectable infectious agent (for example, virus) in the subject) (for example, administering therapeutic substance to the subject). Subject can be the individuality of the risk of developing disease or disorder, such as the individuality of one or more risk factors related to the development or onset of a known disease or disorder. For example, the term " prevention of HIV infection " refers to administering anti-HIV therapeutic substances to the subject without detectable HIV infection. It should be understood that the subject for anti-HIV preventive therapy can be the individuality of the risk of infecting HIV virus. In addition, it should be understood that prevention may not completely prevent the onset of disease or disorder. In some cases, prevention includes reducing the risk of developing disease or disorder. Reducing risk may not completely eliminate the risk of developing disease or disorder.

[0259] With respect to subjects, in some embodiments, the methods of treatment provided herein can be used to treat subjects (e.g., humans, monkeys, dogs, cats, mice) who have been diagnosed with or are suspected of having cancer. In some embodiments, the methods of treatment provided herein can be used to treat subjects (e.g., humans, monkeys, dogs, cats, mice) who have been diagnosed with or are suspected of having a viral infection. As used herein, a subject refers to a mammal, including, for example, a human.

[0260] In some embodiments, the subject may be a person who exhibits one or more symptoms (e.g., a tumor) associated with a cancer or hyperproliferative disease. In some embodiments, the subject may be a person who exhibits one or more symptoms associated with cancer. Any of the cancer treatment methods provided herein can be used to treat cancer at various stages. By way of example, cancer stages include, but are not limited to, early, late, locally advanced, remission, refractory, relapse after remission, and progression. In some embodiments, the subject is in the early stages of cancer. In other embodiments, the subject is in the advanced stages of cancer. In various embodiments, the subject suffers from stage I, II, III, or IV cancer. Optionally, administering the FLT3L-Fc fusion protein once or multiple times together with one or more additional therapeutic agents can promote tumor reduction or shrinkage, reduce or inhibit tumor growth or cancer cell proliferation, and / or induce, increase, or promote tumor cell killing. In some embodiments, the subject is in remission of cancer. Optionally, administering the FLT3L-Fc fusion protein once or multiple times together with one or more additional therapeutic agents can prevent or delay metastasis or recurrence of cancer.

[0261] In some embodiments, the subject can be a person who exhibits one or more symptoms associated with a viral infection (e.g., detectable viral titer). In some embodiments, the subject can be a person who exhibits one or more symptoms associated with a viral infection. Any of the antiviral treatment methods provided herein can be used to treat viral infections at various stages. In some embodiments, the subject is in the early stages of a viral infection. In other embodiments, the subject is in the late stages of a viral infection. In some embodiments, one or more administrations of the FLT3L-Fc fusion protein, optionally together with one or more additional therapeutic agents, can promote a reduction in viral titer in the subject.

[0262] In some embodiments, the subject may be a human at risk or genetically or otherwise predisposed (e.g., having a risk factor) to develop a diagnosed or undiagnosed cancer or hyperproliferative disease. In some embodiments, the subject may be a human at risk or genetically or otherwise predisposed (e.g., having a risk factor) to develop a disease, disorder, or symptom thereof caused by a diagnosed or undiagnosed viral infection.

[0263] As used herein, an "at-risk" individual is an individual at risk of developing a condition to be treated. In some embodiments, a "at-risk" subject is a subject at risk of developing cancer. Generally speaking, a "at-risk" subject may or may not have a detectable disease and may or may not show detectable disease before the treatment methods described herein. "At-risk" means that the individual has one or more so-called risk factors, which are measurable parameters associated with the development of a disease or condition and are known in the art. For example, a subject at risk may have one or more risk factors, which are measurable parameters associated with the development of the cancers described herein. The probability of a subject with one or more of these risk factors developing cancer is higher than that of an individual without these risk factors. Generally speaking, risk factors may include, for example, age, sex, race, diet, previous medical history, the presence of prodromal disease, genetic (e.g., hereditary) considerations, and environmental exposures. In some embodiments, subjects at risk of cancer include, for example, subjects whose relatives have experienced the disease, as well as subjects whose risk is determined by analysis of genetic or biochemical markers. In some embodiments, subjects at risk are at risk of developing symptoms of a viral infection. For example, an individual at risk of AIDS is an individual infected with HIV.

[0264] Additionally, the subject can be a human undergoing one or more standard therapies, such as chemotherapy, radiotherapy, immunotherapy, surgery, or a combination thereof. Thus, one or more kinase inhibitors can be administered before, during, or after the administration of chemotherapy, radiotherapy, immunotherapy, surgery, or a combination thereof.

[0265] In certain embodiments, the subject can be (i) a human who is substantially refractory to at least one chemotherapy, or (ii) a human who has relapsed after treatment with chemotherapy, or both (i) and (ii). In some embodiments, the subject is refractory to at least two, at least three, or at least four chemotherapy treatments (including standard or experimental chemotherapy).

[0266] The FLT3L-Fc fusion protein described herein can be used as a vaccine adjuvant to promote, increase, supplement and / or strengthen the immune response induced by the vaccine. In various embodiments, the vaccine can be an anticancer vaccine, an antiviral vaccine or an antibacterial vaccine. In some embodiments, the anticancer vaccine is a neoantigen vaccine, wherein the neoantigen refers to a class of HLA binding peptides produced by tumor-specific mutations. Exemplary neoantigen anticancer vaccines are described, for example, in the following literature: Ott et al., Nature. 2017 July 13; 547(7662): 217-221; Li et al., Ann Oncol. 2017 December 1; 28(suppl_12): xii11-xii17; Aldous et al., Bioorg Med Chem. 2018 June 1; 26(10): 2842-2849; and Linette et al., Trends Mol Med. 2017 October; 23(10): 869-871. In various embodiments, the vaccine comprises an antiviral vaccine against a virus selected from the group consisting of hepatitis A virus (HAV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), human orthopneumovirus or human respiratory syncytial virus (RSV), human papillomavirus (HPV), varicella zoster virus, measles virus, mumps virus, poliovirus vaccine, influenza virus, paramyxovirus, rotavirus, Zika virus, dengue virus, and Ebola virus. In some embodiments, the vaccine comprises an antibacterial vaccine against a bacterium selected from the group consisting of Mycobacterium tuberculosis, pertussis, tetanus, diphtheria, meningococcus, pneumococcus, Haemophilus influenzae, cholera, typhoid fever, and anthrax. Exemplary anti-cancer vaccines include, but are not limited to, Bacillus Calmette-Guérin. - Live attenuated Mycobacterium bovis for non-muscle invasive bladder cancer; Sipuleucel-T - Dendritic cell (DC) vaccine for metastatic castration-resistant prostate cancer (mCRPC); talimogene laherparepvec (T-VEC or )-Oncolytic virus-based vaccines for advanced melanoma; and recombinant viral prostate cancer vaccines In some embodiments, the anti-cancer vaccine is an antiviral vaccine. In some embodiments, the anti-cancer vaccine is an HPV vaccine. In some embodiments, the HPV vaccine is PRGN-2009 (Precigen; PGEN Therapeutics). In some embodiments, the HPV vaccine is Gardasil or Gardasil-9 (Merck & Co.). In some embodiments, the HPV vaccine is Cervarix (GlaxoSmithKline Biologicals). In some embodiments, the HSV vaccine is HSV529 (Sanofi Pasteur).

[0267] Therefore, in one embodiment, a method for promoting, inducing and / or increasing the amplification and / or proliferation of cells or cell groups expressing fms-related tyrosine kinase 3 (FLT3, CD135) is provided. In some embodiments, these methods include contacting cells or cell groups with an effective amount of a FLT3L-Fc fusion protein as described herein, a homodimer or heterodimer comprising such fusion protein, a polynucleotide encoding such fusion protein, a carrier or lipoplex (such as lipid nanoparticles (LNPs)) comprising such polynucleotides, or a pharmaceutical composition comprising such fusion protein or polynucleotides. As used herein, "lipoplexes" refer to cationic liposomes that are non-viral (synthetic) lipid carriers of DNA. In some embodiments, lipoplexes are lipid nanoparticles (LNPs). As used herein, the term "lipid nanoparticles" refers to one or more spherical nanoparticles having an average diameter between about 10 nanometers and about 1000 nanometers and comprising a solid lipid core matrix that can dissolve lipophilic molecules. In certain embodiments, the lipid core is stabilized by a surfactant (e.g., an emulsifier) ​​and can comprise one or more of the following: triglycerides (e.g., tristearin), diglycerides (e.g., glyceryl behenate), monoglycerides (e.g., glyceryl monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate), including combinations thereof. Lipid nanoparticles are described, for example, in Petrilli et al., Curr Pharm Biotechnol. 15:847-55, 2014; and U.S. Pat. Nos. 6,217,912, 6,881,421, 7,402,573, 7,404,969, 7,550,441, 7,727,969, 8,003,621, 8,691,750, 8,871,509, 9,017,726, 9,173,853, 9,220,779, 9,227,917, and 9,278,130, each of which is incorporated by reference in its entirety.

[0268] In some embodiments, the cell or cell group expressing FLT3 includes dendritic cells (e.g., cDC1 cells and / or cDC2 cells), monocyte-derived dendritic cells (moDC) and / or their progenitor cells. In some embodiments, the cell or cell group expressing FLT3 includes hematopoietic progenitor cells. In some embodiments, hematopoietic progenitor cells include common lymphocyte progenitor cells (CLP), early progenitor cells (EPLM) with lymphocyte and bone marrow potential, granulocyte-monocyt...

Claims

1. A fusion protein comprising: a human fms-related tyrosine kinase 3 ligand extracellular domain operably linked to a crystallizable region of an immunoglobulin fragment, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO:

14.

2. A homodimer comprising two identical fusion proteins according to claim 1.

3. A heterodimer comprising two different fusion proteins, wherein one of the two different fusion proteins is the fusion protein according to claim 1. 4 . The heterologous duplex according to claim 3 , wherein one of the two different fusion proteins is a fusion protein comprising the amino acid sequence of any one of SEQ ID NOs: 1, 6 and 9.

5. A conjugate comprising the fusion protein of claim 1, the homodimer of claim 2, or the heterodimer of claim 3 or 4; the fusion protein, homodimer or heterodimer is attached to a therapeutic agent. The conjugate of claim 5 , wherein the therapeutic agent is covalently linked.

7. The conjugate according to any one of claims 5 to 6, wherein the therapeutic agent is a small organic compound.

8. The conjugate according to any one of claims 5 to 7, wherein the therapeutic agent is an agonist or activator of a toll-like receptor or a stimulator of interferon genes receptor.

9. The conjugate of claim 8, wherein the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist.

10. The conjugate according to claim 9, wherein the TLR7 agonist is selected from the group consisting of GS-9620, DS-0509, LHC-165 and TMX-101, and / or wherein the TLR8 agonist is selected from the group consisting of GS-9688 and NKTR-262.

11. The conjugate of claim 8, wherein the STING receptor agonist or activator is selected from the group consisting of ADU-S100, SB-11285, MK-1454, SR-8291, AdVCA0848, GSK-532, SYN-STING, MSA-1, SR-8291, 5,6-dimethylxanthone-4-acetate, cyclic GAMP and cyclic diAMP.

12. The conjugate of any one of claims 5 to 7, wherein the therapeutic agent is an immune checkpoint inhibitor.

13. The conjugate of claim 12, wherein the immune checkpoint inhibitor is a small molecule inhibitor of CD274, programmed cell death 1 or CTLA4.

14. The conjugate of claim 13, wherein the small molecule inhibitor of CD274 or PDCD1 is selected from the group consisting of GS-4224, GS-4416, INCB086550 and MAX10181.

15. The conjugate of claim 13, wherein the small molecule inhibitor of CTLA4 is BPI-002. A polynucleotide encoding the fusion protein according to claim 1 .

17. The polynucleotide of claim 16, wherein the polynucleotide is selected from the group consisting of DNA, cDNA, RNA or mRNA.

18. The polynucleotide according to any one of claims 16 to 17, comprising the nucleic acid sequence of SEQ ID NO:

54.

19. An expression cassette comprising one or more regulatory sequences operably linked to the polynucleotide of any one of claims 16 to 18.

20. A vector comprising the polynucleotide according to any one of claims 16 to 18 or the expression cassette according to claim 19.

21. The vector according to claim 20, wherein the vector is a plasmid vector or a viral vector.

22. The vector of claim 21, wherein the viral vector comprises an oncolytic viral vector.

23. The vector according to any one of claims 20 to 21, wherein the viral vector comprises a DNA virus or an RNA virus.

24. The vector of any one of claims 21 to 23, wherein the viral vector is from a virus family selected from the group consisting of Adenoviridae, Arenaviridae, Poxviridae, Herpesviridae, Parvoviridae, Reoviridae, Picornaviridae, Paramyxoviridae, Rhabdoviridae, Togaviridae, Enteroviridae.

25. The vector according to claim 24, wherein the Adenoviridae is an adenovirus.

26. The vector of claim 24, wherein the Arenaviridae is selected from the group consisting of Lymphocytic Choriomeningitis Mammalian Arenavirus and Cali Mammalian Arenavirus.

27. The vector according to claim 24, wherein the Poxviridae is vaccinia virus.

28. The vector according to claim 24, wherein the Herpesviridae is a herpes virus.

29. The vector of claim 28, wherein the herpes virus is HSV-1.

30. The vector of claim 24, wherein the Parvoviridae is Parvovirus H1.

31. The vector of claim 24, wherein the Reoviridae is a Reovirus.

32. The vector of claim 24, wherein the Picornaviridae is selected from the group consisting of Coxsackievirus, Seneca Valley virus, and poliovirus.

33. The vector of claim 24, wherein the Paramyxoviridae is selected from the group consisting of Measles virus and Newcastle disease virus.

34. The vector of claim 24, wherein the Rhabdoviridae is vesicular stomatitis virus.

35. The vector of claim 24, wherein the Togaviridae is selected from the group consisting of Alphavirus and Sindbis virus.

36. The vector of claim 24, wherein the Enteroviridae is an Echovirus.

37. A lipid nanoparticle comprising the polynucleotide of any one of claims 16 to 18, the expression cassette of claim 19, or the vector of any one of claims 20 to 36.

38. A cell or cell population comprising a polynucleotide according to any one of claims 16 to 18, an expression cassette according to claim 19, or a vector according to any one of claims 20 to 36, wherein the cell expresses or the cell population expresses a fusion protein according to claim 1, a homodimer according to claim 2, and / or a heterodimer according to any one of claims 3 to 4.

39. The cell or cell population of claim 38, wherein the cell or cell population is a eukaryotic cell.

40. The cell or cell population of any one of claims 38 to 39, wherein the cell or cell population comprises a mammalian cell, an insect cell, a plant cell, or a yeast cell.

41. The cell or cell population of any one of claims 38 to 40, wherein the mammalian cell is a Chinese hamster ovary cell.

42. The cell or cell population of any one of claims 38 to 40, wherein the mammalian cells are human cells.

43. The cell or cell population of claim 42, wherein the cell is a human embryonic kidney cell.

44. The cell or cell population of any one of claims 38-43, wherein the cells primarily sialylate the glycosylation sites in the fusion protein.

45. The cell or cell population of claim 44, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or more of the glycosylation sites in the fusion protein are sialylated.

46. ​​The cell or cell population of any one of claims 44 to 45, wherein the sialylated glycosylation site in the fusion protein comprises 2 to 7 sialic acid residues.

47. The cell or cell population according to claim 46, wherein the sialylated glycosylation site in the fusion protein comprises 3 to 6 sialic acid residues.

48. The cell or cell population according to claim 46, wherein the sialylated glycosylation site in the fusion protein comprises 4 to 5 sialic acid residues.

49. A pharmaceutical composition comprising the fusion protein of claim 1, the homodimer of claim 2, the heterodimer of any one of claims 3 to 4, the conjugate of any one of claims 5 to 15, the polynucleotide of any one of claims 16 to 18, the expression cassette of claim 19, the vector of any one of claims 20 to 36 or the lipid nanoparticle of claim 37 and a pharmaceutically acceptable carrier.

50. The pharmaceutical composition of claim 49, wherein the composition comprises an aqueous formulation.

51. A pharmaceutical composition according to any one of claims 49-50, comprising a fusion protein according to claim 1, a homodimer according to claim 2, a heterodimer according to any one of claims 3 to 4 and / or a conjugate according to any one of claims 5 to 15 at a concentration ranging from 1 mg / ml to 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml or 20 mg / ml.

52. A pharmaceutical composition according to any one of claims 49-50, comprising the fusion protein according to claim 1, the homodimer according to claim 2, the heterodimer according to any one of claims 3 to 4 and / or the conjugate according to any one of claims 5 to 15 at a concentration of 2 mg / ml.

53. A pharmaceutical composition according to any one of claims 49-52, wherein the composition is lyophilized.

54. The pharmaceutical composition of any one of claims 49-53, further comprising one or more additional therapeutic agents.

55. The pharmaceutical composition of any one of claims 49-54, further comprising a second therapeutic agent.

56. The pharmaceutical composition of any one of claims 49-54, further comprising a second therapeutic agent and a third therapeutic agent.

57. A method for promoting, inducing and / or increasing the expansion and / or proliferation of cells or cell populations expressing fms-related tyrosine kinase 3 in vitro, comprising contacting the cells or cell populations with an effective amount of a fusion protein according to claim 1, a homodimer according to claim 2, a heterodimer according to any one of claims 3 to 4, a conjugate according to any one of claims 5 to 15, a polynucleotide according to any one of claims 16 to 18, a vector according to any one of claims 20 to 36, a lipid nanoparticle according to claim 37, or a pharmaceutical composition according to any one of claims 49-56.

58. The method of claim 57, wherein the cells or cell population expressing fms-related tyrosine kinase 3 comprise dendritic cells, monocyte-derived dendritic cells and / or their progenitors.

59. The method of claim 57, wherein the cells or cell population expressing fms-related tyrosine kinase 3 comprise cDC1 cells and / or cDC2 cells.

60. The method of claim 57, wherein the cell or cell population expressing fms-related tyrosine kinase 3 comprises hematopoietic progenitor cells.

61. The method of claim 60, wherein the hematopoietic progenitor cells are selected from the group consisting of common lymphoid progenitor cells, early progenitor cells with lymphoid and myeloid potential, granulocyte-monocyte progenitor cells, monocyte-derived dendritic cell progenitor cells, and lineage - kit + Early multipotent progenitors within the Sca1 compartment.

62. The method of any one of claims 57 to 61, wherein the cell or cell population is contacted in vitro.

63. The method according to any one of claims 57 to 62, wherein conventional dendritic cells are expanded or their proliferation is induced.

64. The method according to any one of claims 57 to 62, wherein cDC1 and / or cDC2 cells are expanded or their proliferation is induced.

65. The method of claim 64, wherein the cDC1 dendritic cells are positive for surface expression of XC motif chemokine receptor 1, thrombomodulin, and 9A containing C-type lectin domain.

66. The method of claim 64, wherein the cDC2 dendritic cells are positive for surface expression of the CD1c molecule.

67. A method for ex vivo expansion of hematopoietic stem cells, comprising culturing hematopoietic stem cells in vitro in the presence of mesenchymal lineage precursors or stem cells and an effective amount of a fusion protein according to claim 1, a homodimer according to claim 2, a heterodimer according to any one of claims 3 to 4, a conjugate according to any one of claims 5 to 15, a polynucleotide according to any one of claims 16 to 18, a vector according to any one of claims 20 to 36, or a lipid nanoparticle according to claim 37, so that hematopoietic stem cells having the phenotype CD34+ are expanded.

68. The method of claim 67, wherein the hematopoietic stem cells are further cultured in the presence of at least one histone deacetylase inhibitor.

69. The method of claim 67 or 68, wherein the HDACi is selected from the group consisting of valproic acid, trichostatin A, DLS3, MS275 and SAHA.

70. The method of any one of claims 67 to 69, wherein the hematopoietic stem cells have the phenotype CD34+, CD90+ or ​​CD34+, CD45RA-, CD49f+ and are expanded at least 5-fold, at least 10-fold, at least 20-fold or at least 40-fold.

71. The method according to any one of claims 67 to 69, further comprising isolating cells having the phenotype CD34+, CD90+ or ​​CD34+, CD45RA-, CD49f+ to provide an enriched cell population having the phenotype CD34+, CD90+ or ​​CD34+, CD45RA-, CD49f+.

72. The method of any one of claims 67 to 71, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide or the pharmaceutical composition is administered to a mammal.

73. A compound for preparing a medicament, comprising or consisting of the fusion protein of claim 1, the homodimer of claim 2, the heterodimer of any one of claims 3 to 4, the conjugate of any one of claims 5 to 15, the polynucleotide of any one of claims 16 to 18, or the vector of any one of claims 20 to 36.

74. Use of the fusion protein of claim 1, the homodimer of claim 2, the heterodimer of any one of claims 3 to 4, the conjugate of any one of claims 5 to 15, the polynucleotide of any one of claims 16 to 18, the vector of any one of claims 20 to 36, the lipid nanoparticle of claim 37, or the pharmaceutical composition of any one of claims 49-56 in the preparation of a medicament for reducing and / or inhibiting the recurrence, growth, proliferation, migration and / or metastasis of cancer cells or cancer cell populations in a subject in need thereof, wherein the subject suffers from colorectal cancer.

75. The use of claim 74, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the carrier, the lipid nanoparticle and / or the pharmaceutical composition is co-administered with one or more additional therapeutic agents.

76. The use according to claim 75, wherein the one or more additional therapeutic agents are selected from the group consisting of: AGEN1884, AGEN1181, AGEN2034, AGEN1307, AGEN2373, AGEN1223 and GS-1423.

77. The use according to any one of claims 74 to 76, wherein the subject suffers from neutropenia or lymphopenia.

78. The use of any one of claims 74 to 77, wherein the subject has received a lymphodepleting chemotherapy regimen.

79. The use of any one of claims 74 to 77, wherein the subject is chemotherapy naive or has not received chemotherapy.

80. The use of any one of claims 74 to 79, wherein the subject has bone marrow cells, or is not depleted of bone marrow cells.

81. The use of any one of claims 74 to 80, wherein the subject does not have a mutation in the gene encoding the fms-related tyrosine kinase 3 receptor that causes or contributes to cancer or is associated with cancer.

82. Use of the fusion protein according to claim 1, the homodimer according to claim 2, the heterodimer according to any one of claims 3 to 4, the conjugate according to any one of claims 5 to 15, the polynucleotide according to any one of claims 16 to 18, the vector according to any one of claims 20 to 36, the lipid nanoparticle according to claim 37, or the pharmaceutical composition according to any one of claims 49-56 in the preparation of a medicament for combined treatment or prevention of human hepatitis B virus infection with a hepatitis B virus vaccine.

83. The use according to claim 82, wherein the hepatitis B virus vaccine is selected from an arenavirus vector expressing hepatitis B surface antigen, an arenavirus vector expressing hepatitis B core antigen and an arenavirus vector expressing hepatitis B virus polymerase.

84. The method of any one of claims 74 to 83, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the carrier, the lipid nanoparticle and / or the pharmaceutical composition is administered systemically or locally.

85. The method of any one of claims 74 to 83, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the carrier, the lipid nanoparticle and / or the pharmaceutical composition is administered intravenously, intratumorally, subcutaneously, intradermally, intramuscularly, intraperitoneally, intravesically, intracranially, intrathecally, intracavitarily or intraventricularly.

86. The use of claim 85, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the carrier, the lipid nanoparticle and / or the pharmaceutical composition and the one or more additional therapeutic agents are administered by the same route of administration.

87. The method of claim 85, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the carrier, the lipid nanoparticle and / or the pharmaceutical composition and the one or more additional therapeutic agents are administered by different routes of administration.

88. The use according to any one of claims 74 to 87, wherein the fusion protein has a serum half-life of at least 7 days.

89. The method of any one of claims 74 to 88, comprising administering the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the carrier, the lipid nanoparticle and / or the pharmaceutical composition multiple times at predetermined time intervals.

90. The method of any one of claims 74 to 89, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the carrier, the lipid nanoparticle and / or the pharmaceutical composition is administered once a week, once every two weeks, once every three weeks, once a month or once every two months or is administered less frequently.

91. The use of claim 90, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the carrier, the lipid nanoparticle and / or the pharmaceutical composition and the one or more additional therapeutic agents are co-administered according to the same schedule.

92. The use of claim 90, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the carrier, the lipid nanoparticle and / or the pharmaceutical composition and the one or more additional therapeutic agents are co-administered according to different schedules.

93. The use according to any one of claims 74 to 92, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the vector, the lipid nanoparticle and / or the pharmaceutical composition is administered at a dose ranging from 0.5 μg / kg to 5000 μg / kg.

94. The use according to any one of claims 74 to 92, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the vector, the lipid nanoparticle and / or the pharmaceutical composition is administered at a dose ranging from 0.1 μg / kg to 100 μg / kg per dose.

95. The use according to any one of claims 74 to 92, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the vector, the lipid nanoparticle and / or the pharmaceutical composition is administered at a dose of 1 μg / kg, 3 μg / kg, 10 μg / kg, 30 μg / kg, 60 μg / kg or 100 μg / kg.

96. The use according to any one of claims 74 to 92, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the vector, the lipid nanoparticle and / or the pharmaceutical composition is administered at a dose of 1 μg / kg per dose.

97. The use according to any one of claims 74 to 92, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the vector, the lipid nanoparticle and / or the pharmaceutical composition is administered at a dose of 3 μg / kg per dose.

98. The use according to any one of claims 74 to 92, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the vector, the lipid nanoparticle and / or the pharmaceutical composition is administered at a dose of 10 μg / kg per dose.

99. The use according to any one of claims 74 to 92, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the vector, the lipid nanoparticle and / or the pharmaceutical composition is administered at a dose of 30 μg / kg per dose.

100. The use according to any one of claims 74 to 92, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the vector, the lipid nanoparticle and / or the pharmaceutical composition is administered at a dose of 60 μg / kg per dose.

101. The use according to any one of claims 74 to 92, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the vector, the lipid nanoparticle and / or the pharmaceutical composition is administered at a dose of 100 μg / kg per dose.

102. The use according to any one of claims 74 to 92, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the carrier, the lipid nanoparticle and / or the pharmaceutical composition is administered at a dose ranging from 0.5 mg to 50 mg.

103. The use according to any one of claims 74 to 92, wherein the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the carrier, the lipid nanoparticle and / or the pharmaceutical composition is administered at a dose of 10 mg per dose.

104. The use according to any one of claims 74 to 103, wherein within 3 weeks of a single administration of the fusion protein, the homodimer, the heterodimer, the conjugate, the polynucleotide, the vector, the lipid nanoparticle and / or the pharmaceutical composition, the cells expressing fms-related tyrosine kinase 3 expand by at least 10 times, 20 times, 50 times, 100 times, 200 times, 300 times, 400 times, 500 times or more.

105. The use according to any one of claims 74 to 104, wherein the cells expressing fms-related tyrosine kinase 3 are expanded in the bone marrow and / or solid tumor of the subject.

106. The use of any one of claims 74 to 105, wherein the subject or the mammal is a human.

107. A kit comprising one or more unit doses of a fusion protein according to claim 1, a homodimer according to claim 2, a heterodimer according to any one of claims 3 to 4, a conjugate according to any one of claims 5 to 15, a polynucleotide according to any one of claims 16 to 18, an expression cassette according to claim 19, a vector according to any one of claims 20 to 36, or a lipid nanoparticle according to claim 37.

108. The kit of claim 107, wherein the one or more unit doses are in a single container.

109. The kit of claim 107, wherein the one or more unit doses are in two or more separate containers.

110. The kit of any one of claims 107 to 109, comprising one or more containers selected from the group consisting of a vial, an ampoule, and a prefilled syringe.

111. The kit of any one of claims 107 to 110, comprising one or more containers comprising an aqueous solution of the fusion protein and / or the homodimer.

112. The kit of claim 111, wherein the aqueous solution comprises the fusion protein and / or the homodimer at a concentration ranging from 1 mg / ml to 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml or 20 mg / ml.

113. The kit of claim 111, wherein the aqueous solution comprises the fusion protein and / or the homodimer at a concentration of 2 mg / ml.

114. The kit of any one of claims 107 to 113, wherein the one or more unit doses are identical.

115. The kit of any one of claims 107 to 113, wherein the one or more unit doses are different.

116. A kit according to any one of claims 107 to 115, wherein each unit dose is in the range of 0.5 mg to 50 mg.

117. The kit according to any one of claims 107 to 116, wherein each unit dose is 10 mg per dose.

118. The kit of any one of claims 107 to 117, further comprising one or more unit doses of one or more additional therapeutic agents.

119. The kit of claim 118, comprising one or more unit doses of one or more therapeutic agents selected from the group consisting of: AGEN1884, AGEN1181, AGEN2034, AGEN1307, AGEN2373, AGEN1223 and GS-1423.

120. The kit of any one of claims 118 to 119, comprising one or more oncolytic viral vectors.

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