FLT3 ligand fusion protein and method of use

Abstract

The present invention provides effectorless immunoglobulin Fc proteins, fusions of effectorless Fc proteins to Flt3-ligand, and methods of using them.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and interest in U.S. Provisional Patent Application No. 63 / 062,713, filed on 7 August 2020, the entirety of which is incorporated herein by reference.

[0002] Sequence List This application includes an electronically filed sequence listing in ASCII format, created on 28 July 2021, which is incorporated herein by reference in its entirety, named P36298-WO_SL.txt, and has a size of 128,413 bytes.

[0003] Field of Invention This invention relates to an Flt3 ligand fusion protein and a method for using the same. [Background technology]

[0004] background Numerous studies have supported the differential role of immune system components in cancer progression (Jochems and Schlom, Exp Biol Med, 236(5):567-579 (2011)). Clinical data suggest that high density of tumor-infiltrating lymphocytes is associated with improved clinical outcomes (Mlecnik et al., Cancer Metastasis Rev.; 30:5-12, (2011)). Tumor immune infiltrates include macrophages, dendritic cells (DCs), mast cells, natural killer (NK) cells, naive and memory lymphocytes, B cells, and effector T cells (T lymphocytes), which are primarily responsible for recognizing antigens expressed by tumor cells and subsequent T cell-mediated destruction of tumor cells.

[0005] Despite antigen presentation by cancer cells and the presence of immune cells potentially capable of responding to tumor cells, the immune system is often unactivated or positively suppressed. Tumors develop several immunomodulatory mechanisms to evade the anti-tumor immune response. The overall outcome is impaired T cell response and induction of apoptosis or CD8 + This involves a decrease in the antitumor immune activity of cytotoxic T cells. Inhibition of antigen-presenting function and dendritic cells (DCs) further contributes to evading antitumor immunity (Gerlini et al. Am.J. Pathol. 165(6), 1853-1863 (2004)).

[0006] Furthermore, the local immunosuppressive nature of the tumor microenvironment may lead to the avoidance of cancer cell subpopulations that do not express target antigens. Therefore, finding approaches that would promote the preservation and / or restoration of the immune system's antitumor activity would be of considerable therapeutic benefit.

[0007] Immune checkpoints are involved in tumor-mediated downregulation of antitumor immunity. T-cell dysfunction has been demonstrated to occur concurrently with the induced expression of inhibitory receptors, CTLA-4 and programmed death 1 polypeptide (PD-1), which are members of the CD28 family of receptors. Nevertheless, despite extensive research in recent years, the success of immunotherapy in clinical practice remains limited. Few treatments are approved by regulatory authorities, and the vast majority of patients do not benefit from them. In recent years, immune checkpoints have been identified as being involved in the downregulation of antitumor immunity and are being used as therapeutic targets. These findings highlight the need to develop novel therapeutic approaches to leverage the immune system against cancer.

[0008] Human Flt3L (Fms-like tyrosine kinase 3 ligand), a type I transmembrane protein that stimulates bone marrow cell proliferation, was cloned in 1994 (Lyman et al., 1994). The use of soluble hFlt3L has been investigated in various preclinical and clinical situations, including stem cell mobilization in bone marrow transplant preparation, cancer immunotherapy such as dendritic cell proliferation, and vaccine adjuvants. However, no pharmaceutical compositions utilizing Flt3L have progressed beyond Phase 2 in clinical trials.

[0009] One challenge is to optimize exposure to Flt3L ligands and identify the optimal dosing regimen while minimizing adverse side effects or potentially undesirable immunological effects. Exposure can be modified, for example, by altering the dosing regimen, changing the dosage, or modifying the pharmacokinetic and / or pharmacodynamic properties of the therapeutic molecule. Flt3L-Fc fusion proteins with beneficial PK / PD properties that are advantageous for enhancing immunotherapy in cancer patients are provided herein. [Overview of the project]

[0010] overview This invention provides effectorless Fc proteins, fusion proteins containing effectorless Fc proteins (including Flt3 ligand fusion proteins containing active Flt3 ligand (Flt3L) fused to effectorless immunoglobulin Fc proteins), and methods for using the same. Methods for using effectorless Fc proteins, fusion proteins containing effectorless Fc proteins, and Flt3L-Fc fusion proteins include the treatment of cancer, particularly cancer in patients undergoing checkpoint immunotherapy. This invention further provides effectorless IgG Fc proteins.

[0011] In one embodiment, an effectorless Fc protein is provided, comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to residue SEQ ID NO: 13, wherein residues 13-17 of SEQ ID NO: 13 comprise the amino acid sequence PVAGP (SEQ ID NO: 20), or residue 76 of SEQ ID NO: 13 is glycine. In another embodiment, the effectorless IgG1 Fc region comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, wherein residues 13-17 of SEQ ID NO: 13 comprise the amino acid sequence PVAGP (SEQ ID NO: 20), and residue 76 of SEQ ID NO: 13 is glycine. In yet another embodiment, the effectorless IgG1 Fc region comprises the same amino acid sequence as SEQ ID NO: 13. For clarity, in some embodiments, if residues 13-17 of SEQ ID NO: 13 contain PVAGP (SEQ ID NO: 20), this may be referred to herein as containing the PVA# variant, and if residue 76 of SEQ ID NO: 13 contains glycine, this may be referred to herein as the N297G mutation, where 297 refers to the EU numbering of the antibody.

[0012] In some embodiments, the effectorless Fc protein comprises the protein sequence of SEQ ID NOs: 2, 4, 5, 6, 13, or 15.

[0013] In yet another embodiment, the effectorless IgG1 Fc protein is attenuated compared to the wild-type IgG1 Fc region containing SEQ ID NO: 12.

[0014] In some embodiments, an antibody is provided comprising an effectorless Fc protein having an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to residue SEQ ID NO: 13, wherein residues 13-17 of SEQ ID NO: 13 contain the amino acid sequence PVAGP (SEQ ID NO: 20), or residue 76 of SEQ ID NO: 13 is glycine.

[0015] In some embodiments, the antibody binds to the Flt3L receptor.

[0016] In some embodiments, the antibody binds to a checkpoint inhibitor protein. In other embodiments, the checkpoint inhibitor protein is PD-L1, PD-1, and / or CTLA-4. In some embodiments, the antibody is a bispecific antibody.

[0017] In some aspects, a heterodimeric protein comprising an effectorless Fc protein of the present disclosure and a second protein is provided. In some embodiments, the effectorless Fc protein and the second protein are covalently bound to each other. In other embodiments, the effectorless Fc protein and the second protein are linked by a disulfide bond.

[0018] In some aspects, an isolated nucleic acid encoding an effectorless Fc protein of the present disclosure, an antibody comprising the effectorless Fc protein, and / or a heterodimeric protein comprising the effectorless Fc protein described herein and above are provided. In some embodiments, the isolated nucleic acid encodes the protein sequence of SEQ ID NO: 2, 4, 5, 6, 13 or 15. In other embodiments, the isolated nucleic acid further encodes a signal sequence at the N-terminus of the effectorless Fc protein. In a preferred example, the isolated nucleic acid encodes the protein sequence of SEQ ID NO: 13.

[0019] In some aspects, a host cell comprising a nucleic acid encoding an effectorless Fc protein of the present disclosure, a fusion protein comprising the effectorless Fc protein, an antibody comprising the effectorless Fc protein, and / or a heterodimeric protein comprising the effectorless Fc protein described herein and above is provided.

[0020] In some embodiments, a method is provided for producing the effectorless Fc protein of the Disclosure, a fusion protein containing the effectorless Fc protein, an antibody containing the effectorless Fc protein, and / or a heterodimer protein containing the effectorless Fc protein, comprising culturing a host cell containing a nucleic acid encoding the effectorless Fc protein of the Disclosure, a fusion protein containing the effectorless Fc protein, an antibody containing the effectorless Fc protein, and / or a heterodimer protein containing the effectorless Fc protein to produce each protein. In some embodiments, the method further comprises recovering the effectorless Fc protein, a fusion protein containing the effectorless Fc protein, an antibody containing the effectorless Fc protein, and / or a heterodimer protein containing the effectorless Fc protein from the host cell. In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In other embodiments, the eukaryotic cell is a CHO cell.

[0021] In some embodiments, a host cell is provided comprising a nucleic acid encoding the effectorless Fc protein of the present disclosure, a fusion protein containing the effectorless Fc protein, an antibody containing the effectorless Fc protein, and / or a heterodimer protein containing the effectorless Fc protein.

[0022] In some embodiments, a method is provided for producing the effectorless Fc protein of the Disclosure, a fusion protein containing the effectorless Fc protein, an antibody containing the effectorless Fc protein, and / or a heterodimer protein containing the effectorless Fc protein, comprising culturing a host cell containing a nucleic acid encoding the effectorless Fc protein of the Disclosure, a fusion protein containing the effectorless Fc protein, an antibody containing the effectorless Fc protein, and / or a heterodimer protein containing the effectorless Fc protein to produce the effectorless Fc protein of the Disclosure, a fusion protein containing the effectorless Fc protein, an antibody containing the effectorless Fc protein, and / or a heterodimer protein containing the effectorless Fc protein. In some embodiments, the method further comprises recovering the effectorless Fc protein, a fusion protein containing the effectorless Fc protein, an antibody containing the effectorless Fc protein, and / or a heterodimer protein containing the effectorless Fc protein from the host cell. In some embodiments, the host cell is a eukaryotic or prokaryotic cell. In other embodiments, the eukaryotic cell is a CHO cell.

[0023] In one embodiment, a fusion protein comprising an effectorless Fc protein (SEQ ID NO: 13 or a variant thereof) and a second protein is provided, wherein the second protein is a ligand for a target protein. In some embodiments, the ligand modulates the target protein when the ligand binds to the target protein. In some embodiments, the second protein is Flt3L. In other embodiments, the target protein is an Flt3L receptor. In a preferred embodiment, the second protein is the N-terminus of the effectorless Fc protein.

[0024] In some embodiments, the fusion protein comprises an Fc protein that is at least about 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, wherein residues 13-17 of SEQ ID NO: 13 comprise the amino acid sequence PVAGP (SEQ ID NO: 20), or residue 76 of SEQ ID NO: 13 is glycine. In other embodiments, the effectorless IgG1 Fc region comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, wherein residues 13-17 of SEQ ID NO: 13 comprise the amino acid sequence PVAGP (SEQ ID NO: 20), and residue 76 of SEQ ID NO: 13 is glycine. In yet another embodiment, the effectorless IgG1 Fc region comprises the same amino acid sequence as SEQ ID NO: 13. For clarity, in some embodiments, if residues 13-17 of SEQ ID NO: 13 contain PVAGP (SEQ ID NO: 20), this may be referred to herein as containing the PVA# variant, and if residue 76 of SEQ ID NO: 13 contains glycine, this may be referred to herein as the N297G mutation, where 297 refers to the EU numbering of the antibody.

[0025] In one embodiment, the fusion protein comprises an Flt3 ligand (Flt3L) and an effectorless IgG1 Fc region, and the Flt3L-Fc fusion protein has attenuated effector function compared to the wild-type IgG1 Fc region containing SEQ ID NO: 12.

[0026] In some embodiments of the Flt3L-Fc fusion protein, Flt3L comprises a protein containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to residues 27-167 of SEQ ID NO: 21. In other embodiments, Flt3L comprises the amino acid sequence of SEQ ID NO: 21, and the amino acid sequence has one, two, or three amino acid substitutions. In other embodiments, the one, two, or three amino acid substitutions are not present in the region of Flt3L that binds to the FcRn protein. In other embodiments, the Flt3L protein comprises the sequence of SEQ ID NO: 22.

[0027] In some embodiments of the Flt3L-Fc fusion protein, Flt3L comprises a protein containing amino acids 27-167, 27-168, 27-169, 27-170, 27-171, 27-172, 27-173, 27-174, 27-175, 27-176, 27-177, 27-178, 27-179, 27-180, 27-181, 27-182, 27-183, 27-184, or 27-185 of SEQ ID NO: 21. In preferred embodiments of the Flt3L-Fc fusion protein, Flt3L comprises an amino acid sequence consisting of amino acids 27-167 or amino acids 27-168 of SEQ ID NO: 21. In other embodiments, Flt3L in the Flt3L-Fc fusion protein does not include amino acid sequences 168-235, 169-235, 170-235, 171-235, 172-235, and 173-235 of SEQ ID NO: 21. In yet another embodiment, Flt3L in the Flt3L-Fc fusion protein does not include amino acid sequences PWSPRPLEATAPTAPQPP (SEQ ID NO: 48), WSPRPLEATAPTAPQPP (SEQ ID NO: 49), SPRPLEATAPTAPQPP (SEQ ID NO: 50), PRPLEATAPTAPQPP (SEQ ID NO: 51), RPLEATAPTAPQPP (SEQ ID NO: 52), or PLEATAPTAPQPP (SEQ ID NO: 53).

[0028] In some embodiments of the Flt3L~Fc fusion protein, Flt3L includes a protein that is at least about 95%, 96%, 97%, 98%, or 99% identical to the protein containing amino acids 27~167, 27~168, 27~169, 27~170, 27~171, 27~172, 27~173, 27~174, 27~175, 27~176, 27~177, 27~178, 27~179, 27~180, 27~181, 27~182, 27~183, 27~184, or 27~185 of SEQ ID NO: 21.

[0029] In some embodiments of the Flt3L~Fc fusion protein, Flt3L includes a protein that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the protein containing amino acids 24-167, 25-167, 26-167, 24-168, 25-168, 26-168, 24-169, 25-169, or 26-169 of SEQ ID NO: 21.

[0030] In some embodiments of the Flt3L-Fc fusion protein, the effectorless IgG1 Fc region includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, wherein residues 13-17 of SEQ ID NO: 13 include the amino acid sequence PVAGP (SEQ ID NO: 20), or residue 76 of SEQ ID NO: 13 is glycine. In other embodiments, the effectorless IgG1 Fc region includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, wherein residues 13-17 of SEQ ID NO: 13 include the amino acid sequence PVAGP (SEQ ID NO: 20), and residue 76 of SEQ ID NO: 13 is glycine. In yet another embodiment, the effectorless IgG1 Fc region includes the same amino acid sequence as SEQ ID NO: 13. For clarity, in some embodiments, if residues 13-17 of SEQ ID NO: 13 contain PVAGP (SEQ ID NO: 20), this may be referred to herein as containing the PVA# variant, and if residue 76 of SEQ ID NO: 13 contains glycine, this may be referred to herein as the N297G mutation, where 297 refers to the EU numbering of the antibody.

[0031] In some embodiments, the Flt3L-Fc fusion protein contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26. In other embodiments, the Flt3L-Fc fusion protein contains the amino acid sequence of SEQ ID NO: 26.

[0032] In some embodiments, the Flt3L-Fc fusion protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26, wherein residues 154-158 of SEQ ID NO: 26 comprise the amino acid sequence PVAGP (SEQ ID NO: 20), and residue 217 of SEQ ID NO: 26 is glycine.

[0033] In some embodiments, the Flt3L-Fc fusion protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25, wherein residues 155-159 of SEQ ID NO: 25 comprise the amino acid sequence PVAGP (SEQ ID NO: 20), and residue 218 of SEQ ID NO: 25 is glycine.

[0034] In some embodiments, the Flt3L-Fc fusion protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32, wherein residues 153-157 of SEQ ID NO: 32 comprise the amino acid sequence PVAGP (SEQ ID NO: 20), and residue 216 of SEQ ID NO: 32 is glycine.

[0035] In some embodiments, the Flt3L-Fc fusion protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33, wherein residues 152-156 of SEQ ID NO: 33 comprise the amino acid sequence PVAGP (SEQ ID NO: 20), and residue 215 of SEQ ID NO: 33 is glycine.

[0036] In some embodiments, the Flt3L-Fc fusion protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34, wherein residues 151-154 of SEQ ID NO: 34 comprise the amino acid sequence PVAGP (SEQ ID NO: 20), and residue 214 of SEQ ID NO: 34 is glycine.

[0037] In some embodiments, the Flt3L-Fc fusion protein contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a protein selected from the group consisting of SEQ ID NOs: 27 to 31 and SEQ ID NOs: 35 to 44.

[0038] In some embodiments, the Flt3L-Fc fusion protein activates antibody-dependent phagocytosis (ADCP) in an in vitro assay at activity levels of approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or less than 99% of the activity level in an in vitro assay with the Flt3L-Fc fusion protein containing wild-type Flt3L fused to wild-type IgG1 Fc (SEQ ID NO: 12). In other embodiments, the ADCP in vitro assay includes the use of primary macrophages as effector cells and cell lines expressing the Flt3L receptor as target cells. In yet another embodiment, the primary macrophages are monocyte-derived macrophages from a healthy human donor. In yet another embodiment, the target cells are human acute lymphoblastic leukemia cells, optionally, SEM cell lines. In some embodiments, activity is measured as percentage phagocytosis (%ADCP), and phagocytosis is determined by measuring the uptake of a visual marker from target cells.

[0039] In some embodiments, the Flt3L-Fc fusion protein activates dendritic cell (DC) proliferation in subjects to which the fusion protein is administered. In other embodiments, DC proliferation is measured in the whole blood of the subject. In yet another embodiment, DC proliferation is measured by flow cytometry or FACS. In some embodiments, DC proliferation is at least 5, 10, 15, 20, 50, 100, 500, 1000, 2000, 5000, or 10,000 times greater than DC proliferation resulting from administration of Flt3L protein that is not fused to a heterologous protein. In other embodiments, the heterologous protein is wild-type human IgG Fc protein. In yet another embodiment, the wild-type human IgG Fc protein is wild-type IgG1, IgG2, IgG3, or IgG4 Fc protein. In some embodiments, the Flt3L protein is not fused to wild-type IgG1 Fc protein or human serum albumin protein. In some embodiments, the subject is a rodent, rabbit, cynomolgus monkey, or human. In other embodiments, the rodent is a mouse or a rat.

[0040] In some embodiments, the Flt3L-Fc fusion protein has increased thermal stability compared to the Flt3L-Fc fusion protein having SEQ ID NO: 14. In other embodiments, thermal stability is measured using differential scanning fluorescence assay. In yet another embodiment, the Flt3L-Fc fusion protein has a melting temperature (Tm) at least 1.5°C, 2°C, 3°C, 4°C, or 5°C higher than the Tm of the Flt3L-Fc fusion protein having SEQ ID NO: 14.

[0041] In some embodiments, the Flt3L-Fc fusion protein contains a single amino acid substitution in the Flt3L domain. In other embodiments, the substitution reduces in vivo immunogenicity but does not reduce functional activity by more than 10%, 20%, or 30%. In other embodiments, the functional activity assay is the proliferation of dendritic cells in animals.

[0042] In some embodiments, isolated nucleic acids encoding the Flt3L-Fc fusion protein described herein and above are provided. In some embodiments, the isolated nucleic acid encodes the protein sequence of SEQ ID NOs. 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44. In other embodiments, the isolated nucleic acid further encodes a signal sequence at the N-terminus of the Flt3L-Fc fusion protein. In a preferred example, the isolated nucleic acid encodes the protein sequence of SEQ ID NO. 45.

[0043] In some embodiments, a host cell is provided comprising a nucleic acid encoding the Flt3L-Fc fusion protein described herein and above.

[0044] In some embodiments, a method is provided for producing an Flt3L-Fc fusion protein, comprising culturing a host cell containing a nucleic acid encoding an effectorless Flt3L-Fc fusion protein so that the Flt3L-Fc fusion protein is produced. In some embodiments, the method further comprises recovering the Flt3L-Fc fusion protein from the host cell. In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In other embodiments, the eukaryotic cell is a CHO cell.

[0045] In some embodiments, a pharmaceutical formulation is provided comprising the Flt3L-Fc fusion protein described herein and a pharmaceutically acceptable carrier.

[0046] In some embodiments, the pharmaceutical formulation further comprises additional therapeutic agents. In yet other embodiments, the additional pharmaceuticals are adjuvants, dendritic cell maturation factors, and / or checkpoint inhibitors.

[0047] In some embodiments, a method is provided for increasing the number of dendritic cells (DCs) in a subject administered with the Flt3L-Fc fusion protein. In some embodiments, the DCs are cDC1 and / or cDC2 cells. In other embodiments, the method comprises administering the Flt3L-Fc fusion protein of the present invention to a subject.

[0048] In some embodiments, the method involves administering Flt3L-Fc fusion protein to a target in doses of approximately 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 40 mg / kg, 0.1 mg / kg to 25 mg / kg, 0.1 mg / kg to 20 mg / kg, 0.1 mg / kg to 15 mg / kg, 0.1 mg / kg to 10 mg / kg, 1 mg / kg to 50 mg / kg, 1 mg / kg to 40 mg / kg, 1 mg / kg to 25 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 15 mg / kg, or 1 mg / kg to 10 mg / kg.

[0049] In some embodiments, the method involves administering the Flt3L fusion protein to the subject approximately once a day, once a week, twice a week, once every two weeks, once every three weeks, once a month, or once every two months.

[0050] In some embodiments, the method involves targeting and administering the Flt3L fusion protein by intravenous, parenteral, intramuscular, or subcutaneous injection.

[0051] In some embodiments, a method for treating cancer is provided, comprising administering the Flt3L-Fc fusion protein described herein to a subject in need thereof. In some embodiments, the method comprises administering a therapeutically effective dose of the Flt3L-Fc fusion protein and administering a therapeutically effective dose of an immune checkpoint inhibitor. In other embodiments, the immune checkpoint inhibitor suppresses or inhibits the effects of PD-1 or PD-L1. In yet another embodiment, the immune checkpoint inhibitor is pembrolizumab, nivolumab, pizilizumab, BMS936559, atezolizumab, or avelumab. In other embodiments, the checkpoint inhibitor is administered before, concurrently with, or after the administration of the Flt3L-Fc fusion protein.

[0052] In some embodiments, the method further includes administering a dendritic cell (DC) maturation factor. In other embodiments, the DC maturation factor is selected from poly-IC, poly-ICLC, DC40 agonists, radiotherapy, and chemotherapy. In other embodiments, the DC maturation factor is administered to the subject before, concurrently with, and / or after administration of the Flt3L-Fc fusion protein. In certain embodiments, the DC maturation factor is radiotherapy. In some embodiments, the method includes administering the Flt3L-Fc fusion protein to the subject after the subject has received the DC maturation factor. In other embodiments, the method includes administering the Flt3L-Fc fusion protein to the subject before the subject has received the DC maturation factor. In yet another embodiment, the method includes administering the Flt3L-Fc fusion protein to the subject approximately simultaneously with the subject receiving the DC maturation factor.

[0053] In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), melanoma, pancreatic ductal adenocarcinoma (PDAC), triple-negative breast cancer (TNBC), non-Hodgkin lymphoma (NHL), colorectal cancer (CRC), breast cancer, bladder cancer, kidney cancer, or a combination thereof.

[0054] In some embodiments, the subjects had been previously treated with checkpoint inhibitors. In other embodiments, the subjects did not respond to treatment with checkpoint inhibitors administered for at least or about 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 year, 1.5 years, or 2 years.

[0055] In some embodiments, subjects were treated with radiotherapy administered for a period of at least or approximately 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 year, 1.5 years, or 2 years.

[0056] In some embodiments, subjects were treated with chemotherapy administered for a period of at least or approximately 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 year, 1.5 years, or 2 years.

[0057] In some embodiments, cancers in the subject are characterized to include immune desert tumors. In other embodiments, cancers are characterized to include tumors with reduced inflammation ("cold tumors") compared to responsive inflammatory tumors ("hot tumors"). [Brief explanation of the drawing]

[0058] [Figure 1] Figure 1 shows different levels of ADCP activity induced by wild-type or variant Fc domain-containing anti-Her2 antibodies. Primary M1 macrophages were incubated with SkBr3 target cells. The x-axis represents antibody concentration, and the y-axis represents percentage phagocytosis. [Figure 2A-E] Figures 2A to 2E show cytokine release by macrophages when anti-Her2 antibodies containing wild-type or variant Fc domains are incubated with primary M1 macrophages and SkBr3 target cells. The x-axis represents antibody concentration, and the y-axis represents pg / ml cytokine. Figure 2A: G-CSF; Figure 2B: GM-CSF; Figure 2C: IL-10; Figure 2D: IL-1ra; Figure 2E: IL-6. [Figure 2F-J]Figures 2F to 2J show cytokine release by macrophages when anti-Her2 antibodies containing wild-type or variant Fc domains are incubated with primary M1 macrophages and SkBr3 target cells. The x-axis represents antibody concentration, and the y-axis represents pg / ml cytokine. Figure 2F: IL-8; Figure 2G: MIP-1α; Figure 2H: MIP-1β; Figure 2I: RANTES; Figure 2J: TNFα. [Figure 2K] Figure 2K shows the ADCC activity of anti-CD20 antibodies containing wild-type and variant Fc proteins. [Figure 2L] Figure 2L shows the ADCC activity of anti-Her2 antibodies containing wild-type and variant Fc proteins. [Figure 3A-C] Figures 3A to 3C show the different characteristics of the Flt3L-FcNG2LH fusion protein regarding aggregation and disulfide bond properties. Analysis of the aggregation and dimerization of the hFLT3L.S163.no.hinge.hIgG1.NG.PVA# protein expressed in CHO cells and HEK293 cells is shown by SDS-PAGE (Figure 3A) and SEC-HPLC (Figure 3B: 293 cells and Figure 3C: HEK297 cells). In Figures 3B to 3C, the x-axis is time and the y-axis is mAU. [Figure 3D-F] Figures 3D to 3F show the different characteristics of the Flt3L-FcNG2LH fusion protein regarding aggregation and disulfide bond properties. Analysis of the aggregation and dimerization of the hFLT3L.P167.5aa.hinge.hIgG1.NG.PVA# protein expressed in CHO cells and HEK293 cells is shown by SDS-PAGE (Figure 3D) and SEC-HPLC (Figure 3E: 293 cells and Figure 3F: HEK297 cells). In Figures 3E to 3F, the x-axis is time and the y-axis is mAU. [Figure 4]Figures 4A and 4B show that Flt3L induces proliferation of OCI-AML5 cells in vitro. Figure 4A shows the dose-response curves of hFLT3L.P167.hIgG1.NG2LH and gCDX-301. The data shown are the mean ± SD of the triple values. The data were normalized to 10 ug / mL of hFLT3L.P167.hIgG1.NG2LH as the maximum (100%) response. Figure 4B shows the EC50 potency of hFLT3L.P167.hIgG1.NG2LH and gCDX-301. The results are shown from five independent experiments using lines representing the mean ± SEM. [Figure 5] Figure 5 shows the serum concentrations of Flt3L-Fc (SEQ ID NO: 28) fusion protein detected in animals administered 0.1 mg / kg, 1 mg / kg, or 10 mg / kg of the fusion protein. [Figure 6] Figures 6A and 6B show dose-dependent cell proliferation of monocytes (Figure 6A) and DCs (Figure 6B) in animals administered with 0.1 mg / kg, 1 mg / kg, or 10 mg / kg of the fusion protein. Anti-gD is a negative control humanized IgG antibody. [Figure 7] Figure 7 shows the plasma concentrations of Flt3L-Fc(NG2LH) in animals administered with 0.1 mg / kg, 1 mg / kg, or 10 mg / kg of the fusion protein. Anti-gD is a negative control humanized IgG antibody. [Figure 8] Figures 8A to 8C show dose-dependent cell proliferation of monocytes (Figure 8A), cDC1 cells (Figure 8B), and cDC2 cells (Figure 8C) in animals administered 0.1 mg / kg, 1 mg / kg, or 10 mg / kg of the fusion protein. Anti-IgD is a negative control humanized IgG antibody. [Figure 9A] Figure 9A shows the immunogenicity of selected Fc-containing proteins. [Figure 9B] Figure 9B shows the immunogenicity of selected Fc-containing proteins. [Figure 10] Figure 10 shows the results of one representative in vitro ADCP assay described in Example 10, comparing the ADCP activity of various Flt3L-Fc proteins. [Modes for carrying out the invention]

[0059] Detailed description of embodiments of the invention I. Definition "Affinity" refers to the sum of the strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by methods common in the art, including those described herein. Specific illustrative descriptions and exemplary embodiments for measuring binding affinity are given below.

[0060] The term "Flt3 ligand," also called "Flt3L," refers to a protein that can bind to the Flt3 receptor (Flt3R) with sufficient affinity to be useful as a diagnostic and / or therapeutic agent when the protein targets the Flt3 receptor. In one embodiment, the degree of binding of the Flt3 ligand to an unrelated non-Flt3R protein is less than 10% of the binding of the protein to Flt3L, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the protein that binds to Flt3R has an affinity of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 nM). -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M. In certain embodiments, Flt3L binds to Flt3R that is conserved among Flt3R from different species.

[0061] As used herein, the term “Flt3L” refers to cleaved soluble Flt3L (e.g., approximately residues 27–185 of SEQ ID NO: 21), but unless otherwise specified, it may also refer to any native Flt3L from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term may encompass “full-length” unprocessed Flt3L, as well as any form of Flt3L resulting from, for example, intracellular processing (e.g., removal of the signal peptide (leader sequence) or cleavage from the TM domain). The term also encompasses naturally occurring variants of Flt3L, such as splice variants or allele variants. The amino acid sequence of an exemplary human Flt3L with an endogenous signal sequence is shown in SEQ ID NO: 21 and is also provided in GenBank accession record P49771, but in other embodiments, the amino acid sequence of a mature Flt3L protein without an exogenous leader sequence is provided as residues 27–167 of SEQ ID NO: 21. A small number of sequence variations, particularly conservative amino acid substitutions of Flt3L that do not affect the function and / or activity of Flt3L (e.g., binding to the Flt3 receptor (GenBank accession number NP_004110)), are also intended by the present invention.

[0062] As used herein, the terms “Flt3L-Fc fusion protein” or “Fc effectorless Flt3L-Fc fusion protein” refer to a fusion protein in which an Flt3L polypeptide is directly or indirectly linked to a variant IgG Fc region, the variant Fc region having reduced effector function compared to its corresponding wild-type Fc region. In any use of the term “effectorless Flt3L-Fc fusion protein,” it should be noted that the term “effectorless” applies to the Fc portion of the fusion protein. In certain preferred embodiments, the Flt3L-Fc fusion protein of the present invention comprises a human Flt3L protein or polypeptide linked to a human IgG Fc region. In certain embodiments, the human Flt3L protein comprises the amino acid sequence of SEQ ID NO: 22. However, it is understood that minor sequence mutations, such as insertions, deletions, substitutions, and especially conservative amino acid substitutions of Flt3L or Fc, which do not significantly affect the function and / or activity of the Flt3L or effectorless Flt3L-Fc fusion protein of the present invention (e.g., SEQ ID NO: 26), are also intended by the present invention. The effectorless Flt3L-Fc fusion protein of the present invention can bind to the Flt3 receptor protein (Flt3), which can lead to downstream signaling of the Flt3 receptor. As used herein, CDX-301 refers to Flt3L having the amino acid sequence of SEQ ID NO: 23.

[0063] The term "antibody" is used here in its broadest sense, encompassing a variety of antibody structures, including (but not limited to) monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0064] An "antibody fragment" refers to a molecule other than an intact antibody, which contains a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0065] The "class" of an antibody refers to the type of constant domain or constant region held by its heavy chain. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0066] "Effector function" refers to the biological activity resulting from the Fc region of an antibody, an isolated Fc region, or an Fc region fused to another protein, where the Fc region varies depending on the isotype of the antibody from which it originates. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, antibody-dependent cell phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. In certain embodiments, the Flt3L-Fc fusion protein exhibits no effector function or detectable effector function. In certain other embodiments, the Flt3L-Fc fusion protein exhibits substantially reduced effector function, e.g., about 50%, 60%, 70%, 80%, or 90% reduced effector function compared to a fusion protein containing the Flt3L protein of the present invention fused to a wild-type Fc protein having SEQ ID NO: 12.

[0067] As used herein, “dendritic cell proliferation,” “dendritic cell growth,” or “increase in the number of dendritic cells” refers to an increase in the number of dendritic cells measured in an in vitro assay or in vivo experiment.

[0068] The "effective dose" of a drug, such as a pharmaceutical preparation, refers to the amount that is effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome.

[0069] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes both the native sequence Fc region and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0070] As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. The T cell activation bispecific antigen-binding molecules of the present invention can induce T cell activation.

[0071] The terms “host cell,” “host cell line,” and “host cell culture” are interchangeable and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include “transformed cells” and “transformed cells,” which include primary transformed cells and offspring derived from primary transformed cells, regardless of passage number. Offspring may include mutations, although they may not be completely identical to the parent cells in terms of nucleic acid content. In this specification, offspring of mutants having the same function or biological activity as those screened or selected in the initially transformed cells are included.

[0072] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0073] "Isolated" proteins or polypeptides are those separated from components of their natural environment. In some embodiments, proteins are purified to a purity of over 95% or over 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purification, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0074] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found inside cells containing nucleic acid molecules, but the nucleic acid molecules are located outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0075] "An isolated nucleic acid encoding an Flt3L-Fc fusion protein" refers to one or more nucleic acid molecules encoding an Flt3L-Fc fusion protein, which may include such nucleic acid molecules in a single vector or separate vectors, and which may be located at one or more locations within a host cell.

[0076] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved in various methods within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the amino acid sequence identity % values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from its source code. The ALIGN-2 program should be compiled for use with UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0077] In situations where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity percentage of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (or, a given amino acid sequence A may be described as having or containing a certain amino acid sequence identity percentage to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y In the formula, X is the number of amino acid residues scored as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0078] The term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient contained therein is effective, and which does not contain any additional components that are unacceptably toxic to the person to whom the preparation is administered.

[0079] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the target substance. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0080] As used herein, “treatment” (and its grammatical variations, e.g., “to treat” or “to treat”) refers to a clinical intervention in an attempt to alter the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinicopathological condition. Desired effects of treatment include preventing the onset or recurrence of the disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, achieving remission or mitigation of the condition, and achieving a recovered or improved prognosis. In some embodiments, the fusion proteins of the present invention are used to delay the onset of the disease or to slow the progression of the disease.

[0081] The term “therapeutic effective dose” means an amount of the protein of the present invention that (i) treats or prevents a particular disease, symptom, or disorder; (ii) reduces, improves, or eliminates one or more symptoms of a particular disease, symptom, or disorder; or (iii) prevents or delays the onset of one or more symptoms of a particular disease, symptom, or disorder as described herein. In the case of cancer, a therapeutic effective dose of a combination of therapeutic agents or drugs as described herein may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow down, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow down, preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or mitigate one or more of the symptoms associated with cancer to some extent. A therapeutic effective dose of a combination of therapeutic agents or drugs as described herein may be cell proliferation inhibitory and / or cytotoxic to the extent that it can prevent the growth of existing cancer cells and / or kill them. With regard to cancer therapy, effectiveness can be measured, for example, by evaluating the time to disease progression (TTP) and / or determining the response rate (RR).

[0082] As used herein, the terms “cancer” and “malignant” refer to or describe physiological conditions in mammals typically characterized by uncontrolled cell growth. “Tumor” includes one or more cancerous cells. Tumors include solid tumors and liquid tumors. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, myelomas, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, malignant brain tumor, melanoma, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, head and neck cancer, and acute myeloid leukemia (AML).

[0083] As used herein, the terms “cold tumor” or “immune desert” refer to cancerous tumors that are low immunogenic or characterized by insufficient induction of tumor-specific immunity and resistance to immunogenic cytotoxicity.

[0084] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of a operably linked nucleic acid. Such vectors are referred to herein as “expression vectors.”

[0085] II. Compositions and Methods A. Effectorless Fc protein In one embodiment, the present invention provides a novel effectorless IgG1 Fc protein with surprisingly reduced effector activity. The Fc protein designed and described herein comprises the amino acid sequence of SEQ ID NO: 2 and is also referred herein to as "FcNG2LH" or "Fc NG PVA#". As shown herein, the FcNG2LH Fc protein exhibits significantly reduced ADCP activity (see, e.g., at least Examples 1 and 10), reduced activation of cytokine release by macrophages (see, e.g., Example 1), and reduced ADCC activity compared to proteins with wild-type IgG1 Fc domains. Also noteworthy is the remarkable increase in thermal stability of the FcNG2LH protein compared to the thermal stability of IgG1 Fc domains containing only the N297G mutation.

[0086] The design of effectorless IgG1 Fc domain proteins has utility in relation to many therapeutic molecules, in addition to its use in the Flt3L-Fc fusion proteins described herein. For example, antibodies designed to bind to and activate molecules (e.g., cell surface receptors) by crosslinking two or more molecules together, antibodies targeting checkpoint inhibitors, and bispecific or multispecific antibodies having at least one arm that binds to and induces ADCC and / or ADCP activity. Those skilled in the art will understand the use of the effectorless Fc protein described herein for the production of fusion proteins, in which the effectorless Fc protein is ligated (e.g., via amino acid bonds) to the N-terminus and / or C-terminus of a second protein, respectively. Preferably, the second protein has the desired therapeutic function. The effectorless Fc domain can prevent adverse reactions such as neutrophilia caused by binding to the IgG Fc receptor. In the field of therapeutic proteins, there is a need for continuous development of methods to improve the control of Fc effector function.

[0087] In one embodiment, the present invention provides an antibody in which the heavy chain constant domain comprises the FcNG2LH protein of SEQ ID NO: 2. In some embodiments, the Flt3L-Fc fusion proteins described herein include immunoglobulins and antibodies. Those skilled in the art can easily imagine a variety of antigen-specific antibody formats that can adapt to and benefit from the presence of NG2LH mutations and the resulting lack or reduction of effector function. For example, the human IgG1 FcNG2LH of this disclosure may be part of a monoclonal antibody, a human antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody. In other words, any immunoglobulin structure that contains or may contain the IgG1 Fc domain.

[0088] B. Exemplary Flt3L-Fc fusion protein In one embodiment, the present invention provides an Flt3L-Fc fusion protein that binds to the Flt3 receptor (Flt3R), wherein the Fc protein has virtually no effector function or significantly reduced effector function compared to the wild-type Fc protein. These fusion proteins are useful in the treatment of cancer with various cancer immunotherapy protocols, particularly when patients do not respond to checkpoint inhibitor immunotherapy or have a limited response to checkpoint inhibitor immunotherapy. The lack of antitumor immune response in "cold tumors" or "immune deserts" is thought to be at least partially due to insufficient or absent antitumor T cell response. Effective antitumor T cell response requires cross-presentation of tumor antigens by dendritic cells (DCs).

[0089] Therefore, one way to enhance the effectiveness of cancer immunotherapy is to increase the number of antigen-presenting DCs within the tumor. It is well known that Flt3L partially functions to increase DCs in a subject through the proliferation, differentiation, development, and recruitment of progenitor cells. Although there is evidence that Flt3L protein administered to healthy subjects can indeed proliferate DCs and hematopoietic stem cells (e.g., Anandasabapathy et al., 2015, Bone Marrow Transplant., 50:924-930), one concern is that recombinant Flt3L in the blood has a relatively short half-life, which may reduce therapeutic efficacy and / or necessitate too frequent administration.

[0090] A well-established strategy for extending the serum half-life of therapeutic proteins is to ligate or fuse them to an immunoglobulin Fc domain (e.g., via a peptide bond, preferably at the N-terminus of the Fc protein) (e.g., Czajkowsky et al., 2012, EMBO, 4:1015-1028; Ha et al., 2016, Front Immunol, 7:394). However, in the case of Flt3L-Fc fusion proteins for use in cancer immunotherapy, for example, having a functional Fc domain that can activate many unpredictable immunological or inflammatory processes in situ can be disadvantageous. Indeed, it is important to minimize the risk of administration resulting in cytokine storms or cytokine release syndrome (CRS). Furthermore, the interaction between the wild-type Fc of the Flt3L-Fc fusion protein domain and the Fc receptor on immune effector cells can lead to phagocytic disruption of dendritic cells (DCs) bound by the Flt3L-Fc fusion protein. It may be important to minimize and control the effect of the Fc region of the Flt3L-Fc fusion protein while maintaining the benefits provided by the Fc region. Described herein is a novel effectorless IgG Fc region, referred to herein as "Fc-NG2LH" or "Fc-PVA# N297G," which has been shown to have remarkably low effector function (see, for example, Example 7) compared to, for example, wild-type IgG1 Fc, IgG1 Fc-N297G, and IgG4 Fc regions. Furthermore, as shown in Example 8, the introduction of the NG2LH variant resulted in an unexpected increase in the thermal stability of the Fc domain and an impact on the half-life in animal serum.

[0091] Further characterization of the Flt3L-Fc fusion protein variants shows that substituting Flt3L W144 significantly increases immunogenicity as determined by in vitro T cell proliferation assays, suggesting that removing the potential oxidative tendency at position 149 of Flt3L (SEQ ID NO: 21) may be detrimental to therapeutic effectorless Flt3L-Fc fusion protein products. However, in contrast, Example 9 shows that substitution of potential glycosylation sites at positions 149 and 151 did not increase immunogenicity as determined by in vitro T cell proliferation assays, suggesting that mutations at these and other positions may be suitable for the production of Flt3L-Fc fusion proteins.

[0092] Importantly, in vivo administration of various doses of the effectorless Flt3L-Fc fusion protein disclosed herein resulted in significant dendritic cell proliferation (see, for example, Example 5). Furthermore, the level of DC proliferation was undoubtedly much greater than that observed with comparable doses of CDX-301.

[0093] The Flt3L-Fc fusion protein contains an Flt3 ligand (Flt3L) protein linked to the IgG1 Fc region, where IgG1 Fc has reduced or no effector function compared to the same Flt3L fused to wild-type IgG1 Fc. The Flt3L portion of the Flt3L-Fc fusion protein can bind to the cell surface Flt3 receptor (Flt3R). The sequence of the Flt3L protein is described in GenBank accession number P49771 (provided herein as Sequence ID No. 21) as a 235-amino acid protein having a signal peptide extending approximately from residues 1 to 26 and a transmembrane domain extending approximately from residues 185 to 205. Although residues 29 to 159 are listed in the GenBank records as an Flt3 ligand, Savvides et al. (Nat Struct Bio, 7:486-491) have shown that residues 27 to 160 alone are sufficient for biological activity. In some embodiments, the disclosure envisions an Flt3 ligand domain containing residues of SEQ ID NO: 21, wherein the N-terminus is any amino acid residue between positions 24 and 30 of SEQ ID NO: 21, and the C-terminus is any amino acid residue between positions 167 and 190 of SEQ ID NO: 21. Savvides et al. (ibid.) showed that polypeptides containing only residues 27-160 (and configured as dimers) are sufficient for ligand functional activity, but the study described in Example 2 below shows the use of an Flt3L protein in which the N-terminus of residue 167 of SEQ ID NO: 21 is terminated and covalently linked to, for example, the Fc N-terminal residue (of SEQ ID NO: 13). It is understood that variants of Flt3L, such as mature Flt3L proteins containing protein sequences having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21 residues 27-167, are also envisioned in the disclosure. Here (for example, in Example 3 below), it was shown that the size of the Flt3 fragment fused to the Fc protein can have a significant effect on the aggregation of the fusion protein.

[0094] In one embodiment, the present invention provides an Flt3L-Fc fusion protein comprising an Flt3L protein having an amino acid sequence at least 95% identical to residues 27-167 of SEQ ID NO: 21, and an IgG Fc molecule at least 95% identical to SEQ ID NO: 13. The IgG Fc molecule exhibits reduced Fc effector function compared to the wild-type IgG1 Fc polypeptide (SEQ ID NO: 12), with the Fc effector function being selected from CDC, ADC, ADCC, and / or ADCP. In some embodiments, the IgG Fc molecule exhibits reduced ADCP activity compared to the wild-type IgG1 Fc polypeptide. While not bound by theory, the low to absent effector function of the Fc(NG2LH) protein is thought to be due to the presence of a unique combination of both the N297G mutation (at position 76 in SEQ ID NO: 13) and the amino acid PVAGP (residues 13-17 in SEQ ID NO: 13) in the lower hinge of the Fc(NG2LH) protein.

[0095] a) Substitution, insertion, and deletion variants In certain embodiments, protein variants having one or more amino acid substitutions are provided. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial variations are shown in Table 1 under the heading "Exemplary Substitutions," and are further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into a fusion protein of interest, and the product can be screened for desired activity, such as reduced immunogenicity or reduced ADCP. [Table 1]

[0096] Amino acids can be classified according to their general side-chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0097] Non-conservative substitution involves swapping one member of one of these classes with one of another.

[0098] Amino acid insertions include amino-terminal and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intra-sequence insertions of one or more amino acid residues. An example of terminal insertion is the Flt3L-Fc fusion protein with an N-terminal methionyl residue. Other insertion variants of the Flt3L-Fc fusion protein molecule include fusion of the N-terminus or C-terminus of the Flt3L-Fc fusion protein with an enzyme (e.g., ADEPT) or a polypeptide that increases the serum half-life of the Flt3L-Fc fusion protein.

[0099] b) Glycosylated variants In certain embodiments, the Flt3L-Fc fusion protein provided herein is modified to increase or decrease the degree to which the Fc portion of the Flt3L-Fc fusion protein is glycosylated. Addition or deletion of glycosylation sites to the Fc domain can be easily achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed.

[0100] With respect to the Fc region, the carbohydrate bound to it can vary. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides that are commonly bound to Asn297 of the CH2 domain of the Fc region by N-bonding. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to the GlcNAc of the "stem" of the branched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the Flt3L-Fc fusion protein of the present invention may be performed to create effectorless Fc variants with specific improved properties.

[0101] In one embodiment, an Fc fusion variant is provided having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans at Asn297 relative to the total amount of all glycan structures attached to Asn297 (e.g., complex structures, hybrid structures, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, as described in International Publication No. 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (Eu numbering of Fc region residues) within the Fc region, although Asn297 may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to slight sequence variations within the antibody. Such fucosylated variants may possess improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.); and U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: U.S. Patent Application Publication No. 2003 / 0157108; International Publication No. 2000 / 61739; International Publication No. 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; International Publication No. 2002 / 0164328; International Publication No. 2004 / 0093621; International Publication No. 2004 / 0 Examples include publications No. 132140; No. 2004 / 0110704; No. 2004 / 0110282; No. 2004 / 0109865; International Publication No. 2003 / 085119; No. 2003 / 084570; No. 2005 / 035586; No. 2005 / 035778; No. 2005 / 053742; No. 2002 / 031140; Okazaki et al. J.Mol.Biol.336:1239-1249(2004); Yamane-Ohnuki et al. Biotech.Bioeng.87:614(2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108 A1, Presta, L; and International Publication No. 2004 / 056312 A1, Adams et al., particularly Example 11), and knockout cell lines, such as those containing the α-1,6-fucosyltransferase gene, FUT8, or knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and International Publication No. 2003 / 085107).

[0102] For example, Fc variants are further provided having a bifid oligosaccharide in which a branched oligosaccharide attached to the Fc region of the fusion protein is bifid by GlcNAc. Such Fc variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in International Publication 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication 2005 / 0123546 (Umana et al.). Also provided are Flt3L-Fc fusion protein variants having at least one galactose residue in the oligosaccharide bound to the Fc region. Such Flt3L-Fc fusion protein (and therefore Fc) variants may have improved CDC function. Such antibody variants are described, for example, in International Publication No. 1997 / 30087 (Patel et al.); International Publication No. 1998 / 58964 (Raju, S.); and International Publication No. 1999 / 22764 (Raju, S.).

[0103] c) Protein derivatives In certain embodiments, the effectorless Fc protein, the antibody or fusion protein containing the effectorless Fc protein, or the Flt3L-Fc fusion protein provided herein may be further modified to include further non-proteinogenic moieties known in the art and readily available. Suitable sites for the derivatization of the Flt3L-Fc fusion protein include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, prolypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers bound to the Fc fusion protein varies, and if multiple polymers are bound, they may be the same molecule or different molecules. In general, the number and / or types of polymers used for derivatization can be determined based on considerations including, but are not limited, the specific properties or functions of the improved Flt3L-Fc fusion protein, and whether the Flt3L-Fc fusion protein derivative will be used therapeutically under defined conditions.

[0104] In another embodiment, a conjugate of an Flt3L-Fc fusion protein and a non-proteinaceous moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the unprotected moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may be of any wavelength and may not harm normal cells, but may include, but is not limited to, wavelengths that heat the non-proteinaceous moiety to a temperature that kills cells adjacent to the fusion protein-non-proteinaceous moiety.

[0105] C. Recombination method and composition Flt3L fusion proteins can be produced using recombinant methods and compositions readily known to those skilled in the art. In one embodiment, an isolated nucleic acid encoding the Flt3L-Fc fusion protein described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the Flt3L and Fc portions of the Flt3L-Fc fusion protein. In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. In a further embodiment, host cells containing such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., transformed using): (1) a vector containing nucleic acids encoding the Flt3L polypeptide and the Fc polypeptide of the present invention. In a preferred embodiment, the nucleic acid encoding the Flt3L polypeptide is upstream of the nucleic acid encoding the Fc polypeptide. Furthermore, the two nucleic acids are in a single operon. In one embodiment, the host cell is a eukaryotic one, e.g., Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method for producing an Flt3L-Fc fusion protein is provided, which includes culturing host cells containing nucleic acids encoding an Flt3L-Fc fusion protein, as provided above, under conditions suitable for the expression of the Flt3L-Fc fusion protein, and optionally recovering the Flt3L-Fc fusion protein from the host cells (or host cell culture medium).

[0106] For recombinant production of the Flt3L-Fc fusion protein, for example, the nucleic acid encoding the Flt3L-Fc fusion protein described above is isolated, inserted into one or more vectors, and further cloned and / or expressed in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the gene encoding the fusion protein).

[0107] Host cells suitable for cloning or expressing effectorless Fc proteins, antibodies or fusion proteins containing Fc proteins, or vectors encoding, for example, Flt3L-Fc fusion proteins, include prokaryotic or eukaryotic cells as described herein. For example, Flt3L-Fc fusion proteins can be produced in bacteria, especially when glycosylation and Fc effector function are not required. For polypeptide expression in bacteria, see, for example, U.S. Patents No. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, Flt3L-Fc fusion proteins can be isolated from bacterial cell paste into a soluble fraction and further purified.

[0108] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable as cloning or expression hosts for vectors encoding Flt3L-Fc fusion proteins, including strains and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of Flt3L-Fc fusion proteins with a partially or completely human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0109] Host cells suitable for the expression of glycosylated Flt3L-Fc fusion proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified and can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0110] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0111] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspensions may be useful. Other examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed with SV40 (COS-7); human fetal kidney lineage (e.g., 293 cells or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical tumor cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); e.g., TRI cells as described in Mather et al., Annals NYAcad. Sci. 383:44-68 (1982); MRC These are 5 cells and FS4 cells. Other useful mammalian host cell lines include DHFR -Examples include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For an overview of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0112] D. Assay The Flt3L-Fc fusion proteins provided herein, which can promote the proliferation of DCs, can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0113] E. Immunoconjugate The present invention also provides an immunoconjugate comprising the Flt3L-Fc protein as defined herein, conjugated with one or more cytotoxic agents such as chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins (e.g., protein toxins, bacterial or fungal enzyme-active toxins of plant or animal origin, or fragments thereof), or radioisotopes.

[0114] In one embodiment, the immunoconjugate of the Flt3L-Fc protein is an antibody that contains maytansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent No. EP0425235B1); auristatins such as monomethyl auristatin drug sites DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,7 See Nos. 80,588 and 7,498,298); drastatin; calicheamycin or its derivatives (US Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman See et al., Cancer Res. 53:3336-3342 (1993); and Cancer Res. 58:2925-2928 (1998); anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters See 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065, among others, which are produced in a manner similar to the production of antibody-drug conjugates (ADCs) conjugated to one or more drugs.

[0115] In another embodiment, the immunoconjugate comprises the Flt3L-Fc protein described herein conjugated to an enzymatically active toxin or a fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecene.

[0116] In another embodiment, the immunoconjugate comprises the Flt3L-Fc protein described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioisotopes are available for the production of radioactive conjugates. Examples include At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 [[ID=十六]]、Sm[[ID=十七]] 153 [[ID=十八]]、Bi[[ID=十九]] 212 [[ID=二十]]、P[[ID=二十一]] 32 [[ID=二十二]]、Pb[[ID=二十三]] 212 [[ID=二十四]]、and radioisotopes of Lu. When used for detection, the radioactive conjugate may include radioactive atoms for scintigraphic examination, such as tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron, etc. [[ID=二十五]] [[ID=二十六]]

[0117] [[ID=二十七]] It should be noted that in the above translation, the Chinese characters in the original text that do not have corresponding English words are retained in pinyin for the sake of accuracy in expressing the original content. In actual patent translation, it is necessary to ensure compliance with relevant laws, regulations and professional norms, and may need to be adjusted according to specific requirements and contexts. If there are any specific requirements or corrections, please feel free to let me know.Conjugates of Flt3L-Fc protein with cytotoxic agents can be prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidylsberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and diactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugate of radioactive nucleotides to antibodies. See International Publication No. 94 / 11026. The linker may be a “cleavable linker” that facilitates the release of cytotoxic drugs within cells. For example, an acid-unstable linker, a peptidase-sensitive linker, a photosensitive linker, a dimethyl linker, or a disulfide-containing linker may be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0118] The immunoconjugates described herein are, but are not limited to, conjugates prepared using commercially available crosslinking reagents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0119] F. Pharmaceutical preparations Pharmaceutical formulations of Flt3L-Fc fusion proteins described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such proteins of a desired degree of purity with one or more pharmaceutically acceptable carriers ("Remington's Pharmaceutical Sciences," 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphates, citrates, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than approximately 10 residues) polypeptides, and serum. Examples of pharmaceutically acceptable carriers herein include, but are not limited to, proteins such as albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include intervening drug dispersants such as soluble neutrally active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins like rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968. In one embodiment, sHASEGP is combined with one or more further glycosaminoglycansases (e.g., chondroitinases).

[0120] The formulations described herein may also contain more than one, preferably complementary, non-adversely affecting, active ingredients necessary for the specific indication being treated. For example, it may be desirable to further provide dendritic cell maturation factors and / or adjuvants. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0121] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation technology or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), or encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such technologies are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0122] A sustained-release preparation may be prepared. A preferred example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing an Fc fusion protein, where these matrices are in the form of molded articles, such as films or microcapsules.

[0123] Preparations used for in vivo administration are generally sterilized. Sterilization can be easily achieved, for example, by filtration using a sterile filtration membrane.

[0124] G. Treatment methods and compositions Any of the Flt3L-Fc proteins provided herein can be used in therapeutic methods.

[0125] In one embodiment, an Flt3L-Fc protein is provided for use as a pharmaceutical. In a further embodiment, an Flt3L-Fc protein is provided for use in cancer. In a particular embodiment, an Flt3L-Fc protein is provided for use in a therapeutic method. In a particular embodiment, the present invention provides an Flt3L-Fc protein for use in a method of treating an individual having cancer, comprising administering an effective amount of the Flt3L-Fc protein to the individual. In such an embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, such as described below. In a further embodiment, the present invention provides an Flt3L-Fc protein for use in the proliferation of dendritic cells. In a particular embodiment, the present invention provides an Flt3L-Fc protein for use in a method of proliferating dendritic cells in an individual, comprising administering an Flt3L-Fc protein effective for proliferating dendritic cells to the individual. The “individual” in any of the above embodiments is preferably a human.

[0126] In a further embodiment, the present invention provides the use of the Flt3L-Fc protein in the manufacture or preparation of pharmaceuticals. In one embodiment, the pharmaceutical is for the treatment of cancer. In a further embodiment, the pharmaceutical is for use in a method of treating cancer, which comprises administering an effective amount of the pharmaceutical to an individual having cancer. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below. In a further embodiment, the pharmaceutical is for the proliferation of dendritic cells. In a further embodiment, the pharmaceutical is for use in a method of proliferation of dendritic cells in an individual, which comprises administering to the individual an effective amount of the pharmaceutical for the proliferation of dendritic cells. The “individual” according to any of the above embodiments may be a human.

[0127] In further embodiments, the present invention provides a method for treating cancer. In one embodiment, the method comprises administering an effective amount of Flt3L-Fc protein to an individual having such cancer. In such an embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, as described below. The “individual” according to any of the above embodiments may be a human.

[0128] In further embodiments, the present invention provides a method for growing dendritic cells in an organism. In one embodiment, the method comprises administering an effective amount of Flt3L-Fc protein to an organism for growing dendritic cells. In one embodiment, the "organism" is a human.

[0129] In a further embodiment, the present invention provides a pharmaceutical formulation comprising, for example, one of the Flt3L-Fc proteins provided herein for use in any of the therapeutic methods described above. In one embodiment, the pharmaceutical formulation comprises one of the Flt3L-Fc proteins provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises one of the Flt3L-Fc proteins provided herein and, for example, at least one additional therapeutic agent described below.

[0130] The Flt3L-Fc protein of the present invention can be used for treatment alone or in combination with other agents. For example, the Flt3L-Fc protein of the present invention can be co-administered with at least one additional therapeutic agent.

[0131] The combination therapies described above include combined administration (containing two or more therapeutic agents in the same or separate formulations) and separate administrations, in which case, administration of the Flt3L-Fc protein of the present invention may be performed before, simultaneously with, and / or after the administration of additional therapeutic agents (may be more than one). In one embodiment, the administration of the Flt3L-Fc protein and the administration of additional therapeutic agents occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days. The Flt3L-Fc protein of the present invention can also be used in combination with radiotherapy.

[0132] The Flt3L-Fc fusion protein of the present invention (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if desired, by intralesional administration in local treatment. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be by any preferred route, e.g., by injection, such as intravenous or subcutaneous injection, depending in part whether the administration is short-term or chronic. Various dosing schedules, including but not limited to single doses, multiple doses at various time points, bolus administration, and pulse infusion, are contemplated herein.

[0133] The Flt3L-Fc fusion protein for use in this invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to the healthcare professional. The Flt3L-Fc fusion protein does not need to be formulated together with one or more reagents currently used to prevent or treat the disease, but may be formulated together as such. The effective amount of such other reagents depends on the amount of Flt3L-Fc fusion protein present in the formulation, the type of disorder or treatment, and the other factors mentioned above. These are generally used by the same dosages and routes of administration as described herein, or at about 1–99% of the dosages described herein, or by any dosage and route that is empirically / clinically deemed appropriate.

[0134] For the prevention or treatment of disease, the appropriate dosage of the Flt3L-Fc fusion protein of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of Flt3L-Fc fusion protein, the severity and course of the disease, whether the fusion protein is administered for preventive or therapeutic purposes, previous treatments, the patient's medical history and response to the Flt3L-Fc fusion protein, and the discretion of the attending physician. The Flt3L-Fc fusion protein is administered appropriately to the patient either in a single dose or over a series of treatments. Depending on the type and severity of the disease, approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of Flt3L-Fc fusion protein may be the initial candidate dose for administration to the patient, for example, by one or more individual doses or by continuous infusion. A typical daily dose may range from approximately 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. In repeated administrations over several days or more, treatment is usually continued, depending on the symptoms, until the desired suppression of disease symptoms occurs. One exemplary dose of Flt3L-Fc fusion protein is in the range of approximately 0.05 mg / kg to approximately 10 mg / kg. Therefore, one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (for example, the patient receives approximately 2 to approximately 20 doses, or for example, approximately 6 doses of the fusion protein). An initial larger dose may be administered, followed by one or more smaller doses. However, other drug regimens may be useful.

[0135] It is understood that any of the above formulations or therapeutic methods may be carried out using the immunoconjugate of the present invention in place of or in addition to the Flt3L-Fc fusion protein.

[0136] H. Articles of Manufacture In another aspect of the present invention, a manufactured article is provided comprising a material useful for the treatment, prevention and / or diagnosis of the above-mentioned disorder. The manufactured article comprises a container and a label or package insert inserted into or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from a variety of materials such as glass or plastic. The container holds the composition to be used alone or in combination with another composition effective for treating, preventing and / or diagnosing the symptom and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). At least one activator in the composition is the Fc fusion protein of the present invention. The label or insert indicates that the composition is used to treat a selected symptom. Furthermore, the manufactured article may comprise (a) a first container containing the composition, wherein the composition comprises the Fc fusion protein of the present invention, and (b) a second container containing the composition, wherein the composition comprises a further cytotoxic agent or other therapeutic agent. The manufactured article in this embodiment of the present invention may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition thereto, the manufactured article may further comprise a second (or third) container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes, etc.

[0137] It will be understood that any of the above products may contain the immunoconjugate of the present invention in place of, or in addition to, the Flt3L-Fc fusion protein. [Examples]

[0138] III. Examples The following are examples of the methods and compositions of the present invention. Considering the general description provided above, it will be understood that various other embodiments may be implemented.

[0139] Example 1. Effectorless Fc protein design The term “NG2LH” refers to a human IgG1 Fc domain containing the IgG1 N297G substitution, which “swaps” lower hinge residues 233–236 of IgG1 (ELLG, SEQ ID NO: 19) with residues 233–236 of human IgG2 (PVA; no amino acid at position 236, also referred to herein as “PVA#”), where the positions are numbered according to the EU index as described in Kabat. The effectorlessness of the NG2LH Fc variant protein (also referred to herein as “PVA# NG”) was first shown in relation to full-length antibodies, compared to wild-type, N297G, and other Fc variant IgG1, as well as IgG2 and IgG4 antibodies, as defined in Table 2 below. [Table 2]

[0140] Full-length antibodies were constructed containing the heavy chain constant region (CH1-CH3) listed in Table 2, which encode the light chain variable domain and heavy chain variable domain of the anti-Her2 antibody (trastuzumab (HERCEPTIN®, Genentech, Inc., South San Francisco, Calif., USA) (US Patent No. 6,407,213 and also mentioned in Lee et al., J. Mol. Biol. (2004), 340(5):1073-93; referred to herein as Sequence ID No. 47). The light chains of these anti-Her2 antibodies contained the natural human C kappa constant region. Nucleic acids encoding the full-length light chain and various full-length heavy chains were cloned into pRK mammalian expression vectors (Eaton et al., 1986, Biochemistry 25:8343-47). The light chain and heavy chain nucleic acid vectors were co-expressed in CHO cells, and the antibodies were purified by protein A affinity chromatography followed by size exclusion chromatography.

[0141] Next, antibodies were tested for their effects on antibody-dependent phagocytosis (ADCP) and antibody-dependent cytokine release (ADCR) using primary macrophages and the SK-BR-3 human breast cancer cell line (ATCC® HTB-30) that overexpress Her2. Anti-gD antibody was used as a negative control antibody. Monocytes were isolated from donor PBMCs, stimulated with 25 ng / ml human MCSF-1, and differentiated into macrophages in T-175 tissue culture flasks for 5-7 days. The day before the ADCP and cytokine release experiments, all macrophages were stimulated with 50 ng / ml IFNγ (R&D Systems 285-IF-100 / CF) to differentiate into M1 macrophages. Macrophages and SK-BR-3 GFP cells were harvested by treatment with acutase (Millipore SCR005). The removed macrophages and SK-BR-3 cells were each divided into 2 x 10⁶ cells. 6 and 0.5x10 6The antibodies were resuspended in culture medium (X-Vivo 10; Lonza 04-743Q, 10% HI FBS; Gibco 10438-026) at a concentration of cells / ml. A 96-well plate map was designed to determine the test conditions (antibody variant and therapeutic concentration). 2x serial dilutions of each antibody variant were prepared in culture medium (6-point 10x dilution series: 2000, 200, 20, 2, 0.2, 0.02 ng / ml). According to the plate map, 50 μl of macrophages and 50 μl of SK-BR-3 were added to each well, yielding 100 kN of macrophages and 25 kN of SK-BR-3 cells in each well. According to the plate map, 100 μL of 2x serial dilution of the antibody was combined with the wells of the 96-well plate. The final antibody treatment concentrations were 1000, 100, 10, 1, 0.1, and 0.01 ng / ml. 100 μL of culture medium was added to three wells containing macrophages and SK-BR-3 cells as a "culture medium only" control treatment. The 96-well plate was centrifuged at 20 × g for 2 minutes and then incubated at 37°C for approximately 24 hours. Cells were centrifuged at 400 × g for 4 minutes, and the supernatant was collected for ADCR analysis by Luminex Reader Millipore Multiplex. Cells were removed from the wells by accutase treatment. Cells in each well were washed once with 200 μL of FACS buffer (BD, 554657). Staining solutions were prepared using anti-CD11b (BD, 555385) and anti-CD14 (BD, 555396) conjugated with Alexa 647 (Invitrogen, Alexa Fluor 647 Antibody Labeling Kit, A20186) (1:100 antibody conjugate in FACS buffer). 50 μL of staining solution was added to each well, and the cells were incubated at 4°C for 30–60 minutes. The cells were washed twice with FACS buffer (200 μL / well). The cells in each well were resuspended in 50 μL of FACS buffer. One plate of cells was analyzed by FACS for ADCP efficiency. The FACS data was analyzed using the software FlowJo. The cells were gated with a Forward and Side Scatter plot, and then GFP expression was plotted on the Y axis and Alexa647 on the X axis.The percentage of cells in each quadrant was calculated. SK-BR-3 cells were mainly in quadrant 1 (Q1, GFP-positive), and macrophages were mainly in quadrant 3 (Q3, Alexa647-positive). The phagocytic percentage for each well was calculated. Phagocytosis % = 100% - (Q1 of each well) / (Average Q1 of 3 control wells containing only culture medium)

[0142] The graph shows the percentage of phagocytosis of the antibody variant relative to the antibody treatment concentration.

[0143] Two plates were analyzed using Luminex (Luminex Corp). As shown in Figure 1, phagocytosis was almost completely absent in the presence of gradually increasing concentrations of LALAPG or NGLH2 Fc anti-Her2 antibody. The highest levels of ADCP were observed in IgG1 wild-type and IgG4 antibody variants. Intermediate levels of phagocytosis were observed for IgG2 wild-type Fc and IgG1 PVA#Fc. Therefore, Figure 1 shows that the IgG1 NG2LH and IgG1 LALAPG Fc domains lack ADCP activity in in vitro assays. Experiments show similar lack of ADCP activity in the presence of IgG1 NG PVAG and IgG1 NG PVA# GSS (data not shown).

[0144] The cytokine release profiles from the ADCP assay are provided in Figures 2A–2J. Consistent with phagocytic data, these results demonstrate that the NG2LH variant does not mediate ADCR by macrophages in the presence of target cells, in contrast to native IgG1, IgG4, and to a lesser extent to N297G.

[0145] These data are in contrast to those from ADCC reporter assays of variants compared to wild-type IgG1 Fc. Variable domains derived from ocrelizumab (anti-CD20) or trastuzumab (anti-Her2) were fused to the wild-type and variant domains described above and tested in ADCC assays. In this assay, NK cells were manipulated as NFAT-RE-luciferase reporter cells to read RcγRIIIa binding, and then assayed in the presence of WIL2S CD20+ cells or SkBr3 Her2+ cells. For both anti-CD20 and anti-Her2 constructs with wild-type or variant domains, all non-wild-type Fc molecules exhibited minimal activity compared to wild-type IgG1 Fc (see Figures 2K and 2L).

[0146] Example 2. Design of Flt3L Fc fusion protein The Flt3L-Fc fusion protein was generated using routine nucleic acid cloning, protein expression, and protein purification techniques.

[0147] Using PCR, cDNA containing nucleotide sequences encoding the Flt3L and Fc portions of the desired chimeric Flt3L-Fc protein (see Table 3, SEQ ID NOs. 10-31) was amplified. For the expression and production of the Flt3L-Fc proteins described in Table 3 (sequences provided in Table 10), cDNA was generated to contain the native signal sequence of human Flt3L (GenBank accession number P49771; residues 1-27 of SEQ ID NO. 21). As an example, the cDNA generated for the expression of the hFLT3L.P167.5aa.hinge.hIgG1.NG.PVA# protein contained a cDNA sequence encoding a fusion protein with the Flt3L signal sequence provided in Table 10 as SEQ ID NO. 45. [Table 3]

[0148] The cDNA encoding the Flt3L-Fc fusion was subcloned into the mammalian expression vector pRK5 (Gorman, et al., DNA and Protein Engineering Techniques 2:1 (1990); U.S. Patent No. 6,232,117) and expressed under the control of the CMV promoter.

[0149] Either CHO or HEK293 cells were transiently transfected with the desired expression construct, grown in appropriate culture medium for 10 or 6 days, respectively, and then harvested for purification.

[0150] Purification of the Flt3L-Fc fusion construct was achieved using HiTrap MabSelect SuRe affinity capture chromatography (GE), followed by Superdex 200 SEC, and finally concentration and buffer exchange by dialysis.

[0151] Example 3. Characterization of the Flt3L-Fc fusion protein The fusion proteins generated as described in Example 2 were characterized with respect to their susceptibility to aggregation in solution and to the reduction and loss of disulfide bonds, and each was analyzed by SDS-PAGE (3-5 ug / well) and HPLC SEC (SuperDex 200; equilibration buffer: 200 mM arginine, 137 mM succinic acid, pH 5.0). Data from experiments using Flt3L-Fc fusion proteins purified from CHO cell cultures are summarized in Table 4 below. Exemplary data from the Flt3L-Fc fusion construct hFLT3L.S163.no.hinge.hIgG1.NG.PVA# (SEQ ID NO: 41), expressed and purified in CHO and HEK293 cells, are shown in Figures 3A to 3C. Data from the Flt3L-Fc fusion construct hFLT3L.P167.5aa.hinge.hIgG1.NG.PVA# (SEQ ID NO: 26), expressed and purified in CHO and HEK293 cells, are shown in Figures 3D to 3F. [Table 4]

[0152] Data analysis of the variants summarized in Table 4 above showed that the number of amino acid residues (bond length) between the last cysteine ​​of the Flt3L molecule and the first cysteine ​​of the Fc(NG2LH) molecule can affect the level of aggregation and / or disulfide bond formation. When the bond length was less than 14 residues, up to 30% of each construct appeared to lack intermolecular hinged disulfide. Bond lengths of 5 amino acid residues or less resulted in very high levels of aggregation. The lowest levels of aggregation were observed for the fusion proteins of SEQ ID NOs. 24, 26, 32, 33, and 34. The lowest levels of incomplete hinged disulfide bonds were observed for the fusion proteins of SEQ ID NOs. 25, 26, and 32, and SEQ ID NOs. 33 and 39 also have relatively low levels of incomplete hinged disulfide. The data observed here were somewhat unexpected, given that the structures of both Flt3L and Fc proteins are well-characterized and have been shown to be able to fold as independent domains. However, it should be noted that each protein domain contains cysteine ​​residues crucial for disulfide bonds and structural integrity. Therefore, the data indicate that the design of Flt3L-Fc(NG2LH) fusion molecules requires careful evaluation of the protein product to ensure sufficient covalent Fc dimerization and minimization or absence of aggregation.

[0153] Example 4. In vitro functional assay for Flt3L activity In vitro assays were performed to evaluate the functional characteristics of the Flt3L-Fc fusion protein compared to the function of the Flt3L protein (without Fc fusion; SEQ ID NO: 23). Using the in vitro assays performed as described herein, the potency of the Flt3L protein in inducing proliferation in human cell lines expressing the Flt3 receptor can be measured.

[0154] Costar 96-well flat-bottom plates (catalog no. 3610 Thermo Fisher) were coated with 100 μL of poly-L-ornithine (catalog no. A-004-C EMD Millipore) overnight at room temperature, washed three times with PBS, and dried. Human OCI-AML5 cells (No. ACC 247, DSMZ) were seeded at 3000 cells / well in 100 μL of assay medium (RPMI1640 containing 5% thermo-inactivated FBS and 1×Glutamax). The Flt3L test product was diluted to 20 μg / mL with assay medium and serially diluted 1:10. 100 μL of the diluted test product was added to each well (in triplicate) to achieve a final starting concentration of 10 μg / mL. The largest and smallest control wells contained either 10 μg / mL of hFLT3L.P167.hIgG1.NG2LH (SEQ ID NO: 26) or assay medium alone. Assay plates were incubated at 37°C in 5% CO2 for 7 days. 100 μL of supernatant was taken from each well, and then 100 μL of Cell Titer Glo (catalog number PR-G7572 Thermo Fisher) was added to each well. The plates were incubated in the dark for 8–10 minutes. Luminescence was measured using a SpectraMax i3 plate reader (Molecular Devices). Raw values ​​were normalized to the maximum and minimum control wells. The dose response of each test was plotted using XLfit (IDBS), and the EC was measured. 50 The values ​​were determined using a four-parameter logistic curve. The graphs shown in Figures 4A and 4B were generated using GraphPad Prism7.

[0155] These data demonstrate that both hFLT3L.P167.hIgG1.NG2LH and gCDX-301 induced dose-dependent proliferation of OCI-AML5 cells in vitro. Average EC 50 The potency values ​​were equivalent: 2.5 ± 0.5 pM for hFLT3L.P167.hIgG1.NG2LH; 2.3 ± 0.5 pM for gCDX-301. Furthermore, hFLT3L.P167.hIgG1.NG2LH and gCDX-301 can induce proliferation of OCI-AML5 cells in vitro with at least equivalent potency.

[0156] Example 5. PKPD study in mice Given the complexity of the immune system, it may be important to demonstrate pharmacodynamics in mammalian subjects that can provide information about this human Fc molecule. PK / PD studies were conducted using mice as described herein.

[0157] This study was approved by Genentech's Institutional Animal Care and Use Committee and conducted using SCID mice. The animals were divided into three groups. Animals in groups 1, 2, and 3 were administered 0.1, 1.0, and 10 mg / kg of FLT3L-Fc (SEQ ID NO: 37--Flt3L.Q159.Fc.NG), respectively. Whole blood was collected at selected time points for cell population counting by FACS. Plasma was collected and assayed using huFlt3L ELISA to determine the amount of test substance in each sample.

[0158] The group-average PK parameters are summarized in Table 5 below. [Table 5]

[0159] Table 5 reports the following NCA parameters: max (Maximum serum concentration observed after the first dose); C max / dose(dose normalized C max );AUC inf (Area under the serum concentration-time curve from day 0 to infinity); AUC inf This includes / dose (dose-normalized AUC); CL (clearance calculated using serum concentration-time curve).

[0160] The results are shown in Figure 5 as a graph.

[0161] In groups 1-3, C maxThe increase was nearly dose-proportional. A dose-proportional increase in AUC was observed, suggesting that target-mediated pharmacokinetics (TMDD) influenced the PK profile in all dose groups. Furthermore, total clearance (CL) was observed to be dose-dependent, suggesting target-mediated CL.

[0162] The mean monocyte and DC counts from peripheral blood samples are shown in Figure 6A (monocytes) and Figure 6B (DCs). In all three groups, strong dose-dependent cell proliferation was observed in peripheral blood at doses of 0.1–10 mg / kg. The initial pharmacokinetics of DC proliferation were similar across doses, but the duration of proliferation was dose / exposure-dependent.

[0163] Example 6. PKPD study in cynomolgus monkeys Further PKPD studies were conducted using various doses of Flt3L-Fc(NG2LH) protein (SEQ ID NO: 26) administered to cynomolgus monkeys. This study was conducted at Charles River Laboratories (Reno, NV) using naive cynomolgus monkeys. The animals were divided into four groups (2 males and 1 female per group). Animals in group 1 were given 10 mg / kg of anti-gD (anti-glycoprotein D human IgG1 antibody with N297G mutation), while animals in groups 2, 3, and 4 were given 0.1, 1.0, and 10 mg / kg of FLT3L-Fc, respectively. Animals in group 2 received a single dose, while animals in the other groups received two doses (on day 1 and day 22 of the study). Whole blood was collected at selected time points for cell population counting by FACS. Serum was collected and assayed using anti-huFlt3L-huFc ELISA to determine the amount of test substance in each serum sample.

[0164] Table 6A below details the medication regimens for each animal group. Table 6B summarizes the group-average PK parameters. [Table 6A] [Table 6B]

[0165] The results are shown in Figure 7 as a graph.

[0166] In groups 2-3, C max The increase was roughly proportional to the dose. Anti-drug antibodies (ADAs) were detected in all doses of animals after day 14, and exposure affected the animals with high ADA titers. AUC after the first dose. 0-21 An increase exceeding dose-proportionality was observed, particularly at ≤1 mg / kg, suggesting that target-mediated pharmacokinetics (TMDD) influenced the PK profile. Furthermore, total clearance (CL) was observed to be dose-dependent, suggesting target-mediated CL. At 10 mg / kg, the anti-GD group and the FLT3L-Fc group had similar PK profiles, with a total CL of 4.92 mL / day / kg for the FLT3L-Fc group.

[0167] The mean monocyte, cDC1, and cDC2 counts for all four groups are shown in Figures 8A and 8C. In FLT3L-Fc-treated animals, strong dose-dependent cell proliferation (monocytes, cDC1, and cDC2) was observed in peripheral blood after the first dose at 0.1–1 mg / kg. Less proliferation was observed at 10 mg / kg. While not theoretically bound, a possible explanation is that high concentrations of Flt3 ligand may interfere with receptor dimerization necessary for proliferation and differentiation. At 1–10 mg / kg, lower but sustained cell proliferation was observed after the second dose (possibly due to the effects of ADA).

[0168] Example 7. FcγR and FcRn binding of Fc(NG2LH) The effector function of the Fc domain can be influenced by specific amino acid changes within the domain. Therefore, the binding of Fc(NG2LH) to FcγR and FcRn receptors was measured using Biacore. For these assays, FcγR was captured on the Biacore chip with an anti-His antibody, or FcRn was immobilized directly on the chip. Data were collected at the end of injection (association period), and relative binding activity (%) was calculated by normalizing the sample value by dividing it by the standard value.

[0169] Table 7 below summarizes the PTD Biocore data. The values ​​represent the relative binding level (%) at 10 ug / ml. [Table 7]

[0170] Example 8. Thermal stability of Fc(NG2LH) The thermal stability of FLT3L ligand fusion proteins is a critical aspect of developing therapeutic biologics. After designing the effectorless Fc(NG2LH) protein, differential scanning fluorescence (DSF) was performed to understand the effect of amino acid changes on the thermal stability of the Fc protein. DSF monitors the thermal unfolding of the protein in the presence of a fluorescent dye and is typically performed using a real-time PCR instrument (e.g., Bio-Rad CFX). SYPRO orange dye (Invitrogen, catalog no. S6650) is diluted 1:20 in PBS. 1 μl of the diluted dye is added to 24 μl of Fab protein (approximately 100 μg / ml) in a well. In a real-time PCR instrument (Bio-Rad CFX), fluorescence intensity is plotted as the temperature increases from 20°C to 100°C, and the inflection point (Tm) of the transition curve is calculated, for example, using Boltzmann's equation. See Nature Protocols, 2007, 2:2212-2221.

[0171] The variant Fc proteins were analyzed in relation to full-length antibodies, specifically as an anti-CD20 antibody derived from ocrelizumab and as an anti-Her antibody derived from trastuzumab. Stability data for these variant antibodies are provided in Table 8 below. Furthermore, the variant Fc proteins were also tested in relation to isolated Fc regions containing the sequences described in Table 2 but lacking the CH1 domain, and therefore starting at the N-terminus with the hinge sequence DKTHT. Stability data for these variant Fc proteins are provided in Table 9 below. [Table 8] [Table 9]

[0172] The data showed that combining the PVA# variant with the N297G substitution resulted in an increase in thermal stability of approximately 2°C compared to the Fc construct with only the N297G mutation, for both full-length IgG and isolated Fc proteins, which was unexpected.

[0173] Example 9. Variant of effectorless Flt3L-Fc fusion protein Experiments were conducted to evaluate the immunogenicity of various Flt3L-Fc fusion proteins. Specifically, the immunogenicity of specific proteins was tested using a T-cell proliferation assay. As shown in Figure 9A, both Flt3L.P167.Fc.NG2LH (SEQ ID NO: 26) and Flt3L.L165.Fc.NG2LH (SEQ ID NO: 33) exhibited detectable immunogenicity. Unexpectedly, significant immunogenicity was detected upon introduction of the Flt3L W144D mutation (SEQ ID NO: 42).

[0174] In another experiment, the immunogenicity of glycosylated variants of the Flt3L protein was evaluated. As shown in Figure 9A, the introduction of N149E, S151D, or S151E mutations did not result in a detectable increase in CD4 T cell proliferation. N149E, S151D, and S151E represent amino acid changes and positions relative to SEQ ID NO: 21.

[0175] Example 10. Effectorless Flt3L-Fc fusion protein in ADCP assay Experiments were conducted to compare the antibody-dependent phagocytic activity (ADCP) of three variants of the human recombinant protein Flt3L-Fc construct: 1) Flt3L-Fc wild-type IgG1 (SEQ ID NO: 27), Flt3L with hIgG1.NG2LH (SEQ ID NO: 26), and 3) Flt3L with hIgG1.N297G (SEQ ID NO: 28). The anti-Flt3 monoclonal antibody EB10.hIgG1 (Piloto et al., 2006, Cancer Res. 66:4843-4851) was used as a positive control. Primary monocyte-derived macrophages from healthy human donors were used as effector cells and SEM cells, and human acute lymphoblastic leukemia cells were used as target cells to perform ADCP assays. In short, primary human macrophages were generated by isolating CD14-positive cells from healthy human PBMCs using positive selection (Miltenyi Biotec Inc, Auburn, California). These cells were cultured in macrophage differentiation medium (RPMI1640, 10% FBS, 1% Glutamax, 1% penicillin / streptomycin, and 20 ng / mL M-CSF (R&D Systems, Minneapolis, MN)) at 37°C in a humidified incubator with 5% CO2. On day 3, the cells were stimulated with 50 ng / mL M-CSF and cultured for a further 4 days. On day 7, the macrophages were stained with 10 μM Cell Trace Violet (Thermo Fisher Scientific, Eugene, OR) and subjected to 1 × 10⁶ assay in ADCP assay medium (IMDM, 10% FBS, 1% Glutamax, 1% penicillin / streptomycin). 6 The cells were diluted to 50 μL / well and added to 96-well low-adhesion U-bottom plates (Costar, Corning, NY). Target SEM cells were incubated in ADCP assay medium at a rate of 2 × 10⁶. 6The antibody was diluted to cells / mL, pre-labeled with pHrodo (Thermo Fisher Scientific, Eugene, OR), and added to assay plates containing macrophages (50 μL / well). Then, 100 μL serial dilutions of the test antibody (Table 1) were added to each well containing macrophages and SEM cells, followed by incubation at 37°C with 5% carbon dioxide for 4.5 hours. The final antibody concentrations for a total of 11 samples per test antibody ranged from 0.457 to 3000 ng / mL after 3-fold serial dilution. After incubation, cells were centrifuged at 1200 rpm for 5 minutes, washed in PBS, and fixed in 4% paraformaldehyde for 10 minutes at 4°C. Cells were analyzed using a flow cytometer (BD Biosciences FACSCanto IVD 10). Phagocytosis was analyzed by FlowJo (Tree Star, Inc.; Ashland, OR). Macrophages in the sample population were gated using Cell Trace Violet fluorescence. Phagocytosis was determined by measuring the percentage of pHrodo Green-positive macrophages. The degree of phagocytosis was normalized by subtracting the percentage of pHrodo Green-positive macrophages from the control condition (absence of antibody). All data points were collected in dice rolls. The percentage of phagocytosis (%ADCP) was plotted against antibody concentration and fitted to a four-parameter model using GraphPad Prism (LaJolla, CA). This procedure was performed in independent experiments using three donors.

[0176] In this study, Flt3-expressing SEM cells were used as target cells. SEM target cells were pre-labeled with pHrodo AM, which fluoresces brightly at acidic pH. As the phagosomes containing the target cells became increasingly acidic, the pHrodo green fluorescence signal increased, which could be detected by flow cytometry. ADCP activity induced by Flt3L with hIgG1.NG2LH Fc (SEQ ID NO: 26, 5.1 mg / mL), Flt3L with hIgG1.N297G (SEQ ID NO: 28, 1.92 mg / mL), Flt3L with WT IgG1 (SEQ ID NO: 27, 2.5 mg / mL), and EB10.hIgG1 (3.17 mg / mL) was investigated and compared in three independent experiments using monocyte-derived macrophages from three different donors.

[0177] All data points were collected in pairs, and the mean % phagocytic activity (%ADCP) was plotted against the concentration of the tested molecule (drug concentration (concertation): 3 ug / mL to 0.457 ng / mL). The data were fitted to a 4-parameter model. Representative dose-response ADCP curves are shown in Figure 10. Due to the inherent limitations of the ADCP assay, in all three experiments, ADCP activity induced by Flt3L and WT IgG1 and EB10.hIgG1 was observed in Flt3-expressing cells. Furthermore, FLT3L-Fc-IgG-N297G induced relatively low ADCP, while FLT3L-Fc-IgG NG2LH showed little to no detectable ADCP activity.

[0178] Further studies can be conducted to measure the ADCP activity of additional Flt3L-Fc fusion proteins, including Fc NG2LH (SEQ ID NO: 13), such as Flt3L-Fc fusion proteins containing one, two, or three amino acid substitutions in the Flt3L portion. Such studies may reveal additional Flt3L-Fc fusion proteins that have little or no ADCP activity but can activate DC proliferation in vivo. [Table 10] JPEG0007847578000012.jpg233170JPEG0007847578000013.jpg238170JPEG0007847578000014.jpg237170 JPEG0007847578000015.jpg236170JPEG0007847578000016.jpg237170JPEG0007847578000017.jpg125170

[0179] Although the above invention has been described in some detail by description and examples for the purpose of clarifying understanding, the description and examples should not be construed as limiting the scope of the invention. All patent and scientific literature disclosures cited herein are expressly incorporated in their entirety by reference.

Claims

1. A Flt3L-Fc fusion protein comprising an effectorless Fc protein, wherein the Flt3L-Fc fusion protein comprises a Flt3 ligand (Flt3L) protein and an effectorless Fc protein, the effectorless Fc protein is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, and the effectorless Fc protein comprises residues 13-17 of SEQ ID NO: 13 containing the amino acid sequence PVAGP (SEQ ID NO: 20), and residue 76 of SEQ ID NO: 13 which is glycine.

2. The Flt3L-Fc fusion protein according to claim 1, wherein the Flt3L protein does not contain the amino acid sequence of PWSPRPLEATAPTAPQPP (SEQ ID NO: 48), WSPPRPLEATAPTAPQPP (SEQ ID NO: 49), SPRPLEATAPTAPQPP (SEQ ID NO: 50), PRPLEATAPTAPQPP (SEQ ID NO: 51), RPLEATAPTAPQPP (SEQ ID NO: 52), or PLEATAPTAPQPP (SEQ ID NO: 53).

3. The Flt3L-Fc fusion protein according to claim 1 or 2, wherein Flt3L comprises a protein having an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

22.

4. The Flt3L-Fc fusion protein according to any one of claims 1 to 3, wherein Flt3L comprises a protein having the amino acid sequence of SEQ ID NO:

22.

5. The Flt3L-Fc fusion protein according to any one of claims 1 to 4, wherein Flt3L comprises a protein that is at least 90% identical to a protein containing amino acids 27-167, 27-168, 27-169, 27-170, 27-171, 27-172, 27-173, 27-174, 27-175, 27-176, 27-177, 27-178, 27-179, 27-180, 27-181, 27-182, 27-183, 27-184, or 27-185 of SEQ ID NO:

21.

6. The Flt3L-Fc fusion protein according to any one of claims 1 to 5, wherein the N-terminus of an effectorless Fc protein is linked to the C-terminus of a Flt3L protein via a peptide bond.

7. The Flt3L-Fc fusion protein according to any one of claims 1 to 6, wherein the fusion protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26, and also comprises an amino acid sequence identical to SEQ ID NO:

13.

8. The Flt3L-Fc fusion protein according to any one of claims 1 to 7, wherein the Flt3L-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:

26.

9. The Flt3L-Fc fusion protein according to any one of claims 1 to 8, wherein the Flt3L protein consists of the amino acid sequence of SEQ ID NO:

22.

10. The Flt3L-Fc fusion protein according to any one of claims 1 to 9, wherein the effectorless Fc consists of the amino acid sequence of SEQ ID NO:

13.

11. The Flt3L-Fc fusion protein according to any one of claims 1 to 10, wherein the Flt3L-Fc fusion protein consists of the amino acid sequence of SEQ ID NO:

26.

12. A Flt3L-Fc fusion protein according to any one of claims 1 to 11, having attenuated Fc effector function compared to the Fc effector function of the wild-type IgG1 Fc region containing SEQ ID NO:

12.

13. The Flt3L-Fc fusion protein according to any one of claims 1 to 12, wherein the Flt3L-Fc fusion protein activates antibody-dependent cell phagocytosis (ADCP) in an in vitro assay at an activity level not exceeding 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the activity level in an in vitro assay using a Flt3L-Fc fusion protein containing wild-type Flt3L fused to wild-type IgG1 Fc (SEQ ID NO: 12).

14. An isolated nucleic acid encoding the Flt3L-Fc fusion protein according to any one of claims 1 to 13.

15. The isolated nucleic acid according to claim 14, further encoding a signal sequence at the N-terminus of the Flt3L-Fc fusion protein.

16. An isolated nucleic acid encoding a Flt3L-Fc fusion protein, wherein the Flt3L-Fc fusion protein contains Sequence ID No.

45.

17. A host cell comprising the nucleic acid described in any one of claims 14 to 16.

18. A method for producing the Flt3L-Fc fusion protein according to any one of claims 1 to 13, comprising culturing the host cell according to claim 17.

19. The method according to claim 18, wherein the host cell is a eukaryotic cell or a prokaryotic cell.

20. The method according to claim 18 or 19, wherein the host cell is Escherichia coli.

21. The method according to claim 18 or 19, wherein the host cell is a CHO cell.

22. A pharmaceutical formulation comprising a Flt3L-Fc fusion protein according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

23. The pharmaceutical formulation according to claim 22, further comprising a second therapeutic agent.

24. The pharmaceutical formulation according to claim 23, wherein the second therapeutic agent is an adjuvant, a dendritic maturation factor, and / or a checkpoint inhibitor.

25. The pharmaceutical preparation according to claim 23 or 24, wherein the second therapeutic agent is a dendritic maturation factor.

26. The pharmaceutical formulation according to claim 23 or 24, wherein the second therapeutic agent is a checkpoint inhibitor.

27. A second therapeutic agent, which is a dendritic maturation factor; and A third type of therapeutic agent, a checkpoint inhibitor. The pharmaceutical preparation according to claim 22, further comprising:

28. The pharmaceutical formulation according to claim 24, 25, or 27, wherein the dendritic maturation factor is selected from polyIC, polyICLC, and DC40.

29. The pharmaceutical formulation according to claim 24, 26, or 27, wherein the checkpoint inhibitor is selected from an antibody that specifically binds to PD-L1, an antibody that specifically binds to PD-1, and an antibody that specifically binds to CTLA-4.

30. A pharmaceutical agent for increasing the number of dendritic cells (DCs) in a target, comprising the Flt3L-Fc fusion protein described in any one of claims 1 to 13.

31. The pharmaceutical product according to claim 30, wherein the Flt3L-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:

26.

32. The pharmaceutical product according to claim 30, wherein the Flt3L-Fc fusion protein consists of the amino acid sequence of SEQ ID NO:

26.

33. A pharmaceutical product according to any one of claims 30 to 32, wherein a dose of 0.1 mg / kg to 50 mg / kg of Flt3L-Fc fusion protein is administered to the target.

34. A pharmaceutical product for treating a target cancer requiring treatment, comprising the Flt3L-Fc fusion protein described in any one of claims 1 to 13 or the pharmaceutical formulation described in any one of claims 22 to 29.

35. The pharmaceutical product according to claim 34, wherein the Flt3L-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:

26.

36. The pharmaceutical product according to claim 34, wherein the Flt3L-Fc fusion protein consists of the amino acid sequence of SEQ ID NO:

26.

37. A pharmaceutical product according to any one of claims 34 to 36, wherein a dose of 0.1 mg / kg to 50 mg / kg of Flt3L-Fc fusion protein is administered to the target.

38. Furthermore, the pharmaceutical product according to any one of claims 34 to 37, wherein a second therapeutic agent is administered to the target.

39. The pharmaceutical product according to claim 38, wherein the second therapeutic agent is an adjuvant, a dendritic maturation factor, and / or a checkpoint inhibitor.

40. A pharmaceutical product according to any one of claims 30 to 39, wherein a dendritic maturation factor is administered.

41. The pharmaceutical product according to claim 40, wherein the dendritic maturation factor is selected from poly-IC, poly-ICLC, DC40, radiotherapy, and chemotherapy.

42. A pharmaceutical product according to any one of claims 30 to 41, wherein an immune checkpoint inhibitor is administered.

43. The pharmaceutical product according to claim 42, wherein the checkpoint inhibitor is administered before, simultaneously with, or after the administration of the Fc effectorless Flt3L-Fc fusion protein.

44. The pharmaceutical product according to claim 42 or 43, wherein the checkpoint inhibitor is selected from an antibody that specifically binds to PD-L1, an antibody that specifically binds to PD-1, and an antibody that specifically binds to CTLA-4.

45. The pharmaceutical product according to any one of claims 42 to 44, wherein the immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, pizilizumab, BMS 936559, atezolizumab, and avelumab.

46. A pharmaceutical product according to any one of claims 30 to 45, wherein a dendritic maturation factor and a checkpoint inhibitor are administered.

47. The pharmaceutical product according to any one of claims 34 to 46, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), melanoma, pancreatic ductal adenocarcinoma (PDAC), triple-negative breast cancer (TNBC), non-Hodgkin lymphoma (NHL), colorectal cancer (CRC), breast cancer, bladder cancer, kidney cancer, or a combination thereof.

48. A pharmaceutical product according to any one of claims 30 to 47, wherein the subject has been previously treated with a checkpoint inhibitor.

49. The pharmacopoeia according to claim 48, wherein the subject has not responded to treatment with a checkpoint inhibitor, and / or has not responded to treatment with a checkpoint inhibitor for a period longer than 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 year, 1.5 years, or 2 years after receiving the last dose of checkpoint immunotherapy.

50. The pharmaceutical product according to any one of claims 34 to 49, wherein the cancer in the target is characterized to include immune desert tumors.

51. An effectorless Fc protein comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13, wherein the effectorless Fc protein comprises residues 13-17 of SEQ ID NO: 13 containing the amino acid sequence PVAGP (SEQ ID NO: 20), and residue 76 of SEQ ID NO: 13 which is glycine.

52. The effectorless Fc protein according to claim 51, comprising the same amino acid sequence as SEQ ID NO: 13.

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