Targeted FLT3l immunoconjugates and methods of use

Abstract

The invention provides an immunoconjugate comprising a mutant FLT3L cytokine and a targeted at least bivalent antibody that binds to CLEC9A, wherein the affinity of the mutant FLT3L is reduced as compared to the wild type FLT3L as measured by surface plasmon resonance (SPR) at 25°Canti and methods of using the same.

Description

[0001] Roche Reference: P39731

[0002] TARGETED FLT3L IMMUNOCONJUGATES AND METHODS OF USE TECHNICAL FIELD

[0003] The present invention relates to targeted FLT3L immunoconjugates and methods of using the same. Particularly the targeted FLT3L immunoconjugates comprise an affinity attenuated FLT3L dimer and an antibody that binds a target cell antigen e.g. CLEC9A. In addition, the invention relates to polynucleotide molecules encoding the immunoconjugates, and vectors and host cells comprising such polynucleotide molecules. The invention further relates to methods for producing the targeted FLT3L immunoconjugate, pharmaceutical composition comprising the same, and uses thereof.

[0004] BACKGROUND

[0005] The growth factor FMS-like tyrosine kinase 3 ligand (FLT3L), which uniquely binds FMS-like tyrosine kinase-3 (FLT3, CD135), is the master cytokine in promoting hematopoietic progenitor commitment to the dendritic cell (DC) lineage as well as DC survival and proliferation in tissues (Liu and Nussenzweig, Eur J Immunol. 2010). The human FLT3 ligand (huFLT3L) occurs in two different protein isoforms: a membrane-spanning form, which is the most common isoform and a soluble protein without a membrane anchor that is released into the circulation from the cell membrane by protease cleavage. The soluble as well as the membrane-bound FLT3L form a non-covalently linked homodimer.

[0006] The FLT3L is expressed by a wide variety of hematopoietic and non-hematopoietic cells in most tissues, including blood-forming organs (i.e. spleen, thymus, peripheral blood and bone marrow). Natural killer (NK) cells and T cells (mainly CD4+) as well as endothelial cells express the highest FLT3L levels (Barry et al., Nat Med. 2018; Solanilla et al., Leukemia. 2000). The wide expression of the ligand is in contrast to the limited expression pattern of the cognate receptor CD 135, which is expressed on hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), immature thymocytes, and steady state DC (Bertho et al., Scand J Immunol. 2000; Gabbianelli et al. Blood 1995; Karsunky et al., J Exp Med. 2003). This expression profile suggests that CD135 expression is the limiting step in determining the tissue-specificity of receptor activation.

[0007] Binding of the FLT3L to CD135 induces the homodimerization of the receptor that results in the phosphorylation of tyrosine kinase domains and subsequently the activation of further downstream effector molecules. The receptor activation is followed by a rapid receptor internalization and degradation (Tsapogas et al., Int J Mol Sci. 2017). Described FLT3L mutations such as L27D or L27P that disrupt the dimerization interface of the ligand, do not fully prevent the formation of dimeric ligands but rather shift the equilibrium from dimeric to monomeric FLT3L. Thereby, the balance between monomeric and dimeric FLT3L is mainly defined by the ligand concentration. This indicates that the reported reduced biological activity of the mutated ligands does not result from the ability of the monomeric FLT3L to induce receptor signaling but rather is the result of an equilibrium shift with less dimeric ligands that induce receptor activation (Graddis et al., J Biol Chem. 1998).

[0008] The human soluble FLT3L displays a high homology to the mouse soluble Flt3L and is cross-reactive to the mouse CD135 receptor. Therefore, human soluble FLT3L have been extensively used to expand mouse DCs in vitro and in vivo. Additionally, recombinant human FLT3L (huFLT3L) has been tested in more than 500 subjects in the clinic, including more than 300 cancer patients. Thus, there is profound pre-clinical and clinical data supporting the use of recombinant huFLT3L in mice and humans to differentiate DCs from their precursors for different medical indications (e.g. stem cell mobilization for transplantation, cancer immunotherapy and as a vaccine adjuvant).

[0009] The currently clinically validated recombinant huFLT3L (CDX-301 - CellDex Therapeutics) requires daily subcutaneous dosing for 7-10 days, but is well tolerated at doses up to 75 pg / kg (Anandasabapathy N. Bone Marrow Transplant. 2015). CDX-301 FLT3L efficiently induces expansion of peripheral monocytes and conventional DCs (eDCs), including cross-presenting conventional dendritic cell type 1 (cDCl) and type 2 (cDC2) and plasmacytoid DCs. This occurs without an appreciable effect on the number of Treg cells, NKT cells, B cells and CD4 or CD8 T cells (Bhardwaj N. Nat Cancer. 2020). Additionally, no anti drug antibody (ADA) incidence has been reported.

[0010]

[0011] However, the recombinant huFLT3L cytokine has some major limitations including: 1) poor PK and needs to be injected every day for 7-10 days in order to induce significant effects on myeloid cell expansion, 2) no single agent efficacy has been observed in cancer patients, and combination with other therapies is required to induce tumor growth remission in some indications and 3) the recombinant FLT3L induces the proliferation of many other cell types besides cDCl differentiation, including cells that are associated with tumor growth progression, such as monocytes and granulocytes, which are reported to be pro-tumorigenic, and contributing to an immunosuppressive tumor microenvironment (Wu et al., Cancers. 2019).

[0012] A cDCl -targeted FLT3L cytokine e.g. CLEC9A-targeted FLT3L immunoconjugate has the potential to overcome these limitations, by shifting the myeloid cell differentiation towards the cDCl subset with minimal effect on other myeloid cell subsets expansion.

[0013] The cDCl subset of dendritic cells is particularly interesting, because of their well-demonstrated and critical role in eliciting CD8+ T cells that can kill tumor cells (Wculek SK, Krummel MF, Sancho D et al. Nat Rev Immunol. 2020). cDCls are, from all the myeloid cells present in the body, the most skillful in antigen cross-presentation, a process of presenting exogenous antigens on MHC class I to activate CD8+ T cells. Tumor-associated cDCls can transport tumor antigen to the draining lymph node and cross-present tumor antigens, resulting in priming and activation of cytotoxic T cells (Bbttcher JP, Reis e Sousa C. Trends Cancer. 2018). Therefore, antigen presentation by cDCls is critical to maximize immune responses against cancer.

[0014] However, a main limitation is that DC1 cells are a rare immune cell subset, corresponding to less than 1% of the total amount of all human peripheral blood leukocytes. In addition, studies in lung cancer patients have demonstrated that these cells are even rarer in lung tumors compared to adjacent healthy lung tissues (Lavin Y., Merad M., Cell, 2017). Therefore, strategies aimed at increasing cDCl abundance in tumors and enhancing their functionality provide attractive new avenues to boost anti-tumor immunity and overcome resistance to cancer immunotherapies.

[0015] A suitable targeting receptor to deliver FLT3L specifically to cDCls is the C-type lectin domain containing 9A (CLEC9A or DGNR-1) due to its exclusive expression on this small population of eDCs, but also present in their corresponding stem cell precursors.

[0016] CLEC9A is a dimeric and well-described member of the membrane bound C-type lectin receptor family. Receptors of this family act as pattern recognition receptors and are involved in the

[0017]

[0018] innate immune response to pathogens. These receptors get quickly internalized upon ligand binding, routing to the endosomes, with the purpose of pathogen clearance and pathogen-associated antigen presentation to T cells. To this date, ligands for CLEC9A have been shown to include actin filaments which are exposed upon necrotic cell death (Zhang JG et al. Immunity. 2012) and complex of F-actin with myosin II greatly improves receptor binding, signaling, and cross-presentation of dead cell associated antigens (Giampazolias E, Reis e Sousa C. et al Cell. 2021). Upon binding of dead cell-associated antigens by CLEC9A, the receptors route to the early endosome, followed by endosomal rupture and release of the processed cargo into the cytosol for the cross-presentation of antigens on MHC class I, and thereby favors dependence of the CD8+ T cell response on crosspriming.

[0019] Expression of CLEC9A is highly restricted to DC1 sub-type in both mouse and human suggesting that the receptor may play a prominent role in regulation of CD8+ T cell responses (Caminschi et al. Blood. 2008; Huysamen et al. J Biol Chem. 2008; Poulin et al. J Exp Med. 2010; Sancho et al. J Clin Invest. 2008). Consistent with that possibility, the receptor is non-redundant in mice for efficient cross-priming of cytotoxic T- cells against dead cell associated antigens, making it the first reported innate immune receptor to bridge DAMP sensing to the induction of T cell immunity (Sancho et al. Nature. 2009).

[0020] WO 2017 / 134301 discloses a variable heavy homodimer (VHH) binding to human CLEC9A (huCLEC9A) and murine CLEC9A.

[0021] Taken together, we believe that targeting FLT3L cytokine to CLEC9A-expressing cDCls can have a drastic impact on the type of myeloid cells that expand and differentiate in cancer patients, preferably increasing the total numbers of highly competent cross-presenting DC Is and ultimately mounting an enhanced anti-tumor immune responses.

[0022] SUMMARY

[0023] The invention provides targeted FLT3L immunoconjugates and methods of using the same. Particularly the targeted FLT3L immunoconjugates comprise an affinity attenuated FLT3L polypeptide and an antibody that binds a target cell antigen e.g. CLEC9A, Clec4C, Clecl2A or CleclOA. The invention also relates to the use of the targeted FLT3L immunoconjugates provided,

[0024]

[0025] in combination with other therapeutic agents, in particular for the use in treating or delaying progression of cancer.

[0026] In one aspect, the invention provides an immunoconjugate comprising a cytokine and an antibody, wherein the cytokine is a mutant FLT3L cytokine dimer with reduced affinity as compared to wild type FLT3L cytokine as measured by SPR at 25°C or cell proliferation assay and wherein the antibody is at least bivalent for a target cell antigen, optionally wherein the target cell antigen is CLEC9A.

[0027] In a certain aspect, the affinity of the mutant FLT3L cytokine compared to the wild type FLT3L cytokine is reduced by at least 1.2 fold, at least 2.9 fold, at least 3 fold, at least 3.2 fold, at least 3.5, at least 5.4, at least 10.8,atleast 11.3,atleast 13.8, at least 20.7, at least 34.1,atleast 47.6, at least 67.7,atleast 73.8,atleast 113.7 or at least 201.7, as measured by SPR at 25°C.

[0028] In a certain aspect, the antibody is an antibody fragment, particularly a Fab fragment, or a scFab fragment.

[0029] In one aspect, the FLT3L cytokine comprises at least one monomer comprising at least one of the substitutions according to EU index of Kabat from the group consisting of H8R, H8A, P10A, P10G, Ill A, Il IV, I11Y, S12A, S12Q, S13A, S13P, S13Q, D14E, N76A, N76H, T77A, T77K, E78A, E78D, K116S, K116E, K116R, H80S, E73S, E58D.

[0030] In one aspect, the cytokine comprises two monomers wherein each of the monomers comprises at least one of the substitutions, optionally wherein each of the monomers comprises one or two of the substitutions.

[0031] In one aspect, the cytokine comprises two monomers each comprising:

[0032] a) one and the same of the substitutions; or

[0033] b) two of the substitutions of PIO and H8R, S13Q and E73S or P10A and E73S.

[0034] In one aspect, the cytokine comprises a first monomer and a second monomer, wherein the first monomer is attached to the antibody and the second monomer is attached to the first monomer and wherein a) the second monomer is II 1 Y when the first monomer is E78A;

[0035]

[0036] b) the second monomer is S13Q when the first monomer is II 1 Y; or

[0037] c) The second monomer is II 1 Y when the first monomer is P10G.

[0038] In one aspect, the FLT3L cytokine comprises any one of SEQ ID NOs: 1-28.

[0039] In one aspect, the FLT3L cytokine dimer comprises a first monomer and a second monomer, wherein the first monomer is attached at its N-terminus to the C-terminus or N-terminus of the antibody via a linker and the second monomer is attached at its N-terminus to the C-terminus of the first monomer via a linker, optionally the linker is a peptide, further optionally the linker between the first and the second monomer comprises the sequence GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 38) and the linker between the first monomer and the antibody comprises the sequence GGGSGGGGS (SEQ ID NO: 39).

[0040] In one aspect, the antibody is an IgG, preferably an IgGl or a VHH attached to an IgGl Fc.

[0041] In a certain aspect, the invention provides one or more isolated polynucleotide(s) encoding the immunoconjugate according to the invention.

[0042] In a certain aspect, the invention provides one or more vectors, particularly expression vectors, comprising the polynucleotide(s) according to the invention.

[0043] In a certain aspect, the invention provides a host cell comprising the polynucleotide(s) or the vector(s) according to the invention.

[0044] In a certain aspect, the invention provides a method of producing an immunoconjugate according to the invention, comprising (a) culturing the host cell according to the invention under conditions suitable for the expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate.

[0045] In a certain aspect, the invention provides an immunoconjugate according to the invention produced by the method according to the invention.

[0046]

[0047] In a certain aspect, the invention provides a pharmaceutical composition comprising the immunoconjugate and a pharmaceutically acceptable carrier according to the invention.

[0048] In a certain aspect, the invention provides the immunoconjugate according to the invention for use as a medicament.

[0049] In a certain aspect, the invention provides the immunoconjugate according to the invention for use in the treatment of a disease.

[0050] In a certain aspect, the invention provides the use of the immunoconjugate according to the invention in the manufacture of a medicament for the treatment of a disease.

[0051] In a certain aspect, the invention provides the immunoconjugate or the use according to the invention, wherein the disease is cancer.

[0052] In a certain aspect, the invention provides a method of increasing immune function, increasing number of cDCl cells, increasing T cell function, increasing B cell function, restoring lymphocyte function, increasing expression of FLT3R receptors, increasing T cell responsiveness, and / or increasing natural killer cell activity in an individual, comprising administering to said individual an effective amount of a composition comprising the immunoconjugate according to the invention in a pharmaceutically acceptable form.

[0053] BRIEF DESCRIPTION OF THE FIGURES

[0054] Figure 1 shows in vitro functionality of CLEC9A VHH-targeted affinity attenuated FLT3L dimers (P10G, P10A, S13Q) as compared to wild type (WT) FLT3L polypeptide.

[0055] Fig. 1A - 1H show targeting efficacy of different CLEC9A-targeted engineered FLT3L polypeptides in a co-culture proliferation assay using 0CI-AML5 wild type cells and huCLEC9A 0CI-AML5 cl.39 cells and measuring the proliferation of both cell lines via flow cytometric assessment of EdU incorporation and assessing the percental huCLEC9A 0CI-AML5 cl.39 cell expansion in culture.

[0056]

[0057] Fig.lA shows induction of proliferation by CLEC9A-targeted wild type FLT3L polypeptide (P1AF7736) compared to DP47-untargeted wild type FLT3L polypeptide (P1AF7535) on OCI-AML5 wild type cells (P1AF7535 white circle and P1AF7736 white triangle) compared to huCLEC9A 0CI-AML5 cl.39 cells (P1AF7535 black circle and P1AF7736 black triangle).

[0058] Fig. IB shows percentage of dose-dependent expansion of huCLEC9A 0CI-AML5 cl.39 cells in mixed culture with 0CI-AML5 wild type cells by CLEC9A-targeted wild type FLT3L polypeptide (P1AF7736) (black triangle) compared to DP47-untargeted wild type FLT3L polypeptide (P1AF7535) (black circle).

[0059] Fig.lC shows induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L polypeptide (P10G) (P1AF7725) compared to DP47-untargeted affinity attenuated FLT3L dimer (P10G) (P1AG1256) 0CI-AML5 wild type cells (P1AG1256 white circle and P1AF7725 white triangle) compared to huCLEC9A 0CI-AML5 cl.39 cells (P1AG1256 black circle and P1AF7725 black triangle).

[0060] Fig. ID shows percentage of dose-dependent expansion of huCLEC9A 0CI-AML5 cl.39 cells in mixed culture with 0CI-AML5 wild type cells by CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) (P1AF7725) (black triangle) compared to DP47-untargeted FLT3L polypeptide (P10G) (P1AG1256) (black circle).

[0061] Fig. IE shows induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (PIO A) (P1AG1249) compared to DP47-untargeted affinity attenuated FLT3L dimer (PIO A) (Pl AG1257) on 0CI-AML5 wild type cells (Pl AG1257 white circle and Pl AG1249 white triangle) compared to huCLEC9A 0CI-AML5 cl.39 cells (P1AG1257 black circle and P1AG1249 black triangle).

[0062] Fig. IF shows percentage of dose-dependent expansion of huCLEC9A 0CI-AML5 cl.39 cells in mixed culture with 0CI-AML5 wild type cells by CLEC9A-targeted affinity attenuated FLT3L dimer (P10A) (P1AG1249) (black triangle) compared to DP47-untargeted affinity attenuated FLT3L dimer (P10A) (P1AG1257) (black circle).

[0063] Fig. 1G shows induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer

[0064]

[0065] (S13Q) (P1AF7727) compared to DP47-untargeted affinity attenuated FLT3L dimer (S13Q) (Pl AG1255) on 0CI-AML5 wild type cells (Pl AG1255 white circle and Pl AF7727 white triangle) compared to huCLEC9A 0CI-AML5 cl.39 cells (P1AG1255 black circle and P1AF7727 black triangle).

[0066] Fig. 1H shows percentage of dose-dependent expansion of huCLEC9A 0CI-AML5 cl.39 cells in mixed culture with 0CI-AML5 wild type cells by CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q) (Pl AF7727) (black triangle) compared to DP47-untargeted affinity attenuated FLT3L dimer (S13Q) (P1AG1255) (black circle).

[0067] Figure 2 shows in vitro functionality of monovalent vs. bivalent targeting of CLEC9A VHH-targeted affinity attenuated FLT3L dimers (P10G, P10A, S13Q).

[0068] Fig. 2A-2D: Targeting efficacy of different bivalent vs. monovalent targeted CLEC9A-FLT3L engineerings in a co-culture proliferation assay using 0CI-AML5 wild type cells and huCLEC9A 0CI-AML5 cl.39 cells and measuring the proliferation of both cell lines via flow cytometric assessment of EdU incorporation and assessing the percentile huCLEC9A 0CI-AML5 cl.39 cell expansion in culture.

[0069] Fig. 2A: Induction of proliferation by bivalent CLEC9A-targeted WT FLT3L (P1AF7736) and bivalent DP47-untargeted WT FLT3L (P1AF7535) (P1AF7736 black circle and P1AF7535 white circle) compared to monovalent CLEC9A-targeted WT FLT3L (Pl AG5898) and monovalent DP47-targeted WT FLT3L (P1AG5906) (P1AG5898 black square and P1AG5906 white square) on huCLEC9A 0CI-AML5 cl.39 cells in coculture with 0CI-AML5 wild type cells.

[0070] Fig. 2B: Induction of proliferation by bivalent CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) (P1AF7725) and bivalent DP47-untargeted affinity attenuated FLT3L dimer (P10G) (P1AG1256) (P1AF7725 black circle and P1AG1256 white circle) compared to monovalent CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) (P1AG5891) and monovalent DP47-untargeted affinity attenuated FLT3L dimer (P10G) (P1AG5903) (P1AG5891 black square and Pl AG5903 white square) on huCLEC9A 0CI-AML5 cl.39 cells in coculture with 0CI-AML5 wild

[0071]

[0072] type cells.

[0073] Fig. 2C: Induction of proliferation by bivalent CLEC9A-targeted affinity attenuated FLT3L dimer (PIO A) (P1AG1249) and bivalent DP47-untargeted affinity attenuated FLT3L dimer (PIO A) (P1AG1257) (P1AG1249 black circle and P1AG1257 white circle) compared to monovalent CLEC9A-targeted affinity attenuated FLT3L dimer (PIO A) (P1AG5899) and monovalent DP47-untargeted affinity attenuated FLT3L dimer (P10A) (P1AG5905) (P1AG5899 black square and Pl AG5905 white square) on huCLEC9A 0CI-AML5 cl.39 cells in coculture with 0CI-AML5 wild type cells.

[0074] Fig. 2D: Induction of proliferation by bivalent CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q) (P1AF7727) and bivalent DP47-untargeted affinity attenuated FLT3L dimer (S13Q) (P1AG1255) (P1AF7727 black circle and P1AG1255 white circle) compared to monovalent CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q) (P1AG5892) and monovalent DP47-untargeted affinity attenuated FLT3L dimer (S13Q) (P1AG5901) (P1AG5892 black square and Pl AG5901 white square) on huCLEC9A 0CI-AML5 cl.39 cells in coculture with 0CI-AML5 wild type cells.

[0075] Fig. 2E: Percentage of CLEC9A+ cell expansion by bivalent CLEC9A-targeted WT FLT3L (P1AF7736) and bivalent DP47-untargeted WT FLT3L (P1AF7535) (P1AF7736 black circle and P1AF7535 white circle) compared to monovalent CLEC9A-targeted WT FLT3L (P1AG5898) and monovalent DP47-targeted WT FLT3L (P1AG5906) (P1AG5898 black square and P1AG5906 white square) in the coculture assay mixing 1% huCLEC9A 0CI-AML5 cl.39 cells to with OCI-AML5 wild type cells.

[0076] Fig. 2F: Percentage of CLEC9A+ cell expansion by bivalent CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) (P1AF7725) and bivalent DP47-untargeted affinity attenuated FLT3L dimer (P10G) (Pl AG1256) (Pl AF7725 black circle and Pl AG1256 white circle) compared to monovalent CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) (P1AG5891) and monovalent DP47-untargeted affinity attenuated FLT3L dimer (P10G) (P1AG5903) (P1AG5891 black square and P1AG5903 white square) in the coculture assay mixing 1% huCLEC9A 0CI-AML5 cl.39 cells to with 0CI-AML5 wild type cells.

[0077]

[0078] Fig. 2G: Percentage of CLEC9A+ cell expansion by bivalent CLEC9A-targeted affinity attenuated FLT3L dimer (P10A) (Pl AG1249) and bivalent DP47-untargeted affinity attenuated FLT3L dimer (P10A) (Pl AG1257) (Pl AG1249 black circle and Pl AG1257 white circle) compared to monovalent CLEC9A-targeted affinity attenuated FLT3L dimer (PIO A) (P1AG5899) and monovalent DP47-untargeted affinity attenuated FLT3L dimer (P10A) (P1AG5905) (P1AG5899 black square and P1AG5905 white square) in the coculture assay mixing 1% huCLEC9A OCI-AML5 cl.39 cells to with 0CI-AML5 wild type cells.

[0079] Fig. 2H: Percentage of CLEC9A+ cell expansion by bivalent CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q) (P1AF7727) and bivalent DP47-untargeted affinity attenuated FLT3L dimer (S13Q) (P1AG1255) (P1AF7727 black circle and P1AG1255 white circle) compared to monovalent CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q) (P1AG5892) and monovalent DP47-untargeted affinity attenuated FLT3L dimer (S13Q) (P1AG5901) (P1AG5892 black square and P1AG5901 white square) in the coculture assay mixing 1% huCLEC9A 0CI-AML5 cl.39 cells to with 0CI-AML5 wild type cells.

[0080] Figure 3 shows in vitro competition to naturally occurring soluble WT FLT3L of CLEC9A VHH-targeted FLT3L engineerings (FLT3L WT, P10G, P10A, S13Q) in mixed culture.

[0081] Fig. 3A-3F: Targeting efficacy of different CLEC9A-FLT3L engineerings in presence of increasing dosages of FLT3L cytokine in a co-culture proliferation assay using 0CI-AML5 wild type cells (99.3% of total cells) and huCLEC9A 0CI-AML5 cl.39 cells (0.7% of total cells) and measuring the proliferation of both cell lines via flow cytometric assessment of EdU incorporation and assessing the percentile huCLEC9A OCI-AML5 cl.39 cell expansion in culture.

[0082] Fig. 3A: Induction of proliferation by CLEC9A-targeted WT FLT3L (P1AF7736) compared to DP47-untargeted WT FLT3L (Pl AF7535) in presence of 50 pg / mL FLT3L cytokine on huCLEC9A OCI-AML5 cl.39 cells (P1AF7535 black circle and P1AF7736 black triangle). Fig.3B: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) (P1AF7725) compared to DP47-untargeted affinity attenuated FLT3L dimer (P10G) (P1AG1256) in presence of 50 pg / mL FLT3L cytokine on huCLEC9A OCI-AML5 cl.39 cells (P1AG1256 black circle and P1AF7725 black triangle).

[0083] Fig. 3C: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q) (P1AF7727) compared to DP47-untargeted affinity attenuated FLT3L dimer (S13Q) (P1AG1255) in presence of 50 pg / mL FLT3L cytokine on huCLEC9A OCI-AML5 cl.39 cells (P1AG1255 black circle and P1AF7727 black triangle).

[0084] Fig. 3D: Induction of proliferation by CLEC9A-targeted WT FLT3L (P1AF7736) compared to DP47-untargeted WT FLT3L (P1AF7535) in presence of 250 pg / mL FLT3L cytokine on huCLEC9A OCI-AML5 cl.39 cells (P1AF7535 black circle and P1AF7736 black triangle).

[0085] Fig. 3E: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) (P1AF7725) compared to DP47-untargeted affinity attenuated FLT3L dimer (P10G) (P1AG1256) in presence of 250 pg / mL FLT3L cytokine on huCLEC9A OCI-AML5 cl.39 cells (P1AG1256 black circle and P1AF7725 black triangle).

[0086] Fig. 3F: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q) (P1AF7727) compared to DP47-untargeted affinity attenuated FLT3L dimer (S13Q) (P1AG1255) in presence of 250 pg / mL FLT3L cytokine on huCLEC9A OCI-AML5 cl.39 cells (P1AG1255 black circle and P1AF7727 black triangle).

[0087] Fig. 3G: Induction of proliferation by CLEC9A-targeted WT FLT3L (P1AF7736) compared to DP47-untargeted WT FLT3L (P1AF7535) in presence of 2500 pg / mL FLT3L cytokine on huCLEC9A OCI-AML5 cl.39 cells (P1AF7535 black circle and P1AF7736 black triangle).

[0088] Fig. 3H: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) (P1AF7725) compared to DP47-untargeted affinity attenuated FLT3L dimer (P10G) (P1AG1256) in presence of 2500 pg / mL FLT3L cytokine on huCLEC9A OCI-AML5 cl.39 cells (P1AG1256 black circle and P1AF7725 black triangle).

[0089]

[0090] Fig. 31: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q) (P1AF7727) compared to DP47-untargeted affinity attenuated FLT3L dimer (S13Q) (P1AG1255) in presence of 2500 pg / mL FLT3L cytokine on huCLEC9A 0CI-AML5 cl.39 cells (P1AG1255 black circle and P1AF7727 black triangle).

[0091] Fig. 3J: Induction of proliferation by CLEC9A-targeted WT FLT3L (P1AF7736) compared to DP47-untargeted WT FLT3L (P1AF7535) in presence of 10000 pg / mL FLT3L cytokine on huCLEC9A 0CI-AML5 cl.39 cells (P1AF7535 black circle and P1AF7736 black triangle).

[0092] Fig. 3K: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) (P1AF7725) compared to DP47-untargeted affinity attenuated FLT3L dimer (P10G) (P1AG1256) in presence of 10000 pg / mL FLT3L cytokine on huCLEC9A OCI-AML5 cl.39 cells (P1AG1256 black circle and P1AF7725 black triangle).

[0093] Fig. 3L: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q) (P1AF7727) compared to DP47-untargeted affinity attenuated FLT3L dimer (S13Q) (P1AG1255) in presence of 10000 pg / mL FLT3L cytokine on huCLEC9A OCI-AML5 cl.39 cells (P1AG1255 black circle and P1AF7727 black triangle).

[0094] Figure 4 shows targeting efficacy of P1AF7736 (WT), P1AF7725 (P10G), P1AF7727 (S13Q), Pl AG1249 (P10A) on OCI-AML5 cells expressing different levels of CLEC9A receptor (cl. 39 vs. cl.25) in the mixed culture assay containing 2% CLEC9A+ cells.

[0095] Figure 5 shows the targeting efficacy of different CLEC9A-targeted affinity attenuated FLT3L dimers in a mixed culture assay using OCI-AML5 wild type cells and huCLEC9A-expressing OCI-AML5 cl.39 cells. Proliferation of both cell lines is measured side-by-side via flow cytometric assessment of EdU incorporation, assessing the specific percentile expansion of huCLEC9A OCI-AML5 cl.39 cells.

[0096] Fig. 5A: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q) P1AF7727 compared to DP47-untargeted affinity attenuated FLT3L dimer (S13Q) P1AG1255 on 0CI-AML5 wildtype cells (Pl AG1255 white circle and P1AF7727 white triangle) and huCLEC9A 0CI-AML5 cl.39 cells (P1AG1255 black circle and P1AF7727 black triangle).

[0097] Fig. 5B: Percentage of dose-dependent expansion of huCLEC9A 0CI-AML5 cl.39 cells by CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q) P1AF7727 (black triangle) compared to DP47-untargeted affinity attenuated FLT3L dimer (S13Q) P1AG1255 (black circle) in mixed culture.

[0098] Fig. 5C: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (E78A-II 1Y) P1AG7542 compared to DP47-untargeted affinity attenuated FLT3L dimer (E78A-I11Y) P1AG7544 on 0CI-AML5 wildtype cells (P1AG7544 white circle and P1AG7542 white triangle) and huCLEC9A 0CI-AML5 cl.39 cells (Pl AG7544 black circle and Pl AG7542 black triangle).

[0099] Fig. 5D: Percentage of dose-dependent expansion of huCLEC9A 0CI-AML5 cl.39 cells by CLEC9A-targeted affinity attenuated FLT3L dimer (E78A-I11Y) P1AF7542 (black triangle) compared to DP47-untargeted affinity attenuated FLT3L dimer (E78A-I11Y) P1AG7544 (black circle) in mixed culture.

[0100] Fig. 5E: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (I11Y-S13Q) P1AG7540 compared to DP47-untargeted affinity attenuated FLT3L dimer (I11Y-S13Q) P1AG7541 on 0CI-AML5 wildtype cells (P1AG7541 white circle and P1AG7540 white triangle) and huCLEC9A 0CI-AML5 cl.39 cells (Pl AG7541 black circle and Pl AG7540 black triangle).

[0101] Fig. 5F: Percentage of dose-dependent expansion of huCLEC9A 0CI-AML5 cl.39 cells by CLEC9A-targeted affinity attenuated FLT3L dimer (I11Y-S13Q) P1AG7540 (black triangle) compared to DP47-untargeted affinity attenuated FLT3L dimer (I11Y-S13Q) P1AG7541 (black circle) in mixed culture.

[0102] Fig. 5G: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (P10G-II 1Y) P1AG7526 compared to DP47-untargeted affinity attenuated FLT3L dimer (P10G-I11Y) P1AG7530 on 0CI-AML5 wildtype cells (P1AG7530 white circle and P1AG7526 white triangle) and huCLEC9A 0CI-AML5 cl.39 cells (Pl AG7530 black circle and Pl AG7526 black triangle).

[0103]

[0104] Fig. 5H: Percentage of dose-dependent expansion of huCLEC9A 0CI-AML5 cl.39 cells by CLEC9A-targeted affinity attenuated FLT3L dimer (I11Y-S13Q) P1AG7540 (black triangle) compared to DP47-untargeted affinity attenuated FLT3L dimer (I11Y-S13Q) P1AG7541 (black circle) in mixed culture.

[0105] Fig. 51: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (P10A H8R-P10A H8R) P1AG8268 compared to DP47-untargeted affinity attenuated FLT3L dimer (P10A H8R-P10A H8R) Pl AG8617 on 0CI-AML5 wildtype cells (Pl AG8617 white circle and Pl AG8268 white triangle) and huCLEC9A 0CI-AML5 cl.39 cells (Pl AG8617 black circle and Pl AG8268 black triangle).

[0106] Fig. 5J: Percentage of dose-dependent expansion of huCLEC9A 0CI-AML5 cl.39 cells by CLEC9A-targeted affinity attenuated FLT3L dimer (P10A H8R-P10A H8R) P1AG8268 (black triangle) compared to DP47-untargeted affinity attenuated FLT3L dimer (P10A H8R-P10A H8R) P1AG8617 (black circle) in mixed culture.

[0107] Fig. 5K: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q E73S-S13Q E73S) P1AG8259 compared to DP47-untargeted affinity attenuated FLT3L dimer (S13Q E73S-S13Q E73S) P1AG8609 on 0CI-AML5 wildtype cells (P1AG8609 white circle and P1AG8259 white triangle) and huCLEC9A 0CI-AML5 cl.39 cells (P1AG8609 black circle and Pl AG8259 black triangle).

[0108] Fig. 5L: Percentage of dose-dependent expansion of huCLEC9A 0CI-AML5 cl.39 cells by CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q E73S-S13Q E73S) P1AG8259 (black triangle) compared to DP47-untargeted affinity attenuated FLT3L dimer (S13Q E73S-S13Q E73S) P1AG8609 (black circle) in mixed culture.

[0109] Fig. 5M: Induction of proliferation by CLEC9A-targeted affinity attenuated FLT3L dimer (P10A E73S-P10A E73S) P1AG8267 compared to DP47-untargeted affinity attenuated FLT3L dimer (P10A E73S-P10A E73S) P1AG8616 on 0CI-AML5 wildtype cells (P1AG8616 white circle and P1AG8267 white triangle) and huCLEC9A 0CI-AML5 cl.39 cells (P1AG8616 black circle and Pl AG8267 black triangle).

[0110]

[0111] Fig. 5N: Percentage of dose-dependent expansion of huCLEC9A 0CI-AML5 cl.39 cells by CLEC9A-targeted affinity attenuated FLT3L dimer (P10A E73S-P10A E73S) P1AG8267 (black triangle) compared to DP47-untargeted affinity attenuated FLT3L dimer (P 10A E73 S-P 10A E73 S) P1AG8616 (black circle) in mixed culture.

[0112] Figure 6 shows the proliferation of different dendritic cell subtypes in vitro from human NSG mice bone marrow cells in a dose response by a 3 fold titration (450-0.003 nM) to: human WT FLT3-ligand N-terminal Fc fusion protein in a human IgGl (Pl AF 1472, white bars), or human WT FLT3-ligand human C-terminal Fc fusion (P1AF2973, black bars, gray border) compared to CLEC9A-targeted human WT FLT3L (P1AF7736, black bars, black border), or CLEC9A-targeted affinity attenuated FLT3L dimer (S13Q) P1AF7727 compared to Fc-untargeted affinity attenuated FLT3L dimer (S13Q) P1AF3017 (bars filled with black dots with a solid or a dashed connecting line, respectively); or CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) P1AF7725 compared to Fc-untargeted affinity attenuated FLT3L dimer (P10G) P1AF3009 (bars filled with black squares with a solid or a dashed connecting line, respectively). The x-axis shows the protein concentration (nM) and the y-axis shows the numbers of Fig. 7A. cDCl, Fig. 7B. cDC2 and Fig. 7C. pDCs in the total of Live huCD45+, Lin-, CD34- and HLADR+ cells. Fig. 7D shows the ratio of cDCl absolute cell counts divided by the sum of the counts of cDC2+pDCs obtained per condition. Graphs are a summary of 1 bone marrow donor, with duplicates. Error bars represent standard deviation of the mean values.

[0113] Figure 7 shows the proliferation of different populations of leukocytes in vivo from human NSG mice bone marrow cells in response to different doses of: human WT FLT3-ligand N-terminal Fc fusion protein in a human IgGl (P1AF1472, white bars) given at 2.5mg / kg; or CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) P1AF7725 compared to DP47-untargeted affinity attenuated FLT3L dimer (P10G) P1AG1256 (bars filled with horizontal lines or vertical lines respectively) given at two different doses: Img / kg or 25ug / kg as indicated in the graphs. The x-axis depicts the description of the groups of mice and corresponding treatments and the y-axis shows the

[0114]

[0115] fold expansion of different populations of leukocytes calculated by dividing the absolute count of cells in treated groups by the number of cells in the control untreated group. Fig. 7A shows a dose dependent fold expansion of cDCl, Fig. 7B monocytes Fig. 7C cDC2 Fig. 7D pDCs and Fig. 7E lymphocytes (CD3+ CD19+ CD56+) calculated by the number of gated cells in the total of Live huCD45+, from each treated groups, in comparison with the vehicle group. Fig. 7F shows the ratio of cDCl absolute cell counts divided by the sum of the counts of cDC2+pDCs+CD14+ Monocytes obtained from the Bone Marrow of mice after the different treatments. Each point represents one individual mouse and bar graphs represent the mean of the counts for each group. Error bars represent standard deviation of the mean values.

[0116] Figure 8A illustrates tumor growth kinetics of subcutaneous MC38 tumor-bearing C57BL / 6 huCLEC9A Tg mice administered intraperitoneally with a PDL-1 5mg / kg 2q7d (square, dashed lines) alone, or in combination with one dose of Img / kg CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) P1AF7725 (closed circle, solid line) or human WT FLT3-ligand N-terminal Fc fusion protein in a human IgGl (P1AF1472, triangles, dotted dash line) in comparison with vehicle group (open square dotted line), at day 0 when tumor volume reached 50 mm3. The x-axis is days post dosing. The y-axis is tumor volume in mm. sup.3. Each data point represents the mean value of 15 animals. Error bars represent standard deviation of the mean values.

[0117] Figure 8B shows the tumor volume relative to the starting point from the in vivo study described above.

[0118] Figure 8C illustrates fold increase of conventional dendritic cell subtype 1 (cDCl) numbers in LNs or tumors of MC38 tumor-bearing C57BL / 6 mice huCLEC9A Tg mice at day 10 after administration of the therapies described above, relative to vehicle group depicted as baseline (dashed line).

[0119] Figure 8D illustrates the ratio of cDCl in comparison with other myeloid cells (including cDC2, pDCs and MDSC) present in the tumors of MC38 tumor-bearing C57BL / 6 mice huCLEC9A Tg mice at day 10 after administration of the therapies described above, relative to vehicle group

[0120]

[0121] depicted as baseline (dashed line).

[0122] Figure 8E illustrates the fold increase of CD8+ T cell numbers in LNs or tumors of MC38 tumorbearing C57BL / 6 mice huCLEC9A Tg mice at day 10 after administration of the therapies described above, and relative to vehicle group depicted as baseline (dashed lines).

[0123] Figure 9 presents various illustrative schematics of the chimeric protein complexes described in this invention. Here, "FLT3L" refers to either the wild-type FLT3L or its mutants as detailed herein; "VHH" denotes the anti-human CLEC9A VHH (SEQ ID NO: 30), as specified herein. The FLT3L is linked to the Fc region via a "linker" as described, and two human Fc domains from IgGl are designed with knob-in-hole mutations and effector knockout (KO) modifications as outlined.

[0124] Figure 9A displays two FLT3L monomers attached to the N-terminal end of an Fc antibody structure (e.g., human WT FLT3-ligand N-terminal Fc fusion protein in a human IgGl (P1AF1472)).

[0125] Figure 9B shows a human IgGl antibody structure with two FLT3L monomers attached to the C-terminal end (e.g., P1AF7535 DP47-untargeted WT FLT3L).

[0126] Figure 9C illustrates two FLT3L monomers attached to the C-terminal end of a VHH antibody fragment (e.g., CLEC9A-targeted affinity attenuated FLT3L dimer (P10G) P1AF7725).

[0127] Figure 9D depicts two FLT3L monomers attached to the C-terminal end of an Fc antibody structure (e.g., human WT FLT3L C-terminal Fc fusion protein in a human IgGl (P1AF2973)).

[0128] Figure 9E shows a single-arm monomeric antibody structure with two FLT3L monomers attached to the C-terminal end (e.g., monovalent DP47-targeted WT FLT3L (Pl AG5906)).

[0129] Figure 9F depicts a single-arm monomeric VHH antibody structure with two FLT3L monomers attached to the C-terminal end (e.g., Monomeric CLEC9A-targeted WT FLT3L (Pl AG5898)).

[0130] Figure 9G shows an antibody structure with two FLT3L monomers connected by a 10 or 20 amino

[0131]

[0132] acid linker, attached to the N-terminal end (e.g., 20 AA GS-linker N-terminal fusion of CLEC9A-targeted FLT3L (I11Y-S13Q) (P1AI6747)).

[0133] Figure 9H depicts different mutation configurations of FLT3L monomers:

[0134] “Single Mutation”: A single amino acid substitution in each FLT3L monomer, labeled "A" (e.g., human H8A variant FLT3L N-terminal Fc fusion protein P1AF3007, where Histidine(8) according to EU index Kabat in FLT3L WT ECD is replaced with Alanine). Two monomers are also called dimer.

[0135] “Double Mutation Symmetric”: Two amino acid substitutions in each FLT3L monomer, labeled "A" & "R" (e.g., CLEC9A-targeted affinity attenuated FLT3L dimer (P10A H8R-P10A H8R) P1AG8268, where Proline(lO) and Histidine(8) in each FLT3L WT ECD monomer are replaced with Alanine and Arginine, respectively). Two monomers are also called dimer.

[0136] “Double Mutation Asymmetric”: A single amino acid substitution in one FLT3L monomer labeled "A" and a different substitution in the other monomer labeled "R" (e.g., CLEC9A-targeted affinity attenuated FLT3L dimer (E78A-I11Y) P1AG7542, where Glutamic acid(78) in one FLT3L monomer is replaced with Alanine, and Isoleucine(l 1) in the other monomer is replaced with Tyrosine). Two monomers are also called dimer.

[0137] DETAILED DESCRIPTION

[0138] I DEFINITIONS

[0139] An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0140] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described in the following.

[0141] The term " Fms-like tyrosine kinase 3 " or "FLT3L" as used herein refers to a protein that is capable of binding the FIt3 receptor with sufficient affinity such that the protein is useful as a diagnostic and / or therapeutic agent in targeting the FLT3 receptor (FLT3R or CD 135).

[0142] The term "Flt3L" as used herein, refers to the cleaved, soluble Flt3L (e.g., approximately residues 27-185 of SEQ ID NO:x), but 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), unless otherwise indicated. The term can encompass "full-length" unprocessed Flt3L as well as any form of Flt3L that results from processing in the cell, e.g. removal of a signal peptide (leader sequence) or cleavage from the TM domain. The term also encompasses naturally occurring variants of Flt3L, e.g., splice variants or allelic variants.

[0143] The term "mutant FLT3L" as used herein is intended to encompass any mutant forms of various forms of the FLT3L polypeptide. The various forms of FLT3L mutants are characterized in having at least one amino acid mutation affecting the interaction of FLT3L with CD135. This mutation may involve substitution, deletion, truncation or modification of the wild-type amino acid residue normally located at that position. Mutants obtained by amino acid substitution are preferred. Unless otherwise indicated, a FLT3L mutant may be referred to herein as a mutant FLT3L polypeptide, a mutant FLT3L peptide sequence, a mutant FLT3L polypeptide, a mutant FLT3L

[0144]

[0145] protein or a mutant FLT3L analog. Affinity attenuated FLT3L polypeptide are also considered to be mutant FLT3L polypeptides.

[0146] A "single-mutant" refers to a variant of a protein in which a single amino acid residue has been altered from its original sequence. This alteration can be a substitution, where one amino acid is replaced by another, a deletion or insertion of a single amino acid.

[0147] A "double mutant" refers to a variant of a protein in which two specific amino acid residues or nucleotides have been altered from their original sequence. These alterations can be substitutions, deletions, or insertions. In the context of protein engineering, a double mutant typically involves two amino acid substitutions within the protein sequence. A “symmetric double mutant” refers to a protein, wherein both mutations occur at the same position in each of two identical or homologous subunits of a protein. A “asymmetric double mutant” refers to a protein wherein both mutations occur at different positions in each of two identical or homologous subunits of a protein (Figure 9H).

[0148] The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g. reduced binding to CD135. Amino acid sequence deletions and insertions include amino- and / or carboxy-terminal deletions and insertions of amino acids. Preferred amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of a FLT3L polypeptide, non-conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Preferred amino acid substitutions include replacing a hydrophobic by a hydrophilic amino acid. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. “Reduced binding”, for example reduced binding to an Fc receptor or CD135, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For clarity, the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction.

[0149] As used herein, a “wild-type” (WT) or “naturally occurring” form of FLT3L is a form of FLT3L that is otherwise the same as the mutant FLT3L polypeptide except that the wild-type form has a wild-type amino acid at each amino acid position of the mutant FLT3L polypeptide. For example, if the FLT3L mutant is the full-length FLT3L (i.e. FLT3L not fused or conjugated to any other molecule), the wild-type form of this mutant is full-length native FLT3L. If the FLT3L mutant is a fusion between FLT3L and another polypeptide encoded downstream of FLT3L (e.g. an antibody chain) the wild-type form of this FLT3L mutant is FLT3L with a wild-type amino acid sequence, fused to the same downstream polypeptide. Furthermore, if the FLT3L mutant is a truncated form of FLT3L (the mutated or modified sequence within the non-truncated portion of FLT3L) then the wild-type form of this FLT3L mutant is a similarly truncated FLT3L that has a wild-type sequence. For the purpose of comparing FLT3L receptor binding affinity or biological activity of various forms of FLT3L mutants to the corresponding wild-type form of FLT3L, the term wild-type encompasses forms of FLT3L comprising one or more amino acid mutation that does not affect FLT3L receptor binding compared to the naturally occurring, native FLT3L. In some embodiments wild-type FLT3L polypeptide for the purpose of the present invention comprises the sequence of SEQ ID NO:29. In certain embodiments according to the invention the wild-type FLT3L polypeptide to which the affinity attenuated FLT3L dimer is compared to comprises the amino acid sequence of SEQ ID NO:29 . In other embodiments the wild-type FLT3L polypeptide to which the affinity attenuated FLT3L dimer is compared comprises the amino acid sequence of SEQ ID NO:29.

[0150] By “fused” is meant that the components (e.g. an antibody and an FLT3L polypeptide) are linked by peptide bonds, either directly or via one or more peptide linkers

[0151] The term “CD 135” as used herein, refers to any native CD135 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed CD 135 as well as any form of CD135 that results from processing in the cell. The term also encompasses naturally occurring variants of CD 135, e.g. splice variants or allelic variants. In certain embodiments CD135 is human CD135. The amino acid sequence of human CD135 is found e.g. in UniProt entry no. P36888.

[0152] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by well established methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).

[0153] The affinity of the mutant or wild-type FLT3L polypeptide for various forms of the FLT3L receptor can be determined in accordance with the method set forth in the WO 2012 / 107417 by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare) and receptor subunits such as may be obtained by recombinant expression (see e.g. Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000)). Alternatively, binding affinity of FLT3L mutants for different forms of the FLT3 receptor may be evaluated using cell lines known to express one or the other such form of the receptor. Specific illustrative and exemplary embodiments for measuring binding affinity are described hereinafter.

[0154] The term “affinity attenuated FLT3L polypeptide” is a FLT3L polypeptide that has decreased receptor agonist activity as compared to the naturally occurring FLT3L polypeptide as measured by surface plasmon resonance (SPR) at 25°C or cell line proliferation. An attenuated FLT3L polypeptide may have at least about 10 fold, at least about 50 fold, at least about 100 fold, at least about 250 fold, at least about 500 fold, at least about 1000 fold or less agonist activity as compared to the naturally occurring polypeptide as measured by surface plasmon resonance (SPR) at 25°C or cell line proliferation.

[0155]

[0156] As used herein, the term “effector cells” refers to a population of lymphocytes that mediate the cytotoxic effects of IL-2. Effector cells include effector T cells such as CD8+cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages / monocytes.

[0157] The term "Flt3L-Fc fusion protein" or alternatively, "Fc-effectorless Flt3L-Fc fusion protein" as used herein refers to a fusion protein in which a Flt3L polypeptide is linked, directly or indirectly, to a variant IgG Fc region wherein the variant Fc region has attenuated effector function relative to its counterpart wildtype Fc region. It is noted that for any use of the term "effectorless Flt3L-Fc fusion protein " the term "effectorless " applies to the Fc portion of the fusion protein. The Flt3L-Fc fusion protein comprises a human Flt3L polypeptide linked to a human IgG Fc region. The effectorless Flt3L-Fc fusion protein can bind to the FIt3 receptor protein (Flt3), which can lead to Flt3 receptor downstream signaling.

[0158] The term “Dendritic cell expansion”, “proliferation of Dendritic cells” or “increase in the number of dendritic cells” as used herein refers to an increase in the number of dendritic cells in an in vitro assay or measured in an in vivo experiment.

[0159] The terms “CLEC9A antibody”, “anti-CLEC9A antibody” and “an antibody that binds to CLEC9A” refer to an antibody that is capable of binding CLEC9A with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CLEC9A. In one aspect, the extent of binding of an anti-CLEC9A antibody to an unrelated, non-CLEC9A protein is less than about 10% of the binding of the antibody to CLEC9A as measured, e.g., by surface plasmon resonance (SPR). In certain aspects, an antibody that binds to CLEC9A has a dissociation constant (KD) of < IpM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., IO’8M or less, e.g., from 10'8M to 10'13M, e.g., from 10'9M to 10'13M). An antibody is said to “specifically bind” to CLEC9A when the antibody has a KD of IpM or less. In certain aspects, an anti-CLEC9A antibody binds to an epitope of CLEC9A that is conserved among CLEC9A from different species.

[0160] By "specific binding" is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigen (e.g. CLEC9A) can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique

[0161]

[0162] (analyzed e.g. on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antibody to an unrelated protein is less than about 10% of the binding of the antibody to the antigen as measured, e.g., by SPR. The antibody comprised in the immunoconjugate described herein specifically binds to CLEC9A.

[0163] As used herein, term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein", "amino acid chain", or any other term used to refer to a chain of two or more amino acids, are included within the definition of "polypeptide", and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.

[0164] By an "isolated" polypeptide or a variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0165] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0166] The term “heavy chain antibody” and “heavy chain-only antibody” and “HCAb” as used herein refer to antibodies devoid of light chains.

[0167] A "single-domain antibody" refers to an antibody fragment consisting of a single monomeric antibody variable domain such as VHHs, nanobodies, VNARs derived from sharks, autonomous VH domains or autonomous VL domains. Single-domain antibodies provide an antigen-binding site which specifically binds to an epitope, i.e. the antigen binding-site is formed solely by the singledomain antibody.

[0168] A variable heavy homodimer (VHH)“VHH” or “VHH domain” or “nanobody” refers to a single-domain antibody derived from the variable domains of heavy chain antibodies from camelids, e.g. camel, dromedary, llama, alpaca, etc. (See Nguyen V.K. et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and for review Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). The antigen-binding site of VHHs is devoid of light chain variable domain. A VHH domain may be humanized.

[0169] The terms “binding site” or “antigen-binding site” as used herein refers to the site, i.e. one or more amino acid residues, of a binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antigen binding domain comprises amino acid residues from the complementarity determining regions (CDRs). An antigen-binding site may be provided by, for-example, one or more variable domains (also called variable regions). In single domain antibodies the antigen-binding site is provided by a single variable domain. Whereas, in a Fab fragment the antigen-binding site is provided by the VH and VL domains.

[0170] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. 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 scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23 : 1126-1136 (2005).

[0171]

[0172] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0173] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG?, IgG?, IgG4, IgAi, and IgA?. In certain aspects, the antibody is of the IgGi isotype. In certain aspects, the antibody is of the IgGi isotype with the P329G, L234A and L235A mutation to reduce Fc-region effector function. In other aspects, the antibody is of the IgG? isotype. In certain aspects, the antibody is of the IgG4isotype with the S228P mutation in the hinge region to improve stability of IgG4antibody. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.

[0174] The term “human FLT3L” as used herein refers to a FLT3L molecule comprising an amino acid sequence of SEQ ID NO: 29 .

[0175] The terms “constant region derived from human origin” or “human constant region” as used in the current application denotes a constant heavy chain region of a human antibody of the subclass IgGi, IgG2, IgG3, or IgG4 and / or a constant light chain kappa or lambda region. Such constant regions are well known in the state of the art and e.g. described by Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also e.g. Johnson, G., and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, numbering of amino acid residues in the constant region is according to the EU numbering system, also called the EU index of Kabat, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0176] “Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody -

[0177]

[0178] dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0179] An “effective amount” of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0180] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present. Amino acid sequences of heavy chains including an Fc region are denoted herein without C-terminal glycine-lysine dipeptide if not indicated otherwise. In one aspect, a heavy chain including an Fc region as specified herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one aspect, a heavy chain including an Fc region as specified herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbering according to EU index). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called 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.

[0181] “Framework” or “FR” refers to variable domain residues other than complementary determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the

[0182]

[0183] following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2- CDR-H2(CDR-L2)-FR3- CDR-H3(CDR-L3)-FR4.

[0184] The terms “full length antibody”, “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0185] The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0186] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigenbinding residues.

[0187] A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one aspect, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one aspect, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

[0188] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody

[0189]

[0190] optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0191] The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”).

[0192] Generally, antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

[0193] (a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0194] (b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35b (Hl), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

[0195] (c) antigen contacts occurring at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (Hl), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).

[0196] Unless otherwise indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0197] An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

[0198] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.

[0199] An “isolated” antibody is one which has been separated from a component of its natural environment. In some aspects, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0200]

[0201] The term “nucleic acid molecule” or “polynucleotide” includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler ert al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2 101 823 Bl).

[0202] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0203] “Isolated nucleic acid encoding an CLEC9A antibody” refers to one or more nucleic acid molecules encoding anti-CLEC9A antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0204]

[0205] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0206] A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0207] “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain.

[0208] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0209]

[0210] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity for the purposes of the alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001 / 007611.

[0211] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W. R. Pearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227-258; and Pearson et. al. (1997) Genomics 46:24-36 and is publicly available from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www. ebi.ac.uk / Tools / sss / fasta. Alternatively, a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used to compare the sequences, using the ggsearch (global proteimprotein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.

[0212] The term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0213]

[0214] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0215] The term “CLEC9A”, as used herein, refers to any native CLEC9A from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed CLEC9A as well as any form of CLEC9A that results from processing in the cell. The term also encompasses naturally occurring variants of CLEC9A, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human CLEC9A is shown in Huysamen C, Willment JA, Dennehy KM, Brown GD. CLEC9A is a novel activation C-type lectin-like receptor expressed on BDCA3+ dendritic cells and a subset of monocytes. J Biol Chem. 2008 Jun 13;283(24):16693-701. doi: 10.1074 / jbc.M709923200. Epub 2008 Apr 11. PMID: 18408006; PMCID: PMC2562446) As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some aspects, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

[0216] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6thed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of

[0217]

[0218] complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0219] The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

[0220] The substitution ID always refers to the FLT3L unless stated otherwise.

[0221] II COMPOSITIONS AND METHODS

[0222] The immunoconjugates according to the present invention comprise a FLT3L polypeptide mutant, wherein the FLT3L polypeptide is affinity attenuated. The use of an affinity attenuated FLT3L dimer in combination with CLEC9A targeted antibody is a novel strategic approach to enhance anti -tumor immunity while mitigating the immunosuppressive effects that can limit the efficacy of cancer immunotherapy. FLT3L is a potent growth factor for the expansion and development of both innate and adaptive immune cells, including monocytes and NK cells, which can suppress the anti -tumor immune response. Combining CLEC9A-targeted antibody with an attenuated FLT3L polypeptide mutant aims to create a more favorable environment for anti-tumor immunity by selectively expanding cDCls cells without concurrently expanding other immune cells that could counteract the therapeutic effects. This combination has the potential to synergistically enhance the immune response against cancer cells while minimizing the expansion of other myeloid cells, associated with traditional high-dose FLT3L therapy.

[0223] affinity attenuated FLT3L dimers were generated by introducing specific mutations at particular amino acid positions within the FLT3L extracellular domain (ECD). By altering key residues that interact with the FLT3 receptor, we were able to increase or decrease the affinity for the FLT3 receptor and translated into better or worse proliferation of cells. Using this strategy we were able to reduce the proliferative activity of the WT FLT3L between 2-1000 fold.

[0224]

[0225] In an effort to identify FLT3L variants with reduced affinity compared to the wild-type FLT3L, we tested single point mutations in the FLT3L extracellular domain (ECD) as well as double point mutations. The double point mutations were designed using two distinct approaches:

[0226] The Symmetric Approach refers to a method that aimed to decrease the affinity on both FLT3L monomers equally.

[0227] The Asymmetric Approach refers to a strategy intended to reduce the affinity on each monomer side of the FLT3L dimer sequentially, analogous to the binding model seen in common gamma-chain cytokines.

[0228] These approaches were utilized to generate affinity-attenuated FLT3L variants, which were then compared to the wild-type FLT3L (SEQ ID NO: 29) to assess their binding affinities.

[0229] A. Exemplary targeted FLT3L Immunoconjugates

[0230] Immunoconjugates according to the invention comprise an affinity attenuated FLT3L dimer and an antibody that is at least bivalent for CLEC9A. Targeted attenuated immunocytokines offer several advantages in therapeutic applications, particularly in the context of cancer immunotherapy and autoimmune diseases: i) reduced systemic toxicity: by having reduced activity on non-target cells, which helps to minimize systemic side effects and toxicity that are often associated with high-dose cytokine therapies, ii) increased specificity: by targeting the immunocytokine to a specific antigen or receptor, it is possible to localize the cytokine's effects to the desired cells or tissues, such as cDCl, while sparing other immune cells responsive to the immunocytokine iii) enhanced therapeutic efficacy: the targeting moiety can enhance the accumulation of the cytokine at the site of disease, potentially increasing the therapeutic efficacy and enabling the use of lower doses compared to nontargeted cytokines, iv) prolonged half-life: the fusion of a cytokine with a targeting moiety, such as an antibody, can increase the molecular size and stability of the cytokine, leading to a longer halflife in circulation and potentially reducing the frequency of dosing, v) modulation of Immune Responses: attenuated immunocytokines can be engineered to preferentially activate or expand specific immune cell subsets, such as cDCls, while avoiding the activation of cells that may suppress the immune response, such as Myeloid-derived suppressor cells (MDSCs) vi) synergy with

[0231]

[0232] other therapies: can be used in combination with other treatments, such as checkpoint inhibitors, chemotherapy, or radiation therapy, to create a more comprehensive and effective treatment regimen.

[0233] Immunoconjugate Formats

[0234] Accordingly, the invention provides an immunoconjugate comprising an affinity attenuated FLT3L dimer as described hereinbefore and an antibody that is at least bivalent for CLEC9A. In one embodiment the affinity attenuated FLT3L dimer comprises a first monomer and a second monomer, wherein the first monomer is attached at its N-terminus to the C-terminus or N-terminus of the antibody via a first linker and the second monomer is fused at its N-terminus to the C-terminus of the first monomer. In one embodiment the linkers are peptides. In a particular embodiment the first linker comprises the amino acid sequence of GGGSGGGGS (SEQ ID NO: 39) and the second linker comprises the amino acid sequence of GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 38). In a specific embodiment the C-terminus and / or the N-terminus of the antibody comprises a first and a second subunit or chain, wherein the first monomer is attached to either of them.

[0235] In some embodiments, the antibody that is at least bivalent for CLEC9A is a VHH molecule or an IgG, more particularly an IgGl, IgG2, IgG3, or IgG4, most preferably an IgGl.

[0236] In some embodiments, the antibody comprises an Fc domain composed of a first and a second subunit, wherein the first monomer is attach to either of the subunits.

[0237] In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an immunoglobulin molecule, particularly a VHH or an IgG class immunoglobulin molecule, more particularly an IgGl subclass immunoglobulin molecule. In certain embodiments the antibody is an antibody fragment. In some embodiments the antibody is a Fab molecule or a scFv molecule. In one embodiment the antibody is a Fab molecule. In another embodiment the antibody is a scFv molecule.

[0238] Exemplary immunoconjugate formats are described in figure 9. In one embodiment, the immunoconjugate according to the present invention comprises an affinity attenuated FLT3L dimer as described herein, and at least one antibody that is bivalent for CLEC9A. In a particular embodiment, said antibody is independently selected from the group consisting of a VHH molecule, an IgG molecule, an Fv molecule, a scFv molecule, and a Fab molecule.

[0239]

[0240] In one embodiment the immunoglobulin molecule is human. In one embodiment, the immunoglobulin molecule comprises a human constant region, e.g. a human CHI, CH2, CH3 and / or CL domain. In one embodiment, the immunoglobulin comprises a human Fc domain, particularly a human IgGl Fc domain. In one embodiment the affinity attenuated FLT3L dimer.

[0241] The affinity attenuated FLT3L dimer may be attached to the antibody directly or through a linker peptide, comprising one or more amino acids, typically about 2-20 amino acids. Linker peptides are known in the art and are described herein. Suitable, non-immunogenic linker peptides include, for example, (G4S)n, (SG4)n, (G4S)n or G4(SG4)n linker peptides, “n” is generally an integer from 1 to 10, typically from 2 to 4. In one embodiment the linker peptide has a length of at least 5 amino acids, in one embodiment a length of 5 to 100, in a further embodiment of 10 to 50 amino acids. In a particular embodiment, the linker peptide has a length of 15 amino acids. In one 25 embodiment the linker peptide is (GxS)n or (GxS)nGm with G=glycine, S=serine, and (x=3, n= 3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m= 0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in a further embodiment x=4 and n=3. In a particular embodiment, the linker between FLT3L monomers is GGGGSGGGGSGGGGSGGGGSGGGGS (5G4S) (SEQ ID NO:38) and the linker between the Fc and FLT3L monomer is GGGSGGGGS (2G4S) (SEQ ID NO:39).

[0242] Antibodies

[0243] The antibody comprised in the immunoconjugate of the invention binds to a cell target antigen, particularly to CLEC9A, more particularly human CLEC9A, and is able to direct the affinity attenuated FLT3L dimer to a target site e.g. where CLEC9A is expressed, particularly to a cDCl cell.

[0244] Suitable antibodies that bind to CLEC9A that may be used in the immunoconjugate of the invention are described in PCT patent application no. PCTZEP2016 / 073248, which is incorporated herein by reference in its entirety.

[0245] In particular, the immunoconjugate of the invention comprises an antibody that is at least bivalent. In one embodiment, the antibody comprised in the immunoconjugate of the invention is

[0246]

[0247] monospecific. In a particular embodiment, the immunoconjugate comprises a single, monospecific antibody, particularly a monospecific immunoglobulin molecule.

[0248] The antibody can be any type of antibody or fragment thereof that retains specific binding to the cell target antigen, particularly CLEC9A, more particularly human CLEC9A. Antibody fragments include, but are not limited to, Fv molecules, scFv molecule, Fab molecule, and F(ab')2 molecules. In particular embodiments, however, the antibody is a full-length antibody. In some embodiments, the antibody comprises an Fc domain, composed of a first and a second subunit. In some embodiments, the antibody is a VHH or an immunoglobulin, particularly an IgG class, more particularly an IgGi subclass immunoglobulin.

[0249] In some embodiments, the antibody is a monoclonal antibody.

[0250] Fc domain

[0251] In particular embodiments, the antibody comprised in the immunconjugates according to the invention comprises an Fc domain, composed of a first and a second subunit. The Fc domain of an antibody consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment the immunoconjugate of the invention comprises not more than one Fc domain.

[0252] In one embodiment the Fc domain of the antibody comprised in the immunoconjugate is an IgG Fc domain. In a particular embodiment the Fc domain is an IgGi Fc domain. In another embodiment the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat EU index numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment the Fc domain is a human Fc domain. In an even more particular embodiment, the Fc domain is a human IgGi Fc domain. An exemplary sequence of a human IgGi Fc region is given in SEQ ID NOs: 35 and 36.

[0253]

[0254] Fc domain modifications promoting heterodimerization

[0255] Immunoconjugates, according to the invention comprise an affinity attenuated FLT3L dimer, particularly an affinity attenuated FLT3L polypeptide single or double mutant, wherein one of the dimers is fused to one or the other of the two subunits of the Fc domain while the other dimer is fused to the first dimer, thus the two subunits of the Fc domain are typically comprised in two nonidentical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the immunoconjugate in recombinant production, it will thus be advantageous to introduce in the Fc domain of the antibody a modification promoting the association of the desired polypeptides.

[0256] Accordingly, in particular embodiments, the Fc domain of the antibody comprised in the immunoconjugate according to the invention comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment said modification is in the CH3 domain of the Fc domain.

[0257] There exist several approaches for modifications in the CH3 domain of the Fc domain in order to enforce heterodimerization, which are well described e.g. in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed).

[0258] In a specific embodiment said modification promoting the association of the first and the second subunit of the Fc domain is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain.

[0259]

[0260] The knob-into-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). Accordingly, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the antibody comprised in the immunoconjugate an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

[0261] Preferably said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0262] Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0263] The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.

[0264] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain (the “knobs” subunit) the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the “hole” subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index).

[0265]

[0266] In yet a further embodiment, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0267] In a particular embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index).

[0268] In some embodiments, the second subunit of the Fc domain additionally comprises the amino acid substitutions H435R and Y436F (numbering according to Kabat EU index).

[0269] In a particular embodiment the affinity attenuated FLT3L dimer is fused (optionally through a linker peptide) to the first subunit of the Fc domain (comprising the “knob” modification). Without wishing to be bound by theory, fusion of the affinity attenuated FLT3L dimer to the knob -containing subunit of the Fc domain will (further) minimize the generation of immunoconjugates comprising two affinity attenuated FLT3L dimers (steric clash of two knob-containing polypeptides).

[0270] Other techniques of CH3 -modification for enforcing the heterodimerization are contemplated as alternatives according to the invention and are described e.g. in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291. In one embodiment the heterodimerization approach described in EP 1870459, is used alternatively. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. One preferred embodiment for the antibody comprised in the immunoconjugate of the invention are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (numbering according to Kabat EU index).

[0271]

[0272] In another embodiment, the antibody comprised in the immunoconjugate of the invention comprises amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numberings according to Kabat EU index).

[0273] In another embodiment, the antibody comprised in the immunoconjugate of the invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said antibody comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numberings according to Kabat EU index).

[0274] In one embodiment, the heterodimerization approach described in WO 2013 / 157953 is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L351D (numberings according to Kabat EU index). In a further embodiment, the first CH3 domain comprises further amino acid mutation L35 IK. In a further embodiment, the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numberings according to Kabat EU index).

[0275] In one embodiment, the heterodimerization approach described in WO 2012 / 058768 is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g. selected from a) T411N, T411R, T411Q, T411K, T411D, T41 IE or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e)N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numberings according to Kabat EU index). In a further

[0276]

[0277] embodiment, a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F. In a further embodiment a first CH3 domain comprises amino acid mutation Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R and S400R (numberings according to Kabat EU index).

[0278] In one embodiment the heterodimerization approach described in WO 2011 / 143545 is used alternatively, e.g. with the amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to Kabat EU index).

[0279] In one embodiment, the heterodimerization approach described in WO 2011 / 090762, which also uses the knobs-into-holes technology described above, is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A. In one embodiment, a first CH3 domain comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (numberings according to Kabat EU index).

[0280] In one embodiment, the antibody comprised in the immunoconjugate or its Fc domain is of IgG? subclass and the heterodimerization approach described in WO 2010 / 129304 is used alternatively. In an alternative embodiment, a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009 / 089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. In one such embodiment, a first CH3 domain comprises amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably K392D or N392D) and a second CH3 domain comprises amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K, D356K, or E357K, and more preferably D399K and E356K). In a further embodiment, the first CH3 domain further comprises amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). In a further embodiment, the first CH3

[0281]

[0282] domain further or alternatively comprises amino acid substitution of K439 and / or K370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to Kabat EU index).

[0283] In yet a further embodiment, the heterodimerization approach described in WO 2007 / 147901 is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (numberings according to Kabat EU index).

[0284] In still another embodiment, the heterodimerization approach described in WO 2007 / 110205 can be used alternatively.

[0285] In one embodiment, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering according to Kabat EU index).

[0286] Fc domain modifications reducing Fc receptor binding and / or effector function

[0287] The Fc domain confers to the immunoconjugate favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the immunoconjugate to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with the FLT3L polypeptide and the long half-life of the immunoconjugate, results in excessive activation of cytokine receptors and severe side effects upon systemic administration.

[0288] Accordingly, in particular embodiments, the Fc domain of the antibody comprised in the immunoconjugate according to the invention exhibits reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a native IgGi Fc domain. In one such embodiment the Fc domain (or the antibody comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgGi Fc domain (or an antibody comprising a native IgGi Fc domain), and / or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native IgGi Fc domain domain (or an antibody comprising a native IgGi Fc domain). In one embodiment, the Fc domain domain (or an antibody comprising said Fc domain) does not substantially bind to an Fc receptor and / or induce effector function. In a particular embodiment the Fc receptor is an Fey receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. In one embodiment the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment the effector function is ADCC. In one embodiment the Fc domain domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgGi Fc domain domain. Substantially similar binding to FcRn is achieved when the Fc domain (or an antibody comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGi Fc domain (or an antibody comprising a native IgGi Fc domain) to FcRn.

[0289] In certain embodiments the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a non-engineered Fc domain. In particular embodiments, the Fc domain of the antibody comprised in the immunoconjugate comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment the antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to an antibody comprising a non-engineered Fc domain. In a particular embodiment the Fc receptor is an Fey receptor. In some embodiments the Fc receptor is a human Fc receptor. In some embodiments the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa,

[0290]

[0291] FcyRI or FcyRIIa, most specifically human FcyRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments binding affinity to a complement component, specifically binding affinity to Clq, is also reduced. In one embodiment binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or an antibody comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or an antibody comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or antibody comprised in the immunoconjugate of the invention comprising said Fc domain, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments the Fc domain of the antibody comprised in the immunoconjugate is engineered to have reduced effector function, as compared to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex -mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or an antibody comprising a nonengineered Fc domain).

[0292] In one embodiment the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or effector function is an amino acid substitution. In one embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index). In a more specific embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index). In some embodiments the Fc domain comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU

[0293]

[0294] index). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. In one embodiment the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index). In one embodiment the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index). In a more specific embodiment the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index). In more particular embodiments the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”). Specifically, in particular embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and the second subunit of the Fc domain the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to Kabat EU index). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. The “P329GLALA” combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgGi Fc domain, as described in PCT publication no. WO 2012 / 130831, which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.

[0295] IgG4antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgGi antibodies. Hence, in some embodiments the Fc domain of the antibody comprised in the immunoconjugate of the invention is an IgG4Fc domain, particularly a human IgG4Fc domain. In one embodiment the IgG4Fc domain comprises amino acid substitutions at position S228, specifically the amino acid substitution S228P (numberings according to Kabat EU index). To further reduce its binding affinity to an Fc receptor and / or its effector function, in one embodiment the IgG4Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numberings according to Kabat EU index). In another

[0296]

[0297] embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numberings according to Kabat EU index). In a particular embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numberings according to Kabat EU index). Such IgG4 Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012 / 130831, incorporated herein by reference in its entirety. In a particular embodiment, the Fc domain exhibiting reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a native IgGi Fc domain, is a human IgGi Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numberings according to Kabat EU index).

[0298] In certain embodiments N-glycosylation of the Fc domain has been eliminated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) (numberings according to Kabat EU index).

[0299] In addition to the Fc domains described hereinabove and in PCT publication no. WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) (numberings according to Kabat EU index). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No.

[0300] 7,332,581).

[0301] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.

[0302] Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. Alternatively, binding affinity of Fc

[0303]

[0304] domains or antibodies comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor. Effector function of an Fc domain, or an antibody comprising an Fc domain, can be measured by methods known in the art. Examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, nonradioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0305] In some embodiments, binding of the Fc domain to a complement component, specifically to Clq, is reduced. Accordingly, in some embodiments wherein the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. Clq binding assays may be carried out to determine whether the Fc domain, or antibody comprising the Fc domain, is able to bind Clq and hence has CDC activity. See e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0306] FcRn binding and in vivo clearance / half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12): 1759-1769 (2006); WO 2013 / 120929).

[0307] B. Recombinant Methods and Compositions

[0308] 1. Polynucleotides

[0309]

[0310] The invention further provides isolated polynucleotides encoding an immunoconjugate as described herein or a fragment thereof. In some embodiments, said fragment is an affinity attenuated FLT3L dimer.

[0311] The polynucleotides encoding immunoconjugates of the invention may be expressed as a single polynucleotide that encodes the entire immunoconjugate or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides that are coexpressed may associate through, e.g., disulfide bonds or other means to form a functional immunoconjugate. For example, the light chain portion of an antibody may be encoded by a separate polynucleotide from the portion of the immunoconjugate comprising the heavy chain portion of the antibody and the affinity attenuated FLT3L dimer. When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the immunoconjugate. In another example, the portion of the immunoconjugate comprising one of the two Fc domain subunits and the affinity attenuated FLT3L dimer could be encoded by a separate polynucleotide from the portion of the immunoconjugate comprising the the other of the two Fc domain subunits. When coexpressed, the Fc domain subunits will associate to form the Fc domain.

[0312] In some embodiments, the isolated polynucleotide encodes the entire immunoconjugate according to the invention as described herein. In other embodiments, the isolated polynucleotide encodes a polypeptide comprised in the immunoconjugate according to the invention as described herein.

[0313] In one embodiment, an isolated polynucleotide of the invention encodes the heavy chain of the antibody comprised in the immunoconjugate (e.g. an immunoglobulin heavy chain), and the affinity attenuated FLT3L dimer. In another embodiment, an isolated polynucleotide of the invention encodes the light chain of the antibody comprised in the immunoconjugate.

[0314] In certain embodiments the polynucleotide or nucleic acid is DNA. In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). RNA of the present invention may be single stranded or double stranded.

[0315]

[0316] 2. Recombinant Methods

[0317] affinity attenuated FLT3L dimers useful in the invention can be prepared by deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. Standard methods that can be used to manipulate DNA are for example described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242. The correct nucleotide changes can be verified for example by sequencing. DNA sequences can be determined by double strand sequencing. In this regard, the nucleotide sequence of native FLT3L has been described by Lyman SD et al. (Cell 75:1157 (1993)) and nucleic acid encoding human FLT3L is available from public depositories such as the American Type Culture Collection (Rockville MD). The sequence of native human FLT3L is shown in SEQ ID NO: 29. Substitution or insertion may involve natural as well as non-natural amino acid residues. Amino acid modification includes well known methods of chemical modification such as the addition of glycosylation sites or carbohydrate attachments, and the like.

[0318] Immunoconjugates of the invention may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production. For recombinant production one or more polynucleotide encoding the immunoconjugate (fragment), e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such polynucleotide may be readily isolated and sequenced using conventional procedures. In one embodiment a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of an immunoconjugate (fragment) along with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis etal., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989);

[0319]

[0320] and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y (1989). The expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which the polynucleotide encoding the immunoconjugate (fragment) (i.e. the coding region) is cloned in operable association with a promoter and / or other transcription or translation control elements. As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' untranslated regions, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g. a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage. In addition, a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the immunoconjugate of the invention, or variant or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, e.g. a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription

[0321]

[0322] termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with intron- A), simian virus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit P-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as inducible promoters (e.g. promoters inducible tetracyclins). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence). The expression cassette may also include other features such as an origin of replication, and / or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno-associated viral (AAV) inverted terminal repeats (ITRs).

[0323] In order to confirm identity and check the integrity of the constructs, mass spectrometry can be performed. Before measurement, the samples are deglycosylated by adding N-Glycosidase F (Roche Diagnostics, Penzberg, Germany). The deglycosylation is performed in 0.1 M sodium phosphate buffer at pH 7.1, at a ratio of 0.14 U / pg antibody. The reaction is incubated for 16 h at 37°C. The deglycosylated constructs are also reduced using 80 mM Dithiothreitol (DTT, Roche Diagnostics, Penzberg, Germany) in 4 M Guanidium chloride at 37°C for 30 min and all the samples (deglycosylated and deglycosylated-reduced) are subsequently separated by reverse-phase chromatography. This is performed using a PLRP-S column (Agilent, Waldbronn, Germany) with mobile phase A containing 0.1% (v / v) formic acid in UPLC grade water, and mobile phase B containing acetonitrile (Fischer Chemical, Schwerte, Germany). The column temperature is 75°C, and a gradient of 25% to 40% mobile phase B was used for separation. MS spectra are acquired

[0324]

[0325] using a MaXis Q-TOF instrument (Bruker Daltonics, Bremen, Germany) controlled by Compass 6.2 software. For data evaluation, in-house-developed software is used.

[0326] Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of the polypeptide. For example, the first 26 amino acids of the full length human FLT3L correspond to the signal peptide MTVLAPAWSPTTYLLLLLLLSSGLSG. This signal peptide is missing in the sequences according to the present invention. In certain embodiments, the native signal peptide, e.g. the FLT3L signal peptide or an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wildtype leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse P-glucuronidase.

[0327] DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the immunoconjugate may be included within or at the ends of the immunoconjugate (fragment) encoding polynucleotide.

[0328] In a further embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments a host cell comprising one or more vectors of the invention is provided. The polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively. In one such embodiment a host cell comprises (e.g. has been transformed or transfected with) one or more vector comprising one or more polynucleotide that encodes the immunoconjugate of the invention. As used herein, the term "host cell" refers to any kind of cellular system which can be engineered to generate the immunoconjugates of the invention or fragments thereof. Host cells suitable for replicating and for supporting expression of immunoconjugates are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the immunoconjugate for clinical applications. Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like. For example, polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See e.g. US PatentNos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3 A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980));

[0329]

[0330] and myeloma cell lines such as YO, NSO, P3X63 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., YO, NSO, Sp20 cell).

[0331] Standard technologies are known in the art to express foreign genes in these systems.

[0332] Standard cell culture techniques are used as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds.), John Wiley & Sons, Inc.

[0333] Cells expressing a affinity attenuated FLT3L dimer fused to either the heavy or the light chain of an antibody may be engineered so as to also express the other of the antibody chains such that the expressed mutant affinity attenuated FLT3L fusion product is an antibody that has both a heavy and a light chain or two heavy chains if the antibody is a VHH.

[0334] In one embodiment, a method of producing an immunoconjugate according to the invention is provided, wherein the method comprises culturing a host cell comprising one or more polynucleotide encoding the immunoconjugate, as provided herein, under conditions suitable for expression of the immunoconjugate, and optionally recovering the immunoconjugate from the host cell (or host cell culture medium).

[0335] In the immunoconjugate of the invention, the affinity attenuated FLT3L dimer may be genetically fused to the antibody, or may be chemically conjugated to the antibody. Genetic fusion of the FLT3L polypeptide to the antibody can be designed such that the FLT3L sequence is fused directly to the polypeptide or indirectly through a linker sequence. The composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy. Particular linker peptides are described herein. Additional sequences may also be included to

[0336]

[0337] incorporate a cleavage site to separate the individual components of the fusion if desired, for example an endopeptidase recognition sequence. In addition, a FLT3L fusion protein may also be synthesized chemically using methods of polypeptide synthesis as is well known in the art (e.g. Merrifield solid phase synthesis), affinity attenuated FLT3L dimers may be chemically conjugated to other molecules, e.g. antibodies, using well known chemical conjugation methods. Bi-functional cross-linking reagents such as homofunctional and heterofunctional cross-linking reagents well known in the art can be used for this purpose. The type of cross-linking reagent to use depends on the nature of the molecule to be coupled to FLT3L and can readily be identified by those skilled in the art. Alternatively, or in addition, mutant affinity attenuated FLT3L and / or the molecule to which it is intended to be conjugated may be chemically derivatized such that the two can be conjugated in a separate reaction as is also well known in the art.

[0338] The immunoconjugates of the invention comprise an antibody. Methods to produce antibodies are well known in the art (see e.g. Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as described in U.S. patent No. 4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy chains and variable light chains (see e.g. U.S. Patent. No. 5,969,108 to McCafferty). Immunoconjugates, antibodies, and methods for producing the same are also described in detail e.g. in PCT publication nos. WO 2011 / 020783, WO 2012 / 107417, and WO 2012 / 146628, each of which are incorporated herein by reference in their entirety.

[0339] Any animal species of antibody may be used in the immunoconjugates of the invention. Nonlimiting antibodies useful in the present invention can be of murine, camel, primate, or human origin. If the immunoconjugate is intended for human use, a chimeric form of antibody may be used wherein the constant regions of the antibody are from a human. A humanized or fully human form of the antibody can also be prepared in accordance with methods well known in the art (see e. g. U.S. Patent No. 5,565,332 to Winter). Humanization may be achieved by various methods including, but not limited to (a) grafting the non -human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b)

[0340]

[0341] grafting only the non-human specificity-determining regions (SDRs or a-CDRs; the residues critical for the antibody-antigen interaction) onto human framework and constant regions, or (c) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues. Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’lAcad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling). Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol.

[0342] 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0343] Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181

[0344]

[0345] and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007 / 0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

[0346] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mousehuman heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

[0347] Human antibodies may also be generated by isolation from human antibody libraries, as described herein.

[0348] Antibodies useful in the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. Methods for screening combinatorial libraries are reviewed, e.g., in Lerner et al. in Nature Reviews 16:498-508 (2016). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Frenzel et al. in mAbs 8:1177-1194 (2016); Bazan et al. in Human Vaccines and Immunotherapeutics 8:1817-1828 (2012) and Zhao et al. in Critical Reviews in Biotechnology 36:276-289 (2016) as well as in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, 2001) and in Marks and Bradbury in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0349]

[0350] In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. in Annual Review of Immunology 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al. vxEMBO Journal 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter in Journal of Molecular Biology 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent Nos.

[0351] 5,750,373; 7,985,840; 7,785,903 and 8,679,490 as well as US Patent Publication Nos.

[0352] 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936. Further examples of methods known in the art for screening combinatorial libraries for antibodies with a desired activity or activities include ribosome and mRNA display, as well as methods for antibody display and selection on bacteria, mammalian cells, insect cells or yeast cells. Methods for yeast surface display are reviewed, e.g., in Scholler et al. in Methods in Molecular Biology 503:135-56 (2012) and in Cherf et al. in Methods in Molecular biology 1319:155-175 (2015) as well as in the Zhao et al. in Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, e.g., in He et al. in Nucleic Acids Research 25:5132-5134 (1997) and in Hanes et al. in PNAS 94:4937-4942 (1997).

[0353] Further chemical modification of the immunoconjugate of the invention may be desirable. For example, problems of immunogenicity and short half-life may be improved by conjugation to substantially straight chain polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG) (see e.g. WO 87 / 00056).

[0354] Immunoconjugates prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis,

[0355]

[0356] affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the immunoconjugate binds. For example, an antibody which specifically binds the affinity attenuated FLT3L dimer may be used. For affinity chromatography purification of immunoconjugates of the invention, a matrix with protein A or protein G may be used. For example, sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate an immunoconjugate essentially as described in the Examples. The purity of the immunoconjugate can be determined by any of a variety of well known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like.

[0357] The NuPAGE® Pre-Cast gel system (Invitrogen) is used according to the manufacturer’s instruction. In particular, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, with NuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels) running buffer is used.

[0358] 3. Compositions, Formulations, and Routes of Administration

[0359] In a further aspect, the invention provides pharmaceutical compositions comprising an immunoconjugate as described herein, e.g., for use in any of the below therapeutic methods. In one embodiment, a pharmaceutical composition comprises any of the immunoconjugates provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises any of the immunoconjugates provided herein and at least one additional therapeutic agent, e.g., as described below.

[0360] Further provided is a method of producing an immunoconjugate of the invention in a form suitable for administration in vivo, the method comprising (a) obtaining an immunoconjugate according to the invention, and (b) formulating the immunoconjugate with at least one pharmaceutically acceptable carrier, whereby a preparation of immunoconjugate is formulated for administration in vivo.

[0361]

[0362] Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of immunoconjugate dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains immunoconjugate and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards or corresponding authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0363] An immunoconjugate of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[0364] Parenteral compositions include those designed for administration by injection, e.g. subcutaneous, intradermal, intralesional, intravenous, intraarterial intramuscular, intrathecal or intraperitoneal

[0365]

[0366] injection. For injection, the immunoconjugates of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the immunoconjugates may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the immunoconjugates of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and / or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng / mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase

[0367]

[0368] the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.

[0369] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

[0370] In addition to the compositions described previously, the immunoconjugates may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the immunoconjugates may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0371] Pharmaceutical compositions comprising the immunoconjugates of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. The immunoconjugates may be formulated into a composition in a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.

[0372] 4. Therapeutic Methods and Compositions

[0373] Any of the immunoconjugates provided herein may be used in therapeutic methods. Immunoconjugates of the invention may be used as immunotherapeutic agents, for example in the treatment of cancers.

[0374] For use in therapeutic methods, immunoconjugates of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0375] Immunoconjugates of the invention may be particularly useful in treating disease states where stimulation of the immune system of the host is beneficial, in particular conditions where an enhanced cellular immune response is desirable. These may include disease states where the host immune response is insufficient or deficient. Disease states for which the immunoconjugates of the invention may be administered comprise, for example, a tumor or infection where a cellular immune response would be a critical mechanism for specific immunity. The immunoconjugates of the invention may be administered per se or in any suitable pharmaceutical composition.

[0376]

[0377] In one aspect, immunoconjugates of the invention for use as a medicament are provided. In further aspects, immunoconjugates of the invention for use in treating a disease are provided. In certain embodiments, immunoconjugates of the invention for use in a method of treatment are provided. In one embodiment, the invention provides an immunoconjugate as described herein for use in the treatment of a disease in an individual in need thereof. In certain embodiments, the invention provides an immunoconjugate for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the immunoconjugate. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In further embodiments, the invention provides an immunoconjugate for use in stimulating the immune system. In certain embodiments, the invention provides an immunoconjugate for use in a method of stimulating the immune system in an individual by administering to the individual an effective amount of the immunoconjugate to stimulate the immune system. An “individual” according to any of the above embodiments is a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in cDCl cells, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of FLT3R receptors, an increase in T cell responsiveness, or an increase in natural killer cell activity, and the like.

[0378] In a further aspect, the invention provides for the use of an immunconjugate of the invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a disease in an individual in need thereof. In one embodiment, the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further embodiment, the medicament is for stimulating the immune system. In a further embodiment, the medicament is for use in a method of stimulating the immune system in an

[0379]

[0380] individual comprising administering to the individual an effective amount of the medicament to stimulate the immune system. An “individual” according to any of the above embodiments may be a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of FLT3 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0381] In a further aspect, the invention provides a method for treating a disease in an individual. In one embodiment, the method comprises administering to an individual having such disease a therapeutically effective amount of an immunoconjugate of the invention. In one embodiment a composition is administered to said invididual, comprising the immunoconjugate of the invention in a pharmaceutically acceptable form. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further aspect, the invention provides a method for stimulating the immune system in an individual, comprising administering to the individual an effective amount of an immunoconjugate to stimulate the immune system. An “individual” according to any of the above embodiments may be a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in cDCl cells, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of FLT3 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0382] In certain embodiments the disease to be treated is a proliferative disorder, particularly cancer. Nonlimiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other

[0383]

[0384] cell proliferation disorders that may be treated using an immunoconjugate of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases. In certain embodiments the cancer is chosen from the group consisting of kidney cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer, prostate cancer and bladder cancer. A skilled artisan readily recognizes that in many cases the immunoconjugates may not provide a cure but may only provide partial benefit. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of immunoconjugate that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount". The subject, patient, or individual in need of treatment is typically a mammal, more specifically a human.

[0385] In some embodiments, an effective amount of an immunoconjugate of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of an immunoconjugates of the invention is administered to an individual for the treatment of disease.

[0386] For the prevention or treatment of disease, the appropriate dosage of an immunoconjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the type of molecule (e.g. comprising an Fc domain or not), the severity and course of the disease, whether the immunoconjugate is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the immunoconjugate, and the discretion of the attending physician. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0387]

[0388] The immunoconjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg / kg to 15 mg / kg (e.g. 0.1 mg / kg - 10 mg / kg) of immunoconjugate can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the immunoconjugate would be in the range from about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, a dose may also comprise from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 milligram / kg / body weight, etc., can be administered, based on the numbers described above. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the immunoconjugate). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0389] The immunoconjugates of the invention will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent a disease condition, the immunoconjugates of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically

[0390]

[0391] effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0392] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

[0393] Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.

[0394] Dosage amount and interval may be adjusted individually to provide plasma levels of the immunoconjugates which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically from about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.

[0395] In cases of local administration or selective uptake, the effective local concentration of the immunoconjugates may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

[0396] A therapeutically effective dose of the immunoconjugates described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of an immunoconjugate can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD50 (the dose lethal to 50% of a population) and the ED50 (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Immunoconjugates that exhibit large therapeutic indices are preferred. In one embodiment, the immunoconjugate according to the present invention exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage lies preferably within a range

[0397]

[0398] of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).

[0399] The attending physician for patients treated with immunoconjugates of the invention would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.

[0400] 5. Other Agents and Treatments

[0401] The immunoconjugates according to the invention may be administered in combination with one or more other agents in therapy. For instance, an immunoconjugate of the invention may be coadministered with at least one additional therapeutic agent. The term "therapeutic agent” encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers. In a particular embodiment, the additional therapeutic agent is an anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti angiogenic agent.

[0402]

[0403] Such other agents are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents depends on the amount of immunoconjugate used, the type of disorder or treatment, and other factors discussed above. The immunoconjugates are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0404] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the immunoconjugate of the invention can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent and / or adjuvant. Immunoconjugates of the invention may also be used in combination with radiation therapy.

[0405] C. Articles of Manufacture

[0406] In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and / or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an immunoconjugate of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an immunoconjugate of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0407] D. Assays

[0408] / . Activity assays

[0409] Several cell-based in vitro assays can be performed to evaluate the activity of the targeted FLT3L immunoconjugates comprising at least one antigen binding domain that binds to a dendritic cell surface receptor.

[0410] For example, an 0CI-AML5 based co-culture assay assessing the antigen binding domain-mediated targeting efficacy in mixed culture via assessment of concomitant proliferation of CLEC9A+ and CLEC9A- 0CI-AML5 cells by flow cytometry, is described in more detail in Example 3.1.

[0411] Furthermore, primary human bone marrow assays, wherein CLEC9A-targeted affinity attenuated FLT3L dimers were tested for targeting efficacy on naturally occurring dendritic cell types (cDCl -targeting efficacy vs. off-target dendritic cell expansion), induced during in vitro cytokine-induced maturation, as described in Example 3.2 and herein below. In certain aspects, an antibody-protein conjugate as reported herein is tested for such biological activity.

[0412] Bone marrow cell suspensions are obtained from humanized NSG animals and depleted of red blood cells with lysis buffer (Pharmy lyse buffer, BD #555899). The resulted bone marrow cells are resuspended in RPMI media (Gibco #72400-021) supplemented with 10 % FBS, Penicillin / Streptomycin, Glutamax, 50 pM Beta Mecaptoethanol and seeded in 96-well culture plates at (0.3 Mio / well). Cultures are incubated for 8 days in the presence of increasing concentrations of CLEC9A fused FLT3L WT or mutants, and the corresponding Fc-Flt3L molecules. At the end of this period, dose effects are evaluated by counting differentiated DC subsets from precursors. Standard flow cytometry staining procedure is performed using CD141, CDlc and

[0413]

[0414] CD123 markers to identify DC1, DC2 and pDC respectively from live CD45+ HLADR+ population. Before staining the cells with the antibodies, lOul Ecounting beads (Life Technologies 01-1234-42) are added to each well. Cells are finally washed twice with cold PBS 0.1% BSA and re-suspended in PBS 0.1% BSA for flow cytometry analysis. Fluorescence is analyzed using a FACS Fortessa (Software FACS Diva). Data is analyzed using GraphPadPrism 7.

[0415]

[0416] EXAMPLES

[0417] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

[0418] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

[0419] Example 1

[0420] Generation of affinity attenuated FLT3L dimers

[0421] 1.1 Preparation, purification and characterization of antigens and screening tools for the characterization of affinity attenuated FLT3L dimers

[0422] 1.1.1 Generation and characterization of OCI-AML5 human CLEC9A-expressing cell line

[0423] Full-length cDNA encoding human CLEC9A (UniProt: Q6UXN8) was subcloned into lentiviral transfer vector under CMV promoter control. Lentiviral particles were prepared by transiently cotransfecting HEK 293 -derived viral production cells (Gibco, # A35347) with transfer plasmid and lentiviral packaging mix (pRSV-Rev, pCgpV, and pCMV-VSV-G) using the LV-MAX Transfection Kit (Gibco, #A35346) according to the manufacturer's protocol. Viral supernatants were harvested 72 h after transfection, filtered through a 0.22 -pm pore size filter and stored at -80°C until use.

[0424] One day before transduction, 1x103 OCI-AML5 cells were seeded per well of a 48-well plate. The next day, the culture medium was replaced by 320 pL of purified lentiviral supernatant and 80 pL fresh culture medium (MEM a (GIBCO, #12571063) supplemented with 20% fetal bovine serum (Gibco, #16140063), 2mM L-Glutamine (Gibco, #25030081) and 10 ng / mL GM-CSF (SIGMA-

[0425]

[0426] ALDRICH, # H5666)). Viral transduction of cells was performed in the presence of 4 pg / mL hexadimethrine bromide and spin-inoculation (2 h, 800*g, 32 °C).

[0427] Three days after transduction, puromycin (Invivogen; #ant-pr-l) was added to 0.25 pg / mL. After initial selection, the cells with human CLEC9A surface expression were sorted by BD FACSAria III cell sorter (BD Biosciences) and cultured to establish stable cell clones. Surface expression and stability was confirmed by flow cytometry analysis using mouse APC-conjugated anti-human CD370 (BioLegend, #353805) over a period of 4 weeks.

[0428] 1.2 Generation of affinity attenuated FLT3L dimers by structural design

[0429] 1.2.1 In silica design of symmetric and asymmetric affinity attenuated FLT3L dimers Based on structural information (Verstraete et al. Blood. 118(l):60— -68 (2011)), point mutations have been introduced into the FLT3L cytokine in order to interfere with its cognate CD135 receptor interaction.

[0430] Table 1 shows the amino acid sequences of the monomers of the generated affinity attenuated FLT3L dimers according to the invention for single mutation dimers (SEQ ID NOs: 1-15) and double mutation symmetric dimers (SEQ ID NOs: 26-28) (also see Figure 10 H). The name of the mutation given by the substitution ID for the double mutation symmetric dimers first mentions the mutation in the first monomer, i.e. the monomer attached to the antibody via the 2G4s linker, and secondly the mutation of the second monomer, i.e. the monomer attached to the first monomer via the 5G4S linker.

[0431] Table 1:

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443] Table 2 shows the amino acid substitutions of the generated affinity attenuated FLT3L dimers according to the invention for double mutation symmetric and asymmetric dimers (also see Figure 9 H). The name of the mutation given by the substitution ID first mentions the mutation in the first monomer, i.e. the monomer attached to the antibody via the 2G4s linker, and secondly the mutation of the second monomer, i.e. the monomer attached to the first monomer via the 5G4S linker.

[0444] Table 2:

[0445]

[0446] Table 3 shows an overview of FLT3L single mutant dimers, wherein the respective FLT3L is cloned and Fc fused via the 2G4S linker (SEQ ID NO: 39) to the N-terminus of human IgGl Fc with LALA PG mutations (SEQ ID NOs: 35 and 36) (also see Figure 9A).

[0447] Table 3:

[0448]

[0449]

[0450]

[0451]

[0452] Table 4 lists more constructs used. The antibodies used are either bivalent or monovalent anti-huCLEC9A VHH CL34 huIgGl with LALA PG knob-into-hole (kh) mutations (SEQ ID NOs:30) as well as corresponding non-targeted controls which are either bivalent or monovalent DP47 huIgGl with LALA PG knob-into-hole (kh) mutations (SEQ ID NOs: 33 and 34), wherein the respective FLT3L is attached to the antibody at the C-terminus via 2G4S linker (SEQ ID NO: 39) and the two FLT3L monomers are connect via a 5G4S linker (SEQ ID NO:38) (also see Figures 9 B, C, E, F and H).

[0453] Table 4:

[0454]

[0455]

[0456]

[0457] Table 5 lists bivalent huCLEC9A - targeted C-terminal fused FLT3L constructs used. The antibodies used in these constructs are anti-huCLEC9A VHH CL34 huIgGl with LALA PG knob-into-hole (kh) mutations (SEQ ID NO: 30). The first monomer of the respective FLT3L dimer is attached at its N-terminus to the antibody’s C-terminus via 2G4S linker (SEQ ID NO:39). The second monomer of the respective FLT3L dimer is attached at its N-terminus to the C-terminus of the first monomer via 5G4S linker (SEQ ID NO:38). The name of the mutation given by the substitution ID first mentions the mutation in the first monomer and secondly the mutation of the second monomer (also see Figures 9 C and H with the detailed representation of the molecules).

[0458]

[0459] Table 5:

[0460]

[0461] Table 6 shows an overview of untargeted FLT3L constructs. Respective FLT3L dimers are attached via 2G4S linker (SEQ ID NO:39) to the N-terminus of the Fc (SEQ ID NOs: 35 or 36) (P1AF1472) (Figure 9A) or the C-terminus of the bivalent human DP47 IgGl (SEQ ID NOs: 33 or 34) (P1AF7535, P1AG1256, P1AG1257, P1AG1255) (Figure 9B) or the C-terminus of the Fc (SEQ ID NOs: 35 or 36) (P1AF2973 (Figure 9D)).

[0462] Table 6:

[0463]

[0464]

[0465] The generated mutants were tested as N-terminal fusions to a human IgGl Fc (SEQ ID NOs: 35 or 36) (Figure 9A), generating molecules symmetrically containing point mutants per monomer (Figure 9H) and were tested for induction of proliferation on AML5 cells in vitro (section 1.2.2). After selection of mutants with differing potential to induce cellular proliferation, CLEC9A-targeted mutants were generated including the CLEC9A-binding VHH (SEQ ID NO: 30) in diverse formats (Figure 9C and 9F) to allow for both, effective targeting to the CLEC9A receptor as well as cytokine agonism to induce cellular proliferation.

[0466] For this, single point mutations were combined either in a symmetrical fashion containing the same mutation per monomer as well as in an asymmetrical fashion, so called double mutants, harboring differing point mutations per monomer (Figure 9H).

[0467] 1.2.2 In vitro Functional assays for symmetric affinity attenuated FLT3L dimers Functional assays for FLt3L were conducted in vitro to evaluate the functional characteristics of the Flt3L-Fc proteins with single point mutations. The OCI-AML5 cell line was employed to examine cellular proliferation in response to FLt3L administration. The cells were cultured in Minimum Essential Medium (MEM) alpha medium (Gibco, #12571063), supplemented with 20% fetal bovine serum, 1% Penicillin-Streptomycin, and 10 ng / ml Granulocyte-Macrophage Colony- Stimulating Factor (GM-CSF) (Peprotech, #300-03).

[0468] For the assays, 30,000 cells were seeded in 50 pl of starvation medium (MEM-alpha, 1% Penicillin-Streptomycin) in a 96-well flat-bottom plate (TPP, #92096) and cultured overnight. The following day, treatment was initiated by adding 50 pl of serially diluted molecules to each well. A 12-step 1:3 dilution series was performed for each molecule, with each treatment conducted in duplicates. The cells were then incubated at 37°C in a 5% CO2 atmosphere for a period of 5 days. For the assay readout, 20 pl of CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, #G3580) was added to each well and incubated for 4 hours. Absorbance was subsequently measured using the Tecan Spark 10M device. Raw values were normalized to the minimum control wells, and fold change was calculated relative to untreated controls.

[0469] A dose-response curve for each test article was plotted using GraphPad Prism 7, and half-maximal effective concentration (EC50) values were determined using a four-parameter logistic curve. The

[0470]

[0471] results are presented in the accompanying table 7 and 8.

[0472] Table 7 shows the proliferative activity (EC50 in nM) of symmetric affinity attenuated N-terminally attached FLT3L double mutation dimers on a hu IgGl FC molecule (Figure 9A), in the AML5 proliferation assay described above:

[0473]

[0474]

[0475] Table 8 shows the proliferative activity (EC50 in nM) of symmetric or asymmetric affinity attenuated C-terminally attached FLT3L double mutation dimers on a huCLEC9A VHH bivalent format molecule (SEQ ID NO:30), in the AML5 proliferation assay described above (also see figure 9C):

[0476]

[0477] 1.2.3 Cloning of CLEC9A-targeted affinity attenuated FLT3L dimer expression plasmids

[0478] For the expression of the constructs as reported herein a transcription unit comprising the following functional elements was used:

[0479] the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A,

[0480] a human heavy chain immunoglobulin 5 ’-untranslated region (5’UTR),

[0481] - a murine immunoglobulin heavy chain signal sequence,

[0482] a nucleic acid encoding the respective polypeptide, and

[0483] the bovine growth hormone polyadenylation sequence (BGH pA).

[0484]

[0485] Beside the expression unit / cassette including the desired gene, the basic / standard mammalian expression plasmid contains

[0486] an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and

[0487] a beta-lactamase gene which confers ampicillin resistance in E. coli.

[0488] 1.2.4 Expression of the CLEC9A-targeted affinity attenuated FLT3L dimers

[0489] Transient expression of the described molecules was performed in suspension-adapted Expi293 (Expi293F™ cells; Thermofisher Scientific) with ExpiFectamine™ 293 Transfection Kit (ExpiFectamine™ 293 Reagent, ExpiFectamine™ 293 Transfection Enhancers 1 und 2).

[0490] Cells were passed, by dilution, at least four times (volume 20-30 ml) after thawing in a 125 ml shake flask (Incubate / Shake at 36.5-37 °C, 7 % CO2, 80-85 % humidity, 80-135 rpm). The cells were then expanded up to 6xl06cells / ml in 0.25-1.8 L culture. Transfection was performed at a cell density around 1.4 - 3.0x106 cells / ml.

[0491] Before transfection 1 mg plasmid-DNA per liter culture was diluted in a final volume of 50 ml per liter culture with Opti-MEM (Gibco). The solution was gently mixed and incubated at room temperature for up to 5 min. For transfections with ExpiFectamine™, 2.7 ml of the ExpiFectamine™ 293 reagent per liter culture were added to 50 ml OptiMEM-solution per liter culture and incubated in a separate tube also for 5 minutes. Then the two solutions were united, mixed gently and incubated at room temperature for 15-20 minutes, and the complete mixture was added to the flask with Expi293 cell culture. The incubation was performed by shaking the flask at 36.5-37 °C, 7 % CO2, 80-85 % humidity, 80-135 rpm for 6 to 7 days. The supernatant was harvested by centrifugation. Thereafter, the cell-free supernatant was filtered through a 0.22 pm bottle top filter and stored in a freezer (-20 °C).

[0492] 1.2.5 Purification of the CLEC9A-targeted affinity attenuated FLT3L dimers

[0493] The described molecule containing culture supernatants were filtered and purified by two chromatographic steps. The antibodies were captured by affinity chromatography using HiTrap MabSelectSuRe (GE Healthcare) equilibrated with PBS (1 mM KH2PO4, 10 mM ISfeHPCU, 137 mM NaCl, 2.7 mM KC1), pH 7.4. Unbound proteins were removed by washing with equilibration buffer, and the described molecule was recovered with 25 mM sodium citrate, pH 3.0 or 100 mM sodium acetate, pH 2.8, and immediately after elution neutralized to pH 6.0 with Tris-HCl, pH 9.0. Subsequently, size exclusion chromatography on a Superdex 200™ column (GE Healthcare) was used as polishing step. The size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. Finally, the described molecule-containing solutions were concentrated with an Amicon Ultra centrifugal unit (Millipore, Billerica, MA), filtered using a 0.22 pm filter and stored at -80 °C.

[0494] Example 2

[0495] Characterization of affinity attenuated FLT3L dimers

[0496] 2.1 Surface plasmon resonance (avidity + affinity)

[0497] 2.1.1 Binding of affinity attenuated N-terminal and C-terminal FLT3L polypeptide mutants to recombinant human FLT3 receptor

[0498] Affinities of different FLT3L mutants to recombinant human Flt-3 / Flk-2 Fc chimera protein (R&D,368-ST / CF) were assessed by surface plasmon resonance (SPR). The SPR experiments were performed on a Biacore T200 at 25 °C with PBS-P+ as running and sample dilution buffer (0.2 M phosphate buffer with 27 mM KC1, 1.37 M NaCl and 0.5% Surfactant P20 (Tween 20), Cytiva, F reiburg / Germany) .

[0499] Anti-PGLALA antibody (Roche, M- 1.7.24 mu!gG2b) or another appropriate capturing system is directly immobilized on a CM5 (Cytiva, Freiburg / Germany) or CM3 (Cytiva, Freiburg / Germany) chip using the standard amine coupling kit (Cytiva, Freiburg / Germany). After activation of the sensor surface with a 1 : 1 mixture of 0.4 M l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1 M N-hydroxysuccinimide (NHS), 20 pg / ml anti-PGLALA (diluted in 10 mM acetate pH 5.0) is injected for 720 s with a flow rate of lOpl / min. After blocking with 1 M ethanol ami ne-HCl pH 8.5, the coupling procedure led to more than 6000 RU capture surface density.

[0500] N-terminal and C-terminal fused FLT3L molecules were captured for 30 s at a flow rate of 5 pl / min with a concentration of 50 nM. Recombinant human Flt-3 / Flk-2 Fc chimera protein

[0501]

[0502] (R&D,368-ST / CF) was injected at a concentration of 42 nM for N-terminal fused FLT3L molecules or 50 nM for C-terminal fused FLT3L molecules and then serially diluted with running buffer in 1 :3 ratio with a flow of 30 pl / min through the flow cells. Association and dissociation were monitored for 180 s and 600 s respectively. The chip surface was then regenerated after every cycle by injection of lOmM NaOH (Cytiva, Freiburg / Germany) for 60 s at a flow of 30 pl / min. Bulk refractive index differences were corrected by subtracting the response obtained on reference flow cell. Binding curves were evaluated using Biacore T200 evaluation software 3.0 (Cytiva, Freiburg / Germany) and for the calculation of binding properties 1:1 Langmuir binding model was used. Affinity constants for the interaction between low-affinity FLT3L polypeptide mutants and recombinant human Flt-3 / Flk-2 Fc chimera protein were determined by fitting to a 1 : 1 Langmuir binding using the BIAeval software (Cytiva, Freiburg / Germany).

[0503] Table 9 shows symmetric affinity attenuated N-terminally attached FLT3L double mutation dimers on a hu IgGl Fc molecule (SEQ ID NOs: 35 or 36) binding to recombinant human FLT3 receptor (R&D,368-ST / CF):

[0504]

[0505]

[0506]

[0507] Table 10 shows symmetric affinity attenuated C-terminally attached FLT3L double mutation dimers on a hu IgGl FC (SEQ ID NOs: 35 or 36) binding to recombinant human FLT3 receptor (R&D,368-ST / CF):

[0508]

[0509]

[0510]

[0511] 2.1.2 Binding of CLEC9A-targeted affinity attenuated C-terminal FLT3L polypeptide double mutants to recombinant human FLT3 receptor

[0512] Affinities of different FLT3L polypeptide mutants to recombinant human Flt-3 / Flk-2 Fc chimera protein (R&D,368-ST / CF) were assessed by surface plasmon resonance (SPR). The SPR experiments were performed on a Biacore T200 (Cytiva, Freiburg / Germany) at 25 °C with PBS-P+ as running and sample dilution buffer (0.2 M phosphate buffer with 27 mM KC1, 1.37 M NaCl and 0.5% Surfactant P20 (Tween 20), Cytiva, Freiburg / Germany).

[0513] After conditioning a Cl sensor chip (Cytiva, Freiburg / Germany) according to the supplier’s instructions, an anti-His antibody was directly immobilized using the standard amine coupling kit (Cytiva, Freiburg / Germany). After activation of the sensor surface with a 1:1 mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1 M N-hydroxysuccinimide (NHS), 2 pg / ml anti-His antibody (diluted in 10 mM acetate pH 4.5) was injected for 720 s with a flow rate of lOpl / min. After blocking with 1 M ethanolamine-HCl pH 8.5, the coupling procedure led to more than 1300 RU capture surface density. Recombinant human Flt-3 / Flk-2 Fc chimera protein (R&D,368-ST / CF) was captured for 30 s at a flow rate of 5 pl / min with a concentration of 50 nM. FLT3L polypeptide mutant is injected at a concentration of 1000 nM and then serially diluted with running buffer in 1:2 ratio with a flow of 30 pl / min through the flow cells. Association and dissociation were monitored for 180 s and 300 s respectively. The chip surface was then regenerated after every cycle by injection of 10 nM glycine pH 1.5 (Cytiva, Freiburg / Germany) for 60 s at a flow of 30 pl / min. Bulk refractive index differences was corrected by subtracting the response obtained on reference flow cell. Binding curves were evaluated using Biacore T200 evaluation software 3.0 (Cytiva, Freiburg / Germany) and for the calculation of binding properties 1 : 1 Langmuir binding model was used.

[0514] Table 11 shows asymmetric affinity attenuated C-terminally attached CLEC9A-targeted FLT3L double mutation dimers on a huIgGl Fc molecule (SEQ ID NO: 35) binding to recombinant human FLT3 receptor (R&D,368-ST / CF):

[0515]

[0516]

[0517] Example 3

[0518] In vitro Functional Characterization of CLEC9A-targeted affinity attenuated FLT3L dimers 3.1 Proliferation assay to assess targeting efficacy of our CLEC9A targeted affinity attenuated FLT3L dimers in mixed culture (flow cytometry analysis)

[0519] As CLEC9A+CD135+ cDCls are only a very small percentage of total CD 135+ cells within the human body, the potential sink of our molecule is significant. To reflect this ratio in a functional in vitro assay and to assess the targeting efficacy of our CLEC9A targeted affinity attenuated FLT3L dimers to induce preferential target-specific proliferation in mixed culture, the following in vitro assay was established. OCI-AML5 cells were mixed with CLEC9A-OCI-AML5 cells (clone 25 or clone 39) in different ratios of 1 :2 up to 1 : 100 and the proliferation of both cell types were assessed side-by-side via EdU incorporation using flow cytometry.

[0520] Briefly, cells were starved in assay medium (MEMa medium containing nucleosides, no Phenol Red, Thermo Fisher , #41061029) overnight before the assay at 37°C, 5% CO2. For this, cells were harvested by centrifugation (300g, 5 minutes) and re-suspended in assay medium at a density of 7.5E+05 cells / mL in a suspension culture flask (Greiner, #661195).

[0521] The next day, cells were harvested for viability staining by centrifugation (300g, 5 minutes) and re-suspended in PBS at a density of 1.0E+06 cells / mL in a suspension culture flask (Greiner, #661195). Next, CellTrace FarRed (Thermo Fisher, #34564; dilution 1 : 10000) was added the OCI-AML5 cells and CellTrace Violet (Thermo Fisher, #34571, dilution 1:1000) was added to the CLEC9A-OCI-AML5 cells followed by a 30 min incubation at 37°C in a water bath. To stop the reaction, equal volume FCS (Gibco, #10500-064) was added for 5min at 37°C in a water bath. Cells were then washed twice with PBS and finally transferred in assay medium at a density of 2.0E+06 cells / mL.

[0522] Next, both cell types were co-cultivated by mixing the two cell suspensions at different ratios ranging from 1:2 up to 1:100 CLEC9A-OCI-AML5 cells to OCI-AML5 cells to a final cell concentration of 2.0E+06 cells / mL in 96-well assay plates (Corning, #3596). Sample dilutions were prepared in assay medium ranging from 900nM to 0.1 pM and added the seeded cells for a 24 h incubation period at 37°C, 5% CO2.

[0523] As a next step, proliferation was assessed via Click-iT™ Plus EdU Alexa Fluor™ 488 flow cytometry kit (Thermo Fisher, #C10633) according to the manufacturer's instructions. In brief, the

[0524]

[0525] EdU stock (Thermo Fisher, #C10633F) was diluted in assay medium to a concentration of lOOpM and added to each well for another 24 h incubation period at 37°C, 5% CO2 at a final concentration of 20pM. Cells were harvested by centrifugation (300g, 3 minutes), washed twice with cold PBS 0.1% BSA, and finally re-suspended in 50pL / well Click-iT fixative (Thermo Fisher, #C10633D) for 15 minutes at RT. To stop the reaction, PBS 0.1% BSA was added and cell were washed again once with cold PBS 0.1% BSA and re-suspended in 20pL / well lx Perm / Wash Buffer (Thermo Fisher, #C10633E) for another 15 minutes at RT.

[0526] Next, the dye solution containing the Alexa Fluor™ 488 picolyle azide (Thermo Fisher, #10633B) was prepared as described in the manufacturer's instructions and 40pL / well were added to each plate for 30 minutes at RT, protected from daylight. Cells were finally washed twice with cold PBS 0.1% BSA and re-suspended in PBS 0.1% BSA for flow cytometry analysis. Fluorescence was analyzed using a FACS Celesta (Software FACS Diva). Data was analyzed using GraphPadPrism 7.

[0527] The results show that CLEC9A-targeted unmodified WT FLT3L molecule P1AF7736 is unable to expand preferentially the CLEC9A+ target cells with the mixed culture in every tested concentration (Figures 1A and IB) compared to the untargeted DP47 control molecule P1AF7535. The EC50 on OCI-AML5 and CLEC9A-OCI-AML5 (clone 39) cells remains as well as the percentage of CLEC9A-OCI-AML5 cells within the culture therefore remains unchanged.

[0528] On the contrary, our molecules Pl AF7725 (P10G), P1AG1249 (P10 A) andPlAF7727 (S13Q) are able to expand preferentially CLEC9A-OCI-AML5 cells within the mixed culture in a dosedependent manner (Figures 1C-1H). EC50 values are depicted in Table 12. All three molecules depict a very potent profile of specific expansion from 2% CLEC9A-OCI-AML5 in the starting culture up to 11.1% CLEC9A-OCI-AML5 cells at optimal concentrations compared to the untargeted DP47 control molecules, resulting in an overall expansion of up to 5-fold (Figures ID, IF and 1H)

[0529] Table 12. EC50 of bivalent CLEC9A targeted N-terminally attached affinity attenuated symmetric FLT3L dimers in the mixed culture assay, measuring the proliferation of both cell lines via flow cytometric assessment of EdU incorporation.

[0530]

[0531]

[0532] Next, we assessed the impact of valency of the CLEC9A-targeting moiety by comparing monovalent to bivalent CLEC9A-targeted affinity attenuated FLT3L mutants side-by side in this assay.

[0533] Figures 2B-2D show that bivalent CLEC9A targeting is superior compared to monovalent CLEC9A targeting for all the tested molecules P1AF7725 (P10G), P1AG1249 (P10A) and P1AF7727 (S13Q) as well as P1AG5891 (P10G), P1AG5899 (PIO A) and P1AG5892 (S13Q), respectively.

[0534] We could again confirm a very potent profile of specific expansion from 1% CLEC9A-OCI-AML5 (clone 39) in the starting culture up to 5% CLEC9A-OCI-AML5 cells at optimal concentrations for all three bivalent CLEC9A-targeted affinity attenuated FLT3L dimers, resulting

[0535]

[0536] in an overall expansion of up to 5-fold (Figures 2F-2H). On the contrary, the monovalent CLEC9A-targeted affinity attenuated FLT3L dimers as well as the untargeted DP47 control molecules remained un-functional (Figures 2F-2H).

[0537] As expected, we could revalidate that neither monovalent (P1AG5898), nor bivalent CLEC9A-targeted WT FLT3L cytokine (P1AF7736) is able to expand preferentially the CLEC9A+ target cells with the mixed culture in every tested concentration (Figures 2A and 2E).

[0538] Naturally occurring FLT3L polypeptide can be found in the serum of healthy individuals ranging from 48.3-173.8 pg / mL (Milne P, et al.. Serum Flt3 ligand is a biomarker of progenitor cell mass and prognosis in acute myeloid leukemia. Blood Adv. 2019 Oct 22;3(20):3052-3061) and blood levels of FLT3L are highly elevated in patients with aplastic anemia or receiving bone marrow-damaging chemotherapy or radiotherapy (5-25 fold), within three months from successful bone marrow transplantation (Wodnar-Filipowicz, A.; et al. Flt3 ligand level reflects hematopoietic progenitor cell function in aplastic anemia and chemotherapy -induced bone marrow aplasia. Blood 1996, 88, 4493-4499., Balog, R.P.; et al. Development of a biodosimeter for radiation triage using novel blood protein biomarker panels in humans and non-human primates. Int. J. Radiat. Biol. 2019, 3, 1-13)..To analyze whether the affinity attenuated molecules are able to compete with these endogenous levels during treatment, we assessed the in vitro functionality of the CLEC9A-targeted affinity attenuated FLT3L dimers P1AF7725 (P10G) and P1AF7727 (S13Q) in competition with increasing dosages of FLT3L polypeptide in the mixed culture assay (Figure 3). In brief, the assay follows the above description, but adds, concomitantly to sample titration addition, increasing doses of FLT3L cytokine (50 pg / mL, 250 pg / mL, 2500 pg / mL and 10.000 pg / mL) to the mixed cell populations.

[0539] Potent specific expansion for both CLEC9A-targeted affinity attenuated FLT3L dimers P1AF7725 (P10G) and P1AF7727 (S13Q) was detectable up to additional 250pg / mL FLT3L (Figures 3B-3C and 3E-3F), started to decrease with 2500pg / mL FLT3L (Figures 3H-3I) and was neutralized at the highest dose of lO.OOOpg / mL FLT3L cytokine (Figures 3K-3L).

[0540] As a control and as expected, CLEC9A-targeted WT FLT3L (P1AF7736) did not show any CLEC9A+ cell expansion at any given concentration and FLT3L addition (Figures 3A, 3D, 3G and 3 J).

[0541]

[0542] To assess the impact of varying levels of CLEC9A expression on the overall efficacy of the targeted molecules, we generated 0CI-AML5 cells with different levels of CLEC9A expression (Example 1.1.1), generating the low CLEC9A-expressing clone 39 and the high CLEC9A-expressing clone 25, harboring approximately 16.000 receptors per cell or 150.000 receptors per cell, respectively. OCI-AML5 WT cells do not naturally express CLEC9A. Naturally occurring primary human dendritic cells best resemble clone 25.

[0543] Figure 4 compares the maximal expansion efficacy at optimal concentrations of CLEC9A-targeted affinity attenuated FLT3L dimers P1AF7725 (P10G), P1AG1249 (P10 A) and P1AF7727 (S13Q) and show targeting efficiency on both, low and high CLEC9A-expressing cells, compared to untargeted DP47 controls. Here again, CLEC9A-targeted WT FLT3L molecule Pl AF7736 serves as a control, showing no specific expansion in either cell line compared to the control. As expected, the maximum efficacy of expansion slightly decreases with lowering numbers of targeting receptors per cell, limiting the pro-proliferative effect of the cytokine mutants. Nevertheless, depict a robust reproducibility of expansion compared to control, also clearly under the level of naturally occurring CLEC9A receptor levels on primary human dendritic cells.

[0544] Figure 5 demonstrates the potency of CLEC9A-specific cell expansion by the CLEC9A-targeted double mutants in the mixed culture assay (Table 13).

[0545] All double mutant molecules depicted a superior targeting efficacy compared to untargeted DP47 control molecules, with variable degrees of efficacy in terms of EC50 values and overall as well as maximum expansion of targeted cells.

[0546] Table 13. EC50 of different symmetric or asymmetric C-terminally attached affinity attenuated FLT3L mutants in the mixed culture assay, measuring the proliferation of both cell lines via flow cytometric assessment of EdU incorporation.

[0547]

[0548]

[0549]

[0550]

[0551]

[0552] 3.2 DC differentiation assay to assess targeting efficacy of CLEC9A targeted affinity attenuated FLT3L dimers on primary human bone marrow

[0553] Since targeting of CLEC9A-FLT3L engineered molecules should result in a more specific DC1 expansion, we tested our CLEC9A targeted affinity attenuated FLT3L dimers in a more physiological model. Bone marrow is a primary source of DC precursors which can proliferate and differentiate in vitro into DCs in presence of Flt3L. Bone marrow cell suspensions were obtained from humanized NSG animals and depleted of red blood cells with lysis buffer (Pharmy lyse buffer, BD #555899). The resulted bone marrow cells were resuspended in RPMI media (Gibco #72400-021) supplemented with 10 % FBS, Penicillin / Streptomycin, Glutamax, 50 pM Beta Mecaptoethanol) and seeded in 96-well culture plates at (0.3 Mio / well). Cultures were incubated for 8 days in the presence of increasing concentrations of CLEC9A fused FLT3L WT or mutants, and the corresponding Fc-Flt3L molecules. At the end of this period, dose effects were evaluated by counting differentiated DC subsets from precursors. Standard flow cytometry staining procedure was performed using CD 141, CDlc and CD 123 markers to identify DC1, DC2 and pDC

[0554]

[0555] respectively from live CD45+ HLADR+ population. Before staining the cells with the antibodies, lOul Ecounting beads (Life Technologies 01-1234-42) were added to each well. Cells were finally washed twice with cold PBS 0.1% BSA and re-suspended in PBS 0.1% BSA for flow cytometry analysis. Fluorescence was analyzed using a FACS Fortessa (Software FACS Diva). Data was analyzed using GraphPadPrism 7.

[0556] As shown in Fig 6, CLEC9A-P10G (P1AF7725) and CLEC9A-S13Q (P1AF7727) induce potent DC1 cell differentiation compared to untreated, untargeted (P1AF3009 and P1AF3017, respectively) and WT Flt3L control (P1AF1472) and depict a more efficient expansion of cDCls over cDC2 as confirmed by the ratio (Fig. 6D), validating the targeting efficacy.

[0557] Example 4

[0558] In vivo Functional Characterization of CLEC9A-targeted affinity attenuated FLT3L dimers 4.1 In vivo model: humanized Mice (huNOG-EXL)

[0559] The in vivo targeting efficiency of huCLEC9A-targeted affinity attenuated FLT3L dimers was evaluated in terms of their ability to induce a specific cDCl expansion in the bone marrow of immunodeficient mice reconstituted with human immune cells (humanized NOG-EXL mice) at 2 different doses. huCLEC9A(VHH)-targeted affinity attenuated FLT3L polypeptide mutants were able to induce cDCl expansion in humanized NOG-EXL as shown by the increase in the fold change of CLEC9A+ CD141+ cDCl cells in comparison to vehicle (untreated) mice or the corresponding untargeted control (DP47) Fig. 7 Treatment with lower a dose of targeted huCLEC9A(VHH)-targeted affinity attenuated FLT3L dimers induces an expansion of cDCl cells, comparable to a 100-times higher dose of untargeted Fc-WT FLT3L (P1AF1472). 25ug / kg of huCLEC9A(VHH)-targeted affinity attenuated FLT3L P10G polypeptide mutant also shows a better shift in the bone marrow myeloid cell content, increasing the numbers of cDCl over other myeloid cell populations (cDC2, pDCs, and monocytes) and inducing minimal expansion of other leukocytes populations, such as T cells, B cells and NK cells (Fig. 7).

[0560]

[0561] 4.2 In vivo model: huCLEC9A transgenic mice - MC38 s.c. tumor model PK / PD The in vivo targeting efficiency of CLEC9A-targeted affinity attenuated FLT3L dimers was also evaluated in terms of their ability to induce tumor growth inhibition in huCLEC9A Transgenic mice (immunocompetent C57B1 / 6 mice engineered to express the huCLEC9A receptor) implanted with MC38 s.c. tumors. Mice were divided into 4 groups: Vehicle (control), aPDLl, aPDLl + CLEC9A-Flt3Ligand P10G (1 mg / kg) or aPDLl + Fc-Flt3L (1 mg / kg). Tumor volume increases over time in all groups, but the combination treatments (aPDLl + CLEC9A-Flt3Ligand P10G and aPDLl + Fc-Flt3L) show a trend towards reduced tumor growth compared to the vehicle and aPDLl alone (Fig. 8A). Comparing the relative tumor volume at the end of the study to the start time point for the same four treatment groups, the combination treatments showed a trend towards lower relative tumor volumes (Fig. 8B). Combination of aPDLl with CLEC9A-targeted affinity attenuated FLT3L dimers showed overall the lowest tumor volume.

[0562] In line with better efficacy and lower tumor volumes, the combination treatments also showed a higher fold increase in cDCl cells in both LNs and tumors compared to aPDLl (Fig. 8C). In comparison with untargeted Fc-WT FLT3L (P1AF1472), the same dose of huCLEC9A(VHH)-targeted affinity attenuated FLT3L P10G polypeptide mutant also shows a better shift in the tumor myeloid cell content, increasing the numbers of cDCl over other myeloid cell populations (cDC2, pDCs, and monocytes) (Fig. 8D).

[0563] Overall, the figure suggests that combining aPDLl with CLEC9A-Flt3 Ligand P10G or Fc-Flt3L enhances anti-tumor immune responses and reduces tumor growth more effectively than aPDLl alone.

[0564]

Claims

1. CLAIMS1. An immunoconjugate comprising a cytokine and an antibody, wherein the cytokine is a mutant FLT3L cytokine dimer with reduced affinity as compared to wild type FLT3L cytokine as measured by SPR at 25°C or cell proliferation assay and wherein the antibody is at least bivalent for a target cell antigen, optionally wherein the target cell antigen is CLEC9A.

2. The immunoconjugate of claim 1, wherein the affinity of the mutant FLT3L cytokine compared to the wild type FLT3L cytokine is reduced by at least 1.2 fold, at least 2.9 fold, at least 3 fold, at least 3.2 fold, at least 3.5, at least 5.4, at least 10.8, at least 11.3, at least 13.8, at least 20.7, at least 34.1, at least 47.6, at least 67.7, at least 73.8, at least 113.7 or at least 201.7, as measured by SPR at 25°C.

3. The immunoconjugate of claim 1 or 2, wherein the antibody is an antibody fragment.

4. The immunoconjugate of claim 3, wherein the fragment is a Fab fragment, optionally a scFab fragment.

5. The immunoconjugate of any one of claims 1 to 4, wherein the FLT3L cytokine comprises at least one monomer comprising at least one of the substitutions according to EU index of Kabat from the group consisting ofH8R, H8A, P10A, P10G, Il 1 A, Il IV, Il 1Y, S12A, S12Q, S13A, S13P, S13Q, D14E, N76A, N76H, T77A, T77K, E78A, E78D, K116S, K116E, K116R, H80S, E73S, E58D.

6. The immunoconjugate of claim 5, wherein the cytokine comprises two monomers wherein each of the monomers comprises at least one of the substitutions, optionally wherein each of the monomers comprises one or two of the substitutions.

7. The immunoconjugate of claim 6, wherein the cytokine comprises two monomers each comprising:8.d) one and the same of the substitutions; or9.e) two of the substitutions of P10 and H8R, S13Q and E73S or P10A and E73S.

8. The immunoconjugate of claim 6, wherein the cytokine comprises a first monomer and a second monomer, wherein the first monomer is attached to the antibody and the second monomer is attached to the first monomer and wherein a) the second monomer is II 1 Y when the first monomer is E78A; b) the second monomer is S13Q when the first monomer is II 1 Y; or11.f) The second monomer is II 1 Y when the first monomer is P10G.

13.

14.

9. The immunoconjugate of any one of claims 1 to 6, wherein the FLT3L cytokine comprises any one of SEQ ID NOs: 1-28.

10. The immunoconjugate of any one of claims 1 to 9, wherein the FLT3L cytokine dimer comprises a first monomer and a second monomer, wherein the first monomer is attached at its N-terminus to the C-terminus or N-terminus of the antibody via a linker and the second monomer is attached at its N-terminus to the C-terminus of the first monomer via a linker, optionally the linker is a peptide, further optionally the linker between the first and the second monomer comprises the sequence GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 38) and the linker between the first monomer and the antibody comprises the sequence GGGSGGGGS (SEQ ID NO: 39).

11. The immunoconjugate of any one of claims 1 to 10, wherein the antibody is an IgG, preferably an IgGl or a VHH attached to an IgGl Fc.

12. One or more isolated polynucleotide(s) encoding the immunoconjugate of any one of claims 1 to 11.

13. One or more vectors, particularly expression vectors, comprising the polynucleotide(s) of claim 11.

14. A host cell comprising the polynucleotide(s) of claim 12 or the vector(s) of claim 13.

15. A method of producing an immunoconjugate according to any one of claims 1 to 11, comprising (a) culturing the host cell of claim 14 under conditions suitable for the expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate.

16. An immunoconjugate according to any one of claims 1 to 11, wherein the immunoconjugate is produced by the method according to claim 15.

17. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1-11 and claim 16 for use as a medicament.

18. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1-11 and claim 16 for use in the treatment of a disease.

19. Use of the immunoconjugate of any one of claims 1 to 11 and 16 in the manufacture of a medicament for the treatment of a disease.

20. The pharmaceutical composition of claim 18 or the use of claim 19, wherein the disease is cancer.

26.

27.

21. A method of increasing immune function, increasing number of cDCl cells, increasing T cell function, increasing B cell function, restoring lymphocyte function, increasing expression of FLT3R receptors, increasing T cell responsiveness, and / or increasing natural killer cell activity in an individual, comprising administering to said individual an effective amount of a composition comprising the immunoconjugate according to the invention in a pharmaceutically acceptable form.28.***29.107

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