Csf1r-based chimeric proteins

Abstract

The present invention relates, in part, to, chimeric proteins which include the extracellular domain of colony stimulating factor 1 receptor (CSF1R) and their use in the treatment of diseases, such as immunotherapies for cancer and/or an inflammatory disease.

What is claimed is: 
     
         1 . A heterologous chimeric protein comprising:
 (a) a first domain comprising a portion of colony stimulating factor 1 receptor (CSF1R) that is capable of binding a CSF1R ligand;   (b) a second domain comprising a portion of CD40 Ligand (CD40L) that is capable of binding a CD40L receptor; and   (c) a linker linking the first domain and the second domain.   
     
     
         2 . The heterologous chimeric protein of  claim 1 , wherein the first domain comprises substantially all of the extracellular domain of CSF1R and the second domain comprises substantially all of the extracellular domain of CD40L. 
     
     
         3 . The heterologous chimeric protein of  claim 1  or  claim 2 , wherein the chimeric protein is capable of inhibiting an immunosuppressive signal. 
     
     
         4 . The heterologous chimeric protein of any one of  claims 1  to  3 , wherein the chimeric protein is capable of:
 (a) reducing or eliminating an immune inhibitory signal when the portion of CSF1R is bound to its ligand and/or 
 (b) increasing or activating an immune stimulatory signal when the portion of CD40L is bound to its receptor. 
 
     
     
         5 . The heterologous chimeric protein of any one of  claims 1  to  4 , wherein the CSF1R ligand is CSF1 or IL-34. 
     
     
         6 . The heterologous chimeric protein of any one of  claims 1  to  5 , wherein the CD40L receptor is CD40. 
     
     
         7 . The heterologous chimeric protein of any one of  claims 1  to  6 , wherein the chimeric protein is capable of contemporaneously binding the CSF1R ligand and the CD40L receptor, wherein the CSF1R ligand is CSF1 or IL-34 and the CD40L receptor is CD40. 
     
     
         8 . The heterologous chimeric protein of any one of  claims 1  to  7 , wherein the chimeric protein is capable of contemporaneously binding recombinant human CD40 and human CSF1 in vitro. 
     
     
         9 . The heterologous chimeric protein of any one of  claims 1  to  8 , wherein the chimeric protein depletes CSF1 and/or IL-34, optionally in the serum. 
     
     
         10 . The heterologous chimeric protein of any one of  claims 1  to  9 , wherein the chimeric protein exhibits enhanced safety profiles and/or reduced toxicity profiles compared to CD40 agonist antibodies and/or CSF1R antagonistic antibodies. 
     
     
         11 . The heterologous chimeric protein of any one of  claims 1  to  10 , wherein the chimeric protein exhibits enhanced anti-tumor effects compared to CD40 agonist antibodies and/or CSF1R antagonistic antibodies. 
     
     
         12 . The heterologous chimeric protein of any one of  claims 1  to  11 , wherein the chimeric protein is capable of increasing or preventing a decrease in a sub-population of CD4+ and/or CD8+ T cells. 
     
     
         13 . The heterologous chimeric protein of any one of  claims 1  to  12 , wherein the chimeric protein is capable of enhancing tumor killing activity by T cells. 
     
     
         14 . The heterologous chimeric protein of any one of  claims 1  to  13 , wherein the chimeric protein is capable of providing a sustained immunomodulatory effect. 
     
     
         15 . The heterologous chimeric protein of any one of  claims 1  to  14 , wherein the chimeric protein is capable of causing activation of antigen presenting cells. 
     
     
         16 . The heterologous chimeric protein of any one of  claims 1  to  15 , wherein the chimeric protein is capable enhancing the ability of antigen presenting cells to present antigen. 
     
     
         17 . The heterologous chimeric protein of any one of  claims 1  to  16 , wherein the chimeric protein shifts the ratio of immune cells in favor of cells that can kill a tumor in opposition to cells that protect tumors. 
     
     
         18 . The heterologous chimeric protein of  claim 17 , wherein the cell that that can kill a tumor is selected from T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, and dendritic cells and wherein the cell that protect tumors is selected from myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs); tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs)). 
     
     
         19 . The heterologous chimeric protein of  claim 18 , wherein the chimeric protein stimulates anti-tumor macrophages and antigen presenting cells, while avoiding induction of MDSC through inhibition of CSF1 and/or IL-34. 
     
     
         20 . The heterologous chimeric protein of any one of  claims 1  to  19 , wherein the chimeric protein increases the ratio of effector T cells to regulatory T cells. 
     
     
         21 . The heterologous chimeric protein of any one of  claims 1  to  20 , wherein the chimeric protein provides a sustained masking effect of immune inhibitory signals. 
     
     
         22 . The heterologous chimeric protein of any one of  claims 1  to  21 , wherein the chimeric protein provides longer on-target (e.g., intra-tumoral) half-life (t 1/2 ) as compared to serum t 1/2  of the chimeric proteins. 
     
     
         23 . The heterologous chimeric protein of any one of  claims 1  to  22 , wherein the chimeric protein reduces toxicities as compared with treatment with antibodies against CSF1R and CD40. 
     
     
         24 . The heterologous chimeric protein of any one of  claims 1  to  23 , wherein the linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, or an antibody sequence. 
     
     
         25 . The heterologous chimeric protein of  claim 24 , wherein the linker comprises hinge-CH2-CH3 Fc domain derived from IgG4. 
     
     
         26 . The heterologous chimeric protein of  claim 25 , wherein the hinge-CH2-CH3 Fc domain is derived from human IgG4. 
     
     
         27 . The heterologous chimeric protein of any one of  claims 1  to  26 , wherein the chimeric protein is expressed by a mammalian host cell as a secretable and functional single polypeptide chain. 
     
     
         28 . The heterologous chimeric protein of any one of  claims 1  to  27 , wherein the portion of CSF1R is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         29 . The heterologous chimeric protein of any one of  claims 1  to  28 , wherein the portion of CD40L is at least 95% identical to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. 
     
     
         30 . The heterologous chimeric protein of any one of  claims 1  to  29 , wherein the linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. 
     
     
         31 . The heterologous chimeric protein of any one of  claims 1  to  30 , wherein the linker comprises one or more joining linkers, such joining linkers independently selected from SEQ ID NOs: 28 to 74. 
     
     
         32 . The heterologous chimeric protein of  claim 31 , wherein the linker comprises two or more joining linkers each joining linker independently selected from SEQ ID NOs: 28 to 74; wherein one joining linker is N terminal to the hinge-CH2-CH3 Fc domain and another joining linker is C terminal to the hinge-CH2-CH3 Fc domain. 
     
     
         33 . The heterologous chimeric protein of any one of  claims 1  to  32 , wherein the chimeric protein is a recombinant fusion protein. 
     
     
         34 . The heterologous chimeric protein of any one of  claims 1  to  33 , wherein the chimeric protein is capable of forming a stable synapse between cells. 
     
     
         35 . The heterologous chimeric protein of  claim 34 , wherein the stable synapse between cells provides spatial orientation that favors tumor reduction. 
     
     
         36 . The heterologous chimeric protein of  claim 34  or  claim 35 , wherein the spatial orientation positions T cells to attack tumor cells and/or sterically prevents a tumor cell from delivering negative signals, including negative signals beyond those masked by the chimeric protein of the invention. 
     
     
         37 . The heterologous chimeric protein of any one of  claims 1  to  36 , wherein binding of either or both of the extracellular domains to its respective binding partner occurs with slow off rates (K off ), which provides a long interaction of a receptor and its ligand. 
     
     
         38 . The heterologous chimeric protein of  claim 37 , wherein the long interaction delivers a longer positive signal effect. 
     
     
         39 . The heterologous chimeric protein of  claim 38 , wherein the longer positive signal effect allows an effector cell to be adequately stimulated for an anti-tumor effect. 
     
     
         40 . The heterologous chimeric protein of any one of  claims 37  to  39 , wherein the long interaction provides T cell proliferation and allows for anti-tumor attack. 
     
     
         41 . The heterologous chimeric protein of any one of  claims 37  to  40 , wherein the long interaction allows sufficient signal transmission to provide release of stimulatory signals. 
     
     
         42 . The heterologous chimeric protein of  claim 41 , wherein the stimulatory signal is a cytokine. 
     
     
         43 . An expression vector, comprising a nucleic acid encoding the chimeric protein of any one of  claims 1  to  42 . 
     
     
         44 . A host cell, comprising the expression vector of  claim 43 . 
     
     
         45 . A pharmaceutical composition, comprising a therapeutically effective amount of the heterologous chimeric protein of any one of  claims 1  to  42 . 
     
     
         46 . A method of treating cancer or an inflammatory disease, comprising administering an effective amount of the pharmaceutical composition of  claim 45  to a subject in need thereof. 
     
     
         47 . A method of modulating a patient's immune response, comprising administering an effective amount of the pharmaceutical composition of  claim 45  to a subject in need thereof. 
     
     
         48 . The method of  claim 46  or  claim 47 , wherein the patient's T cells are activated. 
     
     
         49 . The method of  claim 46  or  claim 47 , wherein the patient has a tumor and one or more tumor cells are prevented from transmitting an immunosuppressive signal. 
     
     
         50 . A method for treating cancer or an inflammatory disease comprising administering an effective amount of a pharmaceutical composition to a subject in need thereof, the pharmaceutical composition comprising a heterologous chimeric protein comprising:
 (a) a first domain comprising a portion of colony stimulating factor 1 receptor (CSF1R) that is capable of binding a CSF1R ligand,   (b) a second domain comprising a portion of CD-40 ligand (CD40L) that is capable of binding an CD40L receptor, and   (c) a linker linking the first domain and the second domain.   
     
     
         51 . The method of  claim 50 , wherein the subject's T cells are activated when bound by the second domain of the heterologous chimeric protein and:
 (a) one or more tumor cells are prevented from transmitting an immunosuppressive signal when bound by the first domain of the heterologous chimeric protein,   (b) a quantifiable cytokine response in the peripheral blood of the subject is achieved, and/or   (c) tumor growth is reduced in the subject in need thereof as compared to a subject treated with CD40 blocking antibodies and/or CSF1 or IL-34 blocking antibodies.   
     
     
         52 . The method of  claim 50  or  claim 51 , wherein the method inhibits CSF1R signaling and inhibits of suppressive myeloid cell populations. 
     
     
         53 . The method of any one of  claims 50  to  52 , wherein the method stimulates CD40 signaling and activates antigen-presenting cells. 
     
     
         54 . The method of any one of  claims 50  to  53 , wherein the method reduces the amount or activity of tumor associated macrophages (TAMs) as compared to untreated subjects or subjects targeting one of CD40/CD40L and CSF1/CSF1R. 
     
     
         55 . The method of any one of  claims 50  to  54 , wherein the method reduces the amount or activity of tumor associated macrophages (TAMs) in the tumor microenvironment (TME) as compared to untreated subjects or subjects targeting one of CD40/CD40L and CSF1/CSF1R. 
     
     
         56 . The method of any one of  claims 50  to  55 , wherein the method reduces the amount or activity of regulatory T cells (Tregs) as compared to untreated subjects or subjects targeting one of CD40/CD40L and CSF1/CSF1R. 
     
     
         57 . The method of any one of  claims 50  to  56 , wherein the method reduces the amount or activity of IL-10 and/or IL-4 as compared to untreated subjects or subjects targeting one of CD40/CD40L and CSF1/CSF1R. 
     
     
         58 . The method any of one of  claims 50  to  57 , wherein the method increases maturation and differentiation of proinflammatory macrophages and dendritic cells as compared to untreated subjects or subjects targeting one of CD40/CD40L and CSF1/CSF1R. 
     
     
         59 . The method of any one of  claims 50  to  58 , wherein the method increases priming of effector T cells in draining lymph nodes of the subject as compared to untreated subjects or subjects targeting one of CD40/CD40L and CSF1/CSF1R. 
     
     
         60 . The method of any one of  claims 50  to  59 , wherein the method causes an overall decrease in immunosuppressive cells and a shift toward a more inflammatory tumor environment as compared to untreated subjects or subjects targeting one of CD40/CD40L and CSF1/CSF1R. 
     
     
         61 . The chimeric protein of any one of  claims 1  to  42  for use as a medicament. 
     
     
         62 . The chimeric protein of any one of  claims 1  to  42  for use in the treatment of cancer. 
     
     
         63 . The chimeric protein of any one of  claims 1  to  42  for use in the treatment of an inflammatory disease. 
     
     
         64 . Use of the chimeric protein of any one of  claims 1  to  42  in the manufacture of a medicament. 
     
     
         65 . A recombinant fusion protein comprising a general structure of:
   N terminus-( a )-( b )-( c )-C terminus,   
       wherein:
 (a) is a first domain comprising an extracellular domain of CSF1R that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2 and is capable of binding a CSF1R ligand, 
 (b) is a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain derived from human IgG4, and optionally a joining linker sequence from SEQ ID 28 to 74, and 
 (c) is a second domain comprising an extracellular domain of CD40 ligand (CD40L) that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 and is capable of binding an CD40L receptor. 
 
     
     
         66 . The recombinant fusion protein of  claim 65 , wherein the linker comprises a sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. 
     
     
         67 . The recombinant fusion protein of  claim 65  or  claim 66  for use as a medicament. 
     
     
         68 . The recombinant fusion protein of any one of  claims 65  to  67  for use in the treatment of cancer. 
     
     
         69 . The recombinant fusion protein of any one of  claims 65  to  68  for use in the treatment of an inflammatory disease. 
     
     
         70 . Use of the recombinant fusion protein of any one of  claims 65  to  69  in the manufacture of a medicament.