Compositions and methods of enhancing anti-tumor response using hybrid neutrophils

Abstract

The present invention relates to compositions and methods that provide novel anti-tumor therapies in cancer. In one aspect, the present invention features a hybrid neutrophil in a non-naturally occurring container, wherein the hybrid neutrophil expresses at least one neutrophil associated molecule selected from the group consisting of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting cell (APC) associated molecule selected from the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89. In another aspect, the present invention features methods of generating a hybrid neutrophil. In still another aspect, the present invention features methods of inhibiting tumor growth in a subject, treating a tumor in a subject, and increasing efficacy of an antibody against a tumor in a subject. The methods comprise (a) administering to the subject an effective amount of an anti-tumor antibody and (b) administering to or generating in the subject an effective amount of a hybrid neutrophil.

1 . A method of generating a hybrid neutrophil, the method comprising contacting a composition comprising a bone marrow (BM) immature CD15-positive (CD15 + ) cell with an amount of tumor conditioned medium, wherein the hybrid neutrophil expresses at least one neutrophil associated molecule selected from the group consisting of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting cell (APC) associated molecule selected from the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89. 
     
     
         2 . A method of generating a hybrid neutrophil, the method comprising contacting a composition comprising a bone marrow (BM) immature CD15-positive (CD15 + ) cell with an amount of interferon γ (IFN-γ) and an amount of granulocyte macrophage colony stimulating factor (GM-CSF), wherein the hybrid neutrophil expresses at least one neutrophil associated molecule selected from the group consisting of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting cell (APC) associated molecule selected from the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89. 
     
     
         3 . A method of generating a hybrid neutrophil, the method comprising contacting a composition comprising a bone marrow (BM) immature CD15-positive (CD15 + ) cell with an amount of an agent that reduces the level of Ikaros polypeptide in the cell and an amount of granulocyte macrophage colony stimulating factor (GM-CSF), wherein the hybrid neutrophil expresses at least one neutrophil associated molecule selected from the group consisting of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting cell (APC) associated molecule selected from the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89. 
     
     
         4 . A method of generating a hybrid neutrophil, the method comprising contacting a composition comprising peripheral blood immature neutrophils with an amount of tumor conditioned medium, wherein the hybrid neutrophil expresses at least one neutrophil associated molecule selected from the group consisting of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting cell (APC) associated molecule selected from the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89. 
     
     
         5 . A method of generating a hybrid neutrophil, the method comprising contacting a composition comprising peripheral blood immature neutrophils with an amount of interferon γ (IFN-γ) and an amount of granulocyte macrophage colony stimulating factor (GM-CSF), wherein the hybrid neutrophil expresses at least one neutrophil associated molecule selected from the group consisting of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting cell (APC) associated molecule selected from the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89. 
     
     
         6 . The method of  claim 5 , wherein the peripheral blood immature neutrophils are mobilized in peripheral blood by contacting peripheral blood with an amount of granulocyte macrophage colony stimulating factor (GM-CSF) or an amount of granulocyte colony stimulating factor (G-CSF). 
     
     
         7 . The method of  claim 2 , wherein the amount of granulocyte macrophage colony stimulating factor (GM-CSF) or the amount of interferon γ (IFN-γ) is at least about 50 pg/ml, at least about 60 pg/ml, at least about 70 pg/ml, at least about 80 pg/ml, at least about 90 pg/ml, or at least about 100 pg/ml. 
     
     
         8 . The method of  claim 3 , wherein the agent is lenalidomide. 
     
     
         9 . The method of  claim 4 , wherein the amount of tumor conditioned medium is about 50% v/v. 
     
     
         10 . The method of  claim 1 , wherein the hybrid neutrophil further expresses at least one molecule selected from the group consisting of: MHC class I, MHC class II, OX40L, 4-1BBL, CD86, CD40, and CCR7. 
     
     
         11 . The method of  claim 1 , wherein the expression level of any one of the molecules is low, intermediate, or high. 
     
     
         12 . The method of  claim 1 , wherein the expression of any one of the molecules is increased relative to expression of the molecule on a canonical tumor-associated neutrophil (TAN). 
     
     
         13 . The method of  claim 1 , wherein the hybrid neutrophil expresses CD14, HLA-DR, CD32, CD64, and CD89. 
     
     
         14 . The method of  claim 10 , wherein the hybrid neutrophil expresses Arg1, MPO, CD66b, CD15, CD14, HLA-DR, MHC class I, OX40L, 4-1BBL, CD86, CD40, CCR7, CD32, CD64, and CD89. 
     
     
         15 . The method of  claim 13 , wherein the expression of CD32 and/or CD64 and/or CD89 is high. 
     
     
         16 . A method of inhibiting tumor growth in a subject, the method comprising
 (a) administering to the subject an effective amount of an anti-tumor antibody or an antigen-binding fragment thereof; and   (b) administering to or generating in the subject an effective amount of a hybrid neutrophil, wherein the hybrid neutrophil expresses at least one neutrophil associated molecule selected from the group consisting of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting cell (APC) associated molecule selected from the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89, thereby inhibiting tumor growth in the subject.   
     
     
         17 . A method of increasing efficacy of an antibody against a tumor in a subject, the method comprising
 (a) administering to the subject an effective amount of an anti-tumor antibody or an antigen-binding fragment thereof; and   (b) administering to or generating in the subject an effective amount of a hybrid neutrophil, wherein the hybrid neutrophil expresses at least one neutrophil associated molecule selected from the group consisting of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting cell (APC) associated molecule selected from the consisting of: CD14, HLA-DR, CD32, CD64, and CD89, thereby increasing efficacy of the antibody against the tumor in the subject.   
     
     
         18 . A method of treating a tumor in a subject, the method comprising
 (a) administering to the subject an effective amount of an anti-tumor antibody or an antigen-binding fragment thereof; and   (b) administering to or generating in the subject an effective amount of a hybrid neutrophil, wherein the hybrid neutrophil expresses at least one neutrophil associated molecule selected from the group consisting of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting cell (APC) associated molecule selected from the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89, thereby treating the tumor in the subject.   
     
     
         19 . The method of  claim 16 , wherein the hybrid neutrophil further expresses at least one molecule selected from the group consisting of: MHC class I, MHC class II, OX40L, 4-1BBL, CD86, CD40, and CCR7. 
     
     
         20 . The method of  claim 16 , wherein the expression of any one of the molecules is low, intermediate, or high. 
     
     
         21 . The method of  claim 16 , wherein the expression of any one of the molecules is increased relative to expression of the molecule on a canonical tumor-associated neutrophil (TAN). 
     
     
         22 . The method of  claim 16 , wherein the hybrid neutrophil expresses CD14, HLA-DR, CD32, CD64, and CD89. 
     
     
         23 . The method of  claim 19 , wherein the hybrid neutrophil expresses Arg1, MPO, CD66b, CD15, CD14, HLA-DR, MHC class I, OX40L, 4-1BBL, CD86, CD40, CCR7, CD32, CD64, and CD89 . 
     
     
         24 . The method of  claim 22 , wherein the expression of CD32 and/or CD64 and/or CD89 is high. 
     
     
         25 . The method of  claim 16 , wherein the anti-tumor antibody is selected from the group consisting of: anti-Her2/neu antibody, rituximab, necitumumab, panitumumab, and cetuximab. 
     
     
         26 . The method of  claim 16 , wherein the step of administering to the subject an effective amount of a hybrid neutrophil increases antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), or effector T cell response in the subject. 
     
     
         27 . The method of  claim 16 , wherein the hybrid neutrophil is generated ex vivo in a biological sample obtained from the subject. 
     
     
         28 . The method of  claim 27 , wherein the biological sample is blood or bone marrow. 
     
     
         29 . The method of  claim 28 , wherein the hybrid neutrophil population is generated by a method according to  claim 1 . 
     
     
         30 . The method of  claim 16 , wherein the hybrid neutrophil is generated in situ in the subject. 
     
     
         31 . The method of  claim 30 , wherein the hybrid neutrophil is generated in situ by administering to the subject an amount of granulocyte macrophage colony stimulating factor (GM-CSF) and at least one agent selected from the group comprised of: IFN-γ and lenalidomide. 
     
     
         32 . The method of  claim 16 , wherein the step of administering to or generating in the subject an effective amount of a hybrid neutrophil is followed by the step of administering to the subject an effective amount of an anti-tumor antibody or an antigen-binding fragment thereof. 
     
     
         33 . The method of  claim 16 , wherein the step of administering to or generating in the subject an effective amount of a hybrid neutrophil is concurrent with the step of administering to the subject an effective amount of an anti-tumor antibody or an antigen-binding fragment thereof. 
     
     
         34 . The method of  claim 16 , wherein the tumor comprises non-small cell lung cancer (NSCLC). 
     
     
         35 . The method of  claim 16 , wherein the subject is human. 
     
     
         36 . A hybrid neutrophil in a non-naturally occurring container, wherein the hybrid neutrophil expresses at least one neutrophil associated molecule selected from the group consisting of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting cell (APC) associated molecule selected from the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89. 
     
     
         37 . The hybrid neutrophil of  claim 36 , wherein the hybrid neutrophil further expresses at least one molecule selected from the group consisting of: MHC class I, MHC class II, OX40L, 4-1BBL, CD86, CD40, and CCR7. 
     
     
         38 . The hybrid neutrophil of  claim 36 , wherein the expression of any one of the molecules is low, intermediate, or high. 
     
     
         39 . The hybrid neutrophil of  claim 36 , wherein the expression of any one of the molecules is increased relative to expression of the molecule on a canonical tumor-associated neutrophil (TAN). 
     
     
         40 . The hybrid neutrophil of  claim 36 , wherein the hybrid neutrophil expresses CD14, HLA-DR, CD32, CD64, and CD89. 
     
     
         41 . The hybrid neutrophil of  claim 37 , wherein the hybrid neutrophil expresses Arg1, MPO, CD66b, CD15, CD14, HLA-DR, MHC class I, OX40L, 4-1BBL, CD86, CD40, CCR7, CD32, CD64, and CD89. 
     
     
         42 . The hybrid neutrophil of  claim 40 , wherein the expression of CD32 and/or CD64 and/or CD89 is high.