Endolysosomal targeting conjugates for improved delivery of cargo molecules to the endolysosomal compartment of target cells

Abstract

Endolysosomal targeting conjugates that are engineered to deliver cargo molecules such as cytotoxic drugs or imaging labels with improved efficiency to late endosomes and/or lysosomes in target cells such as tumor cells are described. The endolysosomal targeting conjugate includes a targeting component and a cargo component. The targeting component is configured to bind to a cell surface molecule of a target cell and the cargo component includes a cargo molecule. The targeting component and the cargo component may be fused by a covalent bond or associated by a non-covalent bond. The targeting component may bind to the cell surface molecule or the cargo component with higher affinity in the extracellular space than in an endolysosomal compartment of the target cell.

1 . An endolysosomal targeting conjugate, comprising:
 a targeting component comprising an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain, wherein the targeting component is configured to bind to a cell surface molecule of a target cell with a lower dissociation constant in an extracellular space than in an endolysosomal compartment of the target cell; and   a cargo component comprising a cargo molecule conjugated to an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain;   wherein   the targeting component is fused directly or indirectly to the cargo component;   upon entry to the endolysosomal compartment, the targeting component is configured to dissociate from the cell surface molecule; and   wherein the endolysosomal targeting conjugate is configured to deliver the cargo molecule to the endolysosomal compartment of the target cell.   
     
     
         2 . (canceled) 
     
     
         3 . The endolysosomal targeting conjugate of  claim 1  wherein the targeting component comprises an antibody, an antibody fragment, an antibody domain, or a nanobody that is configured to bind to the cell surface molecule with a lower dissociation constant at a near neutral pH than at an acidic endolysosomal pH. 
     
     
         4 . (canceled) 
     
     
         5 . The endolysosomal targeting conjugate of  claim 1  wherein the targeting component comprises an antibody, an antibody fragment, an antibody domain, or a nanobody that is configured to bind to a cell surface molecule with a lower dissociation constant at an extracellular Ca2+ concentration than at an endolysosomal Ca2+ concentration. 
     
     
         6 - 7 . (canceled) 
     
     
         8 . The endolysosomal targeting conjugate of  claim 1  wherein the targeting component is a protein, a protein fragment or a protein domain that is configured to bind to a cell surface molecule with a lower dissociation constant at a near neutral pH than at an acidic endolysosomal pH. 
     
     
         9 . (canceled) 
     
     
         10 . The endolysosomal targeting conjugate of  claim 1  wherein the targeting component comprises a protein, a protein fragment or a protein domain that is configured to bind to a cell surface molecule with a lower dissociation constant at an extracellular Ca 2+  concentration than at an endolysosomal Ca 2+  concentration. 
     
     
         11 . (canceled) 
     
     
         12 . The endolysosomal targeting conjugate of  claim 1  wherein the cargo component comprises an antibody, an antibody fragment or an antibody domain comprising an antibody Fc region or a domain of the antibody Fc fragment. 
     
     
         13 . The endolysosomal targeting conjugate of  claim 12  wherein the antibody Fc region or the domain of the antibody Fc fragment is derived from human IgG1. 
     
     
         14 . The endolysosomal targeting conjugate of  claim 1  wherein the cargo component comprises an albumin molecule or a domain of albumin. 
     
     
         15 . The endolysosomal targeting conjugate of  claim 3  wherein the targeting component comprises a Fab fragment or a scFv fragment of a HER2-specific antibody, wherein a heavy chain variable domain of the Fab fragment or the scFv fragment comprises mutations of Ser55 to histidine and Gly57 to glutamic acid. 
     
     
         16 . The endolysosomal targeting conjugate of  claim 3  wherein the targeting component comprises a Fab fragment or a scFv fragment of a HER2-specific antibody, wherein in the Fab fragment or the scFv fragment a heavy chain variable domain comprises a mutation of Ser103 to histidine and a light chain variable domain comprises a mutation of Tyr55 to histidine. 
     
     
         17 - 26 . (canceled) 
     
     
         27 . The endolysosomal targeting conjugate of  claim 1 , wherein the cargo molecule is a cytotoxic drug. 
     
     
         28 . (canceled) 
     
     
         29 . The endolysosomal targeting conjugate of  claim 1  wherein the cargo molecule is an imaging label. 
     
     
         30 . An endolysosomal targeting conjugate, comprising:
 a targeting component comprising an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain, wherein the targeting component is configured to bind to a cell surface molecule of a target cell; and   a cargo component comprising a cargo molecule conjugated to an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain;   wherein   the targeting component is configured to bind to the cargo component with a lower dissociation constant in an extracellular space than in an endolysosomal compartment of the target cell;   upon entry to the endolysosomal compartment, the targeting component is configured to dissociate from the cargo component; and   wherein the endolysosomal targeting conjugate is configured to deliver the cargo molecule to the endolysosomal compartment of the target cell.   
     
     
         31 . The endolysosomal targeting conjugate of  claim 30  wherein the targeting component is configured to bind to the cargo compartment with a lower dissociation constant at near neutral pH than at acidic endosomal pH. 
     
     
         32 . (canceled) 
     
     
         33 . The endolysosomal targeting conjugate of  claim 30  wherein the targeting component is configured to bind to the cargo component with a lower dissociation constant at an extracellular Ca2+ concentration than at an endolysosomal Ca 2+  concentration. 
     
     
         34 - 43 . (canceled) 
     
     
         44 . A method of providing an endolysosomal targeting conjugate for the treatment of cancer comprising the steps of:
 selecting the targeting component of  claim 1 , wherein the targeting component comprises an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain configured to selectively bind to a cell surface molecule on a selected type of tumor target cell, wherein the targeting component is configured to bind to a cell surface molecule with a lower dissociation constant in an extracellular space than in an endolysosomal compartment;   selecting the cargo component of  claim 1 , wherein the cargo molecule comprises a cytotoxic drug having efficacy for suppressing growth of the selected type of tumor target cell; and   providing the endolysosomal targeting conjugate comprising the targeting component fused directly or indirectly to the cargo component.   
     
     
         45 . A method of providing an endolysosomal targeting conjugate for the treatment of cancer comprising the steps of:
 selecting the targeting component of  claim 30 , wherein the targeting component comprises an antibody, an antibody fragment, a nanobody, a protein, a protein fragment, or a protein domain configured to selectively bind to a cell surface molecule on a selected type of tumor target cell;   selecting the cargo component of  claim 30 , wherein the cargo molecule comprises a cytotoxic drug having efficacy for suppressing growth of the selected type of tumor target cell;   wherein   the targeting component is engineered to further comprise a first protein domain;   the cargo component is engineered to further comprise a second protein domain; and   the first protein domain is configured to bind to the second domain with a lower dissociation constant in an extracellular space than in an endolysosomal compartment.   
     
     
         46 - 52 . (canceled) 
     
     
         53 . The endolysosomal targeting conjugate of  claim 1 , wherein the targeting component comprises an antibody, an antibody fragment, an antibody domain, or a nanobody that is configured to bind to human epidermal growth factor receptor 2. 
     
     
         54 . (canceled) 
     
     
         55 . The endolysosomal targeting conjugate of  claim 1 , comprising one or more proteins having an amino acid sequence of at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, or a homolog thereof. 
     
     
         56 - 68 . (canceled) 
     
     
         69 . The endolysosomal targeting conjugate of  claim 1 , wherein the cargo molecule is a drug or other agent that modifies a behavior of the target cell.