Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery
Herein is reported a polypeptide-polynucleotide-complex as therapeutic agent and its use as tool for the targeted delivery of an effector moiety. The polynucleotide part of the complex is essentially resistant to proteolytic and enzymatic degradation in vivo. Additionally the polypeptide part specifically binds to a compound or structure such as a tissue or organ, a process or a disease. Thus, one aspect as reported herein is a polypeptide-polynucleotide-complex comprising a) a polypeptide specifically binding to a target and conjugated to a first member of a binding pair, b) a polynucleotide linker conjugated at its first terminus to the second member of the binding pair, and c) an effector moiety conjugated to a polynucleotide that is complementary to at least a part of the polynucleotide linker.
1. A method of producing a complex comprising the components
a) a first polypeptide that specifically binds to a first target and that is conjugated to a first member of a first binding pair,
b) a second polypeptide that specifically binds to a second target and that is conjugated to a first member of a second binding pair, and
c) a polynucleotide linker conjugated to the second member of the first binding pair and conjugated to the second member of the second binding pair, wherein the polynucleotide linker comprises ss-L-DNA,
comprising the steps of:
i) synthesizing the first polypeptide specifically binding to the first target which is conjugated to the first member of the first binding pair, and synthesizing the second polypeptide specifically binding to the second target which is conjugated to the first member of a second binding pair, respectively,
ii) synthesizing the polynucleotide linker conjugated at its first terminus to the second member of the first binding pair and conjugated at its second terminus to the second member of the second binding pair, and
iii) forming the complex by hybridizing the synthesized components,
wherein the first and second member of the first binding pair comprise the nucleic acid sequences of SEQ ID NO: 5 and SEQ ID NO: 8 or
wherein the first and second member of the second binding pair comprise the nucleic acid sequences of SEQ ID NO: 6 and SEQ ID NO: 7.
2. The method according to claim 1 , wherein the complex further comprises an effector moiety conjugated to a polynucleotide that is complementary to at least a part of the polynucleotide linker ss-L-DNA.
3. The method of claim 2 , wherein the effector moiety is selected from the group consisting of a binding moiety, a labeling moiety and a biologically active moiety.
4. The method according to claim 1 , wherein the first polypeptide is a monovalent antibody or monovalent antibody fragment.
5. The method of claim 1 , wherein the first and second polypeptides bind to the same target and to non-overlapping epitopes on the same target.
6. The method of claim 5 , wherein the polynucleotide linker has an optimal length for synergistic binding of the first and second polypeptides to the non-overlapping epitopes on the same target.
7. The method of claim 1 , wherein the members of the first and second binding pairs are ss-LDNA.
8. The method of claim 1 , wherein the members of the first and second binding pairs are ss-LDNA of 10 to 50 nucleotides in length.
9. The method of claim 1 , wherein the polynucleotide linker has a length of at least 70 nucleotides.
10. The method of claim 1 , wherein the complex is a non-covalent complex.
11. The method of claim 1 ,
wherein the first polypeptide is a FAB′ fragment of the anti-HER2 antibody 2C4 comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 39, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 40, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 41;
the second polypeptide is a FAB′ fragment of the anti-HER2 antibody 4D5 comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 27, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 29, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 31, a HVR-L2 comprising the amino acid sequence of SEQ ID NO: 32, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 33;
the members of the first and the second binding pairs are hybridizing nucleic acids; and
the ss-L-DNA-linker comprises 60 to 100 L-DNA nucleotides.