Tetra-specific, octameric binding agents and antibodies against
Novel, antibody-based binding agents derived from human and camelid immunoglobulins are described. These binding agents recognize and bind with specificity to Clostridium difficile toxin A and/or toxin B and in some cases exhibit toxin neutralizing activity. These binding agents can be used to treat or prevent primary and recurrent CDI. The binding agents include camelid V H H peptide monomers, linked groups of V H H peptide monomers, V H H peptide monomers joined to antibody Fc domains, and V H H peptide monomers joined to IgG antibodies.
1. A tetra-specific, octameric binding agent comprising:
(a) an IgG antibody, two sets of linked first and second VHH peptide monomers, and two sets of linked third and fourth VHH peptide monomers,
wherein the IgG antibody comprises two arms, each arm comprising a heavy chain lacking a variable region and a light chain lacking a variable region, and each chain having an amino terminus,
wherein for each arm of the antibody, one set of linked first and second VHH peptide monomers is joined to the amino terminus of the light chain, and one set of linked third and fourth VHH peptide monomers is joined to the amino terminus of the heavy chain, and wherein the VHH peptide monomers have binding specificity for an epitope of Clostridium difficile toxin A (TcdA) or toxin B (TcdB); or
(b) an antibody Fc domain and two sets of linked first, second, third and fourth VHH peptide monomers,
wherein the antibody Fc domain comprises two arms, each arm comprising hinge, C H 2 and C H 3 regions of an antibody heavy chain, and each arm having an amino terminus,
wherein for each arm of the Fc domain, one set of linked first, second, third and fourth V H H peptide monomers is joined to the amino terminus of the arm, and
wherein the V H H peptide monomers have binding specificity for an epitope of C. difficile toxin A (TcdA) or toxin B (TcdB);
wherein for the binding agents of (a) and (b), the V H H peptide monomers are (i) the 5D V H H monomer (SEQ ID NO: 1), (ii) the E3 V H H monomer (SEQ ID NO:3), (iii) the AA6 V H H monomer (SEQ ID NO:5), and (iv) the AH3 V H H monomer (SEQ ID NO:7);
wherein for the binding agent of (a), the light chain of the binding agent comprises the amino acid sequence set forth in SEQ ID NO:46, and wherein the heavy chain of the binding agent comprises the amino acid sequence set forth in SEQ ID NO:44; and
wherein for the binding agent of (b), the binding agent comprises the amino acid sequence set forth in SEQ ID NO:22.
2. The binding agent of claim 1 , wherein the first, second, third and fourth V H H peptide monomers each has binding specificity for a different epitope.
3. The binding agent of claim 1 , wherein two of the V H H peptide monomers have binding specificity for epitopes of TcdA and two of the V H H peptide monomers have binding specificity for epitopes of TcdB.
4. The binding agent of claim 1 , wherein the V H H peptide monomers independently have binding specificity for an epitope in the glucosyltransferase domain, cysteine protease domain, translocation domain or receptor binding domain of TcdA or TcdB.
5. The binding agent of claim 1 , wherein the binding agent is a binding agent of (a).
6. A method of producing a binding agent comprising culturing an isolated host cell comprising an expression vector comprising an isolated polynucleotide sequence comprising a nucleotide sequence encoding a binding agent of claim 1 under conditions promoting expression of the binding agent, and recovering the binding agent from the cell culture.
7. The binding agent of claim 1 , wherein the binding agent is a binding agent of (b).
8. A pharmaceutical formulation comprising a binding agent of any one of claims 1 , 5 and 7 and a pharmaceutically acceptable carrier or diluent.
9. A method of treating or preventing a disease symptom induced by C. difficile in a subject comprising administering a therapeutically-effective amount of one or more binding agent of any one of claims 1 , 5 and 7 , or pharmaceutical formulation comprising the one or more binding agent and a pharmaceutically acceptable carrier or diluent, to a subject having C. difficile infection or a risk of developing C. difficile infection.
10. A method of neutralizing C. difficile toxin TcdA and/or TcdB in a subject infected by C. difficile comprising administering a therapeutically-effective amount of one or more binding agent of any one of claims 1 , 5 and 7 , or pharmaceutical formulation comprising the one or more binding agent and a pharmaceutically acceptable carrier or diluent, to a subject having C. difficile infection.
11. A method of treating or preventing C. difficile disease in a subject comprising administering a therapeutically-effective amount of one or more binding agent of any one of claims 1 , 5 and 7 , or pharmaceutical formulation comprising the one or more binding agent and a pharmaceutically acceptable carrier or diluent, to a subject having C. difficile infection or a risk of developing C. difficile infection.
12. The method of claim 10 , where the neutralizing is partial or full neutralization.