Immunotoxins for the treatment of diseases related to CMV infection
The present invention relates to the field of cytomegalovirus (CMV) infection. In particular the present invention relates to highly specific immunotoxins useful in treating diseases related to CMV infection. CMV encodes chemokine receptors that undergo constitutive internalization. Thus CMV infected cells can be targeted specifically with immunotoxins with high affinity to CMV encoded constitutively internalizing receptors. This will ensure efficient uptake of the immunotoxin by the CMV infected cell, and thereby ensure the death of the infected cell with a minimum of unwanted toxicity and side effects. Furthermore, the invention relates to a way of inhibiting CMV replication and/or growth by using immunotoxins by targeting constitutively internalizing CMV encoded receptors.
1. An immunotoxin comprising (a) a ligand which is a variant of a CX 3 C chemokine that binds a constitutively internalizing CMV encoded chemokine receptor US28 expressed on a CMV infected cell and (b) a toxin that is cytotoxic to the CMV infected cell, wherein the ligand has increased specificity towards US28 as compared to the parental ligand and the ligand is a variant having at least 80% amino acid sequence identity to amino acids present at positions 25-100 of SEQ ID NO: 2 in the chemokine domain of human CX 3 CL1; and
(i) the ligand is a variant mutated in position 31 of SEQ ID NO: 2, a variant mutated in position 38 of SEQ ID NO: 2, a variant mutated in position 60 of SEQ ID NO: 2, a variant mutated in position 68 of SEQ ID NO: 2, a variant mutated in position 71 of SEQ ID NO: 2, or a variant mutated in position 73 of SEQ ID NO: 2; or
(ii) the ligand is a variant at one or more of amino acid residues in positions 33, 34 and 35 of SEQ ID NO: 2, wherein the one or more residues have been mutated or deleted.
2. The immunotoxin according to claim 1 , wherein the ligand is a chimera between the CX 3 C chemokine and a chemokine selected from the group consisting of CC-chemokines, XC-chemokines and CXC-chemokines.
3. The immunotoxin according to claim 1 , wherein the ligand is a variant which has at least 90% amino acid sequence identity to amino acids present at positions 25-100 of SEQ ID NO: 2 in the chemokine domain of human CX 3 CL1.
4. The immunotoxin according to claim 1 , wherein the ligand is selected from the group consisting of the following variants: a variant mutated in position 31 of SEQ ID NO: 2 to an Alanine, a variant mutated in position 31 of SEQ ID NO: 2 to an Glutamate, a variant in position 38 of SEQ ID NO: 2 to an Alanine, a variant mutated in position 38 of SEQ ID NO: 2 to a Glutamate, a variant mutated in position 60 of SEQ ID NO: 2 to a Alanine, a variant mutated in position 60 of SEQ ID NO: 2 to a Glutamate, a variant mutated in position 68 of SEQ ID NO: 2 to a Alanine, a variant mutated in position 68 of SEQ ID NO: 2 to a Glutamate, a variant mutated in position 71 of SEQ ID NO: 2 to a Alanine, a variant mutated in position 71 of SEQ ID NO: 2 to a Glutamate, a variant mutated in position 71 of SEQ ID NO: 2 to a Glutamine, a variant mutated in position 73 of SEQ ID NO: 2 to a Alanine and a variant mutated in position 73 of SEQ ID NO: 2 to a Leucine.
5. The immunotoxin according to claim 1 , wherein the ligand binds to US28 with a Kd of 10 −8 M or less.
6. The immunotoxin according to claim 5 , wherein the ligand has at least one binding effect selected from those consisting of (i) binding to the US28 receptor with a Kd of less than 10 −9 M; and (ii) binding to the CX 3 CR1 receptor with a Kd of 10 −6 M or more.
7. The immunotoxin according to claim 1 , wherein the toxin is selected from the group consisting of gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria, restrictocin, diphtheria toxin, Pseudomonas exotoxin A and variants thereof.
8. The immunotoxin according to claim 7 , wherein the toxin is Pseudomonas exotoxin A or a variant thereof.
9. The immunotoxin according to claim 8 , wherein the toxin is Pseudomonas exotoxin A variant PE38 KDEL of SEQ ID NO: 9.
10. A pharmaceutical composition comprising an immunotoxin according to claim 1 , or a physiological acceptable salt thereof, and a pharmaceutical acceptable carrier.
11. The pharmaceutical composition according to claim 10 , wherein the composition further comprises at least one anti-viral or immunosuppressive therapeutic agent.
12. A kit for treatment of CMV infection comprising (a) an effective amount of an immunotoxin according to claim 1 and (b) an antiviral or immuno-suppressive therapeutic, wherein (a) and (b) are for simultaneous, separate or sequential administration.
13. The immunotoxin according to claim 1 , wherein the ligand binds to the US28 receptor with a Kd of less than 10 −9 M.
14. The immunotoxin according to claim 1 , wherein the ligand binds to the CX3CR1 receptor with a Kd of 10 −6 M or more.
15. The immunotoxin according to claim 1 , wherein the ligand is selected from the group consisting of the following variants: a variant mutated in position 31 of SEQ ID NO:2 to an Alanine, a variant mutated in position 31 of SEQ ID NO:2 to an Glutamate, a variant in position 38 of SEQ ID NO:2 to an Alanine, a variant mutated in position 38 of SEQ ID NO:2 to a Glutamate, a variant mutated in position 42 of SEQ ID NO:2 to an Alanine, a variant mutated in position 42 of SEQ ID NO:2 to a Glutamate, a variant mutated in position 60 of SEQ ID NO:2 to a Alanine, a variant mutated in position 60 of SEQ ID NO:2 to a Glutamate, a variant mutated in position 61 of SEQ ID NO:2 to a Alanine, a variant mutated in position 61 of SEQ ID NO:2 to a Glutamate, a variant mutated in position 68 of SEQ ID NO:2 to a Alanine, a variant mutated in position 68 of SEQ ID NO:2 to a Glutamate, a variant mutated in position 71 of SEQ ID NO:2 to a Alanine, a variant mutated in position 71 of SEQ ID NO:2 to a Glutamate, a variant mutated in position 71 of SEQ ID NO:2 to a Glutamine, a variant mutated in position 72 of SEQ ID NO:2 to a Alanine, a variant mutated in position 73 of SEQ ID NO:2 to a Alanine and a variant mutated in position 73 of SEQ ID NO:2 to a Leucine.