Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-02-13
2002-08-20
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S252300, C435S320100, C435S471000, C435S069700, C530S387100, C424S094630, C424S178100, C514S002600, C536S023200, C536S023400
Reexamination Certificate
active
06436691
ABSTRACT:
The invention relates to mutant CPB enzymes for use with prodrugs in ADEPT systems.
Abbreviations
Ac
acetyl
ADEPT
antibody directed enzyme prodrug therapy
BOC
tert-butoxycarbonyl
CPB
carboxypeptidase B
DCCI
1,3-dicyclohexylcarbodiimide
DMAP
4-dimethylaminopyridine
DMF
N,N-dimethyl-formamide
DMSO
dimethylsulfoxide
Et
ethyl
EDCI
1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide
HCPB
human CPB
HOBT
1-hydroxybenzotriazole
PCR
polymerase chain reaction
TFA
trifluoroacetic acid
THF
tetrahydrofuran
Targeting of drugs selectively to kill cancer cells in a patient has long been a problem for medical research. ADEPT is one approach to overcome the problem. ADEPT uses a tumour selective agent such as an antibody conjugated to an enzyme. The conjugate is administered to the patient (usually intravenously), allowed to localise at the tumour site(s) and clear from the general circulation. Subsequently a prodrug is administered to the patient which is converted by the enzyme (localised at the tumour sites) into a cytotoxic drug which kills tumour cells. Since one molecule of enzyme can catalyse generation of many cytotoxic drug molecules an amplification effect is produced. Furthermore tumour cells not displaying the antigen recognised by the antibody (tumours usually display microheterogeneity) are also killed by enzymically amplified generation of the cytotoxic drug. A known system uses the procaryotic enzyme carboxypeptidase G2 (CPG2) as the enzyme component (see WO 88/07378).
A further problem with known systems is that repeated administration of the conjugate results in a host immune response rendering the therapy less effective. The antibody component is generally a mouse monclonal antibody which can be humanised using known techniques to reduce immunogenicity. However reduction of the immunogenicity of the enzyme component has proved more problematic. This is because the enzyme component must not be present naturally in the human host circulation otherwise premature conversion of prodrug to cytotoxic drug will occur and little selective toxicity to tumours will be observed.
These problems have been addressed in part by International Patent application WO 95/13095 (Wellcome Foundation). This application proposed ADEPT using mutant mammalian enzymes to activate prodrugs which are not activated by the corresponding native enzyme. However only ADEPT systems using mutants of carboxypeptidase A were enabled in the disclosure.
According to one aspect of the present invention there is provided a conjugate which is substantially non-immunogenic in humans comprising a targeting moiety capable of binding with a tumour associated antigen, the targeting moiety being linked to a mutated form of a carboxypeptidase B (CPB) enzyme capable of converting a prodrug into an antineoplastic drug wherein the prodrug is not significantly convertible into antineoplastic drug in humans by natural unmutated enzyme.
Preferably the targeting moiety is an antibody.
Preferably the antibody is a F(ab′)2 antibody fragment.
Preferably the enzyme is mutated to comprise a polarity change in its active site such that it can turn over a prodrug with a complementary polarity.
Preferably the enzyme is any one of the following pancreatic human CPB mutants:
pancreatic human CPB having amino acid Asp 253 substituted by any one of Arg, Asn, Gln or Lys optionally in combination with any one or more amino acid substitutions selected from:
natural amino acid Gln 54 substituted by any one of Arg, Lys or Asn;
natural amino acid Asp 145 substituted by any one of Val, Leu, Ile or Ala;
natural amino acid Ile 201 substituted by any one of Ser or Thr;
natural amino acid Ser 205 substituted by any one of Asn, Gln, His, Lys or Arg;
natural amino acid Ile 245 substituted by any one of Ser, Thr, Ala, Val, Leu, Asn, Gln, Lys, Arg or His;
natural amino acid Ala 248 substituted by any one of Asn, Gln, Lys, Arg, His, Ser or Thr;
natural amino acid Gly 251 substituted by any one of Thr, Asn, Ser, Gln, His, Lys, Arg, Val, Ile, Leu, Met, Phe, Ala or Norleucine; and
natural amino acid Cys 288 substituted by any one of Ser, Thr, Ala, Val, Leu or Ile.
More preferably the enzyme is any one of the following pancreatic human CPB mutants:
pancreatic human CPB having natural amino acid Asp 253 substituted by any one of Arg or Lys and natural amino acid Gly 251 substituted by any one of Thr, Asn, Ser, Gln, Lys or Val, optionally in combination with any one or more amino acid substitutions selected from:
natural amino acid Gln 54 substituted by Arg;
natural amino acid Asp 145 substituted by Ala;
natural amino acid Ile 201 substituted by Ser;
natural amino acid Ser 205 substituted by Asn;
natural amino acid Ile 245 substituted by any one of Ser, Ala or His;
natural amino acid Ala 248 substituted by any one of His, Ser or Asn; and
natural amino acid Cys 288 substituted by any one of Ser or Ala.
More preferably the enzyme is any one of the following pancreatic human CPB mutants:
pancreatic human CPB having natural amino acid Asp 253 substituted by any one of Arg or Lys and natural amino acid Gly 251 substituted by any one of Thr, Asn or Ser optionally in combination with any one or more amino acid substitutions selected from:
natural amino acid Gln 54 substituted by Arg;
natural amino acid Asp 145 substituted by Ala;
natural amino acid Ile 201 substituted by Ser;
natural amino acid Ser 205 substituted by Asn;
natural amino acid lie 245 substituted by Ala;
natural amino acid Ala 248 substituted by any one of Ser or Asn; and
natural amino acid Cys 288 substituted by Ser.
Especially the enzyme is any one of the following pancreatic human CPB mutants:
D253K; D253R; [G251N, D253K]; [G251T, D253K]; [G251S, D253K]; [G251T, D253R]; [A248S,G251T,D253K]; [A248N,G251N,D253K]; [A248S,G251N,D253K]; or [S205N,G251N,D253K].
According to another aspect of the present invention there is provided a matched two component system designed for use in a host in which the components comprise:
(i) a first component that is a targeting moiety capable of binding with a tumour associated antigen, the targeting moiety being linked to a CPB enzyme capable of converting a prodrug into an antineoplastic drug and;
(ii) a second component that is a prodrug convertible under the influence of the enzyme to the antineoplastic drug;
wherein:
the enzyme is a mutated form of a CPB enzyme;
the first component is substantially non-immunogenic in the host and; the prodrug is not significantly convertible into antineoplastic drug in the host by natural unmutated host enzyme.
The term “the prodrug is not significantly convertible into antineoplastic drug in the host by natural unmutated host enzyme” means that the prodrug does not give undue untargeted toxicity problems on administration to the host.
The term “substantially non-immunogenic” means that the first component (conjugate) can be administered to the host on more than one occasion without causing significant host immune response as would be seen with for example the use of a mouse antibody linked to a bacterial enzyme in a human host.
Preferably the mutated enzyme is based on an enzyme from the same species as the host for which the system is intended for use but the mutated enzyme may be based on a host enzyme from a different species as long as the structure of the enzyme is sufficiently conserved between species so as not to create undue immunogenicity problems.
Preferably the targeting moiety is an antibody, especially an antibody fragment such as for example F(ab′)
2
. Linkage to enzyme for conjugate synthesis may be effected by known methods such as use of heterobifunctional reagents as cross-linkers or by gene fusion or any other suitable method. Antibody may be from the same host (eg use of mouse antibody in mice) or the antibody may be manipulated such that it is not significantly recognised as foreign in the chosen host (eg use of chimeric, CDR grafted or veneered mouse antibodies in humans). Preferably the first component is a conjugate as defined
Blakey David Charles
Davies David Huw
Dowell Robert Ian
Hennam John Frederick
Hennequin Laurent Francois Andre
Achutamurthy Ponnathapu
Zeneca Limited
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