MONOCLONAL ANTIBODIES BINDING MCM5

Abstract

The present invention relates to antibodies that bind to Mcm5. The invention further relates to compositions or kits comprising the antibodies, hybridomas capable of expressing the antibodies, polynucleotides, polypeptides and vectors coding for the antibodies and methods for making the antibodies.

Claims

1-38. (canceled)

39. A monoclonal antibody that specifically binds to Mcm5 which: (a) binds to an epitope having an amino acid sequence of (i) SEQ ID NO: 2; or (ii) SEQ ID NO: 1; or (b) comprises 4B4 CDRH1 which has a sequence of SEQ ID NO: 21, 4B4 CDRH2 which has a sequence of SEQ ID NO: 23, 4B4 CDRH3 which has a sequence of SEQ ID NO: 25, 4B4 CDRL1 which has a sequence of SEQ ID NO: 15, 4B4 CDRL2 which has a sequence of SEQ ID NO: 17, and 4B4 CDRL3 which has a sequence of SEQ ID NO: 19; or (c) comprises 12A7 CDRH1 which has a sequence of SEQ ID NO: 9, 12A7 CDRH2 which has a sequence of SEQ ID NO: 11, 12A7 CDRH3 which has a sequence of SEQ ID NO: 13, 12A7 CDRL1 which has a sequence of SEQ ID NO: 3, 12A7 CDRL2 which has a sequence of SEQ ID NO: 5, and 12A7 CDRL3 which has a sequence of SEQ ID NO: 7.

40. The antibody of claim 39 which has an affinity for Mcm5 in the range of 1-10 nM.

41. The antibody of claim 39(a)(ii) wherein the antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 29, and the antibody comprises a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 27.

42. The antibody of claim 39(c) wherein the antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 29, and the antibody comprises a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 27.

43. The antibody of claim 39(a)(i) wherein the antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 33, and the antibody comprises a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 31.

44. The antibody of claim 39(b) wherein the antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 33, and the antibody comprises a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 31.

45. A kit comprising: (i) a composition comprising a first antibody according to claim 39(a)(i); and (ii) a composition comprising a second antibody according to claim 39(a)(ii).

46. The kit of claim 45 wherein the first antibody and the second antibody both bind to Mcm5 but the first antibody and the second antibody bind to different epitopes of Mcm5; and (a) (i) the first antibody binds to SEQ ID NO: 1 and the second antibody binds to SEQ ID NO: 2; or (ii) the first antibody binds to SEQ ID NO: 2 and the second antibody binds to SEQ ID NO: 1; and/or (b) the first antibody is immobilized to an ELISA plate; and/or (c) the second antibody is immobilized to an ELISA plate; and/or (d) the second antibody is conjugated to a radioactive label, optionally wherein the radioactive label is Europium.sup.3+; and/or (e) the first antibody is conjugated to a radioactive label, optionally wherein the radioactive label is Europium.sup.3+.

47. A method of determining the concentration or amount of Mcm5 in a sample comprising the following steps: (i) providing an immobilized first antibody which is an antibody of claim 39 immobilized to a solid support; (ii) exposing the sample to the immobilized first antibody such that Mcm5 in the sample may bind to the immobilized antibody to provide immobilized Mcm5; (iii) providing a labelled second antibody which is an antibody of claim 39 conjugated to a label; (iv) exposing the immobilized Mcm5 to the labelled second antibody such that the labelled second antibody may bind to the Mcm5; and (v) detecting the concentration of the labelled second antibody; wherein the first antibody and the second antibody both bind to Mcm5 but the first antibody and the second antibody bind to different epitopes of Mcm5.

48. The method of claim 47 wherein the first antibody and the second antibody both bind to Mcm5 but the first antibody and the second antibody bind to different epitopes of Mcm5; and (a) (i) the first antibody binds to SEQ ID NO: 1 and the second antibody binds to SEQ ID NO: 2; or (ii) the first antibody binds to SEQ ID NO: 2 and the second antibody binds to SEQ ID NO: 1; and/or (b) the first antibody is immobilized to an ELISA plate; and/or (c) the second antibody is immobilized to an ELISA plate; and/or (d) the second antibody is conjugated to a radioactive label, optionally wherein the radioactive label is Europium.sup.3+; and/or (e) the first antibody is conjugated to a radioactive label, optionally wherein the radioactive label is Europium

49. The method of claim 47 wherein the step of detecting the concentration of the labelled second antibody comprises immunofluorescent detection of Eu.sup.3+.

50. The method of claim 48 wherein the step of detecting the concentration of the labelled second antibody comprises immunofluorescent detection of Eu.sup.3+.

Description

DETAILED DESCRIPTION

[0062] Antibody

[0063] The present invention provides an antibody. The term antibody can refer to naturally occurring forms or recombinant antibodies such as single-chain antibodies, chimeric antibodies or humanised antibodies. The terms antibody and antibodies may also be considered to encompass fragments of antibodies that can bind to a target protein, such as an Mcm protein like Mcm5. Such fragments may include Fab'.sub.2, F(ab).sub.2, Fv, single chain antibodies or diabodies. In a preferred embodiment antibodies of the invention are naturally occurring, full length antibodies (rather than fragments). In a preferred embodiment the antibody is a mouse antibody (i.e. originally derived from mouse spleen cells).

[0064] In general, antibodies are formed from two heavy chains and two lights chains. Each heavy chain is made up of a heavy chain constant region (CH) and a heavy chain variable region (VH). Similarly each light chain is made up of a light chain constant region (CL) and a light chain variable region (VL). The VH and VL regions comprise complementarity defining regions (CDRs). The CDRs are primarily responsible for specific binding to the target protein.

[0065] An antibody of the invention may be an antibody of any class, particularly IgG, IgM or IgA. In a preferred embodiment the antibody is an IgG antibody.

[0066] The term monoclonal antibody is intended to refer to an antibody that is obtained from a population of substantially similar antibodies, i.e. from a population of antibodies that are identical except for a minority of naturally occurring variants. A population of monoclonal antibodies will bind to the same epitope. Monoclonal antibodies can be produced by a variety of techniques well known to the person skilled in the art and including the methods disclosed in Monoclonal Antibodies: A manual of techniques, H Zola (CRC Press, 1988) and in Monoclonal Hybridoma Antibodies: Techniques and Application, SG Hurrell (CRC Press, 1982). In an embodiment an antibody of the invention is a monoclonal antibody.

[0067] Antibody Target

[0068] Preferably the antibody specifically to Mcm5. Even more preferably the antibody binds to at least one of SEQ ID NO: 1 or SEQ ID NO: 2. Optionally the antibody specifically binds to at least one of SEQ ID NO: 1 or SEQ ID NO: 2.

[0069] Mcm5 is a putative DNA replication licensing factor and a member of the MCM (minichromosome maintenance) protein family. MCM proteins bind to chromatin and are believed to exhibit helicase activity and thus be core components of DNA replication.

[0070] Mcm5 is upregulated in the transition from the G0 to G1/S phase of the cell cycle and may actively participate in cell cycle regulation; Mcm5 can interact with at least two other members of the MCM family.

[0071] MCM proteins 2-7 comprise part of the pre-replication complexes which form on chromatin and which are essential prerequisites, or licensing factors, for subsequent DNA replication. The MCM protein complexes act as replicative helicases and thus are core components of the DNA replication machinery. MCMs are upregulated in the transition from the GO to G1/S phase of the cell cycle and actively participate in cell cycle regulation. The MCM proteins form an annular structure around the chromatin.

[0072] The human Mcm5 gene maps to 22q13.1 and the mature Mcm5 protein consists of 734 amino acids (SEQ ID NO: 35; UNIPROT P33992: HUMAN DNA replication licensing factor MCMS). However, the Mcm5 gene may differ slightly in sequences between individuals. For this reason, the phrase binds to Mcm5 refers to antibodies that bind to SEQ ID NO: 35, but also to antibodies that bind to a polypeptide having sequences at least 90%, 95%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 35. In a preferred embodiment the antibody binds to a polypeptide 100% identical to SEQ ID NO: 35. Furthermore, an antibody of the invention will bind to an epitope (fragment) of Mcm5 (for example SEQ ID NO: 1 or SEQ ID NO: 2). Thus, the term antibody which binds to Mcm5 refers to an antibody that binds to only a single epitope of Mcm5 such as SEQ ID NO: 1 or SEQ ID NO: 2.

[0073] For the purposes of the present invention the term binding affinity refers to the ability of an antibody to bind to its target. For the purposes of the present invention, the term specifically binds' refers to an antibody that binds to a target such as Mcm5 with a binding affinity that is at least 2-fold, 10-fold, 50-fold or 100-fold greater than its binding affinity for binding to another non-target molecule. In an embodiment the non-target molecule is an Mcm protein, other than Mcm5, such as Mcm 2. Preferably, an antibody of the invention is capable of binding to an Mcm protein, optionally Mcm5, with a binding affinity that is at least 2-fold, 10-fold, 50-fold or 100-fold greater than its binding affinity for binding to another non-target molecule. Even more preferably, an antibody of the invention is capable of binding to SEQ ID NO: 1 or SEQ ID NO: 2 with a binding affinity that is at least 2-fold, 10-fold, 50-fold or 100-fold greater than its binding affinity for binding to another non-target molecule.

[0074] A preferred method for the evaluation of binding affinity for Mcm5 is by ELISA. Preferably, an antibody of the invention has an affinity for Mcm5 (measured as an EC50 or 50% maximum binding concentration, as described in Example 2) of 2500 ng/ml or lower, 1500 ng/ml or lower, 1000 ng/ml or lower, 600 ng/ml or lower, 50 ng/ml or lower, 30 ng/ml or lower, 20 ng/ml or lower, or 10 ng/ml or lower. The EC50 will typically be higher than 1 ng/ml and thus the EC50 may be between 1 ng/ml and any of the upper limits specified in the preceding sentence. Other standard assays to evaluate the binding ability of ligands such as antibodies towards targets are known in the art, including for example, Western blots, RIAs, and flow cytometry analysis. The binding kinetics (e.g. binding affinity) of the antibody also can be assessed by standard assays known in the art, such as by Surface Plasmon Resonance (SPR) (e.g. Biacore system) analysis. The affinity constant (KD) for binding to Mcm5 is preferably in the range of 1-10 000 nM, 1-1000 nM, 1-500 nM, 1-100 nM, 1-50 nM or 1-10 nM. The association rate (ka) is preferably in the range of 0.4-3.410.sup.6 1/M. The dissociation rate (kd) is preferably in the range of 1-1010.sup.3 1/s. These values may typically be determined by SPR (Surface Plasmon Resonance).

[0075] Complementary Determining Regions (CDRs)

[0076] An antibody of the invention preferably comprises at least one of the CDRs of antibodies 12A7 or 4B4, i.e. a CDR selected from the group consisting of: [0077] a. 12A7 CDRH1 which has a sequence of SEQ ID NO: 9 or a sequence that differs from SEQ ID NO: 9 by 1, 2 or 3 amino acid substitutions; [0078] b. 12A7 CDRH2 which has a sequence of SEQ ID NO: 11 or a sequence that differs from SEQ ID NO: 11 by 1, 2 or 3 amino acid substitutions; [0079] c. 12A7 CDRH3 which has a sequence of SEQ ID NO: 13 or a sequence that differs from SEQ ID NO: 13 by 1, 2 or 3 amino acid substitutions; [0080] d. 12A7 CDRL1 which has a sequence of SEQ ID NO: 3 or a sequence that differs from SEQ ID NO: 3 by 1, 2 or 3 amino acid substitutions; [0081] e. 12A7 CDRL2 which has a sequence of SEQ ID NO: 5 or a sequence that differs from SEQ ID NO: 5 by 1, 2 or 3 amino acid substitutions; [0082] f. 12A7 CDRL3 which has a sequence of SEQ ID NO: 7 or a sequence that differs from SEQ ID NO: 7 by 1, 2 or 3 amino acid substitutions; [0083] g. 4B4 CDRH1 which has a sequence of SEQ ID NO: 21 or a sequence that differs from SEQ ID NO: 21 by 1, 2 or 3 amino acid substitutions; [0084] h. 4B4 CDRH2 which has a sequence of SEQ ID NO: 23 or a sequence that differs from SEQ ID NO: 23 by 1, 2 or 3 amino acid substitutions; [0085] i. 4B4 CDRH3 which has a sequence of SEQ ID NO: 25 or a sequence that differs from SEQ ID NO: 25 by 1, 2 or 3 amino acid substitutions [0086] j. 4B4 CDRL1 which has a sequence of SEQ ID NO: 15 or a sequence that differs from SEQ ID NO: 15 by 1, 2 or 3 amino acid substitutions; [0087] k. 4B4 CDRL2 which has a sequence of SEQ ID NO: 17 or a sequence that differs from SEQ ID NO: 17 by 1, 2 or 3 amino acid substitutions; and [0088] l. 4B4 CDRL3 which has a sequence of SEQ ID NO: 19 or a sequence that differs from SEQ ID NO: 19 by 1, 2 or 3 amino acid substitutions.

[0089] Antibodies that have the same CDRs as the 4B4 and 12A7 antibodies may differ substantially from the sequences of 4B4 and 12A7 in other regions. Such antibodies may, for example, be antibody fragments.

[0090] The phrase sequence that differs from SEQ ID NO: 3 by a single amino acid substitution refers to the possibility of replacing one amino acid defined in SEQ ID NO: 3 by a different amino acid. Preferably such a replacement is a conservative amino acid substitution. The following eight groups each contain amino acids that are typically conservative substitutions for one another: [0091] 1) Alanine, Glycine; [0092] 2) Aspartic acid, Glutamic acid; [0093] 3) Asparagine, Glutamine; [0094] 4) Arginine, Lysine; [0095] 5) Isoleucine, Leucine, Methionine, Valine; [0096] 6) Phenylalanine, Tyrosine, Tryptophan; [0097] 7) Serine, Threonine; and [0098] 8) Cysteine, Methionine.

[0099] In an embodiment the antibody of the invention comprises at least one CDR from the heavy chain of 12A7 (12A7 CDRH1, 12A7 CDRH2 or 12A7 CDRH3) as well as at least one CDR from the light chain of 12A7 (12A7 CDRL1, 12A7 CDRL2 or 12A7 CDRL3). In a further embodiment the antibody of the invention comprises at least two CDRs from the heavy chain of 12A7 and at least two CDRs from the light chain of 12A7. In a preferred embodiment the antibody of the invention comprises all three CDRs from the heavy chain of 12A7 and/or all three CDRs from the light chain of 12A7. In an embodiment the antibody of the invention comprises 12A7 CDRL1 and 12A7 CDRL2, 12A7 CDRL1 and 12A7 CDRL3, 12A7 CDRL1 and 12A7 CDRH1, 12A7 CDRL1 and 12A7 CDRH2, 12A7 CDRL1 and 12A7 CDRH3, 12A7 CDRL2 and 12A7 CDRL3, 12A7 CDRL2 and 12A7 CDRH1, 12A7 CDRL2 and 12A7 CDRH2, 12A7 CDRL2 and 12A7

[0100] CDRH3, 12A7 CDRL3 and 12A7 CDRH1, 12A7 CDRL3 and 12A7 CDRH2, 12A7 CDRL3 and 12A7 CDRH3, 12A7 CDRH1 and 12A7 CDRH2, 12A7 CDRH1 and 12A7 CDRH3, or 12A7 CDRH2 and 12A7 CDRH3.

[0101] In an embodiment the antibody of the invention comprises at least one CDR from the heavy chain of 4B4 (4B4 CDRH1, 4B4 CDRH2 or 4B4 CDRH3) as well as at least one CDR from the light chain of 4B4 (4B4 CDRL1, 4B4 CDRL2 or 4B4 CDRL3). In a further embodiment the antibody of the invention comprises at least two CDRs from the heavy chain of 4B4 and at least two CDRs from the light chain of 4B4. In a preferred embodiment the antibody of the invention comprises all three CDRs from the heavy chain of 4B4 and/or all three CDRs from the light chain of 4B4. In an embodiment the antibody of the invention comprises 4B4 CDRL1 and 4B4 CDRL2, 4B4 CDRL1 and 4B4 CDRL3, 4B4 CDRL1 and 4B4 CDRH1, 4B4 CDRL1 and 4B4 CDRH2, 4B4 CDRL1 and 4B4 CDRH3, 4B4 CDRL2 and 4B4 CDRL3, 4B4 CDRL2 and 4B4 CDRH1, 4B4 CDRL2 and 4B4 CDRH2, 4B4 CDRL2 and 4B4 CDRH3, 4B4 CDRL3 and 4B4 CDRH1, 4B4 CDRL3 and 4B4 CDRH2, 4B4 CDRL3 and 4B4 CDRH3, 4B4 CDRH1 and 4B4 CDRH2, 4B4 CDRH1 and 4B4 CDRH3, or 4B4 CDRH2 and 4B4 CDRH3.

[0102] In a preferred embodiment the antibody of the invention comprises at least one CDR having a sequence identical to that described in any one of SEQ ID NO: 3 (12A7 CDRL1), SEQ ID NO :5 (12A7 CDRL2), SEQ ID NO: 7 (12A7 CDRL3), SEQ ID NO: 9 (12A7 CDRH1), SEQ ID NO: 11 (12A7 CDRH2), SEQ ID NO: 13 (12A7 CDR H3), SEQ ID NO: 15 (4B4 CDRL1), SEQ ID NO: 17 (4B4 CDRL2), SEQ ID NO: 19 (4B4 CDRL3), SEQ ID NO: 21 (4B4 CDRH1), SEQ ID NO: 23 (4B4 CDRH2) or SEQ ID NO :25 (4B4 CDRH3). In an embodiment where an antibody of the invention comprises 12A7 CDRL2, the 12A7 CDRL2 has the sequence described in SEQ ID NO: 5. In a further embodiment where an antibody of the invention comprises 12A7 CDRL1, the 12A7 CDRL1 has the sequence described in SEQ ID NO: 3. In a further embodiment where an antibody of the invention comprises 12A7 CDRL3, the 12A7 CDRL3 has the sequence described in SEQ ID NO: 7. In a further embodiment where an antibody of the invention comprises 12A7 CDRH1, the 12A7 CDRH1 has the sequence described in SEQ ID NO: 9. In a further embodiment where an antibody of the invention comprises 12A7 CDRH2, the 12A7 CDRH2 has the sequence described in SEQ ID NO: 11. In a further embodiment where an antibody of the invention comprises 12A7 CDRH3, the 12A7 CDRH3 has the sequence described in SEQ ID NO: 13. In a further embodiment where an antibody of the invention comprises 4B4 CDRL1, the 4B4 CDRL1has the sequence described in SEQ ID NO: 15. In a further embodiment where an antibody of the invention comprises 4B4 CDRL2, the 4B4 CDRL2 has the sequence described in SEQ ID NO: 17. In a further embodiment where an antibody of the invention comprises 4B4 CDRL3, the 4B4 CDRL3 has the sequence described in SEQ ID NO: 19. In a further embodiment where an antibody of the invention comprises 4B4 CDRH1, the 4B4 CDRH1 has the sequence described in SEQ ID NO: 21. In a further embodiment where an antibody of the invention comprises 4B4 CDRH2, the 4B4 CDRH2 has the sequence described in SEQ ID NO: 23. In a further embodiment where an antibody of the invention comprises 4B4 CDRH3, the 4B4 CDRH3 has the sequence described in SEQ ID NO: 25.

[0103] Preferably an antibody comprising at least one of the CDRs of 12A7 or 4B4 binds (optionally specifically binds) to Mcm5. Even more preferably an antibody comprising at least one of the CDRs of 12A7 or 4B4 binds (optionally specifically binds) to SEQ ID NO: 1 or SEQ ID NO: 2.

[0104] Heavy and Light Chain Variable Region Sequences

[0105] The antibody of the invention preferably comprises a heavy chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 29 or 33 and/or a light chain variable region sequence having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 27 or 31. Such an antibody is referred to herein as a variant antibody of the invention.

[0106] In an embodiment the antibody comprises a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 29. In a further embodiment the antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 29. In one embodiment the antibody comprises a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 27. In a further embodiment the antibody comprises a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 27. In a further embodiment the antibody comprises a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 27 and a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 29. In a preferred embodiment the antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 29 and a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 27.

[0107] In a further embodiment the antibody comprises a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 33. In a further embodiment the antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 33. In a further embodiment the antibody comprises a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 31. In a further embodiment the antibody comprises a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 31. In a further embodiment the antibody has a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO:33 and a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 31. In a preferred embodiment the antibody has a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 33 and a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 31.

[0108] As is known to the person skilled in the art, antibodies contain multiple regions including framework regions. Deletion or addition of amino acids in the framework regions is unlikely to affect the ability of the antibody to bind to its target. On the other hand, mutations in the CDRs are considerably more likely to affect the ability of an antibody to bind to a target. Thus, in certain embodiments of the invention, variant antibodies have CDRs which are identical to the CDRs of the 12A7 or 4B4 antibodies or have CDRs which vary in only a single amino acid substitution (preferably a conservative amino acid substitution). Variant antibodies of the invention may have framework regions which differ in sequence quite significantly from those described in SEQ ID NO: 27, 29, 31 or 33.

[0109] Optionally, where an antibody of the invention comprises a heavy chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 29, the antibody further comprises at least one of 12A7 CDRH1, 12A7 CDRH2 or 12A7 CDRH3. It is understood by the person skilled in the art that, since target binding specificity is determined by the CDRs, an antibody comprising the CDRs of 12A7 may still bind to Mcm5 even if the remainder of the antibody sequence is quite variable. For this reason where the antibody comprises at least one of 12A7 CDRH1, 12A7 CDRH2 or 12A7 CDRH3 the antibody preferably comprises a heavy chain variable region having a sequence at least 90% identical to SEQ ID NO: 29. In a more preferred embodiment the antibody of the invention comprises 12A7 CDRH1, 12A7 CDRH2 and 12A7 CDRH3 and comprises a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO:29.

[0110] Optionally, where an antibody of the invention comprises a heavy chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 33, the antibody further comprises at least one of 4B4 CDRH1, 4B4 CDRH2 or 4B4 CDRH3. It is understood by the person skilled in the art that, since target binding specificity is determined by the CDRs, an antibody comprising the CDRs of 4B4 may still bind to Mcm5 even if the remainder of the antibody sequence is quite variable. For this reason where the antibody comprises at least one of 4B4 CDRH1, 4B4 CDRH2 or 4B4 CDRH3 the antibody preferably comprises a heavy chain variable region having a sequence at least 90% identical to SEQ ID NO: 33. In a more preferred embodiment the antibody of the invention comprises 4B4 CDRH1, 4B4 CDRH2 and 4B4 CDRH3 and comprises a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 33.

[0111] Optionally, where an antibody of the invention comprises a light chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 27, the antibody further comprises at least one of 12A7 CDRL1, 12A7 CDRL2 or 12A7 CDRL3. It is understood by the person skilled in the art that, since target binding specificity is determined by the CDRs, an antibody comprising the CDRs of 12A7 may still bind to Mcm5 even if the remainder of the antibody sequence is quite variable. For this reason where the antibody comprises at least one of 12A7 CDRL1, 12A7 CDRL2 or 12A7 CDRL3 the antibody preferably comprises a light chain variable region having a sequence at least 90% identical to SEQ ID NO: 27. In a more preferred embodiment the antibody of the invention comprises 12A7 CDRL1, 12A7 CDRL2 and 12A7 CDRL3 and comprises a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 27.

[0112] Optionally, where an antibody of the invention comprises a light chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 31, the antibody further comprises at least one of 4B4 CDRL1, 4B4 CDRL2 or 4B4 CDRL3. It is understood by the person skilled in the art that, since target binding specificity is determined by the CDRs, an antibody comprising the CDRs of 4B4 may still bind to Mcm5 even if the remainder of the antibody sequence is quite variable. For this reason where the antibody comprises at least one of 4B4 CDRL1, 4B4 CDRL2 or 4B4 CDRL3 the antibody preferably comprises a heavy chain variable region having a sequence at least 90% identical to SEQ ID NO: 31. In a more preferred embodiment the antibody of the invention comprises 4B4 CDRL1, 4B4 CDRL2 and 4B4 CDRL3 and comprises a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO:31.

[0113] In a further embodiment of the invention the antibody of the invention comprises:

[0114] (i) 12A7 CDRH1, 12A7 CDRH2 and 12A7 CDRH3;

[0115] (ii) a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 29;

[0116] (iii) 12A7 CDRL1, 12A7 CDRL2 and 12A7 CDRL3; and

[0117] (iv) a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 27.

[0118] In a further embodiment the antibody of the invention comprises:

[0119] (i) 4B4 CDRH1, 4B4 CDRH2 and 4B4 CDRH3;

[0120] (ii) a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 33;

[0121] (iii) 4B4 CDRL1, 4B4 CDRL2 and 4B4 CDRL3; and

[0122] (iv) a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 31.

[0123] An antibody having a heavy chain variable sequence identical to SEQ ID NO: 29 and a light chain variable sequence identical to SEQ ID NO: 27 may be referred to as antibody 12A7. An antibody having a heavy chain variable sequence identical to SEQ ID NO: 33 and a light chain variable sequence identical to SEQ ID NO: 31 may be referred to as an antibody 4B4.

[0124] In some embodiments the variant antibody of the invention will compete for binding to Mcm5 with the 12A7 antibody. Similarly, in some embodiments of the invention the variant antibody of the invention will compete for binding to Mcm5 with the 4B4 antibody.

[0125] For the purposes of the present invention an antibody is at least 90%, 95%, 98%, 99% or 100% identical to a second antibody if the sequences have at least 90%, 95%, 98%, 99% or 100% identity when assessed using ClustalW (Thompson et al., 1994) with the following parameters:

[0126] Pairwise alignment parametersMethod: accurate, Matrix:PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10;

[0127] Multiple alignment parametersMatrix: PAM, Gap open penalty: 10.00, %identity for delay: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specific gap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR.

[0128] It is well within the knowledge of the person skilled in the art how to make variant antibodies of the invention which bind to Mcm5. Such variant antibodies may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50 or 100 substitution or deletion mutations compared to SEQ ID NOs: 27, 29, 31 or 33. Deletion variant antibodies may comprise the deletion of 1, 2, 3, 4, 5 or more amino acids or, in some cases, the deletion of entire regions of SEQ ID NOs: 27, 29, 31 or 33. Substitution variants may comprise the replacement of 1, 2, 3, 4, 5 or more amino acids with the same number of new amino acids.

[0129] Preferably, the variant antibodies of the invention comprise sequences differing from SEQ ID NOs: 27, 29, 31 or 33 by conservative amino acid substitutions (optionally only by conservative amino acid substitutions). The skilled person is well aware that such conservative substitutions are unlikely to alter the binding properties of an antibody.

[0130] Antibodies that Compete with Antibodies of the Invention

[0131] The invention further provides an antibody that competes with an antibody of the invention. For example, the invention provides an antibody that competes with an antibody which:

[0132] (i) binds to a polypeptide having an amino acid sequence of SEQ ID NO: 1 or 2.

[0133] (ii) comprises at least one CDR selected from the group consisting of 12A7 CDRH1, 12A7 CDRH2, 12A7 CDRH3, 12A7 CDRL1, 12A7 CDRL2, 12A7 CDRL3, 4B4 CDRH1, 4B4 CDRH2, 4B4 CDRH3, 4B4 CDRL1, 4B4 CDRL2, and 4B4 CDRL3;

[0134] (iii) comprises a heavy chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 29 or 33; or

[0135] (iv) comprises a light chain variable region sequence having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 27 or 31.

[0136] Preferably the invention provides an antibody that competes with an antibody comprising 12A7 CDRH1, 12A7 CDRH2, 12A7 CDRH3, 12A7 CDRL1, 12A7 CDRL2, and 12A7 CDRL3 or an antibody comprising 4B4 CDRH1, 4B4 CDRH2, 4B4 CDRH3, 4B4 CDRL1, 4B4 CDRL2, and 4B4 CDRL3. Even more preferably the invention provides an antibody that competes with an antibody comprising a heavy chain variable region having a sequence 100% identical to SEQ ID NO: 29 or 33 and a light chain variable region having a sequence 100% identical to SEQ ID NO: 27 or 31. In a most preferred embodiment the invention provides an antibody that competes with antibody 12A7 or antibody 4B4.

[0137] It is within the abilities of the person skilled in the art, to determine whether an antibody competes with another antibody of the invention.

[0138] For example, an ELISA assay may be performed by immobilising the reference antibody, such as a 4B4 or a 12A7 antibody, on an ELISA plate. The plate is then blocked with a suitable blocking agent. An excess of a second antibody is added (the second antibody is the antibody whose ability to compete with the 4B4 or 12A7 antibody is to be assessed for example an antibody of the invention that competes with 4B4 or 12A7). Mcm5 is added. Following a suitable incubation period, the ELISA plate is washed and an Mcm5 detection agent is added to measure the amount of Mcm5 bound to the immobilised reference antibody. If this test is to be used to detect whether an antibody competes with 4B4, a suitable detection reagent is a labelled 12A7 antibody. Similarly if this test is to be used to detect whether an antibody competes with 12A7, a suitable detection reagent is a labelled 4B4 antibody. The concentration at which 50% inhibition occurs is known as the Ki. In a preferred embodiment an antibody that competes with 12A7 or 4B4 binds with a Ki 2-fold, 5-fold, 10-fold, 50-fold or 100-fold lower than an antibody which does not bind to Mcm5.

[0139] In an embodiment an antibody that competes with an antibody of the invention is an antibody that binds to the same epitope (for example SEQ ID NO: 1 or SEQ ID NO: 2). It is possible to determine to which epitope an antibody binds by performing an experiment in which the target protein, such as Mcm5, is digested into fragments (for example see Example 3 below). The affinity with which the 12A7 and 4B4 antibodies to bind to each of the fragments can then be determined. Once the fragment (representing the epitope), to which the first antibody binds with a reasonable affinity, has been determined, the epitope may be synthesised. It is then possible to determine whether a second antibody is capable of binding to the same epitope by measuring the binding affinity of that second antibody to the fragment.

[0140] Labelled Antibodies

[0141] An antibody of the invention may be conjugated to a label (for example Europium.sup.3+ or Horseradish Peroxidase). The label may be directly, attached or may be attached via a linker (such as Adipic Acid Dihyrazide (ADH)).

[0142] The label may be attached by chemical conjugation. Methods of conjugating labels to antibodies are known in the art. For example, carbodiimide conjugation (Bauminger & Wilchek (1980) Methods Enzymol. 70, 151-159) may be used to conjugate labels to antibodies. Other methods for conjugating a label to an antibody can also be used. For example, sodium periodate oxidation followed by reductive alkylation of appropriate reactants can be used, as can glutaraldehyde cross-linking. However, it is recognised that, regardless of which method of producing a conjugate of the invention is selected, a determination must be made that the conjugated antibody maintains its targeting ability and that the conjugated label maintains its function.

[0143] In a preferred embodiment the antibody of the invention is labelled by conjugated to Europium. This may be achieved using an EG&G Wallac DELFIA Eu-labelling kit and following the manufacturer's protocol.

[0144] Compositions

[0145] In further aspects of the invention, compositions are provided comprising an antibody of the invention. Such compositions may further comprise suitable stabilising agents that are capable of stabilising the antibody in solution. For example, the composition may comprise a buffer selected from the group consisting of TAPS (3-{[tris(hydroxymethyl)methyl]amino}propanesulphonic acid), Bicine (N,N-bis(2-hydroxyethyl)glycine), Tris (tris(hydroxymethyl)methylamine), Tricine (N-tris(hydroxymethyl(methylglycine), TAPSO (3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulphonic acid), HEPES (4-2-hydroxyethyl-1-piperazineethanesulphonic acid), phosphate buffer saline and MOPS (3-(N-morpholino)propanesulphonic acid. In a preferred embodiment the composition comprises phosphate-buffered saline at pH 7.6. In a further preferred embodiment the composition comprises phosphate-buffered saline with between 0.01% and 0.09% sodium azide. Preferably, the composition comprises agents suitable for storing the antibodies of the invention at room temperature or at a temperature less than or equal to 15 C., 10 C., 7 C., 5 C., 0 C., 10 C., or 25 C. In a preferred embodiment the composition comprises agents suitable for storing the antibodies of the invention at a temperature between 2 C. and 8 C. In a further embodiment, the composition comprises a stabilising agent such as a sugar, for example lactose or trehalose.

[0146] If the antibody of the invention is conjugated, for example to a Europium label, it is advantageous to include a 7.5% purified BSA stabiliser in the composition.

[0147] Nucleic Acids and Vectors

[0148] The invention also relates to polynucleotides comprising nucleic acid sequences that encode antibodies of the invention. Thus, a polynucleotide of the invention may comprise a nucleic acid sequence that encodes any antibody as described herein. The term nucleic acid molecule and polynucleotide are used interchangeably herein and refer to a polymeric form of nucleotides of any length, deoxyribonucleotides, ribonucleotides, or analogs thereof.

[0149] A nucleic acid which encodes a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5 (amino) terminus and a translation stop codon at the 3 (carboxy) terminus.

[0150] For example, a polynucleotide of the invention may comprise a nucleic acid that encodes an antibody comprising a variable region having a sequence at least 95% identical to SEQ ID NO: 29 or 33, or an antibody comprising a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 27 or 31. A polynucleotide of the invention may encode both a polypeptide having a sequence at least 95% identical to SEQ ID NO: 29 and a polypeptide having a sequence at least 95% identical to SEQ ID NO: 27. Alternatively a polynucleotide of the invention may encode both a polypeptide having a sequence at least 95% identical to SEQ ID NO: 33 and a polypeptide having a sequence at least 95% identical to SEQ ID NO: 31.

[0151] An example polynucleotide of the invention comprises or consists of a nucleic acid sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 or 34. Preferably the polynucleotide of the invention comprises or consists of a nucleic acid sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 28, 30, 32 or 34. Optionally the polynucleotide of the invention comprises a polynucleotide sequence having at least 95% identity to SEQ ID NO: 28 and a polynucleotide sequence having at least 95% identity to SEQ ID NO:30. Optionally the polynucleotide of the invention comprises a polynucleotide sequence having at least 95% identity to SEQ ID NO: 32 and a polynucleotide sequence having at least 95% identity to SEQ ID NO: 34.

[0152] An antibody of the invention may thus be produced from a polynucleotide which encodes, and is capable of expressing, it. Where the antibody comprises two or more chains, a polynucleotide of the invention may encode an antibody light chain, and an antibody heavy chain or both. Two polynucleotides may be provided, one of which encodes an antibody light chain and the other of which encodes the corresponding antibody heavy chain. Such a polynucleotide or pair of polynucleotides may be expressed together such that an antibody of the invention is generated.

[0153] Polynucleotides of the invention can be synthesised according to methods well known in the art, as described by way of example in Sambrook et al (1989, Molecular Cloninga laboratory manual; Cold Spring Harbor Press).

[0154] The invention further provides a vector (such as a plasmid or recombinant viral vector) comprising the polynucleotide of the invention. Typically the vector further comprises a control sequence operably linked to the inserted sequence, thus allowing for expression of the antibody of the invention in vivo. Preferably the vector of the invention further comprises appropriate initiators, promoters, enhances and other elements which may be necessary and which are positioned in the correct orientation, in order to allow for expression of a polypeptide of the invention.

[0155] Hybridoma Cell Lines

[0156] The present invention further provides a hybridoma cell line capable of producing an antibody of the invention. Such a hybridoma cell line will produce the antibody of the invention when maintained in suitable culture conditions.

[0157] It is well within the ability of the person skilled in the art to develop a hybridoma expressing an antibody of the invention. This may be performed by immunising a mammal such as a mouse, rabbit or guinea pig, with an antigen (for example Mcm5). It may be beneficial to include an adjuvant such as Freund's complete adjuvant. The spleen cells of the immunised mammal are removed and fused with myeloma cells to form hybridoma lines which are immortal given appropriate conditions and which secrete antibodies. The hybridomas are separated into single clones and the antibodies secreted by each clone are evaluated for their binding ability to Mcm5 protein.

[0158] Host Cell Lines

[0159] The invention also provides host cells comprising the nucleic acids or vectors of the invention. Such host cells include cells that have been modified to express an antibody of the invention. Such host cells may be eukaryotic cells or prokaryotic cells. For example, the host cells may be mammalian cells, insect cells, or bacterial cells. Particular examples of host cells that may be modified by insertion of vectors or nucleic acids of the invention include mammalian HEK293T, CHO, HeLa, NSO and COS cells. Alternatively bacterial cells such as E.coli may be used.

[0160] Such host cells may be cultured using routine methods to produce an antibody of the invention.

[0161] Antibody Production

[0162] Antibodies of the invention may be produced using any method. In one embodiment, the antibodies are produced directly from a hybridoma cell line. In a further embodiment of the invention, the antibodies are produced recombinantly.

[0163] To produce an antibody from a hybridoma cell line, the cell line can be cultivated in vitro, for example in a vessel such as a cell culture flask or a fermenter, and the antibody can be isolated from the vessel using conventional techniques known in the art. For the purposes of the present invention, an antibody will be considered to have been produced by a hybridoma cell line if the antibody is derived from an antibody which was produced by the hybridoma cell line. For example, it is well within the abilities of the person skilled in the art to obtain an antibody from a hybridoma cell line, sequence the amino acid sequence of the antibody, and transfect cells such as CHO cells or E.coli cells with vectors encoding the amino acid sequence of the antibody. Such antibodies can be considered to have been derived from an antibody which was produced by the hybridoma cell line. In one particular embodiment, antibodies of the invention are produced directly from the hybridoma cell line itself.

[0164] Alternatively antibodies of the invention may be produced using recombinant techniques. The sequence of an antibody of the invention may be used to transfect a host cell line of the invention. The cell lines may be grown in a flask or a fermenter, and the antibody expressed by the cell can be isolated and purified using standard techniques (such as affinity chromatography).

[0165] Once purified the antibodies may then be conjugated to a radio label or immobilised to a solid support. An example of a suitable solid support is an ELISA plate.

[0166] Kits and Methods of Determining the Concentration of Mcm5 in a Sample.

[0167] The antibodies of the invention may be used as part of a kit. For example, the antibodies may be part of a kit for use in detecting the concentration of Mcm5 in a sample, for example a sample from a human, this can allow detection of cancer in a subject from which the sample is taken.

[0168] Alternatively the antibodies of the invention may be used in a method of detecting the concentration or amount of Mcm5 in a sample (also referred to as an Mcm5 detection method of the invention). Preferably such a method comprises the following steps: [0169] (i) providing an immobilised first antibody which is an antibody of the invention immobilised to a solid support; [0170] (ii) exposing the sample to the immobilised first antibody such that Mcm5 in the sample may bind to the immobilised antibody to provide immobilised Mcm5; [0171] (iii) providing a labelled second antibody which is an antibody of the invention conjugated to a label; [0172] (iv) exposing the immobilised Mcm5 to the labelled second antibody such that the labelled second antibody may bind to the Mcm5; and [0173] (v) detecting the concentration of the labelled second antibody.

[0174] Preferably there is a wash step between steps (iv) and (v) to ensure that the second antibody whose concentration is detected in step (v) bound to the Mcm5 in step (iv). Optionally there is a further wash step after step (i) to remove excess first antibody and/or after step (ii) to remove excess Mcm5. Optionally the wash step is performed by exposing the solid support to a wash buffer.

[0175] In one embodiment the antibodies present in a kit or the invention or used in a method of the invention are suitable for detecting a urological cancer such as prostate cancer or bladder cancer.

[0176] Preferably a method of detecting the concentration or amount of Mcm5 in a sample uses, or a kit of the invention comprises, two antibodies of the invention which do not compete with one another (such as the 4B4 and 12A7 antibodies) and preferably bind to different epitopes of the antigen of interest. This can be determined by performing an ELISA, such as that described above. One of the two antibodies may be immobilised onto an ELISA plate. The plate is then blocked with a suitable blocking agent. An excess of the other antibody is then added (the antibody whose ability to compete with the first antibody is to be assessed). The target antigen, such as Mcm5, is then added. Following a suitable incubation period, the ELISA plate is washed and a detection agent capable of binding to the target antigen is added to measure the amount of the target antigen bound to the immobilised antibody. If the two antibodies compete the amount of the target antigen bound to the immobilised antibody will be lower than if the antibodies do not compete (and, optionally, bind to different epitopes). The concentration at which 50% inhibition occurs is known as the Ki. In a preferred embodiment an antibody that competes with another antibody binds with a Ki 2-fold, 5-fold, 10-fold, 50-fold or 100-fold lower than an antibody which does not compete.

[0177] In a preferred embodiment an Mcm5 detection method of the invention uses, or a kit of the invention comprises, a first antibody of the invention and a second antibody of the invention. The first antibody may bind (optionally bind specifically) to SEQ ID NO: 1 and the second antibody may bind (optionally bind specifically) to SEQ ID NO: 2. The first antibody may comprise at least one CDR selected from the group consisting of 12A7 CDRH1, 12A7 CDRH2, 12A7 CDRH3, 12A7 CDRL1, 12A7 CDRL2, and 12A7 CDRL3. The second antibody may comprise at least one CDR selected from the group consisting of 4B4 CDRH1, 4B4 CDRH2, 4B4 CDRH3, 4B4 CDRL1, 4B4 CDRL2 and 4B4 CDRL3.

[0178] Preferably the first antibody comprises 12A7 CDRH1, 12A7 CDRH2, 12A7 CDRH3, 12A7 CDRL1, 12A7 CDRL2, and 12A7 CDRL3. Preferably the second antibody comprises 12A7 CDRH1, 12A7 CDRH2, 12A7 CDRH3, 12A7 CDRL1, 12A7 CDRL2, and 12A7 CDRL3. In an embodiment the first antibody comprises a heavy chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID

[0179] NO: 29 and a light chain variable region sequence having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 27. In an embodiment the second antibody comprises a heavy chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 33 and a light chain variable region sequence having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 31.

[0180] A DELFIA detection system may be used in an Mcm5 detection method of the invention. In such embodiments the first antibody and the second antibody preferably bind different epitopes. In such embodiments one of the antibodies (the first or the second monoclonal antibody) can be immobilised on a plate. A sample to be tested can be added to the plate. The other antibody (the first or the second antibody which was not immobilised to the plate) can then be added. The other antibody should be conjugated to a detection agent such as a Europium label. Time-resolved fluorescent spectroscopy can then be used to determine whether the target antigen is present (by measuring the amount of the labelled antibody that is present).

[0181] Accordingly, in one embodiment of the invention, a kit of the invention comprises a first or second antibody of the invention which is conjugated to a radiolabel, for example Europium. Any method may be used to conjugate the first monoclonal antibody or the second monoclonal antibody to a Europium label. Kits for performing such labelling are available. For example, the first or second monoclonal antibody may be conjugated to Europium using an Eu-NI-ITC chelate.

[0182] In such embodiments a kit of the invention may comprise further components for use in a

[0183] DELFIA detection system. Such further components may include a DELFIA enhancement solution or a DELFIA detection solution

[0184] Definitions

[0185] The terms comprises means includes. Thus, the word comprises and variations such as comprise, and comprising will be understood to imply the inclusion of a stated compound or composition or step, or group of compounds or steps, but not to the exclusion of any other compounds, composition, steps or groups thereof.

[0186] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described aspects and embodiments of the present invention will be apparent to those skilled in the art without departing from the scope of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the following claims.

TABLE-US-00001 TABLE 1 Sequences SEQ ID NO Nucleotide/Polypeptide 1 WDETKGE (epitope to which antibody 12A7 binds) 2 DDRVAIH (epitope to which antibody 4B4 binds 3 12A7 light chain CDR 1 polypeptide sequence 4 12A7 light chain CDR 1 nucleotide sequence 5 12A7 light chain CDR 2 polypeptide sequence 6 12A7 light chain CDR 2 nucleotide sequence 7 12A7 light chain CDR 3 polypeptide sequence 8 12A7 light chain CDR 3 nucleotide sequence 9 12A7 heavy chain CDR 1 polypeptide sequence 10 12A7 heavy chain CDR 1 nucleotide sequence 11 12A7 heavy chain CDR 2 polypeptide sequence 12 12A7 heavy chain CDR 2 nucleotide sequence 13 12A7 heavy chain CDR 3 polypeptide sequence 14 12A7 heavy chain CDR 3 nucleotide sequence 15 4B4 light chain CDR 1 polypeptide sequence 16 4B4 light chain CDR 1 nucleotide sequence 17 4B4 light chain CDR 2 polypeptide sequence 18 4B4 light chain CDR 2 nucleotide sequence 19 4B4 light chain CDR 3 polypeptide sequence 20 4B4 light chain CDR 3 nucleotide sequence 21 4B4 heavy chain CDR 1 polypeptide sequence 22 4B4 heavy chain CDR 1 nucleotide sequence 23 4B4 heavy chain CDR 2 polypeptide sequence 24 4B4 heavy chain CDR 2 nucleotide sequence 25 4B4 heavy chain CDR 3 polypeptide sequence 26 4B4 heavy chain CDR 3 nucleotide sequence 27 12A7 full light chain variable region sequence (polypeptide) 28 12A7 full light chain variable region sequence (nucleotide) 29 12A7 full heavy chain variable region sequence (polypeptide) 30 12A7 full heavy chain variable region sequence (nucleotide) 31 4B4 full light chain variable region sequence (polypeptide) 32 4B4 full light chain variable region sequence (nucleotide) variable region 33 4B4 full heavy chain variable region sequence (polypeptide) 34 4B4 full heavy chain variable region sequence (nucleotide) 35 Mcm5 polypeptide sequence 36 Mcm5 polynucleotide sequence

EXAMPLES

[0187] Cloning of Mcm5

[0188] Stage I: Cloning Strategy

[0189] 1. A verified IMAGE clone containing the human MCM5 cDNA was obtained and used as a PCR template in order to amplify a 648bp fragment of DNA. The forward PCR primer encoded n initiation codon as well as a modified unpatented poly-Histidine tag (HQ).sub.4.

[0190] 2. The PCR fragment was cloned into the inducible expression E. coli vector pTRC99a using a double restriction site strategy. This vector allows inducible expression directed by the hybrid trp/lac trc promoter. Downstream is a lacZ ribosome binding site (RBS) that is situated at an optimized distance from an initiation ATG codon, which is supplied by the NcoI cloning site.

[0191] Stage IIA: Expression and Purification (Small Scale10 ml)

[0192] 3. The recombinant plasmid containing the Mcm5 fragment was transformed into BL21 E. coli (non-DE3) to ensure high yields of expressed protein.

[0193] 4. Several small volume E. coli cultures were induced with IPTG. Cells will be lysed in SDS loading buffer and total protein separated by SDS-PAGE. High expressing clones will be identified by Western blotting using a mouse monoclonal supplied by Urosens.

[0194] Stage IIB: Expression and Purification (Large Scale500 ml)

[0195] 5. The identified clone with high levels of recombinant protein was cultured at the 0.5 L scale and induced with IPTG. Cells were pelleted and stored at 70 C. Cells were disrupted in a denaturing lysis buffer described by Stoeber et al. J Natl Cancer Inst 2002 94 1071-9.

[0196] 6. His-tagged recombinant protein was affinity purified under denaturing conditions on an immobilized metal affinity (IMAC) chromatography column. Protein were eluted using an acidic buffer and dialysed against storage buffer (PBS containing 0.3 SDS)

[0197] 7. Eluted protein was analyzed and quantified by SDS-PAGE and western blotting using a mouse monoclonal supplied by Urosens.

Example 1

MAb Screening ELISA

[0198] Protein A purified MAbs were obtained from the Welcome/CRUK Institute for Cancer and Developmental Biology. These antibodies were screened for their ability to bind to Mcm5.

[0199] An ELISA of recombinant His6-tagged Mcm5 was performed using Ni-NTA His-Sorb 96 well microtitre coated plate wells (Qiagen). The Ni-NTA coated plates, pre-blocked with BSA, ensure an orientated presentation of the 6 His tagged Mcm through Nickel affinity binding to the spacer in comparison to the random presentation of the Mcm5 to the well that would be achieved by passive adsorption plate coating. The Mcm5 at a concentration of 625 ng/ml was Nickel affinity immobilised to Ni-NTA His-Sorb surface (Qiagen) of a 96 well plate at 200 l/well resulting in 125 ng hsMcm5/well. This was done following the manufacturers instructions to apply a His-6 tagged protein or peptide to the plate wells at a titre>100 ng/well. This was done to ensure that the protein or peptide is not limiting to Ab detection. The screening of the MAbs for development of a Mcm5 IFMA was performed with an enzyme linked immunosorbent assay (ELISA) of Mcm5. To screen MAbs, supernatants from hybridoma cultures expressing the MAbs were incubated in the microtitre wells at RT for 3 h and then washed away with an automated 96 well plate washer. The Mcm5 Ag-Ab complexes of the MAbs were detected by a colorimetric ELISA using goat anti-mouse IgG conjugated to HRP, TMB reagent and 1M H.sub.2SO.sub.4 assay stop solution, for the measurement of light absorbance at 450 nanometres (Abs 450 nm). An absence of Non-specific binding (NSB) of goat anti-mouse HRP conjugate to the Ni-NTA His-Sorb surface and Mcm5 was excluded byperforming the ELISA on wells free from MAbs. The degree of NSB of the mouse MAbs being screened to the Ni-NTA coated polystyrene wells was established by performing the ELISA in wells with the omission of Mcm5 wells in parallel to performing the ELISA in wells with the Nickel affinity immobilisation of Mcm5 to the microtitre wells.

[0200] The performance in the colorimetric ELISA of a mouse MAb in the media samples taken from the hybridomas (Table 2) was used for selection of the highlighted clones, 12A7 and 4B4. The selected MAb hybridomas were grown up to produce IgG for purification from the culture supernatant using solid-phase protein-A separation according to the manufacturer's instructions (Amersham Pharmacia Biotech Little Chalfont Buckinghamshire, UK). The 12A7 MAb gave rise to the greatest response to the Mcm5.

TABLE-US-00002 TABLE 2 hybridoma MAb screening ELISA Abs 450-650 results Mcm5 well NSB well Mcm5 specific Mcm5 MAb response response response 12A7 1.404 0.421 0.983 4B4 1.622 1.040 0.582

[0201] MAb Relative Affinities

[0202] Relative affinities of the protein A purified MAbs were determined by normalisation of Ab concentrations and application of the MAbs to the colorimetric screening assay under Ag-limiting conditions. Ag limiting conditions were determined by linear titration of Mcm5 across the Ni-NTA His-Sorb microtitre plate wells. An observed maximal Abs 450 nm of <3.000 is indicative of Ag limiting conditions, because the maximum Abs 450 reading obtainable when Ag is non-limiting is 4.000. The Ag titration was performed using rabbit Mcm5 PAb (101) and Goat anti Rabbit HRP to measure ELISA response. A concentration that gave 25 ng of Mcm5 available for binding to each Ni-NTA well was limiting to the ELISA Abs 450 response of the PAb. The rabbit PAb was chosen to determine Ag-limiting conditions for investigation of the mouse MAbs, as the full array of Mcm5 epitopes are bound by the PAb. This ensured that the MAbs could be investigated in combined incubation studies to determine the degree of epitope competition.

[0203] The concentration of IgG in the protein-A purified MAb solutions was determined by Abs 280 nm measurement in a spectrophotometer and MAb concentrations were normalised by dilution in PBS. The 50% maximum binding concentration was then determined by a linear dilution of the MAbs applied to an ELISA of Nickel affinity Mcm5 wells under Mcm5 Ag-limiting conditions. To demonstrate the plateau of the binding signal to the 25 ng/well hsMcm5rf on the plate the MAb 7A3 was run in the ELISA over a greater concentration range. The 50% maximum binding response for 12A7 was obtained at a concentration of 20 ng/well. 12A7 is shown by its 50% maximum binding concentration to demonstrate the highest relative affinity to hsMcm5. (Table 3).

TABLE-US-00003 TABLE 3 MAb titrations against limiting 25 ng/well antigen couples to Ni-NTA MAb nb/well MAb 4B4 12A7 0 0.086 0.051 0.25 0.108 0.189 2.5 0.464 0.762 25 0.966 1.804 500 1.848 2.808 1000 2.414 3.099

Example 2

Antibody Epitope Competition

[0204] For a two-site immunometric assay two MAbs that are targeted to distinct epitopes are required. In Ag limiting conditions a pair of MAbs competing for the same epitope will provide an ELISA Abs 450 nm measurement equal to the Abs 450 nm signal of the higher relative affinity Ab when it is incubated in the absence of the competing MAb, however, incubation of a pair of MAbs targeted to distinct epitopes will provide an Abs 450 measurement equal to the sum of both the MAbs individual Abs 450 nm responses to the limited Ag available provided there is an absence of steric hindrance. The MAbs 12A7 and 4B4 were normalised to the 50% maximum binding concentration of 20 ng/well of the highest relative affinity MAb 12A7. The MAbs at normalised concentration underwent an ELISA using both individual and paired Ab incubations on the 25 ng/well Mcm5 Ag limiting Ni-NTA plate. The highest Abs 450 nm signal measured from a combined incubation of a pair of the MAbs would be indicative of the most suitable MAbs for use in a two-site IFMA.

[0205] Antibody Epitope Competition Results

[0206] The Ab Ag epitope competition ELISA study results (Table 4) show that the MAb 4B4 is targeted to a distinct epitope compatible for two-site binding with the 12A7. The distinct epitope targeted by 4B4 is demonstrated by the Abs 450 response' being equal to the sum of both MAbs binding to the limiting Ag when 4B4 was incubated in parallel to the other MAbs.

TABLE-US-00004 TABLE 4 Ab Ag epitope competition ELISA study results MAb 20 ng/well Mcm5 25 ng/well incubation Abs 450-650 response 12A7 1.375 4B4 1.244 12A7 + 4B4 2.693

[0207] The MAbs were incubated at a normalised concentration of 20 ng/well, the 50% maximum binding concentration of the MAb with the highest relative affinity 12A7. The optimal MAbs for two-site immunoassay development are highlighted in blue, and their ELISA Abs 450 responses highlighted in yellow.

Example 3

Epitope Mapping

[0208] Materials Used:

TABLE-US-00005 Microarray Content: The sequence of antigen Mcm 5 (aa 367-582) was translated into 15 aa peptides with a peptide-peptide overlap Samples: Mouse monoclonal IgG antibodies 12A7 and 4B4 Washing Buffer: PBS, pH 7.4 with 0.05% Tween 20 (3 1 min after each assay) Blocking Buffer: Rockland blocking buffer MB-070 (30 min before the first assay) Incubation Buffer: PBS, pH 7.4 with 0.05% Tween 20 and 10% Rockland blocking buffer Assay Conditions: Antibody concentrations of 1 g/ml and 10 g/ml in incubation buffer; incubation for 16 h at 4 C. and Secondary Antibody: Goat anti-mouse IgG (H + L) DyLight 680 antibody; Control Antibodies: Monoclonal anti-HA (12CA5)-DyLight680 (1:1000), monoclonal anti-FLAG(M2)- DyLight800 (1:500); staining in Scanner: LI-COR Odyssey Imaging System; scanning offset 0.8 mm, resolution 21 m, scanning intensity red/green of 5/7

[0209] Pre-staining of one of the peptide microarrays was done with the secondary goat anti-mouse IgG (H+L) DyLight680 antibody at a dilution of 1:5000 to investigate background interactions with the antigen-derived peptides that could interfere with the main assays. Subsequent incubation of the peptide microarrays with mouse monoclonal IgG antibodies 12A7 and 4B4 at concentrations of 1 g/ml and 10 g/ml in incubation buffer was followed by staining with the secondary antibody and read-out at a scanning intensity of 5 (red). HA and Flag control peptides framing the peptide arrays were finally stained as internal quality control to confirm the assay quality and the peptide microarray integrity (scanning intensities red/green: 5/7).

[0210] Quantification of spot intensities and peptide annotation were done with PepSlide Analyzer. A software algorithm breaks down fluorescence intensities of each spot into raw, foreground and background signal and calculates the standard deviation of foreground median intensities. Based on averaged foreground median intensities, intensity maps were generated and interactions in the peptide maps highlighted by an intensity color code with red for high and white for low spot intensities.

[0211] We further plotted averaged spot intensities of all assays with the antibody samples against the antigen sequence from the N- to the C-terminus to visualize overall spot intensities and signal to noise ratios. The intensity plots were correlated with peptide and intensity maps as well as with visual inspection of the microarray scans to identify peptides and epitopes that were recognized by the antibody samples. In case it was not clear if a certain amino acid contributed to antibody binding.

[0212] After 15 min pre-swelling in washing buffer and 30 min in blocking buffer, one of the peptide microarrays was initially incubated with the secondary goat anti-mouse IgG (H+L) DyLight680 antibody at a dilution of 1:5000 for 30 min at room temperature to analyze background interactions with the antigen-derived peptides. At a scanning intensity of 5, we did not observe any background due to non-specific binding of the secondary antibody. Data quantification with PepSlide Analyzer was neither possible nor required, since the absence of any spot pattern hampered alignment of the microarray grid.

[0213] The peptide microarrays were incubated with mouse monoclonal antibody 12A7 at concentrations of 1 g/ml and 10 g/ml (top right). After each incubation, staining with the secondary goat anti-mouse IgG (H+L) DyLight680 antibody was followed by read-out at a scanning intensity of 5. We observed a strong and well-defined epitope-like spot pattern formed by a row of neighbored peptides with a consensus motif at excellent signal to noise ratios.

[0214] The final staining of the HA and Flag control peptides framing the peptide microarray gave rise to the expected and well-defined spot pattern and validated the overall peptide microarray integrity.

[0215] Data quantification was followed by generation of peptide and intensity maps as well as of intensity plots for the assays with mouse monoclonal antibody 12A7 at concentrations of 1 g/ml and 10 g/ml; the intensity plot of the latter was leveled to provide a clearer data overview. The signals were based on the epitope-like spot pattern observed in the microarray scan and attributed to peptides with the consensus motif WDETKGE.

[0216] The peptide microarrays were incubated with mouse monoclonal antibody 4B4 at concentrations of 1 g/ml) and 10 g/ml. After each incubation, staining with the secondary goat anti-mouse IgG (H+L) DyLight680 antibody was followed by read-out at a scanning intensity of 5. We observed two strong and well-defined epitope-like spot patterns formed by a row of neighbored peptides with consensus motifs at excellent signal to noise ratios. At an antibody concentration of 10 g/ml the raw intensities of the epitope-like spot pattern were similar but the background interactions were slightly increased indicating a signal saturation of the antibody in the concentration range of 1-10 g/ml. The final staining of the HA and Flag control peptides framing the peptide microarray gave rise to the expected and well-defined spot pattern and validated the overall peptide microarray integrity.

[0217] Data quantification was followed by generation of peptide and intensity maps as well as of intensity plots for the assays with mouse monoclonal antibody 4B4 at concentrations of 1 g/ml and 10 g/ml; the intensity plot of the latter was leveled to provide a clearer data overview. At an antibody concentration of 10 g/ml the raw intensities of the epitope-like spot pattern were similar but the background interactions were slightly increased leading to reduced normalized signal intensities. The signals were based on the epitope-like spot patterns observed in the microarray scan and attributed to peptides with the consensus motifs DDRVAIH and WDETKGE.

TABLE-US-00006 Sequencelisting SEQIDNO:1 WDETKGE SEQIDNO:2 DDRVAIH SEQIDNO:3 QNLVQSNGNTY SEQIDNO:4 CAGAACCTTGTACAAAGTAATGGAAACACCTATTTA SEQIDNO:5 KVS SEQIDNO:6: AAGTTTCCAA SEQIDNO:7: SQSTRVPYT SEQIDNO:8: TCTCAAAGTACACGTGTTCCGTACACA SEQIDNO:9: GFSLSTSGMG SEQIDNO:10: GGGTTTTCACTGAGCACTTCTGGTATGGGT SEQIDNO:11: IFWDDDK SEQIDNO:12: ATTTTCTGGGATGATGACAAG SEQIDNO:13: ARRSDYNYYSMDY SEQIDNO:14: GCGCGGCGAAGTGACTACAATTACTACTCTATGGACTAC SEQIDNO:15: QDIGSS SEQIDNO:16: CAGGACATTGGTAGTAGC SEQIDNO:17: ATS SEQIDNO:18: GCCACATCC SEQIDNO:19: LQYASSPPT SEQIDNO:20: CTACAATATGCTAGTTCTCCTCCGACG SEQIDNO:21: GFTFSNYA SEQIDNO:22: GGATTCACTTTCAGTAACTATGCC SEQIDNO:23: ISRGGSYT SEQIDNO:24: ATTAGTCGTGGTGGTAGTTACACC SEQIDNO:25: ARHGYNYDDGAWFAN SEQIDNO:26: GCAAGACATGGATATAATTACGACGACGGGGCCTGGTTTGCTAAC SEQIDNO:27: DIMLTQSPLSLSVTLGDQASISCRSSQNLVQSNGNTYLTWYLQKPGQSPKVLINKVSNRFY GVPDRFSGSGSGTDFTLRISRVEAEDLGIYFCSQSTRVPYTFGGGTKLEIRR SEQIDNO:28: GATATCATGCTGACCCAATCTCCACTCTCCCTGTCTGTCACTCTTGGAGATCAGGCCTC CATCTCTTGCAGATCTAGTCAGAACCTTGTACAAAGTAATGGAAACACCTATTTAACTT GGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGGTCCTGATCAACAAAGTTTCCAACCG ATTTTATGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTC AGGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTTCTGCTCTCAAAGTACAC GTGTTCCGTACACATTCGGAGGGGGGACCAAGCTGGAAATAAGACG SEQIDNO:29: QQDLQQSGPGILQPTQTLSLTCSFSGFSLSTSGMGVSWIRQSSNMGLEWLAHIFWDDDKRY NPSLRSRLTLSKDTSSSQVFLMITSVSTADSATYYCARRSDYNYYSMDYWGQGTAVTVSS SEQIDNO:30: CAGCAAGATCTGCAGCAGTCTGGCCCTGGGATATTGCAGCCCACCCAGACCCTCAGTC TGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTGAGTTGGATTC GTCAATCTTCAAATATGGGTCTGGAGTGGCTGGCACACATTTTCTGGGATGATGACAA GCGCTATAATCCCTCCCTGAGGAGCCGACTCACGCTCTCCAAGGATACCTCCAGTAGC CAGGTATTTCTCATGATCACCAGTGTGAGTACTGCAGATTCTGCCACATACTACTGTGC GCGGCGAAGTGACTACAATTACTACTCTATGGACTACTGGGGTCAAGGAACCGCAGTC ACCGTCTCCTCA SEQIDNO:31: DIMLTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTIKRLIYATSSLDSGVPKRFS GSRSGSDYSLTISSLESEDFVDYYCLQYASSPPTFGGGTKLEIK SEQIDNO:32: GATATCATGCTGACCCAATCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAGTCAG TCTCACTTGTCGGGCAAGTCAGGACATTGGTAGTAGCTTAAACTGGCTTCAACAGGAA CCAGATGGAACTATTAAACGCCTAATCTACGCCACATCCAGTTTAGATTCTGGTGTCCC CAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCAGCCTT GAGTCTGAAGATTTTGTAGACTATTACTGTCTACAATATGCTAGTTCTCCTCCGACGTT CGGTGGAGGCACCAAGCTGGAAATCAAAC SEQIDNO:33: VKLQESGGGLVKPGGSLKLSCAASGFTFSNYAMSWVRQNPEKRLEWVATISRGGSYTYY PDSVKGRFTISRDNAKNTLYLQMNSLRSEDTAMYFCARHGYNYDDGAWFANWGQGTLV TVSA SEQIDNO:34: TAGGTGAAACTGCAGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAA CTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACTATGCCATGTCTTGGGTTCGCCA GAATCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTCGTGGTGGTAGTTACAC CTACTATCCAGACAGTGTGAAGGGTCGATTCACCATCTCCAGAGACAATGCCAAGAAC ACCCTGTATCTGCAAATGAACAGTCTGAGGTCTGAGGACACGGCCATGTATTTCTGTG CAAGACATGGATATAATTACGACGACGGGGCCTGGTTTGCTAACTGGGGCCAAGGGA CTCTGGTCACTGTCTCTGCA SEQIDNO:35: 102030405060 MSGFDDPGIFYSDSFGGDAQADEGQARKSQLQRRFKEFLRQYRVGTDRTGFTFKYRDELK 708090100110120 RHYNLGEYWIEVEMEDLASFDEDLADYLYKQPAEHLQLLEEAAKEVADEVTRPRPSGEEV 130140150160170180 LQDIQVMLKSDASPSSIRSLKSDMMSHLVKIPGIIIAASAVRAKATRISIQCRSCRNTLT 190200210220230240 NIAMRPGLEGYALPRKCNTDQAGRPKCPLDPYFIMPDKCKCVDFQTLKLQELPDAVPHGE 250260270280290300 MPRHMQLYCDRYLCDKVVPGNRVTIMGIYSIKKFGLTTSRGRDRVGVGIRSSYIRVLGIQ 310320330340350360 VDTDGSGRSFAGAVSPQEEEEFRRLAALPNVYEVISKSIAPSIFGGTDMKKAIACLLFGG 370380390400410420 SRKRLPDGLTRRGDINLLMLGDPGTAKSQLLKFVEKCSPIGVYTSGKGSSAAGLTASVMR 430440450460470480 DPSSRNFIMEGGAMVLADGGVVCIDEFDKMREDDRVAIHEAMEQQTISIAKAGITTTLNS 490500510520530540 RCSVLAAANSVFGRWDETKGEDNIDFMPTILSRFDMIFIVKDEHNEERDVMLAKHVITLH 550560570580590600 VSALTQTQAVEGEIDLAKLKKFIAYCRVKCGPRLSAEAAEKLKNRYIIMRSGARQHERDS 610620630640650660 DRRSSIPITVRQLEAIVRIAEALSKMKLQPFATEADVEEALRLFQVSTLDAALSGTLSGV 670680690700710720 EGFTSQEDQEMLSRIEKQLKRRFAIGSQVSEHSIIKDFTKQKYPEHAIHKVLQLMLRRGE 730 IQHRMQRKVLYRLK SEQIDNO:36 Homosapiensminichromosomemaintenancecomplexcomponent5(MCM5),mRNA NCBIReferenceSequence:NM_006739.3 1 ggaaaaccagaggcgcagtcatgtcgggattcgacgatcctggcattttctacagcgaca 61 gcttcgggggcgacgcccaggccgacgaggggcaggcccgcaaatcgcagctgcagaggc 121 gcttcaaggagttcctgcggcggtaccgagtgggcaccgaccgcacgggcttcaccttca 181 aatacagggatgaactcaagcggcattacaacctgggggagtactggattgaggtggaga 241 tggaggatctggccagctttgatgaggacctggccgactacttgtacaagcagccagccg 301 agcacctgcagctgctggaggaagctgccaaggaggtagctgatgaggtgacccggcccc 361 ggccttctggggaggaggtgctccaggacatccaggtcatgctcaagtcggacgccagcc 421 cttccagcattcgtagcctgaagtcggacatgatgtcacacctggtgaagatccctggca 481 tcatcatcgcggcctctgcggtccgtgccaaggccacccgcatctctatccagtgccgca 541 gctgccgcaacaccctcaccaacattgccatgcgccctggcctcgagggctatgccctgc 601 ccaggaagtgcaacacagatcaggctgggcgccccaaatgcccattggacccgtacttca 661 tcatgcccgacaaatgcaaatgcgtggacttccagaccctgaagctgcaggagctgcctg 721 atgcagtcccccacggggagatgcccagacacatgcagctctactgcgacaggtacctgt 781 gtgacaaggtcgtccctgggaacagggttaccatcatgggcatctactccatcaagaagt 841 ttggcctgactaccagcaggggccgtgacagggtgggcgtgggcatccgaagctcctaca 901 tccgtgtcctgggcatccaggtggacacagatggctctggccgcagctttgctggggccg 961 tgagcccccaggaggaggaggagttccgtcgcctggctgccctcccaaatgtctatgagg 1021 tcatctccaagagcatcgccccctccatctttgggggcacagacatgaagaaggccattg 1081 cctgcctgctctttgggggctcccgaaagaggctccctgatggacttactcgccgaggag 1141 acatcaacctgctgatgctaggggaccctgggacagccaagtcccagcttctgaagtttg 1201 tggagaagtgttctcccattggggtatacacgtctgggaaaggcagcagcgcagctggac 1261 tgacagcctcggtgatgagggacccttcgtcccggaatttcatcatggagggcggagcca 1321 tggtcctggccgatggtggggtcgtctgtattgacgagtttgacaagatgcgagaagatg 1381 accgtgtggcaatccacgaagccatggagcagcagaccatctctatcgccaaggctggga 1441 tcaccaccaccctgaactcccgctgctccgtcctggctgctgccaactcagtgttcggcc 1501 gctgggatgagacgaagggggaggacaacattgacttcatgcccaccatcttgtcgcgct 1561 tcgacatgatcttcatcgtcaaggatgagcacaatgaggagagggatgtgatgctggcca 1621 agcatgtcatcactctgcacgtgagcgcactgacacagacacaggctgtggagggcgaga 1681 ttgacctggccaagctgaagaagtttattgcctactgccgagtgaagtgtggcccccggc 1741 tgtcagcagaggctgcagagaaactgaagaaccgctacatcatcatgcggagcggggccc 1801 gtcagcacgagagggacagtgaccgccgctccagcatccccatcactgtgcggcagctgg 1861 aggccattgtgcgcatcgcggaagccctcagcaagatgaagctgcagcccttcgccacag 1921 aggcagatgtggaggaggccctgcggctcttccaagtgtccacgttggatgctgccttgt 1981 ccggtaccctgtcaggggtggagggcttcaccagccaggaggaccaggagatgctgagcc 2041 gcatcgagaagcagctcaagcgccgctttgccattggctcccaggtgtctgagcacagca 2101 tcatcaaggacttcaccaagcagaaatacccggagcacgccatccacaaggtgctgcagc 2161 tcatgctgcggcgcggcgagatccagcatcgcatgcagcgcaaggttctctaccgcctca 2221 agtgagtcgcgccgcctcactggactcatggactcgcccacgcctcgcccctcctgccgc 2281 tgcctgccattgacaatgttgctgggacctctgcctccccactgcagccctcgaacttcc 2341 caggcaccctcctttctgccccagaggaaggagctgtagtgtcctgctgcctctgggcgc 2401 ccgcctctagcgcggttctgggaagtgtgcttttggcatccgttaataataaagccacgg 2461 tgtgttcaggtaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 2521 aaaaaaaaaaaaaa