METHOD OF PRODUCING AN IMMUNOLIGAND/PAYLOAD CONJUGATE

Abstract

The present invention relates to a method of producing an immunoligand/payload conjugate, which method encompasses conjugating a payload to an immunoligand by means of a sequence-specific transpeptidase, or a catalytic domain thereof.

Claims

1-24. (canceled)

25. A method of treating a pathologic condition in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of an immunoligand/payload conjugate, wherein the immunoligand/payload conjugate is produced by enzymatically conjugating at least one glycine-modified payload to the immunoligand with a sequence-specific sortase or a catalytic domain thereof, wherein the immunoligand is selected from the group consisting of an antibody, a modified antibody format, an antibody derivative or fragment, and an antibody mimetic, and wherein the payload is selected from the group consisting of a cytokine, a radioactive agent, an anti-inflammatory drug, a toxin, and a chemotherapeutic agent.

26. The method of claim 25, wherein the sortase is sortase A.

27. The method of claim 25, wherein the immunoligand/payload conjugate further comprises at least one linker between the immunoligand and the payload, said linker comprising a peptide motif that is a sortase recognition motif, and said linker being conjugated to the C-terminus of at least one peptide chain of the immunoligand.

28. The method according to claim 27, wherein the C-terminal amino acid residue of the sortase recognition motif is replaced by a glycine residue.

29. The method according to claim 28, wherein the sortase recognition motif is LPXTG (SEQ ID NO:27) or NPQTN (SEQ ID NO:28), wherein X represents any amino acid.

30. The method of claim 25, wherein the immunoligand/payload conjugate comprises an antibody/drug conjugate.

31. The method of claim 25, wherein the payload comprises a toxin of molecular weight 2500 Dalton that is cytotoxic to a mammalian cell.

32. The method of claim 31, wherein the toxin is selected from the group consisting of maytansinoids, calicheamicins, Pseudomonas exotoxin PE38, monomethyl Auristatin F (MMAF), monomethyl Auristatin E (MMAE), alpha aminitin, and Diphtheria toxin.

33. The method of claim 25, wherein the payload comprises a chemotherapeutic agent.

34. The method of claim 33, wherein the chemotherapeutic agent is doxorubicin.

35. The method of claim 25, wherein the immunoligand comprises at least two subunits, each being conjugated to a payload.

36. The method of claim 35, wherein the immunoligand with at least two subunits is conjugated to at least two different payloads.

37. The method of claim 36, wherein the different payloads are toxic payloads, each interfering with one or more cellular pathways.

38. The method of claim 35, wherein said immunoligand with at least two subunits comprises a peptide spacer of at least two amino acids appended to the C-terminus of at least one of the two subunits.

39. The method of claim 38, wherein the peptide spacer comprises 2 to 5 amino acids.

40. The method of claim 25, wherein the pathologic condition is a neoplastic disease.

41. The method of claim 40, wherein the neoplastic disease is breast cancer.

42. The method of claim 40, wherein the neoplastic disease is ovarian cancer.

43. The method of claim 25, wherein the subject in need thereof is a mammal.

44. The method of claim 25, wherein the subject in need thereof is a human.

Description

EXPERIMENTS AND FIGURES

[0195] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word comprising does not exclude other elements or steps, and the indefinite article a or an does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0196] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5->3.

Example 1: Cloning of Expression Vectors and Expression of a CD19 Monoclonal Antibody with C-Terminal LPETG Sortase Tag and Additional 6-His and strepII Affinity Purification Tags

[0197] In order to perform the C-terminal conjugation of a payload to an antibody, first a recombinant antibody needs to be expressed that contains C-terminal modifications, including a recognition motif, e.g. for sortase A of Staphylococcus aureus.

[0198] For this, first ORFs for heavy and light chains of an anti-human CD19 specific antibody can be gene synthesized, e.g. at contract research organizations (CROs) offering such gene synthesis services, like e.g. Genscript (www.genscript.com, Piscataway, N.J., USA). As an example, the heavy and light chain sequences of a humanized anti-human CD19 antibody hBU12 can be found in U.S. Pat. No. 8,242,252 B2 under Seq 53 (variant HF) and Seq 58 (variant LG). The V.sub.H and V.sub.L regions of this anti-human CD19 antibody are as follows:

TABLE-US-00002 SEQIDNO1(V.sub.Hcodingregionofhumanizedanti- humanCD19antibodyhBU12): ATGGGATGGAGCTGGATCTTTCTTTTCCTCCTGTCAGGAACTGCAGGTGT CCATTGTCAGGTTCAGCTGCAAGAGTCTGGCCCTGGGTTGGTTAAGCCCT CCCAGACCCTCAGTCTGACTTGTACTGTGTCTGGGGGTTCAATCAGCACT TCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAGGGTCTGGA GTGGATTGGACACATTTGGTGGGATGATGACAAGAGATATAACCCAGCCC TGAAGAGCAGAGTGACAATCTCTGTGGATACCTCCAAGAACCAGTTTAGC CTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCTACTACTGTGC TAGAATGGAACTTTGGTCCTACTATTTTGACTACTGGGGCCAAGGCACCC TTGTCACAGTCTCCTCA Thistranslatestothefollowingaminoacid sequence(SEQIDNO2): MGWSWIFLFLLSGTAGVHCQVQLQESGPGLVKPSQTLSLTCTVSGGSIST SGMGVGWIRQHPGKGLEWIGHIWWDDDKRYNPALKSRVTISVDTSKNQFS LKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSS SEQIDNO3(V.sub.Lcodingregionofhumanizedanti- humanCD19antibodyhBU12) ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTC CAGCAGTGAAATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTCTCTC CAGGGGAAAGGGCTACCCTGAGCTGCAGTGCCAGCTCAAGTGTAAGTTAC ATGCACTGGTACCAGCAGAAGCCAGGGCAGGCTCCCAGACTCCTGATTTA TGACACATCCAAACTGGCTTCTGGTATTCCAGCAAGGTTCAGTGGCAGTG GGTCTGGAACAGATTTTACACTCACAATCAGCAGCCTGGAGCCAGAGGAT GTTGCTGTCTATTACTGTTTTCAGGGGAGTGTATACCCATTCACTTTTGG CCAAGGGACAAAGTTGGAAATCAAA Thistranslatestothefollowingaminoacid sequence(SEQIDNO4): MKLPVRLLVLMFWIPASSSEIVLTQSPATLSLSPGERATLSCSASSSVSY MHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPED VAVYYCFQGSVYPFTFGQGTKLEIK

[0199] These sequences can be fused to human IgG.sub.1 constant heavy and constant light chain regions containing additional C-terminal tags, in order to realize the method disclosed herein.

[0200] In order to realize the invention, the human constant IgG1 heavy chain region can be synthesized with additional 3-codons, encoding an LPETG Staphylococcus aureus sortase A recognition tag, followed by a 6His tag (HHHHHH), a MYC-tag (EQKLISEEDL and a strep II tag (WSHPQFEK) resulting in a sequence, which is as follows:

TABLE-US-00003 SEQIDNO5(humanIgG1heavychainconstant codingregionwithin-frame3extension encodinganLPETGsortasetag,an6xHistagand astrepIItag): AGCACCAAGGGCCCATCTGTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC CTCTGGGGGCACAGCTGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCTG AACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGT GGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAA TCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCT GGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTG TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG TACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAAC CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC CCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTG GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCA CTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAACTGCCCGAGACCG GCCACCACCACCACCACCACGGCGAGCAGAAGCTGATCAGCGAGGAGGAC CTGGGCTGGAGCCACCCCCAGTTCGAGAAGTAG Thistranslatestothefollowingaminoacid sequence(SEQIDNO6,aminoacidsofthetags areunderlined): STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKLPETGHHHHHHGEQKLISEED LGWSHPQFEK*

[0201] Furthermore, the human constant IgG1 kappa light chain region can be synthesized with additional 3-codons, encoding an LPETG Staphylococcus aureus sortase A recognition tag, followed by a 6His tag and a strep II tag (WSHPQFEK) resulting in a sequence, which is as follows:

TABLE-US-00004 SEQIDNO7(humanIgG1kappalightchainconstant codingregionwithin-frame3extensionencoding anLPETGsortasetag,an6xHistag,aMyctag,and astrepIItag): ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTT GAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCA GAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCT CAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCT ACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGC TTCAACAGGGGAGAGTGTCTGCCCGAGACCGGCCACCACCACCACCACCA CGGCGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGCTGGAGCCACCCCC AGTTCGAGAAGTAG Thistranslatestothefollowingaminoacid sequence(SEQIDNO8,aminoacidsofthetags areunderlined): TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGECLPETGHHHHHHGEQKLISEEDLGWSHPQFEK*

[0202] The complete coding regions for LPETG sortase tag, 6His and strepII tagged heavy and light chains of the humanized anti-human CD19 antibody hBU12 are then as follows:

TABLE-US-00005 SEQIDNO9(CompletehumanIgG1V.sub.H-C.sub.Hheavychain codingregionforhBU12withC-terminalLPETG sortasetag,6xHistag,Myctag,andastrepII tag): ATGGGATGGAGCTGGATCTTTCTTTTCCTCCTGTCAGGAACTGCAGGTGT CCATTGTCAGGTTCAGCTGCAAGAGTCTGGCCCTGGGTTGGTTAAGCCCT CCCAGACCCTCAGTCTGACTTGTACTGTGTCTGGGGGTTCAATCAGCACT TCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAGGGTCTGGA GTGGATTGGACACATTTGGTGGGATGATGACAAGAGATATAACCCAGCCC TGAAGAGCAGAGTGACAATCTCTGTGGATACCTCCAAGAACCAGTTTAGC CTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCTACTACTGTGC TAGAATGGAACTTTGGTCCTACTATTTTGACTACTGGGGCCAAGGCACCC TTGTCACAGTCTCCTCAGCTAGCACCAAGGGCCCATCTGTCTTCCCCCTG GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGCCCTGGGCTGCCT GGTCAAGGACTACTTCCCTGAACCTGTGACAGTGTCCTGGAACTCAGGCG CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGG ACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG ACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACC AGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCC TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCG TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCC GGGTAAACTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGA AGCTGATCAGCGAGGAGGACCTGGGCTGGAGCCACCCCCAGTTCGAGAAG TAG Thistranslatestothefollowingaminoacid sequence(SEQIDNO10,aminoacidsofthetags areunderlined): MGWSWIFLFLLSGTAGVHCQVQLQESGPGLVKPSQTLSLTCTVSGGSIST SGMGVGWIRQHPGKGLEWIGHIWWDDDKRYNPALKSRVTISVDTSKNQFS LKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKLPETGHHHHHHGEQKLISEEDLGWSHPQF EK* SEQIDNO11(CompletehumanIgG1V.sub.L-C.sub.Lkappa chaincodingregionforhBU12withC-terminal LPETGsortasetag,6xHistag,Myctag,anda strepIItag): ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTC CAGCAGTGAAATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTCTCTC CAGGGGAAAGGGCTACCCTGAGCTGCAGTGCCAGCTCAAGTGTAAGTTAC ATGCACTGGTACCAGCAGAAGCCAGGGCAGGCTCCCAGACTCCTGATTTA TGACACATCCAAACTGGCTTCTGGTATTCCAGCAAGGTTCAGTGGCAGTG GGTCTGGAACAGATTTTACACTCACAATCAGCAGCCTGGAGCCAGAGGAT GTTGCTGTCTATTACTGTTTTCAGGGGAGTGTATACCCATTCACTTTTGG CCAAGGGACAAAGTTGGAAATCAAAAGAACTGTGGCTGCACCATCTGTCT TCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAA GGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGC AGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCA GGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTCTGC CCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGAAGCTGATCAGC GAGGAGGACCTGGGCTGGAGCCACCCCCAGTTCGAGAAGTAG Thistranslatestothefollowingaminoacid sequence(SEQIDNO12,aminoacidsofthetags areunderlined): MKLPVRLLVLMFWIPASSSEIVLTQSPATLSLSPGERATLSCSASSSVSY MHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPED VAVYYCFQGSVYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGECLPETGHHHHHHGEQKLIS EEDLGWSHPQFEK*

[0203] The coding regions for the heavy and light chains of the anti-human CD19 specific antibody as disclosed in SEQ ID NOs 9 and 11, respectively, can then be synthesized with flanking restriction enzyme sites (e.g. HindIII and NotI) such that they can be cloned into a standard mammalian expression vector, such as pCDNA3.1-hygro (+) (Invitrogen), by standard molecular biology methods known in the art.

[0204] The complete DNA sequence of pCDNA3.1-hygro (+)-IgH chain expression vector for the tagged hBU12 anti-human CD19 antibody will be as follows:

TABLE-US-00006 (codingregionofhumanIgG1V.sub.H-C.sub.HheavychainforhBU12 withC-terminalLPETGsortasetag,6xHistagandastrepIItag underlined,andHindIIIandNotIcloningsitesshaded): SEQIDNO13 GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGC CAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCA AGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCA GATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCA TATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATT TACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGAC GGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA AAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG AGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCC [00001] AGGTGTCCATTGTCAGGTTCAGCTGCAAGAGTCTGGCCCTGGGTTGGTTAAGCCCTCCCAGACCCTCAGTCTGA CTTGTACTGTGTCTGGGGGTTCAATCAGCACTTCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAG GGTCTGGAGTGGATTGGACACATTTGGTGGGATGATGACAAGAGATATAACCCAGCCCTGAAGAGCAGAGTGAC AATCTCTGTGGATACCTCCAAGAACCAGTTTAGCCTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCT ACTACTGTGCTAGAATGGAACTTTGGTCCTACTATTTTGACTACTGGGGCCAAGGCACCCTTGTCACAGTCTCC TCAGCTAGCACCAAGGGCCCATCTGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGC CCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCG GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGT TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAG TCTTCCTCTTCCCCCAAAACCCAAGGAQCACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT CAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC ACAGAAGAGCCTCTCCCTGTCTCCGGGTAAACTGCCCGAGACCGGCCACCACCACCACCACCACGGCGAGCAGA [00002] GCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCG TGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAG CAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATC CCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCC AGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGC TCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGG GTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGAT TTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAA TGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAAT TAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTA GTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGC CCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAG TGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGA TCAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCG TCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATAT GTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGC GCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCAC AGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGAT GCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACAC TACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCG TCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTC GTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGA GGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGC AGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGC ATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATG CGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGA CCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAG CACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGC CGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTT ATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT GGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATC ATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAA AGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAG TCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCG CTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAG GCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAA TCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCC TCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCG CTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGA ACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACT TATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTG AAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGC AGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGG AACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTA AAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTG AGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTT ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGT CTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCT ACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGT TACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGG CCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC GAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCA GCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAG GGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTC TCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAA GTGCCACCTGACGTC

[0205] The complete DNA sequence of pCDNA3.1-hygro (+)-IgL chain expression vector for the tagged hBU12 anti-human CD19 antibody will be as follows:

TABLE-US-00007 (codingregionofhumanIgG1V.sub.L-C.sub.LkappalightchainforhBU12with C-terminalLPETGsortasetag,6xHistag,Myctag,andastrepII tagunderlined,andHindIIIandNotIcloningsitesshaded): SEQIDNO14 GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGC CAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCA AGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCA GATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCA TATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATT TACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGAC GGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA AAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG AGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCC [00003] TGCTTCCAGCAGTGAAATTGTTCTCACCCAGTCTCCAGCAACCCTGTCTCTCTCTCCAGGGGAAAGGGCTACCC TGAGCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGGCAGGCTCCCAGACTC CTGATTTATGACACATCCAAACTGGCTTCTGGTATTCCAGCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTT TACACTCACAATCAGCAGCCTGGAGCCAGAGGATGTTGCTGTCTATTACTGTTTTCAGGGGAGTGTATACCCAT TCACTTTTGGCCAAGGGACAAAGTTGGAAATCAAAAGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAA AGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGG ACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGC GAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTCTGCCCGAGACCGG CCACCACCACCACCACCACGGCGAGCAGAAGCTGATCAGCGAGGAGGACCTGGGCTGGAGCCACCCCCAGTTCG [00004] CCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCT AATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGAC AGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGA AAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGG TTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTC GCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACG GCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTC GCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATC TCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAATGAGCTGATTTAACA AAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAG GCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGC AGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCT AACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCG CCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCG GGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGA GAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTT TCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGT TATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAG CCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTG TTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCA TTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTA TCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGG CCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGC CGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTT CTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGAT CGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTC GATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACA AATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCC CCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGG TTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTT CGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATA AAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCG TCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAAT TCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAA TTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGC GCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCG GCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAA AGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGG CTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAG ATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGT CCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTC GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGA GGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGT ATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGC TGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGA TCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAA AGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTG GTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTG CCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCG CGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGG TCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTA ATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGG TCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTC TTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGT GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGT AACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGA AGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATA TTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

[0206] These constructs allow upon transfection into mammalian cells, like e.g.but not limited to CHO cells, that are typically used for recombinant antibody expression, the expression of the anti-human CD19 specific humanized antibody hBU12 with C-terminal additions of a sortase A tag, a 6His tag, a Myc tag, and a strepII tag at both the IgH and IgL chains.

Example 2: Cloning of Expression Vectors for Monoclonal Antibody with C-Terminal N-Intein Domain of Ssp GyrB 11 Split-Intein with Additional C-Terminal 6His and strepII Affinity Purification Tags

[0207] Similar to the design of expression cassettes and vectors of Staphylococcus aureus sortase A tagged IgG1 heavy and light chains, the coding regions for a C-terminal fusion of N-intein domain of Ssp GyrB 11 split-intein to either the IgH and IgL chain can be designed as follows, in order to gene synthesize the genes by a qualified CRO (e.g. Genscript (www.genscript.com, Piscataway, N.J., USA), with the same elements for the anti-human CD19 antibody as disclosed further above.

[0208] The 150 amino acid sequence of the N-intein domain of Ssp GyrB 11 split-intein can be found in a publication by Appleby et al. (2009), and is as follows:

TABLE-US-00008 SEQIDNO15(N-inteindomainofSspGyrB11 split-intein): CFSGDTLVALTDGRSVSFEQLVEEEKQGKQNFCYTIRHDGSIGVEKIINA RKTKTNAKVIKVTLDNGESIICTPDHKFMLRDGSYKCAMDLTLDDSLMPL HRKISTTEDSGHMEAVLNYNHRIVNIEAVSETIDVYDIEVPHTHNFALAS

[0209] Reverse translation of that amino acid sequence with mammalian codon usage will result in the coding sequence for the N-intein domain of Ssp GyrB 11 split-intein as follows:

TABLE-US-00009 SEQIDNO16(endocingsequenceforN-intein domainofSspGyrB11split-intein): TGCTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGAAGCGTGAG CTTCGAGCAGCTGGTGGAGGAGGAGAAGCAGGGCAAGCAGAACTTCTGCT ACACCATCAGACACGACGGCAGCATCGGCGTGGAGAAGATCATCAACGCC AGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCTGGACAACGG CGAGAGCATCATCTGCACCCCCGACCACAAGTTCATGCTGAGAGACGGCA GCTACAAGTGCGCCATGGACCTGACCCTGGACGACAGCCTGATGCCCCTG CACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATGGAGGCCGTGCT GAACTACAACCACAGAATCGTGAACATCGAGGCCGTGAGCGAGACCATCG ACGTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCCTGGCCAGC

[0210] With this sequence information at hand, the complete IgG1 heavy chain coding region for anti-human CD19 antibody hBU12 with C-terminal extension, comprising the N-intein domain of Ssp GyrB 11 split-intein, followed by a 6His-tag and a strepII tag can be designed as disclosed in SEQ ID NO 17 below:

TABLE-US-00010 ATGAATTTTGGACTGAGGCTGATTTTCCTGGTGCTGACCCTGAAAGGCGT CCAGTGTCAGGTTCAGCTGCAAGAGTCTGGCCCTGGGTTGGTTAAGCCCT CCCAGACCCTCAGTCTGACTTGTACTGTGTCTGGGGGTTCAATCAGCACT TCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAGGGTCTGGA GTGGATTGGACACATTTGGTGGGATGATGACAAGAGATATAACCCAGCCC TGAAGAGCAGAGTGACAATCTCTGTGGATACCTCCAAGAACCAGTTTAGC CTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCTACTACTGTGC TAGAATGGAACTTTGGTCCTACTATTTTGACTACTGGGGCCAAGGCACCC TTGTCACAGTCTCCTCAGCTAGCACCAAGGGCCCATCTGTCTTCCCCCTG GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGCCCTGGGCTGCCT GGTCAAGGACTACTTCCCTGAACCTGTGACAGTGTCCTGGAACTCAGGCG CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA CTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGG ACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCC AAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG ACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACC AGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCC TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCG TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCC GGGTAAATGCTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGAA GCGTGAGCTTCGAGCAGCTGGTGGAGGAGGAGAAGCAGGGCAAGCAGAAC TTCTGCTACACCATCAGACACGACGGCAGCATCGGCGTGGAGAAGATCAT CAACGCCAGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCTGG ACAACGGCGAGAGCATCATCTGCACCCCCGACCACAAGTTCATGCTGAGA GACGGCAGCTACAAGTGCGCCATGGACCTGACCCTGGACGACAGCCTGAT GCCCCTGCACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATGGAGG CCGTGCTGAACTACAACCACAGAATCGTGAACATCGAGGCCGTGAGCGAG ACCATCGACGTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCCT GGCCAGCCACCATCACCATCACCATGGCTGGAGCCACCCCCAGTTCGAGA AGTAG

TABLE-US-00011 ThistranslatestoaminoacidsequenceSEQIDNO18(aminoacidsofthe N-inteindomainareunderlined,6xHistagandstrepIItagareshaded): MNFGLRLIFLVLTLKGVQCQVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGKGLEWIGHIWW DDDKRYNPALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGKCFSGDTLVALTDGRSVSFEQLVEEEKQGKQNFCYTIRHDGSIGVEKIIN ARKTKTNAKVIKVTLDNGESIICTPDHKFMLRDGSYKCAMDLTLDDSLMPLHRKISTTEDSGHMEAVLNYNHRI [00005]

[0211] Likewise, a complete IgG1 kappa light chain coding region for anti-human CD19 antibody hBU12 with C-terminal extension, comprising the N-intein domain of Ssp GyrB 11 split-intein, followed by a 6His-tag and a strepII tag can be designed as disclosed in SEQ ID NO 19 below:

TABLE-US-00012 ATGAATTTTGGACTGAGGCTGATTTTCCTGGTGCTGACCCTGAAAGGCGT CCAGTGTGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTC TAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTTT GATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACC CAAAGTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCA GGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCT GTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGA TCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTGG CTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGC CAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTG CGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA GGGGAGAGTGCTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGA AGCGTGAGCTTCGAGCAGCTGGTGGAGGAGGAGAAGCAGGGCAAGCAGAA CTTCTGCTACACCATCAGACACGACGGCAGCATCGGCGTGGAGAAGATCA TCAACGCCAGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCTG GACAACGGCGAGAGCATCATCTGCACCCCCGACCACAAGTTCATGCTGAG AGACGGCAGCTACAAGTGCGCCATGGACCTGACCCTGGACGACAGCCTGA TGCCCCTGCACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATGGAG GCCGTGCTGAACTACAACCACAGAATCGTGAACATCGAGGCCGTGAGCGA GACCATCGACGTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCC TGGCCAGCCACCATCACCATCACCATGGCTGGAGCCACCCCCAGTTCGAG AAGTAG

TABLE-US-00013 ThistranslatestoaminoacidsequenceSEQIDNO20(aminoacidsofthe N-inteindomainareunderlined,6xHistagandstrepIItagare shaded): MNFGLRLIFLVLTLKGVQCDIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAA SNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECFSGDTLVALTDGRSVSFEQLVEEEKQGKQNFCYTIRHDGSIGVEKIINARKTKTNAKVI KVTLDNGESIICTPDHKFMLRDGSYKCAMDLTLDDSLMPLHRKISTTEDSGHMEAVLNYNHRIVNIEAVSETID [00006]

[0212] The coding regions for the N-intein modified heavy and light chains of the anti-human CD19 specific antibody as disclosed in SEQ ID NOs 17 and 19, respectively, can then be synthesized with flanking restriction enzyme sites (e.g. HindIII and NotI) such that they can be cloned into a standard mammalian expression vector, such as pCDNA3.1-hygro (+) (Invitrogen), by standard molecular biology methods known in the art.

[0213] The complete DNA sequence of pCDNA3.1-hygro (+)-IgH chain expression vector for the N-intein tagged hBU12 anti-human CD19 antibody is then as follows:

TABLE-US-00014 (codingregionofhumanIgG1V.sub.H-C.sub.HheavychainforhBU12withC-terminal N-inteindomainofSspGyrBS11splitintein,followedby6xHistag strepIItag(underlined),andHindIIIandNotIcloningsites(shaded)): SEQIDNO21 GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGC CAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCA AGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCA GATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCA TATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATT TACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGAC GGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA AAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG AGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCC [00007] AGGCGTCCAGTGTCAGGTTCAGCTGCAAGAGTCTGGCCCTGGGTTGGTTAAGCCCTCCCAGACCCTCAGTCTGA CTTGTACTGTGTCTGGGGGTTCAATCAGCACTTCTGGTATGGGTGTAGGCTGGATTAGGCAGCACCCAGGGAAG GGTCTGGAGTGGATTGGACACATTTGGTGGGATGATGACAAGAGATATAACCCAGCCCTGAAGAGCAGAGTGAC AATCTCTGTGGATACCTCCAAGAACCAGTTTAGCCTCAAGCTGTCCAGTGTGACAGCTGCAGATACTGCTGTCT ACTACTGTGCTAGAATGGAACTTTGGTCCTACTATTTTGACTACTGGGGCCAAGGCACCCTTGTCACAGTCTCC TCAGCTAGCACCAAGGGCCCATCTGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCTGC CCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAACCTGTGACAGTGTCCTGGAACTCAGGCGCCCTGACCAGCG GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGT TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAG TCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT CAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC ACAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGCTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGAA GCGTGAGCTTCGAGCAGCTGGTGGAGGAGGAGAAGCAGGGCAAGCAGAACTTCTGCTACACCATCAGACACGAC GGCAGCATCGGCGTGGAGAAGATCATCAACGCCAGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCT GGACAACGGCGAGAGCATCATCTGCACCCCCGACCACAAGTTCATGCTGAGAGACGGCAGCTACAAGTGCGCCA TGGACCTGACCCTGGACGACAGCCTGATGCCCCTGCACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATG GAGGCCGTGCTGAACTACAACCACAGAATCGTGAACATCGAGGCCGTGAGCGAGACCATCGACGTGTACGACAT CGAGGTGCCCCACACCCACAACTTCGCCCTGGCCAGCCACCATCACCATCACCATGGCTGGAGCCACCCCCAGT [00008] TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTT CCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAG GACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGC GGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGG TGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTT CTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTT ACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCT ATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA ACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGG CAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCA GGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCC CCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGG CCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTC CCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGT CGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTG CTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGAT CGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGA GAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCG CTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGC CCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGT GTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTT GGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAAT GGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTT CTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAG GATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAAT TTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTAC ACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGAC GCCCCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAA AGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTT CTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAA ATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATA CCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCAC AATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACAT TAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAA CGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGT TCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAG GAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATA AAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAG GTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAG CGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTT GGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCAC CGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTT TGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCA AAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAAC TTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATA CCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAG TGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAG TTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCA TTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTT CGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATT CTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAG TGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAA AGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGA TGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACA GGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGATTACTCATACTCTTCCTTTTTCA ATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAAC AAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

[0214] The complete DNA sequence of pCDNA3.1-hygro (+)-IgL chain expression vector for the Ssp GyrB S11 N-intein domain tagged hBU12 anti-human CD19 antibody will be as follows:

TABLE-US-00015 (codingregionofhumanIgG1V.sub.L-C.sub.LkappalightchainforhBU12with C-terminalSspGyrBS11N-inteindomain,6xHistagandastrepIItag underlined,andHindIIIandNotIcloningsitesshaded): SEQIDNO22 GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGC CAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCA AGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCA GATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCA TATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATT TACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGAC GGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA AAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG AGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCC [00009] AGGCGTCCAGTGTGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCA TCTCCTGCAAGGCCAGCCAAAGTGTTGATTTTGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGA CAGCCACCCAAAGTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGG GTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAA GTAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTGGCTGCACCATCTGTC TTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTA TCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAG AGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG CTTCAGCGGCGACACCCTGGTGGCCCTGACCGACGGCAGAAGCGTGAGCTTCGAGCAGCTGGTGGAGGAGGAGA AGCAGGGCAAGCAGAACTTCTGCTACACCATCAGACACGACGGCAGCATCGGCGTGGAGAAGATCATCAACGCC AGAAAGACCAAGACCAACGCCAAGGTGATCAAGGTGACCCTGGACAACGGCGAGAGCATCATCTGCACCCCCGA CCACAAGTTCATGCTGAGAGACGGCAGCTACAAGTGCGCCATGGACCTGACCCTGGACGACAGCCTGATGCCCC TGCACAGAAAGATCAGCACCACCGAGGACAGCGGCCACATGGAGGCCGTGCTGAACTACAACCACAGAATCGTG AACATCGAGGCCGTGAGCGAGACCATCGACGTGTACGACATCGAGGTGCCCCACACCCACAACTTCGCCCTGGC [00010] CCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGT GCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTC TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC AGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCC CCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCA GCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCT CTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGG TGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTA ATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATT TTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAAT GTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATT AGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAG TCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCC CCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGT GAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGAT CAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGT CTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATG TCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCG CTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACA GGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATG CGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACT ACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGT CAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCG TGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAG GCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCA GCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCA TTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGC GACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGAC CGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGC ACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCC GGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTA TAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTG GTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCA TGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAA GTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGT CGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA GGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGG CCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAAT CGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCT CGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGC TTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA CCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTT ATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGA AGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTC GGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCA GCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGA ACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAA AAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGA GGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGA TACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTA TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTC TATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTA CAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTT ACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGC CGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTT CTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCG TCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCG AAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAG CATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCT CATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAG TGCCACCTGACGTC

[0215] These pcDNA3.1-hygro(+) based expression vectors disclosed in SEQ ID NOs 21 and 22 allow upon transfection into mammalian cells, like e.g. but not limited to CHO cells, that are typically used for recombinant antibody expression, the expression of the anti-human CD19 specific humanized antibody hBU12 with C-terminal N-intein domain fused, followed by a 6His tag and a strepII tag at both the IgH and IgL chains.

Example 3: Cloning and Expression of Recombinant Sortase a Enzyme from Staphylococcus aureus

[0216] The ORF of Sortase A from Staphylococcus aureus is published in Genbank and can be found under entry: AF162687.1. The aa-sequence in that record reads is shown as SEQ ID NO 23 (amino acid sequence of sortase A from Staphylococcus aureus):

TABLE-US-00016 MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQYDKNVK EQASKDKKQQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRG VSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNET RKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIF VATEVK

[0217] The corresponding nucleotide sequence in this Genbank entry is provided as SEQ ID NO 24:

TABLE-US-00017 ATGAAAAAATGGACAAATCGATTAATGACAATCGCTGGTGTGGTACTTAT CCTAGTGGCAGCATATTTGTTTGCTAAACCACATATCGATAATTATCTTC ACGATAAAGATAAAGATGAAAAGATTGAACAATATGATAAAAATGTAAAA GAACAGGCGAGTAAAGATAAAAAGCAGCAAGCTAAACCTCAAATTCCGAA AGATAAATCGAAAGTGGCAGGCTATATTGAAATTCCAGATGCTGATATTA AAGAACCAGTATATCCAGGACCAGCAACACCTGAACAATTAAATAGAGGT GTAAGCTTTGCAGAAGAAAATGAATCACTAGATGATCAAAATATTTCAAT TGCAGGACACACTTTCATTGACCGTCCGAACTATCAATTTACAAATCTTA AAGCAGCCAAAAAAGGTAGTATGGTGTACTTTAAAGTTGGTAATGAAACA CGTAAGTATAAAATGACAAGTATAAGAGATGTTAAGCCTACAGATGTAGG AGTTCTAGATGAACAAAAAGGTAAAGATAAACAATTAACATTAATTACTT GTGATGATTACAATGAAAAGACAGGCGTTTGGGAAAAACGTAAAATCTTT GTAGCTACAGAAGTCAAATAA

[0218] Technical information with respect to the expression of an enzymatically active fragment of recombinant sortase A in E. coli, comprising amino acids 60-205 with 6His tag are disclosed in reference WO2007/108013A2. The coding region for a 6His tagged version of Staphylococcus aureus sortase A (aa60-205) is provided below as SEQ ID NO 25:

TABLE-US-00018 ATGCAAGCTAAACCTCAAATTCCGAAAGATAAATCGAAAGTGGCAGGCTA TATTGAAATTCCAGATGCTGATATTAAAGAACCAGTATATCCAGGACCAG CAACACCTGAACAATTAAATAGAGGTGTAAGCTTTGCAGAAGAAAATGAA TCACTAGATGATCAAAATATTTCAATTGCAGGACACACTTTCATTGACCG TCCGAACTATCAATTTACAAATCTTAAAGCAGCCAAAAAAGGTAGTATGG TGTACTTTAAAGTTGGTAATGAAACACGTAAGTATAAAATGACAAGTATA AGAGATGTTAAGCCTACAGATGTAGGAGTTCTAGATGAACAAAAAGGTAA AGATAAACAATTAACATTAATTACTTGTGATGATTACAATGAAAAGACAG GCGTTTGGGAAAAACGTAAAATCTTTGTAGCTACAGAAGTCAAACACCAT CACCATCACCATTAA

[0219] This translates to amino acid sequence SEQ ID NO 26:

TABLE-US-00019 MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENE SLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSI RDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVKHH HHHH

[0220] The coding region for the 6His tagged sortase A fragment of Staphylococcus aureus, as provided in SEQ ID NO 25, can be cloned into a standard bacterial expression vector, like e.g. pET29 (Novagen), in order to transform E. coli strain BL21(DE3) (Novagen) and to generate an E. coli clone that can be used for the bacterial production of recombinant sortase A according to standard methods known in the art. In short, E. coli BL21(DE3) transformed with pET29 expression plasmids for sortase A can be cultured at 37 C. in LB medium with 50 g/mL kanamycin until until an OD.sub.600=0.5-0.8 is reached. IPTG can then be added to a final concentration of 0.4 mM and protein expression can be induced for three hours at 30 C. The cells can then be harvested by centrifugation and resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl supplemented with 1 mM MgCl2, 2 units/mL DNAseI (NEB), 260 nM aprotinin, 1.2 M leupeptin, and 1 mM PMSF). Cells can then be lysed by sonication and clarified supernatant can then be purified on Ni-NTA agarose following the manufacturer's instructions.

[0221] Fractions that are of >90% purity, as judged by SDS-PAGE, can then be consolidated and dialyzed against Tris-buffered saline (25 mM Tris pH 7.5, 150 mM NaCl), and the enzyme concentration can be calculated from the measured A.sub.280 using the published extinction coefficient of 17,420 M.sup.1 cm.sup.1. The above-mentioned protocol has been followed and ca. 20 mg of >90% pure recombinant enzymatically active fragment (of ca. 17 kD) sortase A of Staphylococcus aureus has been produced and the analysis of the recombinant protein by SDS-PAGE and Western blotting is disclosed in FIGS. 7A-7B.

Example 4: Expression and Purification of Sortase Tagged or N-Intein Tagged Recombinant Antibodies in CHO Cells

[0222] a.) CHO cell expression: Expression of recombinant IgG1 antibodies from the expression constructs disclosed under Examples 2 and 3 can be achieved by transient transfection using e.g. commercially available CHO expression systems, like the FreeStyle CHO system from Invitrogen following the instructions of the FreeStyle CHO manual.

[0223] In brief, about 1 day prior to transfection, CHO cells shall be seeded at 5-610.sup.6 cells/ml in FreeStyle CHO medium in shaker-flasks in order to expand them at 120 rpm on an orbital shaker at 37 C. in a humidified incubator at 7.5% CO.sub.2 atmosphere. The following day the cells can be transfected, when they reach a density of 1.2-1.510.sup.6/ml. Cells then need to be diluted to 110.sup.6 cells/ml. 30 ml of such a cell suspension then needs to be added to a 125 ml shake flask and 40 g of 1:1 mixed IgH and IgL expression plasmid DNA is added to 600 l OptiPro SF-medium (Invitrogen). At the same time, 40 l of FreeStyle MAX transfection reagent needs to be added to 600 l OptiPro SF-medium, and both samples need to be gently mixed, and incubated for 10 min at room temperature to allow DNA-transfection reagent complexes to form. Then the DNA-transfection reagent mix can be added slowly to the 125 ml CHO cell culture from above and the transfected cells are then grown for up to 6 days at 120 rpm on an orbital shaker at 37 C. in a humidified incubator at 7.5% CO.sub.2 atmosphere. Thereafter, cell culture supernatant can be collected and analyzed for antibody expression titer by appropriate methods known in the art (ELISA, Luminex, etc.).

[0224] b.) Protein A purification: Protein A purification of recombinant antibodies from the CHO cell supernatant can be performed with commercially available protein A sepharose columns (Thermo Fisher, Pierce) according to instructions from the manufacturer.

[0225] In brief, cleared cell culture supernatant is run over a protein A column of appropriate size and capacity equilibrated with PBS. Residual medium is washed with PBS and eventually bound IgG can be eluted with low pH buffer, like 0.1 M citric acid-NaOH, pH 3.0. Eluted IgG should be neutralized immediately with 1/10th volume of 1M Tris/Cl, pH7.4. Combined fractions containing IgG can then be dialized against PBS over night at 4 C.

[0226] The protocols provided in Example 4 provide the skilled person in the art with the instruction to produce sufficient quantities of purified, recombinant antibodies from the constructs disclosed in Examples 1 and 2.

Example 5: Generation of Site-Specifically C-Terminally MMAE Toxic Payload Conjugated Monoclonal Antibodies by Sortase and Split-Intein Mediated Transpeptidation

[0227] Monomethyl Auristatin A toxin coupled to a 5 amino acid glycine stretch and a 6 amino acid SSp GyrB S11 C-int split intein peptide according to the formulas provided below, can be custom ordered from qualified chemistry CROs.

##STR00011##

a.) Toxic MMAE Payload Conjugation of LPETG sortaseA Motif Tagged Recombinant IgG Antibodies

[0228] Conjugation of 5 glycine amino acid modified MMAE toxic payload to LPETG sortase A tagged IgG1 antibody (that can be produced by following Examples 1 and 4) can be achieved by mixing appropriate ratios of LPETG tagged IgG1 antibody with the glycine-modified MMAE toxin disclosed in Formula 1 (e.g. at 1:1 ratio and 50 M concentration) and with recombinant sortase A (production described in Example 3) (e.g. at 5 M concentration), and using physiologic incubation buffer, like e.g.; 5 mM Tris/Cl, 15 mM NaCl, 6 mM CaCl.sub.2), pH 8.0, and incubating at 37 C. to 40 C. for a minimum of 2 hours.

[0229] Efficiency of the conjugation can be monitored by analyzing the absence of the 6His tag and/or the strepII tag after stopping the reaction, e.g. by western-blot analysis or ELISA with anti-His-tag and/or anti strepII tag antibodies.

[0230] Completely conjugated product can be enriched by Nickel-NTA columns, or streptactin column binding, which bind to the 6His tag or strepII tag, respectively, which can only be present in incompletely reacted IgG1 substrate. Final IgG-payload conjugate can eventually be purified using protein A purification as described above.

b.) Toxic MMAE Payload Conjugation of SSp GyrB S11 N-Intein Tagged Recombinant IgG Antibodies

[0231] Conjugation of Ssp GyrB S11 C-intein amino acid modified MMAE toxic payload to N-intein tagged IgG1 antibody (that can be produced by following Examples 2 and 4) can be achieved by mixing appropriate ratios of N-intein tagged IgG1 antibody with the C-intein amino acid-modified MMAE toxin disclosed in Formula 2 (e.g. at 1:10 or 1:25 ratio at 5 M concentration of the IgG antibody) using physiologic incubation buffer, like e.g.; 20 mM Tris/Cl, 250 mM NaCl, 1 mM EDTA, pH 8.5, and incubating at room temperature or at 37 C. a minimum of 4 hours.

[0232] Efficiency of the conjugation can be monitored by analyzing the absence of the 6His tag and/or the strepII tag after stopping the reaction, e.g. by western-blot analysis or ELISA with anti-His-tag and/or anti strepII tag antibodies.

[0233] Completely conjugated product can be enriched by Nickel-NTA columns, or streptactin column binding, which bind to the 6His tag or strepII tag, respectively, which can only be present in incompletely reacted IgG1 substrate. Final IgG-payload conjugate can eventually be purified using protein A purification as described above.

[0234] In summary, the Examples 1-5 disclosed above allow a person skilled in the art to practice the invention of enzymatically conjugating a toxic payload site-specifically to the C-terminus either using sortase A mediated or split-intein mediated transpeptidation.

Example 6: Production of Trastuzumab with C-Terminal GS (Glycine-Serine) Linker, LPETG Sortase Motif and Additional 6-his and Strep II Affinity Purification Tags on Either Heavy or Light Chain

[0235] Antibody expression constructs encoding monoclonal antibody Trastuzumab (Tras) heavy and light chains, either untagged (SEQ ID NOs: 31-34) or C-terminally tagged with GS (glycine-serine) linker, LPETG Sortase tag, 6His tag, and Strep II tag (SEQ ID NOs: 35-38) were generated essentially as described in Example 1. Using these expression constructs, Tras-HC-GS-LHS and Tras-LC-GS-LHS (HC=heavy chain, LC=light chain, GS=glycine-serine, LHS=LPETG-tag+6His-tag+strepII-tag) were produced in CHO cells by co-transfection of the corresponding expression constructs. Tras-HC-GS-LHS is a Trastuzumab variant with an unmodified light chain (SEQ ID NOs: 35-36), and a heavy chain C-terminally tagged with GS (glycine-serine) linker, LPETG Sortase motif, 6His-tag, and strepII-tag (SEQ ID NOs: 33-34). Tras-LC-GS-LHS is a Trastuzumab variant with an unmodified heavy chain (SEQ ID NOs: 31-32), and a light chain C-terminally tagged with GS linker, LPETG Sortase motif, 6His-tag, and strepII-tag (SEQ ID NOs: 37-38). CHO cell transfection and affinity purification of antibodies by proteinA-sepharose chromatography was done essentially as described in Example 4.

Example 7: Sortase A-Mediated Conjugation of Heavy or Light Chain of Trastuzumab with Gly5-Modified DM1 Toxin

[0236] Conjugation reactions containing Gly.sub.5-modified DM1 toxin (ordered from Concortis, San Diego, Calif., U.S., structure see FIG. 14A) and a 17 kD recombinant sortase A fragment from Staphylococcus aureus (see Example 3) were carried out with 10.5 mg of each monoclonal antibody (mAb) (see Example 6) in 1 Sortase buffer (25 mM Tris-HCl, pH8.2; 150 mM NaCl; 7.5 mM CaCl.sub.2)), as shown in Table II, below. The Tras-HC-GS-LHS conjugation reaction was incubated at 25 C. for 2 h; the Tras-LC-GS-LHS conjugation reaction was incubated at 25 C. for 18 h. Each reaction mixture was then passed over a Strep-Tactin Sepharose columns (IBA Life-Sciences, Gttingen, Germany). For this, 1 ml of Strep-Tactin Agarose was packed under gravity into a fitted column and equilibrated with 2 column volumes of equilibration buffer (100 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1 mM EDTA). Each conjugation mixture was passed twice down the same column using gravity flow (to increase residence time on the resin). The resin was washed with an additional column volume of equilibration buffer to maximize conjugate yield and the pool then applied immediately to a protein A column. For this, a 1 ml Protein A HiTrap column was equilibrated with 10 column volumes of buffer (25 mM sodium phosphate pH 7.5). Each conjugation reaction was then applied to an equilibrated column and the column washed with a further 5 column volumes of buffer. Bound conjugate was eluted with 5 column volumes of elution buffer (0.1M succinic acid, pH 2.8) with 1 column volume fractions collected (into tubes containing 25% v/v 1M Tris Base to neutralise the acid) and analysed for protein content. Protein containing fractions were pooled and formulated by G25 column chromatography. For this, NAP 25 columns of an appropriate size for each scale of manufacture were used to formulate the conjugates for long term storage. The columns were equilibrated, loaded and eluted with 10 mM Sodium Succinate pH 5.0, 100 mg/mL Trehalose, 0.1% % w/v Polysorbate 20 (Formulation Buffer for Kadcyla (T-DM1), marketed by Roche/Genentech) according to the manufacturer's instructions.

[0237] The Tras-HC-GS-LHS and Tras-LC-GS-LHS DM1-conjugate yields were, respectively, 8.0 mg (76.2%) and 5.9 mg (56.2%). The major process losses occurred during Protein A and G25 purification, most probably as a result of peak cutting to ensure maximal concentration of the product for each subsequent step or storage.

TABLE-US-00020 TABLE 2 Conjugation conditions for Tras-HC-GS-LHS and Tras-LC-GS-LHS: Final Reaction component HC LC concentration Tras-HC-GS-LHS (5.3 mg/ml) 1981 l 5 M Tras-LC-GS-LHS (5.5 mg/ml) 1911 l 5 M H.sub.20 7775.25 l 7714 l Gly.sub.5-DM1 (1 mM) 1400 l 1400 l 100 M Sortase A (0.85 mg/ml = ca. 43.75 l 175 l 0.156/0.625 M 50 M) 5x Sortase buffer* 2800 l 2800 l 1x

[0238] The drug loading was assessed by Hydrophobic Interaction Chromatography (HIC), and was performed on a TOSOH Butyl-NPR 4.6 mm3.5 cm, 2.5 m column run at 0.8 mL/min with a 12 minute linear gradient between A1.5M (NH.sub.4).sub.2SO.sub.4, 25 mM NaPi, pH=6.950.05 and B75% 25 mM NaPi, pH=6.950.05, 25% IPA. The HIC profiles revealed that for both, Tras-HC-GS-LHS and Tras-LC-GS-LHS, there was no detectable unconjugated mAb left, and a major fraction of each mAb was loaded with 2 drugs (see FIG. 8).

Example 8: In Vitro Toxicity Assay with Sortase A-Mediated Trastuzumab-DM1 Conjugates

[0239] Cytotoxicity of DM1-sortaseA-conjugated Tras-HC-GS-LHS and DM1-sortaseA-conjugated Tras-LC-GS-LHS was investigated and compared to Kadcyla (Roche/Genentech) using SKBR3 cells, a human breast cancer cell line overexpressing the cognate antigen of trastuzumab (Tras) HER-2/neu, and T47D-5R cells, a breast cancer cell line naturally expressing low levels of HER-2/neu, engineered to be devoid of cell surface HER-2/neu (Graus-Porta et al. (1995)). Cells were plated on 96 well plates in 100 l complete DMEM (10000 cells per well). After one day incubation, 50 l medium was carefully removed from each well and replaced by 50 l of 3.5-fold serial dilutions of each ADC in complete DMEM, resulting in ADC concentrations ranging from 20 g/ml to 0.25 ng/ml. Each dilution was done in duplicates or triplicates. After 3 additional days incubation at 37 C. in a humidified incubator at 5% CO.sub.2 atmosphere, plates were removed from the incubator and equilibrated to room temperature. After approximately 30 minutes, 100 l CellTiter-Glo Luminescent Solution (Promega, Cat. No G7570) was added to each well and, after shaking the plates at 450 rpm for 5 min followed by a 10 min incubation without shaking, luminescence was measured on a Tecan Infinity F200 with an integration time of 1 second per well. All three ADCs were highly cytotoxic for the HER-2/neu overexpressing SKBR3 breast cancer cell line, but not for the HER-2/neu-negative T47D-5R breast cancer cell line (see FIGS. 9A-9B). The EC.sub.50 values for Her-2/neu positive breast cancer cell line SKBR3 were: Kadcyla, 32.4 ng/ml; DM1-conjugated Tras-HC-GS-LHS, 45.6 ng/ml; Tras-LC-GS-LHS, 51.4 ng/ml, and thus are within similar range of potency in the in vitro tumor cell killing experiment. Conversely, no specific cellular toxicity was detectable with the Her-2/neu negative breast cancer cell line T47D-5R, demonstrating the functional equivalence of sortaseA, enzymatically conjugated ADC versus traditional, chemically conjugated ADC, when the comparison entails the same targeting antibody and the same toxin (DM1) (FIGS. 9A-9B).

[0240] However, it appears that the lower drug-to antibody ratio of ca. 1.80 (deducted from intergration of the DAR1 and DAR2 peaks in FIGS. 8A-8B) for the Tras-HC-GS-LHS and Tras-LC-GS-LHS sortase A-conjugated ADCs, as compared to the DAR of ca. 3-4, reported for Kadcyla does not translate into a proportionally different cellular cytotoxicity in the in vitro tumor cell killing assays (FIGS. 9A-9B). This unexpected finding may be the result of a more defined and site-specific toxin-antibody conjugation mediated by sortase A in comparison to the less defined, stochastically, chemically conjugated Kadcyla.

Example 9: Optimization of Synchronization of sortaseA Mediated Antibody Heavy Chain and Light Chain Payload Conjugation by Variation of Peptide-Spacer Length Inserted Between C-Terminal End of Antibody Heavy Chain and Light Chain and the sortaseA Recognition Motif

[0241] The influence of peptide-spacer length positioned between the C-terminus of antibody heavy or light chain and LPETG sortase A recognition motif was investigated. For this, antibody heavy chain and light chain expression constructs encoding chimeric CD30-specific mAb Ac10 heavy and light chains (HC sequence derived from US 2008213289A1, Seq1, LC sequence derived from US 2008213289A1, Seq9), C-terminally modified with sequences comprising or not comprising a 2 amino acid GS (glycine-serine) spacer, and comprising a LPETG sortaseA recognition motif, and a strep-II purification tag (SEQ ID NOs: 39-46), have been cloned essentially according to instructions disclosed in Example 1. Using these expression constructs, mAbs Ac10-HC-GS-LHS/LC-GS-LHS and Ac10-HC-LS/LC-LS were produced in CHO cells by co-transfection of the corresponding plasmids. Ac10-HC-GS-LHS/LC-GS-LHS is an Ac10 variant with heavy and light chains modified at the C-termini of each HC and LC with a GS peptide spacer, a LPETG sortaseA motif, a 6His tag, and a strep-II tag (SEQ ID NOs:39-42; Table 3). Ac10-HC-LS/LC-LS is an Ac10 variant with heavy and light chains modified at the C-termini with LPETG Sortase motif and strep-II tag without the 2-peptide GS linker (SEQ ID NOs: 43-46; Table 3). CHO cell transfection and affinity purification of antibodies by protein A-sepharose chromatography was done essentially as described in Example 4.

[0242] To investigate efficiency of conjugation, serial dilutions of Sortase A were used to conjugate penta-glycine-modified FITC (Gly.sub.5-FITC, see Formula 3 below).

##STR00012##

[0243] For this, Gly.sub.5-FITC was sortaseA conjugated to two Ac10 variants in 1 Sortase buffer (25 mM Tris-HCl, pH8.2; 150 mM NaCl; 7.5 mM CaCl.sub.2)), as shown in Table 4. After 4 h at 42 C., reaction products were analyzed by denaturing, reducing SDS-PAGE gel electrophoresis, and FITC was visualized by placing the gels on a UV box (FIGS. 10A-10B). Conjugation to the heavy chain was found to be highly efficient irrespective of the presence absence of the GS-linker between heavy chain C-terminus and LPETG Sortase recognition motif. Unexpectedly, sortaseA mediated conjugation to the light chain was significantly less efficient in comparison to sortaseA mediated heavy chain conjugation. Furthermore, it was surprisingly found that coupling efficiency was dramatically affected by the presence or absence of the 2 peptide GS (glycine-serine) spacer positioned between the C-terminus of the antibody light chains and the LPETG sortaseA recognition motif. Whereas in the presence of the GS-linker, conjugation to the light chain took place with about 5-10 lower efficiency than to the heavy chain, it was about 50-100 less efficient in the absence of a linker. Therefore, it was concluded that increasing the peptide spacer length between the light chain and the LPETG Sortase recognition motif might further improve conjugation efficiency.

[0244] Therefore, the influence of increasing the length of the peptide spacer between light chain and LPETG Sortase A recognition motif on conjugation efficacy was investigated next. Expression constructs encoding mAb Ac10 light chains, C-terminally tagged with LPETG Sortase recognition motif and strep-II purification tag, with a 2 to 5 amino acid linker (SEQ ID NOs: 47-54), were generated essentially as described in Example 1. Using these expression constructs, mAbs Ac10-HC-LS/LC-GS-LS, Ac10-HC-LS/LC-GGS-LS, Ac10-HC-LS/LC-GGGS-LS and Ac10-HC-LS/LC-GGGGS-LS were produced in CHO cells by co-transfection of the corresponding expression constructs. In each of these antibodies, the heavy chain is C-terminally modified with an LPETG Sortase recognition motif and a strep-II purification tag (SEQ ID NOs: 43-44; Table 3). The light chain is C-terminally modified with an LPETG Sortase tag and strep-II tag containing either a GS, GGS, GGGS, or a GGGGS peptide spacer (SEQ ID NOs: 47-54; Table 3) in front of the LPETG motif. CHO cell transfection and affinity purification of antibodies by protein A-sepharose chromatography was done essentially as described in Example 4.

[0245] To investigate conjugation efficiency, serial dilutions of Sortase A were used to conjugate penta-glycine-modified FITC (Glyn-FITC, see Formula 3, above) to the four different Ac10 mAb variants in 1 Sortase buffer (25 mM Tris-HCl, pH8.2; 150 mM NaCl; 7.5 mM CaCl.sub.2)), as shown in Table 5. After 4 h at 42 C., reaction products were analyzed by denaturing, reducing SDS-PAGE gel electrophoresis, and FITC was visualized by placing the gels on a UV box (FIG. 11). As expected, conjugation to the heavy chain was equally efficient in all four antibody variants. In contrast, conjugation to the light chain was improved significantly by increasing peptide-spacer length. Significantly, with the longest peptide-spacer analyzed (GGGGS), light chain conjugation efficiency was equally efficient in comparison to conjugation of the heavy chain, thereby allowing synchronous conjugation of heavy and light chains of an antibody C-terminally modified at both heavy and light chain. It is concluded that this antibody format will facilitate Sortase A-mediated production of homogeneous ADCs loaded with 4 drugs per antibody (DAR4).

TABLE-US-00021 TABLE3 C-terminallymodifiedmonoclonalantibodyAc10 variantsproduced Heavy Light Chain SEQ Chain SEQ modifi- ID modifi- ID Antibody cation NOs cation NOs Ac10-HC-GS- GS-LPETG-G- 39,40 GS-LPETG-G- 41,42 LHS/LC-GS- HHHHHH-G- HHHHHH-G- LHS WSHPQFEK WSHPQFEK Ac10-HC- LPETG-G- 43,44 LPETG-G- 45,46 LS/LC-LS WSHPQFEK WSHPQFEK Ac10-HC- LPETG-G- 43,44 GS-LPETG-G- 47,48 LS/LC-GS-LS WSHPQFEK WSHPQFEK Ac10-HC- LPETG-G- 43,44 GGS-LPETG- 49,50 LS/LC-GGS-LS WSHPQFEK G- WSHPQFEK Ac10-HC- LPETG-G- 43,44 GGGS-LPETG- 51,52 LS/LC-GGGS- WSHPQFEK G- LS WSHPQFEK Ac10-HC- LPETG-G- 43,44 GGGGS- 53,54 LS/LC- WSHPQFEK LPETG-G- GGGGS-LS WSHPQFEK

TABLE-US-00022 TABLE 4 Conjugation conditions for mAbs Ac10-HC-GS-LHS/LC-GS-LHS and Ac10-HC-LS/LC-LS Reaction component 1-8 9-16 Final concentration Ac10-HC-GS-LHS/LC-GS-LHS 10 5 M (3.75 mg/ml = 25 M) Ac10-HC-LS/LC-LS (3.75 10 5 M mg/ml = 25 M) H.sub.20 20 20 Gly.sub.5-FITC (1 mM) 5 5 100 M Sortase A (2x serial dil. of ca. 50 M) 5 5 5 .fwdarw. 0.039 M 5x Sortase buffer 10 10 1x

TABLE-US-00023 TABLE 5 Conjugation conditions for mAbs Ac10-HC-LS/LC-GS-LS, Ac10-HC-LS/LC-GGS-LS, Ac10-HC-LS/LC- GGGS-LS and Ac10-HC-LS/LC-GGGGS-LS. Reaction component 1-7 8-14 15-21 22-28 Final conc. Ac10-HC-LS/LC-GS-LS 10 5 M (3.75 mg/ml = 25 M) Ac10-HC-LS/LC-GGS-LS 10 5 M (3.75 mg/ml = 25 M) Ac10-HC-LS/LC-GGGS-LS 10 5 M (3.75 mg/ml = 25 M) Ac10-HC-LS/LC-GGGGS-LS 10 5 M (3.75 mg/ml = 25 M) H.sub.20 20 20 20 20 Gly.sub.5-FITC (1 mM) 5 5 5 5 100 M Sortase A (2x serial dil. of 5 5 5 5 2.5.fwdarw.0.039 M ca. 25 M) 5x Sortase buffer 10 10 10 10 1x

Example 10: Generation of Homogeneous ADC by strepII-Tag Affinity Purification

[0246] Sortase A mediated conjugation with Gly.sub.5-labeled vc-PAB-MMAE (see Formula 1, Example 5) was performed with anti-CD30 antibody Ac10 modified at the C-termini of either the heavy chains, or the light chains with sequences comprising an LPETG sortase A motif and a strepII-affinity purification tag as provided in Table 6 below:

TABLE-US-00024 TABLE6 C-terminallymodifiedantibodyAc10witheitherHC orLCmodification Heavy Light Chain SEQ Chain SEQ modifi- ID modifi- ID Antibody cation NOs cation NOs Ac10-HC-LS LPETG-G- 43,44 none 29,30 Ac-10-LC WSHPQFEK Ac10-HC none 27,28 GS-LPETG-G- 41,42 Ac10-LC-GS- HHHHHH-G- LHS WSHPQFEK

[0247] The expression vectors encoding the Ac10 heavy or light chain sequences of Table 4 have been constructed essentially as disclosed in Example 1. CHO cell transfection and affinity purification of antibodies by protein A-sepharose chromatography was done essentially as described in Example 4.

[0248] Sortase A mediated conjugation of heavy or light chaing sortase motif tagged anti-CD30 antibodies with Gly.sub.5-labeled vc-PAB-MMAE (see Formula 1, Example 5) was performed essentially according to the protocol provided in Example 7.

[0249] As described further above in the detailed description of the invention, unreacted antibody will retain the C-terminal strep-II affinity purification tag, which can be exploited to enrich fully reacted ADC with DAR2. Analysis of the heavy chain sortase A conjugation with vc-PAB-MMAE toxin via hydrophobicity interaction chromatography (HIC) (FIG. 12A), shows that the majority of the sortase-motif modified heavy chains have been conjugated, but a certain percentage of unreacted substrate (DAR0=drug to antibody ratio=zero), or partially reacted substrate (DAR1=drug to antibody ratio=1) was still detectable by HIC (FIG. 12A).

[0250] Therefore, the protein A purified vc-PAB-MMAE conjugate was passed 4 times times over a StrepTactin affinity column (IBA Sciences, Gttingen, Germany), essentially as described in Example 7, in order to remove unreacted or partially reacted sortase A-modified antibody. FIG. 12B shows that upon several passages of the heterogeneous vc-PAB-MMAE antibody drug conjugate, completely reacted DAR2 ADCs (DAR2=drug to antibody ratio=2) could be highly enriched. This experiment demonstrates the feasibility to utilize additional affinity purification tags added C-terminally to the sortase A LPETG recognition motif to generate homogeneous ADC with a defined drugs per antibody ratio (here DAR2).

Example 11: Synthesis of 5Glycine-Modified Maytansine and Alpha-Amanitin Toxins

[0251] In order to allow conjugation of two different payloads, preferably toxic payloads to a single antibody, modified with different sortase motifs at heavy and light chain C-termini, it is required to modify two different toxins with glycine residues, preferably toxins with different mode of actions, such that a cancer cell targeted with a dual payload conjugated ADC, is attacked with via two different, potentially synergistic routes. The synthesis of two different glycine-modified toxic payloads (here maytansine and alpha-amanitin) satisfying this requirement has been performed and is described herein.

11.1 Synthesis of Glycine-Modified Alpha-Amanitin:

[0252] 30 mg alpha-amanitin (Structure 1) (Sigma-Aldrich, order #A2263) was dissolved in 1 ml anhydrous DMSO. To this solution 19 mg NH-Boc-amino-hexylbromide were added, followed by potassium tert-butoxide (1M solution in THF, 35 l). The reaction mixture was stirred at room temperature for 6 h and more potassium tert-butoxide (1M solution in THF, 20 l) was added. The reaction was kept at room temperature for 16 h. Acetic acid (10 l) was added and the crude mixture was purified by RP-HPLC directly (Sunfire C18 5 3 cm10 cm column, 50 mL/min, 5-50% acetonitrile/water 15 min gradient). The desired fraction was collected and lyophilized to give Structure 2 as a white powder (15 mg), which was treated with TFA/DCM solution (1/1, v/v, 1 ml) for 30 minutes at room temperature. The volatiles were removed under reduced pressure to give Structure 3 as a slightly yellowish gum, which was used in the next step without further purification.

[0253] Fmoc-Gly5-OH (8 mg) was dissolved in anhydrous DMF (0.5 ml). HATU (Sigma-Aldrich, order #445460) (6 mg) was added, followed by DIEA (10 ml) (Sigma-Aldrich, order #496219). The mixture was agitated gently at room temperature for 30 s and then transferred to a solution of compound 3 in DMF (0.5 ml). After 30 mins, LC/MS analysis showed that all of compound 3 was consumed. Piperidine (30 l) was added and the progress of the reaction was monitored by LC/MS. Acetic acid was added to neutralize the reaction after 1 h and the mixture was purified by RP-HPLC (Sunfire C18 5 3 cm25 cm column, 50 mL/min, 2-40% acetonitrile/water 30 min gradient). The fractions were pooled and lyophilized to give structure 5 as a white powder (12 mg). Analytical data for compound 5 is provided in FIG. 13A).

##STR00013##

11.2. Synthesis of Glycine-Modified Maytansine:

[0254] Maytansinol (0.6 g, 1.1 mmol) (Clearsynth Labs, Mumbai, India) was dissolved in anhydrous THF (6 ml) and anhydrous DMF (3 ml) after which 1.2 ml DIEA (Sigma-Aldrich, order #496219) was added. The solution was placed under argon atmosphere. Zinc triflate (1.2 g) and NMeAla NCA (0.7 g) were added in one portion. The mixture was sonicated until the solid was dissolved. The reaction mixture was stirred at room temperature for 2 days and then diluted with ethyl acetate (100 ml). It was washed with saturated NaHCO.sub.3 (aq. solution, 250 ml) and brine (50 ml). The organic layer was dried (over MgSO.sub.4) and concentrated to give the crude maytansinol 3-(S)-alpha-N-methylaminopropionate (8) which was used directly in the next step without further purification.

[0255] Fmoc-Gly5-OH (26 mg) was dissolved in anhydrous DMF (1 ml). HATU (Sigma-Aldrich, order #445460) (19 mg) was added, followed by DIEA (18 L). The mixture was agitated gently at room temperature for 30 s and then transferred to a solution of compound 8 in THF (1 ml). After 30 mins, LC/MS analysis showed that all compound 8 was consumed. Piperidine (40 l) was added and the progress of the reaction was monitored by LC/MS. Ether (40 ml) was added to the reaction after 2 h and the precipitated solid was collected and washed with ether. The crude compound was purified by RP-HPLC (Sunfire C18 5 3 cm10 cm column, 50 ml/min, 10-60% acetonitrile/water 20 min gradient). The fractions were pooled and lyophilized to give compound 10 as a white powder (33 mg). Analytical data for compound 10 is provided in FIG. 13B.

##STR00014##

[0256] Importantly, it is to be noted that in principle, any toxin can be functionalized for sortase mediated enzymatic conjugation, if either 5 glycines (as shown here), or any number of glycine residues greater or equal than one glycine, are attached to the toxins (see FIGS. 14A-14C).

Example 12: In Vivo Tumor Inhibition of Sortase A-Conjugated Trastuzumab-DM1 in SKOV3 Ovarial Carcinoma Xenograft Models

[0257] 510.sup.6 SKOV3 tumor cells in 200 l PBS/Matrigel (1:1 ratio) were implanted subcutaneously into the left flanks of 5-6 weeks old female NMRI nude mice. Primary tumor volumes were monitored by calipering. After a mean tumor volume of 100-200 mm.sup.3 was reached, tumor-bearing animals were randomized into 3 Groups according to tumor sizes (10 animals per group). On the day of randomization (day 0) and on day 21, animals of Groups 1, 2 and 3 were injected intravenously with, respectively, 5 ml/kg PBS, 15 mg/kg Kadcyla, or 15 mg/kg sortase A-conjugated Trastuzumab-DM1. Tumor volumes were measured bi-weekly by calipering (FIG. 15). The study was terminated after 39 days and animals were euthanized according to accepted animal experimentation guidelines.

[0258] In the course of the study, tumors in control animals mock-injected with PBS grew steadily to a volume of approximately 600 mm.sup.3. In contrast, tumors in Kadcyla-treated animals shrank and were essentially undetectable on day 39. Anti-tumor activity of Sortase A-conjugated Trastuzumab-DM1 did not differ significantly from that of commercially available Kadcyla, despite the fact that the sortase-conjugated T-DM1 exhibited a lower drug to antibody ratio of approximately 2, in comparison of a reported DAR of 3.5 of Kadcyla. In combination with the data from Example 8, the results demonstrate that sortase conjugated ADCs, using identical antibody and toxin moiety, have comparable tumor killing activity in comparison to commercially available chemically conjugated Kadcyla in vitro and in vivo, albeit at lower drug to antibody ratio.

Example 13: Sortase A-Mediated Conjugation in Crude CHO Cell Supernatant

[0259] The Trastuzumab variant Tras-HC-LS/LC-GGGGS-LS, consisting of heavy chains C-terminally tagged with LPETG Sortase motif and Strep II purification tag (SEQ ID NOs: 055-056), and light chains C-terminally tagged with a 5 amino acid Gly.sub.4-Ser spacer (GGGGS), LPETG Sortase motif and Strep II tag (SEQ ID NOs: 057-058), was produced in CHO cells essentially as described in Example 4. The resulting serum-free crude cell supernatant contained approximately 157 mg/L Tras-HC-LS/LC-GGGGS-LS and was directly used for conjugation essentially as described in Example 9, by adding Sortase buffer, Gly.sub.5-FITC, and serial dilutions of Sortase A directly to the supernatant. In parallel, Tras-HC-LS/LC-GGGGS-LS purified by protein A affinity chromatography was also conjugated under otherwise identical conditions. After 4 hours at 42 C., the reactions were analyzed by denaturing, reducing SDS-PAGE gel electrophoresis. After visualizing FITC by placing the gel on a UV box, protein was stained using Coomassie Brilliant Blue (FIGS. 16A-16B). The data shows the unexpected finding that Sortase A-mediated conjugation of antibodies in crude cell culture supernatant was as efficient as that of purified antibody. Further, the conjugation reaction was highly specific and none of the protein contaminants present in crude CHO cell supernatant were non-specifically conjugated. Together, these data suggest that the robustness of the Sortase reaction may help facilitate ADC manufacturing by allowing to perform drug conjugation directly after production in CHO cells prior to purification and downstream processing.

FIGURE LEGENDS

[0260] FIGS. 1A-1B: These figures illustrate the principle of the sortase A mediated site-specific payload conjugation to an immunoligand (or binding protein), which can be performed at the N-terminus of a protein (FIG. 1A), or at the C-terminus of the protein (FIG. 1B). In order to achieve N-terminal conjugation, the payload needs to contain a sortase penta-peptide recognition motif (here LPXTG, the recognition motif of sortase A from Staphylococcus aureus (X representing any of the 20 natural amino acids), whereas the N-terminus of the immunoligand/binding protein to be labeled needs to be expressed with an N-terminal extension of minimally 3 glycine residues, here indicated as Gn, (with n>2), that has a free N-terminal amino group (here indicated by the smaller H.sub.2N symbol). Typically 3-5 glycines are used in order to modify a substrate for sortase-mediated conjugation. Addition of recombinant sortas A enzyme from Staphylococcus aureus, as indicated here, then catalyzes the breakage of the peptide bond between the T and the C-terminal G residue in the LPXTG penta-peptide motif and forms a new peptide bond between the N-terminal glycine of the Gn stretch (n>2) and the T residue. The C-terminal G residue of the LPXTG motif (here highlighted in boldface print) is removed in the transpeptidation reaction. (FIG. 1B) Conversely, in order to achieve C-terminal conjugation of a payload to a protein, which is the preferred method for conjugation of payloads, particularly toxins, to antibodies (see FIGS. 6A-6B), the LPXTG sortase recognition penta-peptide motif needs to be added to the C-terminal end of the immunoligand/binding protein (e.g. by recombinant protein expression technology, as described in the Examples), and the payload needs to be modified with a short glycine stretch (Gn, with n>2, typically 3-5 glycines). As described under FIG. 1A, addition of sortase A from Staphylococcus aureus will then catalyze the transpeptidation of the Gn-stretch to the LPXTG motif, whereby the terminal G residue of the LPXTG motif (in boldface) will be removed.

[0261] FIGS. 2A-2B: These figures illustrate the principle of intein (FIG. 2A) and split-intein (FIG. 2B) mediated transpeptidation. (FIG. 2A) Inteins can occur as so-called protein-introns in precursor proteins, where they separate N-terminal and C-terminal parts of a mature protein, which are generally called N-extein and C-extein. The intein protein-intron can catalyze the breakage of the peptide bond between the intein and the C-extein and the formation of a new peptide bond between the N-extein and C-extein by transferring the N-terminal amino acid of the C-extein to the C-terminal amino acid of the N-extein in a transpeptidation reaction. The result of the reaction is the removal of the intein protein-intron from the precursor protein and the generation of a mature protein with a newly created peptide bond between the N-extein and C-intein domains. (FIG. 2B) The intein activity has also been described to be separable into distinct domains, that can be attached to different proteins, for which this intein variation has been termed split-intein. The N-int and C-int domains of the split intein form a non-covalent structural complex, that can perform the same transpeptidation reaction as a contiguous intein, on the attached N-extein and C-extein domains that are then in spatial proximity and part of the complex. The result of the transpeptidation of N-int and C-int split-intein reaction is then a protein trans-splicing, or essentially a protein ligation between the N-extein and C-extein domains, by formation of a novel peptide bond.

[0262] FIGS. 3A-3B: These figures illustrate how particular split inteins that are characterized by either an extremely short C-int domain or an extremely short N-int domain can be used to conjugate any payload to an immunoligand (or binding protein), including small molecular entities, because short amino acid stretches can be synthesize chemically and can easily be attached to small molecular entities by conventional chemical coupling. (FIG. 3A) This part of the illustration shows the use of the Ssp GyrB S11 split intein (described in Appleby et al. (2009)) for the C-terminal conjugation of a payload to an immunoligand/binding protein. Here the C-int domain is only 6 amino acids long and comprises the amino acid sequence GVFVHN, as indicated. However, as there need to be some peptides that are the equivalent of an C-extein domain, additional amino acids need to be added, of which the first one needs to be a serine or cysteine amino acid residue, whereas the remaining amino acids can be chosen. This is indicated by the SX.sub.n symbol, which means that a short amino acid stretch lead at the N-terminal side by serine and followed by n amino acids (n>2, preferably 5), which can be any of the 20 naturally occurring amino acids (therefore indicated as X). Thus, as described in the Example, a short 12 amino acid stretch comprising a 6 amino acid mini C-int domain and 6 amino acid C-ext amino acid stretch are sufficient to allow the N-int/C-int complex to catalyze the transpeptidation from the asparagine-serine peptide bond in the GVFVHN-SX.sub.n (X any amino acid, n>2, preferably 5) to the peptide bond between the N-extein and N-int transition. This will result in a C-terminally conjugated immunoligand/binding protein with the payload attached via the short C-extein amino acid stretch. (FIG. 3B) This part of the illustration shows the use of the Ssp DnaX split intein (described in Song et al. (2012)), which can be separate into a very short, 11 amino acid N-int domain and a 139 aa C-int domain for N-terminal conjugation of a payload to an immunoligand/binding protein. As indicated here, this only requires the synthesis and coupling of a short 11 amino acid N-int domain to any payload (or the addition by recombinant protein technology), which then allows the specific conjugation of the payload to the N-terminus of any immunoligand or protein, that has a 139 amino acid long Ssp DnaX C-int domain fused to the N-terminus. The result of this reaction is then a N-terminally conjugated immunoligand/binding protein. Therefore, like in the case of sortase transpeptidation, where the N- or C-terminal conjugation only depends on the arrangement of the LPXTG and Gn peptide motifs with regard to protein and payload, split inteins can also mediate site-specific N- and C-terminal conjugation of proteins with short peptide modified payloads, and by exploit short mini C-int, or mini N-int peptide domains, like those of Ssp GyrB and Ssp DnaX split inteins, respectively.

[0263] FIGS. 4A-4B: These figures illustrate the utility of adding additional affinity purification and/or detection tags in addition to a sortase tag in the conjugation of payloads to immunoligands. (FIG. 4A) this part of the Figure shows how an additionally added amino acids representing a 6His purification tag (HHHHHH), a Myc-detection tag (EQKLISEEDL) and a strepII affinity purification tag (WSHPQFEK), as described in the Examples are removed in the course of the C-terminal payload conjugation via Staphylococcus aureus sortase A transpeptidase. This allows to select for the conjugated product, if Ni-NTA affinity resins (for the 6His-tag) or streptactin affinity resins (for the strep II-tag) are employed to separate non-conjugated substrate from conjugated product. This combination of tags is only provided by way of Example.

[0264] (FIG. 4B) This Figure illustrates that the use of affinity purification tags is particularly useful to select/purify completely conjugated product in the case of multimeric proteins, like antibodies as illustrated here. As also provided in the examples, antibodies can be modified with specific conjugations sites at heavy and light chains, and if the modification is targeted to the C-termini of IgH and IgL chains, then up to four payloads may be conjugated to the antibody. The addition of (a) further affinity purification tag(s), e.g. as described in FIG. 4A allows to bind incompletely conjugated product, that may only have one, two, or three (as illustrated here) payloads conjugated to the antibody, still bind to the respective affinity purification resin, and can thus easily be separated from the fully payload-conjugated product. This paradigm is of course also applicable to intein-modified immunoligands, and not only to sortase-motif-modified immunoligands, as depicted here.

[0265] FIG. 5: This figure illustrates a variation of the sortase-mediated conjugation that can also be applied, in which the sortase-enzyme is not added as a separate recombinant protein to the sortase tagged immunoligand and glycine-stretch modified payload, but where the enzymatic sortase domain is expressed as a fusion protein C-terminal to the LPXTG sortase tag. The sortase enzyme domain will be inactive as long as it is not incubated with glycine-stretch modified payload (or substrate). As soon as glycine-stretch modified substrate (or here payload) is added to such a construct, the fused sortase domain will catalyze the transpeptidation of glycine-payload substrate to the LPXTG sortase tag, by cleaving the protein between the threonine-4 and glycine-5 position of the LPXTG tag, and thereby removing the sortase enzyme domain with additional affinity purification tags, that can be added optionally, as depicted here. This procedure has the advantage that, similar to the addition of catalytically active split-intein domains, the sortase enzyme domain can be expressed by recombinant protein technology as an integral component of the immunoligand to be conjugated.

[0266] FIGS. 6A-6B: (FIG. 6A) This figure illustrates the use of different transpeptidases (here sortase and split-intein), in order to simultaneously conjugate different payloads to different subunits of a multimeric protein, like e.g., as depicted here, the heavy and the light chains of an antibody. In this selected example, the C-termini of the heavy chains are modified with the N-int domain of Ssp GyrB (as provided in Example 2), while the light chains are modified with the sortase A penta-peptide motif LPXTG (as provided in Example 1, the additional tags are omitted for simplicity). Incubation with a glycine-stretch modified payload A and with a C-int-domain modified payload B and sortase enzyme will allow the simultaneous and selective conjugation of payload B to the heavy chains and payload A to the light chains. If payloads A and B are toxins addressing different cellular pathways, this strategy could generate more potent anti-cancer drugs, as conventional ADCs, only containing a single toxin moiety. (FIG. 6B) This figure illustrates the use of different sortase enzymes (here sortase A and sortase B from Staphylococcus aureus), in order to simultaneously conjugate different payloads to different subunits of a multimeric protein, like e.g., as depicted here, the heavy and the light chains of an antibody. In this selected example, the C-termini of the heavy chains are modified with the pentapeptide recognition motif for sortase B, NPQTN, while the light chains are modified with the sortase A penta-peptide motif LPXTG. Sequential conjugation of glycine-stretch modified payloads A and B with sortase A and sortase B will allow the simultaneous and selective conjugation of payload B to the heavy chains and payload A to the light chains (remaining peptide sequences from LPXTG and NPQTN are omitted in the conjugated structure for simplicity). If payloads A and B are toxins addressing different cellular pathways, this strategy could generate more potent anti-cancer drugs, as conventional ADCs, only containing a single toxin moiety.

[0267] FIGS. 7A-7B: SDS-PAGE (FIG. 7A) and Western-blot (FIG. 7B) analysis of recombinant enzymatically active sortase A fragment of Staphylococcus aureus. (FIG. 7A) Lane 1 in the SDS-PAGE contains BSA (ca. 66.4 kD), Lane M.sub.1 contains protein molecular weight standard of Genscript (Cat.-Nr.: MO0505), Lane 2 contains His-tag purified recombinant sortase A fragment of Staphylococcus aureus. The proteins in the SDS-PAGE are stained with Commassie blue. (FIG. 7B) The Western-blot was developed with an anti-His antibody (Genscript Cat.-Nr.: AO0186). Lane 3 contains His-tag purified recombinant sortase A fragment of Staphylococcus aureus. Lane M.sub.2 contains molecular weight standard of Genscript (Cat.-Nr.: MM0908).

[0268] FIGS. 8A-8B: Hydrophobic Interaction Chromatography (HIC) analysis of DM1-toxin conjugated Tras-HC-GS-LHS (FIG. 8A) and Tras-LC-GS-LHS (FIG. 8B). DAR1 indicates drug to antibody ratio of 1; DAR2 indicates a drug to antibody ratio of 2.

[0269] FIGS. 9A-9B: Dose response of cytotoxic effects of the indicated ADCs on HER2-overexpressing SKBR3 (FIG. 9A) and HER2-negative T47D-5R cells (FIG. 9B). Cells were incubated with serial dilutions of ADCs for 3 days, after which cell viability was detected by CellTiter-Glo Luminescent Solution (Promega). LC: DM1-sortaseA-conjugated Tras-LC-GS-LHS; HC: DM1-sortaseA-conjugated Tras-HC-GS-LHS.

[0270] FIGS. 10A-10B: Sortase A-mediated conjugation of Gly.sub.5-FITC to mAb Ac10 variants with or without GS peptide spacer. Serial dilutions of Sortase A were used to conjugate Gly.sub.5-FITC to mAb Ac10-HC-GS-LHS/LC-GS-LHS (FIG. 10A) and mAb Ac10-HC-LS/LC-LS (FIG. 10B) under otherwise identical conditions. Reaction products were separated by size on denaturing, reducing SDS-PAGE gels. FITC was visualized by placing the gels on a UV box. Sortase A concentrations used were: lanes 1, 9: 50 M; lanes 2, 10: 25 M; lanes 3, 11: 12.5 M; lanes 4, 12: 6.25 M; lanes 5, 13: 3.13 M; lanes 6, 14: 1.56 M; lanes 7, 15: 0.78 M; lanes 8, 16: 0.39 M.

[0271] FIGS. 11A-11B: Influence of peptide spacer length on light chain conjugation efficiency. Serial dilutions of Sortase A were used to conjugate Gly.sub.5-FITC to mAbs Ac10-HC-LS/LC-GS-LS (FIG. 11A, left), Ac10-HC-LS/LC-GGS-LS (FIG. 11A, right), Ac10-HC-LS/LC-GGGS-LS (FIG. 11B, left) and Ac10-HC-LS/LC-GGGGS-LS (FIG. 11B, right) under otherwise identical conditions. Reaction products were separated by size on denaturing, reducing SDS-PAGE gels. FITC was visualized by placing the gels on a UV box. Sortase A concentrations used were: lanes 1, 8, 15, 22: 25 M; lanes 2, 9, 16, 23: 12.5 M; lanes 3, 10, 17, 24: 6.25 M; lanes 4, 11, 18, 25: 3.13 M; lanes 5, 12, 19, 26: 1.56 M; lanes 6, 13, 20, 27: 0.78 M; lanes 7, 14, 21, 28: 0.39 M

[0272] FIGS. 12A-12B: Analysis of sortaseA vc-PAB-MMAE toxin heavy-chain-conjugated ADC of mAb Ac10 by hydrophobicity interaction chromatography (HIC), which is able to differentiate unreacted substrate (DAR0=0 drug to antibody ratio), substrate in which one of the two heavy chains has been conjugated (DAR1=1 drug to antibody ratio), and substrate in which both modified heavy chains have been conjugated (DAR2=2 drugs to antibody ratio), as indicated. Panel A shows the HIC profile after a standard sortase A mediated conjugation of HC modified Ac10 mAb, in which still DAR0 and DAR1 species are detectable, next to the desired DAR2 product. Panel B shows the HIC profile after 4 passes of the ADC preparation analyzed in Panel A over a StrepTactin affinity purification column.

[0273] FIGS. 13A-13B: Analysis of synthesized Gly.sub.5-modified alpha-amanitin toxin (FIG. 13A) and Gly.sub.5-modified maytansin toxin (FIG. 13B). In each of the panels FIG. 13A and FIG. 13B the synthesized structure is provided on top, with the five glycines highlighted by a box. The analysis of each compound by mass spectrometry and reverse-phase HPLC is provided below. a.) The expected mass of the Gly.sub.5-modified alpha-amanitin toxin is 1302.07 D, the observed mass is 1325.38 D, corresponding to Ms+Na.sup.+. The RP-HPLC profile indicates a purity of >95%. b.) The expected mass of the Gly.sub.5-modified maytansine toxin is 991.41 D, the observed mass is 957.69 D, corresponding to Ms+Na.sup.+. The RP-HPLC profile indicates a purity of >95%.

[0274] FIGS. 14A-14C: Structures of 5Glycine (Gly5) modified toxins that either have been synthesized by Concortis, San Diego, Calif., U.S. (structures 1-6, and 9), or that can be synthesized (structures 7 & 8), demonstrating that any toxin can be functionalized for sortase mediated enzymatic conjugation, if either 5 glycines are attached to the toxins (as shown here), or any number of glycine residues greater or equal than one glycine. Glycine-modified toxins can either be synthesized containing additional validated linker/spacer structures as provided in structures 1-3 in FIG. 14A), potentially adding certain additional functionality (e.g. cleavability in certain subcellular compartments) or without additional linkers, as depicted in structures 4-6 in FIG. 14B). If several reactive groups are available at a given toxin, like e.g. in the case of alpha-amanitin toxin, glycine residues can be added to these different groups as exemplified in structures 7-9 in FIG. 14C).

[0275] FIG. 15: Tumor volumes as determined in Example 12. The results demonstrate that sortase conjugated ADCs, using identical antibody and toxin moiety, have comparable tumor killing activity in comparison to commercially available chemically conjugated Kadcyla.

[0276] FIGS. 16A-16B: Gels stained with Coomassie blue as described in example 13 The data shows the unexpected finding that Sortase A-mediated conjugation of antibodies in crude cell culture supernatant was as efficient as that of purified antibody.

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