Site-specific antibody conjugation and its use

Abstract

The present invention relates to a modified antibody comprising a heavy chain and a light chain, wherein the heavy chain or/and the light chain comprises one or more first recognition site(s) for the transglutaminase from Kutzneria albida (KalbTG) or a functionally active variant thereof. The one or more first recognition site(s) are introduced at one or more selected position(s) within the antibody's heavy chain and/or light chain. The invention further relates to one or more nucleic acids encoding the modified antibody according to the invention as well as a covalent conjugate comprising (i) the modified antibody according to the invention and (ii) one or more non-antibody moieties (payload(s)) covalently conjugated to the one or more first recognition site(s) either directly or via a first linker. In certain embodiments, the non-antibody moiety comprises a therapeutic entity and optionally a second linker. The present invention further relates to a method of covalently conjugating a modified antibody according to the invention to non-antibody moieties. In case the non-antibody moiety comprises a therapeutic entity, the present invention further relates to the conjugate of the modified antibody according to the invention and the therapeutic entity as pharmaceutical composition, for use as a medicament as well as for use in treating a disease.

Claims

1. A modified antibody comprising at least an antibody heavy chain, wherein the antibody heavy chain comprises one or two or three or four or more first recognition site(s) for the transglutaminase from Kutzneria albida (KalbTG) at or inserted after one or more of the positions selected independently of each other from the group of positions comprising position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341) and position 401 (HC401) of the antibody heavy chain (numbering according to Kabat).

2. A modified antibody comprising at least an antibody light chain, wherein the antibody light chain comprises one or two or three or four or more first recognition site(s) for the transglutaminase from Kutzneria albida (KalbTG) at or inserted after one or more of the positions selected independently of each other from the group of position comprising position 110 (LC110), position 143 (LC143) and position 214 (LC214) of the antibody light chain (numbering according to Kabat).

3. A modified antibody comprising a heavy chain and a light chain, wherein the heavy chain or/and the light chain comprises one or two or three or four or more first recognition site(s) for the transglutaminase from Kutzneria albida (KalbTG) at or inserted after one or more of the positions selected independently of each other from the group of positions comprising position 110 (LC110), position 143 (LC143) and position 214 (LC214) of the antibody light chain and position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341) and position 401 (HC401) of the antibody heavy chain (numbering according to Kabat).

4-8. (canceled)

9. The modified antibody according to claim 3, wherein the modified antibody comprises in addition to the recognition site(s) for KalbTG the following mutations (numbering according to Kabat): a) L234A, L235A in both Fc-region polypeptides; b) P329G in both Fc-region polypeptides; c) T366W in one Fc-region polypeptide and T366S, L368A, Y407V in the other Fc-region polypeptide; d) S354C in one Fc-region polypeptide and Y349C in the other Fc-region polypeptide; e) a) and b); f) a) and b) and c); or g) a) and b) and c) and d).

10. A modified antibody Fc-region comprising at least one modified antibody heavy chain Fc-region polypeptide, wherein the modified antibody heavy chain Fc-region polypeptide comprises one or two or three or four or more first recognition site(s) for the transglutaminase from Kutzneria albida (KalbTG) at or inserted after one or more of the positions selected independently of each other from the group of positions comprising position 118 (HC118), position 177 (HC177), position 297 (HC297), position 341 (HC341) and position 401 (HC401) of the antibody heavy chain (numbering according to Kabat).

11. The modified antibody according to claim 3, wherein the first recognition sites(s) for KalbTG are independently of each other selected from the group of first recognition sites comprising the amino acid sequences RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15), YRYRQ (SEQ ID NO: 17) and RVRQR (SEQ ID NO: 18).

12-14. (canceled)

15. The modified antibody according to claim 3, wherein the modified antibody comprises an antibody heavy chain constant region with the amino acid sequence of SEQ ID NO: 01 TABLE-US-00031 A.sup.↓STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSS.sup.↓GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN.sup.↓STYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKG.sup.↓QPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD.sup.↓GSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.sup.↓↓, wherein after one or more of the positions identified by an “↓” a first recognition site for KalbTG is inserted, and wherein after the position identified by an “↓↓” a first recognition site for KalbTG is inserted only in case a further first recognition site for KalbTG is inserted at one of the position identified by an “↓”.

16. The modified antibody according to claim 3, wherein the modified antibody comprises an antibody heavy chain constant region with the amino acid sequence of SEQ ID NO: 02 TABLE-US-00032 A.sup.↓STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSS.sup.↓GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN.sup.↓STYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKG.sup.↓QPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD.sup.↓GSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.sup.↓↓, wherein after one or more of the positions identified by an “↓” a first recognition site for KalbTG is inserted, and wherein after the position identified by an “↓↓” a first recognition site for KalbTG is inserted only in case a further first recognition site for KalbTG is inserted at one of the position identified by an “↓”.

17. The modified antibody according to claim 3, wherein the modified antibody comprises an antibody heavy chain constant region with the amino acid sequence of SEQ ID NO: 03 TABLE-US-00033 A.sup.↓STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSS.sup.↓GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVQFNWYVDGVEVHNAKTKPREEQFN.sup.↓STFRVVSVLTVVHQDWLNGKE YKCKVSNKGLPAPIEKTISKTKG.sup.↓QPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD.sup.↓GSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSP G.sup.↓↓, wherein after one or more of the positions identified by an “↓” a first recognition site for KalbTG is inserted, and wherein after the position identified by an “↓↓” a first recognition site for KalbTG is inserted only in case a further first recognition site for KalbTG is inserted at one of the position identified by an “↓↓”.

18. The modified antibody according to claim 3, wherein the modified antibody comprises an antibody heavy chain constant region with the amino acid sequence of SEQ ID NO: 04 TABLE-US-00034 A.sup.↓STKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSS.sup.↓GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRV ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPK SCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVQFKWYVDGVEVHNAKTKPREEQYN.sup.↓STFRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKTKG.sup.↓QPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSD.sup.↓GSFFLYSKLTVDKS RWQQGNIFSCSVMHEALHNRFTQKSLSLSPG.sup.↓↓, wherein after one or more of the positions identified by an “↓” a first recognition site for KalbTG is inserted, and wherein after the position identified by an “↓↓” a first recognition site for KalbTG is inserted only in case a further first recognition site for KalbTG is inserted at one of the position identified by an “↓”.

19. The modified antibody according to claim 3, wherein the modified antibody comprises an antibody heavy chain constant region with the amino acid sequence of SEQ ID NO: 05 TABLE-US-00035 A.sup.↓STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSS.sup.↓GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV SPNMVPHAHHAQAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFN.sup.↓STYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKG.sup.↓QPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD.sup.↓GSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLG.sup.↓↓, wherein after one or more of the positions identified by an “↓” a first recognition site for KalbTG is inserted, and wherein after the position identified by an “↓↓” a first recognition site for KalbTG is inserted only in case a further first recognition site for KalbTG is inserted at one of the position identified by an “↓”.

20. The modified antibody according to claim 3, wherein the modified antibody comprises an antibody heavy chain constant region that has the amino acid sequence of SEQ ID NO: 8.

21. The modified antibody according to claim 3, wherein the modified antibody comprises an antibody heavy chain constant region that has the amino acid sequence of SEQ ID NO: 9.

22. The modified antibody according to claim 3, wherein the modified antibody comprises an antibody heavy chain constant region that has the amino acid sequence of SEQ ID NO: 34.

23. The modified antibody according claim 3, wherein the modified antibody comprises an antibody light chain constant region with the amino acid sequence of SEQ ID NO: 06 TABLE-US-00036 RTV.sup.↓AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE.sup.↓AKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC.sup.↓, wherein after one or more of the positions identified by an “↓” a first recognition site for KalbTG is inserted.

24. The modified antibody according to claim 3, wherein the modified antibody comprises an antibody light chain constant region with the amino acid sequence of SEQ ID NO: 07 TABLE-US-00037 QPK.sup.↓AAPSVTLFPPSSEELQANKATLVCLISDFYPGA.sup.↓VTVAWKADSSPVK AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTEC.sup.↓S wherein after one or more of the positions identified by an “↓” a first recognition site for KalbTG is inserted.

25. The modified antibody according to claim 3, wherein the modified antibody comprises an antibody light chain constant region that has the amino acid sequence set forth in SEQ ID NO: 10.

26-27. (canceled)

28. A covalent conjugate comprising (i) the modified antibody according to claim 3, and (ii) one or more non-antibody domain(s) covalently conjugated to the one or more first recognition site(s) for KalbTG of the modified antibody of (i), wherein the non-antibody domain comprises a second recognition site for KalbTG.

29-31. (canceled)

32. A method of covalently conjugating a modified antibody according to claim 3 to a therapeutic entity, wherein the method comprises a) providing the modified antibody according to claim 3, b) providing a non-antibody domain, wherein the non-antibody domain comprises (i) a therapeutic entity, (ii) a second recognition site for KalbTG, which is complementary to the first recognition site present in the modified antibody provided under (i), and (iii) optionally a second linker between the therapeutic entity and the second recognition site for KalbTG; and c) incubating the modified antibody of a) and the non-antibody domain of b) in the presence of KalbTG or a functionally active variant or fragment thereof and thereby forming an isopeptide bond between the first and the second recognition site for KalbTG, and thereby conjugating the modified antibody to the therapeutic entity.

33. (canceled)

34. The modified antibody according to claim 3, wherein the KalbTG comprises the amino acid sequence (in three letter code) TABLE-US-00038 (SEQ ID NO: 31) Met His Lys Trp Phe Leu Arg Ala Ala Val Val Ala Ala Val Gly Phe Gly Leu Pro Thr Leu Ile Ala Thr Thr Ala Gln Ala Ala Ala Val Ala Ala Pro Thr Pro Arg Ala Pro Leu Ala Pro Pro Leu Ala Glu Asp Arg Ser Tyr Arg Thr Trp Arg Val Glu Asp Tyr Val Glu Ala Trp Glu Arg Tyr His Gly Arg Glu Met Thr Glu Asp Glu Arg Glu Asn Leu Ala Arg Gly Cys Ile Gly Val Thr Val Val Asn Leu Asn Arg Glu Asp Leu Ser Asn Pro Pro Leu Asn Leu Ser Phe Gly Ser Leu Arg Thr Ala Glu Ala Val Gln Ala Ala Leu Asn Lys Ile Val Asp Thr His Pro Ser Pro Ala Gln Tyr Glu Ala Ala Val Ala Lys Asp Pro Ile Leu Lys Arg Leu Lys Asn Val Val Lys Ala Leu Pro Ser Trp Ile Asp Ser Ala Lys Leu Lys Ala Ser Ile Phe Ser Lys Arg Phe Tyr Ser Trp Gln Asn Pro Asp Trp Ser Glu Glu Arg Ala His Thr Thr Tyr Arg Pro Asp Arg Glu Thr Asp Gln Val Asp Met Ser Thr Tyr Arg Tyr Arg Ala Arg Pro Gly Tyr Val Asn Phe Asp Tyr Gly Trp Phe Asp Gln Asp Thr Asn Thr Trp Trp His Ala Asn His Glu Glu Pro Arg Met Val Val Tyr Gln Ser Thr Leu Arg His Tyr Ser Arg Pro Leu Gln Asp Phe Asp Glu Gln Val Phe Thr Val Ala Phe Ala Lys Lys Asp

35. A pharmaceutical composition comprising the covalent conjugate according to claim 28.

36-37. (canceled)

38. A nucleic acid or a composition of nucleic acids encoding the modified antibody according to claim 3.

39. A cell comprising the nucleic acid or the nucleic acid composition according to claim 38.

40. A method for producing the modified antibody according to claim 3 comprising the steps of cultivating a cell according to claim 39, recovering the modified antibody from the cell or/and the cultivation medium, and thereby producing the modified antibody according to claim 3.

41. The modified antibody Fc-region-according to claim 10, wherein the first recognition sites(s) for KalbTG are independently of each other selected from the group of first recognition sites comprising the amino acid sequences RYGQR (SEQ ID NO: 11), RWRQR (SEQ ID NO: 12), YRQRT (SEQ ID NO: 13), IRQRQ (SEQ ID NO: 14), FRYRQ (SEQ ID NO: 15), YRYRQ (SEQ ID NO: 17) and RWRQR (SEQ ID NO: 18).

Description

DESCRIPTION OF THE FIGURES

[0451] FIGS. 1A-1B FIGS. 1A-1B show the overlay of individual elution chromatograms of a hydrophobic interaction chromatography of modified antibodies based on mAb-2 (HER2) with 9 different Q-tag insertion sites, all conjugated to the same single stranded nucleic acid with 15 residues. FIG. 1A illustrates the range of retention times obtained with the different modified antibodies based on mAb-2 (HER2) with 9 different Q-tag insertion sites, wherein the shortest retention time is with the modified antibody having the Q-tag inserted at position HC297 and the longest retention time is with the modified antibody having the Q-tag inserted at position HC446. FIG. 1B shows the same overlay as FIG. 1A but the peaks are annotated with the different positions of the Q-tag insertion sites; additionally the wild-type, i.e. not modified, mAb2 is annotated.

[0452] FIG. 2 illustrates an exemplary reaction for a single step KalbTG-mediated conjugation of a mAb with nucleic acid payload.

EXAMPLES

Example 1

[0453] Recombinant Production of the Modified Antibody According to the Invention

[0454] Gene Synthesis

[0455] Desired gene segments were prepared by chemical synthesis and the synthesized gene fragments were cloned into a suitable vector for expression in HEK293 and Expi293 cells by Twist Bioscience (San Francisco, US).

[0456] Expression of the Modified Antibody in Mammalian Cells

[0457] Antibody production was performed by transient co-transfection of single expression cassette plasmids in HEK293 cells cultivated in F17 Medium (Invitrogen, Carlsbad, CA, USA) or Expi293 cells in Expi293 Expression medium (Thermo Scientific, Waltham, MA, USA), respectively. Transfection was carried out as specified in the manufacturer's instructions with a plasmid ratio of HC:LC expression plasmids=1:1 in case of symmetric standard IgG1 format, or with a plasmid ratio of HC1:HC2:LC expression plasmids=1:1:1 in case of asymmetric knob-into-hole formats. Cell culture supernatants were harvested seven days after transfection. Supernatants were stored at reduced temperature (e.g. −20° C.).

[0458] Quantification of Protein Titer

[0459] The protein titer of supernatant samples was determined by affinity chromatography using a POROS A 20 μm column, 2.1×30 mm (Life Technologies, Carlsbad, CA, USA) on a High Performance Liquid Chromatography system (Ultimate 3000 HPLC system, Thermo Scientific, Waltham, MA, USA). The supernatant was loaded onto the column equilibrated with 0.2 M Na.sub.2HPO.sub.4, pH 7.4, followed by elution with 0.1 M citric acid, 0.2 M NaCl, pH 2.5. Titers were quantified by measuring absorption at 280 nm, and subsequently calculating the protein concentration by comparing the elution peak area (under the curve) of the analyte with a reference standard curve.

[0460] Purification of Modified Antibody from Mammalian Culture Supernatant

[0461] Antibodies in the culture supernatant were captured by Protein A affinity chromatography using a Mab Select SuRe column (GE Healthcare, Chicago, IL, USA), equilibrated with PBS buffer, pH 7.4. Unbound protein was removed by washing with equilibration buffer. The modified antibody was eluted with 50 mM citrate, pH 3.0 and the pH of the eluate was immediately adjusted to pH 7.5 by addition of 2 M Tris, pH 9.0. Size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, IL, USA) in 20 mM Histidine, 140 mM NaCl, pH 6.0 was performed as second purification step. Purified modified antibodies were stored at −80° C.

[0462] Purification of Antibodies Using Mid-Scale Automation (Milan)

[0463] Antibodies were purified in one step using Protein A affinity chromatography as described above, using MabSelectSuRe-Sepharose (Cytiva, Marlborough, MA, USA) on a liquid handling system (Tecan, Männedorf, Switzerland), equipped with columns from Repligen (Waltham, MA, USA). Equilibration, sample loading, and washing steps were performed as described, and 25 mM citrate, pH 3.0 was used to elute the antibodies from the column. The eluted antibody fractions were neutralized with 1.5 M Tris, pH 7.5 and the concentration was determined by measuring the optical density (OD) at 280 nm.

[0464] Overview of Exemplary Antibodies

[0465] Antibody 110:

[0466] Description: [0467] anti-HER2 antibody based on IgG1 subclass with P329G/L234A/L235A mutation; Q-tag insertion into HC after amino acid residue 177 (HC177) (EU numbering); Q-tag and spacer sequence: GGGSYRYRQGGGS (SEQ ID NO: 25)

TABLE-US-00022 Heavy Chain Constant Region Sequence (SEQ ID NO. 27) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGGGSYRYRQGGGSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK Light Chain Constant Domain Sequence (SEQ ID NO. 29) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

[0468] Antibody 113:

[0469] Description: [0470] Anti-HER2 antibody based on IgG1 subclass with P329G/L234A/L235A mutation; Q-tag insertion into HC after amino acid residue 401 (HC401) (EU numbering); Q-tag and spacer sequence: GGGSYRYRQGGGS (SEQ ID NO: 25)

TABLE-US-00023 Heavy Chain Constant Region Sequence (SEQ ID NO. 28) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGGGSYRYRQGGGSGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK Light Chain Constant Domain Sequence (SEQ ID NO. 30) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

TABLE-US-00024 TABLE 1 Expression yield of different antibodies with Q-tags inserted at various positions. yield of at scale [mg] mAb-1 mAb-2 mAb-3 position of (CD163) (HER2) (LeY) Q-tag 24 mL 1 L 24 mL 1 L 24 mL 1 L wild-type, i.e. 4.7 196 4.3 179 0.4 17 without Q-tag (reference) HC118 7.5 313 2.5 104 1.0 42 HC177 7.3 304 2.6 108 1.5 63 HC297 7.7 321 2.9 121 1.6 67 HC341 6.4 267 3.4 142 1.7 71 HC401 4.5 188 2.7 113 1.0 42 HC446 4.3 179 2.8 117 1.3 54 LC110 1.7 70.8 3.0 125 0.4 17 LC143 0.7 29.2 1.1 46 0.4 17 LC214 3.3 138 2.5 104 0.5 21

[0471] The expression of the modified antibody is influenced by the position of the introduced Q-tag. Introduction after position LC110, LC214, HC118, HC177, HC297, HC341 and HC401 resulted in the highest yields.

Example 2

[0472] KalbTG Conjugation of the Modified Antibody According to the Invention to a Fluorescent Dye

[0473] Conjugation Using KalbTG

[0474] Purified antibodies containing a Q-tag were transferred into conjugation buffer (histidine buffer comprising NaCl, pH 8.5) via dialysis. For the KalbTG reaction, the antibody was mixed with K-tagged small molecule (fluorescent dye, 10× molar excess) and KalbTG was added (molar ratio mAb:KalbTG>100:1). The reaction mixture was incubated at 37° C. with shaking, and the reaction was subsequently quenched by adding 10 mM ammonium sulfate to the solution. To remove unconjugated payload and residual enzyme, the conjugated modified antibody was purified by size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, IL, USA) in PBS pH 7.5. The purified conjugates were stored at −80° C.

[0475] Analysis of Conjugates

[0476] Protein quantification was performed with a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). In addition, qualitative DAR measurements were carried out by hydrophobic interaction chromatography as described in Example 4 below. The purity of the conjugates was analyzed by CE-SDS under denaturing and reducing conditions using a Caliper LabChip® GXII Touch™ protein characterization system according to the manufacturer's instructions (Perkin Elmer, Waltham, MA, USA).

[0477] Aggregate content was determined by SEC using a TSKgel UP-SW3000 analytical size-exclusion column (Tosoh Bioscience, Griesheim, Germany), equilibrated with 0.2 M K2HPO.sub.4/KH.sub.2PO.sub.4, 0.25 M KCl, pH 6.2, on a High Performance Liquid Chromatography system (Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, MA, USA).

[0478] The identity of the conjugates was confirmed by ESI-Q-ToF-MS (Bruker maXis 433, Bruker, Billerica, MA, USA). For MS analysis, the samples were deglycosylated using N-glycosidase F (Roche, Basel, Switzerland) and subsequently desalted into 2% formic acid, 40% acetonitrile.

TABLE-US-00025 TABLE 2 Conjugation efficiency of a fluorescent dye to antibody mAb-5 with Q-tags at various positions conjugation efficiency [DAR] mAb-5 (mTfR) position of Q-tag DAR (HIC) HC118 1.8 HC177 1.8 HC297 1.7 HC341 1.8 HC401 2.0 LC110 1.4 LC143 1.3 LC214 1.5

Example 3

[0479] KalbTG Conjugation of the Modified Antibody According to the Invention to a Nucleic Acid

[0480] Synthesis of Antisense Oligonucleotides

[0481] Single-stranded LNA oligonucleotides were synthesized using standard phosphoramidite chemistry. DNA and LNA phosphoramidites and all standard reagents were purchased from Merck KGaA (Darmstadt, Germany). K-tag peptides were custom synthesized by Schafer-N Aps (Copenhagen, Denmark) and Biosyntan (Berlin, Germany).

[0482] Oligonucleotides were synthesized on NittoPhase HL UnyLinker 350 support (Kinovate, Oceanside, CA) on an ÄKTA Oligopilot (GE Healthcare, Brondby, Denmark) at 130 mmol scale. After synthesis, the oligonucleotides were cleaved from the support overnight. The oligonucleotides were purified by ion exchange chromatography and desalted using a Millipore membrane. After lyophilization, the compounds were finally characterized by liquid chromatography-mass spectrometry (reverse phase and electrospray ionization-mass spectrometry).

[0483] The oligonucleotides were conjugated either to the respective linker for conjugating to the K-tag (1-step reaction) or to the acceptor part for Click chemistry conjugation (2-step reaction). The conjugates were then subjected directly to purification by reversed phase HPLC as described below.

[0484] After precipitation of the linker oligonucleotide by 2% lithium perchlorate in acetone, the resulting precipitate was washed with acetone, dried under vacuum and re-dissolved in PBS. 1.5 equivalents of K-tag peptide was dissolved in PBS and added. After 1 hour at room temperature, the reaction mixture was subjected directly to purification by reversed phase HPLC.

[0485] Both reactions above were purified by reversed phase HPLC on a Waters XBridge Peptide BEH C18 OBD Prep Column, 300 Å, 10 μm, 10 mm×150 mm using 0.1 M ammonium acetate and acetonitrile as eluent. Pooled fractions were lyophilized, re-dissolved in water and pH adjusted to pH 7.0 with aqueous NaOH. After a final lyophilization, the compounds were finally characterized by liquid chromatography-mass spectrometry (reverse phase and electrospray ionization-mass spectrometry).

[0486] Enzymatic Conjugation of LNA-ASOs to Antibodies with Q-Tags

[0487] 1-Step Conjugation Using KalbTG

[0488] Purified antibodies containing a Q-tag were transferred into conjugation buffer (histidine buffer comprising about 150 mM chloride ions, pH 7.5) via dialysis. For the KalbTG reaction, the antibody was mixed with excess of K-tagged oligonucleotide and KalbTG (Roche Diagnostics, Mannheim, Germany) was added. The reaction mixture was incubated at 37° C. with shaking, and the reaction was subsequently quenched by adding 10 mM ammonium sulfate to the solution. To remove unconjugated payload and residual enzyme, the conjugated modified antibody was purified by size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, IL, USA) in PBS, 250 mM arginine, pH 7.5. The purified conjugates were stored at −80° C.

[0489] 2-Step Conjugation Using KalbTG and Click Chemistry

[0490] Purified antibodies containing a Q-tag were transferred into conjugation buffer (histidine buffer comprising NaCl, pH 8.5) via dialysis. For the KalbTG reaction, the antibody was mixed with excess of K-tagged linker comprising the first part of Click conjugation (10× molar excess) and KalbTG was added. The reaction mixture was incubated at 37° C. with shaking, and the reaction was subsequently quenched by adding 10 mM ammonium sulfate to the solution. To remove unconjugated linker and residual enzyme, the conjugated modified antibody was purified by size exclusion chromatography using a Superdex 200™ column (GE Healthcare, Chicago, IL, USA) in PBS, 250 mM arginine, pH 7.5. The purified antibody-linker conjugate was added to excess of the oligonucleotide conjugated to the respective other part of Click conjugation in PBS, 250 mM arginine, pH 7.5 and the reaction mixture was incubated over night at room temperature with shaking. The antibody-oligonucleotide conjugate was purified by size exclusion chromatography as described above and purified conjugates were stored at −80° C.

[0491] Analysis of Conjugates

[0492] Quantification of oligonucleotide conjugates was performed by UV/Vis spectrometry at 260, 280, and 350 nm with the SoloVPE system (C Technologies, Bridgewater, NJ, USA). Conjugate concentrations and a quantitative Drug-to-Antibody Ratio (DAR) were calculated with the Lambert-Beer equation. In addition, qualitative DAR measurements were carried out by hydrophobic interaction chromatography as described in Example 4 below. The purity of the conjugates was analyzed by CE-SDS under denaturing and reducing conditions using a Caliper LabChip® GXII Touch™ protein characterization system (Perkin Elmer, Waltham, MA, USA). Aggregate content was determined by SEC using a TSKgel UP-SW3000 analytical size-exclusion column (Tosoh Bioscience, Griesheim, Germany), equilibrated with 0.2 M K.sub.2HPO.sub.4/KH.sub.2PO.sub.4, 0.25 M KCl, pH 6.2, on a High Performance Liquid Chromatography system (Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, MA, USA). The identity of the conjugates was confirmed by ESI-Q-ToF-MS (Bruker maXis 433, Bruker, Billerica, MA, USA). For MS analysis, the samples were deglycosylated using N-glycosidase F (Roche, Basel, Switzerland) and subsequently desalted into 2% formic acid, 40% acetonitrile.

TABLE-US-00026 TABLE 3 Conjugation efficiency of a nucleic acid of 15 or 20 nucleotides to different antibodies with Q-tags at various positions conjugation efficiency [DAR2] mAb-1 mAb-2 mAb-4 (CD163) (HER2) (TfR) position of Q-tag (HIC) (HIC) (UV-vis) (HIC) HC118 1.5 1.9 1.8 n.d. HC177 2.0 1.9 1.8 n.d. HC297 asymmetric n.d. n.d. n.d. 0.9 symmetric 2.0 1.9 1.7 1.9 HC341 2.0 1.8 1.8 n.d HC401 1.9 1.8 1.7 n.d. HC446 asymmetric n.d. n.d. n.d. 0.9 symmetric 2.0 1.9 1.8 1.8 LC110 2.0 1.8 1.7 n.d. LC143 1.9 1.9 1.7 n.d. LC214 asymmetric n.d. n.d. n.d. 1.0 symmetric 2.0 1.8 1.7 n.d. n.d. = note determined

[0493] The conjugation of the modified antibody is influenced by the position of the introduced Q-tag. Introduction after position LC110, LC143, LC214, HC118, HC177, HC297, and HC341 resulted in the best conjugation efficiencies.

TABLE-US-00027 TABLE 4 Expression yields and conjugation efficiencies for modified antibodies with different Q-tags position of Q-tag HC297 HC446 LC143 Q-tag conjugation conjugation conjugation sequence yield efficiency yield efficiency yield efficiency RYGQR  90 1.6/1.5 144 1.8/1.7 42 0.7/0.6 (SEQ ID NO: 11) RWRQR 136 1.7/1.6 114 1.8/1.7 12.2 1.1/0.8 (SEQ ID NO: 12) YRQRT 102 1.7/1.7 0 n.d. 28 1.8/1.7 (SEQ ID NO: 13) IRQRQ 132 3.1/3.1 124 2.8/2.8 44 2.6/2.2 (SEQ ID NO: 14) FRYRQ 136 1.6/1.7 62 1.5/1.3 22 1.7/1.7 (SEQ ID NO: 15) n.d. = not determined

Example 4

[0494] Hydrophobic Interaction Chromatography of KalbTG Conjugated Modified Antibody According to the Invention

[0495] Hydrophobic Interaction Chromatography (HIC) was performed on a High Performance Liquid Chromatography system (Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, MA, USA) using a TSKgel Butyl-NPR column (2.5 μm, 4.6×35 mm, TOSOH Bioscience, Tokyo, Japan) with a flow rate of 1 mL/min. The column was equilibrated with Eluent A (20 mM Na.sub.2HPO.sub.4 dihydrate, 1.5 M (NH.sub.4).sub.2SO.sub.4, pH 7.0) and 60 μg of each sample was loaded onto the column. Subsequently, a gradient between Eluent A and Eluent B (20 mM Na.sub.2HPO.sub.4 dihydrate, 25% (v/v) isopropanol, pH 7.0) was applied.

[0496] Gradient:

TABLE-US-00028 0 min 5% B 0-30 min 5% B->80% B 30-34 min 80% B->100% B 34-44 min 100% B  45-55 min 0% B

[0497] The elution profile was obtained by continuous measurement of the absorbance at 280 nm. Drug to antibody ratios (DAR) were determined by peak integration using Chromeleon 7.2 (Thermo Fisher Scientific, Waltham, MA, USA).

[0498] An exemplary result is shown in FIG. 1.

TABLE-US-00029 TABLE 5 Retention times of modified antibodies conjugated to a nucleic acid of 15 nucleotides (mAb-2) and 20 nucleotides (mAb-4). relative retention time mAb 2 (HER2) mAb 4 (TfR) position of not conjugated to not conjugated to Q-tag conjugated nucleic acid conjugated nucleic acid mAb-2 (wt) 0.246 n.d. 0.023 n.d. (reference) HC118 0.311 0.685 n.d n.d. HC177 0.221 0.647 n.d. n.d. HC297 asymmetric n.d. n.d. 0.023 0.472 symmetric 0.233 0.446 0.070 0.670 HC341 0.269 0.566 n.d. n.d. HC401 0.247 0.634 n.d. n.d. HC446 asymmetric n.d. n.d. 0.015 0.845 symmetric 0.215 0.745 0.001 0.994 LC110 0.402 0.732 n.d. n.d. LC143 0.291 0.691 n.d. n.d. LC214 0.246 0.501 0.020 0.556

[0499] The hydrophilic marker had a retention time of 9.25 min. and the hydrophobic marker had a relative retention time of 25.9 min. (mAb-2) or of 9.00 min. and 24.7 min. (mAb-4), respectively.

[0500] The hydrophobicity of the conjugate is influenced by the position of the introduced Q-tag. Introduction after position LC110, LC214 and HC297 resulted at most in a 2-fold change of non-conjugated to conjugated modified antibody, whereas HC177 had the smallest relative retention time of all tested internal insertion sites.

Example 5

[0501] In Vivo Analysis

[0502] Study Design

[0503] Human FcRn transgenic mice (hFcRn Tg32+/+ mice) were randomly allocated into 12 cohorts (n=3 per cohort) according to the 12 different compounds to be tested.

[0504] All compounds are based on a one-armed anti-transferrin receptor antibody comprising a cognate pair of a full-length antibody light chain and a full-length antibody heavy chain pair with an antibody Fc-region fragment (hinge—CH2-CH3; DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALGA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK; SEQ ID NO: 33).

[0505] The Q-tag had the amino acid sequence GGGSYRYRQGGGS (SEQ ID NO: 25) inserted after the respective position. [0506] Compound 1: reference without conjugated nucleic acid [0507] Compound 2: one nucleic acid conjugated to the Q-tag inserted after position HC118 of the full-length heavy chain Compound 3: one nucleic acid conjugated to the Q-tag inserted after position HC177 of the full-length heavy chain [0508] Compound 4: one nucleic acid conjugated to the Q-tag inserted after position HC295 of the full-length heavy chain [0509] Compound 5: one nucleic acid conjugated to the Q-tag inserted after position HC297 of the full-length heavy chain [0510] Compound 6: one nucleic acid conjugated to the Q-tag inserted after position HC341 of the full-length heavy chain [0511] Compound 7: one nucleic acid conjugated to the Q-tag inserted after position HC401 of the full-length heavy chain [0512] Compound 8: one nucleic acid conjugated to the Q-tag inserted after position HC446 of the full-length heavy chain [0513] Compound 9: one nucleic acid conjugated to the Q-tag inserted after position LC214 [0514] Compound 10: two nucleic acids conjugated to the Q-tag inserted after position HC297 of the full-length heavy chain and the Fc-region fragment [0515] Compound 11: two nucleic acids conjugated to the Q-tag inserted after position HC341 of the full-length heavy chain and the Fc-region fragment [0516] Compound 12: two nucleic acid conjugated to the Q-tag inserted after position HC446 of the full-length heavy chain and the Fc-region fragment

[0517] The compounds were formulated as a solution in 20 mM Histidine (pH 6) and administered intravenously as a single nominal dose of 20 mg/kg of body weight and a dosing volume of 2 mL/kg (exceptionally Compound 1 was administered in a dose volume of 3.3 mL/kg and a dose level of 16.7 mg/kg). The formulations were each injected by slow bolus in one of the two lateral tail veins. Serial microsampling of blood (20 μL/time point/mouse) was performed by tail vein puncture. Blood samples were collected at 5 min, 1 h, 7 h, 24 h, 48 h, 72 h, 96 h and 336 h post dose into K3-EDTA coated Minivette® POCT. Once the Minivette® was filled, blood was immediately transferred into a 0.2 ml PCR Eppendorf Tube® and centrifuged at 4° C., at approx. 10,000×g, for approximately 5 min. For terminal sampling blood was collected by heart puncture and transferred into K3-EDTA coated polypropylene tubes. Plasma samples were stored at −20° C. for further processing and analysis.

[0518] Bioanalysis

[0519] The pharmacokinetics (PK) of the compounds was determined by two separate methods: one method to quantify the antibody component and a second method to quantify the locked-nucleic acid component of the conjugate.

[0520] For determination of the PK in the plasma samples a generic electrochemiluminescense immunoassay (ECLIA) method specific for human CH2 domains on a Cobas® e411 (Roche Diagnostics GmbH, Mannheim, Germany) instrument under non-GLP conditions based on Stubenrauch et al. was used. Briefly, test samples, were added stepwise with a first detection antibody (biotinylated), second detection antibody (ruthenylated) and SA-beads to a detection vessel. The calibration range of the specific standard curves for each of the compounds was 0.69 ng/mL to 1,500 ng/mL assay concentration in 1% C57BL/6 mouse plasma. The analytical sensitivity was 69 ng/mL in 100% plasma. Standard curves, quality controls and sample dilutions were prepared in assay buffer including C57BL/6 mouse plasma resulting in 1% matrix concentration. Plasma samples were analyzed in two different dilutions (1:100 to 1:800).

[0521] The identical plasma samples were analyzed using a hybridization enzyme-linked immunosorbent assay (hELISA) under non-GLP conditions.

[0522] For the hELISA method, standards, quality controls and pre-diluted samples were prepared using the unconjugated nucleic acid as reference. Biotinylated capture oligonucleotide probe and digoxygenylated detection oligonucleotide probe were added for hybridization and transferred to a streptavidin-coated microtiter plate. For detection, polyclonal anti-digoxygenin-POD Fab fragments and TMB solution were used. The color intensity was analyzed photometrically at 450 nm (690 nm reference wavelength). The color intensity was proportional to the analyte concentration in the test sample. The calibration range of the standard curve for 0.8 pM to 111 pM assay concentration in 1% C57BL/6 mouse plasma. For analysis of plasma samples, standard curve, quality controls, all dilutions were prepared in assay buffer including C57BL/6 mouse plasma resulting in 1% matrix concentration. Plasma samples were analyzed in two different dilutions (1:100 to 1:400,000). The analytical sensitivity was 90 pM in 100% plasma.

[0523] Results

TABLE-US-00030 TABLE 6 Determined clearance rates for antibody-nucleic acid conjugates. Clearance Clearance Conjugation antibody part ASO part Site (ml/day/kg) [CV] (ml/day/kg) [CV] Compound 1 23 [4.7%] — Compound 2 83.2 [11.9%] 241 [7.8%] Compound 3 20.6 [17.4%] 92.1 [14.4%] Compound 4 10.8 [25.3%] 117 [15.4%] Compound 5 14.4 [15.5%] 85.7 [9.7%] Compound 6 33.9 [3.9%] 231 [7.9%] Compound 7 29.4 [8.2%] 166 [21.8%] Compound 8 46.2 [13.4%] 279 [4.6%] Compound 9 23.9 [4%] 105 [15.4%] Compound 10 28.6 [14.5%] 145 [15.2%] Compound 11 214 [20.3%] 219 [4.8%] Compound 12 152 [29.7%] 190 [14.7%]

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