WO2025210264A1

WO2025210264A1 - Antibody-drug-conjugates binding napi2b

Info

Publication number: WO2025210264A1
Application number: PCT/EP2025/059355
Authority: WO
Inventors: Stephan DICKGIESSER
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2024-04-04
Filing date: 2025-04-04
Publication date: 2025-10-09
Anticipated expiration: 2026-10-04

Abstract

The present invention relates to ADCs binding to NaPi2b comprising exatecan and a beta-glucuronidase cleavable linker, the ADCs being prepared by transglutaminase conjugation.

Description

Antibody-Drug-Conjugates binding NaPi2b

TECHNICAL FIELD

The present invention relates to NaPi2b binding antibody-drug conjugates.

BACKGROUND

Antibody-drug conjugates or ADCs are a class of biopharmaceutical drugs specifically designed for targeted therapies, particularly in the treatment of cancer (Hamilton GS (September 2015). "Antibody-drug conjugates for cancer therapy: The technological and regulatory challenges of developing drug-biologic hybrids". Biologicals. 43 (5): 318-32).

Structurally, ADCs comprise three covalently linked main components: (i) an antibody component, (ii) a linker, and (iii) a medical drug, often referred to as the "payload".

Upon administration to a patient, the antibody component directs the ADC to the target cells by specifically binding to its corresponding target antigen. Typically, the ADC is then internalized into the cell, e.g. by receptor-mediated endocytosis. Subsequently, the medical drug is released, for example by protease- or pH-dependent linker cleavage or by antibody degradation. The released medical drug then exerts its therapeutic effects within the cell.

In contrast to non-targeted drugs, which reach their site of action through whole-body distribution and passive diffusion, ADCs exhibit a more concentrated distribution at their intended site due to the specific interaction between the antibody component and its target antigen. This targeted delivery allows ADCs to achieve therapeutically effective levels within target cells while requiring smaller dosages, ultimately improving the therapeutic window. The formation of ADCs thus enhances drug specificity and reduces systemic toxicity, enabling the therapeutic use of drugs that may be less suitable or even unsuitable for systemic administration.

Given their specificity, efficacy, and favorable therapeutic window, ADCs are well-suited to address treatment scenarios where conventional therapies may fall short. Unlike traditional chemotherapy, ADCs are designed to selectively target cancer cells while minimizing exposure to healthy cells. This targeted approach is particularly beneficial for treating various solid tumor indications.

Despite the advancements represented by ADCs, there remains a significant need for new therapies to effectively treat many different types of cancer. Thus, there is an ongoing demand for the development of new and improved antibody-drug conjugates. In particular, there is a need for novel antibody-drug conjugates with improved efficacy, specificity, internalization, bystander effect, half-life, pharmacokinetic characteristics, pharmacodynamic properties and/or safety, and for antibody-drug conjugates that can be used broadly and/or for the treatment of further medical indications. Moreover, there is a need for novel antibody-drug conjugates that provide a particularly favourable balance between several or all of the above-mentioned aspects. The present disclosure addresses these needs as described below.

SUMMARY OF THE INVENTION

In the context of the present invention at least three different ADCs have been made and tested.

NaPi2b ADC1 , NaPi2b ADC2 and NaPi2b ADC3 (also referred to herein as ADC1 , ADC2 and ADC3, respectively) have the following amino acid sequences, wherein the light chain sequences are shared between the three ADCs:

The light chain amino acid sequence of NaPi2b ADC1 , NaPi2b ADC2 and NaPi2b ADC3 is:

DIQMTQSPSSLSASVGDRVTITCSASQDIGNFLNWYQQKPGKTVKVLIYYTSSLYSGVPSRF SGSGSGTDYTLTISSLQPEDFATYYCQQYSKLPLTFGQGTKLELKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGECGGTLQSPP (SEQ ID NO: 1)

The complementarity determining regions are exactly as described in US8603474B2. A transglutaminase recognition tag (microbial transglutaminase tag, mTG tag) GGTLQSPP (SEQ ID NO: 5), shown in bold in SEQ ID NO: 1 above, has been added at the C-terminus of the light chain for transglutaminase conjugation. The used bacterial transglutaminase, its expression and purification as well as useful conjugation methods have extensively been described in WQ2023/170239 and WQ2023/170240.

Heavy chain sequence of NaPi2b ADC1 is:

QVQLVQSGAEWKPGASVKMSCKASGYTFTGYNIHWVKQAPGQGLEWIGAIYPGNGDTSY KQKFRGRATLTADTSTSTVYMELSSLRSEDSAVYYCARGETARATFAYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2) The complementarity determining regions are exactly as described in US8603474B2. L234A and L235A (LALA mutation, shown in bold in SEQ ID NO: 2 above) have been introduced to reduce immune effector functions. Such LALA mutation is well known in the art, and is described in Lund et al. ,1992 and in Tamm and Schmidt, 1997 [Lund, J., Pound, J.D., Jones, P.T., Duncan, A.R., Bentley, T., Goodall, M., Levine, B.A., Jefferis, R. and Winter, G. (1992) Mol. Immunol., 29,53-59.; Tamm, A. and Schmidt, R.E. (1997) Int. Rev. Immunol., 16,57-85], Q295 is shown in bold and underlined in SEQ ID NO: 2 above.

The parental, unconjugated antibody of ADC1 (corresponding to the antibody part of ADC1 defined by SEQ ID NO: 1 (light chain) and SEQ ID NO: 2 (heavy chain)) is referred to herein as MAB1.

The heavy chain amino acid sequence of NaPi2b ADC2 is:

QVQLVQSGAEWKPGASVKMSCKASGYTFTGYNIHWVKQAPGQGLEWIGAIYPGNGDTSY KQKFRGRATLTADTSTSTVYMELSSLRSEDSAVYYCARGETARATFAYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDRTHTCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3)

The complementarity determining regions are exactly as described in US8603474B2. L234A and L235A (LALA mutation, shown in bold in SEQ ID NO: 3 above) have been introduced to reduce immune effector functions. Further a K222R mutation (shown in underlined italic style in SEQ ID NO: 3 above) was introduced to optimize transglutaminase conjugation as described in WQ2015/162563. Q295 is shown in bold and underlined in SEQ ID NO: 3 above.

The parental, unconjugated antibody of ADC2 (corresponding to the antibody part of ADC2 defined by SEQ ID NO: 1 (light chain) and SEQ ID NO: 3 (heavy chain)) is referred to herein as MAB2.

The heavy chain amino acid sequence of NaPi2b ADC3 is:

QVQLVQSGAEWKPGASVKMSCKASGYTFTGYNIHWVKQAPGQGLEWIGAIYPGNGDTSY KQKFRGRATLTADTSTSTVYMELSSLRSEDSAVYYCARGETARATFAYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDRTHTCPPCPAPEAAGGPSVF LFPPKPKDTLYIIREPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4)

The complementarity determining regions are exactly as described in US8603474B2. L234A and L235A (LALA mutation, shown in bold in SEQ ID NO: 4 above) have been introduced to reduce immune effector functions. Further a K222R mutation (shown in underlined italic style in SEQ ID NO: 4 above) was introduced to optimize transglutaminase conjugation. In addition, M252Y and S254T and T256E mutations (YTE mutation, shown in bold and double-underlined in SEQ ID NO: 4 above) has been introduced for improved pharmacokinetic properties. Such YTE mutation is described in Acqua, W. F. D., Woods, R. M., Ward, E. S., Palaszynski, S. R., Patel, N. K., Brewah, Y. A., Langermann, S. (2002). The Journal of Immunology, 169(9), 5171— 5180. Q295 is shown in bold and underlined in SEQ ID NO: 4 above.

The parental, unconjugated antibody of ADC3 (corresponding to the antibody part of ADC3 defined by SEQ ID NO: 1 (light chain) and SEQ ID NO: 4 (heavy chain)) is referred to herein as MAB3.

As the skilled person is aware, the light chain and heavy chain of an antibody are generated (by protein expression) from nucleotide sequences encoding the respective amino acid sequences.

The drug-linker structure used in the present invention, particularly in ADC1 , ADC2 and ADC3, is depicted below: The used payload is exatecan. A beta-glucuronidase cleavable linker was used. A triple glycine motif is present for transglutaminase conjugation (see WO2023/170239 and WO2023/170240 for reference).

An ADC of the present invention is depicted below:

The mAB binds to NaPi2b. DAR (drug-antibody ratio) is about 4, i.e. coupling to MTG (microbial transglutaminase) tags at the C-termini of the 2 light chains and to Q295 in the two heavy chains, respectively. On average, about 4 drug molecules are bound to one mAb.

In a preferred embodiment of the present invention the ADC is ADC3.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 : Immunofluorescence analysis to study internalization of NaPi2b monospecific antibody compared to isotype control.

Fig. 2: In vitro anti-tumor activity of an ADC according to the disclosure in OVCAR3 cells.

Fig. 3: In vivo anti-tumor activity in ovarian cancer PDX model OVPF206.

Fig. 4: In vivo anti-tumor activity in ovarian cancer PDX model OVPF070.

Fig. 5: In vivo anti-tumor activity in ovarian cancer PDX model OVPF252.

Fig. 6: In vivo anti-tumor activity in ovarian cancer PDX model OVPF174. Fig. 7: In vivo anti-tumor activity in ovarian cancer PDX model OVPF008.

Fig. 8: In vivo anti-tumor activity in ovarian cancer PDX model OVPF010.

Fig. 9: In vivo anti-tumor activity in ovarian cancer PDX model OVPF159.

Fig. 10: In vivo anti-tumor activity in ovarian cancer PDX model OVPF040.

Fig. 11 : In vivo anti-tumor activity in ovarian cancer PDX model OVPF151.

Fig. 12: In vivo anti-tumor activity in the non-small cell lung cancer (NSCLC) PDX model

CTG-860

Fig. 13: In vivo anti-tumor activity of different ADCs in ovarian cancer PDX model CTG-

0860 (from N.D. Bodyak et al., Mol Cancer Ther (2021) 20 (5): 896-905).

Fig. 14: In vitro potency of indicated ADC2, ADC3, respective isotype control and payload against antigen-positive OVCAR3, NCI-H1781 and HCC78 cell lines. One representative experiment is shown, mean of triplicates ±SD.

Fig. 15: In vitro potency of indicated NaPi2b-ADCs, respective isotype controls and payloads against antigen-positive OVCAR3 and NCI-H1781 cell lines. One representative experiment is shown, mean of triplicates ±SD.

Fig. 16: In vitro potency of indicated NaPi2b-ADCs, respective isotype controls and payloads against antigen-negative SW900, JEG3 and CAOV3 cell lines. One representative experiment is shown, mean of triplicates ±SD.

Fig. 17: In vitro potency of indicated NaPi2b ADCs and respective exatecan, MMAF

(Monomethyl auristatin F) and MMAE (Monomethyl auristatin E) payloads against antigenpositive OVCAR3 and NCI-H1781 cell lines. One representative experiment is shown, mean of triplicates ±SD.

Fig. 18: Potent bystander effect of ADC3 and TUB_NAPI2B_ADC on antigen-negative

SW900 cells in co-culture with antigen-positive OVCAR3 cells. No unspecific effects of ADC3 and TUB_NAPI2B_ADC on SW900 cells alone. Mean of three independent experiments is shown, ±SEM.

Fig. 19: Potent bystander effect of ADC3 on antigen-negative SW900 cells in co-culture with antigen-positive OVCAR3 cells. No unspecific effects of ADC3 on SW900 cells alone. Mean of three independent experiments is shown, ±SEM.

Fig. 20: Internalization of indicated ADCs or antibodies into acidic compartments of

OVCAR3 cells using flow cytometry and secondary labeling with a pH-dependent dye. Mean of three independent experiments is shown, ±SEM. Fig. 21 : In vitro potency of indicated NaPi2b, CDH6 and FRa ADCs, respective isotype controls and payloads against NaPi2b-, CDH6- and FRa-positive 0VCAR3 and FRa-positive JEG3, IGR0V1 and HCC827 cell lines. One representative experiment is shown, mean of triplicates ±SD.

Fig. 22: In vitro potency of indicated NaPi2b and FRa ADCs, respective isotype controls and payloads against JEG3 cell line. One representative experiment is shown, mean of triplicates ±SD.

Fig. 23: In vitro potency of indicated NaPi2b and FRa ADCs, respective isotype controls and payloads against IGR0V1 and HCC827 cell lines. One representative experiment is shown, mean of triplicates ±SD.

Fig. 24: Potent bystander effect of ADC3 on antigen-negative SW900 cells in co-culture with antigen-positive 0VCAR3 cells. No unspecific effects of ADC3 on SW900 cells alone. Mean of three independent experiments is shown, ±SEM.

Fig. 25: Target cancer cell binding of indicated antibodies using flow cytometry. Mean of

MFI PE (Mean Fluorescence Intensity Phycoerythrin) of 3 independent replicates +/-SEM is shown.

Fig. 26: Internalization of indicated NaPi2b ADC or NaPi2b, CDH6 and FRa antibodies into acidic compartments of 0VCAR3 cells using flow cytometry and secondary labeling with a pH-dependent dye. Mean of three independent experiments is shown, ±SEM.

Fig. 27: Anti-tumor effect of ADC3 as measured as tumor volume of OVPF174 PDX model growing on mice treated with three different dose of ADC3 were tested and at 3 different doses (1 mg/kg, 2 mg/kg, 6 mg/kg). All doses were administered intravenously (iv) at day 0, day 14 and day 28 (Q2Wx3).

Fig. 28: Anti-tumor effect of ADC3 as measured as tumor volume of GVPF070 PDX model growing on mice treated with three different dose of ADC3 were tested and at 3 different doses (4 mg/kg, 8 mg/kg, 16 mg/kg ). All doses were administered intravenously (iv) at day 0, day 14 and day 28 (Q2Wx3).

SUMMARY OF SEQUENCES

DETAILED DESCRIPTION OF THE INVENTION

Definitions As used herein "NaPi2b" designates sodium-dependent phosphate transport protein 2b (NaPi2b; also referred to as SLC34A2, NaPi3b, Npt2b; Xu et al., Genomics (1999) 62:281- 284) The protein is normally expressed at the brush border membrane of mammalian small intestine and participates in the transcellular inorganic phosphate (Pi) absorption, contributing to the maintenance of phosphate homeostasis in the body. It is overexpressed in various cancers, including ovarian cancer, non-small cell lung cancer, endometrial cancer and triplenegative breast cancer. NaPi2b has the amino acid sequence MAPWPELGDAQPNPDKYLEGAAGQQPTAPDKSKETNKTDNTEAPVTKIELLPSYSTATLIDE PTEVDDPWNLPTLQDSGIKWSERDTKGKILCFFQGIGRLILLLGFLYFFVCSLDILSSAFQLVG GKMAGQFFSNSSIMSNPLLGLVIGVLVTVLVQSSSTSTSIWSMVSSSLLTVRAAIPIIMGANI GTSITNTIVALMQVGDRSEFRRAFAGATVHDFFNWLSVLVLLPVEVATHYLEIITQLIVESFHF KNGEDAPDLLKVITKPFTKLIVQLDKKVISQIAMNDEKAKNKSLVKIWCKTFTNKTQINVTVPS TANCTSPSLCWTDGIQNWTMKNVTYKENIAKCQHIFVNFHLPDLAVGTILLILSLLVLCGCLIM IVKILGSVLKGQVATVIKKTINTDFPFPFAWLTGYLAILVGAGMTFIVQSSSVFTSALTPLIGIGV ITIERAYPLTLGSNIGTTTTAILAALASPGNALRSSLQIALCHFFFNISGILLWYPIPFTRLPIRMA KGLGN ISAKYRWFAVFYLI I FFFLI PLTVFGLSLAGWRVLVGVGVPVVFI 11 LVLCLRLLQSRCP RVLPKKLQNWNFLPLWMRSLKPWDAVVSKFTGCFQMRCCCCCRVCCRACCLLCDCPKCC RCSKCCEDLEEAQEGQDVPVKAPETFDNITISREAQGEVPASDSKTECTAL (SEQ ID NO: 22).

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, a primate or a human. In embodiments of the invention, the subject (or patient) is a human.

As used herein the term “about” in connection with a numerical value means that the value can vary between +/- 10% of said value.

As used here in unless otherwise specified the concentration of DMSO is given in % (v/v).

The positions of amino acids or mutations in immunoglobulin chains are herein indicated according to Ell numbering.

In an aspect, the present disclosure relates to antibody-drug conjugates (ADCs), also referred to herein as immunoconjugates or, more briefly, conjugates. As used herein, all these terms have the same meaning and are used interchangeably.

As pointed out above, ADCs comprise three covalently linked main components: an antibody, a linker, and a payload.

Antibodies

The antibody used in ADCs of the present disclosure is typically an lgG1 antibody.

Such an antibody includes two light chains and two heavy chains. Each of these chains includes antigen-binding region(s) (i.e. the complete VL and VH domains), as well as light and heavy chain constant domains, as appropriate for the antibody class, wherein the antibody domains remain associated through at least one non-covalent interaction.

As used herein, "antigen" refers to a substance that can specifically bind to the variable region of an antibody.

A "variable region" of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (Sequences of Proteins of Immunological Interest, 5th ed. (1991), editors Kabat et al., National Institutes of Health (Bethesda, USA)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., J. Molec. Biol. (1997), vol. 273, p. 927-948)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

Preferably, the antibody according to the present disclosure binds to NaPi2b.

As used herein, an antibody or immunoconjugate/ADC that "binds to NaPi2b" is an antibody resp. immunoconjugate/ADC that is capable of binding NaPi2b with sufficient affinity that the binding is useful in targeting the antibody resp. immunoconjugate/ADC to a cell expressing the target molecule NaPi2b at its cell surface (e.g. 0VCAR3 cells). In some embodiments, an antibody or immunoconjugate/ADC that "binds to NaPi2b" is an antibody or immunoconjugate/ADC that binds to NaPi2b expressing cells with an apparent affinity (EC50) of 1 x10-7 M or stronger. In some embodiments, an antibody or immunoconjugate/ADC that "binds to NaPi2b" is an antibody or immunoconjugate/ADC that binds to NaPi2b with a KD of 1 X 10’⁷ M or stronger. Preferably, said KD is determined by biolayer interferometry measurements. Preferably, said biolayer interferometry measurements are carried out by immobilizing said antibody or ADC on the biosensor and incubating the biosensor with a solution comprising Napi2b fragment (residues 250-361 , i.e. ESFHFKNGEDAPDLLKVITKPFTKLIVQLDKKVISQIAMNDEKAKNKSLVKIWCKTFTNKTQIN VTVPSTANCTSPSLCWTDGIQNWTMKNVTYKENIAKCQHIFVNFHLPD; SEQ ID NO: 23). Preferably, the measurements are carried out in a buffer of DPBS (Dulbecco's phosphate- buffered saline) comprising 0.1% BSA (bovine serum albumin) and 0.02% Tween-20 at 25 °C.

Preferably, the antibody comprises two light chains and two heavy chains.

Preferred antibodies of the invention that bind to NaPi2b include monoclonal antibodies that comprises an amino acid sequence selected from the group consisting of GGTLQSPP (SEQ ID NO: 5), TLQSG (SEQ ID NO: 6), TLQSPP (SEQ ID NO: 7), GGTLQSG (SEQ ID NO: 8) and TLQSA (SEQ ID NO: 9) and more preferably the amino acid sequence TLQSPP or GGTLQSPP (most preferably the amino acid sequence GGTLQSPP) in at least one and preferably both of its light chain constant regions (CL) and/or in at least one and preferably both of its heavy chain constant regions (CH); and/or comprises a glutamine at position 295 (EU numbering). These amino acid sequences have been found to be suitable to serve as substrate for the microbial transglutaminase (herein also “mTG” or “MTG”), even when incorporated into an antibody light chain constant regions (CL) and/or heavy chain constant regions (CH). These amino acid sequences are therefore also designated herein as “mTG tags”(microbial transglutaminase tags). When included in an antibody’s CL and/or CH chain (preferably at the C-terminus of said chain), then the primary amino group of a linker (preferably a drug linker; but the linker can also include other payloads besides drugs, such as a detectable label or a second antibody) or the primary amino group of a therapeutic agent can react with the glutamine of these aforementioned amino acid sequences of the antibody in the presence of the mTG enzyme:

Acyl donor Acyl accentor

Antibody * ^H2^N-R — T Anti body mTG

Glutamine Amine Isopeptide bond

In the above shown reaction scheme the acyl acceptor is said linker (preferably a drug-linker) or said therapeutic agent that comprises said primary amino group.

Antibodies that can be used in the present invention include an isolated antibody which binds to NaPi2b; and wherein the isolated antibody comprises

(i) at least one light chain constant region (CL) that comprises a sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP and TLQSA (preferably GGTLQSPP) and preferably comprising this sequence at the C-terminus of said light chain constant region; and/or

(ii) at least one heavy chain constant region (CH) comprising one or more of the following amino acid substitutions:

(a) L234A and L235A (LALA mutation);

(b) L234A and L235A and P329G (LALA-PG mutation);

(c) L235A and G237A (LAGA mutation);

(d) M252Y and S254T and T256E (YTE mutation);

(e) K222R; and wherein Eu numbering is used for said amino acid substitutions. Particularly preferred as antibodies according to the present disclosure are those with the following light chain and heavy chain sequences (as disclosed above):

MAB1 : Light chain of SEQ ID NO: 1 ; Heavy chain of SEQ ID NO: 2

MAB2: Light chain of SEQ ID NO: 1 ; Heavy chain of SEQ ID NO: 3

MAB3: Light chain of SEQ ID NO: 1 ; Heavy chain of SEQ ID NO: 4

The amino acid sequences for SEQ ID NO: 1-4 and certain motives, residues and mutations included in these sequences are disclosed above.

Specifically, SEQ ID NO: 1 comprises a transglutaminase recognition tag GGTLQSPP (SEQ ID NO: 5) that has been added at the C-terminus of the light chain for transglutaminase conjugation.

SEQ ID NO: 2 comprises a LALA mutation (L234A and L235A) to reduce immune effector functions. It also comprises the residue Q295 which allows for transglutaminase conjugation, as described below.

SEQ ID NO: 3 comprises a LALA mutation (L234A and L235A), and a K222R mutation to optimize transglutaminase conjugation as described in WO2015/162563. SEQ ID NO: 3 also comprises the residue Q295 for transglutaminase conjugation.

SEQ ID NO: 4 comprises a LALA mutation (L234A and L235A), a K222R mutation, and a YTE mutation (M252Y and S254T and T256E mutations) for improved pharmacokinetic properties. SEQ ID NO: 3 also comprises the residue Q295 for transglutaminase conjugation.

The positions of the above-mentioned residues and mutations are indicated in EU numbering, and their position within the different sequences is indicated in the amino acid listings of the SEQ ID NOs shown above.

As pointed out before, the complementarity determining regions (CDRs) of SEQ ID NO: 1 are exactly as described in US8603474B2. Thus, SEQ ID NO: 1 includes a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17) and a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18).

The complementarity determining regions (CDRs) of SEQ ID NO: 2, 3 and 4 are exactly as described in US8603474B2. Thus, these sequences include a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21). Methods of producing antibodies

Antibodies that can be used for conjugation with a linker or drug linker using the methods of the invention may be obtained or produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Standard techniques of antibody design and preparation are known to a skilled person (see e.g. Antibodies: A Laboratory Manual, 2nd edition (2014), editor Greenfield, Cold Spring Harbor Laboratory Press (U.S.); Antibody Engineering - Methods and Protocols, 2nd edition (2010), editors Nevoltris and Chames, publisher Springer (Germany); Handbook of Therapeutic Antibodies (2014), editors Dubel and Reichert, publisher Wiley-VCH Verlag GmbH & Co. KGaA (Germany); Harper, Methods in Molecular Biology (2013), vol. 1045, p. 41-49).

Knowing the amino acid sequence of a desired antibody, one skilled in the art can readily produce said antibodies or immunoglobulin chains using standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase methods using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, antibodies and immunoglobulin chains of the invention can be produced by recombinant DNA techniques, as is well-known in the art. For example, these polypeptides (e.g. antibodies) can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired polypeptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.

The invention further relates to a method of producing an antibody of the invention, which method comprises the steps consisting of: (i) culturing a transformed host cell; (ii) expressing the antibody; and (iii) recovering the expressed antibody.

Antibodies of the invention can be suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

In some embodiments, a humanized chimeric antibody of the present invention can be produced by obtaining nucleic acid sequences encoding humanized VL and VH regions as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. As the CH domain of a human chimeric antibody, any region which belongs to human immunoglobulin heavy chains may be used, for instance those of IgG class are suitable and any one of subclasses belonging to IgG class, such as lgG1 , lgG2, lgG3 and lgG4, can be used. Also, as the CL of a human chimeric antibody, any region which belongs to human immunoglobulin light chains may be used, and those of kappa class or lambda class can be used.

Methods for producing humanized or chimeric antibodies may involve conventional recombinant DNA and gene transfection techniques are well known in the art (see e.g. Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204, 244).

Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, e. g., Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art including, for example, the technique disclosed in the application W02009/032661 , CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 ; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991 ); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5, 565, 332). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).

A Fab of the present invention can be obtained by treating an antibody of the invention (e.g. an IgG) with a protease, such as papaine. Also, the Fab can be produced by inserting DNA sequences encoding both chains of the Fab of the antibody into a vector for prokaryotic expression, or for eukaryotic expression, and introducing the vector into prokaryotic or eukaryotic cells (as appropriate) to express the Fab.

A F(ab')2 of the present invention can be obtained treating an antibody of the invention (e.g. an IgG) with a protease, pepsin. Also, the F(ab')2 can be produced by binding a Fab' described below via a thioether bond or a disulfide bond.

A Fab' of the present invention can be obtained by treating F(ab')2 of the invention with a reducing agent, such as dithiothreitol. Also, the Fab' can be produced by inserting DNA sequences encoding Fab' chains of the antibody into a vector for prokaryotic expression, or a vector for eukaryotic expression, and introducing the vector into prokaryotic or eukaryotic cells (as appropriate) to perform its expression.

A scFv of the present invention can be produced by taking sequences of the CDRs or VH and VL domains as previously described for the antibody of the invention, then constructing a DNA encoding a scFv fragment, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector into prokaryotic or eukaryotic cells (as appropriate) to express the scFv. To generate a humanized scFv fragment, a well-known technology called CDR grafting may be used, which involves selecting the complementary determining regions (CDRs) according to the invention, and grafting them onto a human scFv fragment framework of known three dimensional structure (see, e. g., W098/45322; WO 87/02671 ; US5,859,205; US5,585,089; US4,816,567; EP0173494).

Modification of the antibodies of the invention

Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.

Modifications and changes may be made in the structure of the antibodies of the present invention, and in the DNA sequences encoding them, and still result in a functional antibody or polypeptide with desirable characteristics.

In making the changes in the amino sequences of polypeptide, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index for the interactive biologic function of a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

A further aspect of the present invention also encompasses function-conservative variants of the polypeptides of the present invention.

For example, certain amino acids may be substituted by other amino acids in a protein structure without appreciable loss of activity. Since the interactive capacity and nature of a protein define its biological functional activity, certain amino acid substitutions can be made in a protein sequence, and of course in its encoding DNA sequence, while nevertheless obtaining a protein with like properties. It is thus contemplated that various changes may be made in the antibody sequences of the invention, or corresponding DNA sequences which encode said polypeptides, without appreciable loss of their biological activity. It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. It is also possible to use well- established technologies, such as alanine-scanning approaches, to identify, in an antibody or polypeptide of the invention, all the amino acids that can be substituted without significant loss of binding to the antigen. Such residues can be qualified as neutral, since they are not involved in antigen binding or in maintaining the structure of the antibody. One or more of these neutral positions can be substituted by alanine or by another amino acid can without changing the main characteristics of the antibody or polypeptide of the invention.

Neutral positions can be seen as positions where any amino acid substitution could be incorporated. Indeed, in the principle of alanine-scanning, alanine is chosen since it this residue does not carry specific structural or chemical features. It is generally admitted that if an alanine can be substituted for a specific amino acid without changing the properties of a protein, many other, if not all amino acid substitutions are likely to be also neutral. In the opposite case where alanine is the wild-type amino acid, if a specific substitution can be shown as neutral, it is likely that other substitutions would also be neutral.

As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take any of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

In some embodiments, the antibody according to the present disclosure is an antibody that has a light chain amino acid sequence that is at least m % identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least m % identical to SEQ ID NO: 4, wherein m is 90%, preferably 95%, more preferably 98% and even more preferably 99%.

Preferably, the substitutions by which said light chain amino acid sequence and the sequence of SEQ ID NO: 1 differ are selected from: substitution between arginine and lysine; substitution between glutamate and aspartate; substitution between serine and threonine; substitution between glutamine and asparagine; substitution between valine, leucine and isoleucine.

Preferably, the substitutions by which said heavy chain amino acid sequence and the sequence of SEQ ID NO: 4 differ are selected from: substitution between arginine and lysine; substitution between glutamate and aspartate; substitution between serine and threonine; substitution between glutamine and asparagine; substitution between valine, leucine and isoleucine. Preferably, the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5),

Preferably, the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21).

Preferably, the heavy chain amino acid sequence comprises the residue Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, the heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering).

If the present disclosure states that a certain sequence A "is at least x % identical" to another sequence B, this is synonymous to the statement that sequence A "has x % identity" to sequence B. The statement reflects a relationship between the two polypeptide sequences A and B determined by comparing the sequences. In general, identity refers to an exact amino acid to amino acid correspondence of the two polypeptide sequences, respectively, over the length of the sequences being compared. For sequences where there is not an exact correspondence, a percentage to which the two sequences are identical may be determined. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so- called local alignment), that is more suitable for sequences of unequal length.

Methods for comparing the identity of two or more sequences are well known in the art. Thus, for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al., 1984), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity between two polypeptide sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (1981) and finds the best single region of similarity between two sequences. Other programs for determining identity sequences are also known in the art, for instance the BLAST family of programs (Altschul S F et al, 1990, Altschul S F et al, 1997, accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson WR, 1990). Preferably, % identity according to the present disclosure is determined according to the BLAST family of programs (Altschul S F et al, 1990, Altschul S F et al, 1997, accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov).

It may be also desirable to modify the antibody of the invention with respect to effector function, e.g. so as to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody, or e.g. to alter the binding to Fc receptors. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing inter-chain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and/or antibody-dependent cellular cytotoxicity (ADCC) (Caron PC. et al. 1992; and Shopes B. 1992). In some embodiments, an antibody of the invention may be an antibody with a modified amino acid sequence that results in reduced or eliminated binding to most Fey receptors, which can reduce uptake and toxicity in normal cells and tissues expressing such receptors, e.g. macrophages, liver sinusoidal cells etc.. An example for such an antibody is one including substitutions of two leucine (L) residues to alanine (A) at position 234 and 235 (i.e. LALA) ; this double substitution has been demonstrated to reduce Fc binding to FcyRs and consequently to decrease ADCC as well to reduce complement binding/activation. Another example for such an antibody is one including the substitution P329G in addition to the L LA double substitution (i.e. PG-LALA; see e.g. Schlothauer et al., Novel human lgG1 and lgG4 Fc-engineered antibodies with completely abolished immune effector functions, Protein Engineering, Design and Selection, Volume 29, Issue 10, October 2016, Pages 457-466). In some embodiments, an antibody of the invention may thus be an antibody having an amino acid sequence that (i) contains e.g. the LALA or the PG-LALA set of substitutions and (ii) is otherwise identical to the amino acid sequence of one of the antibodies of the invention described herein above with reference to the respective SEQ ID NOs.

Another type of amino acid modification of the antibody of the invention may be useful for altering the original glycosylation pattern of the antibody, i.e. by deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody. The presence of either of the tripeptide sequences asparagine-X-serine, and asparagine-X-threonine, where X is any amino acid except proline, creates a potential glycosylation site. Addition or deletion of glycosylation sites to the antibody can conveniently be accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).

Another type of modification involves the removal of sequences identified, either in silico or experimentally, as potentially resulting in degradation products or heterogeneity of antibody preparations. As examples, deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such a deamidation site, in particular Asn-Gly, is present in an antibody or polypeptide, it may therefore be considered to remove the site, typically by conservative substitution to remove one of the implicated residues. Such substitutions in a sequence to remove one or more of the implicated residues are also intended to be encompassed by the present invention.

Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. These procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N-or O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in W087/05330.

Removal of carbohydrate moieties present on the antibody may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N- acetylgalactosamine), while leaving the antibody intact. Chemical deglycosylation is described by Sojahr H. et al. (1987) and by Edge, AS. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura, NR. et al. (1987).

Another type of covalent modification of the antibody comprises linking the antibody to one of a variety of non-proteinaceous polymers, e.g. polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, e.g. in the manner set forth in US Patent Nos. 4,640,835; 4,496,689; 4,301 ,144; 4,670,417; 4,791 ,192 or 4,179,337.

Other amino acid sequence modifications known in the art may also be applied to an antibody of the invention. Immunoconjugates

The present invention provides an immunoconjugate comprising an antibody of the invention (such as MAB1 , MAB2 or MAB3) covalently linked via a linker to at least one growth inhibitory agent or to at least one other agent. Other agents include a detectable label or therapeutic agent. A therapeutic agent preferably is a cytotoxic drug.

The term "growth inhibitory agent" (also referred to as an "anti-proliferative agent") refers to a molecule or compound or composition which inhibits growth of a cell, such as a tumor cell, in vitro and/or in vivo.

In some embodiments, the growth inhibitory agent is a cytotoxic drug (also referred to as a cytotoxic agent).

The term "cytotoxic drug" as used herein refers to a substance that directly or indirectly inhibits or prevents the function of cells and/or causes destruction of the cells. The term "cytotoxic drug" includes e.g. chemotherapeutic agents, enzymes, antibiotics, toxins such as small molecule toxins or enzymatically active toxins, toxoids, vincas, taxanes, maytansinoids or maytansinoid analogs, tomaymycin or pyrrolobenzodiazepine derivatives, cryptophycin derivatives, leptomycin derivatives, auristatin or dolastatin analogs, prodrugs, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA alkylating agents, anti-tubulin agents, CC-1065 and CC-1065 analogs.

Topoisomerase I inhibitors are molecules or compounds that inhibit the human enzyme topoisomerase I which is involved in altering the topology of DNA by catalyzing the transient breaking and rejoining of a single strand of DNA. Topoisomerase I inhibitors are highly toxic to dividing cells e.g. of a mammal. Examples of suitable topoisomerase I inhibitors include camptothecin (CPT) and analogs thereof such as topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan and rubitecan.

In some embodiments, the immunoconjugates of the invention comprise the cytotoxic drug exatecan as the growth inhibitory agent. Exatecan has the chemical name (1 S,9S)-1-Amino- 9-ethyl-5-fluoro-1 ,2,3,9, 12, 15-hexahydro-9-hydroxy-4-methyl-10/7, 13/7- benzo(de)pyrano(3',4':6,7)indolizino(1 ,2-b)quinoline-10,13-dione. Exatecan is represented by the following structural formula:

In further embodiments of the invention, other CPT analogs and other cytotoxic drugs may be used, e.g. as listed above. Examples of some cytotoxic drugs and of methods of conjugation are further given in the application W02008/010101 which is incorporated by reference.

In an immunoconjugate of the present invention, an antibody of the present invention is covalently linked via a linker to the at least one growth inhibitory agent. "Linker", as used herein, means a chemical moiety comprising a covalent bond and/or any chain of atoms that covalently attaches the growth inhibitory agent to the antibody. Linkers are well known in the art and include e.g. disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Conjugation of an antibody of the invention with cytotoxic drugs or other growth inhibitory agents may be performed e.g. using a variety of bifunctional protein coupling agents including but not limited to N-succinimidyl pyridyldithiobutyrate (SPDB), butanoic acid 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1- pyrrolidinyl ester (nitro-SPDB), 4-(Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), N- succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyl (N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)- hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al (1987). Carbon labeled 1-isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to an antibody (WO 94/11026). In embodiments of the present invention, the linker may be a "cleavable linker", which may facilitate release of the cytotoxic drug or other growth inhibitory agent inside of or in the vicinity of a cell, e.g. a tumor cell. In some embodiments, the linker is a linker cleavable in an endosome of a mammalian cell. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase labile linker, a photolabile linker or a disulfide-containing linker (see e.g. U.S. Patent No. 5,208,020) may be used.

When referring to a structural formula representing an immunoconjugate, the following nomenclature is also used herein: a growth inhibitory agent and a linker, taken together, are also referred to as a [(linker)-(growth inhibitory agent)] moiety; for instance, an exatecan molecule and a linker, taken together, are also referred to as a [(I inker)— ( exatecan)] moiety.

In some specific embodiments of the present invention, the linker is a linker cleavable by the human enzyme glucuronidase. For example, an immunoconjugate prepared by the method of the present invention may thus have the following formula, which includes a linker cleavable by glucuronidase: wherein the antibody is an antibody of the invention (preferably a monoclonal antibody), and wherein n is a number of [(linker)-(growth inhibitory agent)] moieties covalently linked to the antibody. In embodiments using the formula above, n is preferably between 3 and 5 and most preferably between 3.5 and 4.5 (i.e. about 4).

The number n is also referred to as "drug-to-antibody ratio" (or "DAR"); this number n is always to be understood as an average number for any given (preparation of an) immunoconjugate.

In the above formula, the chemical structure between the antibody and the growth inhibitory agent is a linker. One of these linkers is also contained in the formula depicted further below.

In any one of the embodiments with linkers cleavable by glucuronidase, as described above, the growth inhibitory agent may be exatecan, for example.

Accordingly, in some embodiments, the present invention provides an immunoconjugate comprising an antibody according to the invention covalently linked via a linker to exatecan, wherein the conjugate has the following formula: wherein n is a number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody. The number n (also referred to as the DAR) may be e.g. between 1 and 10. In embodiments using the formula above, n is preferably between 3 and 5 and most preferably between 3.5 and 4.5 (i.e. about 4).

In some embodiments, the present invention provides an immunoconjugate comprising an antibody according to the invention covalently linked via a linker to exatecan, wherein the conjugate has the following formula: wherein n is a number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody. The number n (also referred to as the DAR) may be e.g. between 1 and 10. In embodiments using the formula above, n is preferably between 3 and 5 and more preferably between 3.5 and 4.5 and most preferably 4. In preparing any of the immunoconjugates described above using the method of the present invention, an antibody against any target may be used.

Accordingly, in some embodiments, the method of the present invention provides an immunoconjugate comprising an antibody according to the invention covalently linked via a linker to exatecan, wherein the conjugate has the following formula:

wherein n is a number of [(linker)-(exatecan)] moieties covalently linked to the antibody. The number n (also referred to as the DAR) may be e.g. between 1 and 10 preferably about 4.

Accordingly, as described above, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 1 and 10.

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence with the CDRs of SEQ ID NO: 16-18 and a heavy chain amino acid sequence with the CDRs of SEQ ID NO: 19-21 is an antibody that binds to NaPi2b. Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b, wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is between 1 and 10.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate

(ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5.

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence with the CDRs of SEQ ID NO: 16-18 and a heavy chain amino acid sequence with the CDRs of SEQ ID NO: 19-21 is an antibody that binds to NaPi2b.

(ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b, wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5. Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate

(ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b, wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4.

(ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is 4.

(ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b, wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is 4. Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate

(ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is between 1 and 10, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b, wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 1 and 10, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

(ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b, wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence with the CDRs of SEQ ID NO: 16-18 and a heavy chain amino acid sequence with the CDRs of SEQ ID NO: 19-21 is an antibody that binds to NaPi2b. As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b, wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b, wherein the antibody has a light chain amino acid sequence comprising a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein the antibody has a heavy chain amino acid sequence comprising a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), and wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 1 and 10.

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4 is an antibody that binds to NaPi2b. Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is between 1 and 10.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5.

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4 is an antibody that binds to NaPi2b. Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4.

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4 is an antibody that binds to NaPi2b. Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is 4.

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4 is an antibody that binds to NaPi2b. Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID

NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is 4. Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody. As the skilled person understands from the present disclosure, the light chain amino acid sequence of SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5).

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4 is an antibody that binds to NaPi2b.

As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein the light chain amino acid sequence of SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands from the present disclosure, the light chain amino acid sequence of SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5).

As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein the light chain amino acid sequence of SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands, Q295 is numbered according to Ell numbering. Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4, wherein the light chain amino acid sequence of SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 3, wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 3 is an antibody that binds to NaPi2b.

As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 3, wherein the light chain amino acid sequence of SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody. Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 3, wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula:

wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 3, wherein the light chain amino acid sequence of SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 3, wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody. As the skilled person understands from the present disclosure, the light chain amino acid sequence of SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5).

As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 3, wherein the light chain amino acid sequence of SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 2, wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 2 is an antibody that binds to NaPi2b.

As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 2, wherein the light chain amino acid sequence of SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 2, wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 2 is an antibody that binds to NaPi2b. As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 2, wherein the light chain amino acid sequence of SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 2, wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 2, wherein the light chain amino acid sequence of SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and wherein the antibody comprises a light chain comprising the variable region of the sequence shown in SEQ ID NO: 1 and and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5) and wherein the antibody comprises a heavy chain comprising the variable region of the sequence shown in SEQ ID NO: 4, wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering).

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and wherein the antibody comprises a light chain comprising the variable region of the sequence shown in SEQ ID NO: 1 and and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5) and wherein the antibody comprises a heavy chain comprising the variable region of the sequence shown in SEQ ID NO: 4, wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and wherein the antibody comprises a light chain comprising the variable region of the sequence shown in SEQ ID NO: 1 and and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5) and wherein the antibody comprises a heavy chain comprising the variable region of the sequence shown in SEQ ID NO: 4, wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and wherein the antibody comprises a light chain comprising the variable region of the sequence shown in SEQ ID NO: 1 and and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5) and wherein the antibody comprises a heavy chain comprising the variable region of the sequence shown in SEQ ID NO: 4, wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and wherein the antibody comprises a light chain comprising the variable region of the sequence shown in SEQ ID NO: 1 and and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5) and wherein the antibody comprises a heavy chain comprising the variable region of the sequence shown in SEQ ID NO: 4, wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (EU numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and wherein the antibody comprises a light chain comprising the variable region of the sequence shown in SEQ ID NO: 1 and and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5) and wherein the antibody comprises a heavy chain comprising the variable region of the sequence shown in SEQ ID NO: 4, wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least 90% identical to SEQ ID NO: 4, wherein the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), and wherein the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least 90% identical to SEQ ID NO: 4, wherein the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), and wherein the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least 90% identical to SEQ ID NO: 4, wherein the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), and wherein the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that is at least 95% identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least 95% identical to SEQ ID NO: 4, wherein the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), and wherein the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that is at least 95% identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least 95% identical to SEQ ID NO: 4, wherein the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), and wherein the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that is at least 95% identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least 95% identical to SEQ ID NO: 4, wherein the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), and wherein the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that is at least 98% identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least 98% identical to SEQ ID NO: 4, wherein the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), and wherein the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that is at least 98% identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least 98% identical to SEQ ID NO: 4, wherein the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), and wherein the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that is at least 98% identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least 98% identical to SEQ ID NO: 4, wherein the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), and wherein the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that is at least 99% identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least 99% identical to SEQ ID NO: 4, wherein the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), and wherein the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that is at least 99% identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least 99% identical to SEQ ID NO: 4, wherein the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), and wherein the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is about 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that is at least 99% identical to SEQ ID NO: 1 and a heavy chain amino acid sequence that is at least 99% identical to SEQ ID NO: 4, wherein the light chain amino acid sequence comprises a CDR1 with the amino acid sequence SASQDIGNFLN (SEQ ID NO: 16), a CDR2 with the amino acid sequence YTSSLYS (SEQ ID NO: 17), a CDR3 with the amino acid sequence QQYSKLPLT (SEQ ID NO: 18) and a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), and wherein the heavy chain amino acid sequence comprises a CDR1 with the amino acid sequence GYTFTGYNIH (SEQ ID NO: 19), a CDR2 with the amino acid sequence AIYPGNGDTSYKQKFR (SEQ ID NO: 20) and a CDR3 with the amino acid sequence GETARATFAY (SEQ ID NO: 21), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Preferably, said heavy chain amino acid sequence comprises the mutations L234A, L235A, K222R, M252Y, S254T and T256E (EU numbering). Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 1 and a heavy chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 4, wherein n is the number of [(I inker)— ( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, preferably about 4, more preferably 4.

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4 (and thus also an antibody that has a light chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 1 and a heavy chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 4), is an antibody that binds to NaPi2b.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 1 and a heavy chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 4, wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, preferably about 4, more preferably 4.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 1 and a heavy chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 4, wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, preferably about 4, more preferably 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands from the present disclosure, the light chain amino acid sequence of SEQ ID NO: 1 (and thus also a light chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 1), comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5).

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence of SEQ ID NO: 1 and a heavy chain amino acid sequence of SEQ ID NO: 4 (and thus also an antibody that has a light chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 1 and a heavy chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 4), is an antibody that binds to NaPi2b. As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 1 and a heavy chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 4, wherein the light chain amino acid sequence that has been generated from a nucleotide sequence encoding SEQ ID NO: 1 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein n is the number of [(I inker)-( exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, preferably about 4, more preferably 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

The present disclosure further provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody has a light chain amino acid sequence of SEQ ID NO: 24 and a heavy chain amino acid sequence of SEQ ID NO: 25, wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, preferably about 4, more preferably 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 in the two heavy chains of the antibody.

As the skilled person understands from the present disclosure, the light chain amino acid sequence of SEQ ID NO: 24 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5).

As the skilled person understands from the present disclosure, an antibody that has a light chain amino acid sequence of SEQ ID NO: 24 and a heavy chain amino acid sequence of SEQ ID NO: 25 is an antibody that binds to NaPi2b.

As the skilled person understands, Q295 is numbered according to Ell numbering.

Moreover, the present disclosure provides an immunoconjugate/antibody-drug conjugate (ADC), comprising an antibody covalently linked via a linker to exatecan, wherein the immunoconjugate/ADC has the following formula: wherein the antibody binds to NaPi2b and has a light chain amino acid sequence of SEQ ID NO: 24 and a heavy chain amino acid sequence of SEQ ID NO: 25, wherein the light chain amino acid sequence of SEQ ID NO: 24 comprises a microbial transglutaminase tag GGTLQSPP (SEQ ID NO: 5), wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody, and wherein n is between 3.5 and 4.5, preferably about 4, more preferably 4, wherein the [(linker)-(exatecan)] moieties are coupled to the microbial transglutaminase tags at the C-termini of the two light chains and to Q295 (Ell numbering) in the two heavy chains of the antibody.

Suitable methods for preparing an immunoconjugate are well known in the art (see e.g. Hermanson G. T., Bioconjugate Techniques, Third Edition, 2013, Academic Press). For instance, methods of conjugating a cytotoxic drug to an antibody via a linker that attaches covalently to cysteine residues of interchain disulfide bridges of the antibody are well known. The immunoconjugates of the invention may be prepared by in vitro methods, e.g. as described herein below and in the Examples; preferably they can be prepared by a method according to the present invention.

In general, an immunoconjugate can be obtained e.g. by a process comprising the steps of:

(i) preparing a compound comprising the linker and the growth inhibitory agent (e.g. cytotoxic drug), also referred to herein as a “drug-linker compound”;

(ii) bringing into contact an optionally buffered aqueous solution of an antibody with a solution of the drug-linker compound;

(iii) then optionally separating the conjugate which was formed in (ii) from the unreacted antibody and/or drug-linker compound.

The aqueous solution of antibody can be buffered with buffers such as e.g. histidine, potassium phosphate, acetate, citrate or N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer may be chosen depending upon the nature of the antibody. The drug-linker compound can be dissolved e.g. in an organic polar solvent such as dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA).

For conjugation to the cysteine residues of an antibody, the antibody is subjected to reduction (e.g. using TCEP) before step (ii). Suitable reduction conditions to reduce only the interchain disulfide bonds are known in the art.

The reaction temperature for conjugation is usually between 20 and 40°C. The reaction time can vary and is typically from 1 to 24 hours. The reaction between the antibody and the druglinker compound can be monitored by size exclusion chromatography (SEC) with a refractometric and/or UV detector. If the conjugate yield is too low, the reaction time can be extended.

A number of different chromatography methods can be used by the person skilled in the art in order to perform the separation of step (iii): the conjugate can be purified e.g. by SEC, adsorption chromatography (such as ion exchange chromatography, I EC), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed-support chromatography such as hydroxyapatite chromatography, or high performance liquid chromatography (HPLC) such as reverse-phase HPLC. Purification by dialysis or filtration or diafiltration can also be used.

After step (ii) and/or (iii), the conjugate-containing solution can be subjected to an additional step (iv) of purification e.g. by chromatography, ultrafiltration and/or diafiltration. Such an additional step of purification e.g. by chromatography, ultrafiltration and/or diafiltration can also be performed with the antibody-containing solution after the reduction reaction, in cases where reduction is performed prior to conjugation.

The conjugate is recovered at the end of such a process in an aqueous solution. The drug-to- antibody ratio (DAR) is a number that can vary with the nature of the antibody and of the druglinker compound used along with the experimental conditions used for the conjugation (such as the ratio (drug-linker compound)/(antibody), the reaction time, the nature of the solvent and of the cosolvent if any). Thus, the contact between the antibody and the drug-linker compound can lead to a mixture comprising several conjugates differing from one another by different drug-to-antibody ratios. The DAR that is determined is thus an average value.

An exemplary method which can be used to determine the DAR consists of measuring spectrophotometrically the ratio of the absorbance at of a solution of purified conjugate at AD and 280 nm. 280 nm is a wavelength generally used for measuring protein concentration, such as antibody concentration. The wavelength AD is selected so as to allow discriminating the drug from the antibody, i.e. as readily known to the skilled person, AD is a wavelength at which the drug has a high absorbance and AD is sufficiently remote from 280 nm to avoid substantial overlap in the absorbance peaks of the drug and antibody. For instance, AD may be selected as being 370 nm for exatecan (or for camptothecin or other camptothecin analogs), or 252 nm for maytansinoid molecules.

A method of DAR calculation may be derived e.g. from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science: The absorbances for the conjugate at AD (AAD) and at 280 nm (A280) are measured either on the monomeric peak of the size exclusion chromatography (SEC) analysis (allowing to calculate the "DAR(SEC)" parameter) or using a classic spectrophotometer apparatus (allowing to calculate the "DAR(UV)" parameter). The absorbances can be expressed as follows:

AAD = (CD X £D D) + (CA X EA D)

A280 = (CD X £D28O) + (CA X £A28O) wherein : • CD and CA are respectively the concentrations in the solution of the drug and of the antibody

• £DAD and ED28O are respectively the molar extinction coefficients of the drug at AD and 280 nm

• EAAD and £A28O are respectively the molar extinction coefficients of the antibody at AD and 280 nm.

Resolution of these two equations with two unknowns leads to the following equations:

CD = [( EA280 X AAD) - (£AAD X A280)] I [(£DAD X EA28O) - ( SAAD X ED28O)]

CA ” [A280 - (CD X £D28O)] I £A2SO

The average DAR is then calculated from the ratio of the drug concentration to that of the antibody: DAR = CD / CA.

An alternative method for preparing an immunoconjugate is described in the following, and this is a method of the present invention. This method is a preferred method for the preparation of immunoconjugates/ADCs according to the present disclosure. The method can in particular be used for antibodies that comprise an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP and TLQSA in at least one of its light chain constant regions (CL) and/or in at least one of its heavy chain constant regions (CH). Thus, a further aspect of the invention relates to a method for producing an antibody-linker-conjugate comprising the steps:

(1) providing an antibody that comprises an amino acid sequence selected from the group consisting of GGTLQSPP, TLQSG, TLQSPP and TLQSA and more preferably the amino acid sequence TLQSPP or GGTLQSPP (most preferably the sequence GGTLQSPP) in at least one and preferably both of its light chain constant regions (CL) and/or in at least one and preferably both of its heavy chain constant regions (CH);

(2) mixing together in a reaction buffer at least the following components:

(a) said antibody provided in step (1);

(b) a microbial transglutaminase preferably a transglutaminase comprising the amino acid sequence

MGSGSGSGTGEEKRSYAETHRLTADDVDDINALNESAPAASSAGPSFRAPDSDE RVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTEEQRE WLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEFEGRV AKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALRNEDA RSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRKYGDP EAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKTVWTH GNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYWTFVPKSWNTAPDKVT QGWP (SEQ ID NO: 10) or

FRAPDSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQ QMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGE TRAEFEGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANG NDALRNEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGS

SDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEA DADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKS WNTAPDKVTQGWPLE (SEQ ID NO: 11) or

SDKRKYGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEA DADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYVVTFVPKS WNTAPDKVTQGWP (SEQ ID NO: 12) or

DSDERVTPPAEPLDRMPDPYRPSYGRAETIVNNYIRKWQQVYSHRDGRKQQMTE EQREWLSYGCVGVTWVNSGQYPTNRLAFAFFDEDKYKNELKNGRPRSGETRAEF EGRVAKDSFDEAKGFQRARDVASVMNKALENAHDEGAYLDNLKKELANGNDALR NEDARSPFYSALRNTPSFKDRNGGNHDPSKMKAVIYSKHFWSGQDRSGSSDKRK

YGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKT VWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYWTFVPKSWNTAP DKVTQGWPLE (SEQ ID NO: 13); or

YGDPEAFRPDRGTGLVDMSRDRNIPRSPTSPGESFVNFDYGWFGAQTEADADKT VWTHGNHYHAPNGSLGAMHVYESKFRNWSDGYSDFDRGAYWTFVPKSWNTAP DKVTQGWP (SEQ ID NO: 14) and

(c) a linker that comprises an FhN-moiety capable of reacting with the antibody from step (1) in the presence of said transglutaminase and wherein the linker is preferably a drug-linker where said linker is covalently attached to a drug;

(3) separating the antibody-linker-conjugate produced in step (2) from unreacted linker and from said transglutaminase preferably by subjecting said mixture from step (2) to a sizeexclusion chromatography. In one embodiment, the transglutaminase is encoded by the polynucleotide

ATGGGTAGCGGTAGCGGTTCAGGCACCGGTGAAGAAAAACGTAGCTATGCAGAAACC CATCGTCTGACCGCAGATGATGTTGATGATATTAATGCACTGAATGAAAGCGCACCGG CAGCAAGCAGCGCAGGTCCGAGCTTTCGTGCACCGGATAGTGATGAACGTGTTACCCC TCCGGCAGAACCGCTGGATCGTATGCCGGATCCGTATCGTCCGAGCTATGGTCGTGCC GAAACCATTGTTAATAACTATATTCGTAAGTGGCAGCAGGTTTATAGCCATCGTGATGGT CGTAAACAGCAGATGACCGAAGAACAGCGTGAATGGCTGAGTTATGGTTGTGTTGGTGT TACCTGGGTTAATAGCGGTCAGTATCCGACCAATCGTCTGGCATTTGCATTTTTCGATGA GGACAAATACAAGAACGAGCTGAAAAATGGTCGTCCGCGTAGCGGTGAAACCCGTGCA GAATTTGAAGGTCGTGTTGCAAAAGATAGCTTTGATGAAGCAAAAGGTTTTCAGCGTGC ACGTGATGTTGCAAGCGTTATGAATAAAGCACTGGAAAATGCACATGATGAAGGTGCCT ATCTGGACAACCTGAAAAAAGAACTGGCAAATGGTAATGATGCCCTGCGTAATGAAGAT GCACGTAGCCCGTTTTATAGCGCACTGCGTAATACCCCGAGCTTTAAAGATCGTAATGG TGGTAATCATGACCCGAGCAAAATGAAAGCAGTGATCTATAGCAAACACTTTTGGAGCG GTCAGGATCGTAGTGGTAGCAGCGATAAACGTAAATATGGTGATCCGGAAGCATTTCGT CCGGATCGTGGCACCGGTCTGGTTGATATGAGCCGTGATCGTAATATTCCGCGTAGTC CGACCAGTCCGGGTGAAAGCTTTGTTAATTTTGATTATGGTTGGTTTGGCGCACAGACC GAAGCAGATGCAGATAAAACCGTTTGGACCCATGGCAATCATTATCATGCACCGAATGG

TAGCCTGGGTGCAATGCATGTTTATGAAAGCAAATTTCGCAATTGGAGCGACGGCTATA GCGATTTTGATCGTGGTGCCTATGTTGTTACCTTTGTTCCGAAAAGCTGGAATACCGCTC CGGATAAAGTTACCCAAGGTTGGCCG7AA (SEQ ID NO: 15).

In a preferred embodiment of the method, said reaction buffer is 7 % DMSO, 24 mM HEPES, pH 7.0.

In the method of the invention, said linker is a linker having the formula wherein R is the remainder of the linker and may optionally also comprise a drug, whereby the drug is preferably exatecan. In more preferred embodiments of the method of the invention the linker is NH2-GGG-beta-glucuronide.

In a exemplary embodiment of the method of the invention in step (2) the mixture comprises the following drug-linker:

In a further embodiment of the method of the invention, the mixture in step (2) comprises 5 molar equivalents of linker or drug-linker, respectively, per conjugation site, wherein a conjugation site is a sequence GGTLQSPP, TLQSG, TLQSPP or TLQSA comprised in the light chain constant regions (CL) and/or in the heavy chain constant region (CH) of said antibody.

A further aspect of the invention relates to an antibody-linker conjugate producible according to the method of the invention.

Exemplary methods of the invention for preparing an immunoconjugate are described in the Examples.

Drug-linker compounds

The present disclosure also provides compounds comprising a linker and a growth inhibitory agent (e.g. a cytotoxic drug), also referred to herein as “drug-linker compounds”. For instance, the present disclosure provides a compound of the following formula: or a physiologically acceptable salt thereof; the compound according to formula XA is also referred to herein as “drug-linker compound 1-M”, “compound DL1-M” or “DL1-M”.

These drug-linker compounds may be used to prepare immunoconjugates using the method of the invention as described herein.

The exemplary drug-linker compounds disclosed may be prepared by chemical synthesis, for instance as described in the Examples further below.

Pharmaceutical compositions

The ADCs of the invention may be combined with pharmaceutically acceptable carriers, diluents and/or excipients, and optionally with sustained-release matrices including but not limited to the classes of biodegradable polymers, non-biodegradable polymers, lipids or sugars, to form pharmaceutical compositions.

Thus, another aspect of the invention relates to a pharmaceutical composition comprising an immunoconjugate of the invention and a pharmaceutically acceptable carrier, diluent and/or excipient.

Methods for preparing pharmaceutical compositions are known to a skilled person in the art (Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press).

The term "pharmaceutically acceptable" designates that said carrier, diluent or excipient is a non-toxic, inert material that is compatible with the other ingredients of the pharmaceutical composition and not harmful to the patient that the pharmaceutical composition is administered to, such that it can be used in a pharmaceutical product. Substances suitable as carriers, diluents or excipients in pharmaceutical compositions are known to a skilled person in the art (Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press). The pharmaceutical composition may further include e.g. additional adjuvants, antioxidants, buffering agents, bulking agents, colorants, emulsifiers, fillers, flavoring agents, preservatives, stabilizers, suspending agents and/or other customary pharmaceutical auxiliaries.

In some embodiments, said pharmaceutical composition further includes at least one additional adjuvant, antioxidant, buffering agent, bulking agent, colorant, emulsifier, filler, flavoring agent, preservative, stabilizer, suspending agent and/or other customary pharmaceutical auxiliary.

Therapeutic methods and uses

The ADCs disclosed herein will be useful for targeting said drug (e.g. growth inhibitory agent) to the target cells (e.g. cancerous cells) expressing or over-expressing the antigen (preferably a cell-surface antigen) that the antibodies bind to. Accordingly, the ADCs of the present disclosure, such as ADC1 , ADC2 and ADC3, will be useful for targeting the exatecan payload of these ADCs to cancer cells expressing or overexpressing NaPi2b at their cell surface.

An aspect of the invention relates to a method of treating cancer, comprising administering a therapeutically effective amount of the immunoconjugate or pharmaceutical composition of the invention to a subject in need thereof.

As used herein, "treatment" of a disease and "treating" a disease refers to the process of providing a subject with a pharmaceutical treatment, e.g., the administration of a drug, such that said disease is alleviated, reduced, minimized, halted or even healed, and/or such that the chances of a relapse into the disease are reduced or a relapse into the disease is even prevented.

The use of compounds in the treatment of diseases is known to a skilled person in the art. Thus, the skilled person is aware that the components of the compound, in particular the targeting moiety of the immunoconjugate/ADC, must be selected appropriately in order to allow for successful treatment. For example, for treatment of a specific cancer, the targeting moiety of the immunoconjugate/ADC must be selected such that binding of the targeting moiety to its target site directs the immunoconjugate/ADC to said cancer. Specifically, the NaPi2b-binding antibody of an immunoconjugate/ADC according to the present disclosure will direct the immunoconjugate/ADC of the present disclosure to cancer cells expressing NaPi2b at their cell surface. Accordingly the immunoconjugate/ADC of the present disclosure is specifically suited for the treatment of cancers that express NaPi2b at their cell surface. Cytotoxic effects will then be achieved by the exatecan payload of the immunoconjugate/ADC of the present disclosure.

Efficacy of the treatment with an antibody or immunoconjugate or pharmaceutical composition according to the invention may be readily assayed in vivo, for instance in a mouse model of cancer and by measuring e.g. changes in tumor volume between treated and control groups, % tumor regression, partial regression or complete regression.

In another aspect, the present disclosure relates to a method of treating a disease in a patient in need thereof, said method comprising administering an immunoconjugate/ADC according to the present disclosure to said patient. Preferably, said disease is cancer.

In another aspect, the present disclosure relates to method of treating cancer in a patient in need thereof, said method comprising administering to said patient a therapeutically effective amount of an immunoconjugate/ADC according to the present disclosure.

By "therapeutically effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease. The effective amount of active agent(s) (e.g., a compound according to the present disclosure) used for therapeutic treatment of a disease according to the present disclosure varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as a "therapeutically effective" amount.

The term "patient", as used herein, refers preferably to a human.

In another aspect, the present disclosure relates to an immunoconjugate/ADC according to the present disclosure for use as a medicament.

In another aspect, the present disclosure relates to an immunoconjugate/ADC according to the present disclosure for the treatment of cancer.

In another aspect, the present disclosure relates to an immunoconjugate/ADC according to the present disclosure for use in the treatment of cancer.

In another aspect, the present disclosure relates to the use of an immunoconjugate/ADC according to the present disclosure for the manufacture of a medicament for the treatment of cancer. Preferably, said medicament is prepared for administration to a human. In another aspect, the present disclosure relates to a pharmaceutical composition for use in the treatment of cancer, said pharmaceutical composition comprising an immunoconjugate/ADC according to the present disclosure.

In some embodiments, the cancer in any of the above-described aspects is a solid cancer.

In some embodiments, the cancer in any of the above-described aspects is a cancer that expresses NaPi2b at its cell surface. Preferably, said cancer expresses NaPi2b at such a level at its cell surface that the ADC according to the invention can be targeted to the cells of said cancer.

In some embodiments, the cancer in any of the above-described aspects is a cancer that overexpresses NaPi2b at its cell surface. Preferably, said cancer overexpresses NaPi2b at such a level at its cell surface that the ADC according to the invention can be targeted to the cells of said cancer.

In some embodiments, the cancer in any of the above-described aspects is selected from the group consisting of ovarian cancer, NSCLC (non-small cell lung cancer), thyroid cancer, breast cancer (preferably triple-negative breast cancer (TNBC)), endometrial cancer, cervical cancer and pancreatic cancer (preferably pancreatic ductal adenocarcinoma (PDAC)). In some embodiments, the cancer in any of the above-described aspects is selected from the group consisting of ovarian cancer, NSCLC (non-small cell lung cancer), thyroid cancer, breast cancer (preferably triple-negative breast cancer (TNBC)) and endometrial cancer. In some embodiments, the cancer in any of the above-described aspects is selected from the group consisting of ovarian cancer, NSCLC (non-small cell lung cancer), breast cancer (preferably triple-negative breast cancer (TNBC)) and endometrial cancer. In some embodiments, the cancer in any of the above-described aspects is ovarian cancer or NSCLC. In some embodiments, the cancer in any of the above-described aspects is triple-negative breast cancer (TNBC). In some embodiments, the cancer in any of the above-described aspects is endometrial cancer. In some embodiments, the cancer in any of the above-described aspects is ovarian cancer. In some embodiments, the cancer in any of the above-described aspects is NSCLC. Also disclosed with regard to the above-described subject matter is the following:

[1] Antibody-drug conjugate (ADC), wherein the ADC is wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody; and wherein n is preferably between 1 and 10 and more preferably about 4, and the antibody binds to NaPi2b.

[2] An ADC according to [1] with the antibody portion of the ADC having a light chain amino acid sequence of SEQ ID NO:1 and a heavy chain amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, preferably SEQ ID NO:4.

[3] A pharmaceutical composition comprising the ADC of [1] or [2],

[4] An ADC according to [1] or [2] for the treatment of cancer, preferably ovarian cancer or NSCLC

EXAMPLES

The following examples describe the preparation and characterization of ADCs according to the present disclosure, as well as related compounds and methods, along with comparative disclosure. It is understood that various embodiments of the disclosure reflected in the examples may be practiced, given the general description provided above. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. Example 1 : Synthesis of a drug-linker compound with glucuronide-based linker

The synthetic route to compound 9).

The synthetic route to compound 11 (also referred to herein as drug-linker compound 1 -M (DL1-M) Protocol of chemical preparation to compound 9

Step 1 : Compound 1

1

To a stirred solution of (2S,3S,4S,5R,6R)-3,4,5-Triacetoxy-6-bromo-tetrahydro-pyran-2- carboxylic acid methyl ester (8.30 g; 20.90 mmol; 1.00 eq.) and 4-Hydroxy-3-nitro- benzaldehyde (5.24 g; 31.35 mmol; 1.50 eq.) in Acetonitrile (83.00 ml; 10.00 V) was added Silver(l) oxide (9.69 g; 41.80 mmol; 2.00 eq.). The reaction mixture was stirred at RT for 16 h. The reaction mixture was filtered through celite. The filtrate was concentrated under vacuum to get solid. The solid was dissolved in EtOAc and washed with 10% aqueous solution of NaHCOs to remove excess 4-Hydroxy-3-nitro-benzaldehyde. The organic layer was concentrated under vacuum to get compound 1 as sand colour solid.

Yield: 9.0 g

Percentage Yield: 89.1%

Analytical data: NMR: ¹H-NMR (400 MHz, DMSO-d6): 9.98 (s,1 H), 8.46 (s, 1 H), 8.25-8.21 (m, 1 H),7.64 (d, J = 11.60Hz, 1 H), 5.94 (d, J= 10.00 Hz, 1 H), 5.51-5.44 (m, 1 H), 5.20-5.09 (m, 2H),4.80 (d, J = 13.20 Hz, 1 H), 3.64 (s,3H), 2.09 (s, 9H).

Step 2: Compound 2 To a stirred solution of compound 1 (9.00 g; 18.62 mmol; 1.00 eq.) in Propan-2-ol (33.00 ml; 3.67 V) and CHCI₃ (167.00 ml; 18.56 V) were added silica gel 60-120 (3.60 g; 112.09 mmol; 6.02 eq.) followed by sodium borohydride (1.80 g; 46.55 mmol; 2.50 eq.). The reaction mixture was stirred for 1 h at RT. After completion, the reaction mixture was quenched with cooled H2O and filtered through celite. The filtrate was extracted with Dichloromethane and dried over Na2SC>4. The solvent was concentrated to get compound 2 as off-white powder.

Yield: 8.70 g

Percentage Yield: 92.4%

Analytical data

LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5 pm; Mobile phase A: 0.1% HCOOH in H2O:

ACN (95:5); B: ACN

RT (min): 2.05; M+H: 503.2, Purity: 96.6%

Step 3: Compound 3

To a stirred solution of compound 2 (8.70 g; 17.21 mmol; 1.00 eq.) in ethyl acetate (100.00 ml; 11.49 V) and THF (100.00 ml; 11.49 V) was added Palladium on carbon (10% w/w) (2.50 g; 2.35 mmol; 0.14 eq.). The reaction mixture stirred for 3 h at RT under hydrogen atmosphere. After completion, the reaction mixture was filtered off through celite. The solvent was concentrated under vacuum to get compound 3 as off-white solid.

Yield: 8.5 g

Percentage Yield: 100%

Analytical data:

LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5 pm; Mobile phase A: 0.1% HCOOH in H2O: ACN (95:5); B: ACN RT (min): 1.73; M+H: 456.10, Purity: 95.1%

Step 4: Compound 4

3 4

To a stirred solution of compound 3 (10.00 g; 20.89 mmol; 1.00 eq.) and (9H-Fluoren-9- ylmethoxycarbonylamino)-acetic acid (7.60 g; 25.06 mmol; 1.20 eq.) in DCM (250.00 ml; 25.00

V) was added 2-Ethoxy-2H-quinoline-1 -carboxylic acid ethyl ester (15.65 g; 62.66 mmol; 3.00 eq.) at 0 °C. The reaction mixture was stirred for 16h at RT. After completion, solvent was removed under reduced pressure to get a crude product. The crude product was purified by column chromatography (56% EtOAc: petroleum ether) to get compound with purity 80%. The compound was purified further by washings with30% EtOAc and pet ether to get compound 4 as white solid.

Yield: 8.5 g

Percentage Yield: 50.7%

Analytical data: LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5 pm; Mobile phase A: 0.1% HCOOH in H₂O: ACN (95:5); B: ACN

RT (min): 3.03; M+H: 735.2, Purity: 81.9 % Step 5: compound 5

To a stirred solution of compound 4 (2.00 g; 2.49 mmol; 1.00 eq.) in THF (40.00 ml; 20.00 V) at 0 °C, were added Carbonic acid bis-(4-nitro-phenyl) ester (3.06 g; 9.97 mmol; 4.00 eq.) and DIPEA (4.40 ml; 24.92 mmol; 10.00 eq.). The reaction mixture was stirred at RT for 12 h. After completion of the reaction, reaction mixture was concentrated under vacuum. The crude product was purified by column chromatography using silica gel (230-400) and pet ether / ethyl acetate as an eluent to afford compound 5 as pale yellow solid.

Yield: 2.0 g

Percentage Yield: 84.6%

Analytical data:

LCMS: Column: X-Bridge C8(50X4.6) mm, 3.5pm; Mobile phase: A: 0.1% TFA in MilliQ water; B: ACN

RT (min): 3.24; M+H: 900.20, Purity: 94.9%

Step 6: Compound 6

Compound 5 (1 ,369 g; 1 ,00 eq.) was dissolved in N,N-dimethylformamide (15,00 ml), Exatecan mesylate (679,7 mg; 1 ,00 eq.), 4-methylmorpholine for synthesis (0,422 ml; 3,00 eq.) and 1- Hydroxybenzotriazol (172,8 mg; 1 ,00 eq.) were added. The reaction mixture was stirred at room temperature for overnight. After the stirring time the reaction suspension was changed to a brown solution. The reaction was monitored by LC-MS, which showed a complete conversion of the starting material. The reaction mixture was purified via RP flash chromatography. The product containing fractions were combined, concentrated in vacuo and lyophilized overnight to afford compound 6 as an yellow solid.

Yield: 1.59 g

Percentage Yield: 87.5%

Analytical data:

LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H₂O in ACN; T: 40 °C; Flow: 3,3 ml/min; MS: 100-2000, amu positive; 1% ->100% B: 0 ->2,0min; 100% B: 2,0 ->2,5 min

RT (min): 1.95; M+H: 1196.40, Purity: 84.4%.

Step 7: Compound 7

Compound 6 (1 ,586 g; 1 ,00 eq.) was dissolved in tetrahydrofuran (50,00 ml) and a solution (0.1M) of LiOH (contains Lithium hydroxide hydrate (281 ,77 mg; 6,00 eq.) in water (67,100 ml)) was added dropwise at 0°C. The pH value was checked during the addition. The pH should not exceed 10. The addition of the solution of LiOH was completed after 1.5 hours. The reaction was monitored by LC-MS, which showed a complete conversion of the starting material. The reaction was quenched with citric acid solution, pH adjusted to 5. The reaction mixture was concentrated under reduced pressure. The crude was purified by prep. HPLC. The product containing fractions were combined and lyophilized to afford Compound 7 as a dark yellow solid.

Yield: 728 mg

Percentage Yield: 54.8% Analytical data:

LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H₂O; B: 0.04% HCOOH and 1% H₂O in ACN; T: 40 °C; Flow: 3,3 ml/min; MS: 100-2000, amu positive; 1% ->100% B: 0 ->2,0min; 100% B: 2,0 ->2,5 min

RT (min): 1.68; M+H: 1056.30, Purity: 98.5%.

Step 8: Compound 8

Compound 7 (728,000 mg; 1 ,00 eq.) was dissolved in N,N-dimethylformamide (20,00 ml). Piperidine (136,513 pl; 2,00 eq.) was added and the solution was stirred at RT for totally 4 hours. The reaction was monitored by LC-MS, which showed a complete conversion of the starting material. The reaction mixture was concentrated under reduced pressure and the crude product was purified by RP flash chromatography. The product containing fractions were combined, the solvent was removed partially and it was lyophilized overnight to afford compound 8 as an yellow solid.

Yield: 706 mg

Percentage Yield: 100%

Analytical data:

RT (min): 1.22; M+H: 834.30, Purity: 97.6%. Step 9: Compound 9

To a solution of compound 8 (854 mg; 1,00 eq.) in dimethylformamid (30,00 ml) were added N-ethyldiisopropylamine (149,234 pl; 1,00 eq.) and 3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)- propionic acid 2,5-dioxo-pyrrolidin-1-yl ester (233,61 mg; 1,00 eq.). The reaction mixture was stirred at RT for 3 hours. The reaction was monitored by LC-MS, which showed a complete conversion of the starting material. The reaction mixture was concentrated under reduced pressure and the crude product was by RP flash chromatography. The product containing fractions were combined, concentrated and lyophilized to give the desired produce with a purity of 91%. This material was again purified by RP chromatography to give compound 9 as an yellow solid.

Yield: 580 mg

Percentage Yield: 60.1%

Analytical data:

RT (min): 1.38; M+H: 985.30, Purity: 90% (the other 10% of isomer can be removed by HPLC) ¹H NMR (500 MHz, DMSO-cfe) 613.10-12.44 (m, 1H), 9.08 (s, 1H), 8.32 (t, J= 5.8 Hz, 1H), 8.16 (s, 1H), 8.02 (d, J= 8.8 Hz, 1H), 7.76 (d, J= 10.9 Hz, 1H), 7.31 (s, 1H), 7.15-7.09 (m, 2H), 6.98 (s, 2H), 5.48-5.38 (m, 2H), 5.32-5.22 (m, 3H), 5.11 -5.01 (m, 2H), 4.87 (d, J = 7.6 Hz, 1H), 3.92 - 3.88 (m, 1H), 3.89 - 3.84 (m, 2H), 3.65 - 3.61 (m, 2H), 3.46 - 3.41 (m, 1H), 3.42 - 3.37 (m, 1H), 3.38 - 3.31 (m, 1H), 3.28 - 3.20 (m, 1H), 3.15 - 3.07 (m, 1H), 2.48 -2.44 (m, 2H), 2.38 (s, 3H), 2.24-2.13 (m, 2H), 1.94-1.80 (m, 2H), 0.88 (t, J= 7.3 Hz, 3H). Protocol of chemical preparation to compound 11

Step 1 : Compound 10

To a solution of compound 8 (289.00 mg; 1.00 eq) in dimethyl formamide (10.00 ml) were added N-ethyl diisopropylamino (0.104 ml, 2.00 eq) and 2,5-dioxopyrrolidin-1-yl (tert- butoxycarbonyl)glycylglycinate (105.70 mg; 1 .00 eq). The reaction mixture was stirred at room temperature for 1 hour and the reaction was mirrored by LC-MS. After completion, the solvent was removed in vacuo and the crude product was purified by prep. HPLC over a Sunfire column. The fractions containing product were combined and lyophilized to afford compound 10 trifluoroacetic acid salt as a yellow solid.

Yield: 162.00 mg, 0.14 mmol.

Percentage yield: 44.8 %

Analytical data:

LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1 % H2O in ACN; T: 40 °C; Flow: 3,3 ml/min; MS: 100-2000, amu positive; 1% ->100% B: 0 ->2,0min; 100% B: 2,0 ->2,5 min

RT (min): 1.41 ; M+H: 1048.1 , Purity: 97.9%

Step 2: Compound 11

To a suspension of compound 10 (162.00 mg; 1.00 eq) in dichloromethane (5.00 ml) was added 4.0 M hydrogen chloride solution in 1,4-dioxane (682.43 pl; 20.00 eq) and it was stirred at room temperature for 3.5 hours and the reaction was monitored by LC-MS. After completion, the reaction mixture was concentrated in vacuo and the crude product was purified by prep. HPLC. The fractions containing product were combined and lyophilized to afford compound 11 trifluoroacetic acid salt as a yellow solid.

Yield: 113 mg; 0.11 mmol.

Precent yield: 77.0 %

Analytical data:

LCMS: Column: Chromolith HR RP-18e (50-4,6 mm); Mobile phase A: 0.05% HCOOH in H2O; B: 0.04% HCOOH and 1% H2O in ACN; T: 40 °C; Flow: 3,3 ml/min; MS: 100-2000, amu positive; 1% ->100% B: 0 ->2,0min; 100% B: 2,0 ->2,5 min

RT (min): 1.22; M+H: 948.0, M+2H: 474.8; Purity: 98.8%

¹H NMR (700 MHz, DMSO-d6) 612.93- 12.79 (m, 1H), 9.14 (s, 1H), 8.61 (t, J = 5.8 Hz, 1H), 8.41 (t, J = 5.9 Hz, 1H), 8.14 (s, 1H), 8.05 (d, J = 8.8 Hz, 1H), 8.03-7.93 (m, 3H), 7.79 (d, J = 10.8 Hz, 1H), 7.31 (s, 1H), 7.13 (s,2H), 6.58-6.47 (m, 1 H), 5.80 - 5.61 (m, 1 H), 5.48 - 5.40 (m, 2H), 5.31 - 5.24 (m, 3H), 5.08 (d, J = 12.2 Hz, 1H), 5.03 (d, J = 12.3 Hz, 1H), 4.91 (d, J = 7.7 Hz, 1 H), 3.96 (d, J = 5.8 Hz, 2H), 3.95 - 3.86 (m, 3H), 3.62 (q, J = 5.8 Hz, 2H), 3.43 (t, J = 9.3 Hz, 1H), 3.41 (t, J = 8.5 Hz, 1H), 3.35 (t, J = 9.0 Hz, 1H), 3.27-3.20 (m, 1H), 3.15-3.09 (m, 1H), 2.38 (s, 3H), 2.23-2.13 (m, 2H), 1.93-1.81 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). Example 2: Expression and purification of antibodies

DNA sequences encoding for antibodies mAb1 , mAb2 and mAb3 (also referred to herein as MAB1 , MAB2 and MAB3, respectively) were synthesized and cloned onto pTT5 plasmids for recombinant expression at GeneArt (Life Technologies). Produced plasmids were used for transient transfection and recombinant protein expression in shaking flasks using the ExpiCHO expression system (GibcoTM, Thermo Fisher Scientific Inc.). Seven days post transfection, supernatants were harvested and expressed antibodies purified using a standard stepwise process including protein A affinity chromatography (HiTrap MabSelect SuRe columns, Cytiva) and size-exclusion chromatography (HiLoad Superdex 200 pg columns, Cytiva).

Example 3: Exemplary ADC preparation and initial characterization

Monoclonal antibodies (mAb) were stored at -80°C. Prior conjugation, mAbs were thawed at RT and buffer was exchanged to 24 mM HEPES, pH 7.0 using HiTrap Desalting columns in combination with an Akta liquid chromatography (LC) system (Cytiva). To couple the drug linker (DL) to the antibody, a microbial transglutaminase (mTG) was used. The reaction setup was typically as follows: 5 mg/ml mAb, 5 molar equivalents of drug-linker per conjugation site, 20 ll/rnl mTG, 3-4 % DMSO, 24 mM HEPES, pH 7.0. The reaction was carried out at 37°C for 18-20 h. ADCs were separated from DL and mTG via size exclusion chromatography (SEC). Prior to SEC purification, NaCI concentration of the samples was adjusted to 100 mM using a 5 M NaCI stock solution. SEC was carried out using a HiLoad Superdex 200 26/60 or 26/40 Increase column in combination with an Akta LC system (Cytiva) at a flow rate of 2.5 ml/min and 20 mM Histidine, 80 mM NaCI, pH 5.5 as running buffer. Fractionated samples containing the ADC material were pooled and concentrated to 8 mg/ml using Amicon Ultra 15 50K centrifugal (Millipore) followed by a final buffer exchange into 10 mM Histidine, 40 mM NaCI, 6% Trehalose, 0.05% TWEEN, pH 5.5 using HiTrap Desalting columns in combination with an Akta LC system (Cytiva). Final bulk drug substance (BDS) was filtered through a 0.22 pm sterile filter unit (Millipore) and shock frozen in liquid nitrogen till further use.

ADCs were characterized as described in WO2023/170239 and WO2023/170240. Quality attributes of exemplary batches are summarized below.

ADC1 ADC2

ADC3

Further molecules used in the present studies MAB1: The parental, unconjugated antibody of ADC1 (corresponding to the part of ADC1 defined by SEQ ID NO: 1 (light chain) and SEQ ID NO: 2 (heavy chain)).

MAB2: The parental, unconjugated antibody of ADC2 (corresponding to the part of ADC2 defined by SEQ ID NO: 1 (light chain) and SEQ ID NO: 3 (heavy chain)).

MAB3: The parental, unconjugated antibody of ADC3 (corresponding to the part of ADC3 defined by SEQ ID NO: 1 (light chain) and SEQ ID NO: 4 (heavy chain)). MAB4_7: An antibody with the same sequence as MAB1 , but without the light chain transglutaminase recognition tag and with three allotypic mutations (heavy chain R214K, E356D, M358L).

MAB5_6: An antibody with the same properties as MAB4_7, but with YTE mutations (heavy chain M252Y, S254T, T256E).

MAB8: An antibody with the same properties as MAB3, but with three allotypic mutations (heavy chain R214K, E356D, M358L); light chain: SEQ ID NO: 24; heavy chain: SEQ ID NO: 25.

ADC4: MAB4_7 conjugated with compound 9 via a 2-step approach as described in Dickgiesser et al., Bioconjugate Chemistry, 2020 (10.1021/acs.bioconjchem.0c00061). First, cysteamine (2-aminoethanthiol) is attached primarily to the heavy chain position Q295 via transglutaminase conjugation. Second, compound 9 is attached to cysteamine serving as a reactive spacer resulting in drug-to-antibody ratio of ~2.

ADC5: MAB5_6 conjugated with compound 9 as described for ADC4.

ADC6: MAB5_6 conjugated with compound 9 after full reduction of interchain disulfide bonds resulting in a drug-to-antibody ratio of ~8.

ADC7: MAB4_7 conjugated with compound 9 after full reduction of interchain disulfide bonds resulting in a drug-to-antibody ratio of ~8.

ADC8: MAB8 conjugated in the same manner and with the same drug-linker compound 11 as ADC1-3 to a drug-to-antibody ratio of ~4.

ZW_NAPI2B_MAB: NaPi2b-targeting antibody as described in published international patent application W02024/082056A1 , Sequences No. 61 and 62.

ZW_NAPI2B_ADC: NaPi2b-targeting ADC produced from antibody ZW_NAPI2B_MAB as described for ‘v38591-MC-GGFG-AM-Compound 139’ in W02024/082055A1 with a drug-to- antibody ratio of ~4.

ZW Payload: Payload of ZW_NAPI2B_ADC (ZD06519, i.e. Compound 139 in WG2024/082055A1).

TUB_NAPI2B_MAB: NaPi2b-targeting antibody as described in WO2024/083925A2, Sequences No. 6 and 7. TUB_NAPI2B_ADC: NaPi2b-targeting ADC produced from antibody TUB_NAPI2B_MAB as described for ‘AV25-P5(PEG24)-VC-PAB-Exatecan’ in WO2024/083925A2.

DS_CDH6_MAB: Cadherin-6 targeting unconjugated antibody of raludotatug deruxtecan with sequences according to WHO recommended INN: List 89 (WHO Drug Information, Vol. 37, No. 1 , 2023).

DS_CDH6_ADC: Cadherin-6 targeting ADC raludotatug deruxtecan with sequences and druglinker according to WHO recommended INN: List 89 (WHO Drug Information, Vol. 37, No. 1 , 2023) with a drug-to-antibody ratio of ~8.

DS payload: Payload of DS_CDH6_ADC (DXd).

AZ_FRA_MAB: Folate receptor alpha (FOLRI)-targeting unconjugated antibody as described in WO2023/169896A1 (Sequences No. 49 and 50) and WO2023/170216A1 (AB1370049).

AZ_FRA_ADC: Folate receptor alpha (FOLRI)-targeting ADC consisting of AZ_FRA_MAB conjugated with 8 drug-linkers SG3932 as described in WO2023/169896A1 (Sequences No. 49 and 50) or WO2023/170216A1 (AB1370049-SG3932)

AZ Payload: Payload of AZ_FRA_ADC (AZ14170132, see WO2023/169896A1).

PRO_FRA_MAB: Folate receptor alpha (FOLRI)-targeting unconjugated antibody of rinatabart sesutecan with sequences according to WHO proposed INN: List 130 (WHO Drug Information, Vol. 37, No. 4, 2023).

PRO_FRA_ADC: Folate receptor alpha (FOLRI)-targeting ADC rinatabart sesutecan with sequences and drug-linker according to WHO proposed INN: List 130 (WHO Drug Information, Vol. 37, No. 4, 2023) with a drug-to-antibody ratio of ~8.

LIFA_NAPI2B_MMAE_ADC: NaPi2b-targeting ADC consisting of an antibody of Sequences No. 80 and 81 from US8535675B2 conjugated with mc-vc-PAB-MMAE (CAS. Nr. : 646502-53- 6), DAR 3.5-4.

UPI_NAPI2B_MMAE_ADC: NaPi2b-targeting ADC consisting of the antibody Upifitamab according to the sequences given in WHO Recommended INN: List 85 (WHO Drug Information, Vol. 35, No. 1 , 2021) conjugated with mc-vc-PAB-MMAE (CAS. Nr. : 646502-53- 6), DAR 4. UPI_NAPI2B_MMAF_ADC: NaPi2b-targeting ADC consisting of the antibody Upifitamab according to the sequences given in WHO Recommended INN: List 85 (WHO Drug Information, Vol. 35, No. 1 , 2021) conjugated with MC-Monomethyl Auristatin F (CAS. Nr. : 863971-19-1), DAR 3.8.

ADC2_ISOTYPE_MAB: CD20-targeting isotype control antibody with a transglutaminase recognition tag GGTLQSPP fused to the C-termini of the light chain.

ADC2_ISOTYPE_ADC: ADC2_ISOTYPE_MAB conjugated with the same drug-linker compound 11 as ADC1-3 to a drug-to-antibody ratio of ~4.

DS_ISOTYPE_ADC: CD20-targeting isotype control antibody conjugated with the same druglinker as DS_CDH6_ADC to a drug-to-antibody ratio of ~8.

AZ_ISOTYPE_ADC: Digoxigenin-binding isotype control antibody with heavy chain mutations L234A/L235A conjugated with the same drug-linker as AZ_FRA_ADC to a drug-to-antibody ratio of 6.

PRO_ISOTYPE_ADC: Digoxigenin-binding isotype control antibody with heavy chain mutations L234A/L235A conjugated with the same drug-linker as PRO_FRA_ADC to a drug- to-antibody ratio of ~8.

ZW_ISOTYPE_ADC: ADC: Digoxigenin-binding isotype control antibody with heavy chain mutations L234A/L235A conjugated with the same drug-linker as ZW_NAPI2B_ADC to a drug- to-antibody ratio of ~4.

TUB_ISOTYPE_ADC: Digoxigenin-binding isotype control antibody with heavy chain mutations L234A/L235A conjugated with the same drug-linker as TUB_NAPI2B_ADC to a drug-to-antibody ratio of ~8.

Pharmacokinetic studies in FcRn transgenic (Tg 276 model) mice

Pharmacokinetic studies in FcRn transgenic (Tg 276 model) mice were performed following single intravenous administration of 3 mg/Kg of MAB4_7, MAB5_6, ADC5, ADC4, ADC6, ADC7 and ADC8. Samples were taken 1 , 6, 24, 48, 72, 168, 240 and 336 hours after dosing for 6 animals per group.

Total antibody was measured as analyte. Data were pooled for PK analysis, see Table 1. Mab MAB5_6 with YTE showed lower clearance and longer half-life than MAB4_7 with-no YTE.

The half-life extension is maintained in the ADC format, too where YTE variants ADC5, ADC6, ADC8 showed lower clearance and longer half-life of corresponding YTE variants ADC4 and ADC7.

PK studies comparing TGase-conjugated DAR 2 and 4 vs cysteine-conjugated DAR8 exatecan ADCs with and without YTE set of mutation reveal:

• Favorable PK of TGase-conjugated ADCs

• Favorable PK of ADCs with YTE mutations

• Best PK of YTE-bearing TGase-conjugated DAR 4 ADC with PK being very similar to unconjugated parental antibody

Table 1 : Key pharmacokinetic parameters from studies in transgenic human FcRn TG276 mice

Pharmacokinetic studies in FcRn transgenic (Tg32 model) mice

Pharmacokinetic studies in FcRn transgenic (Tg32 model) mice were performed following single intravenous administration of 3 or 5 mg/Kg of ADC1 , ADC3, TUB_NAPI2B_ADC and ZW_NAPI2B_ADC. Samples were taken at 0.167, 4, 24, 48, 96, 168, 240, 336, 504, and 672 hours after dosing for 3 animals for treatment group. Total antibody was measured as analyte. Data were pooled for PK analysis, see Table 2. The lowest clearance and longer half-life were calculated for ADC3.

Table 2: PK parameters of final candidates in transgenic human FcRn Tg32 mice

Favorable PK properties (clearance in human FcRn mice: 0.221 resp. 0.214 mL/h/kg) enable at least Q3W or less frequent administration for ADCs according to the present disclosure, in particular ADC3.

Example 4: Analysis of cellular internalization and effects on cancer cell line viability in vitro /In vivo anti-tumor activity in PDX models

Internalization into ovarian cancer cells (OVCAR3)

Internalization of the NaPi2b monospecific antibody compared to isotype control was examined by immunofluorescence analysis in cultured OVCAR3 ovarian cancer cells. Antibodies were labeled with secondary Fab fragment conjugated to a pH-dependent fluorophore. As can be seen from the staining in Fig. 1 , round nuclei are surrounded by smaller fluorescent structures which indicate internalization into acidic compartments

(NaPi2b antibody). These structures are not present around nuclei of the same cell line incubated with an isotype control antibody. Thus, in this experiment effective internalization of NaPi2b monospecific antibody compared to isotype control was observed.

Effects on Cell Viability

The effects on cell viability in OVCAR3 cells was tested with ADC2 as an exemplary ADC according to the present disclosure by a standard viability assay as also described in Example 5 (section "Viability Assay"). Inhibition of ovarian cancer cell viability with sub-nanomolar potency (OVCAR3 0.08 nM) was observed (Fig. 2).

Thus, these experiments showed effective internalization of the ADCs of the present disclosure into acidic compartments and strong effects on cancer cell line viability in vitro.

In vivo anti-tumor activity in PDX models

The antitumor activity of the ADCs of the present disclosure was further studied in vivo in PDX (patient-derived xenograft) models for different types of cancer. The experiment was essentially carried out as described in Example 12, the treatment was 8 mg/kg q2w x3 (8 mg/kg of ADC resp. vehicle control were administered every 2 weeks for a total of three administrations).

Ovarian cancer PDX models

Results obtained with different PDX models for ovarian cancer are shown in Fig. 3-11 (squares: NaPi2b ADC2; circles: Control).

Overall strong anti-tumor activity was observed in all PDX models for ovarian cancer. The models OVPF040 and OVPF151 grew very slowly. This may have an impact on the activity of the exatecan ADCs.

Non-small cell lung cancer (NSCLC) PDX model

Results obtained with the PDX model for non-small cell lung cancer (NSCLC) CTG-860 are shown in Fig. 12.

Strong anti-tumor activity was observed. As can be seen from Fig. 13 (from N.D. Bodyak et al., Mol Cancer Ther (2021) 20 (5): 896-905), NaPi2b exatecan DAR4 ADC is also more efficacious than former NaPi2b ADC clinical candidates (XMT-1536 Dolaflexin, DAR11 ; Lifastuzumab vedotin, DAR4; for details see N.D. Bodyak et al., Mol Cancer Ther (2021) 20 (5): 896-905) in the same model when indirectly comparing doses reflecting relevant and expected tolerated exposures (10 mg/kg qw x3 for internal ADC). Summary of Key Molecule Parameters

The key molecule parameters of ADC3 are given below:

Example 5: Effects of NaPi2b ADCs on Cancer Cell Viability

Viability Assay

Cytotoxicity effects of ADCs on cancer cell lines were measured by cell viability assays. Cells were seeded in a volume of 90 pL in 96-well plates the day before treatment. Test compounds (ADCs or free payloads) were formulated at 10-fold the starting concentration in cell culture medium. Test compounds were serial diluted (1 :4) and 10 pL of each dilution was added to the cells in triplicates. Plates were cultured at 37 °C in a CO2 incubator for six days. For cell viability measurement, Cell Titer-Gio® reagent (Promega™ Corp, Madison, Wl) was added to each well, and plates processed according to the manufacturer’s instructions. Luminescence signals were measured using a Varioskan plate reader (Thermo Fisher). Luminescence readings were converted to % viability relative to untreated cells. Data was fitted with nonlinear regression analysis, using log (inhibitor) vs. response, variable slope, 4-parameter fit equation using Genedata Screener or GraphPad Prism. Data is shown as % relative cell viability vs. molar compound concentration, error bars indicating standard deviation (SD) of duplicates or triplicates. Geometric mean values of IC50s derived from multiple experiments were calculated.

ADC2 and ADC3 potently inhibit cancer cell viability

Using the method as described in the section "Viability Assay" above, the human cancer cell lines OVCAR3, NCI-H1781 , HCC78 and SW900 were used to compare effects of ADC2, ADC3, ADC2_IS0TYPE_ADC and/or exatecan payload on cancer cells with NaPi2b expression compared to effects on cancer cells lacking NaPi2b expression (SW900).

ADC2 inhibited OVCAR3, NCI-H1781 and HCC78 cancer cell viability with high potency of 0.08 nM, 2.7 nM and 0.66 nM, respectively. ADC3 inhibited OVCAR3, NCI-H1781 and HCC78 cancer cell viability with high potency of 0.10 nM, 1.8 nM and 0.54 nM, respectively (Figure 14). ADC3 minimally affected (-16%) the viability of SW900 lacking NaPi2b expression and effects were comparable to the isotype control ADC (ADC2_ISOTYPE_ADC) (Figure 16). For cell lines with NaPi2b expression, there was a high differentiation between the ADC2 and ADC3 isotype control (ADC2_ISOTYPE_ADC) compared to ADC2 and ADC3. Exatecan, the payload of ADC2 and ADC3, effectively inhibited cancer cell line viability of OVCAR3, NCI- H1781 , HCC78 and SW900 cell lines (Figure 14, Figure 16; see also below Table 3).

In conclusion, ADC2 and ADC3 inhibit the viability of cancer cell lines with high potency and high specificity.

Comparison with other NaPi2b-directed ADCs

Using the method as described in the section "Viability Assay" above, the human cancer cell lines OVCAR3, NCI-H1781 , SW900, JEG3 and CAOV3 were used to compare effects of ADC3, ADC2_ISOTYPE_ADC, exatecan, ZW_NAPI2B_ADC, ZW_ISOTYPE_ADC, ZW payload, TUB_NAPI2B_ADC and/or TUB_ISOTYPE_ADC on cancer cells with NaPi2b expression compared to effects on cancer cells lacking NaPi2b expression (SW900, JEG3, CAO 3).

ADC3 inhibited both NaPi2b-positve cancer cell lines, OVCAR3 and NCI-H1781 , viability with high potency of 0.07 nM and 7.5 nM, respectively.

ZW_NAPI2B_ADC cancer cell line viability inhibition was restricted to OVCAR3 (0.01 nM) while for NCI-H1781 the potency was low (42 nM) despite sub-nanomolar potency of the ZW payload (0.91 nM).

TUB_NAPI2B_ADC inhibited both NaPi2b-positve cancer cell lines, OVCAR3 and NCI-H1781 , viability with high potency of 0.02 nM and 0.11 nM, respectively.

ADC3 and ZW_NAPI2B_ADC minimally effected the viability of cell lines lacking NaPi2b expression (SW900, JEG3, CAOV3). However, compared to ZW_NAPI2B_ADC, ADC3 was more potent against NaPi2b-positive NCI-H1781.

In conclusion, ADC3 provides a favorable balance between high potency and minimal unspecific effects on target-negative cells compared to ZW_NAPI2B_ADC and TUB_NAPI2B_ADC. Table 3: Potency of indicated ADCs, isotype controls and free payload against multiple human cell lines. Maximal effects compared to untreated controls at the highest tested compound concentration are indicated below. Geometric mean of IC50 and mean percent effect are shown from 2 to 4 independent experiments.

Comparison to non-TOP1 inhibitor payload anti-NaPi2b ADCs

Using the method as described in the section "Viability Assay" above, the human cancer cell lines OVCAR3 and NCI-H1781 were used to compare the effects of ADC3, UPI_NAPI2B_MMAE_ADC, UPI_NAPI2B_MMAF_ADC, LIFA_NAPI2B_MMAE_ADC, exatecan payload, MMAE payload and MMAF payload on cancer cells with NaPi2b expression.

ADC3 inhibited both NaPi2b-positve cancer cell lines, OVCAR3 and NCI-H1781, viability with high potency of 0.09 nM and 1.6 nM, respectively.

Both, UPI_NAPI2B_MMAE_ADC and UPI_NAPI2B_MMAF_ADC inhibited both NaPi2b- positve cancer cell lines, OVCAR3 and NCI-H1781 , viability with comparable high potency of 0.01 nM and 0.34 nM to 0.42 nM, respectively. Compared to ADC3, LIFA_NAPI2B_MMAE_ADC inhibited 0VCAR3 and NCI-H1781 cancer cell line viability with lower potency of 0.17 nM and 8.3 nM, respectively.

Table 4: Potency of indicated ADCs and respective free exatecan, MMAF and/or MMAE payloads against antigen-positive OVCAR3 and NCI-H1781 cell lines. Maximal effects compared to untreated controls at the highest tested compound concentration are indicated below. Geometric mean of IC50 and mean percent effect are shown of 3 independent experiments.

Example 6: Bystander Effect

Bystander Assay

An image-based assay to determine the bystander effect on antigen-negative cells in coculture with antigen-positive cells was carried out as follows:

Cytotoxicity effects of ADCs on antigen-negative cancer cell lines in co-culture with antigenpositive cancer cell lines were measured by bystander assays. One thousand NaPi2b-negative SW900 cells were seeded in co-culture experiments with 3000 NaPi2b-positive OVCAR3 cells per well. As a control, 1000 SW900 cells only were seeded in parallel. Cells were seeded in a total volume of 90 pL in 96-well plates the day before treatment. Test compounds were formulated at 10-fold the final indicated concentrations in cell culture medium and 10 pL was added to the cells in duplicates. Plates were cultured at 37 °C in a CO2 incubator for six days. Prior to immunofluorescence staining, medium was removed and cells were treated with 100% methanol (-20°C) for 30 minutes. After methanol removal and one PBS wash step, cells were treated with 2.5% paraformaldehyde (PFA) with 0.2% Triton X-100 in PBS for 15 minutes at room temperature. After solution removal and one PBS wash step, cells were treated with 3% fetal bovine serum (FBS) 1 1 mM EDTA 10.2% Tween 1 0.1% sodium azide in PBS for at least one hour at room temperature. Antigen-positive and antigen-negative cells were discriminated by immunofluorescence staining with 10 pg/mL human anti-MUC16 (mAb1) primary antibody and 1 :2000 dilution of donkey anti-human IgG fluorescently (phycoerythrin) labeled secondary antibody (Jackson ImmunoResearch #709-116-149). Cells were identified by nuclei staining using 1 pg/mL Hoechst 34580 (Invitrogen #21486) dye. Staining was carried out in 3% FBS / 1 mM EDTA / 0.2% Tween /0.1% sodium azide PBS solutions for 30 minutes at room temperature. Secondary antibody staining was combined with Hoechst dye staining. Between and after staining steps, cells were washed thrice with PBS.

Plates were imaged with the confocal quantitative image cytometer CQ1 (Yokogawa® Electric Corporation, Tokyo, Japan). Analysis was adapted from the CQ1 software (Yokogawa) template “Nucleus and pseudo-Cell body” and FCS export files were analyzed using FlowJo (BD). Based on staining or absence of staining with fluorescently labeled antibody around the nucleus, antigen-positive and antigen-negative cells were distinguished and quantified. Bar graphs show the number of identified antigen-positive and antigen-negative cells per treatment condition.

Bystander effect of different NaPi2b ADCs

The potential of ADC3, ADC2_ISOTYPE_ADC, exatecan, ZW_NAPI2B_ADC, ZW_ISOTYPE_ADC, TUB_NAPI2B_ADC and/or TUB_ISOTYPE_ADC to mediate a bystander effect against antigen-negative cells in close proximity to antigen-positive cells was evaluated in bystander assays.

ADC3 showed a potent bystander effect against NaPi2b-negative SW900 cells in the presence of NaPi2b-positive OVCAR3 (Figure 18). At the tested concentrations in mono-culture viability assays, ADC3 treatment resulted in maximal effects on OVCAR3 cells (Figure 14) while no effect on SW900 (Figure 16) was observed. In line with these findings, no non-specific effect of ADC3 was observed in the bystander assay setup for SW900 only controls (Figure 18). However, in the co-culture with target-positive OVCAR3 cells, an effective bystander effect by ADC3 on target-negative SW900 cells was observed at all tested concentrations.

Exatecan effectively inhibits cancer cell viability of cancer cell lines independent of NaPi2b expression (Figure 14, Figure 16). In line with these findings and included as a positive control, exatecan potently reduced the cell numbers in the SW900 cells only and in the co-culture of 0VCAR3 cells with SW900 cells.

ZW_NAPI2B_ADC did not result in unspecific effects on target-negative SW900 cells at the tested concentrations. In contrast to ADC3, in the co-culture with target-positive 0VCAR3 cells, no bystander effect by ZW_NAPI2B_ADC on target-negative SW900 cells was observed at the tested concentrations.

TUB_NAPI2B_ADC did not result in unspecific effects on target-negative SW900 cells at the tested concentrations. Like ADC3, in the co-culture with target-positive 0VCAR3 cells, an effective bystander effect by TUB_NAPI2B_ADC on target-negative SW900 cells was observed at the tested concentrations. The slightly higher bystander effect of TUB_NAPI2B_ADC compared to ADC3 may be related to the higher DAR of 8 compared to 4, respectively.

None of the respective isotype control ADCs (ADC2_ISOTYPE_ADC, ZW_ISOTYPE_ADC, TUB_ISOTYPE_ADC) resulted in unspecific effects on target-negative cells only or in cocultures.

In conclusion, ADC3 and TUB_NAPI2B_ADC mediate a potent bystander effect while no bystander effect was detected for ZW_NAPI2B_ADC.

Bystander effect compared to microtubule inhibitor (MTi) payload ADCs

The potential of ADC3, ADC2_ISOTYPE_ADC, exatecan payload, UPI_NAPI2B_MMAE_ADC, UPI_ISOTYPE_MMAE_ADC, UPI_NAPI2B_MMAF_ADC and LIFA_NAPI2B_MMAE_ADC to mediate a bystander effect against antigen-negative cells in close proximity to antigen-positive cells was evaluated in bystander assays (Figure 19).

In line with data discussed above, ADC3 showed a potent bystander effect against NaPi2b- negative SW900 cells in the presence of NaPi2b-positive 0VCAR3.

In contrast to ADC3, UPI_NAPI2B_MMAE_ADC, UPI_NAPI2B_MMAF_ADC, LIFA_NAPI2B_MMAE_ADC did not mediate a potent bystander effect. The higher bystander effect of ADC3 compared to UPI_NAPI2B_MMAE_ADC, UPI_NAPI2B_MMAF_ADC, LIFA_NAPI2B_MMAE_ADC is more pronounced at a concentration of 2.5 nM, a concentration at which ADC3, UPI_NAPI2B_MMAE_ADC, UPI_NAPI2B_MMAF_ADC,

LIFA_NAPI2B_MMAE_ADC resulted in maximal effects on 0VCAR3 cells in mono-culture (Figure 17, Table 4).

For UPI_NAPI2B_MMAF_ADC, no bystander effect was detected which may be due to the non-permeable payload moiety released from mc-MMAF linker-payload ADCs. Example 7: Cell Internalization

Image-based internalization assay

ADC internalization by antigen-positive cancer cell lines was measured by internalization assays. Ten thousand NaPi2b-positive 0VCAR3 cells were seeded in a total volume of 90 pL in 96-well plates the day before treatment. Cells were identified by nuclei staining using 0.5 pg/mL Hoechst 34580 (Invitrogen #21486) dye. Hoechst 34580 was formulated at 10-fold of the final concentration of 0.5 pg/mL in cell culture medium and 10 pL was added to the cells. Plates were cultured at 37 °C in a CO2 incubator for 30 minutes. Prior to antibody treatment, Hoechst 34580 solution was removed and replaced by 90 pL cell culture medium. Test compounds were formulated at 20-fold of the final concentration of 2.5E-8 M in cell culture medium and incubated with the same amount of 20-fold concentrated Zenon™ pHrodo™ i FL Red Human IgG Labeling Reagent (Invitrogen #Z25612) at room temperature for 5 minutes in the dark. Ten pL of the test compound / pHrodo solution was added to the cells in 90 pL cell culture medium in duplicates. Plates were cultured at 37 °C in a CO2 incubator for two days. Plates were imaged for their pHrodo intensity after 0.5h, 2h, 4h, 6h, 24h and 48h with the confocal quantitative image cytometer CQ1 (Yokogawa® Electric Corporation, Tokyo, Japan). Analysis was adapted from the CQ1 software (Yokogawa) template “Nucleus and pseudo-Cell body” and FCS export files were analyzed using FlowJo (BD). Bar graphs show the pHrodo intensity of identified cells per treatment condition.

More effective internalization of ADC3 and mAb3 compared to other NaPi2b-directed ADCs

Using an image-based assay as described above with secondary-labeling of antibodies or ADCs with a pH-dependent dye (pHRodo), the internalization into cancer cell lines was investigated (Figure 20). mAb3 (the antibody moiety of ADC3) and ADC3 showed effective internalization into OVCAR3 cancer cells compared to unstained controls (untreated w/o pHRodo), controls containing the secondary label only (untreated) or ADC2_ISOTYPE_MAB. Both, mAb3 and ADC3, showed higher maximal internalization signals compared to ZW_NAPI2B_MAB and ZW_NAPI2B_MAB.

Example 8: Effects on Cancer Cell Viability in Comparison With Further ADCs

Using the method as described in the section "Viability Assay" above, the human cancer cell lines OVCAR3, JEG3, IGROV1 and HCC827 were used to compare the effects of ADC3, ADC2_ISOTYPE_ADC, exatecan, DS_CDH6_ADC (directed against cadherin 6), DS_ISOTYPE_ADC, DS payload, PRO_FRA_ADC (directed against folate receptor alpha), PRO_ISOTYPE_ADC, AZ_FRA_ADC (directed against folate receptor alpha), AZ_ISOTYPE_ADC and/or AZ payload on cancer cells with NaPi2b expression (0VCAR3) compared to the effects on cancer cells lacking NaPi2b expression (SW900, JEG3, CA0V3) (Table 5, Figure 21-23).

ADC3 inhibited NaPi2b-positve cancer cell line OVCAR3 viability with high potency of 0.01 nM.

DS_CDH6_ADC inhibited OVCAR3 cancer cell line viability with high potency of 3.8 pM. While there is a clear differentiation of DS_CDH6_ADC and DS_ISOTYPE_ADC indicating a specific effect, the magnitude of unspecific effects by DS_ISOTYPE_ADC on OVCAR3 cells is high (potency of 1.6 nM).

PRO_FRA_ADC inhibited JEG3 cancer cell line viability with a potency of 7.7 nM with limited differentiation from its isotype control (PRO_ISOTYPE_ADC).

AZ_FRA_ADC inhibited OVCAR3 cancer cell line viability with a potency of 4.7 nM with differentiation from its isotype control (AZ_ISOTYPE_ADC). Maximal effects of AZ_FRA_ADC were lower than PRO_FRA_ADC on JEG3 cells with -64% and -98%. In contrast to AZ_FRA_ADC, PRO_FRA_ADC showed limited effects on OVCAR3 cells (potency of 11 nM) without any differentiation to the isotype control (AZ_ISOTYPE_ADC).

Despite binding by the antibody moieties of PRO_FRA_ADC and AZ_FRA_ADC to IGROV1 and HCC827 cells (Figure 23), no specific viability inhibition of these cancer cells was observed (Figure 21).

Table 5: Potency of indicated ADCs, isotype controls and free payload against multiple human cell lines. Maximal effects compared to untreated controls at the highest tested compound concentration are indicated below. Geometric mean of IC50, N=3 and mean of percent effect are shown.

Example 9: Bystander Effect in Comparison With Further ADCs

The potential of ADC3, ADC2_ISOTYPE_ADC, exatecan, DS_CDH6_ADC, DS_ISOTYPE_ADC, PRO_FRA_ADC, PRO_ISOTYPE_ADC, AZ_FRA_ADC and AZ_ISOTYPE_ADC to mediate a bystander effect against antigen-negative cells in close proximity to antigen-positive cells was evaluated by bystander assays as described in the section "Bystander Assay" above (Figure 24). In line with data discussed above, ADC3 showed a potent bystander effect against NaPi2b- negative SW900 cells in the presence of NaPi2b-positive 0VCAR3.

DS_CDH6_ADC effectively reduced the number of 0VCAR3 cells, however, no bystander effect on SW900 was observed in co-culture. At the highest tested concentration of 10 nM, DS_CDH6_ADC did result in a slight reduction of SW900 cells. However, using this condition the isotype control (DS_ISOTYPE_ADC) also reduced the number of SW900 cells. Even though DS_ISOTYPE_ADC resulted in reduction of OVCAR3 cells in the co-culture, it can be concluded that DS_CDH6_ADC does not mediate a potent bystander effect.

PRO_FRA_ADC resulted in a slight reduction of OVCAR3 cells in co-culture with SW900 cells compared to its isotype control (PRO_ISOTYPE_ADC). At 10 nM the reduction of OVCAR3 cells by PRO_FRA_ADC treatment was most pronounced, however, no effect on SW900 cells was observed.

AZ_FRA_ADC resulted in a slight reduction of OVCAR3 cells in co-culture with SW900 cells compared to its isotype control (AZ_ISOTYPE_ADC). At 10 nM the reduction of OVCAR3 cells by AZ_FRA_ADC treatment was most pronounced, however, no effect on SW900 cells was observed. Compared to PRO_FRA_ADC which is directed against the same target (FRa), AZ_FRA_ADC showed a more specific reduction of OVCAR3 cells in the co-culture with 10 nM ADC treatment, as indicated by the better differentiating effect compared to its isotype control (AZ_ISOTYPE_ADC).

In conclusion, ADC3 mediates a potent bystander effect in the co-culture of OVCAR3 and SW900 cells while no bystander effect was detected for DS_CDH6_ADC, PRO_FRA_ADC and AZ_FRA_ADC.

Example 10: mAb3 and non-NaPi2b-directed ADC binding to different cancer cell lines

Flow cytometry assay

Binding of antibodies to cancer cell lines was determined by titrating the antibody on cell lines and measuring the median fluorescence intensity (MFI) of the cells after staining with an fluorescently-labeled secondary anti-human Fc (Jackson Immuno Research #709-116-098) or anti-human IgG antibody (Jackson ImmunoResearch #711-116-152). Antibody binding capacity was conducted using Quantum Simply Cellular anti-Human IgG Kit (Bangs Laboratories #816B) according to the manufacture’s instructions. Target cell binding and antigen-binding capacity determination

Using flow cytometry as described in the section "Flow cytometry assay" above, effective binding of mAb3 to OVCAR3 cancer cells was confirmed. As can be seen from Fig. 25, maximal binding signals for mAb3 were higher than for PRO_FRA_MAB and AZ_FRA_MAB. mAb3 did not bind to NaPi2b-negative cell lines JEG3, IGR0V1 and HCC827.

For PRO_FRA_MAB and AZ_FRA_MAB, binding to JEG3, IGR0V1 and HCC827 was confirmed.

Example 11 : Effective internalization of ADC3 and mAb3 compared to non-NaPi2b- di reefed competitors

Using an image-based assay with secondary-labeling of antibodies or ADCs with a pH- dependent dye (pHRodo), the internalization into cancer cell lines was investigated. MAB3 and ADC3 showed more effective internalization into 0VCAR3 cancer cells compared to DS_CDH6_MAB, PRO_FRA_MAB and AZ_FRA_MAB (Figure 26).

Example 12: In Vivo Efficacy Study in Ovarian cancer PDX model OVPF174

32 NU/NU female mice (Vr:NU-Foxn1 nu, 5-6 weeks old, Beijing Vital River Laboratories) received 3-6 pieces tumor fragment (10-20 mg/piece) of a patient derived xenograft model (OVPF174, Ovarian cancer, poorly differentiated primary carcinoma) mixed with 15-50 l Matrigel subcutaneously into the right flank area.

Tumor growth and body weight was measured twice weekly using caliper. Tumor volume was calculated using the formula TV = L x Wx W/ 2.

When tumor volume reached 100-300 mm3, mice were divided into 4 groups (N=8) based on the tumor volume to ensure each group had the same mean tumor volume (Day 0). Assigned animals were then treated three times every 14 days (Q2Wx3) intravenously with either vehicle (10 mM Histidine, 8 % Trehalose, 5 mM Methionine, 0.05 % Polysorbate 20, pH 5.5) or ADC3 at dosing levels of 1 , 2, and 6 mg/kg.

Tumor growth was followed for 91 days (Figure 27).

The statistical analysis revealed that significant tumor growth inhibition was achieved compared to vehicle treated group for all tested dosing levels of ADC3 (Table 6). Among the different tested dosing levels of ADC3, a statistically significant different treatment effect on tumor growth was achieved for 2 or 6 mg/kg compared to 1 mg/kg but not between 2 and 6 mg/kg (Table 6). Statistical analysis of tumor volumes between treatment groups was done using repeated measures data in a mixed-effects model followed by Tukey's multiple comparisons test (GraphPad Prism 10.2.1).

All mice in this study were used in accordance with the requirements for the humane care and use of animals and approved by LIDE Institutional Animal Care and Use Committee (IACUC).

Table 6:

Example 13: In Vivo Efficacy Study in Ovarian cancer PDX model QVPF070

32 NU/NU female mice (Vr:NU-Foxn1 nu, 5-6 weeks old, Beijing Vital River Laboratories) received 3-6 pieces tumor fragment (10-20 mg/piece) of a patient derived xenograft model (OVPF070, Ovarian cancer, poorly to moderate differentiated primary carcinoma) mixed with 15-50 pl Matrigel subcutaneously into the right flank area.

When tumor volume reached 100-300 mm3, mice were divided into 4 groups (N=8) based on the tumor volume to ensure each group had the same mean tumor volume (Day 0). Assigned animals were then treated three times every 14 days (Q2Wx3) intravenously with either vehicle (10 mM Histidine, 8 % Trehalose, 5 mM Methionine, 0.05 % Polysorbate 20, pH 5.5) or ADC3 at dosing levels of 4, 8, and 16 mg/kg.

Tumor growth was followed for 94 days (Figure 28).

The statistical analysis revealed that significant tumor growth inhibition was achieved compared to vehicle treated group for tested dosing levels of ADC3 at 8 & 16 mg/kg (Table 7). A significant dose-dependent different treatment effect on tumor growth was achieved among all tested dosing levels of ADC3 (Table 7).

Statistical analysis of tumor volumes between treatment groups was done using repeated measures data in a mixed-effects model followed by Tukey's multiple comparisons test (GraphPad Prism 10.2.1).

Table 7:

Example 14: Pharmacokinetic (PK) Studies in Cynomolgus Monkeys

ADC3 was administered in Cynomolgus monkeys to assess pharmacokinetic properties in non-human primates. The test item was administered intravenously at 3 mg/kg and conjugated antibody and conjugated payload have been measured as analytes.

By measuring conjugated antibody, the concentration of antibody with an attached payload is measured. For such measurements, a three-step immunoassay sandwich method was applied. A Biotinylated anti-exatecan antibody was used as capture reagent and an labelled anti-human kappa light chain antibody was used as detection reagent.

By measuring conjugated payload, the concentration of payload with an attached antibody is measured. For such measurements, the ADC was captured with streptavidin magnetic beads, contaminants were washed away, the payload was released by protease digestion and the resulting mixture analyzed by LC-MS/MS. Samples were taken at 0, 0.167, 2, 6, 24, 48, 96, 120, 168, 240, 336, 504, 672, 840 and 1008 hours after injection. Data were pooled for PK analysis, see Table 8.

ADC3 exhibited an outstandingly low clearance and long half life. Table 8:

Claims

1. Antibody-drug conjugate (ADC), wherein the ADC is wherein n is the number of [(linker)-(exatecan)] moieties covalently linked to the antibody; and wherein n is preferably between 1 and 10 and more preferably about 4, and the antibody binds to NaPi2b.

2. An ADC according to claim 1 with the antibody portion of the ADC having a light chain amino acid sequence of SEQ ID NO:1 and a heavy chain amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, preferably SEQ ID NO:4.

3. A pharmaceutical composition comprising the ADC of claims 1 or 2.

4. An ADC according to claims 1 or 2 for the treatment of cancer, preferably ovarian cancer or NSCLC.